Disclosure of potential conflicts of interest is found at the end of this article.
Author contributions: M.L.: concept and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript; V.F.: collection and/or assembly of data, data analysis and interpretation, manuscript writing; P.R.D.: concept and design, data analysis and interpretation, final approval of manuscript.
First published online in STEM CELLSEXPRESS January 7, 2011.
Human adipose-derived stem cells (ASCs) have become an increasing interest to both stem cell biologists and clinicians because of their potential to differentiate into adipogenic, osteogenic, chondrogenic, and other mesenchymal lineages, as well as other clinically useful properties attributed to them, such as stimulation of angiogenesis and suppression of inflammation. ASCs have already been used in a number of clinical trials, and some successful outcomes have been reported, especially in tissue reconstruction. However, a critical review of the literature reveals considerable uncertainty about the true clinical potential of human ASC. First, the surgical needs that ASC might answer remain relatively few, given the current difficulties in scaling up ASC-based tissue engineering to a clinically useful volume. Second, the differentiation of ASC into cell lineages apart from adipocytes has not been conclusively demonstrated in many studies due to the use of rather simplistic approaches to the confirmation of differentiation, such as the use of nonspecific histological dyes, or a small number of molecular markers of uncertain significance. Third, the ASC prepared from human lipoaspirate for different studies differ in purity and molecular phenotype, with many studies using cell preparations that are likely to contain heterogeneous populations of cells, making it uncertain whether ASC themselves are responsible for effects observed. Hence, while one clinical application already looks convincing, the full clinical potential of ASC awaits much deeper investigation of their fundamental biology. STEM CELLS 2011,29:404–411
Multipotent mesenchymal stem cells (MSCs) can be isolated from various tissue sources in adults. As shown in Figure 1, adipose-derived stem cells (ASCs) can be derived from human adipose tissue, which can be harvested by direct excision or more commonly from lipoaspirate, the discarded tissue following liposuction surgery. The tissue is washed and red blood cells removed. Digestion with collagenase is performed and the tissue is centrifuged to obtain the cell pellet, known as the stromal vascular fraction (SVF). Research over the past decade has shown the SVF of human adipose tissue to include multipotent MSCs as well as endothelial cells [1, 2], mirroring earlier observations in mice . The MSC within adipose tissue have the potential to form bone, cartilage, and muscle, as well as fat [1, 2, 4, 5] (Fig. 1). These stem cells have been variously termed preadipocytes, stromal cells, processed lipoaspirate cells, multipotent adipose-derived stem cells, and adipose-derived adult stem cells. However, at a consensus conference of the International Fat Applied Technology Society, the term “adipose-derived stem cells” was recommended for consistency between research groups .
Table 1. Summary of data from reviewed papers
The ability of ASC to differentiate down different mesenchymal lineages has led to interest in their clinical use . Indeed, as described below and summarized in Table 1, a considerable number of clinical trials using ASC have already been reported, in a very diverse range of contexts. However, two questions hang over many of these clinical applications. The first is whether the use of ASC is likely to be practical and effective in each of these clinical contexts, especially for applications such as reconstructive surgery that require relatively high volumes of tissue. The second question is whether current knowledge of the biology of ASC is sufficient to justify these clinical applications, especially given the differences in the quality of the available evidence that ASC can be driven to differentiate into each of the relevant cell types. Below we review the published evidence regarding differentiation of human ASC into common mesenchymal lineages and reflect on the strength of support for the various clinical applications of ASC that have been proposed to date.
DEFINITION OF ASC
The molecular phenotype of ASC has only recently been clarified [8, 9], following many years of referring to any cultured SVF cells as ASC. Freshly isolated SVF contains a mixture of cells, which not only includes ASC but also contains endothelial cells, smooth muscle cells, pericytes, fibroblasts, and circulating cell types such as leukocytes, hematopoietic stem cells, or endothelial progenitor cells . One of the most significant issues limiting the interpretation of results and clinical progression of ASC research is the lack of standardization between research groups in defining what they mean by ASC. Some authors use the entire unpurified SVF in their experiments, and this is also common practice in clinical trials. However, it has been suggested that even a low fraction of contaminating cells such as hematopoietic stem cells could account for some of the differentiated cell populations seen in some ASC experiments . For work that makes claims about ASC properties, rather than those of an admitted mixture such as SVF, it seems reasonable to advocate for purification of the ASC from the SVF. ASC purification has most often been performed by prolonged culture of SVF, relying on the ability of ASC to outcompete other cell populations under the culture conditions over time. These culture protocols have usually started with a 24-hour adhesion period before washing away nonadherent cells; more recent experimentation has demonstrated that forceful washing after 60 minutes results in a significant increase in the proportion of the adherent cells expressing stem cell markers . Alternatively, cell sorting based on cell surface markers expressed by ASC may allow purification of cell subpopulations which have higher chondrogenic or osteogenic potential . Regardless of purification method, techniques such as flow cytometry can be used to confirm the homogeneity of the cells used in subsequent experiments and applications. Standardization of ASC characterization would allow direct comparison of results across all research groups and better definition of the clinical potential for ASC versus any of the other cell populations present in SVF.
USE OF ASC TO GENERATE ADIPOSE TISSUE
This is an area of strong clinical demand in reconstructive and cosmetic surgery. The first use of fat grafts for visible defects was by Neuber, reported in 1893 . Since this time, surgeons have been conducting autologous fat grafts by traditional methods of suction-assisted adipocyte harvest, table top processing in the operating room and reinjection of the prepared fat. However, the longevity of these grafts is still somewhat unpredictable, as reabsorption occurs in most grafts to a variable degree, presumably due to poor revascularization and therefore subsequent death of the fully differentiated adipocytes originally injected. The discovery of ASC has led to great interest in using these cells to provide a regenerating source of adipocytes for fat grafting.
Unsurprisingly there is good evidence of the ability of ASC to differentiate into mature adipocytes, when exposed to a medium containing steroids (to promote terminal differentiation), a cyclic AMP inducer, and fatty acids [2, 5]. Confirmation of differentiation into adipocytes has usually been performed by staining with oil red O or Nile red. These dyes stain cytoplasmic lipid droplets red, specifically labeling cells performing a defining function of adipocytes. Human adult fibroblasts do not possess this capacity (our own unpublished observations), in contrast to the well-described ability of the partially transformed murine fibroblast line 3T3 to differentiate into adipocytes (“3T3-L1”).
Expression of adipocyte-specific genes has also been used to demonstrate ASC differentiation into adipocytes. The genes lipoprotein lipase (LPL) and fatty acid binding protein 4 (FABP4, also known as αFABP) are well-established markers that are required for fatty acid metabolism [15, 16]. Both of these genes represent effector molecules for a change in cell function to lipid accumulation. In our hands, FABP4 expression has proven a reliable and sensitive indicator of adipocyte differentiation, being detected at very low levels by real time quantitative polymerase chain reaction (RT-qPCR) in undifferentiated ASC or human fibroblasts but strongly expressed within 14 days of exposure to adipocyte induction medium (data not published).
As the differentiation of ASC into adipocytes is not in any doubt, progression of related clinical treatments and trials is further advanced than for other differentiation lineages. Many clinical trials aim to improve the results of fat grafting for contour deformities or breast augmentation. Some current research is focused on a strategy known as cell-assisted lipotransfer, in which the SVF is isolated from half of the aspirated fat and recombined with the other half prior to reinjection. This is said to improve the take of larger volume reinjections, such as for breast augmentation or treatment of contour deformities [17, 18]. Automated systems for the processing of harvested fat and enrichment of subsequent fat grafts with the obtained ASC have already been manufactured and are in clinical use in Europe and Asia. One such company has recently completed enrolment of a 70 patient clinical trial in Europe to assess safety, graft volume retention and satisfaction with the procedure , although full results are not due for publication until 2011. Enrichment of autologous fat grafts with additional autologous ASC could potentially improve the long-term “take” of the fat graft, but the trade off is the longer operative time to complete the SVF isolation prior to grafting and additional costs involved.
Other relevant clinical trials of ASC in fat grafts include their use in the treatment of chest wall radiation necrosis and for the production of larger constructs to reconstruct mastectomy or congenital defects [20–22]. The former trial  showed excellent clinical outcomes with injection of fat grafts into the chest wall but lacked any controls to prove that these results were mediated by ASC. The latter trial , using a device called Neopec to provide a vascularized environment to encourage growth of a large volume of adipose tissue, is due to start recruiting women in 2010. Clearly, fat grafting is already a practical application of ASC for which there is strong clinical demand. However, it is interesting to note that in this field, speed of preparation is of the essence for optimal surgical practice, so coarse preparations from lipoaspirate such as SVF are acceptable, because they are quick to prepare in theater, and will still provide ASC into the graft, even though they are also likely to contain other cells. Hence, for some surgical applications, purity of ASC may be sacrificed in favor of surgical convenience.
USE OF ASC TO GENERATE BONE
There is still a strong clinical need to generate bone for the repair of large osseous defects. Traditionally, nonvascularized bone grafts are considered suitable for the repair of bone defects smaller than 6 cm, whereas vascularized bone grafts are needed to reliably heal larger areas of bone loss . The main clinical needs for engineered bone are therefore to either facilitate bony union with nonvascularized bone grafts in larger defects or to engineer bone of specific three-dimensional (3D) shapes for reconstructive purposes. Although some authors have imagined being able to engineer bone on a scale above cubic centimeter for implantable repair, this is likely to come at significant costs of both time and expense which may not be tolerable in the practical world of modern surgery. As an alternative, some researchers believe that progenitor cells might be implanted with biomaterials to encourage differentiation in vivo , so called, “facilitated endogenous repair,” to avoid the necessity for extended ex vivo culturing of ASCs. ASC differentiation within 3D scaffolds might also enable functional testing such as load bearing and stress testing to provide clinically useful demonstrations of functional bone. Combined with immunohistochemical (IHC) analysis of bone-specific protein markers in comparison with human bone tissue, this is potentially the most conclusive way to demonstrate genuine bone can be generated from human ASC.
Demonstration of ASC differentiation into an osteogenic lineage is more complex than adipogenic differentiation. The simplest method reported is staining for calcified extracellular matrix components. Alizarin red, historically an important dye once used to produce British soldiers' “Redcoats,” is now commonly being used to evaluate the presence of calcium-rich deposits produced by cells in culture, along with naphthol fast blue or similar to stain for alkaline phosphatase (ALP) enzyme activity . However, these methods do not specifically demonstrate differentiation of ASC into an osteogenic lineage. Simple tissue culture manipulations such as adding calcium to normal ASC growth medium has been shown to produce a positive result for Alizarin red staining in as little as 7 days . In our hands, the same treatment causes positive Alizarin red staining in human dermal fibroblasts, confirming this stain's lack of specificity for the osteogenic lineage.
Gene or protein expression profiles specific for osteoblasts are also lacking. Gene markers commonly assessed are runt-related transcription factor 2 (RUNX2) , transcriptional co-activator with PDZ-binding motif (TAZ or WWTR1) , osteocalcin (BGLAP), osteopontin (SPP1), type I collagen, and ALP . However, generally these have not been presented in comparison with expression levels in human bone, so data are often hard to interpret. Collagen I and ALP are clearly not specific for bone, and in our hands, RUNX2 is expressed in negative control cell lines such as human dermal fibroblasts. Some of the most comprehensive analysis of osteogenic differentiation of human ASC published to date included assay of ALP activity, Alizarin red staining, and analysis of mRNA expression for markers including SPP1 and BGLAP [25, 29]. The combination of bone-associated markers and the lack of reliance on nonspecific histological stains is reassuring and it seems likely that osteogenic differentiation was induced by the protocol reported, which included vitamin D3, β-glycerophosphate, and ascorbate-2-phosphate.
Clinical trials of osteogenesis in ASC followed murine studies, which appear promising for bone formation [30, 31]. However, the first human case study of ASC osteogenesis involved transplantation of both the entire SVF cell pellet (not purified ASC) and traditional bone graft simultaneously . This limited the conclusions that could be drawn regarding the source of the osteoneogenesis in this case. Subsequently, tissue engineering strategies have been employed in Finland to create a neomaxilla for a 65-year-old man who underwent maxillectomy . This reconstruction involved three operations over a 9-month period, using a β-tricalcium phosphate-filled titanium scaffold and cultured autologous ASC for osteogenesis. Bone growth in this case was sufficient to subsequently allow osseointegration of dental implants. However, the paucity of clinical trials, or even case reports, of ASC-induced osteogenesis speaks to the challenges of 3D bone generation in terms of time, cost, and labor intensity. Clearly, osteogenesis is possible, but producing clinically relevant volumes and strengths of bone in the in vitro environment is likely to remain challenging.
USE OF ASC TO GENERATE CARTILAGE
There is considerable hope that ASC might be used to generate cartilage for clinical use in the treatment of degenerative joints. Osteoarthritis (OA) is the most common form of joint disease and is increasing in prevalence and cost to society. A recent study from Norway showed an average incidence rate of 12.8%, being greater in women and older people . The rates of hip and knee joint replacements have been increasing in recent years and are predicted to continue to rise rapidly . Treatment with cartilage regeneration rather than joint replacement could potentially have major societal and economic benefits. An important clinical difference with bone is that relatively small pieces of autologous cartilage may be useful in joint repair, as native cartilage is poorly vascularized and even small defects do not heal well; in contrast, there is limited clinical demand for small pieces of autologous bone.
Overall, demonstration of chondrogenic differentiation of ASC faces the same problems as osteogenic differentiation. The most basic method used to demonstrate ASC differentiation into a chondrogenic lineage is staining for increased expression of proteoglycans using Alcian Blue or Safranin-O . In a similar manner to calcified extracellular matrix staining of osteogenic differentiated ASC, this type of positive staining does not demonstrate differentiation of ASC into cells capable of forming cartilage tissue, it simply shows that the cells are increasing expression of proteoglycans.
Genes commonly used for confirmation of chondrogenic differentiation analysis include type I collagen (COL1A1), type II collagen (COL2A1), type X collagen (COL10A1), cartilage oligomeric matrix protein , and aggrecan core protein. Type I collagen is involved in fibril formation in bone, tendons, and ligaments and therefore, is clearly not a specific marker for either bone or cartilage. Similarly, although highly expressed in cartilage tissue, many collagen-based markers can also be found in tissues other than cartilage, particularly, bone and sometimes skeletal muscle, emphasizing the importance of quantitative comparisons between different mesenchymal tissues.
Confirmation of chondrogenic differentiation would ideally include mRNA analysis of differentiated cells for expression of cartilage-specific transcripts and analysis of the extracellular matrix produced by differentiated cells for cartilage-specific proteins. Most of these methods are well described in a recent publication by Estes et al. . One of the most comprehensive analyses of human ASC chondrogenic differentiation was recently published by Kim and coworkers . They analyzed their samples with Safranin-O to confirm increased expression of proteoglycans. mRNA expression of COL1A1, COL2A1, COL10A1, RUNX2, and SOX9 was analyzed by RT-qPCR. Protein expression of RUNX2, type I, type II, and type X collagen was analyzed by IHC. Most importantly, for protein expression analysis, they included articular cartilage as a positive control. Based on their results, type II and type X collagen appear to be useful markers for chondrogenic differentiation.
Differentiation of ASC to functional cartilage that withstands mechanical load and stress as well as native cartilage does would be extremely valuable clinically. Similar to bone, 3D culture of human ASC differentiated into chondrocytes would allow the physical characteristics of the putative cartilage to be tested. To date, there is little evidence of successful 3D cartilage generation in vitro beyond techniques that generate clusters of a few thousand cells. Indeed, when ASCs were cultured as spheroids, they showed even less evidence of generating cartilage than bone marrow mesenchymal stem cells . Evidence is also lacking for cartilage formation by ASC introduced in vivo. Nathan and coworkers looked at the ability of ASC to reconstruct a femoral condyle defect in New Zealand white rabbits . They found that cartilage repair by ASC was more effective than the results from replacement of the osteochondral plug by 24 weeks; nevertheless, the results fell short of intact cartilage at all time points . ASCs have recently been used for the treatment of OA in dogs  and rheumatoid arthritis in humans [43, 44]. The long-term follow-up of the subjects in these studies will be of great interest in determining the durability of the symptomatic responses reported and the likely mechanism of action. Given the lack of evidence to date of structural improvement in the cartilage of the joints, it seems likely that the symptomatic benefits seen in these trials may relate to the anti-inflammatory properties of ASC rather than chondrogenic differentiation. In summary, cartilage reconstruction by ASC has not yet been demonstrated.
USE OF ASC FOR DIFFERENTIATION ALONG OTHER LINEAGES
Coronary heart disease (CHD) is a major cause of morbidity and mortality in the Western world, with nearly 8% of American adults over 20 years of age estimated to have some form of CHD, according to the American Heart Association . Trials are underway in Europe to treat acute myocardial infarction and chronic myocardial ischemia by injection of ASC into the coronary arteries . However, the mechanism of action in these situations remains unclear. Although myogenic differentiation of ASC has been demonstrated, it may be impossible to discover whether any potential results from this trial are due to ASC spontaneously differentiating into cardiac muscle to replace the injured tissue or due to the anti-inflammatory effects of ASC allowing inherent healing or regeneration to occur. However, the myogenic potential of ASC may be harvested in the treatment of Duchenne muscular dystrophy (DMD), an inherited genetic disorder characterized by progressive degeneration of skeletal muscle . In vivo murine studies have shown that the implantation of ASC into dystrophin-deficient, immunocompetent mice resulted in restoration of dystrophin expression, both in the muscle at the site of injection and in adjacent muscles over the long-term . In vitro studies show that ASC can fuse with DMD myoblasts, generating dystrophin and skeletal myotubules to re-establish dystrophin expression . Further animal studies will be required before human trials can be considered, but it appears that ASC have the potential to be useful as stem cell therapy for muscular diseases.
Similar to cartilage regeneration, nerve regeneration by stem cells would be extremely valuable clinically, as neurons have limited capacity to self-renew. Research into the transdifferentiation of ASC poses even greater levels of difficulty than mesenchymal differentiation. There is some evidence that ASC may differentiate along neuroectodermal and endodermal lineages, but in general, transdifferentiation is inefficient and the function of the resulting cells is often low compared with true lineage cells [48–50]. Differentiation into neuronal cells has been attempted, and whereas, some researchers have shown that phenotypically similar cells can be produced [51, 52], there is as yet no evidence of nerve function occurring in these cells. It seems logical that, if stem cell populations for these other cells types are available, these populations should be preferentially used over ASC. As several researchers have published their work using neural crest stem cells for nerve regeneration [53, 54], it is likely that the use of ASC in this field has limited application.
The recent International Federation of Adipose Therapeutics and Science meeting in Dallas, Texas (October 22–24, 2010) demonstrated some of the many novel uses for lipoaspirate, SVF, and ASC that are currently being investigated to solve clinical problems. These included the use of ASC to provide stromal paracrine support for pancreatic islet cells in vitro, before their clinical use in treating type I diabetes mellitus; ASC as a potential treatment for Alzheimer's disease; and harnessing the anti-inflammatory effects of ASC to treat multiple sclerosis (MS). To date, MS treatment has largely involved intrathecal and intravenous infusions of unpurified SVF rather than purified ASC , limiting the conclusions that can be drawn regarding the role of ASC in mediating any clinical effects. Researchers in Spain are currently recruiting for a Phase I/II randomized trial injecting two different doses of ASC intravenously to assess the safety and potential efficacy in MS . The same facility is also performing a Phase I/II trial of ASC intra-arterial injection into the popliteal artery for treatment of critical limb ischemia , although the trial is currently suspended. We await the outcomes of these trials with interest.
The burgeoning of human ASC research may appear to indicate that we are far advanced in our understanding of ASC and our ability to manipulate them. However, the current literature is often difficult to interpret due to uncertainty about the cells being used, that is, whether pure ASCs or mixtures of cells including ASCs, and the use of assays of uncertain significance to “confirm” differentiation down particular lineages. Methods used to differentiate ASC down these different lineages are still relatively generic and unsophisticated in many publications, and a lack of control cells such as differentiated mesenchymal cells can make it difficult to determine whether “differentiation” observed is unique to ASC or a property shared with other mesenchymal cells. The field would clearly benefit from more standardized disclosure of the experiments conducted, as proposed in Figure 2. Such disclosure might include better characterization of the starting cell populations, especially the degree of purity of the ASC and their cell surface molecular profile by flow cytometry; more rigorous sets of molecular markers to confirm lineage commitment and integration of standardized methods for their detection at the protein or mRNA level, based on positive control tissues such as bone; wider use of control mesenchymal cells or tissue such as differentiated human fibroblasts to confirm unique properties of ASC; and functional assays that link through to clinical applications, such as the ability to accumulate lipid for adipocytes or to generate 3D connective tissue for other lineages. Only with reliable, standardized basic science research can real clinical progression be achieved. We look forward to see ASC in vitro research translated to beneficial clinical outcomes in the near future.
DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
The authors indicate no potential conflicts of interest.