StemCell Therapy

Abstract

Adipose-derived stem cells (ADSCs) are a subset of mesenchymal stem cells (MSCs) that possess many of the same regenerative properties as other MSCs. However, the ubiquitous presence of ADSCs and their ease of access in human tissue have led to a burgeoning field of research. The plastic surgeon is uniquely positioned to harness this technology because of the relative frequency in which they perform procedures such as liposuction and autologous fat grafting. This review examines the current landscape of ADSC isolation and identification, summarizes the current applications of ADSCs in the field of plastic surgery, discusses the risks associated with their use, current barriers to universal clinical translatability, and surveys the latest research which may help to overcome these obstacles.

Recent advances in regenerative medicine, in particular the discovery of multipotent, easily accessible stem cells such as adipose-derived stem cells (ADSCs), have provided the opportunity of using autologous stem cell transplants as regenerative therapies. The field of plastic surgery, centred on the restoration and enhancement of the body, is logically positioned to utilize such new technologies focused on the repair and replacement of diseased cells and tissues [1]. The ability of stem cells to self-renew, to secrete trophic factors and to differentiate into different cell types has allowed for the development of more flexible therapies to redefine the classic autologous tissue transplant and offer more customizable treatment options. ADSCs are being utilized for a variety of different applications in plastic surgery [2-11], and as our understanding of the basic science of stem cells continues to develop, the plastic surgeon should be prepared for the translational and clinical implications of this progress.

Adipose-derived stem cells are particularly useful as they can be easily harvested with minimal donor site morbidity and have a differentiation potential similar to other MSCs [12, 13]. In addition, ADSCs have higher yields and greater proliferative rates in culture when compared to bone marrow stromal cells [14-16]. The discovery that ADSCs are not only precursors to adipocytes but also are multipotent progenitors to a variety of cells [17] including osteoblasts, chondrocytes, myocytes, epithelial cells and neuronal cells [18], creates the potential to treat a variety of tissue defects from a single, easily accessible autologous cell source.

Adult stem cell research has made significant strides as a therapeutic modality in recent years. However, there remain significant barriers to the safe and efficacious use of stem cell therapies. With regard to ADSCs, this includes better defining the source population of multipotent cells, optimizing the isolation of these cells in compliance with regulatory standards, and better understanding the behaviour of ADSCs in their transplanted niche. The purpose of this review is to (i) explore the utilization of ADSCs in plastic surgery, (ii) describe the current limitations of ADSC treatments with regard to developing translatable clinical therapies and (iii) describe certain techniques used in our laboratory that may help overcome these barriers. Understanding the current status of clinical ADSC treatments and defining the challenges ahead may bring us closer to achieving desired outcome while minimizing unwanted side effects with these therapies.

The most commonly published method of ADSC isolation involves enzymatic digestion of lipoaspirate to release the stromal vascular fraction (SVF) of cells which include stromal & endothelial cells, pericytes, various white blood cells, red blood cells and stem/progenitor cells [19]. The enzyme preparations used to achieve this fraction include dispase, trypsin and more commonly collagenase. In our laboratory, we take freshly harvested lipoaspirate and wash it with sterile 1% PBS until golden in colour. The adipose tissue is then digested with 0.01% collagenase/PBS solution at a ratio of 1ml of enzyme solution to 1cm3 of adipose tissue. This mixture is incubated at 37C with intermittent agitation until it becomes cloudy (usually 30min.). The infranatant is then carefully aspirated, transferred to 50ml conical tubes and centrifuged at 706g for 8min. The supernatant is discarded and resulting pellet, the SVF, is resuspended in control media [DMEM supplemented with 10% foetal bovine serum (FBS), 500IU penicillin and 500g streptomycin; Mediatech, Manassas, VA, USA]. The cells are then counted and plated in uncoated T75 flasks at a concentration of 1106 cells. Consistently, 20mg of lipoaspirate is ample tissue to harvest an adequate yield of SVF (>1107 cells).

In 2006, the International Society for Cellular Therapy (ICTS) defined a set of minimal criteria for identifying cells as ADSCs. These include plastic adherence while maintained in standard culture conditions, expression of CD73, CD90 and CD105 while lacking the expression of CD45, CD34, CD14 or CD11b, CD79 or CD19 and HLA-DR surface molecules [20]. In conjunction with the International Federation for Adipose Therapeutics and Science in 2013, the ICTS has denoted additional surface markers CD13, CD29 and CD44 as being constitutively expressed at >80% on the surface of ADSCs, while CD31, CD45 and CD235a are the primary negative markers that should be expressed on less than 2% of the cells [19]. Ultimately, the viability of the isolated cells should exceed 70% and the presence of at least two positive and two negative markers are necessary for foundational phenotyping. Finally, ADSCs must possess the ability to differentiate into osteoblasts, adipocytes and chondroblasts.

Identification of ADSCs in our laboratory is accomplished by labelling our plastic-adherent cells with a mesenchymal stem cell (MSC) phenotyping kit after the second passage (Miltenyi Biotec Inc, Auburn, CA, USA). Cells are analysed using a C6 Accuri Flow Cytometer (BD Biosciences, San Jose, CA, USA) which demonstrate positive staining for CD90 (81.3%), CD105 (86.6%) and CD73 (99.9%) and negative staining for CD14, CD20, CD34 and CD45 (1.97% Fig.1). To complete the identification of our ADSCs, we culture these cells in adipogenic, osteogenic, or chondrogenic conditions provided in commercially available kits (Cyagen Biosciences Inc., Sunnyvale, CA, USA). Cells subjected to adipogenic or osteogenic conditions reveal lipid droplets or calcium synthesis after staining with Oil Red O or Alizarin Red S, respectively, after fixation in 4% formalin. Cells subjected to chondrogenic conditions reveal proteoglycan synthesis upon staining with Alcian Blue after paraffin embedding (Fig.2). The ease at which ADSCs can be isolated has led to rapid and widespread translational applications.

Figure1. Flow cytometry analysis of isolated ADSCs after collagenase method. Cells stained (A) 81.3% positive for CD90, (B) 99.9% positive for CD73, (C) 86.6% positive for CD105 and (D) 1.97% positive for CD14, CD20, CD34 and CD45.

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Figure2. Undifferentated and differentiated ADSCs visualized using microscopy. Original magnification, 10. (A) Control stain uADSCs stained with Oil Red O (other controls not shown). (B) Staining with Alcian Blue revealing presence of chondroblasts. (C) Staining with Oil Red O revealing presence of adipocytes. (D) Staining with Alizarin Red S revealing presence of osteoblasts.

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A number of groups have described the isolation of ADSCs using non-enzymatic methods. Studies show that ADSCs reside in the infranatant layer of the suction canister after liposuction and that these cells can be expanded ex vivo. And while these cells exhibit phenotypic and differentiation potential similar to ADSCs isolated via collagenase digestion, their presence is significantly lower with reported yields ranging from a 3- to 19-fold decrease in comparison [21-24]. Interestingly, it has been found that multiple variables, including medical comorbidities of the patient, location adipose tissue stores, and the method in which this tissue is harvested, all affect the properties of the ADSCs therein. For example, diabetic patient fat stores have been found to contain fewer ADSCs with a reduced phenotypic expression profile and ability to proliferate [3]. The anatomical location of adipose tissue harvest also appears to have an effect on the yield and characteristics of the isolated ADSCs [25, 26]. More recently, Gnanasegaran etal. demonstrated that the gene expression levels and tendency towards specific germ layer differentiation is affected by whether the fat is harvested via liposuction versus lipectomy [27].

In Europe, ADSCs are considered Advanced Therapy Medicinal Products, as defined by the European Union (European Commission) 1394/2007 which contains rules for authorization, supervision, and pharmacovigilance regarding the summary of product characteristics, labelling, and packaging of Advanced Therapy Medicinal Products that are prepared commercially and in academic institutions [68]. This regulation refers to the European good manufacturing process (eGMP) rules [69]. The process of converting protocols, including collagenase-processed ADSCs, into a process that is compliant with eGMP requires assays that have had careful consideration of all the risks and benefits for the patient end user. As a result, the general recommendation on the use of enzyme-processed CAL in the clinical setting is not prohibited as this technique has been demonstrated to provide satisfying results in terms of long-term outcome, most likely because of the dramatic release of angiogenic growth factors and the differentiation of ADSCs into adipocytes and vascular endothelial cells [5, 10, 11].

In the United States, the Food and Drug Administration (FDA) regulates Human Cells and Tissue-Based Products (HCT/P) intended for human transplant and maintains two levels of classifications: 361 and 351 products. HCT/P 361 encompasses tissue (e.g. bone, ligaments, vein grafts, etc.) and their related procedures that take place in the same operative session, all of which fall under the jurisdiction of practice of medicine which is governed by state medical boards and professional societies; not the FDA. HCT/P 351, on the other hand, includes drugs/biologics (e.g. cultured cells, lymphocyte immune therapy, cell therapy involving the transfer of genetic material, etc.) which is fully governed by FDA [70, 71]. Regulation 21 CFR 1271 directly demonstrates the FDA's position on enzymatically isolated adipose stem cells derived from SVF for reconstructive purposes as beyond the scope of minimal manipulation and therefore, a drug [72]. Thus, the practical implication is the need for any surgeon who wishes to use ADSCs isolated via collagenase to submit an Investigational New Drug application to the FDA and have an approved Institutional Review Board with the referring Institution.

Given the time, expense and complexity of the regulatory issues surrounding ADSCs intended for transplantation, it is evident that U.S. physicians are discouraged to perform any cell-supplemented lipotransfer techniques in the current commonly accepted practices. Furthermore, automated devices for separating adipose stem cells are regulated as class III medical devices by the FDA, and currently, none are approved for human use in the United States. Kolle etal. demonstrated that CAL, when supplemented with ADSCs expanded ex vivo after collagenase digestion, yields superior results when compared to lipotransfer alone [38]. The FDA restrictions that would preclude such a study to be conducted in the United States prompt an impetus to develop methods for CAL that results in minimal manipulation of source adipose tissue.

In 2006, Yoshimura etal. described a cell population in the liposuction aspirate fluid that exhibited similar phenotypic properties to ADSCs harvested in the traditional manner (collagenase) from processed lipoaspirate cells; however, the yield was reduced by athird when comparing to the two methods [23]. Since that time, additional studies have been published touting the benefits of non-enzymatic ADSC isolation. In 2010, Francis etal. described a method of ADSC Rapid Isolation in ~30min. that excluded the use of collagenase, however, a significant disadvantage of this study was the low yield of ~250,000 cells from a starting volume of ~250ml liposuction aspirate fluid [21]. Zeng etal. describe a rapid and efficient form of non-enzymatic ADSC isolation in which adipose tissue is cut into tiny pieces and placed in culture flasks with 100% FBS in which the plastic-adherent cells were allowed to expand over a period of days [24]. One obvious downside to this method is the requirement to expand the cell population in calf serum. Most recently, Shah etal. describe aform of non-enzymatic ADSC isolation combining the cells of the liposuction aspirate fluid with the cells captured from the processed lipoaspirate tissue wash that is typically discarded prior to collagenase digestion [22]. They observed significant improvement in MSC-related phenotypic markers and similar adipogenic and osteogenic differentiation characteristics. While their isolation time was cut by one-third, they observed a 19-fold decrease in ADSC isolation when compared to the traditional method. In our laboratory, we have adopted a very similar protocol of non-enzymatic isolation that includes processing the processed lipoaspirate effluent. The primary difference in our protocol, however, is the method of plating cells. While Shah etal. plate the entire SVF pellets in T175 flasks, we resuspend our pellets in culture media and then plate the cells at specific concentrations. In one experiment for example, we plated the SVF pellet after collagenase digestion at a concentration of 5105 in a T75 flask. Concurrently, we plated the SVF pellet obtained after non-enzymatic isolation at 2106. After 6days of culture, these two flasks appeared nearly identical in terms of confluence, correlating to a fourfold decrease in ADSC harvest when using the latter method. The two cell populations were then analysed under flow cytometry as previously described. There is little difference in the phenotypic expression between the two populations as demonstrated by >80% expression of CD90, CD73 and CD105 and <5% expression of CD14, CD20, CD 34 and CD45 (Fig.3).

Figure3. Flow cytometry analysis of isolated ADSCs after rapid isolation (no collagenase). Cells stained (A) 85.8% positive for CD90, (B) 99.9% positive for CD73, (C) 99.4% positive for CD105 and (D) 3.79% positive for CD14, CD20, CD34 and CD45. (E) Collagenase-isolated ADSCs after 6days of primary culture seeded at 5105 in T75 flask. (F) Rapid isolation ADSCs after 6days primary culture seeded at 2106 in T75 flask.

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Most convincingly, Kolle etal. demonstrated a clear benefit to CAL over lipotransfer alone. They isolated and expanded ADSCs ex vivo from human cases followed by lipotransfer to the cases arms with or without ADSC supplementation. They demonstrated a 65% improvement in fat graft survival after 4months in the experimental group [38]. The major drawback to their experimental model was that to achieve these results, the 34ml of lipotransfer was supplemented with 6.5108 ADSCs or 2000 times the physiological level [38]. The methods of rapid isolation, previously mentioned, demonstrate the ability to isolate ADSCs without the aid of enzymatic digestion, but at a cost of greatly reduced yields. There is significant doubt that ADSCs used at such low concentrations would serve for any clinical benefit. As previously discussed, ex vivo expansion of ADSCs is not practical for application in the United States or other principalities with strict regulations. Therein lies an impetus to discover innovative methods of ADSC isolation and characterization of the regenerative components of the SVF that might yield similar results to concentrated ADSCs alone.

There is promise in capitalizing on the plastic-adherent properties of ADSCs as a form of non-enzymatic isolation. The same group that first described the isolation of cells from the LAF, Doi etal., has demonstrated that an adherent column of rayonpolyethylene non-woven fabrics may also be used to isolate ADSCs, though at an inferior yield to the traditional method [73]. Further advancements in harnessing the plastic-adherent properties of these cells are clearly needed as Buschmann etal. demonstrated that 3050% of ADSCs remain in suspension after 24hrs of primary culture [74].

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