MMG 445 Basic Biotechnology (2011) 7:24-30

MMG445.5213335

Prospering on the Fat of the Land: Adipose-derived stem cells as an industrially-viable resource for regenerative treatment
Alex Hoekstra Department of Microbiology and Molecular Genetics, Michigan State University Correspondence: hoekst42@msu.edu The field of regenerative medicine can be defined by its unique objectives: to restore and recover rather than merely repair or preserve damaged tissue. Stem cells offer a means of achieving this goal, but broadly-applicable scalability has been limited by numerous challenges including availability, purity, tissue development potential and transplant-related immunogenicity. This review will address these issues through a specific examination of a unique subset of adult stem cells, derived from adipose tissue. Abbreviations ASC - Adipose-derived Stem Cell, ESC - Embryonic Stem Cell, iPSC - Induced Pluripotent Stem Cell, MSC - Mesenchymal Stem Cell

The application of stem cells represents an enormous potential for serving a market in regenerative treatments. The ability to restore full function to damaged tissue, beyond the normal capabilities of the body, is a highly-valued development in the biotechnological realm with far-reaching implications. The goals of these regenerative treatments can be achieved through the application of stem cells [1-12]. Stem cells can be defined as primitive cells that have the capacity to differentiate into a variety of tissue types when induced to do so. Stem cells offer the potential to restore function to damaged tissue by way of inducing the growth of new tissue which can be specialized for the function of that which was damaged. At present, the widespread availability of stem cells as an effective treatment for human tissue damage and disease is hindered by a number of technical challenges. Industrial viability requires consistency, purity, efficacy, cost-effective scalability and non-patient-specificity in order to address the needs of a significant market. Stem cell therapy has thus far faced barriers on all of these fronts. The abundance, availability and purity, the efficiency and complexity of differentiation, and the patient-specificity related to stem cells have been the subject of research attempting to address these barriers to their broad commercialization as a therapeutic agent [7-12].

Introduction

Distinctions between stem cell Varieties

Stem cells are characterized both by their source and potency (their ability to differentiate into multiple tissue types). The varieties that offer the greatest potential are embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and adult stem cells. ESCs and iPSCs have been the focus of numerous studies [13-15] because both varieties are pluripotent and abe to selfreproduce. Both, however, are limited in abundance, availability (involving both biological and ethical constraints) and patient-specificity with regard to immunogenicity [9,16]. Mesenchymal stem cells (MSCs), also known as
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One variety of stem cells that exhibits a number of unique features contributing to the practicality of its use on an industrial scale is derived from human adipose tissue. Called adipose-derived stem cells (ASCs), this variety of cells expresses a number of unique characteristics which qualify them as an optimal resource for regenerative tissue applications on an industrial (as distinguished from an individual, patient-specific) scale. Included among these unique features are factors contributing to abundance and availability of the stem cells [1-8,9], their capacity to differentiate into numerous tissue types and their low immunogenicity [8,9].

adult stem cells, on the other hand, offer availability and immunocompatibility while maintaining multipotentiality. MSCs have been isolated from a variety of human tissues, including bone marrow, dental pulp and adipose tissue [8,9]. Adipose-derived stem cells (ASCs) possess a number of unique advantages that contribute to their potential for widespread (industrial-scale) implementation. Surface marker proteins are used to distinguish stem cell types. Regardless of the methods for isolation, culture or Figgrowth time, ASCs express similar proteins as previously-established bone marrow-derived MSCs [9,10].

Stem cell characteristics necessary for clinical application

In order to be considered a viable candidate resource for clinical regenerative therapies, the following characteristics have been proposed as criteria for stem cells [8,9,11].

1. Able to be collected in abundant quantities (millions to billions of cells); 2. Able to be collected with a minimally invasive procedure;

The protocols for isolation of MSCs from human adipose tissue have been demonstrated and replicated by numerous laboratories. Briefly, cells are extracted and isolated from a tissue specimen using processes that allow for up-scaling. These techniques involve chemicals that are commonly available (such as phosphate-buffered saline [PBS], collagenase, fetal bovine serum [FBS] and modified Eagle medium [MEM]) [11]. This process itself represents a model that appears suitable for largescale, high-yield expansion to meet an industrialscale demand.

procedures per year. Each lipoaspiration procedure routinely produces from 100 mL to more than 3 L of adipose tissue [11]. Adipose tissue has a density of approximately 0.9 g/mL [17], thus a single lipoaspiration procedure can yield from between 90 and 2,700 grams of raw adipose tissue. On average, each gram of adipose tissue yields approximately 5 x 103 stem cells [9]. ASCs can be characterized as highly abundant relative to alternative stem cell varieties.
Hoekstra A

Isolation of MSCs from human adipose tissue

Abundance of MSCs derived from bone marrow and dental pulp is limited. Collection of stem cells from these tissues can be painful and yield only relatively small quantities of viable cells. Adipose tissue, in contrast, is not in short supply. Per gram, adipose tissue yields a 500-fold greater number of MSCs than bone marrow [9]. Further, the collection of adipose tissue from a human subject in substantial quantities can be done with a minimally-invasive, low-risk procedure that offers little discomfort to patients. Lipoaspiration (or liposuction) is currently performed across the United States at a frequency of nearly 400,000

Abundance and Availability

4. Able to be transplanted to either an autologous (self) or allogenic (non-self) host in a safe and effective manner.

3. Able to differentiate into a variety of tissue types in a regulatable and reproducible manner;

ESCs convey totipotency (a capacity to differentiate into virtually all cell lineages) and iPSCs convey pluripotency (a more limited, but still extensive range of potential differentiation, including endodermal, mesodermal and exodermal tissues). Adult stem cells such as MSCs were previously thought to be limited in their range of development into the cell line of their origin. This attribute is termed multipotency which had been used to characterize adult stem cells has since been replaced by pluripotency [11]. MSCs have demonstrated the ability to mature into a variety of tissues but are still regarded as multipotent. Induced differentiation of MSCs has resulted in osteogenesis (generation of bone tissue), chondrogenesis (cartilage tissue), myogenesis (muscle tissue) and adipogenesis (fat tissue). ASCs have been widely demonstrated to possess the ability to yield all of these mesenchymal cell lineages [8-10,11], and have been further determined to possess the ability to differentiate into non-mesenchymal tissues such as neuron-like, endothelial, epithelial, hematopoetic and pancreatic cells [9]. These features are summarized in Table 1.

Potentiality and Differentiation

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Adipose-derived stem cells Table 1. A Comparison of stem cell varieties Potency Totipotent Stem cell name Embryonic stem cells (ESCs) Source Embryonic tissue Potential lineages Endoderm Ectoderm Mesoderm Extra-embryonic

Pluripotent Pluripotent

Induced pluripotent stem cells (iPSCs) Mesenchymal stem cells (MSCs)

Adult somatic tissue

In vitro differentiation of ASCs can be accomplished through treatment with induction factors, outlined in Table 2. In vivo application of ASCs is accomplished through either autologous or allogenic transplantation of isolated stem cells into the damaged tissue.

Bone marrow Endoderm Dental pulp Ectoderm Cord blood Mesoderm Placenta Adipose tissue Stem cell varieties, their respective sources and potential lineage pathways. Examples of ectodermal tissues include nervous tissue, enamel and the epidermis. Endodermal tissue comprises of the innermost layer of cells in an organ, including the lining of the gastrointestinal, respiratory, urinary and auditory tracts, as well as the lining of the follicles of endocrine organs. Mesodermal tissue comprises the middle layer of cells (existing between the endoderm and the endoderm), and consists of the primary tissues of the muscular, circulatory, skeletal, and excretory, as well as connective tissues. Extraembryonic tissue comprises of the structure surrounding a developing embryo, including the placenta [18].

In order to meet the demands of a developed market for the application of stem cells in regenerative medicine, stem cell lines must not only be available in quantities above what is directly isolatable from tissue specimens, but also applicable for both autologous (self) and allogenic (non-self) transplantation. This requires first the suppression of surface markers and antigens on cells that would otherwise trigger an immune response, and finally expansion of stem cell numbers through large-scale culture.

Scalability

Contamination of stem cell lines with unidentified and undesired cells is another significant challenge confronting their clinical implementation. The presence of undefined cells reduces the efficacy of induced differentiation and further contributes to unintended differentiation of both ESCs and iPSCs [14]. Isolation of pure stem cells has proven to be a challenge for ESCs, iPSCs and adult stem cells. Due to the relatively high abundance of stem cells in

Purity

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Numerous studies have demonstrated that ASCs can be propagated in vitro [8,9,16,17,19-21]. Approximate doubling time of ASCs in vitro has been reported to be between 2 and 4 days, depending upon the origin including age, tissue type (brown vs. white adipose tissue), the methods of extraction, isolation and culture [9]. Bioreactor technology until now has principally been 3D scaffolding, which is effective but costly [18-21]. Emerging bioreactor technologies involve the use of homogenous liquid phase culture and may provide a less expensive means of high-volume expansion [19-21]. These reactors could provide a direct means of not only expanding the number of stem cells, but also a means of large-volume in vitro differentiation through chemical treatment (as per the induction factors listed in Table 2).

adipose tissue and the efficacy of isolation methods, ASCs represent a unique opportunity for establishing high-purity cultures. Due to a high abundance of stem cells within an adipose tissue sample, the effect of unintended loss of stem cells resultant of purification processes is minimized. The purity of ASCs can be determined by fluorescent microscopy, which requires significantly fewer cells than the more commonly-employed purity assessment through flow cytometry [16,17].

Culturing, large-scale expansion and storage

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Hoekstra A Table 2. ACS lineage differentiation induction factors Cell Type Induction Factors Adipocyte Chondrocyte Osteocyte Endothelial Dexamethasone, isobutyl methylxanthine, indomethacin, insulin, thiazolidinedione Ascorbic acid, bone morphogenetic protein 6, dexamethasone, insulin, transforming growth factor-beta Ascorbic acid, bone morphogenetic protein 2, dexamethasone, 1,25 dihydroxy vitamin D3 Proprietary medium (EGM-2-MV; Cambrex) containing ascorbate, epidermal growth factor, basic fibroblast growth factor, hydrocortisone Dexamethasone, horse serum Transferrin, IL-3, IL-6, VEGF Butylated hydroxyanisole, valproic acid, insulin

Myocyte Cardiomyocyte Neuron-like

Unlike both ESCs and iPSCs, which have demonstrated immunogenicity and rejection in both autogenic and allogenic transplantations [16], MSCs exhibit low immunogenicity after allogenic transplantation. Imanishi et. al [19] showed that the

Prolonged cultures (lasting greater than four months) revealed the preservation of ASC telomere length, implying that passage-related (age-related) degeneration is minimal [8,11]. Collas et al. reported successful maintenance of pure ASC lines for a period of over six months without any agerelated defects in cellular activity [22,23]. On the other hand, at least one laboratory reported ASCs cultured for more than four months exhibited chromosomal abnormalities and induced a high rate of tumors when implanted in immunodeficient mice [8]. The conflicting results indicate that while there does appear to be a means of sustaining cultures long-term without inducing abnormalities or defects, this method is not yet widely applied and requires further investigation. Bunnell et al. [11] also describe a method of cryopreservation. The results of these studies together indicate that cultured cells, under the correct conditions, can be sustained and preserved for future use. Also worth noting is the diminished need for culture of ASCs due to their relatively high abundance when taken directly from tissue specimens. As mentioned above, adipose tissue yields roughly 4500 CFU-F of stem cells per milliliter of original tissue sample (in contrast, bone marrow yields as little as 100 CFU-F per milliliter) [10,17]. This diminishes the need for culturing in order to establish quantities sufficient for clinical application.

(Non) Immunogenicity

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level of inflammatory cytokines in living hearts that received allogenic transplants of MSCs as a treatment for acute myocardial infarction returned to nearnormal levels only seven days post-transplant and remained at normal levels over the following weeks [19]. MSCs express fewer surface marker proteins and antigens that induce host immune response and rejection. Passaged ASCs, in contrast to newlyisolated cells, express diminished numbers of surface antigens and fail to induce immune reactions when co-cultured with allogenic immune cells [12]. Expression of surface proteins within the major histocompatibility complex class signifies the potential of ACSs for allogenic transplantation without immunogenic response [10]. Successful human treatment for radiation-induced tissue damage as well as for acute Graft Versus Host Disease (GVHD) using allogenically-transplanted ASCs [9] indicates that these cells convey immunosuppressive qualities in humans, reaffirming their suitability in non-self transplantation, free of immunogenic response or host rejection. In vivo application of ASCs has demonstrated successful restoration of tissue function in these patients, who remained free from side-effects and immunogenic host response after a series of follow-ups lasting a period of 40 months [9]. Such in vivo studies are, as yet, limited in the human model, but extensive study has taken place to affirm the allogenic immunocompatibility of adipose stem cells in murine models [20,21]. Transplantation of xenogenic (human) ASCs into mice resulted in suppression of inflammatory and immune responses, reducing the stimulation of numerous cytokines and chemokines [20] (Figure 1A) and inhibiting the activity of host macrophages [20,21] (Figure 1B).
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Similar experiments involved the application of autogenic or allogenic mouse ASCs, and resulted in similar suppression of immunogenicity [21] (Figure 2). These studies indicate that ASCs represent high
Adipose-derived stem cells

immunocompatibility (in autologous, allogenic and xenogeneic [20,21](non-species) transplantation) and are thus an attractive candidate for broadlyuseable therapeutic application.

Figure 1. Gonzlez et. al [20,21] demonstrated the inhibition of inflammatory cytokines (A) and macrophage activity (B) in mice with induced arthritis through the treatment with human adipose-derived stem cells (hASC).

Figure 2. Gonzlez et. al [20] demonstrated inhibition of cytokine activity in mice with induced arthritis when treated with mouse adipose-derived stem cells (mASC) (A). Further, assessment of arthritic symptoms revealed that similar success was achieved through both autologous and allogenic transplantation (B). 28 MMG 445 Basic Biotechnology

Stem cells are the principle resource for regenerative medicine, which has the distinct goal of restoring full function of damaged tissue using the native developmental processes of an individual. The implications of this manner of treatment are far-reaching, but limitations in stem cell technology have thus far inhibited industrial development. Adipose-derived stem cells in particular demonstrate a variety of highly-valuable characteristics with respect to the goal of developing clinical application beyond isolated and individualized treatments. Available in relatively high abundance and expandable via sustained culture, ASCs exhibit some of the properties necessary for industrial scaling. Further, the tissue lineages that have been successfully developed through ASCs represent a group of tissues of medical importance. Perhaps most significantly, ASCs express immunosuppressive properties including host cytokine level reduction and

Conclusion

A great many thanks are due to Professors George Garrity and Clive Waldron, as well as to my fellow students enrolled in the MMG 445 course who have all

Acknowledgements

macrophage inhibition, as well as cell surface antigen reduction, making them highly suitable for both autologous and allogenic transplantation without host immune response or rejection. Still, challenges remain before large-scale manufacturing of stem cells can be employed for widespread clinical use. As with any scaled-up manufacturing method, purity on numerous levels must be accounted for (from serum-free culture media to sterile bioreactor conditions) in order to ensure quality and consistency of stem cell production. The optimal bioreactor conditions must yet be determined before large-scale stem cell differentiation and expansion can be achieved without structurally complex and costly bioprocessing systems. With further use, the effects of long-term culture and storage will become apparent, but must be clarified before use in a fullscale clinical setting.
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