REGENERATIVE MEDICINE Concise Review: Adipose-Derived Stem Cells as a Novel Tool for Future Regenerative Medicine HIROSHI MIZUNO,a MORIKUNI TOBITA,b A. CAGRI UYSALc a
Department of Plastic and Reconstructive Surgery, Juntendo University School of Medicine, Tokyo, Japan; Department of Dentistry, Japan Self Defense Force Hospital Yokosuka, Yokosuka, Japan; cDepartment of Plastic and Reconstructive Surgery, Baskent University Hospital, Ankara, Turkey b
Key Words. Adipose-derived stem cells • Regenerative medicine • Proliferation • Differentiation • Clinical trials • Growth factors
ABSTRACT The potential use of stem cell-based therapies for the repair and regeneration of various tissues and organs offers a paradigm shift that may provide alternative therapeutic solutions for a number of diseases. The use of either embryonic stem cells (ESCs) or induced pluripotent stem cells in clinical situations is limited due to cell regulations and to technical and ethical considerations involved in the genetic manipulation of human ESCs, even though these cells are, theoretically, highly beneficial. Mesenchymal stem cells seem to be an ideal population of stem cells for practical regenerative medicine, because they are not subjected to the same restrictions. In particular, large number of adipose-derived stem cells (ASCs) can be easily harvested from adipose tissue. Furthermore, recent
basic research and preclinical studies have revealed that the use of ASCs in regenerative medicine is not limited to mesodermal tissue but extends to both ectodermal and endodermal tissues and organs, although ASCs originate from mesodermal lineages. Based on this background knowledge, the primary purpose of this concise review is to summarize and describe the underlying biology of ASCs and their proliferation and differentiation capacities, together with current preclinical and clinical data from a variety of medical fields regarding the use of ASCs in regenerative medicine. In addition, future directions for ASCs in terms of cell-based therapies and regenerative medicine are discussed. STEM CELLS 2012;30:804– 810
Disclosure of potential conflicts of interest is found at the end of this article.
INTRODUCTION Promising bioengineering technologies, such as tissue engineering, which is an interdisciplinary field involving physicians, engineers, and scientists, may provide novel tools for reconstructive surgery. A number of conditions, such organ failure, tissue loss due to trauma, cancer abrasion, and congenital structural anomalies, can be treated by current clinical procedures/ surgical strategies including organ transplantation, autologous tissue transfer, and the use of artificial materials; however, these treatments have potential limitations, including organ shortages, damage to healthy parts of the body during treatment, allergic reactions, and immune rejection. Recent developments in the emerging field of stem cell science, stem cell-associated growth factors, and regenerative medicine may allow the use of stem cells to repair tissue damage and, eventually, to replace organs. Stem cell candidates include embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and postnatal adult stem cells. ESCs are capable of extensive self-renewal and expansion and have the potential to differentiate into any type of somatic tissue [1]. These traits make human ESCs promising for future
use in regenerative medicine. iPSCs are derived from differentiated cells such as skin fibroblasts and appear to have the same potential and properties; however, their generation is not dependent upon a source of embryos [2]. As such, although the therapeutic potential of both ESCs and iPSCs is enormous due to their auto-reproducibility and pluripotentiality, there are still some limitations to their practical use: for example, cellular regulation of teratoma formation, ethical considerations, immune concerns regarding ESCs, and difficulties with genetic manipulation with respect to iPSCs [1, 3]. In contrast, postnatal adult stem cells are, by their nature, immunocompatible, and there are no ethical concerns related to their use. Multipotent mesenchymal stem cells (MSCs) are nonhematopoietic cells of mesodermal derivation that are present in a number of postnatal organs and connective tissues. Recently, MSCs, with similar characteristics to bone marrowderived MSCs, have been isolated from different tissue sources including trabecular bone [4], periosteum [5], synovial membrane [6], skeletal muscle [7], skin [8], pericytes [9], peripheral blood [10], deciduous teeth [11], periodontal ligament [12], and umbilical cord [13, 14]. Although the stem cell population derived from these sources are valuable,
Author contributions: H.M.: conception and design, financial support, provision of study materials, manuscript writing, and final approval of manuscript; M.T.: administrative support and correction and/or assembly of data; A.C.U.: correction and/or assembly of data and data analysis and interpretation. Correspondence: Hiroshi Mizuno, M.D., Department of Plastic and Reconstructive Surgery, Juntendo University School of Medicine, 2-1-1 Hongo Bunkyo-ku, Tokyo 1138421, Japan. Telephone: 81-3-5802-1107; Fax: 81-3-5689-7813; e-mail:
[email protected]. jp Received January 7, 2011; accepted for publication February 15, 2012; first published online in STEM CELLS EXPRESS March 13, C AlphaMed Press 1066-5099/2012/$30.00/0 doi: 10.1002/ 2012; available online without subscription thorugh the open access option. V stem.1076
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common problems include low number of harvested cells and limited amount of harvested tissues. Therefore, almost all adult-derived stem/progenitor cells require at least some degree of ex vivo expansion or further manipulation before they are used preclinically or clinically to satisfy efficacy and safety requirements. In contrast, the multipotent stem cells within adipose tissue, termed adipose-derived stem cells (ASCs) [15], are one of the most promising stem cell population identified thus far, since human adipose tissue is ubiquitous and easily obtained in large quantities with little donor site morbidity or patient discomfort. Therefore, the use of autologous ASCs as both research tools and as cellular therapeutics is feasible and has been shown to be both safe and efficacious in preclinical and clinical studies of injury and disease [16, 17]. To date, a number of scientific publications have described the underlying biology of ASCs, preclinical studies for the use of ASCs in regenerative medicine in various fields have been performed, and the efficacy of ASCs has been determined in several clinical trials. Therefore, the aim of this concise review is to highlight recent progress in ASC biology, trends in preclinical and clinical studies, and future directions for the use of ASCs with particular reference to the field of regenerative medicine.
LOCALIZATION AND CELLULAR CHARACTERIZATION OF ASCS Adipose tissue is composed mainly of fat cells organized into lobules [18]. It is a highly complex tissue consisting of mature adipocytes, which constitute more than 90% of the tissue volume, and a stromal vascular fraction (SVF), which includes preadipocytes, fibroblasts, vascular smooth muscle cells, endothelial cells, resident monocytes/macrophages, lymphocytes, and ASCs [19, 20]. Several articles have shown that many of the characteristics of ASCs differ according to the location of the harvested adipose tissue. ASCs harvested from superficial abdominal regions are significantly more resistant to apoptosis than ASCs harvested from the upper arm, medial thigh, trochanteric, and superficial deep abdominal depots [21]. The density of stem cell reserves varies within adipose tissue and is a function of location, type, and species (e.g., human vs. murine). In white adipose tissue, for example, ASC yields are greater in subcutaneous depots compared with visceral fat, with the highest concentrations in arm adipose tissue depots and the greatest plasticity in ASCs isolated from inguinal adipose tissue depots [22]. Likewise, it has been reported that ASCs have been found within the brown adipose tissue depots found distributed in white fat depots present in the body and that they easily undergo skeletal myogenic differentiation [23]. Phenotypically, the characterization of ASCs is still in its infancy. To date, all attempts to establish an exact definition of the ASC phenotype and to discriminate clearly between these cells and fibroblasts have been unsuccessful. Freshly isolated SVF cells are a heterogeneous cell population that includes putative ASCs (CD31, CD34þ/, CD45, CD90þ, CD105, and CD146), endothelial (progenitor) cells (CD31þ, CD34þ, CD45, CD90þ, CD105, and CD146þ), vascular smooth muscle cells or pericytes (CD31, CD34þ/, CD45, CD90þ, CD105, and CD146þ), and hematopoietic cells (CD45þ) in uncultured conditions [24]. Additionally, compared with ASCs from later passages, freshly isolated SVF cells and early passage ASCs www.StemCells.com
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express higher levels of CD117 (c-kit), human leukocyte antigen-DR (HLA-DR), and stem cell-associated markers such as CD34, along with lower levels of stromal cell markers such as CD13, CD29 (b1 integrin), CD44, CD63, CD73, CD90, CD105, and CD166 (Table 1) [14, 24–40]. While the consequences of the decrease in CD34 expression in later passage ASCs are not clear, there is at least one study demonstrating that CD34 expression can be maintained during 20 weeks of culture [36]. Furthermore, several studies have shown that CD34þ ASCs have a greater proliferative capacity, while CD34 ASCs are more plastic [25, 41]. Additionally, there are two reports demonstrating that CD106 was slightly expressed in cultured ASCs [31, 33]. As indicated, ASCs share many cell surface markers with pericytes and bone marrow-MSCs. Except for those mentioned above, the pericyte markers expressed by ASCs include smooth muscle b-actin, platelet-derived growth factor (PDGF) receptor-b, and neuro-glial proteoglycan 2 [25], while the markers shared by ASCs and MSCs include CD13, CD29, CD44, CD58, and CD166. The exact location of ASCs within adipose tissue remains elusive; however, several studies have shown that ASCs may exist within the perivascular tissue since they express similar cell surface antigens to pericytes [42, 43]. Culturing of these cells eventually results in the appearance of a relatively homogenous population of mesodermal or MSCs [15]. However, it is clear that the different techniques used for harvesting ASCs greatly affect the proportions of various cell types that accompany them. Moreover, cell culture conditions markedly affect ASC gene expression profiles, in particular, the medium used and the mechanophysiological environment (e.g., three-dimensional culture, the imposition of mechanical force on the cells, and the degree of oxygenation). ASCs exhibit telomerase activity to some extent, although it is lower than that in cancer cell lines, which indicates that ASCs have the capacity for self-renewal and proliferation [44]. Finally, Puissant et al. [45]. have reported on the lack of HLA-DR expression and the immunosuppressive properties of human ASCs. Based on such findings, Fang et al. published preliminary data showing that severe steroid-refractory acute graft-versus-host disease (GVHD) could be treated with human ASCs from HLA-mismatched donors [46].
ISOLATION
OF
ASCS FROM ADIPOSE TISSUE
The fact that stem cell yields are greater from adipose tissue than from other stem cell reservoirs is another significant factor in their suitability for use in regenerative medicine. Routinely, 1 107 adipose stromal/stem cells have been isolated from 300 ml of lipoaspirate, with greater than 95% purity [47]. In other words, the average frequency of ASCs in processed lipoaspirate is 2% of nucleated cells, and the yield of ASCs is approximately 5,000 fibroblast colony-forming units (CFU-F) per gram of adipose tissue, compared with estimates of approximately 100–1,000 CFU-F per milliliter of bone marrow [48]. Subcutaneous adipose tissue samples can generally be obtained under local anesthesia. Current methods used for isolating ASCs rely on collagenase digestion followed by centrifugal separation to isolate the SVFs from primary adipocytes. They display a fibroblast-like morphology and lack the intercellular lipid droplets seen in adipocytes. Isolated ASCs are typically expanded in monolayer culture on standard tissue culture plastics with a basal medium containing 10% fetal bovine serum [49].
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Table 1. Characterization of freshly isolated human stromal vascular fractions from adipose tissue and mesenchymal stem cells SVFs CD markers
Noncultured ASCs
CD9 CD10 CD13 CD19 CD24 CD29 CD31 CD34 CD44 CD45 CD49b CD58 CD71 CD73 CD90 CD105 CD106 CD117 CD140b CD146 CD151 CD166 CD271 Ref.
þ þ þ þ þ þ þ NA þ þ þ þ þ/ þ NA þ/ þ/ [24, 25, 29, 30, 34, 35, 36]
Bloodderived cells
Endothelial cells
Pericytes
NA NA NA NA NA NA þ/ NA þ NA NA NA NA NA þ/ NA NA NA NA NA NA NA [36]
NA NA NA NA NA NA þ þ NA NA NA NA NA þ þ/ þ þ NA þ NA þ NA [24, 36]
NA NA NA NA NA NA NA NA NA NA NA þ NA NA NA þ NA NA NA [24, 36]
Cultured ASCs (Passage 3)
Cultured ASCs (Passage 4)
BM-MSCs
þ þ/ þ NA þ þ/ þ þ NA þ þ þ þ þ/ NA NA þ þ þ NA [14, 29, 31, 33, 35, 36, 40]
þ/ þ/ þ NA þ þ þ NA þ þ þ þ NA NA NA þ þ [14, 29, 34, 35, 37, 39]
þ þ þ NA NA þ þ þ þ NA þ þ þ þ þ NA þ NA þ þ [25, 26, 28, 30, 32]
Cultured BM-MSCs
NA NA NA NA NA þ þ NA þ þ þ þ þ NA þ þ NA þ [26–28, 38]
Abbreviations: ASCs, adipose-derived stem cells; BM-MSCs, bone marrow-derived mesenchymal stem cells; NA, not available; SVF, stromal vascular fraction.
THE ROLE OF SOLUBLE FACTORS FROM ASCS IN REGENERATIVE MEDICINE Previous reports have suggested that the beneficial effects of bone marrow-derived MSC-based therapy, such as angiogenesis, anti-inflammation, and antiapoptosis, are largely mediated by the trophic actions of cytokines and growth factors secreted by the bone marrow-derived MSCs rather than by the differentiation of MSCs into local tissue cell types [50]. Similarly for ASCs, it has been shown that the beneficial impact on different organs/tissues within the human body may be due to soluble factors produced by ASCs rather than their differentiation capability toward different mature lineages [51]. The ASCs secretome has the potential to be a powerful tool for use in future approaches to develop cell-/tissue-based therapeutics for regenerative medicine. A number of papers have described the secretory profiles of preadipocytes, ASCs, or adipose tissue, which were determined using enzyme-linked immunoabsorbent assays or related techniques [52, 53]. Analyses of the soluble factors released from human ASCs have revealed that cultured ASCs, at relatively early passages, secrete hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), transforming growth factor-b, insulin-like growth factor (IGF)-1, basic fibroblast growth factor (bFGF), granulocyte-macrophage colony-stimulating factor, tumor necrosis factor (TNF)-a, interleukin-6, 7, 8, and 11, adiponectin, angiotensin, cathepsin D, pentraxin, pregnancy zone protein, retinol-binding protein, and CXCL12 [52–54]. Moreover, it has also been shown that the soluble factors secreted by ASCs can be modulated by exposure to different agents [53, 54]; for example, HGF expression is increased af-
ter the cells have been exposed to bFGF, epidermal growth factor (EGF), or ascorbic acid. Thus, it may be that when ASCs are transplanted into inflammatory or ischemic regions, they actively secrete these growth factors, thereby significantly promoting wound healing and tissue repair.
PROLIFERATION CAPACITY
OF
ASCS
The proliferation capacity of ASCs seems to be greater than that of bone marrow-derived MSCs. Previous reports have shown that the doubling times of ASCs during the logarithmic phase of growth range from 40 to 120 hours [15, 55, 56] are influenced by donor age, type (white or brown adipose tissue) and location (subcutaneous or visceral) of the adipose tissue, the harvesting procedure, culture conditions, plating density and media formulations [35, 56]. The younger the donor, the greater the proliferation and cell adhesion of the ASCs, while cells gradually lose their proliferative capacity with passaging [56]. Based on b-galactosidase activity, senescence in ASCs is similar to that in bone marrow-derived MSCs [55]. ASCs are generally considered to be stable throughout long-term culture, as it was reported that even ASCs that had passed more than 100 population doublings had a normal diploid karyotype [57]. On the other hand, one report suggests that human ASCs undergo malignant transformation when passaged for more than 4 months [58]; although recent reports show that spontaneous transformation of MSCs may apparently be due to cross-contamination with malignant cell lines such as fibrosarcoma and osteosarcoma [59, 60]. Since the issue of spontaneous ASCs transformation is still controversial, further experiments and discussion are required, and
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Table 2. Clinical trials using stromal vascular fractions and adipose-derived stem cells Conditions
Design
Study type
Results
Coronary disease
Autologous SVFs
Phase I
Ongoing
Peripheral vascular diseases cardiovascular diseases Limb ischemia
Autologous SVFs
Phases I and II
Recruiting
ASCs-coated ePTTE vascular graft Autologous ASCs Autologous ASCs
Phases I and II
Recruiting
Phases I and II
Ref.
NCT00426868a NCT00442806a NCT01211028a
Autologous SVFs and ASCs Allogenic ASCs
Prospective Phase III Case report Case report Phases I and II
Liver cirrhosis Diabetes mellitus
Autologous adipose derived stromal cells Autologous ASCs
Safety study Phases I and II
Recruiting Completed Recruiting Completed Recruiting Terminated Completed Completed 24 patients Nine patients Recruiting Recruiting Completed Suspended Unknown
Lipodystrophy Buerger’s disease Spinal cord Injury Fecal incontinence Progressive hemifacial atrophy Romberg’s disease The repair of large osseous defects Degenerative arthritis Articular cartilage lesion of the femoral condyle Breast reconstruction Breast augmentation Soft tissue augmentation (craniofacial) Radiation ulcers Craniofacial bone reconstruction GVHD
Liposuction material enriched with ASCs Autologous ASCs Autologous ASCs Autologous cultured ASCs Autologous ASCs
Phase I Phases I and II Phase I Phase I Phase II
Ongoing Recruiting Completed Terminated Completed
NCT01305863a NCT01257776a NCT01314092a NCT00992485a NCT01011244a NCT00992485a NCT01011244a NCT01378390a NCT01020825a NCT00475410a [71] [72] NCT00999115a NCT01157650a NCT01372969a NCT00913289a NCT00703599a NCT00703612a NCT00715546a NCT01302015a NCT01274975a NCT01011686a NCT01309061a
ASCs with scaffolds Autologous ASCs Autologous cultured ASCs
Prospective Phases I and II Phases I and II
Recruiting Recruiting Recruiting
NCT01218945a NCT01300598a NCT01399749a
Autologous Autologous Autologous Autologous Autologous Autologous
Phase IV Case report Case report Case report Case report Case report
Completed 403 patients 58 patients 30 patients Two patients Two patients
NCT00616135a [73] [74] [75, 76] [77, 78] [46]
Fistula
SVFs adipose derived stromal cells adipose derived stromal cells SVFs cultured ASCs ASCs
The trial listed in this table correspond to the use of such fraction. a Identifier on clinical trials website: http://clinicaltrials.gov/ct2/results?term ¼ adiposeþderivedþcells.Abbreviations: ASCs, adipose derived stem cells; ePTTE, expanded polytetrafluoroethylene; GVHD, graft-versus-host disease; SVFs, stromal vascular fraction.
careful manipulation of ASCs and long-term observation of patients after clinical application are essential. The proliferation of ASCs can be stimulated by a single growth factor such as FGF-2, EGF, IGF-1, or TNF-a [61, 62]. FGF-2, in particular, is an effective growth-stimulating factor and is required for the long-term propagation and self-renewal of ASCs via the extracellular signal-related kinase (ERK) 1/2 signaling pathway [63]. The proliferation of ASCs can also be stimulated by PDGF via Jun amino-termanal kinase (JNK) activation [64] and by Oncostatin M via activation of the microtubule-associated protein kinase/ERK and the JAK3/STAT1 pathways [65]. ASCs proliferation is also reported to be enhanced by multiple growth factors, which can include any of the single growth factors mentioned above supplemented by thrombin-activated platelet-rich plasma [66], human platelet lysate [67], and human thrombin [68].
DIFFERENTIATION POTENTIAL OF ASCS TOWARD A VARIETY OF LINEAGES To date, there have been numerous scientific publications demonstrating that ASCs possess the potential to differentiate www.StemCells.com
toward a variety of cell lineages both in vitro and in vivo [69], and some of the research has been passed on to patients in the form of clinical trials (Table 2). Although ASCs are of mesodermal origin, it is now clear that they can differentiate into ectoderm and endoderm lineage cells as well as mesoderm [70]. Therefore, due to space limitations and the fact that there are several published reviews documenting the mesengenic potential of ASCs, this chapter highlights those reports describing the ectodermal and endodermal differentiation of ASCs. As far as the differentiation into cells of the mesodermal lineages and regeneration of mesodermal tissues are concerned, ASCs can differentiate into adipogenic [79–81], osteogenic [82], chondrogenic [82–84], myogenic [85], cardiomyogenic [86, 87], angiogenic [52, 88], tenogenic [89], and periodontogenic lineages [90], and tissue regeneration studies with suitable scaffolds and growth factors in appropriate external environment have been carried out [77, 78]. There have been a few studies describing the differentiation of ASCs toward cells of the ectodermal lineage; for example, one study reported that ASCs cultured in monolayers with retinoic acid or on a fibrin matrix in the presence of EGF express the epithelial markers, cytokeratin 18 or E-cadherin
808
and cytokeratin 8, respectively [91, 92]. In another study, ASCs exposed to vasoactive intestinal peptide were induced to form retinal pigmented cells, which are of ectodermal origin, as indicated by the expression of retinal pigment epithelium (RPE) markers, namely bestrophin, cytokeratins 8 and 18, and RPE 65 [93]. It has also been demonstrated that ASCs can differentiate into neuronal or neuronal precursor cells, both morphologically and functionally, under appropriate culture conditions [94]. Historically, neural tissue has been regarded as having poor regenerative capacity, but recent advances in the growing fields of tissue engineering and regenerative medicine have raised new hopes for the treatment of nerve injuries and neurodegenerative disorders. Although clinical trials have not yet been performed in the fields of neurology and neurosurgery, several interesting preclinical studies have been conducted, particularly for the treatment of brain stroke and spinal cord injury. Following a stroke due to brain ischemia or hemorrhage, the i.v. administration of ASCs resulted in both functional and histological improvement, as confirmed by a reduction in brain water content, brain atrophy, and glial proliferation in a hemorrhagic stroke model [95, 96]. In addition, recent studies have revealed that when ASCs are administered intravenously in a spinal cord injury model, they migrate to the injured cord, partially differentiate into neurons and oligodendrocytes, and eventually restore locomotor functions [97]. Finally, it has been shown that ASCs can differentiate into endoderm lineage cells. Several reports have shown that ASCs have the potential to differentiate into hepatocytes as indicated by the presence of HGF and FGF-1 and 4 [98, 99]. In theory, ASCs could be used to reduce liver inflammation and treat liver fibrosis by differentiating directly into hepatocytes or by secreting factors such as angiogenic, antiapoptotic, anti-inflammatory, and antifibrotic factors. In addition to hepatic differentiation, the exposure of ASCs to nicotinamide, activin-A, exendin-4, HGF, and pentagastrin resulted in the production of pancreatic-like ASCs capable of insulin, glucagons, and somatostatin secretion [100, 101].
OTHER THERAPEUTIC APPLICATIONS OF ASCS Wound healing is a complex process that involves the coordinated efforts of many types of cells and the cytokines they release. Recent breakthroughs in understanding the roles played by ASCs in wound healing and tissue regeneration have provided new options for treating difficult wounds. Preclinical studies in a murine model have shown that the topical administration of autologous ASCs, in conjunction with a type I collagen sponge matrix, into a diabetic animal accelerated the healing of diabetic ulcers [102]. In addition, a clinical study on the treatment of radiation-induced tissue damage using human ASCs showed progressive improvement in tissue hydration and new vessel formation [75, 76]. It has been speculated that the healing mechanism is due to the release of several growth factors, including VEGF and HGF, and to the subsequent angiogenesis and proliferation of keratinocytes or dermal fibroblasts. Furthermore, complex fistulas in patients suffering from Crohn’s and non-Crohn’s disease were healed following the direct injection of ASCs into the tract wall together with a fibrin glue sealant, and no adverse effects were observed [71, 72]. Free fat to the body is likely to be absorbed due to the lack of an adequate blood supply. The concept of cell-assisted
Adipose-Derived Stem Cells for Regenerative Medicine
lipotransfer, which involves ASC-enriched fat grafting by adding ASCs isolated from the other sources of adipose tissue, has been successfully used in the clinic for soft tissue augmentation in a number of patients [73, 74]. Another potential application of ASCs involves the harnessing of their immunomodulatory effects to treat allergic diseases, autoimmune diseases, and GVHD. In a rat allergic rhinitis model, for example, intravenously injected ASCs migrated to the surface of the nasal mucosa, inhibited eosinophilic inflammation, modulated T-cell activity, and eventually reduced the allergic symptoms [103]. At the immunoglobulin level, IgE and the ratio of IgG1/IgG2a, representing the Th2 immune response, were significantly decreased by the i.v. administration of ASCs, while IgG2a levels, which indicated a Th1 immune response, were significantly increased by ASCs. In addition to allergic rhinitis, collagen-induced arthritis in DBA/1 mice was also reduced by the systemic administration of ASCs through a similar mechanism involving the suppression of effector T cells [104]. It has also been shown that ASCs can ameliorate steroid-resistant severe GVHD after hematopoietic stem cell transplantation [46], and further studies are needed to examine the mechanism by which this occurs and to determine the long-term effects of such treatment.
FUTURE DIRECTIONS ASCs are classified as adult multipotent stem cells and, as such, their multipotency is limited compared with ESCs and iPSCs. From a practical standpoint, however, numerous investigators have examined the practicalities of using ASCs in overcoming the current limitations in the various fields of regenerative medicine. So far, relatively few clinical trials in a limited number of research areas have been conducted to assess the therapeutic potential of ASCs compared with the large number of published preclinical studies [105]. In addition, there are several points that remain unclear with regard to ASCs; first, there is evidence that the differentiation potential of ASCs may depend on the anatomic location of the fat and the donor’s gender and age [25]; second, the key molecular events and transcription factors that initially allocate the ASCs to a specific lineage are still unknown, although their lineage-specific differentiation into cells of mesodermal origin is well understood at the molecular level. Despite these limitations, ASCs have practical advantages in clinical medicine and their use has become more realistic [106] because adipose tissue, the primary source of ASCs, is abundant and easy to obtain with less donor site morbidity. Further preclinical and clinical studies are needed to determine whether ASC-based therapies can fulfill expectations and can be used successfully to treat disorders for which current medical and surgical therapies are either ineffective or impractical.
DISCLOSURE
OF OF
POTENTIAL CONFLICTS INTEREST
The authors indicate no potential conflicts of interest.
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REFERENCES
28 29
1 2 3 4 5
6 7 8 9 10
11 12 13
14 15 16 17 18 19 20 21 22 23 24 25 26
27
Lenoir N. Europe confronts the embryonic stem cell research challenge. Science 2000;287:1425–1427. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006;126:663–676. Ben-David U, Benvenisty N. The tumorigenicity of human embryonic and induced pluripotent stem cells. Nat Rev Cancer 2011;11: 268–277. Song L, Young NJ, Webb NE et al. Origin and characterization of multipotential mesenchymal stem cells derived from adult human trabecular bone. Stem Cells Dev 2005;14:712–721. Choi YS, Noh SE, Lim SM et al. Multipotency and growth characteristic of periosteum-derived progenitor cells for chondrogenic, osteogenic, and adipogenic differentiation. Biotechnol Lett 2008;30: 593–601. De Bari C, Dell’Accio F, Tylzanowski P et al. Multipotent mesenchymal stem cells from adult human synovial membrane. Arthritis Rheum 2001;44:1928–1942. Dodson MV, Hausman GJ, Guan L et al. Skeletal muscle stem cells from animals I. Basic cell biology. Int J Biol Sci 2010;6: 465–474. Belicchi M, Pisati F, Lopa R et al. Human skin-derived stem cells migrate throughout forebrain and differentiate into astrocytes after injection into adult mouse brain. J Neurosci Res 2004;77:475–486. Feng J, Mantesso A, Sharpe PT. Perivascular cells as mesenchymal stem cells. Expert Opin Biol Ther 2010;10:1441–1451. Shi M, Ishikawa M, Kamei N et al. Acceleration of skeletal muscle regeneration in a rat skeletal muscle injury model by local injection of human peripheral blood-derived cd133-positive cells. Stem Cells 2009;27:949–960. Miura M, Gronthos S, Zhao M et al. Shed: Stem cells from human exfoliated deciduous teeth. Proc Natl Acad Sci USA 2003;100: 5807–5812. Seo BM, Miura M, Gronthos S et al. Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet 2004; 364:149–155. Baksh D, Yao R, Tuan RS. Comparison of proliferative and multilineage differentiation potential of human mesenchymal stem cells derived from umbilical cord and bone marrow. Stem Cells 2007;25: 1384–1392. Musina RA, Bekchanova ES, Sukhikh GT. Comparison of mesenchymal stem cells obtained from different human tissues. Bull Exp Biol Med 2005;139:504–509. Zuk PA, Zhu M, Mizuno H et al. Multilineage cells from human adipose tissue: Implications for cell-based therapies. Tissue Eng 2001;7: 211–228. Gimble JM, Katz AJ, Bunnell BA. Adipose-derived stem cells for regenerative medicine. Circ Res 2007;100:1249–1260. Tobita M, Orbay H, Mizuno H. Adipose-derived stem cells: Current findings and future perspectives. Discov Med 2011;11:160–170. Yoshimura K, Suga H, Eto H. Adipose-derived stem/progenitor cells: Roles in adipose tissue remodeling and potential use for soft tissue augmentation. Regen Med 2009;4:265–273. Weisberg SP, McCann D, Desai M et al. Obesity is associated with macrophage accumulation in adipose tissue. J Clin Invest 2003;112: 1796–1808. Xu H, Barnes GT, Yang Q et al. Chronic inflammation in fat plays a crucial role in the development of obesity-related insulin resistance. J Clin Invest 2003;112:1821–1830. Schipper BM, Marra KG, Zhang W et al. Regional anatomic and age effects on cell function of human adipose-derived stem cells. Ann Plast Surg 2008;60:538–544. Prunet-Marcassus B, Cousin B, Caton D et al. From heterogeneity to plasticity in adipose tissues: Site-specific differences. Exp Cell Res 2006;312:727–736. Seale P, Bjork B, Yang W et al. Prdm16 controls a brown fat/skeletal muscle switch. Nature 2008;454:961–967. Zimmerlin L, Donnenberg VS, Pfeifer ME et al. Stromal vascular progenitors in adult human adipose tissue. Cytometry A 2010;77: 22–30. Bailey AM, Kapur S, Katz AJ. Characterization of adipose-derived stem cells: An update. Curr Stem Cell Res Ther 2010;5:95–102. Basciano L, Nemos C, Foliguet B et al. Long term culture of mesenchymal stem cells in hypoxia promotes a genetic program maintaining their undifferentiated and multipotent status. BMC Cell Biol 2011;12:12. Bernardo ME, Zaffaroni N, Novara F et al. Human bone marrow derived mesenchymal stem cells do not undergo transformation after long-term in vitro culture and do not exhibit telomere maintenance mechanisms. Cancer Res 2007;67:9142–9149.
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45
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Deans RJ, Moseley AB. Mesenchymal stem cells: Biology and potential clinical uses. Exp Hematol 2000;28:875–884. Folgiero V, Migliano E, Tedesco M et al. Purification and characterization of adipose-derived stem cells from patients with lipoaspirate transplant. Cell Transplant 2010;19:1225–1235. Freitas CS, Dalmau SR. Multiple sources of non-embryonic multipotent stem cells: Processed lipoaspirates and dermis as promising alternatives to bone-marrow-derived cell therapies. Cell Tissue Res 2006;325:403–411. Gronthos S, Franklin DM, Leddy HA et al. Surface protein characterization of human adipose tissue-derived stromal cells. J Cell Physiol 2001;189:54–63. Jones EA, English A, Kinsey SE et al. Optimization of a flow cytometry-based protocol for detection and phenotypic characterization of multipotent mesenchymal stromal cells from human bone marrow. Cytometry B Clin Cytom 2006;70:391–399. Katz AJ, Tholpady A, Tholpady SS et al. Cell surface and transcriptional characterization of human adipose-derived adherent stromal (hadas) cells. Stem Cells 2005;23:412–423. Meyerrose TE, De Ugarte DA, Hofling AA et al. In vivo distribution of human adipose-derived mesenchymal stem cells in novel xenotransplantation models. Stem Cells 2007;25:220–227. Mitchell JB, McIntosh K, Zvonic S et al. Immunophenotype of human adipose-derived cells: Temporal changes in stromal-associated and stem cell-associated markers. Stem Cells 2006;24:376–385. Yoshimura K, Shigeura T, Matsumoto D et al. Characterization of freshly isolated and cultured cells derived from the fatty and fluid portions of liposuction aspirates. J Cell Physiol 2006;208:64–76. Alipour R, Sadeghi F, Hashemi-Beni B et al. Phenotypic characterizations and comparison of adult dental stem cells with adiposederived stem cells. Int J Prev Med 2010;1:164–171. Delorme B, Ringe J, Gallay N et al. Specific plasma membrane protein phenotype of culture-amplified and native human bone marrow mesenchymal stem cells. Blood 2008;111:2631–2635. Kim YJ, Yu JM, Joo HJ et al. Role of cd9 in proliferation and proangiogenic action of human adipose-derived mesenchymal stem cells. Pflugers Arch 2007;455:283–296. Wagner W, Wein F, Seckinger A et al. Comparative characteristics of mesenchymal stem cells from human bone marrow, adipose tissue, and umbilical cord blood. Exp Hematol 2005;33:1402–1416. Suga H, Matsumoto D, Eto H et al. Functional implications of cd34 expression in human adipose-derived stem/progenitor cells. Stem Cells Dev 2009;18:1201–1210. Tang W, Zeve D, Suh JM et al. White fat progenitor cells reside in the adipose vasculature. Science 2008;322:583–586. Traktuev DO, Merfeld-Clauss S, Li J et al. A population of multipotent cd34-positive adipose stromal cells share pericyte and mesenchymal surface markers, reside in a periendothelial location, and stabilize endothelial networks. Circ Res 2008;102:77–85. Jeon BG, Kumar BM, Kang EJ et al. Characterization and comparison of telomere length, telomerase and reverse transcriptase activity and gene expression in human mesenchymal stem cells and cancer cells of various origins. Cell Tissue Res 2011;345:149–161. Puissant B, Barreau C, Bourin P et al. Immunomodulatory effect of human adipose tissue-derived adult stem cells: Comparison with bone marrow mesenchymal stem cells. Br J Haematol 2005;129: 118–129. Fang B, Song Y, Liao L et al. Favorable response to human adipose tissue-derived mesenchymal stem cells in steroid-refractory acute graft-versus-host disease. Transplant Proc 2007;39:3358–3362. Boquest AC, Shahdadfar A, Brinchmann JE et al. Isolation of stromal stem cells from human adipose tissue. Methods Mol Biol 2006; 325:35–46. Strem BM, Hicok KC, Zhu M et al. Multipotential differentiation of adipose tissue-derived stem cells. Keio J Med 2005;54:132–141. Sterodimas A, de Faria J, Nicaretta B et al. Tissue engineering with adipose-derived stem cells (adscs): Current and future applications. J Plast Reconstr Aesthet Surg 2010;63:1886–1892. Van Poll D, Parekkadan B, Borel Rinkes I et al. Mesenchymal stem cell therapy for protection and repair of injured vital organs. Cell Mol Bioeng 2008;1:42–50. Kim WS, Park BS, Sung JH. Protective role of adipose-derived stem cells and their soluble factors in photoaging. Arch Dermatol Res 2009;301:329–336. Rehman J, Traktuev D, Li J et al. Secretion of angiogenic and antiapoptotic factors by human adipose stromal cells. Circulation 2004; 109:1292–1298. Salgado AJ, Reis RL, Sousa NJ et al. Adipose tissue derived stem cells secretome: Soluble factors and their roles in regenerative medicine. Curr Stem Cell Res Ther 2010;5:103–110. Kilroy GE, Foster SJ, Wu X et al. Cytokine profile of human adipose-derived stem cells: Expression of angiogenic, hematopoietic, and pro-inflammatory factors. J Cell Physiol 2007;212:702–709.
Adipose-Derived Stem Cells for Regenerative Medicine
810
55 56 57 58 59 60
61
62
63 64
65 66 67
68 69 70 71 72
73 74 75
76 77 78 79
De Ugarte DA, Morizono K, Elbarbary A et al. Comparison of multi-lineage cells from human adipose tissue and bone marrow. Cells Tissues Organs 2003;174:101–109. Izadpanah R, Trygg C, Patel B et al. Biologic properties of mesenchymal stem cells derived from bone marrow and adipose tissue. J Cell Biochem 2006;99:1285–1297. Rodriguez AM, Elabd C, Delteil F et al. Adipocyte differentiation of multipotent cells established from human adipose tissue. Biochem Biophys Res Commun 2004;315:255–263. Rubio D, Garcia-Castro J, Martin MC et al. Spontaneous human adult stem cell transformation. Cancer Res 2005;65:3035–3039. Garcia S, Bernad A, Martin MC et al. Pitfalls in spontaneous in vitro transformation of human mesenchymal stem cells. Exp Cell Res 2010;316:1648–1650. Torsvik A, Rosland GV, Svendsen A et al. Spontaneous malignant transformation of human mesenchymal stem cells reflects cross-contamination: Putting the research field on track—letter. Cancer Res 2010;70:6393–6396. Hebert TL, Wu X, Yu G et al. Culture effects of epidermal growth factor (egf) and basic fibroblast growth factor (bfgf) on cryopreserved human adipose-derived stromal/stem cell proliferation and adipogenesis. J Tissue Eng Regen Med 2009;3:553–561. Kras KM, Hausman DB, Martin RJ. Tumor necrosis factor-alpha stimulates cell proliferation in adipose tissue-derived stromal-vascular cell culture: Promotion of adipose tissue expansion by paracrine growth factors. Obes Res 2000;8:186–193. Zaragosi LE, Ailhaud G, Dani C. Autocrine fibroblast growth factor 2 signaling is critical for self-renewal of human multipotent adiposederived stem cells. Stem Cells 2006;24:2412–2419. Kang YJ, Jeon ES, Song HY et al. Role of c-jun N-terminal kinase in the pdgf-induced proliferation and migration of human adipose tissue-derived mesenchymal stem cells. J Cell Biochem 2005;95: 1135–1145. Song HY, Jeon ES, Jung JS et al. Oncostatin m induces proliferation of human adipose tissue-derived mesenchymal stem cells. Int J Biochem Cell Biol 2005;37:2357–2365. Kakudo N, Minakata T, Mitsui T et al. Proliferation-promoting effect of platelet-rich plasma on human adipose-derived stem cells and human dermal fibroblasts. Plast Reconstr Surg 2008;122:1352–1360. Blande IS, Bassaneze V, Lavini-Ramos C et al. Adipose tissue mesenchymal stem cell expansion in animal serum-free medium supplemented with autologous human platelet lysate. Transfusion 2009;49: 2680–2685. Freyberg S, Song YH, Muehlberg F et al. Thrombin peptide (tp508) promotes adipose tissue-derived stem cell proliferation via pi3 kinase/akt pathway. J Vasc Res 2009;46:98–102. Mizuno H. Adipose-derived stem and stromal cells for cell-based therapy: Current status of preclinical studies and clinical trials. Curr Opin Mol Ther 2010;12:442–449. Zuk PA. The adipose-derived stem cell: Looking back and looking ahead. Mol Biol Cell 2010;21:1783–1787. Garcia-Olmo D, Herreros D, Pascual I et al. Expanded adiposederived stem cells for the treatment of complex perianal fistula: A phase ii clinical trial. Dis Colon Rectum 2009;52:79–86. Garcia-Olmo D, Herreros D, Pascual M et al. Treatment of enterocutaneous fistula in Crohn’s disease with adipose-derived stem cells: A comparison of protocols with and without cell expansion. Int J Colorectal Dis 2009;24:27–30. Yoshimura K, Sato K, Aoi N et al. Cell-assisted lipotransfer for cosmetic breast augmentation: Supportive use of adipose-derived stem/ stromal cells. Aesthetic Plast Surg 2008;32:48–55; discussion 56–47. Yoshimura K, Sato K, Aoi N et al. Cell-assisted lipotransfer for facial lipoatrophy: Efficacy of clinical use of adipose-derived stem cells. Dermatol Surg 2008;34:1178–1185. Rigotti G, Marchi A, Galie M et al. Clinical treatment of radiotherapy tissue damage by lipoaspirate transplant: A healing process mediated by adipose-derived adult stem cells. Plast Reconstr Surg 2007;119:1409–1422; discussion 1423–1404. Akita S, Akino K, Hirano A et al. Mesenchymal stem cell therapy for cutaneous radiation syndrome. Health Phys 2010;98:858–862. Mesimaki K, Lindroos B, Tornwall J et al. Novel maxillary reconstruction with ectopic bone formation by gmp adipose stem cells. Int J Oral Maxillofac Surg 2009;38:201–209. Lendeckel S, Jodicke A, Christophis P et al. Autologous stem cells (adipose) and fibrin glue used to treat widespread traumatic calvarial defects: Case report. J Craniomaxillofac Surg 2004;32:370–373. Brayfield CA, Marra KG, Rubin JP. Adipose tissue regeneration. Curr Stem Cell Res Ther 2010;5:116–121.
80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101
102
103 104 105 106
Cherubino M, Marra KG. Adipose-derived stem cells for soft tissue reconstruction. Regen Med 2009;4:109–117. Rubin JP, Marra KG. Soft tissue reconstruction. Methods Mol Biol 2011;702:395–400. Dragoo JL, Samimi B, Zhu M et al. Tissue-engineered cartilage and bone using stem cells from human infrapatellar fat pads. J Bone Joint Surg Br 2003;85:740–747. Estes BT, Diekman BO, Gimble JM et al. Isolation of adiposederived stem cells and their induction to a chondrogenic phenotype. Nat Protoc 2010;5:1294–1311. Estes BT, Guilak F. Three-dimensional culture systems to induce chondrogenesis of adipose-derived stem cells. Methods Mol Biol 2011;702:201–217. Mizuno H, Zuk PA, Zhu M et al. Myogenic differentiation by human processed lipoaspirate cells. Plast Reconstr Surg 2002;109:199–209; discussion 210–191. Lee WC, Sepulveda JL, Rubin JP et al. Cardiomyogenic differentiation potential of human adipose precursor cells. Int J Cardiol 2009; 133:399–401. Planat-Benard V, Menard C, Andre M et al. Spontaneous cardiomyocyte differentiation from adipose tissue stroma cells. Circ Res 2004; 94:223–229. Cherubino M, Rubin JP, Miljkovic N et al. Adipose-derived stem cells for wound healing applications. Ann Plast Surg 2011;66:210–215. Uysal AC, Mizuno H. Tendon regeneration and repair with adipose derived stem cells. Curr Stem Cell Res Ther 2010;5:161–167. Tobita M, Mizuno H. Periodontal disease and periodontal tissue regeneration. Curr Stem Cell Res Ther 2010;5:168–174. Brzoska M, Geiger H, Gauer S et al. Epithelial differentiation of human adipose tissue-derived adult stem cells. Biochem Biophys Res Commun 2005;330:142–150. Long JL, Zuk P, Berke GS et al. Epithelial differentiation of adipose-derived stem cells for laryngeal tissue engineering. Laryngoscope 2010;120:125–131. Vossmerbaeumer U, Ohnesorge S, Kuehl S et al. Retinal pigment epithelial phenotype induced in human adipose tissue-derived mesenchymal stromal cells. Cytotherapy 2009;11:177–188. Safford KM, Safford SD, Gimble JM et al. Characterization of neuronal/glial differentiation of murine adipose-derived adult stromal cells. Exp Neurol 2004;187:319–328. Ikegame Y, Yamashita K, Hayashi S et al. Comparison of mesenchymal stem cells from adipose tissue and bone marrow for ischemic stroke therapy. Cytotherapy 2011;13:675–685. Kim JM, Lee ST, Chu K et al. Systemic transplantation of human adipose stem cells attenuated cerebral inflammation and degeneration in a hemorrhagic stroke model. Brain Res 2007;1183:43–50. Kang SK, Shin MJ, Jung JS et al. Autologous adipose tissue-derived stromal cells for treatment of spinal cord injury. Stem Cells Dev 2006;15:583–594. Aurich H, Sgodda M, Kaltwasser P et al. Hepatocyte differentiation of mesenchymal stem cells from human adipose tissue in vitro promotes hepatic integration in vivo. Gut 2009;58:570–581. Banas A, Teratani T, Yamamoto Y et al. Rapid hepatic fate specification of adipose-derived stem cells and their therapeutic potential for liver failure. J Gastroenterol Hepatol 2009;24:70–77. Chandra V, Swetha G, Phadnis S et al. Generation of pancreatic hormone-expressing islet-like cell aggregates from murine adipose tissue-derived stem cells. Stem Cells 2009;27:1941–1953. Timper K, Seboek D, Eberhardt M et al. Human adipose tissuederived mesenchymal stem cells differentiate into insulin, somatostatin, and glucagon expressing cells. Biochem Biophys Res Commun 2006;341:1135–1140. Nambu M, Kishimoto S, Nakamura S et al. Accelerated wound healing in healing-impaired db/db mice by autologous adipose tissuederived stromal cells combined with atelocollagen matrix. Ann Plast Surg 2009;62:317–321. Cho KS, Roh HJ. Immunomodulatory effects of adipose-derived stem cells in airway allergic diseases. Curr Stem Cell Res Ther 2010;5:111–115. Gonzalez MA, Gonzalez-Rey E, Rico L et al. Treatment of experimental arthritis by inducing immune tolerance with human adipose-derived mesenchymal stem cells. Arthritis Rheum 2009;60:1006–1019. Gimble JM, Guilak F, Bunnell BA. Clinical and preclinical translation of cell-based therapies using adipose tissue-derived cells. Stem Cell Res Ther 2010;1:19. Gimble JM, Bunnell BA, Chiu ES et al. Concise review: Adiposederived stromal vascular fraction cells and stem cells: Let’s not get lost in translation. Stem Cells 2011;29:749–754.
