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Sickle cell anemia (SCA) is a monogenic disease of high mortality, affecting millions of people worldwide. There is no broad, effective, and safe definitive treatment for SCA, so the palliative treatments are the most used. The establishment of an in vitro model allows better understanding of how the disease occurs, besides allowing the development of more effective tests and treatments. In this context, iPSC technology is a powerful tool for basic research and disease modeling, and a promise for finding and screening more effective and safe drugs, besides the possibility of use in regenerative medicine. This work obtained a model for study and treatment of SCA using iPSC. Then, episomal vectors were used for reprogramming peripheral blood mononuclear cells to obtain integration-free iPSC. Cells were collected from patients treated with hydroxyurea and without treatment. The iPSCP Bscd lines were characterized for pluripotent and differentiation potential. The iPSC lines were differentiated into HSC, so that we obtained a dynamic and efficient protocol of CD34+CD45+ cells production. We offer a valuable tool for a better understanding of how SCA occurs, in addition to making possible the development of more effective drugs and treatments and providing better understanding of widely used treatments, such as hydroxyurea.
Sickle cell anemia (SCA) is one of the most common hereditary hematological diseases in the world, reaching a significant proportion of the population in different countries. It is particularly common among people whose ancestors came from sub-Saharan Africa, South America, Cuba, Central America, Saudi Arabia, India, and Mediterranean countries such as Turkey, Greece, and Italy. In the United States, the disease affects about 72,000 people and occurs in about 1 in 500 African Americans born and every 1 in 10001400 Hispanic Americans born [1] (WHO, http://www.who.int/). In Latin America, 8% of Afro-descendents have the mutated gene, which occurs in 1 every 10004000 Hispanic-American births [2]. In Brazil, it is the most prevalent hereditary disease, with about 1 carrier per 1500 born, with 700 to 1000 new cases per year; it is estimated that there are more than 2 million HbS gene carriers and more than 3000 affected with the homozygous form (Ministry of Health, http://www.saude.gov.br). Described in 1910 by Herrick [3], SCA is a hereditary, monogenic, autosomal codominant inheritance, resulting from a recessive mutation in the -globin gene, located in the chromosomal region 11p15.5. Replacement of a single nucleotide changes the codon of the sixth amino acid, from glutamic acid to valine (GAG GTG: Glu6Val). This mutation causes an abnormal hemoglobin, called hemoglobin S (HbS) [4, 5]. It manifests with injuries in several organs, causing high morbidity and mortality, approximately 3.4% of deaths in affected children under 5 years [6]. Infections are the main cause of morbidity and mortality in SCA, particularly in childhood [7].
Although monogenic, being defined by a single change in a specific nucleotide of genomic DNA, the clinical manifestations of SCA are extremely variable among individuals; while some patients have a very serious condition and are subject to numerous complications and frequent hospitalizations, with a high mortality rate, others present a more benign, in some cases, almost asymptomatic evolution. Hereditary and acquired factors contribute to this clinical variability, such as fetal hemoglobin (HbF) levels and socioeconomic status. However, these factors relate to more severe forms or not but do not explain these variations in their entirety.
Some available treatments include, for example, the use of hydroxyurea, the first drug approved for the treatment of sickle cell anemia. This chemotherapeutic agent acts by reactivating the production of fetal hemoglobin (HbF), a form present in newborns, and recent studies show an increase in patient survival [8]. However, the use of this drug, which only controls the symptoms, can cause side effects such as myelosuppression, particularly the granulocytic series, and the possibility of increasing the risk of tumor development, which increases even more with the long time of use [9]. The only potentially curative treatment for sickle cell anemia is hematopoietic stem cell (HSC) transplantation, with the goal of replacing the patients bone marrow with cells without the mutation [10]. However, this is a risky procedure with high morbidity and mortality, which presents the risk of developing graft versus host disease (GVHD), making it recommended only for the most severe cases.
Induced pluripotent stem cell (iPSC) can originate any cell type and represent an alternative source to derive patient-specific blood cells. IPSC technology emerged in 2006 as a powerful tool for basic research, tissue differentiation research, and disease modeling and a promise for future clinical applications to find and screen new, more effective and safe drugs, besides the possibility of use in regenerative medicine, in the production of patient-specific cells for cell therapy. As iPSC can be expanded indefinitely in vitro and can differentiate into hematopoietic cells with blood reconstitution capacity [11, 12], this technology provides a great chance to improve the results of bone marrow transplantation, since it provides an unlimited number of immunologically compatible HSCs [13, 14]. Patients with monogenic hematological diseases, such as SCA, would be the major beneficiaries of the bone marrow transplantation procedure based on iPSC. The treatment of SCA in murine model using iPSC corrected by gene editing provided a proof of concept that the clinical application of iPSC to treat genetic blood disorders is possible.
Although iPSCs were initially obtained from fibroblasts using retroviral vectors, multiple strategies were developed for generation of iPSC without integration from fibroblasts and other cell types, including blood cells [15, 16]. The most used cells in the reprogramming have been the skin fibroblasts with the use of viral vectors [1720], whose protocol to obtain iPSC is very well defined. However, the need for skin biopsies and the expansion of these cells by multiple in vitro passages represent a challenge that must be overcome to make iPSC technology widely applicable. Evidence obtained from several recent studies indicates that other human somatic cells can be used to generate human iPSCs, such as keratinocytes, hepatocytes, neural progenitor cells, and adipose tissue stem cells, among others [21]; but obtaining large amounts of these cell types is still a very time-consuming procedure. An easily accessible tissue that can be obtained using less invasive procedures is peripheral blood. Circulating T cells can be easily obtained from peripheral blood and can be induced to proliferate by stimulation with cytokines [22]. However, the use of these cells for reprogramming should be avoided because they are cells that do not have their genome intact, due to somatic rearrangements, creating prereorganized V(D)J DNA segments. Recently, episomal plasmid-based protocols have demonstrated the generation of integration-free iPSC from human bone marrow and umbilical cord cells [23, 24]. Although reprogramming of peripheral blood cells has a lower efficiency [23], it represents a much more affordable and abundant source of patient cells for reprogramming without the need for extensive maintenance in culture [25].
The iPSC has been used as an experimental platform for in vitro disease model. Several groups have demonstrated that cell types related to a disease are differentiated from iPSC and can faithfully reproduce the disease phenotypes [2629]. The discovery of iPSCs has opened up a new possibility for the development of in vitro disease models in humans for the investigation of pathophysiology and for aiding drug development [3034], and these, in the short term, are the most important aspects of this technology. The potential use of iPSC as a treatment of diseases has been proposed and tested in animal models in vitro and in vivo with promising results [3537].
With the iPSC field progressing so rapidly, the next challenge will be to demonstrate the functional utility of iPSC-derived cells in preclinical models of various human diseases and eventually move that technology to the clinic [38]. In recent years, great progress has been made in the development of hematopoietic differentiation systems and the production of major blood cell types from human pluripotent stem cells (hPSCs) [14]. Even so, the generation of hematopoietic cells with robust and long-term reconstitution potential remains a major challenge.
In this study, we explored the use of a nonintegrating episomal vetor to reprogram adult blood cells from sickle cell anemia patients and establish a study and treatment model for SCA, through the establishment of an efficient protocol for obtaining hematopoietic progenitor cells. iPSC generated can be used in the future to correct the -globin gene mutation and differentiate into hematopoietic cells. The methodology developed in this study has potential applications in iPSC banking and in disease modeling for other genetic disease, so in the future, these iPSCs could be used in regenerative medicine. Establishing an in vitro model of the disease allows a better understanding of how the disease occurs, the possible causes of the clinical differences demonstrated by the patients affected, and the development of new tests and more effective treatments against the disease. IPSCs, while still unsuitable for clinical use, have the potential to revolutionize the way we study human development, generate life-threatening disease models, and eventually how we treat patients.
The cells reprogrammed in this study were obtained through peripheral blood collection of sickle cell anemia patients from blood center of Ribeirao Preto, after acceptance and signature of the informed consent. The protocol was analyzed and approved by the local ethics committee (number: 486.426). The inclusion criteria were age over 18 years, homozygous for the S allele, and absence of treatment with hydroxyurea. About 12mL of blood was collected from a superficial vein of the arm, after antisepsis of the puncture site, by a qualified professional. Peripheral blood MNCs were separated by Ficoll-Hypaque Premium (GE Healthcare) density gradient, according to the manufacturers instructions and as described [39, 40]. The cells were freezed at a concentration of 1107 cells per vial in fetal bovine serum (Hyclone) supplemented with 20% dimethyl sulfoxide [DMSO (Sigma-Aldrich)]. Of the MNC obtained, 1-2106 was cultured with StemSpan medium (STEMCELL Technologies) supplemented with the following cytokines: 100ng/mL stem cell factor (SCF), 10ng/mL IL-3, 2U/mL erythropoietin (EPO), 40ng/mL insulin-like growth factor 1 (IGF-1) (all from Peprotech), and 1g/mL dexamethasone (Sigma-Aldrich). The cells were counted, and the medium was changed every 3 days. The day 12 and day 14 cultured cells were used for reprogramming. This culture condition favors the enrichment erythroblastic population and does not support the growth of the lymphoid population. After 3 days of thawing and after 12 days of expansion, the cells were evaluated for lymphoid cell markers and erythroblast/erythroid cell markers by flow cytometry.
The MNCs cultured in MNC medium for 1214 days were used for the reprogramming process by the transfection of the episomal plasmids, pEB-C5, expressing five factors (OCT4, SOX2, KLF4, C-MYC, and LIN28), and pEB-Tg, expressing SV40 large T antigen, in 2.5106 cells [33]. These episomal plasmids are available from Addgene (plasmid numbers 28213 [pEB-C5] and 28220 [pEB-Tg]). The transfection was performed using the Lonza human CD34+ cell nucleofector kit (Lonza) on the Amaxa nucleofector II device (Lonza), with a total of 2.5106 cells at a cell suspension of 100 L combined with 8g of DNA of the pEB-C5 plasmid (1g/L) and 2g of DNA of the pEB-Tg plasmid (1g/L). After transfection, the cells were plated back in the expansion medium in one well of a 12-well plate to allow recovery and were incubated at 37C, 5% CO2, 5% O2. Two days later, they were plated onto standard plates coated with feeder cells, with the culture medium changing to ESC medium [20% knockout serum replacement, 2mM L-glutamine, 0.1mM nonessential amino acids, 0.1mM -mercaptoethanol, 50U/mL penicillin, 50g/mL streptomycin, and 10ng/mL basic fibroblast growth factor (bFGF) in Knockout Dulbeccos modified Eagles medium (Knockout DMEM) (all from Gibco)] the following day. At this point, we added sodium butyrate (NaB) (0.25mM) (Sigma-Aldrich) to the cultures to enhance iPSC derivation. The medium was changed every other day, until small colonies begin to appear, when the medium started being changed every day. On day 9 after transfection, the cells started to being fed with conditioned medium (CM). The use of CM is necessary at this time to maintain colony growth. For the production of CM, we used a 12-well plate with mitotically inactivated MEFs plated at a density of ~100,000 cells per well with ESC medium. Every day, we collected and replaced the ESC medium. CM was collected daily for 4-5 days, as long as the MEF morphology was acceptable.
During days 911 after transfection, colonies with ESC-like morphology start to become visible and, during days 14-15, large colonies were picked, expanded, and examined for pluripotency markers. The colonies were picked manually under an inverted microscope after treatment with 0.5mM EDTA. After the first picking, the human iPSCs were maintained and expanded in mTeSR1 medium (STEMCELL Technologies) on matrix Matrigel (Corning). The expanded colonies were evaluated for pluripotency and self-renewal characteristics and differentiation potential.
Total RNA from iPSC, from MNC, and from ESC was purified with Trizol reagent (Invitrogen) and treated with DNase using the RNeasy mini kit (Qiagen, Hilden, Germany). Two micrograms of total RNA was used for reverse transcription reaction using the high-capacity cDNA reverse transcription kit (Applied Biosystems) according to the manufacturers instructions. Quantitative polymerase chain reaction (qPCR) was performed by TaqMan (Applied Biosystems) and analyzed with the 7500 real-time PCR system (Applied Biosystems). All experiments were performed in duplicate, and a nontemplate control (lacking cDNA template) was included in each assay. Gene expression was normalized relative to that from endogenous gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and -actin (Applied Biosystems). The primer sets used for detecting ESC marker gene expression were the pluripotency markers OCT4, SOX2, and NANOG (Applied Biosystems).
Reprogrammed cells were fixed in 4% paraformaldehyde (Merck) for 20 minutes at room temperature, washed twice with phosphate-buffered saline, and then permeabilized with 0.2% Triton X-100 (Sigma-Aldrich) for 1 hour at room temperature. The cells were blocked for 1 hour with 5% bovine serum albumin and 2% goat serum in phosphate-buffered saline solution. Samples were incubated at room temperature for 1 hour with primary antibodies against antistage-specific embryonic antigen 4 (SSEA4) (STEMCELL Technologies), octamer-binding transcription factor 4 (OCT4) (Chemicon), sex-determining region Y-box 2 (SOX2), and NANOG homeobox (NANOG). The secondary antibodies used were AlexaFluor 564 mouse antibodies (Molecular Probes, Invitrogen), incubated at room temperature for 1 hour. The nucleus was stained with 4,6-diamidino-2-phenylindole (VYSIS). Cells were visualized using a confocal laser scanning microscope (LSM 710; Carl Zeiss) with objective lens 63 in oil immersion, having a numerical aperture of 1.4. Argon laser of 590nm was used to excite the surface marked with secondary antibodies, and the emission was measured at 617nm. Image analysis and colocalization studies were carried out using the ZEN 2008 system confocal software (ZEN, version 2.5).
The immunophenotypic characterization was perfomed by flow cytometry using the following monoclonal antibodies: NANOG-fluorescein isothiocyanate (FITC), OCT3,4-phycoerythrin (PE), and SOX2-PE (Pharmingen).
Cells were harvested with StemPro Accuttase Cell Dissociation Reagent (Gibco) and were incubated with the antibodies following the manufacturers instructions. Nonspecific immunoglobulin G of the corresponding class served as the negative control. Cell suspensions were analyzed on FACSort flow cytometer (Becton-Dickinson) using CellQuest software.
Alkaline phosphatase (AP) staining was performed with the AP leukocyte alkaline phosphatase kit (Sigma-Aldrich) according to the manufacturers instructions. High levels of AP expression indicate undifferentiated cells with self-renewal potential. Reddish pink-stained cells were classified as positive for AP expression [41].
For spontaneous differentiation through embryoid body (EB) formation, reprogrammed cells were harvested by 0.5mM EDTA treatment. The cell clumps were transferred to a 6-well low adhesion plate in mTeSR1 medium (STEMCELL Technologies). After 5 days in suspension culture, EBs were transferred to a 6-well gelatin-coated plates (0.1%) and cultured in DMEM suplemmented with 10% FBS, 1% antibiotic-antimycotic, and 2mM L-glutamine (all from Gibco) for 15 additional days, changing medium every other day. This condition allows EB cells to spontaneously differentiate in vitro. After 15 days, the cells were collected for RNA extraction and qPCR analyses. For detecting the diferentiation potential in the formed EB, we used Nestin, alpha-smooth muscle actin (-SMA) and alpha-fetoprotein (AFP) (Applied biosystems) markers for the identification of cells from ectoderm, mesoderm, and endoderm, respectively.
The evaluation of the differentiation potential was performed in vivo by the teratoma formation assay in immunodeficient mice. Approximately 2106 iPSC clumps were subcutaneously injected into NOD/SCID Gamma mice. After approximately 1012 weeks, mice were sacrificed and tissues were analyzed for tumor formation. The histological processing of the teratoma, preserved in 10% formalin for 24 hours, comprised a dehydration battery followed by clarification and immersion and inclusion in paraffin. The sections were fixed, clarified, and dehydrated for staining with hematoxylin and eosin for morphological analysis. The sections were subjected to a new dehydration and clarification, and the slides were set up with Entellan (Merck Millipore) adhesive gel.
The sections for immunohistochemistry assay were incubated at 50C overnight. For immunostaining, the sections passed through a battery of clarification and dehydration. The antigen retrieval was performed with citrate buffer (10mM, pH 6), and peroxidase blockade was performed with 3% H2O2 diluted in methanol. Blocking of nonspecific binding was performed with PBS-0.25% Tween +5% BSA. The antibody was applied, diluted in 0.1% PBS-Triton + 1% BSA, and incubated overnight. The biotinylated secondary antibody [Universal Dako Cytomation Labelled Streptavidin-Biotin System, Horseradish Peroxidase (Dako)] was applied and incubated, followed by applying the tertiary antibody streptavidin (supplied with the kit described above). The DAB (supplied with the kit described above) was diluted and applied, followed by the application of hematoxylin. The sections were subjected to a new dehydration and clarification, and the slides were set up with Entellan (Merck Millipore) adhesive gel. Sections were analyzed by immersion microscopy.
We used the TaqMan hPSC Scorecard (Applied Biosystems) methodology, which contains specially formulated gene expression assays for the evalution of iPSC and CTE, to confirm the self-renewal potential and predict the differentiation potential of the lineages generated. Total RNA from iPSC, from MNC, and from ESC was purified with Trizol reagent (Invitrogen) and treated with DNase using the RNeasy mini kit (Qiagen). One microgram of total RNA was used for reverse transcription reaction using the high-capacity cDNA reverse transcription kit (Applied Biosystems) according to the manufacturers instructions, and the amplification conditions are 25C for 10 minutes, 37C for 120 minutes, and 85C for 5 minutes. The prepared cDNA samples were diluted with nuclease-free water and 2X TaqMan Fast Advanced Master Mix, according to the manufacturers instructions. The mixture was distributed on the 96-well plate and, after sealing and centrifugation, the plate was cycled in equipment suitable for fast thermocycling following the amplification parameters: 1 cycle at 50C for 20 seconds and 40 cycles at 95C for 1 second and 60C for 20 seconds. Gene expression data were analyzed using the hPSC Scorecard analysis software available online (http://lifetechnologies.com/scorecarddata).
To evaluate the iPSC DNA for the presence of spontaneous integrations of the episomal plasmids used in the generation of the cells, we performed the screening through PCR with two sets of primers designed from sequences pre-established in literature [42] and synthesized for amplification and identification of each one of the vectors used in cell generation. The analyzed cells were iPSC generated at different passages, which were dissociated by enzymatic treatment with the StemPro Accutase dissociation reagent (Gibco). The cell suspension was collected, washed with PBS (Gibco), and used for DNA extraction with Dneasy Blood & Tissue kit (Qiagen), according to the manufacturers instructions. After extraction, the DNA was quantified in spectrophotometer (Nanodrop-Spectrophotometer ND-1000) at 260nm.
As positive control, we used expanded MNCs which were nucleofected with the two vectors, pEB-C5 and pEB-Tg, as described in item above. Transfected cells were incubated for for 48 hours and then were collected for DNA extraction as described above. The set of primers are shown in Table 1.
For the amplification reaction, we used 10pmols of each primer, 1L of 10mM dNTP, 5L of 10 buffer (200mM Tris, pH 8.4, 500mM KCl) (Invitrogen), 1.5L of 50mM MgCl2 (Invitrogen), and 0.2L of 5U/L Taq DNA Polymerase (Invitrogen) in a final volume of 50L. The reactions were thermocycled in MyCycler Thermal Cycler (Bio-Rad) with the following amplification program: 94C for 10 minutes; 35 cycles of 94C for 45 seconds, Ta appropriate for each set of primer for 45 seconds and 72C for 90 seconds; a final extension of 72C for 7 minutes. The amplification evaluation was performed by agarose gel electrophoresis 1.5% stained with ethidium bromide.
For the molecular identification of the mutation responsible for the sickle cell phenotype, the generated iPSC lines were subjected to amplification of the -globin gene (HBB) and subsequently submitted to sequencing. The cells used were the iPSC lines generated and a negative control of the reaction without any sample. The primers used in the amplification reactions were HBB forward (5-GAAGAGCCAAGGACAGGTAC-3) and HBB reverse (5-CAACTTCATCCACGTTCACC-3).
For the amplification reaction of the HBB gene, we used 0.06g of DNA, 1L dNTP 10mmol/L (Invitrogen), HBB forward 10pmols, HBB reverse 10pmols, 5L of 10 enzyme buffer (200mmol/L Tris, pH 8.4, 500mmol/L KCl) (Invitrogen), 2L of MgCl2 (50mmol/L) (Invitrogen), and 0.2L of Taq DNA polymerase (5U/L) (Invitrogen) in a final volume of 50L. This reaction was thermocycled in MyCycler Thermal Cycler (Bio-Rad) with an amplification program of 94C for 10 minutes; 35 cycles of 94C for 45 seconds, 55C for 45 seconds, and 72C for 90 seconds; and a final extension of 72C for 7 minutes.
To verify the amplification of the HBB gene, 1.5% agarose gel electrophoresis stained with ethidium bromide was performed.
The amplified samples were submitted to sequencing of the HBB gene to locate the Glu6Val mutation responsible for the sickle cell phenotype. For the sequencing of a sample of each generated iPSC line, a new PCR reaction was performed under the same conditions described above. The product of this first PCR reaction for sequencing was subjected to a second reaction in which 1l of this was added to 2l of Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and 5pmols of primer HBB forward or 5pmols of primer HBB reverse, in a final volume of 10L. These reactions, made in triplicate each, were thermocycled in the MyCycler Thermal Cycler (Bio-Rad), using the amplification program of 95C for 1 minute, 25 cycles of 95C for 10 seconds, 51C for 5 seconds, and 60C for 4 minutes. Then, a precipitation of the PCR product following the ABI/Isopropanol (MERCK) protocol was performed, for the elimination of unincorporated dNTPs, ddNTPs, and enzymes. The samples were submitted to electrophoresis in the automatic sequencer ABI 3500XL Genetic analyzer (capillary electrophoresis system), using Sangers method, based on chain termination chemistry with dideoxynucleotides (ddNTPs). In the form of an electropherogram, the DNA sequences generated were directly sent to a computer connected to the sequencing apparatus. These electropherograms were interpreted by the software DNA Sequencing Analyzer 4.0 and converted into DNA sequences, which were later analyzed using the ChromasPro program.
The iPSCs obtained were differentiated into hematopoietic progenitors/hematopoietic stem cells (HSC) using a combination of available protocols [43, 44]. Initially, iPSCs underwent an adaptation step to enzymatic picking with StemPro Accutase dissociation reagent (Gibco) supplemented with ROCk inhibitor (Tocris) at 37C. The cell suspension was washed with PBS (Gibco), counted using Neubauers chamber and 0.4% Trypan Blue solution (Gibco), and plated at high cell concentration. This culture was maintained for at least 4-5 passages until identification of complete adaptation, that is, when cells after individualization by treatment with dissociation reagent are again grouped into colonies.
After adaptation, the cells were again dissociated and counted, and approximately 4000 cells were plated per well in a low-adhesion 96-well plate in STEMdiff APEL (STEMCELL Technologies) differentiation medium supplemented with the BMP4 (10ng/mL) (Peprotech) and bFGF (10ng/mL) (Peprotech) cytokines, in addition to the ROCK inhibitor (10mM) (Tocris), in a final volume of 50L per well. The plates were centrifuged at 300 g for 5 minutes to form aggregates and incubated at 37C and 5% CO2. The experiment was conducted for 14 days, with fresh medium additions every 3 days. The fresh medium was supplemented with the cytokines BMP4 (10ng/mL) (Peprotech), bFGF (10ng/mL) (Peprotech), VEGF (1020ng/mL) (Peprotech), and SCF (50100ng/mL) (Peprotech). On differentiation induction days 0, 4, 8, 11, and 14, cells were collected for evaluation of pluripotency and hematopoietic progenitors markers by flow cytometry. For the analyses, the EBs were collected, centrifuged at 160 g for 4 minutes, and dissociated with StemPro Accutase (Gibco) dissociation reagent at 37C for about 1520 minutes. The dissociated cells were homogenized using 22-gauge needle, centrifuged at 250 g for 4 minutes, and resuspended in 1mL PBS (Gibco), and an aliquot was took out for counting.
On day 11 of hematopoietic induction, EBs were also collected for colony-forming cell (CFC) assay in methylcellulose. For the assay, 2105 cells (140L) were homogenized with 3mL of methylcellulose medium with recombinant cytokines [MethoCult H4434 classic (StemCell)]. The mixture was distributed in 3 plates of 35mm, 1mL/plate, which were incubated at 37C for about 15 days, when they could be analyzed for colony formation.
MNCs were isolated from six patients with sickle cell anemia (SCA), after diagnostic confirmation of SCA, four males and two females, three in treatment with hydroxyurea (HU) and three who were not under treatment with HU, ranging in age from 19 to 32 years. The Ficoll-Hypaque processing of the samples resulted in a yield of about 13107 cells. Cells were frozen at a concentration of 5106 cells/vial; each patient sample generated approximately 26 vials. Figure 1 shows a schedule of the reprogramming process used to generate iPSC from MNC.
The MNCs were expanded in StemSpan medium containing a combination of SCF, IL-3, IGF-1, EPO, and dexamethasone for 1214 days for lymphocyte depletion and erithroblast expansion. This expansion was aimed at reducing the risks of reprogramming a cell of the lymphoid lineage. The expansion comprised about 1214 days, with medium changes and counts every 3 days. For the cells to maintain adequate growth and to have no limitation of their growth, they were maintained at a concentration of 2106 cells/mL. Cell growth was assessed throughout the expansion process. MNCs from patients treated with HU showed lower cellular expansion than those from untreated patients (Figure 2). Patient samples not treated with HU showed a cell growth of 55% (19.5) relative to the initial cell percentage. In cells of patients treated with HU, not even the initial cell population was reached after consecutive falls on days 3 and 6 of culture in specific medium. After 3 days of MNCs thawing and after 12 days of expansion in specific medium, cells were evaluated for lymphoid cell markers and erythroblast/erythroid cell markers by flow cytometry (Figure 3). It was found that a large population resemble cells of the erythroblast/erythroid line, expressing high levels of CD71 (88.5%5.5%) and CD235a (85.3%6.3%) that are not found at the beginning of the expansion (CD71: 3.5%0.5%, CD235a: 3.7%0.17%). There was also a decrease in the cell population expressing T cell markers [CD4 (4.6%1.6%) and CD8 (3%2.4%)], B cells [CD19 (0.9%0.2%)], and common leukocyte markers [CD45 (16%6%)], when compared to the cells at the start of induction. Initial expression of these markers in the cells was high for T cells [CD4 (54%3.7%) and CD8 (30%5%)], B cells [CD19 (6.7%0.7%)], and leukocytes [CD45 (99.8%0.02%)] (data were expressed as mean standard deviation).
On day 12 or 14, the expanded cells (2.5106) were transfected with episomal vectors carrying mouse OCT4, SOX2, KLF4, C-MYC, LIN28, and SV40 large T. The first colonies were observed aproximately 9 days after transfection and picked up by days 14-15. Large changes were observed in the morphology of cells that changed from small, rounded, individualized cells that grow in suspension to epithelial cell morphology, characterized by small, juxtaposed, colony-growing ESC-like cells (Figure 4). Six patient samples were submitted to erythroblast expansion followed by transfection with plasmids for reprogramming. From these samples, we obtained 4 pluripotent colonies, 3 samples from patients not being treated with HU and only 1 patient sample being treated with HU. The only patient sample on treatment with HU that was reprogrammed (PBscd08HU) had a low number of colonies (only 4), which impacted in reprogramming efficiency (0.0011%) (Table 2). The other two samples from patients on HU treatment (PBscd06HU and PBscd07HU) were submitted to erythroblast expansion and transfection; however, after extensive culture in ESC medium, they did not present colony formation. Table 2 shows the reprogramming efficiency of MNCs by episomal vectors and summarizes all details of the 6 experiments. Regarding reprogramming, we found that cells derived from patients not treated with hydroxyurea had an 11-fold improvement in the generation of colonies compared to cells derived from treated patients.
The lineage generated from MNCs from patients on treatment with HU, iPSC PBscd08HU generated 4 colonies that were used to clonal picking due to the low number of them. For the isolation of the clones, one colony at a time was picked and transferred to a 24-well plate, one colony per well, previously covered with matrix and with mTeSR1 medium. Three clones of the iPSC line PBscd08HU, clones 02, 03, and 04, were generated after expansion. These clones, as well as the colonies of the other lines obtained, were expanded in matrix and after successive passages were characterized for the pluripotent and differentiation potential.
PBscd iPSCs, generated as described above, robustly proliferated under human iPSC/ESC culture conditions, remaining undifferentiatiated and morphologically alike for more than 100 passages. PBscd iPSC colonies showed a morphology of juxtaposed cells of growth in colonies, characteristic of human pluripotent stem cells (Figure 5). All the samples used to reprogram show colonies with ESC-like morphology.
The complete characterization was performed for the first lineage generated, iPSC PBscd01, to certify that the method could generate pluripotent colonies. This characterization included the evaluation of the pluripotency by flow cytometry, qRT-PCR, immunocytochemistry, alkaline phosphatase assay, assessment of the differentiation potential in vitro through the formation of embryoid bodies, differentiation potential prediction by the TaqMan hPSC Scorecard methodology, in addition to the teratoma formation assay for the assessment of the differentiation potential in vivo, and evaluation of the sections by immunohistochemistry and HE. For the following lines, the pluripotent potential was characterized by evaluation of the markers by flow cytometry and the characterization of the potential of differentiation was performed through the formation of teratomas. Table 3 summarizes the characterization made with each iPSC line.
The iPSC PBscd01 flow cytometry characterization showed that approximately 73% of the cells are OCT4 positive, 77% of the cells express NANOG, and 81% are positive for SOX2 (Figure 6(a)). The MNCscd01 flow cytometry characterization with pluripotency markers showed that the labelling is specific to iPSC (Figure 6(a)). qRT-PCR assay was used to quantify the expression of the pluripotent genes OCT4, SOX2, and NANOG. The iPSC PBscd01 express 5000, 120,000, and 600 times more OCT4, SOX2, and Nanog, respectively, than the parental line MNCscd01, and even higher levels of expression than those observed in the hESC samples (Figure 6(b)). Immunostaining of iPSC PBscd01 colonies also showed expression of pluripotency markers OCT4, SOX2, and NANOG (Figure 6(c)). The iPSC PBscd01 also demonstrated high expression of alkaline phosphatase, characterized by reddish pink or purplish red cells when fixed and stained, indicating that they are undifferentiated cells with self-renewal potential (Figures 7(b) and 7(c)), different from the somatic cells and fibroblasts, used as negative control (Figure 7(a)).
For the other generated iPSC PBscd lines (iPSC PBscd02, iPSC PBscd03, iPSC PBscd08cl02HU, iPSC PBscd08cl03HU, and iPSC PBscd08cl04HU), we evaluated the pluripotent potential by flow cytometry characterization of pluripotency markers OCT4, SOX2, NANOG, and SSEA-4 (Figure 8). IPSC PBscd02 showed 86% of the cells positive for NANOG, 81% positive for OCT4, and 89% positive for SOX2; iPSC PBscd03 showed positivity for NANOG in 87.6% of the cells, for OCT4 in 46% of the cells, and for SOX2 in 85% of the cells (Figure 8(a)). The clone 02 of iPSC PBscd08HU showed 60% of the cells positive for the surface marker SSEA-4, 93% positive for NANOG, 92% positive for OCT4, and 94.5% positive for SOX2; the clone 03 of iPSC PBscd08HU showed positivity for SSEA-4 in 81.8% of the cells, for NANOG in 94.6% of the cells, for OCT4 in 61.3% of the cells, and for SOX2 in 99% of the cells; and the clone 04 of iPSC PBscd08HU showed 80.3% of the cells positive for SSEA-4, 97.3% positive for NANOG, 75.4% positive for OCT4, and 99% positive for SOX2 (Figure 8(b)). Thus, all lines showed high percentages of positive cells for pluripotency markers, confirming the pluripotent potential of these cells.
We further investigated whether PB iPSC can be induced to differentiate into cells of different lineages in culture. IPSC PBscd01 could form spherical structures called embryoid bodies (EB) when cultured under specific conditions (Figure 9(a), I-II). EBs are spherical structures formed in vitro from pluripotent cells, which harbor the development of various cell types and tissues. After the EB formation, the cells were cultured for adhesion and spontaneous differentiation (Figure 9(a), III-IV). Gene amplification by quantitative real-time PCR demonstrated that after differentiation, the cells express ectoderm marker, Nestin; the endoderm marker, AFP; and the mesoderm marker, SMA, at higher levels than ESC H1 and MNCs (Figure 9(b)).
IPSC PBscd01 were also evaluated for in vivo differentiation ability by the teratoma formation assay. Teratomas are benign tumors formed by several cell types and tissues, considered an essential tool to evidence the efficiency of iPSC generation. Sections of the teratomas formed from the iPSC PBscd01 were processed and immunostained for cell/tissue identification of the 3 germ layers (Figure 10). We identified cells from the endodermal line, members of respiratory and glandular epithelial tissues by AFP marking (Figure 10(a), III-IV), cells of the mesodermal lineage, present in cartilage and muscle tissue by -SMA marking (Figure 10(a), V-VI), and cells of the ectodermal lineage such as neural tissue (Figure 10(b), I) by positivity in marking with Nestin (Figure 10(b), II-III). The other iPSC PBscd lines were also evaluated for differentiation ability through teratoma formation assay. We also performed an immunohistochemistry (IHC) evaluation of the iPSC line PBscd08cl02HU (Figure 11), as this was a clonal line, differing from the others and the iPSC PBscd01, also evaluated by IHQ. The evaluation of the iPSC PBscd08cl02HU teratoma sections by IHC revealed the presence of cells/tissues of the 3 germ layers, evidenced by the -SMA immunostaining positivity, identifying elongated cells like muscle fibers, of mesodermal origin (Figure 11(a), IIV), AFP immunostaining positivity, identifying respiratory and guandular epithelial cells of endodermal origin (Figure 11(a), I and VVII), besides the positivity for Nestin evidencing neural tissue that is of ectodermal origin (Figure 11(b)). The other lines, iPSC PBscd02 and iPSC PBscd03, were analyzed by staining with hematoxylin and eosin (Figure 12). In the iPSC line PBscd02, we observed cells derived from the endoderm, such as columnar epithelium with apparently secretive characteristics (Figure 12, I), and mesoderm, such as chondrocytes, adipocytes, osteocytes, and muscle cells (Figure 12, II-III). In the iPSC line PBscd03, we observed tissues derived from the mesoderm, such as chondrocytes, adipocytes, and osteocytes (Figure 12, IV), and the ectoderm as pigmetal epithelium composed of melanocytes (Figure 12, V).
The TaqMan hPSC Scorecard (Applied Biosystems) test, which contains specially formulated gene expression assays for the evaluation of iPSC and ESC, was performed as the final test for pluripotency and differentiation potential. The cells used were the undifferentiated iPSC PBscd01 and after spontaneous differentiation and formation of EB (EB iPSCscd01). For the test, RNA of both cells was extracted and quantified, and 1g of the total RNA was used to obtain the cDNA in RT-PCR reaction. Then, the cDNA produced from both samples was used in qPCR reaction, one sample per plate, for evaluation of self-renewal potential and prediction of differentiation potential. Each plate provides a panel of pluripotency and differentiation probes, comprising the ectodermal, mesodermal, and endodermal lineage. Gene expression data were analyzed with the help of the online hPSC Scorecard analysis software.
The analysis confirmed that the iPSC PBscd01 are undifferentiated, with characteristics of self-renewal and pluripotent potential, with high expression of SOX2, and intermediate expression of NANOG and OCT4, besides high expression of CXCL5, LCK, a consensus marker of hESC, and NR5A2 which is a highly expressed gene in hESC (Figures 13(b) and 13(c), bottom line). The analysis also showed that after spontaneous differentiation, the cells give rise to cells from the 3 germ layers (Figure 13(a)), with the genes of negatively regulated self-renewal and pluripotency genes and positively regulated differentiation genes. Many highly expressed ectodermal genes, many highly expressed endoderm genes, but mainly many highly expressed mesoderm genes have been found. Of the 22 mesodermal tissue-related genes, 21 showed high expression in the EB from iPSC PBscd01 and 1 showed intermediate expression (Figure 13(c), upper line).
Together, these data demonstrate that PBscd iPSCs are morphologically, phenotypically, and functionally like pluripotent stem cells and they have potential to differentiate into different tissues from three germ layers.
The iPSC PBscd were screened for the occurrence of spontaneous integrations of the vectors used for reprogramming using two sets of primers specific to each vector. Initially, we performed a test of the primers with the DNA of the pEB-C5 and pEB-Tg vectors extracted with the same kit used for the other extractions, serially diluted 107 to 10 times. The dilution curve shows us how many copies of the vectors the primers can efficiently identify in the cell. A 1% agarose gel electrophoresis was performed, resulting in a vector dilution curve (Figure 14). The curve demonstrates that the primers are efficient at identifying the pEB-C5 vector up to 104 copies and the pEB-Tg vector up to 102 copies.
For the screening of the integration of the pEB-C5 vector, a reaction was performed with at least 2 DNA samples from each line at different passages. As a positive control, the DNA extracted from the expanded and transfected parental MNCs was collected after 48 hours of nucleofection. A 1.5% agarose gel electrophoresis stained with ethidium bromide (Figure 15) was performed. The gel showed amplification of the vector only in the positive control MNC PBscd. The fragment of about 244bp shows that the vector was still present in the cell 48 hours postnucleofection. After the reprogramming and expansion of the cells by some passages, the vector was eliminated. For the pEB-Tg vector, a reaction was performed with the DNA samples from the iPSC PBscd lines. The same positive control described above was used. A 1.5% agarose gel electrophoresis stained with ethidium bromide (Figure 16(a)) was performed. The gel showed amplification of the pEB-Tg vector in the positive control MNC PBscd, showing that the vector was present in the cells 48 hours postnucleofection. However, amplification of the pEB-Tg vector on iPSC PBscd01 P5 (Figure 16(a), line 3) was identified. To evaluate if the vector was eliminated after cultivation for further passages, a new amplification was performed only with the DNA of the lineage of which there was positivity (iPSC PBscd01), in the passages P5 and P10. A 1.5% agarose gel electrophoresis stained with ethidium bromide (Figure 16(b)) showed amplification of the pEB-Tg vector in the positive control MNC PBscd and in the iPSC line PBscd01 P5 (Figure 16(b), line 2), as seen in the previous gel. However, the gel did not show amplification for iPSC PBscd01 P10 (Figure 16(b), line 3), showing that the vector previously found in the iPSC line PBscd01 P5 was deleted after a few culture passages.
For molecular identification of the mutation responsible for the sickle cell phenotype, the generated iPSC DNA was amplified for the -globin (HBB) gene with specific primers and the fragments were then sequenced to screen the mutation. A reaction was performed using a DNA sample from each generated iPSC PBscd line, and then electrophoresis was performed on 1.5% agarose gel stained with ethidium bromide (Figure 17(a)). The gel showed amplification of the HBB gene in cell lines used, without contamination or nonspecific amplifications.
Sequencing of the amplified samples in ABI 3500XL Genetic analyzer automatic sequencer was performed to locate the mutation responsible for the sickle cell phenotype. A sample of each line of iPSC PBscd generated was used. The electropherograms arranged in Figure 17(b) were obtained after analysis in ChromasPro software. The HbA sequence is shown above the sequence of the HbS mutation lines at codon 6 (GAG to GTG).
Throughout this work, the hematopoietic differentiation underwent an extensive standardization to obtain an efficient protocol, which is summarized in the scheme of Figure 18(a). For this, the iPSC colonies were submitted to a process of adaptation to the enzymatic picking, so that after their individualization, the cells were regrouped in colonies and did not suffer great losses due to cell death (Figure 18(b)). The cells take at least 5 passages so that they are adapted to the enzymatic passage, but this time can vary according to the lineage employed. These adapted cells were used in the differentiation experiment to produce hematopoietic progenitors. The cells started to aggregate from the centrifugation and culture without adherence. On day D+2, it was already possible to verify the formation of EB, with cells surrounding that did not aggregate. The monitoring of EB showed a subtle initial growth with these structures becoming noticeably more compact. On day D+8, it was possible to verify the presence of small cells near the EB, and from day D+11, a marked growth of the EB was observed, with the experiment being finalized on day D+14, with the presence of small cells around the EBs (Figure 19).
Prior to initiating the differentiation experiment, the differentiated cells at day 0 of differentiation (D+0) were evaluated for pluripotency markers and showed a high percentage of cells positive for OCT4 (87%), SOX2 (98%), and NANOG (87%) (Figure 20). After 4 days of differentiation induction (D+4), cells were evaluated for the same pluripotency markers (Figure 20) and flow cytometry evaluations showed a drop in OCT4 (7%), SOX2 (46%), and NANOG (2%) markers. After this short differentiation induction (D+4), the EBs were also evaluated for some hematopoietic markers, such as CD31, CD144, CD34, CD43, KDR, and CD235a (Figure 21) and we noticed the emergence of still very discrete hematopoietic markers such as CD34 (3.4%) a hematopoietic progenitor cell marker, and CD43 (3.4%), a T cell and myeloid lineage cell marker. Cells were negative for KDR, a marker that shows commitment with the mesodermal lineage, but 19% of the cells were CD235a positive (glycophorin A), which is a marker of primitive erythroid differentiation. The EB collected also did not present positivity for CD31 (0.6%) and CD144 (0.05%) markers, which when expressed in conjunction with CD34, characterize endothelial cells with hemogenic endothelium potential. After further 4 days of induction (D+8), new EBs were collected for differentiation evaluation with a panel of markers (CD31/CD144/CD45/CD34, CD43/KDR/CD235a/CD34, and CD34/CD135/CD117/CD38) (Figure 21). After 8 days (D+8) of induction, we observed an increase in the expression of differentiation markers, with double positives as CD34+CD45+ (39%), CD38+CD117+ (40.1%), CD34+CD235a+ (15%), and CD34+ CD43+ (8.7%), in addition to high expression of hematopoietic markers such as CD45 (77%), CD34 (58%), and CD117 (84%). Cells at D+8 were also screened for the presence of common myeloid progenitors (CMP) by the CD34+CD117+CD135+CD38+ pattern, that was not found, and the presence of megakaryocyte erythroid progenitors (MEP) by the CD34+CD135CD117+CD38+ pattern, identified in the differentiating cells. After further 3 days of induction (D+11), new cells were taken for differentiation evaluation, using the same panel of D+8 (CD31/CD144/CD45/CD34, CD43/KDR/CD235a/CD34, and CD38/CD135/CD117/CD34) (Figures 21 and 22). A decrease in hematopoietic markers was observed, with some markers losing expression, such as CD31 (0%), CD45 (0.64%), and CD38 (0%); some that had low expression had the marking lost as CD144 (0%), CD135 (0%), and KDR (0%), and others had a drastic fall in the percentage of positive cells, such as CD34 (27.4%), CD43 (10%), and CD235a (4.95%). EBs also did not show double positivity for CD34/CD45 as verified on D+8 and only 3% of the cells showed marking for CD34+CD235a+. Methocult H4434 classic cells at D+11 were used for the CFC assay in methylcellulose (Methocult H4434 Classic) in triplicate, and after 15 days of induction, no colonies were found. On the last day of the differentiation experiment, after 14 days of induction (D+14), some individualized cells were visible in the supernatant, which were collected along with the EB and separated for individual analysis (Figure 22). Most hematopoietic progenitor markers had been lost, with only CD34-positive cells remaining in the EB (24.4%) and in the supernatant (30.3%).
The development of the hematopoietic differentiation protocol is summarized in the Table 4, which shows the percentage of positive cells for each marker on specific days of differentiation. The table summarizes the enrichment of the population with hematopoietic progenitor cells, with a peak at day 8 and subsequent fall after that period.
In this study, we report that integration-free iPSC can be efficiently generated from human adult PB MNC of patients with sickle cell anemia, after brief suspension culture system, in 3-4 weeks by nucleofection of episomal vectors expressing the 5 factors OCT4, SOX2, C-MYC, KLF4, and LIN28 as a single polycistronic unit and expressing the SV40 large T antigen. These vectors are based on a well-established technology in which the inclusion of the EBNA1 gene and the OriP sequence of the Epstein-Barr virus allow a plasmid, after a single transfection, to replicate extrachromosomally, as a circular episome, in several cell types [45]. The stimulation of Tg in reprogramming was observed by other groups using different vectors and cell types [46, 47]. Nucleoporation is a method of transferring genetic material to mammalian cells considered difficult to transfect, such as stem cells and primary cells [48]. Based on the physical electroporation method, nucleoporation uses a combination of electrical parameters with specific reagents for each cell type, so that the genetic material to be transfected is transferred directly to the nucleus and cellular cytoplasm. In contrast, other commonly used nonviral transfection methods depend on cell division for the transfer of DNA to the nucleus, so nucleofection provides the possibility of transfecting even nondividing cells such as neurons and resting blood cells [49, 50]. Prior to the introduction of this technology, the transfer of efficient genetic material into primary cells was restricted to the use of viral vectors, which typically involves disadvantages as security risks due to genome insertion, complexity, and high costs.
Despite being the most used source, in the present work, the SV40 large T antigen episomal vector and the episomal vector expressing the 5 reprogramming factors in a single molecule (~18kb) were not able to reprogram skin fibroblasts derived from patients with sickle cell anemia (data not shown). The nucleofection process is a nonviral process of nucleic acid transfer of all sizes. However, the transfection efficiency of large sequences falls considerably with increasing plasmid size, due to low cell viability [51], and this toxicity occurs only when the cells are exposed to the electrical pulses [52]. More precisely, Lesueur et al. [52] have shown that the observed drop in survival and transfection efficiency are directly linked to the physical size of each individual plasmid molecule, that is, a single copy of a large plasmid is more toxic and difficult to transfect than a single copy of a small plasmid or the number of copies of a small plasmid equivalent in mass to the single copy of the large plasmid. An option for our study would be the use of a different set of plasmids expressing the reprogramming factors, but divided into more than one molecule, to reduce their physical size, thus reducing the toxic effects to nucleofected cells.
Peripheral blood is a more advantageous source of cells than dermal fibroblasts, the most widely used source, but it requires skin biopsies, an invasive procedure that requires a long time of previous culture (more than 4 weeks). In contrast, adequate amounts of MNCs for reprogramming can be obtained from a few milliliters of peripheral blood, so that a simple venipuncture would be more acceptable to patients and donors, especially pediatric patients. The reprogramming of MNCs was simple and efficient. MNCs were cultured in specific medium with a combination of interleukins for depletion of the lymphoid population and expansion of the erythroblast/erithroid population. This stage of expansion not only drives cells into the cell cycle, so that we get a proliferative cell population, but also gives the cells certain epigenetic states that are more easily reprogrammed [42]. Chou et al. [42] have identified a DNA methylation profile of these proliferating cells more like ESC and iPSC than to nonhematopoietic cells of similar age, such as fibroblasts and endothelial cells, which could contribute to high efficiency in obtaining iPSC from these blood cells after only one transfection.
After culturing for about 12 days in a specific medium, we were able to identify the enrichment of the erythroblast/erythroid cell line population, especially regarding CD71 (transferrin receptor) levels, erythroid precursor marker [53], and CD235a (glycophorin A), an erythroid marker [54]; we also identified a decrease in the cell population expressing T cell markers such as CD4 and CD8, glycoproteins found on the surface of helper and cytotoxic T cells, respectively, as well as being found on the surface of other cells of the immune system such as monocytes, macrophages, dendritic cells, natural killer cells, among others [55]. We also identified a drop in the cell population expressing B cell markers, such as CD19 [56], and the common leukocyte antigen CD45 marker, present on the surface of most human leukocytes, such as monocytes, lymphocytes, and eosinophils, but absent in erythrocytes and platelets [57]. This variation shows that the expansion with medium and specific cytokines for lymphoid cell depletion and erythroblast/erythroid cell induction was successful in reducing the chances of reprogramming a lymphoid cell. Lymphocytes are known to lack their intact genome because of somatic rearrangements or V(D)J recombination, responsible for diversity in the repertoire of antibodies and T cell receptors [58]. IPSC obtained from nonlymphoid cells presenting their intact genome may be more suitable for therapeutic purposes, as the absence of such recombined DNA segments in the iPSC genome may eliminate some safety issues, issues raised by the observations that mice derived from reprogrammed T cells had a high incidence of T cell lymphoma [59].
The expanding cells from patients undergoing hydroxyurea treatment had an impact on cell growth, so that after consecutive falls in the first 6 days of expansion, the cells were not able to reach the initial cell population, ending on the 12th day of culture with, on average, fewer cells than at the beginning of the expansion. Hydroxyurea or hydroxycarbamide is a cytotoxic agent frequently used to treat SCA to reduce the number and frequency of pain episodes, vaso-occlusive crises, episodes of acute chest syndrome, and hospitalizations by raising fetal hemoglobin (HbF) levels [60, 61]. Its mechanism of action is the inhibition of the ribonucleotide reductase enzyme (RNR), reducing intracellular deoxynucleotide triphosphate and acting as a specific agent of phase S with inhibition of DNA synthesis and eventual cellular cytotoxicity. Its effect on HbF synthesis is attributed to the premature impairment of erythroid progenitors due to its cytotoxic suppression effects and to cell stress signaling that affects the kinetics and physiology of erythropoiesis and leads to the recruitment of erythroid progenitors with high levels of HbF [10]. Its prolonged use is associated with myelosuppression, due to its cytotoxic effects, but these same effects are beneficial for the treatment of SCA, because it reduces the production, not only of erythroid progenitors but of neutrophils and reticulocytes, which promote vaso-occlusion through adhesion to vascular endothelium, and platelets, an important mediator of inflammation [60, 62]. The effects of low cell growth found in our study can be attributed to the cytoreductive effect of hydroxyurea, which concentrates on leukocytes and, especially on erythrocytes, cells whose growth is conditioned by the cocktail of cytokines added to the culture medium.
We also identified that low cell growth found had an impact on the reprogramming of PBscd08 in iPSC, since these cells derived from patients treated with hydroxyurea did not reach the minimum number of cells ideal for the transfection of the reprogramming factors. However, hydroxyurea influenced reprogramming not only by impacting cell growth but also the number of colonies obtained; cells derived from untreated patients showed an 11-fold increase in the number of colonies obtained compared to cells from treated patients. The greatest impact on the number of colonies obtained from cells from patients treated with hydroxyurea occurred because in the cells PBscd06 and PBscd07, despite reaching the ideal number of cells for nucleoporation, no colony was obtained after culture in ESC medium. The low reprogramming efficiency of the cells of patients treated with hydroxyurea may be linked to differences in the epigenetic pattern of these cells, since hydroxyurea, as a drug that alters DNA synthesis, can lead to changes in DNA methylation pattern [63]. Although the effects of hydroxyurea are related to cell cycle inhibition leading to the activation of stress erythropoiesis, the precise mechanism by which hydroxyurea induces HbF is not fully understood. Expressions of erythroid genes of the -globin and -globin loci are regulated by a complex series of epigenetic and molecular processes during development, and some epigenetic mechanisms of HbF regulation in proposed hemoglobinopathies include methylation and histone deacetylation [64]. Compared with fetal erythroid cells, the promoter region of -globin in adult erythroid cells is highly methylated, and this hypermethylation has been inversely related to HbF expression, and although hydroxyurea has not been identified as a hypomethylating agent, studies suggest a decrease in the methylation of the -globin gene in association with exposure to hydroxyurea [65, 66]. Although more experiments are needed, our results together with others cited above reinforce the need for future studies to investigate epigenetic and molecular processes as potential mechanisms of HbF expression and induction.
The iPSC PBscd lines obtained could differentiate into cells/tissues from the 3 germ layers, characteristic of pluripotent stem cells (PSCs). ESC and iPSC are defined by their potential for pluripotent differentiation and unlimited self-renewal ability [67], so that this ability to become any somatic cell type found in the human body has gained significant attention and interest in fields of cell biology and regenerative medicine [68]. In the study of these cells, quality control analyses are crucial, and pluripotency tests continue to be key components of any research project. Analyses commonly used to test such potential include quantitative PCR that search for upregulated pluripotency genes, immunocytochemistry for the detection of pluripotency markers, and formation of embryoid bodies to test the ability of differentiation into cells/tissues of the 3 germ layers in vitro [69]. However, the most effective test to evaluate the ability of PSCs to form tissues of all 3 germ layers in vivo is in the form of encapsulated tumors called teratomas [7072]. The lines obtained from iPSC PBscd could form these structures that harbor the formation of several cell types derived from tissues with mesodermal, endodermal, or ectodermal origin, when infused in immunodeficient mice. In vivo teratoma formation is considered the most accurate pluripotency test because it provides more reliable and comprehensive confirmation than testing cells in simplified and artificial systems such as culture plates [69].
Although the teratoma formation test is considered a test of high confidence and comprehensiveness, some questions are still unanswered regarding the equivalence between ESC and iPSC, making the selection and characterization of these cells increasingly important. For example, it has been described that human iPSCs depart from ESC in the expression of hundreds of genes [73], in their overall patterns of DNA methylation [74] and in their neural differentiation properties [75]. In addition, some recent studies have argued that some iPSC lineages may retain a certain epigenetic memory inherited from the somatic cells from which they originated, so that they exhibit reduced differentiation efficiencies toward a specific cell type [75, 76].
To access the nature of the epigenetic variations that exist between hPSCs, Bock et al. [77] performed 3 tests with 20 established ESC lines and 12 characterized iPSC lines, and from the results, they could identify specific genes and predict the propensity of differentiation of each lineage in the 3 germinative layers. These 3 tests were combined into a bioinformatics platform that allows high-throughput predictions of the quality and utility of any iPSC lineage [77]. This platform, in scorecard form, was applied to one of our blood cell lineages, both in undifferentiated iPSC and after spontaneous differentiation in vitro. The undifferentiated iPSC analysis showed a positive regulatory pattern of CXCL5, LCK, and SOX2, a known pluripotency gene used to generate iPSC, which is at the center of the pluripotency regulatory network [78]. The lymphocyte-specific p56 protein tyrosine kinase (LCK) is considered a self-renewal marker because it was identified in the network of protein-protein interaction genes with high expression in ESC and iPSC compared to somatic cells: LCKHM89CD133SOX2 [79]. On the other hand, CXCL5 (chemokine (CXC motif) ligand 5) is actively regulated by FGF2 (or bFGF), inducing the mobilization of hematopoietic stem cells (HSCs) from the endosteal niche or bone marrow into the peripheral blood [80, 81]. Thus, the daily addition of the cytokine -FGF given by the daily exchange of the culture medium made the expression levels of CXCL5 increase considerably. Our iPSC PBscd also demonstrated positive regulation of the nuclear receptor NR5A2, previously related to the reprogramming of murine somatic cells in iPSC, replacing OCT4 [82]. Heng et al. [82] demonstrated that NR5A2 shares a lot of common gene targets with SOX2 and KLF4, suggesting that all three transcription factors work in combination with reprogramming.
The analyses of EB formed by the spontaneous differentiation of one of the iPSC PBscd lines demonstrated a shutdown of genes related to self-renewal and an upregulation of genes related to the 3 germ layers, mesoderm, endoderm, and ectoderm. However, we could observe that the genes related to the mesoderm were more expressed and 95.5% were upregulated, which did not occur with the other groups (Ecto: 64%, Endo: 61.5%). Retention of a donor cell memory in iPSCs seems to make it easier to redifferentiate them in the donor cell type than in another cell type [8385]. Kim et al. [83] demonstrated that iPSCs derived from both blood and dermal cells have distinct potentials for both hemopoietic and osteogenic direction, respectively. IPSCs derived from blood cells form hemopoietic colonies more easily, while iPSCs from dermal cells form more colonies when differentiating in the osteogenic direction [83]. However, some studies have demonstrated that a unique pattern of methylation and/or gene expression characteristic for low-pass iPSC is not stable but gradually reverts to the pattern of ESC gene expression as consequence of culture passages [85, 86]. In the case of iPSC PBscd, the finding that identifies a possible memory of the blood cells from which the iPSCs were derived could facilitate hematopoietic differentiation, which would be an advantage, considering how complex this differentiation may be. One of the overexpressed genes in EB from iPSC PBscd that stood out from the mesodermal lineage was SNAI2, also known as SLUG, a zinc finger transcriptional repressor that downregulates E-cadherin expression in neural crest premigratory cells, from which induces epithelial cells to lose the strong binding characteristic to acquire a mesenchymal phenotype [epithelial-mesenchymal transition (EMT)], allowing gastrulation in the embryonic development [87]. The transition from a mesenchymal state to an epithelial state (MET) is known as a mandatory phase during the early stages of reprogramming [88, 89], as well as the reverse path, EMT is essential for efficient differentiation, whose occurrence in EB reflects the EMT observed during gastrulation in the human development [90].
In summary, the Scorecard methodology allowed us a rapid lineage-specific characterization and allowed us to identify how well the iPSC PBscd can differentiate into mesodermal lineages such as hematopoietic tissue and in the 3 germ layers. All the information provided by the test requires a more thorough evaluation, and all derived lineages in the same way must be compared to each other to identify lineage-specific variations.
Using PCR (2 sets of specific primers), we did not detect the vector DNA either as episomes or anywhere in the iPSC genome analyzed, after about 10 passages. Similar results were found by Chou et al. [42], where plasmid DNA became undetectable after 1012 passages. The use of the plasmidial system for reprogramming as well as being a nonintegrative system also provides some advantages over other nonintegrative or nonviral methods, such as ease of preparation and stability, different from proteins and mRNA, which require repetitive transfer of multiple proteins and mRNA daily for up to 17 days [91, 92].
We have developed a simple and efficient method of obtaining hematopoietic progenitor cells from the combination of a methodology of forced cell aggregation and formation of EB with induction through the addition of specific cytokines. Davis et al. [93] demonstrated the role of BMP4 in inducing mesoderm from ESC, which is also dependent on Activin A and bFGF signaling [94, 95], and several studies have shown the role of BMP4, SCF, bFGF, and VEGF in promoting hematopoiesis from ESC [9699].
Cells after induction did not show KDR marking and showed positivity to 31 only after 8 days of induction. However, CD31, which was initially identified in endothelial cells and platelets [100], is also present in leukocytes [101], which could also explain the presence of this marker in differentiating cells. The hemogenic endothelial phenotype was defined as KDR+CD117+CD45 cells [102104] and demonstrated that these cells give rise to multilinear hematopoietic progenitors which are KDRCD117+CD45+ cells and can be distinguished from mature blood cell types having the KDRCD31CD45+. On day 8 of differentiation induction, we observed KDRCD45+ populations, both CD117+ and CD31; however, before day 8, we did not observe KDR+ cells. The KDR receptor appears in cells at an early stage of mesodermal differentiation [105] and may not have been detected because we did not evaluate every day of differentiation; after the initiation of the differentiation, we only performed the evaluation after 4 days of induction, and after another 4 days, and given the rapid production characteristic of progenitor cells (only 8 days), this marking may have been lost.
On day 8 of differentiation induction, we observed that the cells presented as a CD34+CD45+ and CD34+CD43+ population. The CD34 marker can be expressed in a wide variety of cell types, so that their use alone may not be sufficient to identify a pure population of hematopoietic progenitors from hPSC [106]. Our population demonstrated CD43 positivity, a marker expressed in the T cell cytoplasm and in myeloid lineage cells, whose marking clearly separates CD43+ hematopoietic colony-forming progenitors from CD43CD31+ endothelial cells and CD43CD31 cells with mesenchymal characteristics [107, 108]. In cultures of hPSC differentiated with OP9, the first CD43+ cells were detected within VE-cadherin+ cells near day 4 of differentiation [107109]. These cells, CD41a and expressing the erythroid marker CD235a (glycophorin A) and low levels of CD43, were defined as angiogenic hematopoietic progenitors, which maintained the ability to produce endothelial cells [109, 110]. On day 4 of differentiation, our cells expressed CD235a (glycophorin A) and low CD43 levels; however, we did not evaluate the CD41a marker on day 4. On day 11, we performed the methylcellulose assay, but the cell had already lost its progenitor cell potential, leaving only the CD34 marking at higher levels.
We could not observe many morphological changes happening during the differentiation process because this process occurred in the EB. However, different from monolayer differentiation protocols using stromal cells such as OP9 cells, EBs can mimic a microenvironment more favorable to development and differentiation. Differentiation from EB can be considered more like what happens in the hematopoietic niche, supporting the development of hematopoietic progenitor cells, since the process of hematopoietic differentiation requires intrinsic and extrinsic signaling and hierarchical organization of hematopoietic precursors [111].
In summary, the results of the differentiation show that the cells, obtained in greater quantity on day 8 of differentiation, are hematopoietic progenitor cells coming from a hemogenic endothelium. However, further analysis must be performed to prove this pathway of induction. Thus, our data show that iPSCs are also capable of differentiating into hematopoietic progenitor cells and our method of differentiation has shown promise.
This work demonstrated the establishment of iPSC lineages derived from patient cells with sickle cell anemia, that is, a cell with a genetic background that is able to self-renew indefinitely and can differentiate in vitro in any cell type. Thus, we provide a valuable tool for a better understanding of how the disease occurs and the possible causes for its clinical discrepancies among the patients affected, in addition to making possible the development of new drugs and more effective treatments for the disease and to provide a better understanding of the widely used treatments available, such as hydroxyurea. The development of an efficient protocol to obtain hematopoietic progenitors, besides contributing to the concept of modeling the disease, allows us a better understanding of the process of hematopoietic differentiation, a complex and not yet fully clarified system. The great difficulty in establishing robust protocols for hematopoietic differentiation is mainly due to the lack of understanding of this process that occurs in the microenvironment of the bone marrow and the difficulty to artificially mimic this complex microenvironment in the culture plate.
The authors declare that they have no conflict of interest.
The authors thank Prof. Dr. Andreia Machado Leopoldino for the valuable personal support in immunohistochemical analysis and Dr. Simone Kashima Haddad for her valuable support and insightful feedback. The authors also thank Patricia Viana and the Laboratory of Flow Cytometry for the help and discussions about the flow cytometry analysis. Special thanks to Sandra Navarro Bresciani for her valuable support. This work was supported by Fundao de Amparo Pesquisa do Estado de So Paulo (FAPESP) (process number 2013/08135-2), Conselho Nacional de Desenvolvimento Cientfico e Tecnolgico (CNPq) (process number 141058/2016-1), Coordenao de Aperfeioamento de Pessoal de Nvel Superior (CAPES), and Blood Center of Ribeirao Preto, Brazil.
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Induced Pluripotent Stem Cell for the Study and Treatment of ... - Hindawi
What Happens When Everyone Realises We Can Live Much Longer? We May Find Out As Soon As 2025 Forbes
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What Happens When Everyone Realises We Can Live Much Longer? We May Find Out As Soon As 2025 - Forbes
INTERNATIONAL STEM CELL CORP Management's Discussion and Analysis of Financial Condition and Results of Operations (form 10-Q) Marketscreener.com
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3D Cell Culture Market stands at revenue of US$ 1.15 Bn in 2022, and is predicted to surge at a CAGR of 9.8% to hit worth of US$ 2.67 Bn during...
YUBO INTERNATIONAL BIOTECH LTD Management's Discussion and Analysis of Financial Condition and Results of Operations. (form 10-Q) Marketscreener.com
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ACTINIUM PHARMACEUTICALS, INC. MANAGEMENT'S DISCUSSION AND ANALYSIS OF FINANCIAL CONDITION AND RESULTS OF OPERATION (form 10-Q) Marketscreener.com
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Stem cell supplements are usually composed of natural materials and ingredients that helps support your bodys stem cells.
Stem cells, which can be easily described as the bodys building blocks or raw-material cells, are the cells responsible for all healing and growth processes in the body (1).
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Actif Stem Cell Mega Support is one of the most complete and advanced formulas on the market, using 15 factors to support stem cell growthand decrease DNA damage.It has been clinically proven to support stem cell renewal, boost cognitive function, and assist in circulation while decreasing the signs of aging and offering regeneration to your system.
Their enhanced formula includes L-carnosine for neurons, L-leucine to boost muscles and prevent fatigue, organic methylfolate to enhance the metabolism of cells. Actif Stem Cell Mega Support is also non-GMO, gluten free, and made in the USA, in GMP (Good Manufacturing Process) certified facilities. For these reasons, its our #1 pick.
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Stem Cell Worx Intra-oral Spray, is formulated by scientists and biochemists to activate your own adult stem cells instead of inserting new ones. The spray provides a 95% absorption rate versus the 20% average from pills and capsules.Over 50% of this product is protein and it has one of the highest content formulas on the market in terms of natural immune factors.
Its manufactured in the USA using GMP certified facilities that have been inspected and approved by the FDA.When it comes to independent clinical studies, this product has several to back up its claims (which can be found on the companys website).
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Enzymedica Stem XCellis a potent, research-backed formula that is designed to help boost and protect your stem cells from free radicals.This formula contains powerful stem cell supporting ingredients including glutathione, SOD, and alpha-lipoic acid along with 6 more enzymes that are designed to enhance the effectiveness and potency of the pure Stem XCell formula.
Their patented NT020 blend has been studied extensively and proven to promote health and growth in your bodys cells. Enzymedica Stem XCell contains no fillers andis vegetarian friendly. The company also supports the Autism Hope Alliance, Vitamin Angels, and Green Mountain Energy.
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Enzyme Science Stem XCell Proincludes antioxidants likegreen tea, red wine, and blueberry in a special and powerful formulathat helps boost immune and cellular health while preventing oxidative damage from free radicals.This product is designed with a patented NT-020 blend that was developed by researchers and scientists and studied for the ability to maintain and produce stem cells.
Enzyme Science Stem XCell Procontains no egg, soy, yeast, dairy, preservatives, salt, sucrose, casein, potato, rice, corn, wheat, nuts, artificial colors, or flavors. It is also gluten free and doesnt use any ingredients that are produced using biotechnology.
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Nu-Derm Cell Biotic Complete Complex offers powerful gastrointestinal and anti-aging support for older adults through the use of. DE111TM, lactobacillus rhamnosus, lactobacillus casei, and more.
Each capsule contains 5.75 billion probiotic bacteria that will help improve your digestive health without a prescription. Nu-Derm Cell Biotic Complete Complex is made in the USA and manufactured using GMP (Good Manufacturing Practice).
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Regenexx Advanced Stem Cell Support Formula has an 8 in 1 regenerative formula thatcan help your joint mobility. This supplement is tested in vitro using real human mesenchymal stem cells. The laboratory studies showed that these supplements help to maintain a healthy cell environment thanks to the special formula.
Regenexx Advanced Stem Cell Support Formula is oneof the rare stem cell supplements that is drinkable and comes in delicious flavors like strawberry and banana.
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Healthycell AC-11 repairs your DNA using Amazonian Uncaria tomentosa, Cats claw, and other natural ingredients. Over 40 peer-reviewed studies and nearly 20 years of research have backed this supplement up and it has 10 US patents.
Healthycell AC-11 isvertically integrated, sustainably sourced, located from the Peruvian Amazon Rainforest origin, and extracted in Brazil. It has no preservatives, no fillers, no flow agents, and no GMOs.
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BioXcell Stem Cell Enhanceris 100% AFA (Aphanizomenon flos-aquae) with blue green algae.This natural product has been shown to provide a variety of fantastic benefits for your health.
It helps regenerate your DNA, restore broken stem cells and increase adult cell metabolism, decrease intestinal problems, and keeps your mental health boosted as you have improved cognition and memory.
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Digestacute Autoimmune-X Advanced Formula isrecommended by some of the nations most respected immune restoration practitioners, including Dr. C. Nomal Shealy, who is the President of the American Holistic Medical Association.It is organically grown and does not contain any soy, GMOs, fillers, L-glutamine, and other ingredients.
Digestacute Autoimmune-X Advanced Formula is cruelty-free and does not test on animals.
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Stem Vida StemAliveis made in the USA and is 100% natural, non-stimulating and caffeine free.Stem Vida StemAlive rejuvenates your your stem cells and repairs your tissues and organs.
The first thing we looked at, when formulating our rankings for the best stem cell supplements, was the clinical trials. If a product was not backed by clinical trial data, it did not meet our purity requirement and was thus axed. All of the products on our list are independently tested and examined by scientists. In the case of Healthycell, over 2 decades of clinical study has gone into ensuring this product was safe and effective.
Another requirement we looked at was the ingredients. Making sure that the ingredients were all natural and pure, while still being potent, was very important. Organic and biologically sourced ingredients, like those found inActif Stem Cell Mega SupportandDigestacute Autoimmune-X Advanced Formula,are crucial when it comes to healthy and reliable stem cell supplements.
We also looked at the manufacturing process. Many low-quality supplements will come from China, which can be damaging and even dangerous to ones health especially when something as potent as stem cells are involved. It was important to us that products only originated from the USA and were manufactured in certified labs. Youll find on our list thatStem Cell Worxwas manufactured in an FDA-certified lab, andEnzymedicais manufactured in GMP-certified labs, among others.
Another thing we looked at was the cost. Stem cell supplements can be costly, especially ones with quality materials. However, the supplement industry has a bad habit of creating 10-20x markups, so we know that very expensive supplements are not necessarily always the best. As such, we tried to balance our rankings between price, quality, and affordability to ensure everyone was able to afford stem cell supplements.
1. Stem cell supplements can favorably alter gene expression.This translates to regulating and lowering the possibility of cells replicating in error. Gene expression is given a chance to mutate positively or harmfully each time that a cell divides. By taking stem cell supplements, you can tip the scales in your favor and lessen the chance that stem cells will divide poorly(2).
In addition, stem cell supplements can also help your stem cells replicate more frequently (3).
Because the stem cells are positively linked with a reduction in the signs of aging, causing your stem cells to replicate more often will lead to a better aging process, fewer side effects associated with aging, and overall better quality of life.
Having more stem cells can lead to looking and feeling younger for a more extended time. Stem cells are directly related to an adult bodys ability to heal from injury and natural degradation as a result of telomere shortening, otherwise known as aging.
2.The stem sell supplement vitaminD can support those who have multiple sclerosis. As an autoimmune disease that hinders the ability of nerve cells to send signals from one place to another, multiple sclerosis is thought to be currently incurable.However, vitamin D supplementation may possess the ability to reverse nerve cell damage caused by multiple sclerosis (4).
While this is not technically curing multiple sclerosis, this does offer victims of the autoimmune disease a fighting chance and can potentially extend their lifespan to natural levels so they can live out the rest of their life normally. In addition, it may provide patients with enough time for a cure to be found in the future.
3. Certain stem cell supplements may improve stem cells in the brain. During one study, animals were provided with blueberry, vitamin D, green tea, and carnosine as stem cell supplements. This resulted in a reduction of inflammatory effects, such as pro-inflammatory cytokines, that improved brain function and health overall (5).
In addition, this indicates that nutritional supplements can improve the overall environment for brain stem cells and lower the risk that they will change into sick or diseased cells in the future. As functioning stem cells are directly related to greater overall health, particularly when it comes to neurological function, this indicates that better brain health can safely be attributed as a benefit to stem cell supplements.
A healthier brain is already at a lower risk of suffering from neurodegenerative diseases in the future (6).
In this way, stem cell supplements can be seen as effective agents which reduce the likelihood of a person developing a neurodegenerative disease.
4. Stem cells supplements can provide improved cognitive and memory function.A study during which certain animals were provided with various nutritional supplements showed that sufficient supplementation could cause the proliferation of new stem cells (7).
This is likely due to an increase in overall healthy brain cells as described above but is specific enough that it warrants its own attention.Memory loss is a major concern for anyone leaving middle-age. Taking good nutritional supplements to assist with stem cell reproduction and function is a good way to combat this effect.
5. Stem cellsupplementscan lead to increased energy and endurance.As stem cells are chiefly responsible for the restoration of the body, those who supplement their stem cells with the right nutritional additives can expect greater recovery time from workouts and greater energy reserves to draw upon during times of stress or excess energy release (8).
This effect can allow people to build muscle more quickly or remain fit for longer; even as they start to ascend in age. The bodys physical capabilities naturally decline as it grows older, but increased stem cell production can truncate this effect or delay it for longer than normal. This can lead to improved physical ability and function.
6. Stem cell supplements can provide faster recovery times from injuries or sicknesses.Those who suffer from chronic illnesses or from major bodily injuries can expect faster recovery times if they have an excess of stem cells(9).
Because stem cells are responsible for physical repair, having extra cells may lead to quicker overall recovery time and fewer long-term side effects as a result of the initial injury.In addition, a plethora of reparative stem cells can lead to fewer mistakes during the healing process or a better healing process in general.
While certain major injuries have a tendency to cause permanent damage or lower function depending on injury location, excess stem cells, bolstered by a good nutritional environment, may result in near-perfect healing of the wounded area. This effect has applications for cancer patients or victims of regular physical trauma from everyday accidents.
7. Stem cells supplements may delay the onset of aging.Regular aging side effects, ranging from common symptoms like the wrinkling of the skin to serious problems like diseases that affect the elderly, may be truncated or reduced thanks to healthy stem cell abundance.
Taking stem cell supplements will make your stem cells more plentiful and more effective in general. Both of these will result in a gentler aging process than is normal. Although stem cells cannot stop the aging process permanently, lessening the negative aspects of growing older will allow people to enjoy their later years more fully and allow them to maintain healthy lifestyles for longer. In many cases, aging side effects are exacerbated as older people become less active or stop making healthy choices as a result of chronic pain or discomfort. By remaining healthy for longer, this potentially destructive loop is delayed as well.
8. Stem cell supplements can lead to more effective stem cell activity, preventing the body from being as vulnerable to certain illnesses.The human body is naturally under attack from various viruses and other diseases at all times. While the immune system is normally quite effective at keeping out these potential threats, minor damage incurred from daily activity or normal cell replication can breach these defenses.
Having help from your stem cells can allow these gaps to be closed more quickly and lessen the chances of various viruses infiltrating a bodily system. While stem cells cannot directly stop diseases from entering a body, they can promote a greater immune response and a faster recovery time in the events that it does become infected (10).
9. Stem cell supplements can promote healthy weight due tothebodys greater ability to maintain itself.It is still important to maintain a good exercise routine and dietary guidelines, but many people struggle with staying within a healthy weight range even when keeping these factors in consideration.
Stem cells already work to keep a healthy body in a homeostatic condition, where will function most optimally. Boosting the number of stem cells in the bloodstream beyond their regular number will improve this effect and make it harder for your body to fall out of balance (11).
In addition, stem cells ability to improve muscle growth will allow for easier exercise improvements and consistency (12). Because stem cells passively boost overall energy, keeping up a healthy exercise routine will be more achievable for many people.
Combined with a good exercise routine and smart nutritional practices, lots of people can experience a body within a healthy weight much more easily.
10. Stem cells improved through supplements can help increase alertness. Research shows that stem cells that grow healthily and in good environments, boost brain function and cognitive effectiveness, this directly translates to a better attention span and greater alertness (13).
The possible uses for improved attention span and alertness are limitless. People suffering from chronic fatigue or who have to work a job during the nighttime hours can benefit from being more awake and being able to think more clearly than before. This can be particularly effective for professions such as doctors for emergency response personnel, where their jobs and the lives of others often depend on their alertness levels.
Greater attention span can also assist people who suffer from attention-based disorders, such as ADD, who may face greater challenges in life due to their condition.
1. Stem cell supplements may be harmful for the liver. Stem cell supplements which use blue-green algae as one of their key ingredients may impose certain side effects on their users (14).
While most people should be able to take blue-green algae without worry, some blue-green algae products may contain certain contaminants that can damage the liver. These negative components can be things like harmful bacteria, certain toxic metals, or microcystins.
2. Children should not take stem cell supplements in any form but especially those which contain blue-green algae. Children are generally more sensitive to blue-green algae products and may react negatively to ingesting or absorbing the substance in any way (15).
3. Stem cell supplements can cause IBS like symptoms. If contaminated blue-green algae are consumed by a supplement user, they may experience liver damage, weakness, rapid heartbeat, shock, thirst, vomiting, nausea, or stomach pain. More extreme reactions may result in death, so it is critical that anyone considering taking a stem cell supplement product ensures that the product uses cleared and contamination-free blue-green algae.
4. Women who are pregnant should avoid blue-green algae stem cell supplementation products due to the unforeseen side effects that stem cell production may have on an unborn fetus. Pregnant women already have more stem cells in their body due to the developing baby in their uterus and adding more stem cells to this process may complicate factors or lead to unforeseen developments that can cause negative health effects for either the mother or the child (16).
5, Finally, all boosts to stem cell production can inadvertently cause cancer, even in people that have not developed cancer in their life.The inherent risk of stem cells is that they can become any type of cell at all, including cancer cells(17).
Those with a family history of cancer should be careful when considering taking a stem cell supplement as this may increase their chances of developing cancer inadvertently.
Most stem cell supplements are taken either pill or spray form. Regardless of whichever method is used, dosages should not go above 500 mg taken twice daily, or 1000 mg in total per day. Depending on the exact product used, some stem cell supplements will have all of this limit reached with a single pill or tablet while others will require two or three tablets or capsules to reach 500-1000 mg.
Sprays are somewhat different. Recommended dosage varies by product and is harder to measure due to the application method used. However, users should avoid spraying too much of the supplement on to their skin at any one time. This will prevent oversaturation of compounds (such as blue-green algae) in the body and give the body time to adapt to the improvement of its stem cells.
How long does it take for stem cell supplements to work?The length of time between supplement administration and results varies greatly from product to product. This is because every persons stem cells are unique and will respond to boosting from supplementation differently. However, stem cell production occurs all the time, so results should not take very long to experience in some capacity.
What does stem cell pills do? Stem cell therapy, also known as regenerative medicine, promotes the repair response of diseased, dysfunctional, or injured tissue using stem cells or their derivatives.
Does fasting increase stem cells? Yes, MIT researchers have found that fastingdramatically improves stem cells ability to regenerate, in both aged and young mice.
Where do stem cells come from?Stem cells that are boosted by supplements come from bone marrow or other organs which may possess stem cell niches (18). These niches are where reparative stem cells reside in advance of damage for a particular organ or part of the body. In this way, stem cells are always around where they are needed to begin the healing process.
How long do stem cells live? Some stem cells have lasted five months and others for more than three years.
Do supplements create new stem cells?No. Stem cells are technically never truly replicated. This is because stem cells transform into the cells required for correct healing and restoration of damaged tissue (19).
Therefore, stem cells dont really replicate: they produce new cells which then perform the required task to ensure bodily health.New stem cells are made in the bone marrow or in stem cell niches throughout various internal organs or tissues (20).
Stem cell supplements boost stem cells by improving their effectiveness or creating positive environments where they can flourish and remain healthier for longer.Stem cells that are more effective have greater health benefits on the body, which is why supplements are effective.
Do stem cell supplements and therapywork for back pain? Yes, stem cell therapy may have the potential to be an alternative to invasive spine surgery.
Are stem cell supplements safe?Stem cell supplements do not have negative side effects as of yet. There have been no recorded medical issues as a result of ingesting or absorbing stem cell supplements. However, stem cells may cause cancer on their own, so promoting stem cell growth and activity may cause cancer down the road.
Overall, stem cell supplements by themselves are not dangerous or harmful. Children should not take stem cell supplements due to their developmental progress and an increase in possible side effects.
Does insurance pay for stem cell supplements and therapy? Medicare also does not cover stem cell injections. To be clear, proven bone marrow transplants/hematopoietic stem cell therapies such as for leukemia, which are established therapies covered by insurance, are a different story.
How long can you live after a stem cell transplant? A stem cell transplant may help you live longer. In some cases, it can even cure blood cancers. About 50,000 transplantations are performed yearly, with the number increasing 10% to 20% each year. More than 20,000 people have now lived five years or longer after having a stem cell transplant.
Does radiation kill stem cells? Radiation therapy and chemotherapy aimed at killing cancer cells may have the undesirable effect of helping to create cancer stem cells, which are thought to be particularly adept at generating new tumors and are especially resistant to treatment, researchers say.
How much are stem cell injections? The cost can vary, but is usually around $3,000 to inject one body area and $5000 for two areas. The final cost of the treatments will ultimately be determined by what particular injections are being done. Stem cell therapy and fat grafts are typically not covered by your insurance company.
How do stem cells start? Stem cells start off as a basic, undeveloped cell, that is sent to an area in need of new material. Once there, stem cells become the required cell to ensure proper organ or tissue function. Stem cells can become muscle cells, skin cells, blood cells, brain cells, or more. Because of this unique transformative ability, stem cells possess unparalleled healing capabilities.
The body naturally wears itself down from daily activity and from experiencing various damages and illnesses. Over time, these maladies pile up and impair function or lead to a lower quality of life. Ingesting or absorbing stem cell supplements can lessen these effects and improve many aspects of living.
How do stem cells affect gene expression? Stem cell supplements can favorably alter gene expression, lowering the possibility of cells replicating in error. In addition, stem cell supplements can also help your stem cells replicate more frequently which can lead to a better aging process, and a better quality of life.
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Top 10 Best Stem Cell Supplement Brands - Healthtrends
Human stem cells are essential for the growth and maintenance of our organs, bones, and systems. They are also amazing tools of discovery for scientists at the Institute for Stem Cell and Regenerative Medicine and researchers around the world studying how to stop diseases. However, predatory businesses across the country are misusing the term stem cells to market unapproved, unproven, and unsafe procedures that are often expensive and largely ineffective. Its important to understand what stem cell therapy really means.
Lets start by creating two categories of stem cell therapies approved (by the FDA) and unapproved. Whether a stem cell therapy is approved or unapproved has critical implications for the science, effectiveness, and safety of the procedure.
(In addition to blood stem cell transplants), the FDA lists a limited number of additional approved products on its website.)
More recently, hundreds of businesses around the country referring to themselves as clinics have begun marketing various versions of stem cell therapy that promise to help patients with serious conditions like Parkinsons disease and more common ailments like joint pain. In reality, most of these types of stem cell therapy do not use stem cells at all. Rather, they remove tissues that presumably contains adult stem cells from one body part and inject those cells into another part of the body.
Furthermore, there is no proof that any stem cell therapy offered by stem cell clinics is effective or safe. Unlike FDA-approved procedures, which are subject to years of rigorous trials, unapproved treatments marketed directly to patients are developed and performed with little oversight. While stem cell clinics often tout testimonials from satisfied customers, there has never been a large-scale clinical trial to demonstrate that the perceived benefits of a stem cell therapy arent the result of a placebo effect. In recent years, the FDA has begun to expand regulations and enforcement of these clinics.
Thanks to decades of data, we know much more about the effectiveness of blood stem cell transplants. We also know they are not instant cures. While the procedure itself only lasts a few hours, recovery can take weeks. During this period, patients are monitored closely by physicians and nurses for side effects and for evidence of recovery.
There are side effects associated with approved and unapproved stem cell therapies. The possible side effects of blood stem cell transplants are detailed on the Cancer.org website. Patients considering an unapproved stem cell therapy should be aware that these procedures carry serious risks and that these risks may not be managed by a qualified care team. Injecting even a persons own tissue in a different body part has resulted in severe illness and, in some cases, blindness.
Therapies offered by stem cell clinics come with financial risk as well. Because these procedures are generally not covered by insurance, people seeking treatment are required to pay large out-of-pocket fees with no guarantee of improved health.
In their advertising, stem cell clinics promise unsubstantiated relief or even cures for everything from knee pain to Parkinsons disease, often taking advantage of vulnerable individuals who may feel they have nowhere else to turn. In reality, there is no strong evidence to back up claims that any stem cell therapy works let alone has lasting benefits.
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How Does Stem Cell Therapy Work and What Are the Risks? | ISCRM
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Stem Cell Wellness Kit
The new report by Expert Market Research titled, GlobalHuman Embryonic Stem Cell MarketReport and Forecast 2022-2027, gives an in-depth analysis of the global human embryonic stem cell market, assessing the market based on its segments like applications and major regions. The report tracks the latest trends in the industry and studies their impact on the overall market. It also assesses the market dynamics, covering the key demand and price indicators, along with analysing the market based on the SWOT and Porters Five Forces models.
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The key highlights of the report include:https://bit.ly/3A1uYjO
Market Overview (2017-2027)
Historical Market Size (2020): USD 0.7 billion Forecast CAGR (2022-2027): 10%
The human embryonic stem cell market is being driven by the thriving medical sector. The rising demand for embryonic stem cells can be attributed to the increasing prevalence of chronic diseases around the world owing to the rising adoption of unhealthy and sedentary lifestyle among the youth and middle-class population. The increased risk of premature death as a result of chronic diseases is a growing concern. Therefore, human embryonic stem cells are gaining popularity in the medical sector. Furthermore, the increase in research grants and private as well as public funding for the development of effective and safe stem cell therapy products is further aiding the market growth. The rising investments from key players towards enhancing human embryonic cell therapy is expected to aid the market growth in the forecast period. In post-COVID days, as the various sectors recover from the negative impacts of the pandemic, human embryonic stem cells are likely to witness a rise in demand.
Industry Definition and Major Segments
Human embryonic stem cells, also known as human embryonic stem cells are self-replicating cells derived from human fetal tissue or human embryos that develop into tissues and cells of 3 primary layers. Furthermore, human embryonic stem cells are pluripotent and are roughly 3-5 days old. It is highly versatile, as it may split into new stem cells and even transform into any type of cell in the human body, allowing it to regenerate or repair any diseased organ or tissue.
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The human embryonic stem cell market, on the basis of application, can be segmented into:
Regenerative Medicine Stem Cell Biology Research Tissue Engineering Toxicology Testing
The regional markets for human embryonic stem cell include:
North America Europe Asia Pacific Latin America Middle East and Africa
Among these, North America represents a significant share of the human embryonic stem cell market.
Market Trends
The rising population along with the rapidly evolving medical infrastructure of emerging economies like India and China is expected to provide an impetus to the human embryonic stem cell market. Furthermore, technological advancements and increasing research and development investments and initiatives are expected to generate opportunities in the market. Researchers and scientists are increasingly leaning toward the transformation of human embryonic stem cells into a number of mature cell types that represent various tissues and organs in the body, as embryonic cells provide unequalled data relating to a variety of disorders. The increasing efforts by the governments of various nations towards enhancing human embryonic stem cell therapy is likely to be another key trend bolstering the market growth in the forecast period.
Key Market Players
The major players in the market Astellas Pharma Inc, Stemcell Technologies Inc., Biotime INC, Thermo Fisher Scientific, Inc., among others. The report covers the market shares, capacities, plant turnarounds, expansions, investments and mergers and acquisitions, among other latest developments of these market players.
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Expert Market Research (EMR) is a leading market research and business intelligence company, ensuring its clients remain at the vanguard of their industries by providing them with exhaustive and actionable market data through its syndicated and custom market reports, covering over 15 major industry domains. The companys expansive and ever-growing database of reports, which are constantly updated, includes reports from industry verticals like chemicals and materials, food and beverages, energy and mining, technology and media, consumer goods, pharmaceuticals, agriculture, and packaging.
EMR leverages its state-of-the-art technological and analytical tools, along with the expertise of its highly skilled team of over 100 analysts and more than 3000 consultants, to help its clients, ranging from Fortune 1,000 companies to small and medium-sized enterprises, easily grasp the expansive industry data and help them in formulating market and business strategies, which ensure that they remain ahead of the curve.
Kangstem Biotech said Monday that it has voluntarily withdrawn a phase 1 and 2a clinical trial investigational new drug (IND) approval for "FURESTEM-OA Kit Inj.," a candidate material for stem cell-based osteoarthritis (OA) treatment.
The company decided to withdraw its plans after determining that it required further data reinforcement concerning establishing a cell bank for clinical trial drugs after the government started enforcing the "Advanced Regenerative Medicine and Advanced Biopharmaceuticals Safety and Support Act."
The Ministry of Food and Drug Safety had requested the results of the adventitious virus-negative test from Kangstem Biotech. The test proves that even when the test drug used in clinical trials is manufactured using a cell bank, the quality and safety are the same, and there is no scientific risk factor.
Accordingly, the company confirmed that there is no adventitious virus by completing the virus test by the qPCR test method following the ICH (International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use) regulations at Korean institutions.
Also, the company entrusted the test to the additional culture method of Charles River, an American consignment testing institution.
However, the company decided to voluntarily withdraw its IND approval, confirming it would be difficult to complete the test and analysis to secure additional data within the administrative processing period required for review of the clinical trial plan approval period.
"The IND application for the FURESTEM-OA Kit was for the first clinical trial for a stem cell-based fusion drug under the Advanced Regenerative Medicine and Advanced Biopharmaceuticals Safety and Support Act," Kangstems Clinical Development Division Director Bae Yo-han said. "Therefore, the IND approval process was somewhat delayed as the Ministry of Food and Drug Safety had to review its safety from various angles thoroughly."
During the delay period, additional test data that did not need to be initially submitted became a requirement, Bae added.
However, Bae stressed that the Ministry of Food and Drug Safety also believes that there are no additional problems in the clinical trial plan itself, other than a review on securing safety related to adventitious factors by ingredients used in the manufacturing process of the drug.
"Therefore, the company is aiming to re-apply for the phase 1 and 2a IND of the FURESTEM-OA Kit in July at the earliest and get approval within October," he said.
Due to the company's explanation, the company's shares rebounded on Wednesday after dropping about 5 percent the previous day.
As of 1:40 p.m. Tuesday, the company's stock price stood at 2,860 won ($2.22) per share, up 2.33 percent from the previous trading day.
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Kangstem Biotech withdraws trial application for stem cell-based osteoarthritis treatment - KBR
SEATTLE, June 13, 2022 (GLOBE NEWSWIRE) -- Sana Biotechnology, Inc. ( SANA), a company focused on creating and delivering engineered cells as medicines, today announced that the company will present data from its hypoimmune platform at the International Society for Stem Cell Research (ISSCR) 2022 Annual Meeting taking place from Wednesday, June 15 through Sunday, June 19 in San Francisco.
About Sana BiotechnologySana Biotechnology, Inc. is focused on creating and delivering engineered cells as medicines for patients. We share a vision of repairing and controlling genes, replacing missing or damaged cells, and making our therapies broadly available to patients. We are a passionate group of people working together to create an enduring company that changes how the world treats disease. Sana has operations in Seattle, Cambridge, South San Francisco, and Rochester.
Cautionary Note Regarding Forward-Looking StatementsThis press release contains forward-looking statements about Sana Biotechnology, Inc. (the Company, we, us, or our) within the meaning of the federal securities laws, including those related to the companys vision, progress, and business plans, the Companys participation at the ISSCR Annual Meeting, and the subject matter of the Companys presentations and data being presented at ISSCR Annual Meeting. All statements other than statements of historical facts contained in this press release, including, among others, statements regarding the Companys strategy, expectations, cash runway and future financial condition, future operations, and prospects, are forward-looking statements. In some cases, you can identify forward-looking statements by terminology such as aim, anticipate, assume, believe, contemplate, continue, could, design, due, estimate, expect, goal, intend, may, objective, plan, positioned, potential, predict, seek, should, target, will, would and other similar expressions that are predictions of or indicate future events and future trends, or the negative of these terms or other comparable terminology. The Company has based these forward-looking statements largely on its current expectations, estimates, forecasts and projections about future events and financial trends that it believes may affect its financial condition, results of operations, business strategy and financial needs. In light of the significant uncertainties in these forward-looking statements, you should not rely upon forward-looking statements as predictions of future events. These statements are subject to risks and uncertainties that could cause the actual results to vary materially, including, among others, the risks inherent in drug development such as those associated with the initiation, cost, timing, progress and results of the Companys current and future research and development programs, preclinical and clinical trials, as well as the economic, market and social disruptions due to the ongoing COVID-19 public health crisis. For a detailed discussion of the risk factors that could affect the Companys actual results, please refer to the risk factors identified in the Companys SEC reports, including but not limited to its Quarterly Report on Form 10-Q dated May 10, 2022. Except as required by law, the Company undertakes no obligation to update publicly any forward-looking statements for any reason.
Investor Relations & Media:Nicole Keith[emailprotected][emailprotected]
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Sana Biotechnology Announces Multiple Preclinical Data Presentations to Showcase Its Hypoimmune Platform, Including in Type 1 Diabetes, at the...
Dzierzak, E. & Philipsen, S. Erythropoiesis: Development and differentiation. Cold Spring Harb. Perspect. Med. 3, a011601a011601 (2013).
PubMed PubMed Central Article CAS Google Scholar
Hattangadi, S. M., Wong, P., Zhang, L., Flygare, J. & Lodish, H. F. From stem cell to red cell: Regulation of erythropoiesis at multiple levels by multiple proteins, RNAs, and chromatin modifications. Blood 118, 62586268 (2011).
CAS PubMed PubMed Central Article Google Scholar
Mei, Y., Liu, Y. & Ji, P. Understanding terminal erythropoiesis: An update on chromatin condensation, enucleation, and reticulocyte maturation. Blood Rev. 46, 100740 (2021).
CAS PubMed Article Google Scholar
Steinman, R. A. Cell cycle regulators and hematopoiesis. Oncogene 21, 34033413 (2002).
CAS PubMed Article Google Scholar
Walkley, C. R., Sankaran, V. G. & Orkin, S. H. Rb and hematopoiesis: Stem cells to anemia. Cell Div. 3, 13 (2008).
PubMed PubMed Central Article CAS Google Scholar
Teixeira, L. K. & Reed, S. I. Ubiquitin ligases and cell cycle control. Annu. Rev. Biochem. 82, 387414 (2013).
CAS PubMed Article Google Scholar
Zhou, Z., He, M., Shah, A. A. & Wan, Y. Insights into APC/C: From cellular function to diseases and therapeutics. Cell Div. 11, 118 (2016).
Article CAS Google Scholar
Chang, L. & Barford, D. Insights into the anaphase-promoting complex: A molecular machine that regulates mitosis. Curr. Opin. Struct. Biol. 29, 19 (2014).
CAS PubMed Article Google Scholar
Eguren, M., Manchado, E. & Malumbres, M. Non-mitotic functions of the anaphase-promoting complex. Semin. Cell Dev. Biol. 22, 572578 (2011).
CAS PubMed Article Google Scholar
Qiao, X., Zhang, L., Gamper, A. M., Fujita, T. & Wan, Y. APC/C-Cdh1: From cell cycle to cellular differentiation and genomic integrity. Cell Cycle 9, 39043912 (2010).
CAS PubMed PubMed Central Article Google Scholar
Wsch, R., Robbins, J. A. & Cross, F. R. The emerging role of APC/CCdh1 in controlling differentiation, genomic stability and tumor suppression. Oncogene 29, 110 (2010).
PubMed Article CAS Google Scholar
Engelbert, D., Schnerch, D., Baumgarten, A. & Wsch, R. The ubiquitin ligase APCCdh1 is required to maintain genome integrity in primary human cells. Oncogene 27, 907917 (2008).
CAS PubMed Article Google Scholar
Garca-Higuera, I. et al. Genomic stability and tumour suppression by the APC/C cofactor Cdh1. Nat. Cell Biol. 10, 802811 (2008).
PubMed Article CAS Google Scholar
Sigl, R. et al. Loss of the mammalian APC/C activator FZR1 shortens G1 and lengthens S phase but has little effect on exit from mitosis. J. Cell Sci. 122, 42084217 (2009).
CAS PubMed Article Google Scholar
Yuan, X., Srividhya, J., De Luca, T., Lee, J.-H.E. & Pomerening, J. R. Uncovering the role of APC-Cdh1 in generating the dynamics of S-phase onset. Mol. Biol. Cell 25, 441456 (2014).
PubMed PubMed Central Article Google Scholar
Greil, C. et al. The role of APC/CCdh1 in replication stress and origin of genomic instability. Oncogene 35, 30623070 (2016).
CAS PubMed Article Google Scholar
Garzn, J. et al. Shortage of dNTPs underlies altered replication dynamics and DNA breakage in the absence of the APC/C cofactor Cdh1. Oncogene 36, 58085818 (2017).
PubMed Article CAS Google Scholar
Kimata, Y. APC/C ubiquitin ligase: Coupling cellular differentiation to G1/G0 phase in multicellular systems. Trends Cell Biol. 29, 591603 (2019).
CAS PubMed Article Google Scholar
Sigrist, S. J. & Lehner, C. F. Drosophila fizzy-related down-regulates mitotic cyclins and is required for cell proliferation arrest and entry into endocycles. Cell 90, 671681 (1997).
CAS PubMed Article Google Scholar
Delgado-Esteban, M., Garca-Higuera, I., Maestre, C., Moreno, S. & Almeida, A. APC/C-Cdh1 coordinates neurogenesis and cortical size during development. Nat. Commun. 4, 2879 (2013).
ADS PubMed Article CAS Google Scholar
Eguren, M. et al. The APC/C cofactor Cdh1 prevents replicative stress and p53-dependent cell death in neural progenitors. Nat. Commun. 4, 2880 (2013).
ADS PubMed Article CAS Google Scholar
Holt, J. E. et al. The APC/C activator FZR1 is essential for meiotic prophase I in mice. Development 141, 13541365 (2014).
CAS PubMed Article Google Scholar
Li, W., Wu, G. & Wan, Y. The dual effects of Cdh1/APC in myogenesis. FASEB J. 21, 36063617 (2007).
CAS PubMed Article Google Scholar
Wu, G. et al. The anaphase-promoting complex coordinates initiation of lens differentiation D. Mol. Biol. Cell 18, 10181029 (2007).
CAS PubMed PubMed Central Article Google Scholar
Koulnis, M. et al. Identification and analysis of mouse erythroid progenitors using the CD71/TER119 flow-cytometric assay. J. Vis. Exp. https://doi.org/10.3791/2809 (2011).
Article PubMed PubMed Central Google Scholar
de Boer, J. et al. Transgenic mice with hematopoietic and lymphoid specific expression of Cre. Eur. J. Immunol. 33, 314325 (2003).
PubMed Article Google Scholar
Bennett, L. F., Liao, C. & Paulson, R. F. Stress Erythropoiesis Model Systems 91102 (Springer, 2018). https://doi.org/10.1007/978-1-4939-7428-3_5.
Book Google Scholar
Swartz, K. L. et al. E2F2 promotes nuclear condensation and enucleation of terminally differentiated erythroblasts. Mol. Cell. Biol. 37, 110 (2017).
Article Google Scholar
Farrs, J. et al. PARP-2 sustains erythropoiesis in mice by limiting replicative stress in erythroid progenitors. Cell Death Differ. 22, 11441157 (2015).
PubMed Article CAS Google Scholar
Liu, J. et al. Quantitative analysis of murine terminal erythroid differentiation in vivo: Novel method to study normal and disordered erythropoiesis. Blood 121, e43e49 (2013).
CAS PubMed PubMed Central Article Google Scholar
Kozar, K. et al. Mouse development and cell proliferation in the absence of D-cyclins. Cell 118, 477491 (2004).
CAS PubMed Article Google Scholar
Sankaran, V. G. et al. Cyclin D3 coordinates the cell cycle during differentiation to regulate erythrocyte size and number. Genes Dev. 26, 20752087 (2012).
CAS PubMed PubMed Central Article Google Scholar
Spike, B. T. & Macleod, K. F. The Rb tumor suppressor in stress responses and hematopoietic homeostasis. Cell Cycle 4, 4245 (2005).
CAS PubMed Article Google Scholar
Sankaran, V. G., Orkin, S. H. & Walkley, C. R. Rb intrinsically promotes erythropoiesis by coupling cell cycle exit with mitochondrial biogenesis. Genes Dev. 22, 463475 (2008).
CAS PubMed PubMed Central Article Google Scholar
Kinross, K. M., Clark, A. J., Iazzolino, R. M. & Humbert, P. O. E2f4 regulates fetal erythropoiesis through the promotion of cellular proliferation. Blood 108, 886895 (2006).
CAS PubMed Article Google Scholar
Hu, T. et al. Concomitant inactivation of Rb and E2f8 in hematopoietic stem cells synergizes to induce severe anemia. Blood 119, 45324542 (2012).
CAS PubMed PubMed Central Article Google Scholar
Ghazaryan, S. et al. Inactivation of Rb and E2f8 synergizes to trigger stressed DNA replication during erythroid terminal differentiation. Mol. Cell. Biol. 34, 28332847 (2014).
PubMed PubMed Central Article CAS Google Scholar
Alvarez, S. et al. Replication stress caused by low MCM expression limits fetal erythropoiesis and hematopoietic stem cell functionality. Nat. Commun. 6, 8548 (2015).
ADS CAS PubMed Article Google Scholar
Ishizawa, J. et al. The cell cycle regulator Cdh1 controls the pool sizes of hematopoietic stem cells and mature lineage progenitors by protecting from genotoxic stress. Cancer Sci. 102, 967974 (2011).
CAS PubMed Article Google Scholar
He, C. et al. c-myc in the hematopoietic lineage is crucial for its angiogenic function in the mouse embryo. Development 135, 24672477 (2008).
CAS PubMed Article Google Scholar
Dirlam, A., Spike, B. T. & Macleod, K. F. Deregulated E2f2 underlies cell cycle and maturation defects in retinoblastoma null erythroblasts. Mol. Cell. Biol. 27, 87138728 (2007).
CAS PubMed PubMed Central Article Google Scholar
Cohen, M. et al. Unbiased transcriptome signature of in vivo cell proliferation reveals pro- and antiproliferative gene networks. Cell Cycle 12, 29923000 (2013).
CAS PubMed PubMed Central Article Google Scholar
Boekhout, M. et al. Feedback regulation between atypical E2Fs and APC/C-Cdh1 coordinates cell cycle progression. EMBO Rep. 17, 414427 (2016).
CAS PubMed PubMed Central Article Google Scholar
Clijsters, L. et al. Cyclin F controls cell-cycle transcriptional outputs by directing the degradation of the three activator E2Fs. Mol. Cell 74, 12641277 (2019).
CAS PubMed PubMed Central Article Google Scholar
Yuan, R. et al. Cyclin F-dependent degradation of E2F7 is critical for DNA repair and G2-phase progression. EMBO J. 38, 20 (2019).
Google Scholar
Wasserman, D. et al. Cell cycle oscillators underlying orderly proteolysis of E2F8. Mol. Biol. Cell 31, 725740 (2020).
CAS PubMed PubMed Central Article Google Scholar
Choudhury, R. et al. APC/C and SCF(cyclin F) constitute a reciprocal feedback circuit controlling S-phase entry. Cell Rep. 16, 33593372 (2016).
CAS PubMed PubMed Central Article Google Scholar
Angulo-Ibez, M. et al. Erk5 contributes to maintaining the balance of cellular nucleotide levels and erythropoiesis. Cell Cycle 14, 38643876 (2015).
PubMed PubMed Central Article CAS Google Scholar
Bracken, A. P., Ciro, M., Cocito, A. & Helin, K. E2F target genes: Unraveling the biology. Trends Biochem. Sci. 29, 409417 (2004).
CAS PubMed Article Google Scholar
Hayashi, S., Lewis, P., Pevny, L. & McMahon, A. P. Efficient gene modulation in mouse epiblast using a Sox2Cre transgenic mouse strain. Mech. Dev. 119, S97S101 (2002).
PubMed Article Google Scholar
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Efficient terminal erythroid differentiation requires the APC/C cofactor Cdh1 to limit replicative stress in erythroblasts | Scientific Reports -...
MELBOURNE, Australia--(BUSINESS WIRE)--Propanc Biopharma, Inc. (OTCQB: PPCB) (Propanc or the Company), a biopharmaceutical company developing novel cancer treatments for patients suffering from recurring and metastatic cancer, today announced that the results from a small trial of just 18 rectal cancer patients in complete remission using an immunotherapy called dostarlimab are impressive, whilst acknowledging theres more work to be done. Chief Scientific Officer and Co-Founder, Dr Julian Kenyon MD, MB, ChB, believes that the fields biggest challenge remains that immunotherapies work inconsistently across cancers. Oncologists estimate a response rate of 20% across cancer types, according to the Wall Street Journal (WSJ). The drugs can wipe out cancers from some people, but fail to work for others. It is also uncertain whether the cancer may eventually return once a patient is in remission, even after a prolonged period of time.
Immunotherapies like dostarlimab, known as a checkpoint inhibitor, seek to inhibit key regulators of the immune system that when stimulated, reduces the bodys immune response to fight cancer. Given that immunotherapies target specific gene sequences, it often means they can encounter resistance, due to mutations that occur and genetic variation even within the primary tumor of a patient. As a result, Dr Cercek, from Memorial Sloan Kettering, who conducted the study for dostarlimab, estimates only 10% of rectal cancer patients and about 4% of all cancers will respond to treatment, according to the WSJ.
For many cancers, multiple factors can drive growth, making it hard to effectively match one biomarker, or a particular gene sequence, to a single drug. On the other hand, a therapeutic approach like our lead product candidate, PRP, which alters the characteristics of the cancer cell, by enforcing it to express proteins it normally wouldnt, means the treatment is less likely to encounter resistance through mutations, which is what we have observed in the lab as well as in clinical practice, said Dr Julian Kenyon.
In addition to specifically selecting the 18 rectal cancer patients according to their genetic biomarker, the trial included patients that were pre-metastatic, where tumors were locally advanced in one area, but not spread to other organs. This means patients identified with metastatic cancer were excluded from the trial. Therefore, the treatment and prevention of metastatic cancer, the main cause of patient death for sufferers, still remains the unsolved, final frontier. Cancer stem cells, which are the cells responsible for spreading to other parts of the body, remains a key focus for Dr Kenyon.
Dr Kenyon said, PRP is a proenzyme treatment that targets and eradicates cancer stem cells by altering multiple pathways of a cancerous cell rather than a single genetic sequence. Weve observed that once they are treated with PRP, the effects are irreversible and are more easily recognizable by the immune system, therefore potentially improving the response rates of standard approaches like immunotherapy, to overcome advanced cancers. We look forward to testing the utility of PRP with these approaches as we further advance into the clinic.
PRP is a mixture of two proenzymes, trypsinogen and chymotrypsinogen from bovine pancreas administered by intravenous injection. A synergistic ratio of 1:6 inhibits growth of most tumor cells. Examples include kidney, ovarian, breast, brain, prostate, colorectal, lung, liver, uterine and skin cancers.
About Propanc Biopharma, Inc.
Propanc Biopharma, Inc. (the Company) is developing a novel approach to prevent recurrence and metastasis of solid tumors by using pancreatic proenzymes that target and eradicate cancer stem cells in patients suffering from pancreatic, ovarian and colorectal cancers. For more information, please visit http://www.propanc.com.
The Companys novel proenzyme therapy is based on the science that enzymes stimulate biological reactions in the body, especially enzymes secreted by the pancreas. These pancreatic enzymes could represent the bodys primary defense against cancer.
To view the Companys Mechanism of Action video on its anti-cancer lead product candidate, PRP, please click on the following link: http://www.propanc.com/news-media/video
Forward-Looking Statements
All statements other than statements of historical facts contained in this press release are forward-looking statements, which may often, but not always, be identified by the use of such words as may, might, will, will likely result, would, should, estimate, plan, project, forecast, intend, expect, anticipate, believe, seek, continue, target or the negative of such terms or other similar expressions. These statements involve known and unknown risks, uncertainties and other factors, which may cause actual results, performance or achievements to differ materially from those expressed or implied by such statements. These factors include uncertainties as to the Companys ability to continue as a going concern absent new debt or equity financings; the Companys current reliance on substantial debt financing that it is unable to repay in cash; the Companys ability to successfully remediate material weaknesses in its internal controls; the Companys ability to reach research and development milestones as planned and within proposed budgets; the Companys ability to control costs; the Companys ability to obtain adequate new financing on reasonable terms; the Companys ability to successfully initiate and complete clinical trials and its ability to successful develop PRP, its lead product candidate; the Companys ability to obtain and maintain patent protection; the Companys ability to recruit employees and directors with accounting and finance expertise; the Companys dependence on third parties for services; the Companys dependence on key executives; the impact of government regulations, including FDA regulations; the impact of any future litigation; the availability of capital; changes in economic conditions, competition; and other risks, including, but not limited to, those described in the Companys periodic reports that are filed with the Securities and Exchange Commission and available on its website at http://www.sec.gov. These forward-looking statements speak only as of the date hereof and the Company disclaims any obligations to update these statements except as may be required by law.
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Propanc Biopharma's CSO Hails Dostarlimab's Impressive Results Whilst Acknowledging More Work to Be Done in the Fight Against Cancer - Business Wire
DURHAM, N.C.--(BUSINESS WIRE)--Precision BioSciences, Inc. (Nasdaq: DTIL), a clinical stage gene editing company developing ARCUS-based ex vivo allogeneic CAR T and in vivo gene editing therapies, today announced it has entered into an exclusive worldwide in vivo gene editing research and development collaboration and license agreement with Novartis Pharma AG (the Agreement). As part of the Agreement, Precision will develop a custom ARCUS nuclease that will be designed to insert, in vivo, a therapeutic transgene at a safe harbor location in the genome as a potential one-time transformative treatment option for diseases including certain hemoglobinopathies such as sickle cell disease and beta thalassemia.
Under the terms of the Agreement, Precision will develop an ARCUS nuclease and conduct in vitro characterization, with Novartis then assuming responsibility for all subsequent research, development, manufacturing and commercialization activities. Novartis will receive an exclusive license to the custom ARCUS nuclease developed by Precision for Novartis to further develop as a potential in vivo treatment option for sickle cell disease and beta thalassemia. Precision will receive an upfront payment of $75 million and is eligible to receive up to an aggregate amount of approximately $1.4 billion in additional payments for future milestones. Precision is also eligible to receive certain research funding and, should Novartis successfully commercialize a therapy from the collaboration, tiered royalties ranging from the mid-single digits to low-double digits on product sales.
We are excited to collaborate with Novartis to bring together the precision and versatility of ARCUS genome editing with Novartis gene therapy expertise and commitment to developing one-time, potentially transformative treatment for hard-to-treat inherited blood disorders, said Michael Amoroso, Chief Executive Officer at Precision BioSciences. This collaboration will build on the unique gene insertion capabilities of ARCUS and illustrates its utility as a premium genome editing platform for potential in vivo drug development. With this Agreement, Precision, either alone or with world-class partners, will have active in vivo gene editing programs for targeted gene insertion and gene deletions in hematopoietic stem cells, liver, muscle and the central nervous system showcasing the distinctive versatility of ARCUS.
We identify here a collaborative opportunity to imagine a unique therapeutic option for patients with hemoglobinopathies, such as sickle cell disease and beta thalassemia a potential one-time treatment administered directly to the patient that would overcome many of the hurdles present today with other therapeutic technologies, said Jay Bradner, President of the Novartis Institutes for Biomedical Research (NIBR), the Novartis innovation engine. We look forward to working with Precision and leveraging the ARCUS technology platform, which could bring a differentiated approach to the treatment of patients with hemoglobinopathies."
The in vivo gene editing approach that we are pursuing for sickle cell disease could have a number of significant advantages over other ex vivo gene therapies currently in development, said Derek Jantz, Ph.D., Chief Scientific Officer and Co-Founder of Precision BioSciences. Perhaps most importantly, it could open the door to treating patients in geographies where stem cell transplant is not a realistic option. We believe that the unique characteristics of the ARCUS platform, particularly its ability to target gene insertion with high efficiency, make it the ideal choice for this project, and we look forward to working with our partners at Novartis to bring this novel therapy to patients.
Upon completion of the transaction, Precision expects that existing cash and cash equivalents, expected operational receipts, and available credit will be sufficient to fund its operating expenses and capital expenditure requirements into Q2 2024.
Precision BioSciences Conference Call and Webcast Information
Precision's management team will host a conference call and webcast tomorrow, June 22, 2022, at 8:00 AM ET to discuss the collaboration. The dial-in conference call numbers for domestic and international callers are (866)-996-7202 and (270)-215-9609, respectively. The conference ID number for the call is 6252688. Participants may access the live webcast on Precision's website https://investor.precisionbiosciences.com/events-and-presentations in the Investors page under Events and Presentations. An archived replay of the webcast will be available on Precision's website.
About ARCUS and Safe harbor ARCUS Nucleases
ARCUS is a proprietary genome editing technology discovered and developed by scientists at Precision BioSciences. It uses sequence-specific DNA-cutting enzymes, or nucleases, that are designed to either insert (knock-in), remove (knock-out), or repair DNA of living cells and organisms. ARCUS is based on a naturally occurring genome editing enzyme, I-CreI, that evolved in the algae Chlamydomonas reinhardtii to make highly specific cuts in cellular DNA. Precision's platform and products are protected by a comprehensive portfolio including nearly 100 patents to date.
Precision can use an ARCUS nuclease to add a healthy copy of a gene (or payload) to a persons genome. The healthy copy of the gene can be inserted at its usual site within the genome, replacing the mutated, disease-causing copy. Alternatively, an ARCUS nuclease can be used to insert a healthy copy of the gene at another site within the genome called a safe harbor that enables production of the healthy gene product without otherwise affecting the patients DNA of gene expression patterns.
About Sickle Cell Disease and Beta Thalassemia
Sickle cell disease (SCD) is a complex genetic disorder that affects the structure and function of hemoglobin, reduces the ability of red blood cells to transport oxygen efficiently and, early on, progresses to a chronic vascular disease.1-4 The disease can lead to acute episodes of pain known as sickle cell pain crises, or vaso-occlusive crises, as well as life-threatening complications.5-7 The condition affects 20 million people worldwide.8 Approximately 80% of individuals with SCD globally live in sub-Saharan Africa and it is estimated that approximately 1,000 children in Africa are born with SCD every day and more than half will die before they reach five.9,10 SCD is also a multisystem disorder and the most common genetic disease in the United States, affecting 1 in 500 African Americans. About 1 in 12 African Americans carry the autosomal recessive mutation, and approximately 300,000 infants are born with sickle cell anemia annually.11 Even with todays best available care, SCD continues to drive premature deaths and disability as this lifelong illness often takes an extreme emotional, physical, and financial toll on patients and their families.12,13
Beta thalassemia is also an inherited blood disorder characterized by reduced levels of functional hemoglobin.14 The condition has three main forms minor, intermedia and major, which indicate the severity of the disease.14 While the symptoms and severity of beta thalassemia varies greatly from one person to another, a beta thalassemia major diagnosis is usually made during the first two years of life and individuals require regular blood transfusions and lifelong medical care to survive.14 Though the disorder is relatively rare in the United States, it is one of the most common autosomal recessive disorders in the world.14 The incidence of symptomatic cases is estimated to be approximately 1 in 100,000 individuals in the general population.14, 15 The frequency of beta-thalassemia mutations varies by regions of the world with the highest prevalence in the Mediterranean, the Middle-East, and Southeast and Central Asia. Approximately 68,000 children are born with beta-thalassemia.16
About Precision BioSciences, Inc.
Precision BioSciences, Inc. is a clinical stage biotechnology company dedicated to improving life (DTIL) with its novel and proprietary ARCUS genome editing platform. ARCUS is a highly precise and versatile genome editing platform that was designed with therapeutic safety, delivery, and control in mind. Using ARCUS, the Companys pipeline consists of multiple ex vivo off-the-shelf CAR T immunotherapy clinical candidates and several in vivo gene editing candidates designed to cure genetic and infectious diseases where no adequate treatments exist. For more information about Precision BioSciences, please visit http://www.precisionbiosciences.com.
Forward-Looking Statements
This press release contains forward-looking statements, as may any related presentations, within the meaning of the Private Securities Litigation Reform Act of 1995. All statements contained in this herein and in any related presentation that do not relate to matters of historical fact should be considered forward-looking statements, including, without limitation, statements regarding the goal of providing a one time, potentially curative treatment for certain hemoglobinopathies, the success of the collaboration with Novartis, including the receipt of any milestone, royalty, or other payments pursuant to and the satisfaction of obligations under the Agreement, clinical and regulatory development and expected efficacy and benefit of our platform and product candidates, expectations about our operational initiatives and business strategy, expectations about achievement of key milestones, and expected cash runway. In some cases, you can identify forward-looking statements by terms such as aim, anticipate, approach, believe, contemplate, could, estimate, expect, goal, intend, look, may, mission, plan, potential, predict, project, should, target, will, would, or the negative thereof and similar words and expressions. Forward-looking statements are based on managements current expectations, beliefs and assumptions and on information currently available to us. Such statements are subject to a number of known and unknown risks, uncertainties and assumptions, and actual results may differ materially from those expressed or implied in the forward-looking statements due to various important factors, including, but not limited to: our ability to become profitable; our ability to procure sufficient funding and requirements under our current debt instruments and effects of restrictions thereunder; risks associated with raising additional capital; our operating expenses and our ability to predict what those expenses will be; our limited operating history; the success of our programs and product candidates in which we expend our resources; our limited ability or inability to assess the safety and efficacy of our product candidates; our dependence on our ARCUS technology; the initiation, cost, timing, progress, achievement of milestones and results of research and development activities, preclinical studies and clinical trials; public perception about genome editing technology and its applications; competition in the genome editing, biopharmaceutical, and biotechnology fields; our or our collaborators ability to identify, develop and commercialize product candidates; pending and potential liability lawsuits and penalties against us or our collaborators related to our technology and our product candidates; the U.S. and foreign regulatory landscape applicable to our and our collaborators development of product candidates; our or our collaborators ability to obtain and maintain regulatory approval of our product candidates, and any related restrictions, limitations and/or warnings in the label of an approved product candidate; our or our collaborators ability to advance product candidates into, and successfully design, implement and complete, clinical or field trials; potential manufacturing problems associated with the development or commercialization of any of our product candidates; our ability to obtain an adequate supply of T cells from qualified donors; our ability to achieve our anticipated operating efficiencies at our manufacturing facility; delays or difficulties in our and our collaborators ability to enroll patients; changes in interim top-line and initial data that we announce or publish; if our product candidates do not work as intended or cause undesirable side effects; risks associated with applicable healthcare, data protection, privacy and security regulations and our compliance therewith; the rate and degree of market acceptance of any of our product candidates; the success of our existing collaboration agreements, and our ability to enter into new collaboration arrangements; our current and future relationships with and reliance on third parties including suppliers and manufacturers; our ability to obtain and maintain intellectual property protection for our technology and any of our product candidates; potential litigation relating to infringement or misappropriation of intellectual property rights; our ability to effectively manage the growth of our operations; our ability to attract, retain, and motivate key executives and personnel; market and economic conditions; effects of system failures and security breaches; effects of natural and manmade disasters, public health emergencies and other natural catastrophic events; effects of COVID-19 pandemic and variants thereof, or any pandemic, epidemic or outbreak of an infectious disease; insurance expenses and exposure to uninsured liabilities; effects of tax rules; risks related to ownership of our common stock and other important factors discussed under the caption Risk Factors in our Quarterly Report on Form 10-Q for the quarterly period ended March 31, 2022, as any such factors may be updated from time to time in our other filings with the SEC, which are accessible on the SECs website at http://www.sec.gov and the Investors page of our website under SEC Filings at investor.precisionbiosciences.com.
References
1 Saraf SL, et al. Paediatr Respir Rev. 2014;15(1):4-12.2 Stuart MJ, et al. Lancet. 2004;364(9442):1343-1360.3 National Institutes of Health (NIH). Sickle cell disease. Bethesda, MD. U.S. National Library of Medicine. 2018:1-7.4 Conran N, Franco-Penteado CF, Costa FF. Hemoglobin. 2009;33(1):1-16.5 Ballas SK, et al. Blood. 2012;120(18):3647-3656.6 Elmariah H, et al. Am J Hematol. 2014(5):530-535.7 Steinberg M. Management of sickle cell disease. N Engl J Med. 1999;340(13):1021-1030.8 National Heart Lung and Blood Institute: What Is Sickle Cell Disease? 9 Odame I. Perspective: We need a global solution. Nature. 2014 Nov;515(7526):S1010 Scott D. Grosse, Isaac Odame, Hani K. Atrash, et al. Sickle Cell Disease in Africa: A Neglected Cause of Early Childhood Mortality. American Journal of Preventive Medicine 41, no. S4 (December 2011): S398-40511 Sedrak A, Kondamudi NP. Sickle Cell Disease. [Updated 2021 Nov 7]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 Jan-.12 Sanger M, Jordan L, Pruthi S, et al. Cognitive deficits are associated with unemployment in adults with sickle cell anemia. Journal of Clinical and Experimental Neuropsychology. 2016;38(6):661-671.13 Anim M, Osafo J, Yirdong F. Prevalence of psychological symptoms among adults with sickle cell disease in Korie-Bu Teaching Hospital, Ghana. BMC Psychology. 2016;4(53):1-9.14 NORD Rare Disease Database: Beta Thalassemia 15 Galanello R, Origa R. Orphanet J Rare Dis. 2010;5:1116 Needs T, Gonzalez-Mosquera LF, Lynch DT. Beta Thalassemia. [Updated 2022 May 8]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 Jan-.
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Precision BioSciences Announces In Vivo Gene Editing Collaboration with Novartis to Develop Potentially Curative Treatment for Disorders Including...
The last decade of immunotherapy progress was based on decades of prior research, including other forms of immunotherapy.
Until recent years, cancer treatment revolved around surgery, chemotherapy, and radiation. But the FDA approval of ipilimumab (Yervoy) in 2011 led to a fourth leg of that treatment stool: immunotherapy. This enabled new treatment paradigms, sometimes with shocking levels of success.
The types of immunotherapy treatments available are proliferating, with approved immune checkpoint inhibitors (ICIs) and cellular therapies like chimeric antigen receptor (CAR) T cells as well as other modalities in the research and discovery phases. Some even include more established approaches like vaccines that are being revisited with new information and iterations.
The last decade of immunotherapy progress was based on decades of prior research, including other forms of immunotherapy. The Bacillus Calmette-Gurin vaccine, used to prevent tuberculosis for a century, has also been used as an immunotherapy to treat nonmuscle invasive bladder cancer since 1990.1 And rituximab (Rituxan), a monoclonal antibody therapy approved in 1997 for B-cell malignancies, is seen by some as an early immunotherapy as well.2
What many clinicians think of in terms of immunotherapy, however, are treatments targeting CTLA-4 and PD-1/PD-L1 pathways, brought from the bench by James P. Allison, PhD, and Tasuku Honjo, PhD, respectively, leading to a Nobel Prize awarded jointly to them in 2018.3
Immune responses are tightly controlled by T cells, and these T cells have on/off switches that help control their responses, according to Padmanee Sharma, MD, PhD, a professor in the Department of Genitourinary Medical Oncology in the Division of Cancer Medicine and the scientific director of the James P. Allison Institute at The University of Texas MD Anderson Cancer Center in Houston. Previously, she said, clinicians were not aware of the off switches. Allison showed that CTLA-4 was an inhibitory pathway and that by blocking it, the T cells could stay longer to eradicate the tumors.
With 8 ICIs approved for immunotherapy in hematological and solid tumors,4 researchers are not only investigating newer forms of therapy, but also combining them to fi nd more effective and durable treatments and introducing them into earlier lines of treatment (TIMELINE). Current research is also attempting to predict who will respond to which therapy based on current and emerging biomarkers.
Ipilimumab, which kicked off the current era of cancer immunotherapy treatment with FDA approval in 2011, targets CTLA-4 for newly diagnosed or previously treated unresectable or metastatic melanoma.5 Ipilimumab blocks CTLA-4, removing its inhibitory signals. This allows the T cells to activate and launch an immune response to the tumors antigens.
CTLA-4 is basically the fi rst inhibitory pathway that comes up on the T cells, Sharma said. CTLA-4 is a member of an immunoglobulin-related receptor family responsible for some immune regulation aspects of T cells.6 It is thought to regulate T-cell proliferation mostly in lymph nodes, early in an immune response, by having an inhibitory role.7
What ipilimumab really did and what the immune checkpoint inhibitors really did is they opened up this whole different way to approach the immune system, Elizabeth Buchbinder, MD, a medical oncologist at Dana-Farber Cancer Institute and an assistant professor of medicine at Harvard Medical School in Boston, Massachusetts, said. Ipilimumab provided amazing durable responses in patients with melanoma with widely metastatic disease, some of whom were alive 10 years later, she said.
The PD-1 and PD-L1 blockades build on ipilimumabs success. Like CTLA-4, PD-1 is a negative regulator of T-cell immune function, inhibiting the target to increase immune system activation. PD-1 suppresses T cells mostly in the peripheral tissues.7 As of November 2021, 8 ICIs have been approved that target CTLA-4, PD-1, and PD-L1 pathways and treat 18 types of cancer.3
AntiPD-1 inhibitors
The percentage of people who benefi tted from ipilimumab was on the low side, Buchbinder said, with only an 11% response rate and 20% of people doing well long term in clinical trials. With PD-1 inhibition, however, there was approximately a 40% response rate and many more patients doing well long term, as demonstrated in clinical trials. So [PD-1 inhibition is] both far more effective and also less toxic, Buchbinder said.
When choosing an agent in the PD-1 class, we dont need to differentiate them. Theyre all antiPD-1, Sharma explained. There arent any data to indicate that patients will respond any differently to pembrolizumab [Keytruda] vs nivolumab [Opdivo]. The mechanism of action for both drugs [is] exactly the same.
Instead, clinicians should consider the FDA approvals for each drugs indications and combinations. But from a scientific standpoint, theres no distinguishing between [them], Sharma said.
AntiPD-L1 inhibitors
PD-1 and PD-L1 targeting drugs were found to work beyond melanoma and kidney cancer, the early indications for treatments targeting the CTLA-4 pathway, Buchbinder said. That was a huge opening up of this fi eld to all of these other cancers, like lung cancer, head and neck cancer, GI [gastrointestinal] cancer, breast [cancer], and beyond, she said.
Before receiving these immunotherapies, patients may need to show PD-1 or PD-L1 expression, although this may not identify all patients who can benefi t from the treatments. Researchers continue to try to identify additional and better biomarkers to indicate which patients may respond.13
In March, the FDA approved the newest ICI, nivolumab and relatlimab-rmbw (Opdualag), for adult and pediatric patients (12 years and older) with unresectable or metastatic melanoma. 3 Nivolumab is a PD-1 inhibitor, and relatlimab blocks LAG3 proteins on immune cells. It is being tested in a lot of other tumors, Buchbinder noted.
Another target in the discovery phase is T cell immunoglobulin and mucin domain 3, which is a checkpoint receptor expressed by many immune cells and leukemic stem cells.14 It is activated by several ligands and is being tested in different cancer types.
Also in clinical trials are tumor-infiltrating lymphocytes (TIL) that recognize cancer cells as abnormal, entering the tumor to kill the cells. TILs already recognize the targets because they originate from the tumor itself.15 Although they need to be expanded, they are not the same as CAR T cells, which must be engineered to recognize the targets.
In addition, older therapies are experiencing a resurgence, with research underway to make interleukin 2 (IL-2) help cytokines function better. That work is trying to optimize what those cytokines do in the body and the immune system, Buchbinder said. There are so many areas where the goal of the therapy is activation of the immune system.
One of these areas includes a return to vaccines. In earlier vaccine therapy, We had no idea that while we were giving therapy to turn on the cells, we were also rapidly turning off the cells because an on switch will automatically drive an off switch for the immune system, Sharma said. The yin and the yang of the immune response is very important to understand because when the immune response is driven in one direction, it will always try to control itself. With that in mind, newer vaccines might work better if given in combination with an antiCTLA-4, for example, to block the inhibitory pathways, she said.
Vaccines are taking many forms, including the mRNA vaccine used for COVID-19, peptide vaccines that include a tiny bit of protein that is expected to be expressed on the tumor surface, and vaccines constructed from dendritic cells, which stimulate T cells, Buchbinder said.
There are also viral therapies injected directly into tumor vaccines, such as talimogene laherparepvec (Imlygic) approved in 2015 for the treatment of some patients with metastatic melanoma that cannot be surgically removed.16 It is a is a modifi ed herpes virus directly injected into the tumor to bring about a local immune response, Buchbinder said.
According to Sharma, approximately 60 targets are currently being evaluated for immunotherapy development.
The FDA has approved 2 CAR T-cell therapies, both in 2017: tisagenlecleucel (Kymriah) for patients 25 years and younger with relapsed B-cell precursor acute lymphoblastic leukemia17 and axicabtagene ciloleucel (Yescarta) for the treatment of adult patients with large B-cell lymphoma that is refractory to fi rst-line chemoimmunotherapy or that relapses within 12 months of fi rst-line chemoimmunotherapy.18 These treatments involve collecting T cells from the patient and engineering them to express CARs that recognize the patients cancer cells. The cells are then enlarged and infused back into the patient, where they can target the antigen- expressing cancer cells. CARs have been shown to greatly improve clinical response and disease remission in some patients.19
I think CAR T cells are clearly building on the concept that T cells are the soldiers of immune response. They are basically engineering the cell to have an antibody that recognizes a specifi c antigen, Sharma said, adding that its important to ensure the targeted antigen is part of the cancer.
CAR T cells have had limited effectiveness in treating solid tumors, given the low T-cell infiltration and immunosuppressive environment that challenges the immune system from successfully reaching and killing solid tumor cancer cells.20
Natural killer (NK) cells are another cell type being researched to attempt tumor eradication, and this therapy is in the early stages, according to Sharma. CAR NK cells can be generated from allogenic donors, making them more attractive as off the shelf treatments compared with CAR T cells, which are collected from the patient. As of early 2021, more than 500 CAR T-cell trials and 17 CAR T-cell/NK-cell trials were in the works globally.21
A major consideration when choosing any treatment, including immunotherapies, is the adverse event (AE) profile. Immunotherapy drugs have different AEs than oncology treatments like chemotherapy or radiation. [With immunotherapy,] what we see is infl ammation because youre turning on the immune system in such a powerful way, Sharma said. Inflammatory reactions include a skin rash or dermatitis, infl ammation in the colon (colitis and diarrhea), and/or infl ammation in the lung with pneumonitis. Clinicians are now aware of these AEs and can monitor them closely, stopping therapy if needed to control them before they become severe, Sharma said.
Toxicities with ipilimumab can be severe, and patients requiring hospital admission might need high-dose steroids, Buchbinder noted. Common AEs for the CTLA-4 inhibitor are typically GI related, including diarrhea, colitis, and hepatitis. Some patients may experience fatigue or a small rash, but most generally make it through treatment with minimal AEs.
The stronger AEs with ipilimumab can be seen from a trial comparing ipilimumab plus nivolumab to nivolumab and relatlimab. Almost 60% of patients experienced AEs with the ipilimumab combination vs 20% in the latter group.17
PD-1 and PD-L1 inhibition typically involve AEs that cause lung issues rather than GI. The types of organ systems affected by immunotherapy AEs can vary based upon which checkpoint inhibitor you use but in some ways, the mechanism by which these occur is very similar, Buchbinder said. Its all an overactivation of the immune system leading to infl ammation in an organ, and there are very few organs that we have not seen toxicity from immunotherapy.
Buchbinder noted that cellular therapies can cause more severe AEs, such as cytokine release syndrome (CRS). Patients can get very sick very quickly, she said, because the therapies given with the cellsincluding the chemotherapy given before and the IL-2 given aftercause most of the AEs. With a lot of the injection therapies, the AEs are related to delivery method, like injection-site issues, but there are also potential systemic AEs like fever, chills, and reactions someone would get to a virus. Its really a huge range in terms of the different [adverse] effects, Buchbinder said.
CRS is the most common AE of CAR T-cell therapy, and it is caused by large numbers of T cells activating, which releases inflammatory cytokines. Although this demonstrates that the therapy is working, it can cause worrisome symptoms. The CRS and the related neurotoxicity can be treated with tocilizumab (Actemra).
One question in the immunotherapy world is whether the development of immune-related AEs predicts a positive or negative response to treatment. With melanoma, we think the data have been very tricky, Buchbinder said. Early trials appeared to show a higher response rate for patients who developed severe symptoms, but as trials developed, that signal was not always there. I think the overall impression is that yes, severe AEs are associated with a better response, she said. A cosmetic AE that clinicians who treat melanoma are excited to see, she said, is vitiligo. It suggests that the immune system is attacking normal melanocytes and that it is attacking cancer cells as well. Those patients generally do far better than patients who dont get vitiligo.
A meta-analysis of 30 studies on the topic, including 4971 individuals, showed that patients who developed immune-related AEs experienced an overall survival benefi t and a progression-free survival benefi t using ICI therapy compared with those who did not. The authors stated that more studies are needed and that the results are controversial.22
Melanoma has been the proving ground for ICIs, Buchbinder said, But now the bar is higher in terms of immunotherapy.
ICIs are now being tested in more immuneresistant tumors. Although there are huge hurdles in terms of some cancers where its going to be hard for immune therapy to do muchlike pancreatic cancer or prostate cancerthere are still diseases where theres opportunity and a possibility that the correct approach or combination might get to some great therapy for those diseases, Buchbinder said
Immunotherapies are being combined with conventional therapies to better integrate treatment. We dont see cancer as a death sentence anymore, Sharma said. We really do see a lot of hope, [and patients with cancer] should be encouraged to discuss immunotherapy with their physician either in a clinical trial or an FDA-approved agent. If you do have a response, its a pretty phenomenal response.
REFERENCES:
1. Lobo N, Brooks NA, Zlotta AR, et al. 100 years of Bacillus Calmette- Gurin immunotherapy: from cattle to COVID-19. Nat Rev Urol. 2021;18(10):611-622. doi:10.1038/s41585-021-00481-1
2. Pierpont TM, Limper CB, Richards KL. Past, present, and future of rituximab-the worlds fi rst oncology monoclonal antibody therapy. Front Oncol. 2018;8:163. doi:10.3389/fonc.2018.00163
3. Kruger S, Ilmer M, Kobold S, et al. Advances in cancer immunotherapy 2019 - latest trends. J Exp Clin Cancer Res. 2019;38(1):268. doi:10.1186/s13046-019-1266-0
4. Lee JB, Kim HR, Ha SJ. Immune checkpoint inhibitors in 10 years: contribution of basic research and clinical application in cancer immunotherapy. Immune Netw. 2022;22(1):e2. doi:10.4110/in.2022.22.e2
5. FDA approves Yervoy (ipilimumab) for the treatment of patients with newly diagnosed or previously-treated unresectable or metastatic melanoma, the deadliest form of skin cancer. News release. Bristol Myers Squibb. March 25, 2011. Accessed May 11, 2022. https://bit.ly/3PFp7q2
6. Rowshanravan B, Halliday N, Sansom DM. CTLA-4: a moving target in immunotherapy. Blood. 2018;131(1):58-67. doi:10.1182/ blood-2017-06-741033
7. Buchbinder EI, Desai A. CTLA-4 and PD-1 pathways: similarities, differences, and implications of their inhibition. Am J Clin Oncol. 2016;39(1):98-106. doi:10.1097/COC.0000000000000239
8. Keown A. Keytruda approvals: a timeline. BioSpace. Aug 13, 2019. Accessed May 11, 2022. https://bit.ly/3yHvfrL
9. Stewart J. Opdivo FDA approval history. Drugs.com. Updated March 15, 2022. Accessed May 20, 2022. https://bit.ly/3lnmtar
10. Markham A, Duggan S. Cemiplimab: fi rst global approval. Drugs. 2018;78(17):1841-1846. doi:10.1007/s40265-018-1012-5
11. FDA grants accelerated approval to dostarlimab-gxly for dMMr endometrial cancer. FDA. Updated April 22, 2021. Accessed May 20, 2022. https://bit.ly/38BSJns
12. Pierpont TM, Limper CB, Richards KL. Past, present, and future of rituximab-the worlds first oncology monoclonal antibody therapy. Front Oncol. 2018;8:163. doi:10.3389/fonc.2018.00163
13. Opdualag becomes fi rst FDA-approved immunotherapy to target LAG-3. National Cancer Institute. April 6, 2022. Accessed May 11, 2022. https://bit.ly/3FZWaAp
14. Acharya N, Sabatos-Peyton C, Anderson AC. TIM-3 finds its place in the cancer immunotherapy landscape. J Immunother Cancer. 2020;8(1):e000911. doi:10.1136/jitc-2020-000911
15. Boldt C. TIL Therapy: 6 things to know. MD Anderson Cancer Center. April 15, 2021. Accessed May 11, 2022. https://bit.ly/3wmguJb
16. FDA approves talimogene laherparepvec to treat metastatic melanoma. National Cancer Institute. November 25, 2015. Accessed May 20, 2022. https://bit.ly/3woTDwA
17. OLeary MC, Lu X, Huang Y, et al. FDA approval summary: tisagenlecleucel for treatment of patients with relapsed or refractory B-cell precursor acute lymphoblastic leukemia. Clin Cancer Res. 2019;25(4):1142-1146. doi:10.1158/1078-0432.CCR-18-2035
18. FDA approves CAR-T cell therapy to treat adults with certain types of large B-cell lymphoma. News release. FDA. Oct. 18, 2017. Accessed May 11, 2022. https://bit.ly/3wpECL1
19. FDA approves fi rst CAR T-cell therapy the evolution of CAR T-cell therapy. Cell Culture Dish. October 24, 2017. Accessed May 10, 2022. https:// bit.ly/3LlDD2B
20. Albinger N, Hartmann J, Ullrich E. Current status and perspective of CAR-T and CAR-NK cell therapy trials in Germany. Gene Ther. 2021;28:513-527. doi:10.1038/s41434-021-00246-w
21. Ahmad A, Uddin S, Steinhoff M. CAR-T cell therapies: an overview of clinical studies supporting their approved use against acute lymphoblastic leukemia and large B-cell lymphomas. Int J Mol Sci. 2020;21(11):3906. doi:10.3390/ijms21113906
22. Zhou X, Yao Z, Yang H, Liang N, Zhang X, Zhang F. Are immune-related adverse events associated with the efficacy of immune checkpoint inhibitors in patients with cancer? a systematic review and meta-analysis. BMC Med. 2020;18(1):87. doi:10.1186/s12916-020-01549-2
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10 Years of Immunotherapy: Advances, Innovations, and Better Patient Outcomes - Targeted Oncology
The moral status of the embryo is a controversial and complex issue. The main viewpoints are outlined below.
1. The embryo has full moral status from fertilization onwardsEither the embryo is viewed as a person whilst it is still an embryo, or it is seen as a potential person. The criteria for personhood are notoriously unclear; different people define what makes a person in different ways.
Development from a fertilized egg into to baby is a continuous process and any attempt to pinpoint when personhood begins is arbitrary. A human embryo is a human being in the embryonic stage, just as an infant is a human being in the infant stage. Although an embryo does not currently have the characteristics of a person, it will become a person and should be given the respect and dignity of a person.
An early embryo that has not yet been implanted into the uterus does not have the psychological, emotional or physical properties that we associate with being a person. It therefore does not have any interests to be protected and we can use it for the benefit of patients (who ARE persons).
The embryo cannot develop into a child without being transferred to a womans uterus. It needs external help to develop. Even then, the probability that embryos used for in vitro fertilization will develop into full-term successful births is low. Something that could potentially become a person should not be treated as if it actually were a person. A candidate for president is a potential president, but he or she does not have the rights of a president and should not be treated as a president.
2. There is a cut-off point at 14 days after fertilizationSome people argue that a human embryo deserves special protection from around day 14 after fertilization because:
3. The embryo has increasing status as it developsAn embryo deserves some protection from the moment the sperm fertilizes the egg, and its moral status increases as it becomes more human-like.
There are several stages of development that could be given increasing moral status:
1. Implantation of the embryo into the uterus wall around six days after fertilization.2. Appearance of the primitive streak the beginnings of the nervous system at around 14 days.3. The phase when the baby could survive if born prematurely.4. Birth.
If a life is lost, we tend to feel differently about it depending on the stage of the lost life. A fertilized egg before implantation in the uterus could be granted a lesser degree of respect than a human fetus or a born baby.
More than half of all fertilized eggs are lost due to natural causes. If the natural process involves such loss, then using some embryos in stem cell research should not worry us either.
We protect a persons life and interests not because they are valuable from the point of view of the universe, but because they are important to the person concerned. Whatever moral status the human embryo has for us, the life that it lives has a value to the embryo itself.
If we judge the moral status of the embryo from its age, then we are making arbitrary decisions about who is human. For example, even if we say formation of the nervous system marks the start of personhood, we still would not say a patient who has lost nerve cells in a stroke has become less human. (But there is a difference between losing some nerve cells and losing the complete nervous system - or never having had a nervous system).
If we are not sure whether a fertilized egg should be considered a human being, then we should not destroy it. A hunter does not shoot if he is not sure whether his target is a deer or a man.
4. The embryo has no moral status at allAn embryo is organic material with a status no different from other body parts.
Fertilized human eggs are just parts of other peoples bodies until they have developed enough to survive independently. The only respect due to blastocysts is the respect that should be shown to other peoples property. If we destroy a blastocyst before implantation into the uterus we do not harm it because it has no beliefs, desires, expectations, aims or purposes to be harmed.
By taking embryonic stem cells out of an early embryo, we prevent the embryo from developing in its normal way. This means it is prevented from becoming what it was programmed to become a human being.
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Embryonic Stem Cell Research: An Ethical Dilemma
Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have the potential to differentiate into various types of cells including skeletal muscle cells. The approach of converting ESCs/iPSCs into skeletal muscle cells offers hope for patients afflicted with the skeletal muscle diseases such as the Duchenne muscular dystrophy (DMD). Patient-derived iPSCs are an especially ideal cell source to obtain an unlimited number of myogenic cells that escape immune rejection after engraftment. Currently, there are several approaches to induce differentiation of ESCs and iPSCs to skeletal muscle. A key to the generation of skeletal muscle cells from ESCs/iPSCs is the mimicking of embryonic mesodermal induction followed by myogenic induction. Thus, current approaches of skeletal muscle cell induction of ESCs/iPSCs utilize techniques including overexpression of myogenic transcription factors such as MyoD or Pax3, using small molecules to induce mesodermal cells followed by myogenic progenitor cells, and utilizing epigenetic myogenic memory existing in muscle cell-derived iPSCs. This review summarizes the current methods used in myogenic differentiation and highlights areas of recent improvement.
Duchenne muscular dystrophy (DMD) is a genetic disease affecting approximately 1 in 3500 male live births [1]. It results in progressive degeneration of skeletal muscle causing complete paralysis, respiratory and cardiac complications, and ultimately death. Normal symptoms include the delay of motor milestones including the ability to sit and stand independently. DMD is caused by an absence of functional dystrophin protein and skeletal muscle stem cells, as well as the exhaustion of satellite cells following many rounds of muscle degeneration and regeneration [2]. The dystrophin gene is primarily responsible for connecting and maintaining the stability of the cytoskeleton of muscle fibers during contraction and relaxation. Despite the low frequency of occurrence, this disease is incurable and will cause debilitation of the muscle and eventual death in 20 to 30 year olds with recessive X-linked form of muscular dystrophy. Although there are no current treatments developed for DMD, there are several experimental therapies such as stem cell therapies.
Skeletal muscle is known to be a regenerative tissue in the body. This muscle regeneration is mediated by muscle satellite cells, a stem cell population for skeletal muscle [3, 4]. Although satellite cells exhibit some multipotential differentiation capabilities [5], their primary differentiation fate is skeletal muscle cells in normal muscle regeneration. Ex vivo expanded satellite cell-derived myoblasts can be integrated into muscle fibers following injection into damaged muscle, acting as a proof-of-concept of myoblast-mediated cell therapy for muscular dystrophies [69]. However, severe limitations exist in relation to human therapy. The number of available satellite cells or myoblasts from human biopsies is limited. In addition, the poor cell survival and low contribution of transplanted cells have hindered practical application in patients [6, 8, 9]. Human-induced pluripotent stem cells (hiPSCs) are adult cells that have been genetically reprogrammed to an embryonic stem cell- (ESC-) like state by being forced to express genes and factors important for maintaining the defining properties of ESCs. hiPSCs can be generated from a wide variety of somatic cells [10, 11]. They have the ability to self-renew and successfully turn into any type of cells. With their ability to capture genetic diversity of DMD in an accessible culture system, hiPSCs represent an attractive source for generating myogenic cells for drug screening.
The ESC/iPSC differentiation follows the steps of embryonic development. The origin of skeletal muscle precursor cells comes from the mesodermal lineage, which give rise to skeletal muscle, cardiac muscle, bone, and blood cells. Mesoderm subsequently undergoes unsegmented presomitic mesoderm followed by segmented compartments termed somites from anterior to caudal direction. Dermomyotome is an epithelial cell layer making up the dorsal part of the somite underneath the ectoderm. Dermomyotome expresses Pax3 and Pax7 and gives rise to dermis, skeletal muscle cells, endothelial cells, and vascular smooth muscle [12]. Dermomyotome also serves as a tissue for secreted signaling molecules to the neural tube, notochord, and sclerotome [13, 14]. Upon signals from the neural tube and notochord, the dorsomedial lip of dermomyotome initiates and expresses skeletal muscle-specific transcription factors such as MyoD and Myf5 to differentiate into myogenic cells termed myoblasts. Myoblasts then migrate beneath the dermomyotome to form myotome. Eventually, these myoblasts fuse with each other to form embryonic muscle fibers. ESCs/iPSCs mimic these steps toward differentiation of skeletal muscle cells. Many studies utilize methods of overexpression of muscle-related transcription factors such as MyoD or Pax3 [15], or the addition of small molecules which activate or inhibit myogenic signaling during development. Several studies show that iPSCs retain a bias to form their cell type of origin due to an epigenetic memory [1619], although other papers indicate that such epigenetic memory is erased during the reprogramming processes [2022]. Therefore, this phenomenon is not completely understood at the moment. In light of these developments, we have recently established mouse myoblast-derived iPSCs capable of unlimited expansion [23]. Our data demonstrates that these iPSCs show higher myogenic differentiation potential compared to fibroblast-derived iPSCs. Thus, myogenic precursor cells generated from human myoblast-derived iPSCs expanded ex vivo should provide an attractive cell source for DMD therapy. However, since DMD is a systemic muscle disease, systemic delivery of myoblasts needs to be established for efficient cell-based therapy.
During developmental myogenesis, presomitic mesoderm is first formed by Mesogenin1 upregulation, which is a master regulator of presomitic mesoderm [24]. Then, the paired box transcription factor Pax3 gene begins to be expressed from presomitic mesoderm to dermomyotome [25]. Following Pax3 expression, Pax7 is also expressed in the dermomyotome [26], and then Myf5 and MyoD, skeletal muscle-specific transcription factor genes, begin to be expressed in the dorsomedial lip of the dermomyotome in order to give rise to myoblasts which migrate beneath the dermomyotome to form the myotome. Subsequently, Mrf4 and Myogenin, other skeletal muscle-specific transcription factor genes, followed by skeletal muscle structural genes such as myosin heavy chain (MyHC), are expressed in the myotome for myogenic terminal differentiation (Figure 1) [27, 28]. Pax3 directly and indirectly regulates Myf5 expression in order to induce myotomal cells. Dorsal neural tube-derived Wnt proteins and floor plate cells in neural tube and notochord-derived sonic hedgehog (Shh) positively regulate myotome formation [13, 29]. Neural crest cells migrating from dorsal neural tubes are also involved in myotome formation: Migrating neural crest cells come across the dorsomedial lip of the dermomyotome, and neural crest cell-expressing Delta1 is transiently able to activate Notch1 in the dermomyotome, resulting in conversion of Pax3/7(+) myogenic progenitor cells into MyoD/Myf5(+) myotomal myoblasts [30, 31]. By contrast, bone morphogenetic proteins (BMPs) secreted from lateral plate mesoderm are a negative regulator for the myotome formation by maintaining Pax3/Pax7(+) myogenic progenitor cells [29, 32]. Pax3 also regulates cell migration of myogenic progenitor cells from ventrolateral lip of dermomyotome to the limb bud [33]. Pax3 mutant mice lack limb muscle but trunk muscle development is relatively normal [34]. Pax3/Pax7 double knockout mice display failed generation of myogenic cells, suggesting that Pax3 and Pax7 are critical for proper embryonic myogenesis [35]. Therefore, both Pax3 and Pax7 are also considered master transcription factors for the specification of myogenic progenitor cells. Importantly, MyoD was identified as the first master transcription factor for myogenic specification since MyoD is directly able to reprogram nonmuscle cell type to myogenic lineage when overexpressed [3638]. In addition, genetic ablation of MyoD family gene(s) via a homologous gene recombination technique causes severe myogenic developmental or regeneration defects [3945]. Finally, genetic ablation of combinatory MyoD family genes demonstrates that MyoD/:Myf5/:MRF4/ mice do not form any skeletal muscle during embryogenesis, indicating the essential roles in skeletal muscle development of MyoD family genes [28, 46]. It was proven that Pax3 also possesses myogenic specification capability since ectopic expression of Pax3 is sufficient to induce myogenic programs in both paraxial and lateral plate mesoderm as well as in the neural tube during chicken embryogenesis [47]. In addition, genetic ablation of Pax3 and Myf5 display complete defects of body skeletal muscle formation during mouse embryogenesis [48]. Finally, overexpression of Pax7 can convert CD45(+)Sca-1(+) hematopoietic cells into skeletal muscle cells [49]. From these notions, overexpression of myogenic master transcription factors such as MyoD or Pax3 has become the major strategy for myogenic induction in nonmuscle cells, including ES/iPSCs.
The overexpression of MyoD approach to induce myogenic cells from mESCs was first described by Dekel et al. in 1992. This has been a standard approach for the myogenic induction from pluripotent stem cells (Table 1). Ozasa et al. first utilized Tet-Off systems for MyoD overexpression in mESCs and showed desmin(+) and MyHC(+) myotubes in vitro [50]. Warren et al. transfected synthetic MyoD mRNA in to hiPSCs for 3 days, which resulted in myogenic differentiation (around 40%) with expression of myogenin and MyHC [51]. Tanaka et al. utilized a PiggyBac transposon system to overexpress MyoD in hiPSCs. The PiggyBac transposon system allows cDNAs to stably integrate into the genome for efficient gene expression. After integration, around 70 to 90% of myogenic cells were induced in hiPSC cultures within 5 days [52]. This study also utilized Miyoshi myopathy patient-derived hiPSCs for the MyoD-mediated myogenic differentiation. Miyoshi myopathy is a congenital distal myopathy caused by defective muscle membrane repair due to mutations in dysferlin gene. The patient-derived hiPSC-myogenic cells will be able to provide the opportunity for therapeutic drug screening. Abujarour et al. also established a model of patient-derived skeletal muscle cells which express NCAM, myogenin, and MyHC by doxycycline-inducible overexpression of MyoD in DMD patient-derived hiPSCs [53]. Interestingly, MyoD-induced iPSCs also showed suppression of pluripotent genes such as Nanog and a transient increase in the gene expression levels of T (Brachyury T), Pax3, and Pax7, which belong to paraxial mesodermal/myogenic progenitor genes, upstream genes of myogenesis. It is possible that low levels of MyoD activity in hiPSCs may initially suppress their pluripotent state while failing to induce myogenic programs, which may result in transient paraxial mesodermal induction. Supporting this idea, BAF60C, a SWI/SNF component that is involved in chromatin remodeling and binds to MyoD, is required to induce full myogenic program in MyoD-overexpressing hESCs [54]. Overexpression of MyoD alone in hESC can only induce some paraxial mesodermal genes such as Brachyury T, mesogenin, and Mesp1 but not myogenic genes. Co-overexpression of MyoD and BAF60C was now able to induce myogenic program but not paraxial mesodermal gene expression, indicating that there are different epigenetic landscapes between pluripotent ESCs/iPSCs and differentiating ESC/iPSCs in which MyoD is more accessible to DNA targets than those in pluripotent cells. The authors then argued that without specific chromatin modifiers, only committed cells give rise to myogenic cells by MyoD. These results strongly indicate that nuclear landscapes are important for cell homogeneity for the specific cell differentiation in ESC/iPSC cultures. Similar observations were seen in overexpression of MyoD in P19 embryonal carcinoma stem cells, which can induce paraxial mesodermal genes including Meox1, Pax3, Pax7, Six1, and Eya2 followed by muscle-specific genes. However, these MyoD-induced paraxial mesodermal genes were mediated by direct MyoD binding to their regulatory regions, which was proven by chromatin immunoprecipitation (ChIP) assays, indicating the novel role for MyoD in paraxial mesodermal cell induction [55].
hESCs/iPSCs have been differentiated into myofibers by overexpression of MyoD, and this method is considered an excellent in vitro model for human skeletal muscle diseases for muscle functional tests, therapeutic drug screening, and genetic corrections such as exon skipping and DNA editing. Shoji et al. have shown that DMD patient-derived iPSCs were used for myogenic differentiation via PiggyBac-mediated MyoD overexpression. These myogenic cells were treated with morpholinos for exon-skipping strategies for dystrophin gene correction and showed muscle functional improvement [56]. Li et al. have shown that patient-derived hiPSC gene correction by TALEN and CRISPR-Cas9 systems, and these genetically corrected hiPSCs were used for myogenic differentiation via overexpression of MyoD [57]. This work also revealed that the TALEN and CRISPR-Cas9-mediated exon 44 knock-in approach in the dystrophin gene has high efficiency in gene-editing methods for DMD patient-derived cells in which the exon 44 is missing in the genome.
Along this line of the strategy, Darabi et al. first performed overexpression of Pax3 gene, which can be activated by treatment with doxycycline in mESCs, and showed efficient induction of MyoD/Myf5(+) skeletal myoblasts in EB cultures [15]. Upon removing doxycycline, these myogenic cells underwent MyHC(+) myotubes. However, teratoma formation was observed after EB cell transplantation into cardiotoxin-injured regenerating skeletal muscle in Rag2/:C/ immunodeficient mice [15]. This indicates that myogenic cell cultures induced by Pax3 in mESCs still contain some undifferentiated cells which gave rise to teratomas. To overcome this problem, the same authors separated paraxial mesodermal cells from Pax3-induced EB cells by FACS using antibodies against cell surface markers as PDGFR(+)Flk-1() cell populations. After cell sorting, isolated Pax3-induced paraxial mesodermal cells were successfully engrafted and contributed to regenerating muscle in mdx:Rag2/:C/ DMD model immunodeficient mice without any teratoma formations. Darabi et al. also showed successful myogenic induction in mESCs and hES/iPSCs by overexpression of Pax7 [58, 59]. Pax3 and Pax7 are not only expressed in myogenic progenitor cells. They are also expressed in neural tube and neural crest cell-derived cells including a part of cardiac cell types in developmental stage, suggesting that further purification to skeletal muscle cell lineage is crucial for therapeutic applications for muscle diseases including DMD.
Taken together, overexpression of myogenic master transcription factors such as MyoD or Pax3/Pax7 is an excellent strategy for myogenic induction in hESCs and hiPSCs, which can be utilized for in vitro muscle disease models for their functional test and drug screening. However, for the safe stem cell therapy, it is essential to maintain the good cellular and genetic qualities of hESC/hiPSC-derived myogenic cells before transplantation. Therefore, random integration sites of overexpression vectors for myogenic master transcription factors and inappropriate expression control of these transgenes may diminish the safety of using these induced myogenic cells for therapeutic stem cell transplantation.
Stepwise induction protocols utilizing small molecules and growth factors have been established as alternative myogenic induction approaches and a more applicable method for therapeutic situations. As described above, during embryonic myogenesis, somites and dermomyotomes receive secreted signals such as Wnts, Notch ligands, Shh, FGF, BMP, and retinoic acid (RA) with morphogen gradients from surrounding tissues in order to induce the formation of myogenic cells (Figure 2). The canonical Wnt signaling pathway has been shown to play essential roles in the development of myogenesis. In mouse embryogenesis, Wnt1 and Wnt3a secreted from the dorsal neural tube can promote myogenic differentiation of dorsomedial dermomyotome via activation of Myf5 [31, 32, 60]. Wnt3a is able to stabilize -catenin which associates with TCF/LEF transcription factors that bind to the enhancer region of Myf5 during myogenesis [61]. Other Wnt proteins, Wnt6 and Wnt7a, which emerge from the surface ectoderm, induce MyoD [62]. BMP functions as an inhibitor of myogenesis by suppression of some myogenic gene expressions. In the lateral mesoderm, BMP4 is able to increase Pax3 expression which delays Myf5 expression in order to maintain an undifferentiated myogenic progenitor state [63]. Therefore, Wnts and BMPs regulate myogenic development by antagonizing each other for myogenic transcription factor gene expression [64, 65]. Wnt also induces Noggin expression to antagonize BMP signals in the dorsomedial lip of the dermomyotome [66]. In this region, MyoD expression level is increased, which causes myotome formation. Notch signaling plays essential roles for cell-cell communication to specify the different cells in developmental stages. During myotome formation, Notch is expressed in dermomyotome, and Notch1 and Notch2 are expressed in dorsomedial lip of dermomyotome. Delta1, a Notch ligand, is expressed in neural crest cells which transiently interact with myogenic progenitor cells in dorsomedial lip of dermomyotome via Notch1 and 2. This contact induces expression of the Myf5 or MyoD gene in the myogenic progenitor cells followed by myotome formation. The loss of function of Delta1 in the neural crest displays delaying skeletal muscle formation [67]. Knockdown of Notch genes or use of a dominant-negative form of mastermind, a Notch transcriptional coactivator, clearly shows dramatically decrease of Myf5 and MyHC(+) myogenic cells. Interestingly, induction of Notch intracellular domain (NICD), a constitutive active form of Notch, can promote myogenesis, while continuous expression of NICD prevents terminal differentiation. Taken together, transient and timely activation of Notch is crucial for myotome formation from dermomyotome [30].
Current studies for myogenic differentiation of ESCs/iPSCs have utilized supplementation with some growth factors and small molecules, which would mimic the myogenic development described above in combination with embryoid body (EB) aggregation and FACS separation of mesodermal cells (Table 2). To induce paraxial mesoderm cells from mESCs, Sakurai et al. utilized BMP4 in serum-free cultures [68]. Three days after treatment with BMP4, mESCs could be differentiated into primitive streak mesodermal-like cells, but the continuous treatment with BMP4 turned the ESCs into osteogenic cells. Therefore, they used LiCl after treatment with BMP4 to enhance Wnt signaling, which is able to induce myogenic differentiation. After treatment with LiCl, PDGFR(+) E-cadherin() paraxial mesodermal cells were sorted by FACS. These sorted cells were cultured with IGF, HGF, and FGF for two weeks in order to induce myogenic differentiation. Hwang et al. have shown that treatment with Wnt3a efficiently promotes skeletal muscle differentiation of hESCs [69]. hESCs were cultured to form EB for 9 days followed by differentiation of EBs for additional 7 days, and then PDGFR(+) cells were sorted by FACS. These PDGFR(+) cells were cultured with Wnt3a for additional 14 days. Consequently, these Wnt3a-treated cells display significantly increased myogenic transcription factors and structural proteins at both mRNA and protein levels. An interesting approach to identify key molecules that induce myogenic cells was reported by Xu et al. [70]. They utilized reporter systems in zebrafish embryos to display myogenic progenitor cell induction and myogenic differentiation in order to identify small compounds for myogenic induction. Myf5-GFP marks myogenic progenitor cells, while myosin light polypeptide 2 (mylz2)-mCherry marks terminally differentiated muscle cells. They found that a mixed cocktail containing GSK3 inhibitor, bFGF, and forskolin has the potential to induce robust myogenic induction in hiPSCs. GSK3 inhibitors act as a canonical Wnt signaling activator via stabilizing -catenin protein, which is crucial for inducing mesodermal cells. Forskolin activates adenylyl cyclase, which then stimulates cAMP signaling. cAMP response element-binding protein (CREB) is able to stimulate cell proliferation of primary myoblasts in vitro, suggesting that the forskolin-cAMP-CREB pathway may help myogenic cell expansion [71], However the precise mechanisms for CREB-mediated myogenic cell expansion remain unclear. The adenylyl cyclase signaling cascade leads to CREB activation [71]. During embryogenesis, phosphorylated CREB has been found at dorsal somite and dermomyotome. CREB gene knockout mice display significantly decreased Myf5 and MyoD expressions in myotomes. While activation of Wnt1 or Wnt7a promotes Pax3, Myf5, and MyoD expressions, inhibition of CREB eliminates these Wnt-mediated myogenic gene expressions without altering the Wnt canonical pathway, suggesting that CREB-induced myogenic activation may be mediated through noncanonical Wnt pathways. Several groups also utilized GSK3 inhibitors for inducing mesodermal cells from ESCs and iPSCs [72, 73]. These mesodermal cell-like cells were expanded by treatment with bFGF, and then ITS (insulin/transferrin/selenite) or N2 medium were used to induce myogenic differentiation. Finally, bFGF is a stimulator for myogenic cell proliferation. Caron et al. demonstrated that hESCs treated with GSK3 inhibitor, ascorbic acid, Alk5 inhibitor, dexamethasone, EGF, and insulin generated around 80% of Pax3(+) myogenic precursor cells in 10 days [74]. Treatment with SB431542, an inhibitor of Alk4, 5, and 7, PDGF, bFGF, oncostatin, and IGF was able to induce these Pax3(+) myogenic precursor cells into around 5060% of MyoD(+) myoblasts in an additional 8 days. For the final step, treatment with insulin, necrosulfonamide, an inhibitor of necrosis, oncostatin, and ascorbic acid was able to induce these myoblasts into myotubes in an additional 8 days. Importantly, the same authors utilized ESCs from human facioscapulohumeral muscular dystrophy (FSHD) to demonstrate the myogenic characterization after myogenic induction by using the protocol described above. Hosoyama et al. have shown that hESCs/iPSCs with high concentrations of bFGF and EGF in combination with cell aggregation, termed EZ spheres, efficiently give rise to myogenic cells [75]. After 6-week culture, around 4050% of cells expressed Pax7, MyoD, or myogenin. However, the authors also showed that EZ spheres included around 30% of Tuj1(+) neural cells. Therefore, the authors discussed the utilization of molecules for activation of mesodermal and myogenic signaling pathways such as BMPs and Wnts.
Taken together, it is likely that the induced cell populations from ESCs/iPSCs may contain other cell types such as neural cells or cardiac cells because neural cells share similar transcription factor gene expression with myogenic cells such as Pax3, and cardiac cells also develop from mesodermal cells. To overcome this limitation, Chal et al. treated ESCs/iPSCs with BMP4 inhibitor, which prevents ESCs/iPSCs from differentiating into lateral mesodermal cells [76, 77]. To identify what genes are involved in myogenic differentiation in vivo, they performed a microarray analysis which compared samples of dissected fragments in mouse embryos, which are able to separate tail bud, presomitic mesoderm, and somite regions. From microarray data, the authors focused on Mesogenin1 (Msgn1) and Pax3 genes. Importantly, they utilized three lineage tracing reporters, Msgn1-repV (Mesogenin1-Venus) marking posterior somitic mesoderm, Pax3-GFP marking anterior somitic mesoderm and myogenic cells, and Myog-repV (Myogenin-Venus) marking differentiated myocytes, allowing the authors to readily detect different differentiation stages during ESC/iPSC cultures. Treatment with GSK3 inhibitors and then BMP inhibitors in ESC cultures induced Msgn1(+) somitic mesoderm with 45 to 65% efficiencies, Pax3(+) anterior somitic mesoderm with 30 to 50% efficiencies, and myogenin(+) myogenic cells with 25 to 30% efficiencies. Furthermore, the authors examined differentiation of mdx ESCs into skeletal muscle cells and revealed abnormal branching myofibers. Current protocols were also published and described more details for hiPSC differentiation [77].
Some nonmuscle cell populations such as mesoangioblasts have the potential to differentiate into skeletal muscle [6]. Mesoangioblasts were originally isolated from embryonic mouse dorsal aorta as vessel-associated pericyte-like cells, which have the ability to differentiate into a myogenic lineage in vitro and in vivo [6, 78]. Mesoangioblasts possess an advantage for the clinical cell-based treatment because they can be injected through an intra-arterial route to systemically deliver cells, which is crucial for therapeutic cell transplantation for muscular dystrophies [79]. Tedesco et al. successfully generated human iPSC-derived mesoangioblast-like stem/progenitor cells called HIDEMs by stepwise protocols without FACS sorting [80, 81]. They displayed similar gene expression profiles as embryonic mesoangioblasts. However, HIDEMs do not spontaneously differentiate into skeletal muscle cells, and thus, the authors utilized overexpression of MyoD to differentiate into skeletal muscle cells. Similar to mesoangioblasts, HIDEM-derived myogenic cells could be delivered to injured muscle via intramuscular and intra-arterial routes. Furthermore, HIDEMs have been generated from hiPSCs derived from limb-girdle muscular dystrophy (LGMD) type 2D patients and used for gene correction and cell transplantation experiments for the potential therapeutic application.
Myogenic precursor cells derived from ESCs/iPSCs by various methods may contain nonmuscle cells. Therefore, further purification is mandatory for therapeutic applications. Barberi et al. isolated CD73(+) multipotent mesenchymal precursor cells from hESCs by FACS, and these cells underwent differentiation into fat, cartilage, bone, and skeletal muscle cells [82]. Barberi et al. also demonstrated that hESCs cultured on OP9 stroma cells generated around 5% of CD73(+) adult mesenchymal stem cell-like cells [83]. After FACS, these CD73(+) mesenchymal stem cell-like cells were cultured with ITS medium for 4 weeks and then gave rise to NCAM(+) myogenic cells. After FACS sorting, these NCAM(+) myogenic cells were purified by FACS and transplanted into immunodeficient mice to show their myogenic contribution to regenerating muscle.
It has been shown that many genes are associated with myogenesis. In addition, exhaustive analysis, such as microarray, RNA-seq, and single cell RNA-seq supplies much gene information in many different stages. Chal et al. showed key signaling factors by microarray from presomitic somite, somite, and tail bud cells [76]. They found that initial Wnt signaling has important roles for somite differentiation. Furthermore, mapping differentiated hESCs by single cell RNA-seq analysis is useful to characterize each differentiated stage [84].
As shown above, cell sorting of mesodermal progenitor cells, mesenchymal precursor cells, or myogenic cells is a powerful tool to obtain pure myogenic populations from differentiated pluripotent cells. Sakurai et al. have been able to induce PDGFR(+)Flk-1() mesodermal progenitor cells by FACS followed by myogenic differentiation [85]. Chang et al. and Mizuno et al. have been able to sort SMC-2.6(+) myogenic cells from mouse ESCs/iPSCs [86, 87]. These SMC-2.6(+) myogenic cells were successfully engrafted into mouse regenerating skeletal muscle. However, this SMC-2.6 antibody only recognizes mouse myogenic cells but not human myogenic cells [86, 88]. Therefore, Borchin et al. have shown that hiPSC-derived myogenic cells differentiated into c-met(+)CXCR4(+)ACHR(+) cells, displaying that over 95% of sorted cells are Pax7(+) myogenic cells [72]. Taken together, current myogenic induction protocols utilizing small molecules and growth factors, with or without myogenic transcription factors, have been largely improved in the last 5 years. It is crucial to standardize the induction protocols in the near future to obtain sufficient myogenic cell conversion from pluripotent stem cells.
Recent work demonstrated that cells inherit a stable genetic program partly through various epigenetic marks, such as DNA methylation and histone modifications. This cellular memory needs to be erased during genetic reprogramming, and the cellular program reverted to that of an earlier developmental stage [16, 22, 89]. However, iPSCs retaining an epigenetic memory of their origin can readily differentiate into their original tissues [1619, 90100]. This phenomenon becomes a double-edged sword for the reprogramming process since the retention of epigenetic memory may reduce the quality of pluripotency while increasing the differentiation efficiency into their original tissues. DNA methylation levels are relatively low in the pluripotent stem cells compared to the high levels of DNA methylation seen in somatic cells [101]. Global DNA demethylation is required for the reprogramming process [102]. In the context of these observations, recent work demonstrates that activation-induced cytidine deaminase AID/AICDA contributing to the DNA demethylation can stabilize stem-cell phenotypes by removing epigenetic memory of pluripotent genes. This directly deaminates 5-methylcytosine in concert with base-excision repair to exchange cytosine in genomic DNA [103]. MicroRNA-155 has been identified as a key player for the retention of epigenetic memory during in vitro differentiation of hematopoietic progenitor cell-derived iPSCs toward hematopoietic progenitors [104]. iPSCs that maintained high levels of miR-155 expression tend to differentiate into the original somatic population more efficiently.
Recently, we generated murine skeletal muscle cell-derived iPSCs (myoblast-derived iPSCs) [23] and compared the efficiency of differentiation of myogenic progenitor cells between myoblast-derived iPSCs and fibroblast-derived iPSCs. After EB cultures, more satellite cell/myogenic progenitor cell differentiation occurred in myoblast-derived iPSCs than that in fibroblast-derived-iPSCs (unpublished observation and Figure 3), suggesting that myoblast-derived iPSCs are potential myogenic and satellite cell sources for DMD and other muscular dystrophy therapies (Figure 4). We also noticed that MyoD gene suppression by Oct4 is required for reprogramming in myoblasts to produce iPSCs (Figure 3) [23]. During overexpression of Oct4, Oct4 first binds to the Oct4 consensus sequence located in two MyoD enhancers (a core enhancer and distal regulatory region) [105107] preceding occupancy at the promoter in myoblasts in order to suppress MyoD gene expression. Interestingly, Oct4 binding to the MyoD core enhancer allows for establishment of a bivalent state in MyoD promoter as a poised state, marked by active (H3K4me3) and repressive (H3K27me3) modifications in fibroblasts, one of the characteristics of stem cells (Figure 3) [23, 108]. It should be investigated whether the similar bivalent state is also established in Oct4-expressing myoblasts during reprogramming process from myoblasts to pluripotent stem cells. It remains to be elucidated whether Oct4-mediated myogenic repression only relies on repression of MyoD expression or is just a general phenomenon of functional antagonism between Oct4 and MyoD on activation of muscle genes. Nevertheless, myoblast-derived iPSCs will enable us to produce an unlimited number of myogenic cells, including satellite cells that could form the basis of novel treatments for DMD and other muscular dystrophies (Figure 4).
There are pros and cons of transgene-free small molecule-mediated myogenic induction protocols. In the transgene-mediated induction protocols, integration of the transgene in the host genome may lead to risk for insertional mutagenesis. To circumvent this issue, there is an obvious advantage for transgene-free induction protocols. Some key molecules such as Wnt, FGF, and BMP have used signaling pathways to induce myogenic differentiation of ES/iPSCs. However, these molecules are also involved in induction of other types of cell lineages, which makes it difficult for ES/iPSCs to induce pure myogenic cell populations in vitro. By contrast, transgene-mediated myogenic induction is able to dictate desired specific cell lineages. In any case, it is necessary to intensively investigate these myogenic induction protocols for the efficient and safe stem cell therapy for patients.
For skeletal muscle diseases, patient-derived hiPSCs, which possess the ability to differentiate into myogenic progenitor cells followed by myotubes, can be a useful tool for drug screening and personalized medicine in clinical practice. However, there are still limitations for utilizing hiPSC-derived myogenic cells for regenerative medicine. For cell-based transplantation therapies such as a clinical situation, animal-free defined medium is essential for stem cell culture and skeletal muscle cell differentiation. Therefore, such animal-free defined medium needs to be established for optimal myogenic differentiation from hiPSCs. Gene correction in DMD patient iPSCs by TALENs and CRISPR-Cas9 systems are promising therapeutic approaches for stem cell transplantation. However, there are still problems for DNA-editing-mediated stem cell therapy such as safety and efficacy. Since iPSC-derived differentiated myotubes do not proliferate, they are not suited for cell transplantation. Therefore, a proper culture method needs to be established for hiPSCs in order to maintain cells in proliferating the myogenic precursor cell stage in vitro in order to expand cells to large quantities of transplantable cells for DMD and other muscular dystrophies. For other issues, it is essential to establish methods to separate ES/iPSC-derived pure skeletal muscle precursor cells from other cell types for safe stem cell therapy that excludes tumorigenic risks of contamination with undifferentiated cells. In the near future, these obstacles will be taken away for more efficient and safe stem cell therapy for DMD and other muscular dystrophies.
The authors declare that they have no conflicts of interest.
This work was supported by the NIH R01 (1R01AR062142) and NIH R21 (1R21AR070319). The authors thank Conor Burke-Smith and Neeladri Chowdhury for critical reading.
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Skeletal Muscle Cell Induction from Pluripotent Stem Cells
Peter J. Embi, MD1,2, Matthew E. Levy, PhD3, Allison L. Naleway, PhD4, Palak Patel, MBBS5, Manjusha Gaglani, MBBS6, Karthik Natarajan, PhD7,8, Kristin Dascomb, MD, PhD9, Toan C. Ong, PhD10, Nicola P. Klein, MD, PhD11, I-Chia Liao, MPH6, Shaun J. Grannis, MD2,12, Jungmi Han7, Edward Stenehjem, MD9, Margaret M. Dunne, MSc3, Ned Lewis, MPH11, Stephanie A. Irving, MHS4, Suchitra Rao, MBBS10, Charlene McEvoy, MD13, Catherine H. Bozio, PhD5, Kempapura Murthy, MBBS6, Brian E. Dixon, PhD12,14, Nancy Grisel, MPP9, Duck-Hye Yang, PhD3, Kristin Goddard, MPH11, Anupam B. Kharbanda, MD15, Sue Reynolds, PhD5, Chandni Raiyani, MPH6, William F. Fadel, PhD12,14, Julie Arndorfer, MPH9, Elizabeth A. Rowley, DrPH3, Bruce Fireman, MA11, Jill Ferdinands, PhD5, Nimish R. Valvi, DrPH12, Sarah W. Ball, ScD3, Ousseny Zerbo, PhD11, Eric P. Griggs, MPH5, Patrick K. Mitchell, ScD3, Rachael M. Porter, MPH5, Salome A. Kiduko, MPH3, Lenee Blanton, MPH5, Yan Zhuang, PhD3, Andrea Steffens, MPH5, Sarah E. Reese, PhD3, Natalie Olson, MPH5, Jeremiah Williams, MPH5, Monica Dickerson, MPH5, Meredith McMorrow, MD5, Stephanie J. Schrag, DPhil5, Jennifer R. Verani, MD5, Alicia M. Fry, MD5, Eduardo Azziz-Baumgartner, MD5, Michelle A. Barron, MD10, Mark G. Thompson, PhD5 and Malini B. DeSilva, MD13
1Regenstrief Institute, Indianapolis, Indiana; 2Indiana University School of Medicine, Indianapolis, Indiana; 3Westat, Rockville, Maryland; 4Center for Health Research, Kaiser Permanente Northwest, Portland, Oregon; 5CDC COVID-19 Response Team; 6Baylor Scott & White Health, Texas A&M University College of Medicine, Temple, Texas; 7Department of Biomedical Informatics, Columbia University, New York, New York; 8New York Presbyterian Hospital, New York, New York; 9Division of Infectious Diseases and Clinical Epidemiology, Intermountain Healthcare, Salt Lake City, Utah; 10School of Medicine, University of Colorado, Anschutz Medical Campus, Aurora, Colorado; 11Kaiser Permanente Vaccine Study Center, Kaiser Permanente Northern California, Oakland, California; 12Center for Biomedical Informatics, Regenstrief Institute, Indianapolis, Indiana; 13HealthPartners Institute, Minneapolis, Minnesota; 14Fairbanks School of Public Health, Indiana University, Indianapolis, Indiana; 15Children's Minnesota, Minneapolis, Minnesota.
Corresponding author Peter J. Embi, pembi@regenstrief.org.
All authors have completed and submitted the International Committee of Medical Journal Editors form for disclosure of potential conflicts of interest. Allison L. Naleway reports institutional support from Pfizer outside the submitted work. Anupam B. Kharbanda reports institutional support through HealthPartners to Children's Minnesota for VISION. Charlene McEvoy reports institutional support from AstraZeneca for the AZD1222 COVID-19 vaccine trial. Jill Ferdinands reports travel support from Institute for Influenza Epidemiology, funded in part by Sanofi Pasteur. Nicola P. Klein reports institutional support from Pfizer for COVID-19 vaccine clinical trials and institutional support from Pfizer, Merck, GlaxoSmithKline, Sanofi Pasteur, and Protein Sciences (now Sanofi Pasteur) outside the submitted work. Suchitra Rao reports grant support from GlaxoSmithKline and Biofire Diagnostics. No other potential conflicts of interest were disclosed.
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