StemCell Therapy

Proc Natl Acad Sci U S A. 2000 Aug 15; 97(17): 95559560.

Immunology

Departments of *Medicine and Pathology, Stanford University School of Medicine, Stanford, CA 94305

Contributed by Irving L. Weissman

Engraftment of allogeneic bone marrow (BM) has been shown to induce tolerance to organs genotypically matched with the BM donor. Immune reconstitution after BM transplantation therefore involves re-establishment of a T cell pool tolerant to antigens present on both donor and host tissues. However, how hematopoietic grafts exert their influence over the regenerating immune system is not completely understood. Prior studies suggest that education of the newly arising T cell pool involves distinct contributions from donor and host stromal elements. Specifically, negative selection is thought to be mediated primarily by donor BM-derived antigen-presenting cells, whereas positive selection is dictated by radio-resistant host-derived thymic stromal cells. In this report we studied the effect of highly purified allogeneic hematopoietic stem cells (HSCs) on organ transplantation tolerance induction and immune reconstitution. In contrast to engraftment of BM that results in near-complete donor T cell chimerism, HSC engraftment results in mixed T cell chimerism. Nonetheless we observed that HSC grafts induce tolerance to donor-matched neonatal heart grafts, and one way the HSC grafts alter host immune responses is via deletion of newly arising donor as well as radiation-resistant host T cells. Furthermore, using an in vivo assay of graft rejection to study positive selection we made the unexpected observation that T cells in chimeric mice rejected grafts only in the context of the donor MHC type. These latter findings conflict with the conventionally held view that radio-resistant host elements primarily dictate positive selection.

Keywords: bone marrow transplantation, MHC restriction, mice

Transplantation of allogeneic bone marrow (BM) is known to alter immune responses in recipients so that tolerance is established to tissues matched with the genotype of the BM donors (13). Thus, the process of regeneration of the hematopoietic system involves the re-establishment of parameters that identify self- from nonself-antigens. The way in which BM grafts affect these changes is not completely understood. However, because T cells control antigen-specific immune responses the pathways that lead to regeneration of the peripheral T cell pool are central to immune reconstitution. T cell development after BM transplantation (BMT) is thought to recapitulate normal T cell ontogeny, which begins with the migration of BM-derived hematopoietic stem cells (HSCs) or more differentiated progenitors to the thymus (4). Within the thymus, under the influence of a specialized stromal microenvironment, progenitor T cells expand, differentiate, and undergo the rigorous processes of positive and negative selection (58). Positive selection results in survival of T cells with antigen receptors that corecognize self-MHC molecules plus foreign peptides. T cells whose receptors do not detect self-MHC molecules die, presumably by failure to receive critical differentiating signals. Negative selection involves the removal of potentially autoreactive T cells that interact too well with self-MHC molecules plus self-peptides.

Classic BM and thymus grafting studies by Zinkernagel et al. (9) and Bevan and Fink (10, 11) showed that the radio-resistant elements in the host thymus dictate MHC restriction of killer T cells. They proposed, and many experiments followed to support, the notion that these positively selecting elements in the thymus are epithelial cells (5, 6, 8). Subsequent studies refined these observations by tracking T cell development via expression of V type or expression of a single transgenic T cell receptor and showed that both CD8+ and CD4+ T cells are likely to be positively selected on a subpopulation of epithelial cells located in the thymic cortex (5, 6, 8). In contrast, negative selection primarily is mediated by BM-derived antigen-presenting cells (APCs) (7, 12, 13). The absoluteness with which these stromal components dictate the selection processes continues to be challenged by discordant observations (1417). In the setting of an MHC-mismatched allogeneic BMT, this schema of T cell selection predicts that the resultant host will be immunodeficient, insofar as the developing cells will be educated in the thymus to respond to antigens in the context of host MHC type, but will encounter BM-derived APCs in the periphery with the donor MHC type.

In the studies presented here we examined the issues of tolerance induction and immune reconstitution after transplantation of highly purified MHC-disparate HSCs in mice. HSCs are devoid of contaminating differentiated cell populations and thus, unlike most radiation BM chimeras, the effects of the donated immune system that arises from the HSC grafts are solely the result of de novo hematopoiesis. The HSC-transplanted mice also differ from BM chimeras because the former retain a significant proportion of radio-resistant host T cells (18). We found that HSCs induce tolerance to donor-matched organs and that such grafts can mediate negative selection of both developing donor T cells and residual T cells from the host. Furthermore, we made the unexpected observation that analysis of MHC restriction by an in vivo assay suggests that in chimeric mice the donor, not the host-type MHC, predominates in controlling heart graft rejection, a measure of T cell responsiveness. These studies, and the studies by Zinkernagel and Althage (17), reopen the issues of how, where, and on which cell types developing T cells learn MHC restriction and suggest that immunoincompetence in the post-BMT setting, a known clinical problem, is not completely explained by disparity between the MHC type of the donor versus the host.

Three different C57BL/Ka congenic mouse lines were used as donors or recipients. C57BL/Ka were mice H-2b, Thy-1.2, CD45.2; congenic Thy-1.1 mice were H-2b, Thy-1.1, CD45.2 (C57BL/Ka.Thy-1.1) and designated as BA throughout the text; and congenic CD45.1 mice were H-2b, Thy-1.1, CD45.1 (C57BL/Ka.Thy-1.1.CD45.1) and designated BA.CD45.1 throughout the text. HSC or BM recipients were 7- to 10-week-old BALB/c (H-2d, Thy 1.2), BALB/k (H-2k, Thy 1.2), or C57BL/Ka mice. HSC and BM donors were BA or AKR/J mice (H-2k, Thy 1.1). For the neonatal heart transplantation experiments donors were 1- to 24-h-old neonates derived from BA, BA.CD45.1, BALB/c, C3H.SW (H-2b) or DBA.2 (H-2d) strain mice. All mice were bred and maintained at Stanford University.

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Purified hematopoietic stem cell grafts induce tolerance ...

It's really more personal than consequential. Some people believe that life starts when an egg is fertilized and some think that life starts right when a fetus leaves the womb. Whatever you may think the whole stem cell research commotion is like liberal vs. conservative type thing. Stem cell research, if achieved, can eventually provide organs to people who require it when one is hard to come by.The whole concept is much more complex than that but that's the basics. It's really a struggle between your personal views of life.

It sounds like your teacher is against the idea. In my opinion I would do one of several approaches. 1. you can ignore your teachers comment and rewrite the essay (do not recommend) 2. approach the principle and tell him/her to review your essay and tell him/her that you wrote your essay based on your views. 3. Confront your teacher and state that stem cell research does not have negative aspects other than the remarks of few people and that you shouldn't be required to rewrite the essay.

If you decide to rewrite your essay explain the different views that people favor.

* If you want a more in depth explanation then respond to this post.

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What are some negative aspects of stem cell research ...

Hematopoietic stem cells (HSCs) are the blood cells that give rise to all the other blood cells and are derived from mesoderm. They are located in the red bone marrow, which is contained in the core of most bones.

They give rise to the myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells), and lymphoid lineages (T-cells, B-cells, NK-cells). The definition of hematopoietic stem cells has changed in the last two decades. The hematopoietic tissue contains cells with long-term and short-term regeneration capacities and committed multipotent, oligopotent, and unipotent progenitors. HSCs constitute 1:10.000 of cells in myeloid tissue.

HSCs are a heterogeneous population. Three classes of stem cells exist, distinguished by their ratio of lymphoid to myeloid progeny (L/M) in blood. Myeloid-biased (My-bi) HSC have low L/M ratio (between 0 and 3), whereas lymphoid-biased (Ly-bi) HSC show a large ratio (>10). The third category consists of the balanced (Bala) HSC, whose L/M ratio is between 3 and 10. Only the myeloid-biased and -balanced HSCs have durable self-renewal properties. In addition, serial transplantation experiments have shown that each subtype preferentially re-creates its blood cell type distribution, suggesting an inherited epigenetic program for each subtype.

HSC studies through much of the past half century have led to a much deeper understanding. More recent advances have resulted in the use of HSC transplants in the treatment of cancers and other immune system disorders.[1]

HSCs are found in the bone marrow of adults, specially in the pelvis, femur, and sternum. They are also found in umbilical cord blood and, in small numbers, in peripheral blood.[2]

Stem and progenitor cells can be taken from the pelvis, at the iliac crest, using a needle and syringe.[3] The cells can be removed as liquid (to perform a smear to look at the cell morphology) or they can be removed via a core biopsy (to maintain the architecture or relationship of the cells to each other and to the bone).[citation needed]

In order to harvest stem cells from the circulating peripheral blood, blood donors are injected with a cytokine, such as granulocyte-colony stimulating factor (G-CSF), that induces cells to leave the bone marrow and circulate in the blood vessels.[citation needed]

In mammalian embryology, the first definitive HSCs are detected in the AGM (aorta-gonad-mesonephros), and then massively expanded in the fetal liver prior to colonising the bone marrow before birth.[4]

HSCs can replenish all blood cell types (i.e., are multipotent) and self-renew. A small number of HSCs can expand to generate a very large number of daughter HSCs. This phenomenon is used in bone marrow transplantation, when a small number of HSCs reconstitute the hematopoietic system. This process indicates that, subsequent to bone marrow transplantation, symmetrical cell divisions into two daughter HSCs must occur.

Stem cell self-renewal is thought to occur in the stem cell niche in the bone marrow, and it is reasonable to assume that key signals present in this niche will be important in self-renewal. There is much interest in the environmental and molecular requirements for HSC self-renewal, as understanding the ability of HSC to replenish themselves will eventually allow the generation of expanded populations of HSC in vitro that can be used therapeutically.

Link:
Hematopoietic stem cell - Wikipedia, the free encyclopedia

Side effects can happen with any type of treatment, but everyones experience is different. Some people may have many side effects. Others have few or none at all.

Side effects can happen any time during, immediately after or days to months after stem cell transplant. Short-term, or acute, side effects generally develop during the first 100 days after a stem cell transplant. Long-term, or chronic, side effects generally develop 100 or more days after the transplant. Most side effects go away on their own or can be treated, but some side effects can last a long time or become permanent.

Side effects of a stem cell transplant will depend mainly on:

It is hard to say exactly which side effects a child will have, how long they will last and when the child will recover. A childs body seems to handle chemotherapy better than an adults body. Children usually have less severe side effects and will often recover from them faster than adults.

A stem cell transplant is a very complex procedure. Side effects of stem cell transplant can be very serious or even life-threatening. The healthcare team watches people receiving a stem cell transplant very closely. They will take measures to prevent side effects and will quickly deal with any side effects that develop.

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Side effects of stem cell transplant - Canadian Cancer Society

If you just started to study hematopoiesis you can always see in the protocols indication to so-called lineage-negative population in bone marrow.

Lineage-positive (Lin+) cells are a mix of all cells expressing mature cell lineage markers. For example for mouse bone marrow: Mac-1 for myeloid cells, CD4 and CD8 for T-cells, CD19 or B220 for B-cells, Ter-119 for erythrocytes, ect. The rest of the cells are lineage-negative (Lin-) they are not stained by the lineage antibodies. All stem and progenitor cell activity was identified withing Lin- population, but not in Lin+ cells.

Lin- populations are very heterogeneous and contain a small % of true stem cells, most of the cells are progenitors;

Lin Ab mix ready to use - mouse: BioLegend FITC or PE Anti-Mouse Lineage Cocktail With Isotype Control Caltag/Invitrogen Mouse Lineage Mixture, Hamster/Rat Anti-Mouse biotin / Alexa Fluor 488 human: eBioscience Human Hematopoietic Lineage Flow Cocktail (FITC)

kits for isolation Lin- population: mouse: Miltenyi Biotech -Lineage Cell Depletion Kit R&D Systems MagCellect Mouse Hematopoietic Cell Lineage Depletion Kit Stem Cell Technologies Customize your kit! for human and mouse human: Miltenyi Biotech -Lineage Cell Depletion Kit

biothinylated anti-mouse Ab: CD4, CD8, B220, IL-7R, Ter119, Gr-1, Mac1 from Adult mouse hematopoietic stem cells: purification and single-cell assays. Nature Protocols 2007; 1: 2979

******************** I have experience with commercial and hand-made customizable Lin Ab mixtures and kits. Please let us know if you have any trouble isolating a clean Lin- population, and wed love your feedback on products and kits youve used either successfully or unsuccessfully.

Tagged as: cell separation, FACS, flow cytometry, HSC isolation, lineage, MACS

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What is lineage-negative cells? - Stem Cell Assays

by Amanda J. Harper

(Medical Xpress)New research from The Ohio State University Comprehensive Cancer Center Arthur G. James Cancer Hospital and Richard J. Solove Research Institute (OSUCCC James) suggests that it may be the quality of cancer stem cells rather than their quantity that leads to better survival in certain patients with oral cancer.

The researchers investigated cancer stem cell numbers in oral cancers associated with human papillomavirus (HPV) and in oral cancers not associated with the virus. Typically, patients with HPV-positive oral cancer respond better to therapy and have a more promising prognosis than patients with HPV-negative tumors. The latter are usually associated with tobacco and alcohol use.

The OSUCCC James team's findings, published in the journal Cancer, suggest that relying on the number of cancer stem cells in a tumor might inaccurately estimate the potential for the tumor's recurrence or progression.

"We show that high levels of cancer stem cells are not necessarily associated with a worse prognosis in head and neck cancer, a finding that could have far-reaching implications for patient care," says principal investigator Quintin Pan, PhD, associate professor of otolaryngology and scientist with the OSUCCC James Experimental Therapeutics Program.

Head and neck cancer is the sixth most common cancer worldwide, with an estimated 600,000 cases diagnosed annually. Although the disease is often linked to alcohol and tobacco use, cancer-causing types of HPV are a major risk factor for the malignancy, and cases of HPV-associated oral cancers have tripled in the past 30 years.

Cancer stem cells make up only a small percent of the malignant cells within a tumor. When these cells divide, they can produce either more cancer stem cells or the nondividing malignant cells that constitute the bulk of a tumor.

Research has shown that cancer stem cells are highly resistant to chemotherapy and radiation and those cancer stem cells that survive treatment cause tumor recurrence. For these reasons, it is thought that tumors with high numbers of cancer stem cells are more likely to recur.

In this study, Pan and his OSUCCC James collaborators hypothesized that patients with HPV-positive tumors had better outcomes because their tumors had fewer cancer stem cells than tumors with HPV-negative tumors. They discovered just the opposite, however.

Comparing numbers of cancer stem cells in human tumor samples and in oral-cancer cell lines, they found that the HPV-positive samples had 2.4 to 62.6 times more cancer stem cells than did the HPV-negative samples.

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Number of cancer stem cells might not predict outcome in ...

Stem Cell Research

Stem cells can now be grown and transformed into specialized cells with characteristics consistent with cells of various tissues such as muscles or nerves through cell culture. Stem cells are distinctive from any other adult cell of the body in that they are capable of continuous mitotic divisions and self-renewal over long periods without undergoing the process of differentiation.

Positives of Stem Cell Research

Medical researchers believe that stem cell therapy has the potential to dramatically change the treatment of human disease. A number of adult stem cell therapies already exist, particularly bone marrow transplants that are used to treat leukemia. Stem cells can be engineered to replicate various specialized cells--those in the brain, liver and skin and have the potential to treat vast numbers of illnesses.

In the future, medical researchers anticipate being able to use technologies derived from stem cell research to treat a wider variety of diseases including cancer, Parkinson's disease, spinal cord injuries, Amyotrophic lateral sclerosis, multiple sclerosis, and muscle damage, amongst a number of other impairments and conditions.

Negatives of Stem Cell Research

Most misconceptions revolving around stem cell research are ethical in nature. Many people are against embryonic stem cell use because extracting stem cells from an embryo destroys it. However, recently, it has been shown in principle that adult stem cell lines can be manipulated to generate embryonic-like stem cell lines using a single-cell biopsy similar to that used in preimplantation genetic diagnosis that may allow stem cell creation without embryonic destruction.

Opponents of the research argue that embryonic stem cell technologies are a slippery slope to reproductive cloning and can fundamentally devalue human life. Those in the pro-life movement argue that a human embryo is a human life and is therefore entitled to protection.

Well, above are some points in favor of and against stem cell research!

By: Trinity

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Positives And Negatives Of Stem Cell Research

Cells Weekly is a digest of the most interesting news and events in stem cell research, cell therapy and regenerative medicine. Cells Weekly is posted every Sunday night!

This week was a week of ISSCR 2015, so read all news from the conference here.

1. The price of gene therapy trial failure A year ago, Celladon became the first gene therapy company, which received designation by US FDA Breakthrough Therapy for their MYDICAR platform in heart failure. It means very good data from Phase 1 trial and impressive efficacy in early Phase 2. Despite the recognition and all excitement, a year later, MYDICAR failed to deliver efficacy as result of Phase 2. The fate of the company was unclear. This week, the company warned their investor about potential termination suspension of MYDICAR and other pre-clinical programs, sale/ merger of the company or liquidation:

We are aggressively pursuing that course, said Paul Cleveland, president and chief executive officer of Celladon. If we are unable to identify a merger or sale that provides superior value to our shareholders, we will move forward with a liquidation and distribution of net cash to shareholders. The Company also announced a second reduction in its workforce, with approximately half of the employees not previously notified of termination of employment being expected to depart in the third quarter.

Very bad news for the field! Many lessons to learn

2. Heart peacemaker activity turned on by light The most interesting study from this week came from 2 Israeli scientists. They were able to modulate cardiac pacing in vivo, using optogenetic approach. AVV vector with light-sensitive protein transgene was injected directly into myocardium:

This allowed optogenetic pacing of the hearts at different beating frequencies with blue-light illumination both in vivo and in isolated perfused hearts. Optical mapping confirmed that the source of the new pacemaker activity was the site of ChR2 transgene delivery. Notably, diffuse illumination of hearts where the ChR2 transgene was delivered to several ventricular sites resulted in electrical synchronization

This is fantastic! The author Lior Gepstein says:

Our work is the first to suggest a non-electrical approach to cardiac resynchronization therapy, Gepstein said. Before this, there have been a number of elegant gene therapy and cell therapy approaches for generating biological pacemakers that can pace the heart from a single spot. However it was impossible to use such approaches to activate the heart simultaneously from a number of sites for resynchronization therapy.

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Stem Cell Assays Reproducible Research on Stem Cells

Abstract Introduction

The control of differentiation of mesenchymal stromal/stem cells (MSCs) is crucial for tissue engineering strategies employing MSCs. The purpose of this study was to investigate whether the transcriptional co-factor Yes-associated protein (YAP) regulates chondrogenic differentiation of MSCs.

Expression of total YAP, its paralogue transcriptional co-activator with PDZ-binding motif (TAZ), and individual YAP transcript variants during in vitro chondrogenesis of human MSCs was determined by quantitative reverse transcription polymerase chain reaction (RT-PCR). YAP expression was confirmed by western blotting. To determine the effect of high YAP activity on chondrogenesis, C3H10T1/2 MSC-like cells were transduced with human (h)YAP and treated in micromass with bone morphogenetic protein-2 (BMP-2). Chondrogenic differentiation was assessed by alcian blue staining and expression of chondrocyte-lineage genes. BMP signalling was determined by detection of pSmad1,5,8 by western blotting and expression of BMP target genes by quantitative RT-PCR. Finally, YAP and pYAP were detected in mouse embryo hindlimbs by immunohistochemistry.

YAP, but not TAZ, was downregulated during in vitro chondrogenesis of human MSCs. One of the YAP transcript variants, however, was upregulated in high-density micromass culture. Overexpression of hYAP in murine C3H10T1/2 MSCs inhibited chondrogenic differentiation. High YAP activity in these cells decreased Smad1,5,8 phosphorylation and expression of the BMP target genes Inhibitor of DNA binding/differentiation (Id)1, Id2 and Id3 in response to BMP-2. In developing mouse limbs, Yap was nuclear in the perichondrium while mostly phosphorylated and cytosolic in cells of the cartilage anlage, suggesting downregulation of Yap co-transcriptional activity during physiological chondrogenesis in vivo.

Our findings indicate that YAP is a negative regulator of chondrogenic differentiation of MSCs. Downregulation of YAP is required for chondrogenesis through derepression of chondrogenic signalling. Therapeutic targeting of YAP to promote cartilage repair and prevent secondary osteoarthritis is an exciting prospect in rheumatology.

Symptomatic joint surface defects require treatment to achieve repair and attempt prevention of secondary osteoarthritis (OA). Biological repair of the joint surface is becoming a clinical reality. Autologous chondrocyte implantation remains the gold standard of cell therapy for cartilage repair; however, chondrocyte preparations are known to be difficult to manufacture robustly because chondrocytes in culture have a limited lifespan and undergo de-differentiation, thereby losing their ability to form cartilage [1],[2].

Mesenchymal stromal/stem cells (MSCs), present in bone marrow [3] and connective tissues such as periosteum [4],[5] and synovium [6],[7], are attractive alternative cells for the repair of articular cartilage due to their easy access and culture expansion and their capacity to form cartilage [8]. To fully harness the therapeutic value of these cells for cartilage repair, an in-depth understanding of the molecular regulation of chondrogenesis is essential.

Yes-associated protein (YAP; gene symbol YAP1) is a key transcriptional co-factor that has been implicated in recent years in the regulation of stem cell fate [9]. YAP and its paralogue transcriptional co-activator with PDZ-binding motif (TAZ) shuttle between the cytoplasm and the nucleus and interact with transcription factors to regulate their activity. Uncontrolled activity of YAP causes tissue overgrowth due to modulation of stem cell proliferation in multiple tissues and organs, including liver [10],[11], intestine [11], brain [12], and epidermis [13], and we have shown that YAP increases proliferation in muscle satellite cells [14].

YAP is regulated by the Hippo pathway, comprising the kinases Mst1/2 (mammalian Ste20-like; a class II GC kinase) and Lats1/2 (large tumour suppressor; an Ndr kinase). Activation of the Hippo pathway, for example through cell-cell contact [15] or GPCR signalling [16], leads to phosphorylation of YAP on specific serine residues, most notably Ser127. Phosphorylation at Ser127 promotes its cytosolic retention and proteasomal degradation [10],[17]. In addition, YAP is regulated by actomyosin cytoskeletal tension, thereby acting as transducer of mechanical cues exerted by extracellular matrix (ECM) stiffness and cell shape, with a stiff ECM and cell spreading increasing YAP activity [18].

YAP and its paralogue TAZ have been shown to be key factors in the regulation of MSC lineage commitment, with low YAP/TAZ activity promoting adipogenesis, while high YAP/TAZ activity drives MSCs towards osteogenesis [19]-[21]. The role of Yap in chondrogenesis is less clear and the mechanism of how YAP modulates chondrogenesis is not known.

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Yes-associated protein (YAP) is a negative regulator of ...

Treatment

The Stem Cell treatment performed at our clinics is a painless medical procedure where Stem Cells (cellular building blocks) are usually administered intravenously and subcutaneously (under the skin). The whole procedure takes approximately one hour and has no known negative side effects.

Following the treatment, the Fetal Stem Cells will travel throughout the body, detecting damaged cells and tissue and attempts to restore them. The Fetal Stem Cells can also stimulate existing normal cells and tissues to operate at a higher level of function, boosting the bodys own repair mechanisms to aid in the healing process. These highly adaptive cells then remain in the body, continually locating and repairing any damage they encounter.

As with any medical treatment, safety should be of the highest priority. The Stem Cells used in our treatment undergo extensive screening for possible infection and impurities. Utilizing tests more sophisticated than those regularly used in the United States for Stem Cell research and transplant. Our testing process ensures we use only the healthiest cells to enable the safest and most effective Fetal Stem Cell treatment possible. And, unlike other types of Stem Cells, there is no danger of the bodys rejection of Fetal Stem Cells due to the fact they have no antigenicity (cellular fingerprint). This unique quality eliminates the need for drugs used to suppress the immune system, which can leave a patient exposed to serious infections.

With over 3000 patients treated, Stem Cell Of America has achieved positive results with a wide variety of illnesses, conditions and injuries. Often, in cases where the diseases continued to worsen, our patients have reported substantial improvements following the Stem Cell treatment.

Patients have experienced favorable developments such as reduction or elimination of pain, increased strength and mobility, improved cognitive function, higher tolerance for chemotherapy, and quicker healing and recovery.

To view follow up letters from patients, please visit the patient experiences page on our website.

All statements, opinions, and advice on this page is provided for educational information only. It is not a substitute for proper medical diagnosis and care. Like all medical treatments and procedures, results may significantly vary and positive results may not always be achieved. Please contact us so we may evaluate your specific case.

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Stem Cell Of America | Breakthrough Stem Cell Treatment

The California stem cell agency this week received good marks for changes made by its new president, but it is also being told that it needs to improve how it tracks potential royalties and how it prevents grant reviewer conflicts of interest.

A "performance audit" by Moss-Adams, a Seattle business consulting firm, made 12 recommendations for the $3 billion research enterprise. One of the 12 was to implement the unfulfilled recommendations made by Moss-Adams three years ago. Seven of the 24 from that audit still need more action, the firm said.

On Thursday, the agency's governing board is scheduled to discuss the latest audit at a meeting in Berkeley. The study is required by state law every three years. The agency's scientific performance, however, is specifically excluded from being examined. Moss-Adams is scheduled to receive $230,000 from the agency for the audit, which was for the 2013-14 year.

On Sunday, the California Stem Cell Report covered the deficiencies involving disclosures of the financial interests of grant reviewers.

Other areas of concern included the need for better tracking of intellectual property that could mean royalties for the state, more timely review of progress reports from grantees, more timely, formal evaluation of employees and keeping up-to-date on technology related to grant management and agency efficiency.

Under the subject of "commendations," Moss-Adams said that CIRM had "many strengths." The consultant said the agency has made "significant strides" in three areas: the grant management system, grants process improvements and "organizational culture."

The grants process comment referred to CIRM 2.0, the fast-track funding program initiated by Randy Mills since he became president a year ago. The organizational culture commendation also involved Mills' efforts, but touched indirectly and delicately on the resignation of Robert Klein as chairman and the election of Jonathan Thomas to replace him in June of 2011.

The audit found significant deficiencies involving the treatment of CIRM employees, some of which have been addressed in a positive way already by Mills. One example cited by the audit involved performance evaluations that are tied to pay increases. It said that evaluations that were scheduled to occur in 2013-14 did not actually take place until January of this year.

Moss-Adams said the agency also needs to do better in monitoring and protecting its intellectual property (IP), which could generate royalties. Without tight tracking of the IP and inventions funded by CIRM research, the state could lose out on revenue. Backers of Proposition 71 told voters in 2004 that the state could receive more than $1 billion in royalties from CIRM research. So far, none has resulted.

Moss-Adams said that royalties are now more possible because the agency is backing late stage research that is more likely to make it into the market place.

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California Stem Cell Report

Highlights

Donor age and prolonged cell culture time reduce reprogramming efficiency

Upregulation of the p21 associates with the donor age and time in culture

Knockdown of p21 restored iPSC generation in long-term passaged fibroblasts

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSC) by the forced expression of the transcription factors OCT4, SOX2, KLF4 and c-MYC. Pluripotent reprogramming appears as a slow and inefficient process because of genetic and epigenetic barriers of somatic cells. In this report, we have extended previous observations concerning donor age and passage number of human fibroblasts as critical determinants of the efficiency of iPSC induction. Human fibroblasts from 11 different donors of variable age were reprogrammed by ectopic expression of reprogramming factors. Although all fibroblasts gave rise to iPSC colonies, the reprogramming efficiency correlated negatively and declined rapidly with increasing donor age. In addition, the late passage fibroblasts gave less reprogrammed colonies than the early passage cell counterparts, a finding associated with the cellular senescence-induced up regulation of p21. Knockdown of p21 restored iPSC generation even in long-term passaged fibroblasts of an old donor, highlighting the central role of the p53/p21 pathway in cellular senescence induced by both donor age and culture time.

Reprogramming rejuvenates aged somatic cells back into the pluripotent state (Takahashi et al., 2007andTakahashi and Yamanaka, 2006). The developmental plasticity of induced pluripotent stem cells (iPSC) demonstrated the potential for regenerative therapies of human diseases (Braam et al., 2013, Song et al., 2012andYu et al., 2012). Various types of somatic cells have been successfully used for iPSC derivation, including for instance skin fibroblasts, blood cells and myoblasts (Seki et al., 2010, Trokovic et al., 2013, Trokovic et al., 2014andYu et al., 2007). Alternative methods for iPSC derivation have been intensively developed to avoid the integration of transgenes, including reprogramming induced by Sendai virus, mRNA, episomal vectors or small molecules (Hou et al., 2013, Nishimura et al., 2011, Warren et al., 2010andZhou et al., 2009). Although methods for iPSC derivation have been intensively developed, most current technologies are still inefficient, which may be due to intrinsic barriers in the ability of cells to undergo a rapid shift in their proliferative rate (Hanna et al., 2009andSmith et al., 2010).

Multiple factors are known to contribute to the efficiency of iPSC generation (Park et al., 2014). For example, differentiation state of the starting cell is a significant factor, since progenitors and stem cells give higher reprogramming efficiency than terminally differentiated cells (Eminli et al., 2009). There is also evidence for varying efficiency for different types of somatic cells from the same donor (Streckfuss-Bomeke et al., 2013). In addition cellular senescence has been shown to affect the reprogramming efficiency (Banito et al., 2009, Kawamura et al., 2009, Li et al., 2009, Marin et al., 2009andUtikal et al., 2009). Cellular senescence increases with age and one of its hallmarks is the irreversible cell cycle arrest through the activation of the p53/p21 and p16 pathways (Campisi and d'Adda di Fagagna, 2007andNarita et al., 2003). These findings suggest that intrinsic properties of somatic cells determine the reprogramming efficiency.

Donor age has been shown to have an effect on reprogramming efficiency of murine cells (Wang et al., 2011). Contrary to what has been observed in mice, donor age was suggested not to impair the reprogramming efficiency of human cells (Somers et al., 2010) and iPSC have been successfully derived even from the fibroblasts of centenarians (Lapasset et al., 2011). However, there are no reports on the combined effect of age and culture time on reprogramming efficiency of human cells.

The aim of this report was to evaluate the independent and combined impact of donor age and passage number on the pluripotent reprogramming efficiency of human dermal fibroblasts. Gene expression profiles of selected genes and telomere lengths of starting fibroblasts were analysed in order to identify potential factors behind distinct reprogramming efficiencies. We found that the reprogramming efficiency of human dermal fibroblasts is synergistically affected by donor age and culture time, both inducing cellular senescence through the p53/ p21 pathway.

Donors or their guardians provided their written informed consent for participation. Coordinating Ethics Committee of Helsinki and Uusimaa Hospital District approved generation and use of human iPSC (statement nr. 423/13/03/00/08) on April 2009. Human dermal fibroblasts and foreskin fibroblasts (HFF; CRL-2429; ATCC) were used for reprogramming (Table1).

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Combined negative effect of donor age and time in culture ...

Abstract Introduction

Triple-negative breast cancer (TNBC) is a heterogeneous group of tumours in which chemotherapy, the current mainstay of systemic treatment, is often initially beneficial but with a high risk of relapse and metastasis. There is currently no means of predicting which TNBC will relapse. We tested the hypothesis that the biological properties of normal stem cells are re-activated in tumour metastasis and that, therefore, the activation of normal mammary stem cell-associated gene sets in primary TNBC would be highly prognostic for relapse and metastasis.

Mammary basal stem and myoepithelial cells were isolated by flow cytometry and tested in low-dose transplant assays. Gene expression microarrays were used to establish expression profiles of the stem and myoepithelial populations; these were compared to each other and to our previously established mammary epithelial gene expression profiles. Stem cell genes were classified by Gene Ontology (GO) analysis and the expression of a subset analysed in the stem cell population at single cell resolution. Activation of stem cell genes was interrogated across different breast cancer cohorts and within specific subtypes and tested for clinical prognostic power.

A set of 323 genes was identified that was expressed significantly more highly in the purified basal stem cells compared to all other cells of the mammary epithelium. A total of 109 out of 323 genes had been associated with stem cell features in at least one other study in addition to our own, providing further support for their involvement in the biology of this cell type. GO analysis demonstrated an enrichment of these genes for an association with cell migration, cytoskeletal regulation and tissue morphogenesis, consistent with a role in invasion and metastasis. Single cell resolution analysis showed that individual cells co-expressed both epithelial- and mesenchymal-associated genes/proteins. Most strikingly, we demonstrated that strong activity of this stem cell gene set in TNBCs identified those tumours most likely to rapidly progress to metastasis.

Our findings support the hypothesis that the biological properties of normal stem cells are drivers of metastasis and that these properties can be used to stratify patients with a highly heterogeneous disease such as TNBC.

Breast cancer is a highly heterogeneous disease broadly classified on the basis of clinical parameters such as size, grade and node status, as well as histopathological criteria, primarily expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) [1]. While defined targeted therapeutic strategies have been developed for patients with ER+/PR+ and HER2+ diseases, chemotherapy is currently the mainstay of systemic treatment for triple-negative (ER/PR/HER2) breast cancer (TNBC) patients, which represents approximately 20% of all breast cancers [2]. Clinically, TNBC encompasses a heterogeneous group of aggressive tumours with poor prognosis [1],[3]-[7], partly due to high recurrence within the first years and limited targeted therapy options. Although chemotherapy is often initially beneficial in these tumours, especially in the neoadjuvant setting, many TNBCs have a high risk of relapse [8]. Since there is currently no means of predicting which TNBC will relapse, identification of subpopulations of TNBC that are most at risk is vital for the clinical management of these breast cancer patients.

Strong evidence is emerging supporting the hypothesis that cancer stem cells with similar features to normal tissue stem cells are resistant to standard chemotherapy and drive tumour regrowth after therapy finishes [9]. We hypothesised that biological properties of normal stem cells are reactivated in tumour cells to facilitate metastasis. Genes expressed in stem cells of the normal mammary gland might therefore carry prognostic information for relapse and metastasis in breast cancer. However, the development of such gene sets depends on the ability to isolate highly pure stem cells for analysis.

The mammary epithelium consists of two main layers, the luminal and basal layers. The luminal layer consists of ER- cells (mainly proliferative progenitors) and ER+ cells (mainly non-proliferative differentiated cells). The basal layer consists of myoepithelial cells (MYOs) and mammary stem cells (MaSCs), the latter characterised by their robust outgrowth activity in the cleared fat pad transplant assay. The relationship between these populations is summarised in Additional file 1A. Previous studies have analysed total basal breast epithelial cells, without further purification of the minority stem cell fraction [10] or used a dye label-retention strategy to identify asymmetrically dividing cells (putative stem cells) in non-adherent mammosphere cultures [11]. Only one previous study has attempted to freshly purify basal stem cells and compare their gene expression profile to MYOs [12]; however, that study identified only four genes expressed >2-fold more highly in stem cells compared to MYOs, and none of these achieved statistical significance. Here, we have defined the first gene signature specific for highly purified, freshly isolated MaSCs and further enriched the stem cell specificity by excluding basal-associated genes common to both the stem and myoepithelial populations. Pathway analysis revealed that this signature was enriched in genes associated with cell migration, adhesion and tissue morphogenesis. Single cell resolution gene expression analysis showed that the stem cell population included cells that expressed both epithelial- and mesenchymal-associated genes. Strikingly, when the expression of the stem cell gene signature was interrogated in two large independent TNBC cohorts, tumours with an activated stem cell signature showed a higher propensity to relapse in the first years after diagnosis in comparison to TNBC with lower activation scores for the stem cell gene signature. In contrast, in three large independent ER+ breast cancer data sets, an activated stem cell signature identified tumours least likely to metastasise. The prognostic power of the stem cell gene signature when applied to expression profiling of total tumour material implies that in poor prognosis TNBC the cancer stem cell-like genetic programme is not restricted to a minority cell population but rather is driving the behaviour of the bulk of tumour cells.

Our findings show that the biology of normal MaSCs, as reflected in their gene expression profiles, is highly relevant for understanding the drivers of aggressive disease in TNBC.

All animal work was carried out under UK Home Office project and personal licences following local ethical approval by the Institute of Cancer Research Animal Ethics Committee and in accordance with local and national guidelines. Single cells were prepared from fourth mammary fat pads of 8- to 10-week-old virgin female FVB mice as described [13] and stained with anti-CD24-FITC, anti-Sca-1-APC, anti-CD45-PE-Cy7, anti-CD49f-PE-Cy5 and anti-c-Kit-PE. Mammary epithelial cell subpopulations were defined as shown in Figure1 and Additional file 1.

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Mouse mammary stem cells express prognostic markers for ...

Download Ten Problems with Embryonic Stem Cell Research PDF

Embryonic stem cells are the basic building blocks for some 260 types of cells in the body and can become anything: heart, muscle, brain, skin, blood. Researchers hope that by guiding stem cells in the laboratory into specific cell types, they can be used to treat diabetes, Parkinson's disease, heart disease, or other disorders. The primary clinical source is the aborted fetus and unused embryos currently housed in frozen storage at IVF facilities. A developed stem cell line comes from a single embryo, becoming a colony of cells that reproduces indefinitely. Consider now the following ten problems with Embryonic Stem Cell Research (ESCR).

1. The issue of who or what

As the nation sits embroiled over the battle of where to draw the line on ESCR, the real issue that truly divides us is whether embryonic stems represent a who or a what. In other words, are we talking about people or property?

Since Roe v. Wade we have not been willing or able as a nation to address the issue. As a result, those who oppose ESCR and those who support it will never reach an acceptable point of compromise. Still, in the midst of the flurry of all this biotechnology and all the problems it presents, there is some very good news that has been overlooked by almost everyone. Ready? Cloning proves scientifically that life begins at conceptiona position to which the author and most Christians philosophically already adhere.

Additionally, the insights provided by cloning technology destroy the scientific and legal basis of distinguishing a preembryo from an embryo, the popular distinction made at 14 days after conception. This is significant because this distinction determines the handling and treatment of human life less than 14 days old, which is so basic to all ESCR.

In short, our understanding of embryonic development as provided by cloning technology could force not only those who participate in ESCR specifically, but also those who participate in in-vitro fertilization (IVF) procedures generally, to recognize there is no real preembryoembryo distinction and that all human life begins at conception. Therefore, as a nation, we should rightly adjust the moral and legal treatment and status of all embryos to people not property from the point of conception.

2. The deliberate misuse of terminology in defining stem cells

Proponents of ESCR often use the term pluripotent. This word intends to imply that the ESC cannot make or reform the outer layer of the embryo called the trophoblast. The trophoblast is required for implantation of the embryo into the uterus. This is a distinction used by proponents of ESCR to imply a fully formed implantable embryo cannot and does not reform after the original embryo is sacrificed. This is significant because to isolate the stem cells, scientists peel away the trophoblast or skin of the embryo much like the peel of an orange. They then discharge the contents of the embryo into a petri dish.

At this stage of development, the stem cells that comprise almost the entire inner body of the early embryo look and function very similar to one another. Once put into the petri dish, the un-programmed cells can be manipulated to multiply and divide endlessly into specific cell types. The question regarding use of the term pluripotent is whether stem cells emptied into the petri dish can reform the trophoblast creating an implantable embryo of the originally sacrificed embryo?

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Ten Problems with Embryonic Stem Cell Research | The ...

Stephen Barrett, M.D.

Stem cell therapy is certainly a promising area for research. Stem cells have the ability to give rise to many specialized cells in an organism. Certain types of stem cells are already used to restore blood-forming and immune system function after high-dose chemotherapy for some types of cancer, and several other restorative uses have been demonstrated. The broadest potential application is the generation of cells and tissues that could be used to repair or replace damaged organs. If scientists can learn how to control stem cell conversion into new, functionally mature cells, doctors might be able to cure many diseases for which therapy is currently inadequate [1,2]. However, the claims made by commercial promoters go way beyond what is now likely and should be regarded with extreme skepticism. The main commercial sources have included Embryonic Tissues Center in the Ukraine; Stem Cell of America (formerly called Medra, Inc) in Mexico; the Brain Therapeutics Medical Clinic (formerly called the Health Restoration Medical Center and the Brain Cell Therapeutic Clinic) in Mission Viejo, California; the Vita Nova Clinic in Barbados; and the Beijing Xishan Institute for Neuroregeneration and Functional Recovery in Beijing, China.

The Embryonic Tissues Center (EmCell) appears to be the oldest commercial source of embryonic stem cell therapy. Its proprietors, Alexander Smikodub, M.D., Ph.D., and Alexey Karpenko, M.D., Ph.D., are described as professors at National Medical University. The EmCell Web site claims:

How credible are these claims? How are the cells prepared? Are steps taken to ensure that they are not infectious? How was it determined that patients have no side effects? Does the clinic follow its patients and keep score? Have enough cancer patients to determine 5-year survival rates? Have Smikodub and Karpenko published their results? Do their theories and methodology make sense?

The ALS Therapy Development Foundation has been monitoring claims that fetal stem cell infusions might be effective against amyotropic lateral sclerosis (Lou Gehrig's disease). Its Web site states that two American physicians (Mitchell Ghen, D.O., and Dan Cosgrove, M.D.) have treated patients in a "new and untested way," but so far no conclusions could be drawn about effectiveness. Foundation documents also note that (a) some patients have experienced flu-like symptoms, (b) three patients have had dark-colored urine that may signify hemolytic anemia and/or kidney damage, and (c) it is not clear whether the stem cells are actually surviving long enough to have an effect [10,11]. In March 2003, the FDA seized records at Ghen's clinic and Cosgrove said he had stopped offering the treatment [12]. Cryobanks International, which had supplied the cells to Ghen and Cosgrove, stopped doing so after the FDA contacted them [13].

The ALS Foundation has also investigated the Cell Therapy Clinic by talking with a staff physician, sending a detailed follow-up questionnaire, and talking with several former patients. The Foundation's report states:

In August 2003, I did Medline searches to see whether Smikodub or Karpenko had published any reports about their patients in peer-reviewed medical journals. I found none that appeared relevant to the curative claims described above.

The chief American commercializer of embryonic stem cell therapy is William C. Rader, M.D., a psychiatrist in Malibu, California, who used to run Rader Institute clinics that specialized in treating eating disorders. For $25,000 (wired in advance), Rader will arrange for treatment at his Mexican clinic. In the past, he has also done business under the names Mediquest Ltd., Czech Foundation, Dulcinea Institute, Ltd., and Medra, Inc. A message posted to the Yahoo StemCells group indicates that before he opened his own clinic (in 1997 in the Bahamas), Rader escorted patients to the Ukraine clinic. Like EmCell, Rader has claimed that his fetal stem cell treatment is not antigenic and has no side effects. In a 1997 document, he stated:

Because fetal cells uniquely do not have antigenicity, they can be given to anyone with no reaction, no rejection, immunusuppressive drug therapy, or any side effects whatsoever. When a patient receives fetal fresh cell therapy (usually given intravenously over a few hours. . . ), the first action of cells is to stimulate the cells already present in the recipient's system, making them more potent. Then they actually replace the recipient's immune cells and, eventually engraft, which means they actually continually grow more fetal cells, resulting in a new and stronger immune system [15].

With respect to cancer, Rader has claimed that his treatment enables chemotherapy and radiation to continue longer and virtually eliminate their side effects [15]. Medra's "Factsheet" claimed:

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The Shady Side of Embryonic Stem Cell Therapy

Cell Therapy & Stem Cell Boosting

Stem Cells have been used in medical applications for over 40 years. In most countries the use of these stem cells are an approved method of treating various hematological conditions such as Leukemia and Aplastic Anemia.

Stem cells are biological cells found in all multi-cellular organisms, that can divide through mitosis and differentiate into diverse specialized cell types and can self renew to produce more stem cells. In mammals, there are two broad types of stem cells: embryonic stem cells that are isolated from the inner cell mass of blastocysts, and adult stem cells that are found in various tissues. In adult organisms, stem cells and progenitor cells act as a repair system for the body, replenishing adult tissues.

- Plasticity: Potential to change into other cell types like nerve cells

- Homing: To travel to the site of tissue damage

- Engraftment: To unite with other tissues

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Stem Cell Therapy Info - Stem Cell Treatment in Thailand ...

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Cell Isolation Products, Cell Culture Media, Cell Research

, are multipotent stem cells that can differentiate into a variety of cell types. Cell types that MSCs have been shown to differentiate into in vitro or in vivo include osteoblasts, chondrocytes, myocytes, adipocytes, and, as described lately, beta-pancreatic islets cells.

MSCs are rare in bone marrow, representing ~1 in 10,000 nucleated cells. Although not immortal, they have the ability to expand manyfold in culture while retaining their growth and multilineage potential. MSCs are identified by the expression of many molecules including CD105 (SH2) and CD73 (SH3/4) and are negative for the hematopoietic markers CD34, CD45, and CD14.

The properties of MSCs make these cells potentially ideal candidates for tissue engineering. It has been shown that MSCs, when transplanted systemically, are able to migrate to sites of injury in animals, suggesting that MSCs possess migratory capacity. However, the mechanisms underlying the migration of these cells remain unclear. Chemokine receptors and their ligands and adhesion molecules play an important role in tissue-specific homing of leukocytes and have also been implicated in trafficking of hematopoietic precursors into and through tissue. Several studies have reported the functional expression of various chemokine receptors and adhesion molecules on human MSCs. Harnessing the migratory potential of MSCs by modulating their chemokine-chemokine receptor interactions may be a powerful way to increase their ability to correct inherited disorders of mesenchymal tissues or facilitate tissue repair in vivo.

Mesenchymal stem cells are characterized morphologically by a small cell body with a few cell processes that are long and thin. The cell body contains a large, round nucleus with a prominent nucleolus which is surrounded by finely dispersed chromatin particles, giving the nucleus a clear appearance. The remainder of the cell body contains a small amount of Golgi apparatus, rough endoplasmic reticulum, mitochondria, and polyribosomes. The cells, which are long and thin, are widely dispersed and the adjacent extracellular matrix is populated by a few reticular fibrils but is devoid of the other types of collagen fibrils.

This website serves as a single key resource for all up to date information on Mesenchymal Stem Cell research. It provides links to current papers, protocols, and information about providers of MSC research products.

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Mesenchymal Stem Cells

Abstract Introduction

The existence of cancer stem cells (CSCs) has been associated with tumor initiation, therapy resistance, tumor relapse, angiogenesis, and metastasis. Curcumin, a plant ployphenol, has several anti-tumor effects and has been shown to target CSCs. Here, we aimed at evaluating (i) the mechanisms underlying the aggravated migration potential of breast CSCs (bCSCs) and (ii) the effects of curcumin in modulating the same.

The migratory behavior of MCF-7 bCSCs was assessed by using cell adhesion, spreading, transwell migration, and three-dimensional invasion assays. Stem cell characteristics were studied by using flow cytometry. The effects of curcumin on bCSCs were deciphered by cell viability assay, Western blotting, confocal microscopy, and small interfering RNA (siRNA)-mediated gene silencing. Evaluations of samples of patients with breast cancer were performed by using immunohistochemistry and flow cytometry.

Here, we report that bCSCs are endowed with aggravated migration property due to the inherent suppression of the tumor suppressor, E-cadherin, which is restored by curcumin. A search for the underlying mechanism revealed that, in bCSCs, higher nuclear translocation of beta-catenin (i) decreases E-cadherin/beta-catenin complex formation and membrane retention of beta-catenin, (ii) upregulates the expression of its epithelial-mesenchymal transition (EMT)-promoting target genes (including Slug), and thereby (iii) downregulates E-cadherin transcription to subsequently promote EMT and migration of these bCSCs. In contrast, curcumin inhibits beta-catenin nuclear translocation, thus impeding trans-activation of Slug. As a consequence, E-cadherin expression is restored, thereby increasing E-cadherin/beta-catenin complex formation and cytosolic retention of more beta-catenin to finally suppress EMT and migration of bCSCs.

Cumulatively, our findings disclose that curcumin inhibits bCSC migration by amplifying E-cadherin/beta-catenin negative feedback loop.

Breast cancer is the most common form of cancer diagnosed in women. In 2013, breast cancer accounted for 29% of all new cancer cases and 14% of all cancer deaths among women worldwide [1]. Breast cancer-related mortality is associated with the development of metastatic potential of the primary tumor [2]. Given this high rate of incidence and mortality, it is critical to understand the mechanisms behind metastasis and identify new targets for therapy. For the last few decades, various modalities of cancer therapy were being investigated. But the disease has remained unconquered, largely because of its invasive nature.

Amidst the research efforts to better understand cancer progression, there has been increasing evidence that hints at a role for a subpopulation of tumorigenic cancer cells, termed cancer stem cells (CSCs), in metastasis formation [3]. CSCs are characterized by their preferential ability to initiate and propagate tumor growth and their selective capacity for self-renewal and differentiation into less tumorigenic cancer cells [4]. There are reports which demonstrate that CSCs are enriched among circulating tumor cells in the peripheral blood of patients with breast cancer [5]. Moreover, recent studies show that epithelial-mesenchymal transition (EMT), an early step of tumor cell migration, can induce differentiated cancer cells into a CSC-like state [6]. These observations have established a functional link between CSCs and EMT and suggest that CSCs may underlie local and distant metastases by acquiring mesenchymal features which would greatly facilitate systemic dissemination from the primary tumor mass [7]. Taken together, these studies suggest that CSCs may be a critical factor in the metastatic cascade. Now, the incurability of the malignancy of the disease raises the question of whether conventional anti-cancer therapies target the correct cells since the actual culprits appear to be evasive of current treatment modalities.

Studies focusing on the early steps in the metastatic cascade, such as EMT and altered cell adhesion and motility, have demonstrated that aggressive cancer progression is correlated with the loss of epithelial characteristics and the gain of migratory and mesenchymal phenotype [8], for which downregulation of E-cadherin is a fundamental event [9]. A transcriptional consequence of the presence of E-cadherin in epithelial cells can be inferred from the normal association of E-cadherin with -catenin in adherens junctions. This association prevents -catenin transfer to the nucleus and impedes its role as a transcriptional activator, which occurs through its interaction mainly with the TCF (T-cell factor)-LEF (lymphoid enhancer factor) family of transcription factors but also with other DNA-binding proteins [10]. Accordingly, the involvement of -catenin signaling in EMTs during tumor invasion has been established [11]. Aberrant expression of -catenin has been reported to induce malignant pathways in normal cells [12]. In fact, -catenin acts as an oncogene and modulates transcription of genes to drive cancer initiation, progression, survival, and relapse [12]. All of the existing information regarding abnormal expression and function of -catenin in cancer makes it a putative drug target [12] since its targeting will negatively affect both tumor metastasis and stem cell maintenance. Transcriptional target genes of -catenin involve several EMT-promoting genes, including Slug. Expression of Slug has been shown to be associated with breast tumor recurrence and metastasis [13-15]. Pro-migratory transcription factor Slug (EMT-TF), which can repress E-cadherin, triggers the steps of desmosomal disruption, cell spreading, and partial separation at cell-cell borders, which comprise the first and necessary phase of the EMT process [16].

Recently, the use of natural phytochemicals to impede tumor metastasis via multiple targets that regulate the migration potential of tumor cells has gained immense importance [17]. In this regard, curcumin, a dietary polyphenol, has been studied extensively as a chemopreventive agent in a variety of cancers, including those of the breast, liver, prostate, hematological, gastrointestinal, and colorectal cancers, and as an inhibitor of metastasis [18]. In a recent report, curcumin was shown to selectively inhibit the growth and self-renewal of breast CSCs (bCSCs) [19]. However, there are no reports regarding the contribution of curcumin in bCSC migration.

The present study describes (i) the mechanisms governing the augmented migration potential of bCSCs, which (ii) possibly associates with tumor aggressiveness and is largely attributable to the inherent downregulation of the anti-migratory tumor suppressor protein, E-cadherin, in bCSCs, and (iii) the role of curcumin in modulating the same. A search for the upstream mechanism revealed higher nuclear translocation and transcriptional activity of -catenin resulting from disruption of E-cadherin/-catenin complex formation in bCSCs in comparison with non-stem tumor cells. Upregulation of nuclear -catenin resulted in the augmentation of Slug gene expression that, in turn, repressed E-cadherin expression. In contrast, exposure to curcumin inhibited the nuclear translocation of -catenin, thereby hampering the activation of its EMT-promoting target genes, including Slug. Resultant upregulation of E-cadherin led to increase in E-cadherin/-catenin complex formation, which further inhibited nuclear translocation of -catenin. As a consequence, the E-cadherin/-catenin negative feedback loop was amplified upon curcumin exposure, which reportedly inhibits EMT on one hand and promotes cell-cell adherens junction formation on the other. These results suggest that curcumin-mediated inhibition of bCSC migration may be a possible way for achieving CSC-targeted therapy to better fight invasive breast cancers.

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Stem Cell Research & Therapy | Full text | Curcumin ...

Schneider BP, Winer EP, Foulkes WD, Garber J, Perou CM, Richardson A, Sledge GW, Carey LA: Triple-negative breast cancer: risk factors to potential targets.

Clin Cancer Res 2008, 14:8010-8018. PubMedAbstract | PublisherFullText

Goss PE, Ingle JN, Ales-Martinez JE, Cheung AM, Chlebowski RT, Wactawski-Wende J, McTiernan A, Robbins J, Johnson KC, Martin LW, Winquist E, Sarto GE, Garber JE, Fabian CJ, Pujol P, Maunsell E, Farmer P, Gelmon KA, Tu D, Richardson H: Exemestane for breast-cancer prevention in postmenopausal women.

New Engl J Med 2011, 364:2381-2391. PubMedAbstract | PublisherFullText

Rosen JM, Jordan CT: The increasing complexity of the cancer stem cell paradigm.

Science 2009, 324:1670-1673. PubMedAbstract | PublisherFullText | PubMedCentralFullText

Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF: Prospective identification of tumorigenic breast cancer cells.

Proc Natl Acad Sci USA 2003, 100:3983-3988. PubMedAbstract | PublisherFullText | PubMedCentralFullText

Charafe-Jauffret E, Ginestier C, Iovino F, Wicinski J, Cervera N, Finetti P, Hur MH, Diebel ME, Monville F, Dutcher J, Brown M, Viens P, Xerri L, Bertucci F, Stassi G, Dontu G, Birnbaum D, Wicha MS: Breast cancer cell lines contain functional cancer stem cells with metastatic capacity and a distinct molecular signature.

Cancer Res 2009, 69:1302-1313. PubMedAbstract | PublisherFullText | PubMedCentralFullText

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Breast Cancer Research | Full text | Cancer stem cell ...