Concise Review: Adipose-Derived Stem Cells as a Novel Tool for Future Regenerative Medicine†‡§
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.
Disclosure of potential conflicts of interest is found at the end of this article.
First published online in STEM CELLSEXPRESS March 13, 2012; available online without subscription thorugh the open access option.
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
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 . 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 . 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 marrow-derived MSCs, have been isolated from different tissue sources including trabecular bone , periosteum , synovial membrane , skeletal muscle , skin , pericytes , peripheral blood , deciduous teeth , periodontal ligament , and umbilical cord [13, 14]. Although the stem cell population derived from these sources are valuable, 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) , 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 . 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 . 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 . 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 .
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−, CD34+/−, CD45−, CD90+, CD105−, and CD146−), endothelial (progenitor) cells (CD31+, CD34+, CD45−, CD90+, CD105−, and CD146+), vascular smooth muscle cells or pericytes (CD31−, CD34+/−, CD45−, CD90+, CD105−, and CD146+), and hematopoietic cells (CD45+) in uncultured conditions . Additionally, compared with ASCs from later passages, freshly isolated SVF cells and early passage ASCs 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 (β1 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 . Furthermore, several studies have shown that CD34+ 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].
Table 1. Characterization of freshly isolated human stromal vascular fractions from adipose tissue and mesenchymal stem cells
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 β-actin, platelet-derived growth factor (PDGF) receptor-β, and neuro-glial proteoglycan 2 , 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 . 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 . Finally, Puissant et al. . 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 .
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 . 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 .
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 .
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 . 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 . 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-β, insulin-like growth factor (IGF)-1, basic fibroblast growth factor (bFGF), granulocyte-macrophage colony-stimulating factor, tumor necrosis factor (TNF)-α, 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 after 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 . Based on β-galactosidase activity, senescence in ASCs is similar to that in bone marrow-derived MSCs .
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 . On the other hand, one report suggests that human ASCs undergo malignant transformation when passaged for more than 4 months ; 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 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-α [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 . The proliferation of ASCs can also be stimulated by PDGF via Jun amino-termanal kinase (JNK) activation  and by Oncostatin M via activation of the microtubule-associated protein kinase/ERK and the JAK3/STAT1 pathways . 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 , human platelet lysate , and human thrombin .
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 toward a variety of cell lineages both in vitro and in vivo , 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 . 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.
Table 2. Clinical trials using stromal vascular fractions and adipose-derived stem cells
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 , chondrogenic [82–84], myogenic , cardiomyogenic [86, 87], angiogenic [52, 88], tenogenic , and periodontogenic lineages , 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 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 .
It has also been demonstrated that ASCs can differentiate into neuronal or neuronal precursor cells, both morphologically and functionally, under appropriate culture conditions . 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 .
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 . 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 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 . 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 . It has also been shown that ASCs can ameliorate steroid-resistant severe GVHD after hematopoietic stem cell transplantation , and further studies are needed to examine the mechanism by which this occurs and to determine the long-term effects of such treatment.
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 . 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 ; 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  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 POTENTIAL CONFLICTS OF INTEREST
The authors indicate no potential conflicts of interest.