Adipose tissue stem cells meet preadipocyte commitment: going back to the future.
Adipocyte commitment and differentiation are complex processes, which can be Adipose tissue contains adipocytes in addition to a wide population of cells, such as muscle cells, mesenchymal stem cells (MSCs), and adipose precursor cells. .. preadipocytes are an excellent model for the study of adipocyte-related . Dynamic upregulation of CD24 in pre-adipocytes promotes adipogenesis If comparing 2 samples, the Student's t-test was used (Excel, version ). Adipose tissue stem cells meet preadipocyte commitment: going. Stem cell commitment to the adipose lineage produces preadipocytes that, Resident pluripotent stem cells in the vascular stroma of adipose tissue and .. We thank Kathleen Anuzis for excellent technical assistance. .. Adipose tissue stem cells meet preadipocyte commitment: going back to the future.
Some of these different studies may appear conflicting, particularly as they relate to exact cell types that APCs represent as well as their localization in vivo i. In our view, most of these data are not mutually exclusive, since we cannot rule out the possibility that multiple populations may exist.
Here we present each population as it was identified and characterized; however, it is important to note that there is likely significant overlap in the reported precursor populations.
Furthermore, stable APC populations with intermediate phenotypes may exist, reflecting an adipose precursor hierarchy e. Alternatively, each precursor population may exhibit a specialized role in adipose biology. The exact precursor population drawn on to undergo adipogenesis may depend on the sex, location, age, or proadipogenic stimulus. At the very least, the studies described here indicate that adipose precursors, much like the adipocytes themselves, are likely heterogeneous.
The vast complexity of the adipose lineage is increasingly more apparent as lineage tracing results continue to be reported. As such, additional distinct precursor populations are likely to emerge. Studies pertaining to dermal and bone marrow stem cells indicate that one should not assume that the sole function of APCs is to give rise to adipocytes.
Cells similar or related to the APC populations described here have also been implicated in the regulation of inflammation, angiogenesis, and stem cell maintenance through cytokine production.
A major challenge going forward will be to determine the exact requirement of the various precursor populations described here.
This will be best achieved by the development of genetic tools for temporal control of gene expression in these populations. The development of genetic tools and FACS-based approaches to isolate adipose precursors gives plenty of reason for optimism in this growing field; however, there are still significant limitations to the current approaches that must be addressed going forward.
In fact, some of these limitations may explain the inherent discrepancies in published lineage tracing results. Notably, many lineage tracing studies have used transgenic lines in which Cre activity is constitutive. The use of constitutively active Cre lines can be limiting in a number of ways.
When bred to a Cre-dependent Rosa26R reporter strain, reporter expression is subsequently activated in promoter-expressing cells and maintained in all descending cells; this occurs regardless of whether descending cells continue to express the promoter of interest. Therefore, it becomes difficult to directly assess whether adipogenesis originates from cells actively expressing the promoter of interest during development.
Moreover, the lack of temporal control over Cre activity prevents the use of these strains to determine the contribution of putative precursors to adipocyte hyperplasia under specific physiological conditions postnatally. Another important limitation is in the nature of the promoters ultimately driving Cre expression.
This can become an issue in attempts to manipulate gene expression in different precursor populations, as confounding nonadipose phenotypes may emerge that indirectly affect adipogenesis. Unfortunately, a single marker that unambiguously identifies native APCs has yet to be identified. Both systems have been powerful in this field; however, they, too, have concerning limitations.
Tamoxifen is a well-known inhibitor of estrogen receptor activity. The estrogen receptor itself plays an important role in adipose tissue remodeling Davis et al. At certain doses, tamoxifen can elicit detrimental effects on adipose tissue and remain localized in the adipocyte nucleus well beyond removal of the ligand Ye et al. These effects include rapid cell death followed by adipose regeneration via de novo adipocyte differentiation.
Moreover, Kloting and colleagues Hesselbarth et al. Notably, the investigators observed that tamoxifen treatment triggers substantial browning of the subcutaneous depot. There are also adverse effects of chronic doxycycline treatment on metabolism. Auwerx and colleagues Chatzispyrou et al. Doxycycline can also impact the gut microbiota in humans; the direct impact of this on adipose biology is unclear but potentially significant Angelakis et al.
Furthermore, doxycycline can regulate the function of matrix metalloproteinases Stechmiller et al. Nevertheless, we are presently unaware of data that would suggest that short-term doxycycline exposure directly impacts adipose tissue turnover in a manner that would confound lineage tracing studies.
Moreover, doxycycline leaves the system shortly after removal of doxycycline-containing food or water; expression of the widely used TRE-Cre transgene turns off within 24 h of removing doxycycline Wang et al.
This precise and verifiable temporal control over Cre expression is essential for lineage tracing analysis. As such, it is our position that the Tet-on systems are preferable over the tamoxifen-based Cre induction systems when performing adipose lineage tracing studies.
At the very least, great caution must be taken in interpreting lineage tracing results when using tamoxifen; its action on the estrogen receptor in adipose tissue and its potential to trigger cell turnover must be considered.
Our discussion here focused on recent advancements in murine adipose precursor biology. In Vivo Muscle Regeneration Original models for muscle regeneration focused on skeletal muscle formation using established murine models of dystrophy and muscle derived stem cells for review see [ ].
Not surprisingly, the myogenic differentiation capacity of bone marrow MSCs has been explored [ 10]. However, functional recovery in dystrophin-deficient mice has not been observed with MSCs [ ].
Unlike MSCs, recent studies using ASCs have reported some exciting results—that the implantation of human ASCs without immunosuppression into murine models of dystrophy can yield good engraftment levels and improvements in muscle function, thus allowing researchers to make more accurate conclusions about the myogenic differentiation of human ASCs in vivo.
Pioneering work from Rodriguez et al. Good engraftment with ASCs located around muscle fibers is also reported upon the injection of human ASCs virally engineered to overexpress MyoD [ ]. However, both studies use a rather rare population of human ASCs, termed hMADs, produced through rapid adherence of ASCs to tissue culture polystyrene, followed by culture for up to doublings. Therefore, it cannot be confirmed that these ASCs have undergone some atypical transformation making them more apt to undergo myogenesis in comparison to ASCs maintained for less time in culture.
However, there are additional studies that seem to confirm the findings of these hMAD cells. ASCs, induced to differentiate in vitro then injected into mdx mice, preferentially home to injured muscle and differentiate, producing transient and sequential peaks in MyoD, myogenin, myosin, and dystrophin, with each differentiation marker colocalizing with human b2-macroglobulin, suggesting the engraftment and direct differentiation of the human ASC within the muscle [ ].
Like Vieira et al. However, these researchers suggest that their results are due to the fusion of the ASC with the host muscle, a finding that may be substantiated by that of Goudenege, who observe ASCs within the muscles fibers themselves [ ] and by that of de la Garza-Rodea et al.
Whether the ASC differentiates within muscle de novo, fuses with the host muscle to drive regeneration or is involved in a combination of both, these studies do provide support for the use of human ASCs in the repair of skeletal muscle tissue. Moreover, these stem cells may be used without a substantial host immune response. While saw the use of human ASCs in the regeneration of skeletal muscle, this year also saw their use in smooth muscle differentiation in vivo with the injection of human ASCs into athymic rats and mice, resulting in their incorporation into the smooth muscle of the bladder [ ].
This incorporation and resulting smooth muscle regeneration was eventually confirmed in this group to be due to differentiation and not to fusion of the ASC to host cells [ ], suggesting that ASCs may be capable of in vivo smooth muscle formation. Similar incorporation into bladder muscle and improvements in bladder function and smooth muscle content have been reported in models of stress urinary incontinence, with confirmation of ASC differentiation into smooth muscle cells being reported .
Increased smooth muscle bundles, together with formation of stratified urothelium, have been observed upon injection of ASCs preinduced to the smooth muscle lineage [ ]. In addition to this work, results from Zhao et al. Specifically, the injection of rat ASCs seeded onto PLGA scaffolds designed for the controlled release of NGF results in significant improvements in smooth muscle development, abdominal leak point pressure, and retrograde urethral perfusion pressure when compared to controls.
More interestingly perhaps may be their finding that there are also improvements in ganglia formation within these treated urethras, suggesting that the injected ASCs, plus NGF, work together to produce innervated smooth muscle.
This finding has also been observed upon the injection of rabbit ASCs seeded onto bladder acellular matrix grafts [ ]. However, it is possible that these observed improvements in smooth muscle differentiation and function are due to paracrine actions of the ASC, decreasing apoptosis and maintaining vascular supply as proposed in models of diabetic bladder dysfunction [ ].
In a study by Fotuhi et al. Moreover, these ASC treated hearts required extrastimuli to induce an arrythmia, suggesting that ASCs could be used in the treatment of cardiac muscle infarcts [ ]. With in vitro studies confirming the cardiomyogenic potential of these stem cells, infarct treatment could be mediated through the differentiation of ASCs into cardiomyocytes. Previous work on bone marrow MSCs supports this theory, as implantation of these stem cells has been shown to improve heart function in several model systems .
However, there is a debate on whether the ASC contributes directly to cardiac muscle regeneration or supports this event through the production of angiogenic growth factors and cytokines. In support of vascularization, bone marrow mononuclear cells and endothelial progenitors are known to improve cardiac function by incorporating into newly forming capillaries and releasing angiogenic factors .
Similar events may also be induced by ASCs. In support of this, Beitnes and coworkers show significant improvement in left ventricle ejection fraction LVEFdecreases in infarct sizes, and increases in vascularization when human ASCs are injected into infarcts in nude rats. Moreover, they specifically observe an absence of ASC engraftment, suggesting paracrine action [ ]. Finally, endothelial cells created from induced pluripotent ASCs iPSCs and injected into murine infarcts are found localized specifically around, but not directly integrated into, newly formed microvessels near the regenerating infarct region, suggesting paracrine action of these cells on the host vasculature [ ].
Furthermore, these iPSC-derived ECs are specifically found to release pro-angiogenic factors in the ischemic environment of the heart. However, the possibility of direct differentiation of ASCs into cardiomyocytes cannot be discounted. Furthermore, while Zhang et al. Finally, real-time tracking of labeled ASCs transplanted into infarct regions via implanted fibrin scaffolds also confirms costaining of the ASC label with cardiac troponin in the infarct region [ ]. Therefore, cardiac muscle regeneration by ASCs may be a combination of both direct transdifferentiation of ASCs into cardiomyocytes combined with their promotion of vascularization.
In Vivo Endoderm Regeneration With in vitro studies suggesting hepatic potential for ASCs, translational models have attempted to confirm this capacity using parameters such as production of albumin protein together with functional assays.
Initial studies in show increased engraftment of human ASCs into regenerating livers following partial hepatectomy [ ]. Indicative of liver regeneration, preinduced human ASCs, loaded onto PLGA scaffolds and implanted into the peritoneum of hepatectomized rats, not only survive on-scaffold at least 14 days after implantation but aslo, like their in vitro counterparts, exhibit increased expression of liver-specific genes [ ].
However, this study is unable to confirm differentiation at the functional level. Similarly, induced human ASCs transplanted into CCl4-injured livers in nude mice not only increase their production of albumin protein but also restore liver functions such as ammonia and purine metabolism and decrease liver injury markers, such as alanine aminotransferase and aspartate aminotransferase activity [ ].
However, the authors do point out that they are unable to rule out ASC fusion with endogenous hepatocytes. Finally, whether ASCs need preinduction to increase liver regeneration is called into question as Ruiz et al. In addition, GFP-labelled MSCs injected into the subarachnoid space of the lumbar spine have been found on the surface and within the parenchyma of a spinal cord lesion, suggesting that adult stem cells may be used to repair nervous tissue injuries [ ].
Like these MSC studies, early studies with ASCs used simple transplantation into nervous tissue injuries followed by histologic analysis and functional assessment of recovery. Improvement of deficits by transplanted ASCs has been reported in models of middle cerebral occlusion or ischemic stroke, where infarct size can be decreased upon ASC administration and sensimotor dysfunction improved [ — ], spinal cord contusion injury, where GFP-labelled canine ASCs are able to improve pelvic limb function significantly versus controls, together with nerve conduction velocity [ ] and peripheral nerve gaps, where ASCs seeded into acellular nerve grafts promote sciatic nerve regeneration and functional recovery in some cases at levels comparable to autografts .
However, differentiation into glial cells may be more likely as Ryu et al. As such, current in vivo studies have begun to explore the regenerative capacity of ASCs predifferentiated into Schwann cells SCsrather than hoping for the direct in situ differentiation of the ASCs into neurons. In support of this, rats implanted with ASC-derived SCs show not only significant locomotor function recovery compared with untreated ASCs but also a reduction in gliosis [ ].
Rat ASCs, differentiated to SCs and transplanted into denervated nerves, survive and maintain their differentiated state for up to 10 weeks, forming myelin sheaths and expressing key glial cell markers [ ]. When examined histologically, increased axonal regeneration comparable to implantation of primary SCs can be observed using these stem cells [ ]. While early in vitro studies suggest a neurogenic potential for ASCs, the majority of in vivo studies fail to show direct differentiation in situ of the transplanted ASCs into neurons.
In addition, extremely low levels ASC differentiation into mature neurons is noted in a model of cerebral cortex injury [ ]. So while the transplantation of ASCs may be successful in improving neural function, it is likely due to a supportive role in tissue healing.
InZhao et al.
Adipose-Derived Stem Cells in Tissue Regeneration: A Review
A similar hypothesis has been put forth by Wei et al. In vivo protection of cerebellar granule neurons from apoptosis has also been observed using ASC-conditioned media [ ]. In addition, injection of ASCs or ASC lysates into models of cavernous nerve injury is found to significantly decrease fibrosis and improve erectile function [ ]. In support of this, functional deficits in a model of middle cerebral artery occlusion can also be dramatically improved using ASC transduced to overexpress BDNF [ ].
Moreover, enhanced nerve fiber growth is observed in models of mice limb reinnervation using ASCs preinduced toward the neural lineage thus enhancing their production of brain-derived neurotrophic factor BNDF [ ]. In vivo ectodermal capacity of ASCs is also indicated by their regeneration of epithelial tissues. ASCs, combined with scaffolds, and implanted into rat tracheal defects show the development of a pseudostratified epithelium with goblet cells .
Increased collagen content is measured upon subcutaneous implantation of rat ASCs seeded on decellularized dermal matrix [ ]. Furthermore, Trottier et al. With their ability to differentiate into epithelial and dermal layers when seeded onto hydrogels , the ASC may replace dermal fibroblasts in the treatment of skin wounds and disorders.
However, as with other in vivo regenerative models, studies have yet to confirm whether these ASCs directly contribute to tissue formation through differentiation or support differentiation through the production of soluble factors. A study using GFP-labelled human ASCs seeded into silk fibroin-chitosan scaffolds and implanted into full-thickness skin defects finds increased wound closure [ ]. In addition, this study shows coexpression of both the GFP marker and those of epidermal epithelial cells, suggesting that wound closure is due, in part, to the differentiation of the ASC.
However, wounds also show increased microvascular density and ASC differentiation into endothelial cells, possibly indicating that wound closure is due also to vascularization. Conditioned medium made from a mixture of ASCs and dermal fibroblasts can also increase keratinocyte proliferation and migration  and increased levels of HGF and KGF can be found in the media, suggesting a role for these factors [ ]. Similar to this, stromal layers derived from human ASCs can induce the proliferation and expression of appropriate epidermal keratins when overlaid with keratinocytes, leading the authors to conclude that the ASC can be used to create a biomimetic stroma capable of stimulating epidermal development [ ].
Autologous ASCs have also been found to favor epidermal healing in porcine models of cutaneous radiation injury [ ] and the effects of ASC-conditioned media on aged fibroblasts have been studied as a means of developing antiaging strategies [ ]. The Role of Vascularization It has long been known that vascularization is critical to tissue healing.
The in vitro differentiation of ASCs to endothelial cells is not under dispute as they quickly and easily form vessel-like structures in Matrigel substrates that assume endothelial function [, ]. Consistent with this, vessel formation has been observed in several ASC models of cardiac infarct treatment, epithelial regeneration, and neural tissue healing discussed above.
In addition, multiple studies of ischemia describe increased vascularization following ASC administration [, — ]. Hemodynamic abnormalities in pulmonary arterial hypertension may be reversed using ASCs—a finding thought to be attributable to their induction of angiogenesis and increased formation of small, pulmonary arteries [ ]. Increased capillary densities and higher blood flow have been reported in several studies using ASCs for the healing of skin wounds and improvement of skin flap survival .
While tissue damage associated with ischemia has been well described, it is important to note that the reperfusion period is also associated with damage.
Amelioration of IR damage has been described previously using MSCs, cardiac stem cells and induced pluripotent stem cells [ — ]. Transendocardial injections of ASCs in a minipig model of IR injury results in long-lasting improvements in cardiac function, along with increased angiogenesis and vasculogenesis [ ]. In support of this, Chen et al.
Increased antioxidant marker levels versus controls i. In vivo, GFP-labelled ASCs can improve the vasculature in excisional wounds in normal and diabetic rats and are found to coexpress CD31, suggesting their endothelial differentiation [ ].
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Such results suggest that ASC administration can result in increased vessel formation through their direct differentiation. For example, both in vitro and in vivo studies suggest that the ASC drives endothelial differentiation and stabilizes it through paracrine action [ ].
As a candidate for mediating vessel formation, the most obvious paracrine factor is VEGF and many studies propose that it is the secretion of VEGF as the underlying reason for the improved vascularization by the ASCs [,— ]. In addition, Gao et al. Increased VEGF expression, together with increased collagen density and microvascular density, is also measured in fill-thickness rat skin grafts injected with ASCs [ ].
VEGF secretion by ASCs is significantly upregulated in vitro upon metabolic induction of ischemia [ ] and ischemic limbs in diabetic nude mice treated with ASCs show earlier and more abundant neovessel formation, together with increased levels of plasma VEGF [ ]. Inhibition of VEGF secretion by ASCs through RNA interference RNAifollowed by their transplantation into syngeneic models of small-for-size liver injury results in significant disturbances to graft microcirculation, serum liver functional parameters, and graft survival [ ].
From these studies, it can be concluded that the ASC is capable of increasing vascularization in regenerating tissues—most likely through a combined action of direct endothelial differentiation and paracrine action. The Role of Inflammation Successful tissue regeneration is also reliant upon control of inflammation. In light of the numerous similarities found between bone marrow MSCs and ASCs, it may be that ASCs are also capable of modulating host immunity through immunosuppression.
Furthermore, ASC-mediated immunosuppression has been observed in numerous in vitro experiments utilizing the gold-standard mixed lymphocyte reaction MLR system [ — ]. With regards to in vivo model systems, reduced inflammatory infiltration and airspace enlargement results upon the systemic administration of human ASCs to murine models of emphysema. This can be explained by enrichment of certain cell types during culture where diverse parameters such as composition of basal media, supplements, plating substrates or cell confluence could have an impact on this issue.
Culture of ASCs with myogenic differentiation media has resulted in ASCs adopting an elongated morphology, similar to differentiating myoblasts, and expression of early MyoD1, myogenin and late myosin heavy chain markers of muscle differentiation Zuk et al.
These and other works show variable differentiation efficiencies in terms of onset and fusogenic ability, which may be the result of using different inductive media Table 1. Co-culture experiments with myoblasts have shown that secreted factors and cell-to-cell contacts induce ASCs myogenic conversion and fusion with mouse C2C12 and primary myoblasts Lee and Kemp, ; Di Rocco et al.
Variations in fusogenic ability were observed when hASCs were treated with myogenic differentiation media before co-culture, and the highest efficiency was obtained when ASCs were already at late stages of myogenic differentiation and expressing myosin heavy chain MyHC and dystrophin Eom et al.
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Importantly, for regenerative purposes as a treatment for dystrophies, ASCs should also be able to fuse with dystrophic myotubes, for this reason, Vieira and colleagues showed that in vitro human ASCs could indeed fuse with DMD-derived myoblasts to form myotubes that recovered dystrophin expression Vieira et al. Therefore, these experiments support the use of allogeneic ASCs not only for their conversion towards the myogenic lineage and thus contributing to counteract muscle loss, but also as providers of WT dystrophin and other potential beneficial factors that may be missing in dystrophic muscles.
The use of coatings and hydrogels has shown to enhance myogenic differentiation from ASCs Choi et al. However, at present, a universal media composition for a robust myogenic differentiation of ASCs is missing. Table 1 summarizes the results showed in these works and indicates the composition of differentiation media used in each of them.
In vivo Skeletal Muscle Differentiation Potential of ASCs All these in vitro works indicate that different stimuli can potentiate and promote ASCs differentiation towards myogenic lineage, ranging from hormones and growth factors present in media or secreted by cellsto cell-to-cell contacts or even by other mechanophysical inputs such as plating surfaces.
However, whether ASCs could contribute to myogenic regeneration in vivo was not clear until the work of Rodriguez et al. In this work, the authors characterized in vitro human ASCs from different young donors and prior to transplantation in mdx mice, they identified two multipotent populations, one characterized to be fast adherent and expandable in vitro for more than passages chosen for transplantation experimentsand a slower adherent population that showed senescent features upon long term culture.
Transplanted animals showed expression of dystrophin at several days post-transplantation, and human nuclei elegantly revealed by FISH with a specific probe for human centromeres were observed in central and peripheral locations of muscles, indicating that human-derived ASCs contributed to the regenerative process. After 6 months, dystrophin expression was well distributed along muscle fibers, and even present in adjacent gastrocnemius muscle, suggesting that cell migration from injection site to other dystrophic muscles could have occurred or that dystrophin delivery was achieved by another unknown mechanism.
Furthermore, in immunocompetent mice, regeneration took place successfully with no evidence of CD3 positive-lymphocytic infiltration. In contrast, hASC from early passages that express HLA-I antigens elicited an immune response in the host and could not restore dystrophin expression. Whether hASCs contributed to regeneration not only by fusion but also by converting themselves to myogenic lineage in vivo was not clear, although in vitro conversion of ASCs to myogenic lineage suggests that both mechanisms fusion to myotubes and also myogenic conversion of ASC themselves could have occurred.
In this regard, the work of Liu et al. In this work, a subset of hASCs expressing endothelial marker Flk1 were transplanted by two different methods in two different regeneration models: They both resulted in successful engraftment, partial recovery of sarcolemmal expression of dystrophin, decreased necrosis after 4 weeks and lower levels of muscle creatinine kinase in blood after 12 post-transplantation weeks.
Another work Vieira et al. Different from in vitro co-culture experiments where highest fusion was observed using hASCs differentiated to late myogenic stages Eom et al. Also, it is possible that myogenic-differentiated ASCs are not able to sustain a proliferating pool of cells that could give cycles of regeneration upon time, which may be required to achieve robust and successful engraftments.
These authors also reported that inflammatory process shown by those dystrophic muscles from SJL mice, did not improve upon hASC transplantation, in contrast to other works, which have reported a beneficial immunoprotective effect of ASC transplantation, for instance in rat brains upon a hemorrhagic stroke Kim et al. Nevertheless, mice showed improved muscle functionality in several motor ability tests up to 6 months of transplantation.
To assess the viability of cell transplantation approaches in humans, it is crucial to validate whether they can be feasible in larger animal models. This issue is important in order to predict pathogenesis and treatment outcomes. An alternative bigger model than mice and rats for muscle regeneration is the golden retriever muscular dystrophy GRMD dog.
This model reproduces full spectrum of human DMD and it has been used for successful transplantation studies with systemically injected mesoangioblasts, which is a type of mesenchymal stem cell present in walls of large vessels.
In these experiments, dystrophin expression was recovered as well as partial muscle functionality Sampaolesi et al. However, the dogs were immunosuppressed and treated with steroids, which have been shown a beneficial impact on muscle functionality. For this reason, Vieira et al. Expression of human dystrophin was validated up to 6 months after transplantation but could not be found after 12 months, suggesting that hASCs were not able to replenish the stem cell pool.
Unfortunately, histopathological features were not improved and beneficial effects on disease symptoms were difficult to be concluded. Therefore, from these data one can speculate that perhaps multiple injections, for instance every 6 months, and larger amounts of cells may be required to maintain exogenous dystrophin expression at levels that give a positive functional output.
Surviving animals did not present adverse immune responses or other complications after 24 months, which might indicate ASC transplantation to be a safe procedure. In contrast, locally injected hASC were not able to engraft successfully, opposite to what had been described for mdx mice Rodriguez et al. Table 1 summarizes the results showed in these works and indicates quantifications of ASCs contribution to muscle differentiation and regeneration.
MyoD-Driven Conversion of ASCs From all these works it is suggested that ASCs can be reprogrammed to myogenic lineage in vitro and in vivo by extracellular cues, highlighting a possible therapeutic use of ASCs as a new source of myogenic progenitors. In order to increase levels of myogenic conversion, Goudenege et al.
MyoD1-pioneering ability was suggested years ago by Weintraub and colleagues, who ectopically expressed MyoD1 in fibroblasts, fat, liver and nerve cell lines resulting in activation of myogenic gene expression Weintraub et al. This data argues in favor of manipulating ASCs in vitro with transcriptional factors to force ASCs commitment towards myogenic lineage, since using these manipulated ASCs result in a higher contribution to muscle regeneration. Epigenetics of ASC-Derived Myocytes During adult stem cell differentiation as well as in the progression from a pluripotent to a multipotent state, expression of proliferative and pluripotent genes is erased, and expression of differentiation or lineage-specific genes from previously silent loci is activated.
This process is controlled by concerted action of signaling cues with ubiquitous and cell-specific transcription factors that govern a changing epigenetic landscape during cellular differentiation. In other words, distinct epigenetic signatures can be associated to a precise cellular stage Hussein et al. DNA methylation at CpG sites is one of best-studied epigenetic modifications, which has been associated to gene repression Heard et al. Therefore, to verify a certain lineage reprograming, conversion, or proper differentiation, instead of monitoring expression of a few markers of these processes, global characterization of gene expression and epigenetic patterns seems to be a more complete approach to validate whether a full or partial conversion has been achieved.
Berdasco and colleagues pursued this technique and characterized global DNA methylation status of myogenic lineages derived from hASC by Infinium methylation arrays interrogating 27, CpG sites Berdasco et al. Their results indicate that methylome of ASCs-derived myocytes is more similar to the one of undifferentiated ASCs than to the one of primary myocytes from human biopsies.