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Myogenic program induction in mature fat tissue (with MyoD expression)
Experimental Cell Research 308 (2005) 300 – 308 www.elsevier.com/locate/yexcr
Research Article
Myogenic program induction in mature fat tissue (with MyoD expression) Y.C. Kocaefea,b,*, D. Israelia, M. Ozgucb, O. Danosa, L. Garciaa,* a
Genethon CNRS UMR8115 1 bis rue de l’Internationale 91002 Evry cedex, France Hacettepe University, Faculty of Medicine, Department of Medical Biology, Ankara, Turkey
b
Received 5 July 2004, revised version received 9 March 2005 Available online 25 May 2005
Abstract MyoD exerts a master transcriptional control over the myogenic differentiation cascade. Here, we study different approaches to induce myogenic transdifferentiation in mature adipocytes utilizing MyoD gene transfer. Organotypic cultures of fat tissue and a long-term culture of in vitro differentiated adipocytes deduced that MyoD provoked morphological changes in mature adipocytes that can be summarized as loss of fat content, acquisition of a fusiform shape and eventual fusion with committed neighbor cells. In vivo, MyoD gene transfer into rat interscapular and inguinal fat pads demonstrated that while structural proteins of muscle lineage were expressed, they co-existed with specific adipocyte proteins. Expression of these proteins diminished over time likewise the fat content. The transdifferentiation process initiated by MyoD did not require cell cycle progression and was well tolerated by the fully differentiated and mature adipocytes. D 2005 Elsevier Inc. All rights reserved. Keywords: MyoD; Myogenic conversion; Transdifferentiation; Muscle dystrophy; Myogenic fusion
Introduction The switching of a differentiated cell type from one lineage to another is called transdifferentiation. Although this phenomenon is a common process in embryonic development, it is a rare event in adulthood, and is referred as metaplasia under pathologic conditions. In addition to various examples in drosophila [1], induction of transdifferentiation in tissues and organs through experimental intervention in mammals has also been demonstrated [2– 4]. Master transcription factors of specific transcription cascades are the key factors for ignition of differentiation processes. MyoD, a helix –loop –helix DNA binding transcription factor [5], is regarded as the master control over the myogenic differentiation cascade [6,7]. It binds to the enhancers of muscle specific genes through a homodimer or heterodimers
* Corresponding authors. Y.C. Kocaefe is to be contacted at Hacettepe University, Faculty of Medicine, Department of Medical Biology 06100, Ankara, Turkey. Fax: +90 312 3096060. L. Garcia, Genethon 1 bis rue de l’Internationale 91002 Evry, France. Fax: +33 1 60 778698. E-mail addresses: [email protected] (Y.C. Kocaefe), [email protected] (L. Garcia). 0014-4827/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2005.03.038
of positive acting transcriptional activators [8,9]. The downstream effectors of MyoD include other myogenic regulation factors (MRFs) such as Myf5, Myogenin, and MRF-4 [10,11]. The forced expression of MyoD triggers myogenesis in a wide range of mesenchymal cells and cell lines, a phenomenon referred to as ‘‘myogenic conversion’’ [12 – 14]. Although this phenomenon was initially shown on adipocyte cell line [12], transdifferentiation of differentiated mature adipocytes into muscle lineage has not yet been challenged. The adipogenic transcription program is initiated by PPARg and C/EBPa factors. Ectopic expression of either of these factors or upregulation of PPARg by activators can induce the adipogenic program in committed myoblasts [15] or fibroblasts [16]. While the fatty infiltration is the hallmark of the end point of many muscle diseases such as Duchenne muscular dystrophy, the origin of the adipocytes infiltrating the degenerating muscle is still unknown. Gene replacement therapy for the treatment of muscle dystrophies would apply to early-diagnosed cases before muscle deterioration. Once the fatty degeneration is established, there is no more contractile tissue left in the muscle to be cured by approaches of gene replacement therapy. However, if the conversion of the adipocytes
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residing in the degenerated muscle into myogenic program may be achieved under the frame of an organized muscle, it is conceivable that this could possibly maintain the contractile function. In this study, we have tested the possibility to induce the transdifferentiation of mature fat tissue into muscle utilizing MyoD gene transfer. We have challenged myogenic conversion in adipocytes in three different setups. Firstly, we have designed a system to obtain mature differentiated adipocytes from primary preadipocytes. Yet, to this day, no method has been described to isolate and cultivate pure primary mature adipocytes for long term. To overcome this, we have secondly utilized cultures of organ explants of fat pads, which could be maintained up to several weeks. This allowed us to compare various viral vectors to assess targeting of mature adipocytes. Lastly, we investigated the consequences of MyoD gene transfer in fat pads of rats in vivo.
Materials and methods Gene transfer vectors (i) 1st-generation serotype 5 adenovirus vector, (ii) serotype 2 adeno-associated virus (AAV), and (iii) secondgeneration lentiviral vector pseudotyped with VSVg (vesicular stomatitis virus g protein) envelope were employed to express EGFP protein driven by CMV promoter except the last, where the PGK promoter was used instead. The production methods have been described previously elsewhere [17 – 19]. We also used the same adenoviral gene expression vector for the delivery of mouse MyoD cDNA [genebank accession: 6996931] under the control of the CMV promoter. Downstream to the MyoD cDNA, the expression cassette also included an IRES (Internal Ribosomal Entry Site) and an EGFP reporter to follow the transgene expression (Clontech Laboratories, Paolo Alto, CA). Organ culture The interscapular and inguinal fat tissues of 6-month-old Sprague –Dawley rats were resected with special care to avoid contamination by surrounding superficial muscles. Tissues were then grossly dissected with a scalpel into small pieces with a diameter of a few millimeters. Afterward, samples were placed in culture flasks with adipogenic induction medium for 24 h. This medium consisted of 1:1 F12:DMEM (Gibco, France) supplemented with 10% fetal calf serum, 0.5 mM IBMX, 10 Ag/ml insulin, 1 AM dexamethasone, and 0.2 nM T3 (Sigma, France) [20]. The induction medium was removed gradually starting from the 1st day by replacing partially with an adipogenic maintenance medium in which fetal calf serum was reduced to 4% and did not contain IBMX, dexamethasone, and T3.
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Cultures were kept in 5% CO2 in 37-C incubator. The medium was partially refreshed every 2 days. The tissues were transducted at day 1 with either adenoviral or adenoassociated viral and lentiviral expression vectors harboring EGFP or MyoD. Tissues were investigated on a daily basis with fluorescent microscopy and fixated at different time points with 4% paraformaldehyde for further confocal microscopy analysis. Differentiating preadipocytes and long-term culture The inguinal and interscapular fat pads were dissected as above and were minced with scalpel into smaller pieces under dissection microscope while special care was taken to remove the vascular structures and connective tissue. The minced adipose tissue is digested with 0.5 v/w of collagenase B and dispase II (Roche Diagnostics, Mannheim, Germany) at 37-C for 1 h with gentle agitation. After washing twice and filtering through 40-Am filters, the cells were plated at near confluency in pre-treated (described in the Results and discussion section) Lab-Tek glass chambers (Nunc, Roskilde, Denmark) with 105 cells for each chamber of 1 cm2 surface area with adipogenic induction medium. Every 2 days, the medium was replaced partially with adipogenic maintenance medium. It was possible to observe the first inclusions of lipid accumulation after the first week. The preadipocytes acquire a multilocular view by their 4th week and only after the 10th to 12th week, the multilocular lipid vesicles fuse to give the cell the look of a mature monolocular adipocyte, gaining a stability in their shape and size. Animals and surgical procedures Six- to nine-month-old (mean weight 240 T 50 g) male Sprague – Dawley rats were used in the study. All animal procedures were performed according to an institutionapproved protocol and under strict biological containment. The animals were placed on a regimen of 50 Ag of cyclophosphamide delivered intraperitoneally (IP) every 72 h during the course of the experiment. The interscapular or the inguinal skin incised following 70 Ag of IP ketamine anesthesia and fat pads were revealed. A total of 50 Al (7 1010 physical particles) viral vector preparation was injected in an infiltrating manner at 18 to 20 different locations on the target fat tissues. The skin was sutured back after the intervention. At 2-, 3-, 4-, and 5-week time points, injected rats were sacrificed via pentobarbital overdose. Transducted fat tissues were resected, mounted, and fresh frozen for further analysis. Immunohistochemistry The explanted tissues from rats at different time points were investigated for the expression of muscle-specific proteins after cryosectioning. The tissue-specific antibodies used were monoclonal anti-desmin (Sigma, France), mono-
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clonal myosin heavy chain (MHC) (Novocastra, France), monoclonal anti-a-actinin (Abcam, Cambridge, UK), and guinea pig polyclonal; anti-perilipin and anti-adipophilin (Progen, Heidelberg, Germany). Transgene expression has been confirmed via polyclonal MyoD antibody (Santacruz Biotech, France). Immunostaining procedure was conducted according to antibody producers’ directions and analyzed by Bio-Rad multiphoton confocal microscopy (Bio-Rad, Hercules, USA).
Results and discussion Organotypic fat tissue explants and long-term culture of differentiated preadipocytes The main obstacle working with adipocytes is to isolate them alive and to maintain them for a sufficient period of time. Organ culture of adipose tissue has long been accepted as the model of choice for biochemistry experiments where mature adipocytes and their activities can be traced in an isolated environment for a short time [20,21]. This setup is adequate to observe the impact of a short-term modulator or the outcome of environmental changes on the adipocyte metabolism. In our hands, we could keep the explanted mature adipocytes alive for up to 3 weeks. We observed no significant difference in maintaining adipocytes from either interscapular or inguinal fat pads. During the first week, adipocytes tended to lose their fat content rapidly. This is a well-known phenomenon which has been inaccurately named ‘‘dedifferentiation’’ [22]. There is a significant number of studies using preadipocytes and/or adipofibroblasts to investigate adipogenic commitment and differentiation [23]. Although these cells could easily be handled, they naturally harbor a significant plasticity unless they are fully differentiated. We utilized an alternative approach of differentiating the primary preadipocytes to obtain mature fully differentiated adipocytes. Once the adipogenic differentiation was induced, the precursors underwent commitment and started accumulating fat vesicles (Figs. 1a– d). On the long course of incubation, the average time for these adipocytes to achieve the monolocular mature phenotype took at least 10 to 12 weeks. After the 12th week, the adipocytes were stable in size and shape in culture (Figs. 1e, f). As the adipocytes grow and acquire more fat, they tend to lose their adhesion capabilities and eventually break loose and start floating. This was the main obstacle of this method and generally occurred as the cells achieved the monolocular shape. One approach to overcome this problem was to grow the preadipocytes to confluency before differentiation, fix them with ethanol, wash repeatedly with PBS, and achieve a surface for a second round of plating and differentiation. In our hands, this approach enhanced the adhesion properties and gave better results compared to collagen-coated tissue culture chambers.
Gene transfer into mature adipocytes In the course of this study, one of the primary questions was which mode of gene transfer has to be employed to target mature adipocytes. As very little information was available in the literature, we conducted a triplicate set of experiments to assess the tropism of adenoviral (1stgeneration serotype 5), adeno-associated (serotype 2) and lentiviral (self-inactivating 2nd generation) EGFP expression vectors for the mature adipocytes. Special care was taken to observe adipocytes via epifluorescence microscopy, as they tend to diffract the light by their nature and can easily cause misinterpretation of the GFP signal. The organotypic tissue culture provided a stable short-term system to follow the reporter expression. Indeed, the organ culture of adipose tissue revealed that primary mature adipocytes could be kept alive up to 20 days maximally. Starting from the third week, it was possible to observe cells undergoing apoptosis by their morphology, as well as necrosis in the center of the tissue piece. As it was not possible to assess the exact number of cells in pieces of explanted fat tissue, viral vectors were added into medium arbitrarily at various amounts ranging from 1 108 to 7 1010 physical particles/ml. Although this approach did not achieve a meticulous titration assay, it was sufficient to sort out the best vector system for our application that was the adenoviral vectors. During the 3-week follow-up of tissues transducted with adenovirus, the GFP expression could be visualized starting from the 24th hour within the mature adipocytes (Fig. 2a). As the promoter we used had no tissue specificity, GFP was also observed in a wide variety of stromal cells, vascular structures, and endothelia within the tissue. The observations on the lentiviral-transduced tissues revealed that numerous small cells residing between the adipocytes did uptake the vector whereas no expression could be observed in mature adipocytes. Starting from the 1st week of observation, these small cells began to accumulate fat deposits (Fig. 2b). Taking into account that these organotypic cultures were carried out in the presence of adipogenic induction factors (IBMX, dexamethasone, Insulin), it is likely that lentiviral-targeted cells were the preadipocytes, which differentiated later in the course of the culture. For the adeno-associated viral vector, we observed GFP expression limited to stromal cells in between the adipocytes. Contrary to cells transduced with lentiviral vector, these cells did not differentiate into adipocytes (Figs. 2c). We concluded that in our system, adenoviral vectors were the most appropriate. The in vitro differentiated mature adipocytes also enabled us to make a further assessment of the transduction efficiency with an EGFP expression vector. The differentiation was initiated with approximately 105 cells and some of the adipocytes detached and lost on the course of the long process. The 2 109 physical particles yields 100% transduction of the non-adipocyte cells in the longterm culture but only 10 to 15% of the mature adipocytes
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Fig. 1. Differentiation and maintenance of preadipocytes in long-term tissue cultures. Panels a to f show primary preadipocytes from rat interscapular fat pads at 0, 1, 4, 8, 12, and 15 weeks, respectively. Note that the adipocytes are rather flat until the 8th week, and then acquire a round shape with increasing volume and transition to monolocular morphology. After week 12, adipocytes were stable in their size and shape. Scale bar represents 20 Am.
were observed to be GFP (+). Increasing the mount of viral vector did not enhance the ratio further. It has been previously demonstrated that stable expression of the adenovirus receptor CAR in 3T3-L1 cell line provided a 100-fold increase in the transfection efficiency via adenoviral vectors [24]. This observation suggests that the limited expression of CAR on mature adipocytes might be the obstacle restricting transfection. Myogenic conversion of mature adipocytes using Adenoviral MyoD vectors MyoD-transduced adipocytes in organotypic cultures were fixed in 4% PFA and traced under confocal microscopy. Multiple z-stack acquisition also enabled to achieve 3-D reconstitution of the images. The GFP reporter in the MyoD expression cassette permitted to trace the transduced adipocytes in living organotypic cultures. While the signal was generally weak due to the thin cytoplasm, it was
possible to observe the GFP in living mature adipocytes starting from 48 h after Adeno-MyoD transduction. Beginning from day 15, mature adipocytes underwent a number of significant morphologic changes; they developed polarity while acquiring a lemon-like shape with protruding tentaclelike extensions from poles (Fig. 3a). The control adipocytes transduced with GFP-only expressing adenoviral vectors did not demonstrate such changes. Neither the virus nor the promoter we have utilized in this study have any specificity for adipose tissue. As a consequence of that, some mesenchymal cells, fibroblasts, and perivascular pluripotent cells residing in the tissue, which were transduced, also committed to myogenic differentiation. As expected, these non-adipocyte cells harbored more plasticity and started to fuse rapidly from the 7th day onward as thin myotubes. The discrimination between these newly forming myotubes and the adipocytes were made by one simple criterion, which is ‘‘cytoplasmic monoloculated fat droplet’’. As a highly hydrophilic globular protein, the GFP was excluded from
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Fig. 2. Explanted adipocytes in organotypic cultures transduced with various EGFP expression vectors. GFP expression in mature adipocytes could be observed 2 days after transduction with recombinant adenoviral vectors (a). Lentiviral vectors only transduced some small cells surrounding mature adipocytes, which started accumulating fat droplets and presented a multilocular preadipocyte phenotype on the course of organotypic culture. Thirteen days after transduction, these preadipocytes are clearly distinguishable with their fat droplets as indicated by arrowheads (b). The adeno-associated viral vector targeted small stromal cells that did not demonstrate any fat deposits at the end of the second week (c). Scale bar represents 20 AM.
this fat droplet and concentrate in the thin cytoplasm of the adipocyte. Starting from 15th day after transduction, it was possible to observe the adipocytes that were undergoing morphological changes beginning to fuse with other committed cells in their neighborhood (Fig. 3b). It is clear
Fig. 3. Morphologic changes of mature adipocytes in organotypic culture conditions after transduction with 2 109 physical particles/ml of AdenoMyoD expression vector. By 15 days after transduction, adipocytes (TA) acquired polarity and developed tendril-like extensions from poles. With time, these extensions stretched out (arrow) to nearby myotubes (mt) (a). Further in the course of these changes, with the advancement of the loss of fat content, eventual fusion to a neighbor myotube could be observed (b). Immunostaining of MyoD appears in yellow, stars indicate fat droplets.
that, in order for an adipocyte to achieve this, it needs a remarkable time period and some loss of cytoplasmic fat content. The lack of vector specificity allowed us to observe the fusion of adipocytes with other plastic stromal cells in the neighborhood. Please refer to online supplementary documentation for animated 3-D images of these structures. Since explants of primary mature adipocytes couldn’t be kept further than 3 weeks, we assessed the effect of MyoD on mature adipocytes derived from in vitro differentiated primary preadipocytes. After the 12th week of differentiation, upcoming mature adipocytes were transduced with Adenoviral MyoD expression vector as described before. Consistent with the organotypic observations, transduced adipocytes developed polarity after the first week, which progressed to tendril-like extensions. They eventually started to fuse to neighbor cells expressing MyoD (Fig. 4) after the second week. It was also possible to observe that although keeping their round shape, some adipocytes harbored multiple nuclei showing that this fusiform morphological change is not necessary for their fusion (please refer to online Supplementary material in Appendix A). This approach of differentiating preadipocytes in long-term tissue culture conditions provided mature monolocular adipocytes with stability in tissue culture, amenable to modification and stable observation on the long run. The abovementioned morphological changes were observed in about 50% of the transduced mature adipocytes but subsequent fusion could be observed in roughly 10% of them. It was difficult to perform immunostaining on these
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Fig. 4. Adipocytes differentiated and grown to maturity in long-term tissue culture displaying morphological changes subsequent to transduction with Adenoviral MyoD expression vector (2 109 physical particles/ml). Transduced adipocytes (T) acquired a fusiform shape and polarity in 10 days. Note the morphological difference to the non-transduced neighbor (nT) (a). This alteration in morphology is followed by subsequent progress to fusion with transduced neighbor cells (b) in 19 days. Scale bar represents 20 Am.
cells due to their extremely fragile nature. Even very mild fixation or permeabilization deformed or detached them. Desmin expression could be observed in adipocytes demonstrating morphological changes and fusion at 7 days after the transduction (data not shown). In vivo MyoD gene transfer to mature adipocytes in fat pads The interscapular and inguinal fat pads of the rats have been revealed and observed after sacrificing. The viral vector injected tissue appeared harder on the gross examination. Beginning from the 2-week time points, musclespecific proteins could be demonstrated within the transduced adipocytes. The expression of the transgene and muscle-specific proteins has been studied via immunostaining (Fig. 5). The adipocytes were discriminated specifically by immunostaining for perilipin and adipophilin which are structural proteins surrounding the fat lobule. Desmin, which is expressed as an early marker on the myogenic differentiation program, was the first muscle-specific protein observed within the adipocytes. It could be detected in the thin cytoplasm of some adipocytes within 7 to 10 days of transduction (Figs. 5a, d– g). To assess the later stages of the
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differentiation, we have checked for the presence of myosin heavy chain (Figs. 5b, h) and sarcomeric a-actinin (Figs. 5c, i –k). The presence of these two structural muscle proteins further supports the progression of the myogenic program in mature adipocytes. The lace-like staining patterns of MHC and sarcomeric a-actinin proteins suggest a nascent sarcomeric organization. a-Actinin and MHC proteins were visible by immunostaining starting about 1 week after the onset of desmin expression in adipocytes. Although the exact time-course observations could not be made due to variations between samples, it was evident that the intensity of the staining patterns of MHC and a-actinin increased over time. The mature monoloculated adipocytes transduced with MyoD vectors seemed to lose their fat content with time, acquiring a thicker cytoplasm. Likewise, on the course of advancement of the myogenic transdifferentiation, the perilipin staining of transduced adipocytes was much fainter than their untransduced neighbors (Figs. 5i, j). In addition, adipophilin staining was never visible in any adipocyte undergoing myogenic conversion, indicating a regression from adipogenic differentiation program (Fig. 5k). It has been previously demonstrated that adipocytes undergoing lipolysis under the effect of TNFa downregulated their perilipin content [25]. This phenomenon is explained by perilipin’s providing more accessibility to lipases functioning in the breakdown of the fat content [26]. None of the above observations were made with adenoviral-EGFP expression vectors injected to time-matched control animals and the myogenic conversion of the adipocytes was observed in every animal that was injected with the MyoD expression vector. We have conducted our experiments on both interscapular and inguinal fat pads as these pads can easily be reached and manipulated. Although there is a consensus in literature that while the inguinal fat pads represent more of white adipose tissue (WAT) and interscapular fat is referred as brown adipose tissue (BAT), the latter is composed of mixed lobules of multiloculated brown adipocytes surrounded by large (80 – 100 Am diameter) monoloculated white adipocytes. As brown fat tends to decrease and be replaced by white fat with age, we especially choose to use older rats to achieve a more heterogeneous adipocyte population. We observed no difference in both time points and patterns of expression of muscle proteins between these two tissues. The diameter of the fat deposit in a MyoDtransducted adipocyte seemed to decrease over time while the cytoplasm thickness increased. This is concordant with our experiments proposing that while the myogenic transcription program is ongoing, the adipogenic transcription program diminishes. This may be regarded as a dedifferentiation process where adipocytes are digressing from their original transcription program and adopting a new fate (Fig. 6). Consequently, the MyoD-induced program might be regarded as dominant to the adipogenic program. Unfortunately, adenoviral vectors are not the best choice for a long-term in vivo experiment due to the inflammatory
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Fig. 5. In vivo myogenic conversion in fat pads via Adenoviral MyoD gene delivery. Confocal analysis of muscle-specific proteins (green staining) in adipocytes from inguinal fat pads 3 weeks after viral vector injection: desmin (a), MHC (b) and a-actinin (c). The adipocyte-specific protein perilipin is shown in red. Myogenic conversion could also be achieved in interscapular adipocytes after vector injections (d – l). Ten days after vector delivery, desmin expression could be detected in the thin cytoplasm of large adipocytes (asterisk) (d). A higher magnification of a single adipocyte is provided in panel e. At a much later time point (5 weeks), cells expressing desmin had little fat content and much thicker cytoplasm indicating a massive loss of fat (f—arrowheads) compared to an earlier time point (d, e) and untransduced neighbors (f—stars). A higher magnification is provided in panel g. MHC can be detected at about 2 weeks after transduction (h). At 3 weeks, transduced adipocytes expressing MHC displayed a mixed population with varying amount of fat deposits (stars). a-Actinin staining in 4-week samples reveals a lace-like pattern resembling sarcomeric organization (green). The perilipin staining of these adipocytes (stars) were much fainter compared to their untransduced neighbors (arrowheads) (i, j). Interestingly, these adipocytes undergoing myogenic conversion were also almost negative for adipophilin (red staining) compared to untransduced neighbor adipocytes (stars) (k). Even though seldom, it was possible to observe MyoD-transduced adipocytes undergoing fusion (arrowhead) (l), nuclear staining of MyoD in red and MHC in green. Nuclei were counterstained with Dapi. Scale bars represent 10 Am in panels e, i, j; 50 Am in panel f and 20 Am elsewhere.
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Fig. 6. Selected adipocytes representing different time points (from left to right; 10, 12, 15, 21, 22, 28, 35, 42 days, respectively) illustrating the putative course of the myogenic transdifferentiation process. MHC staining in green, nuclei are counterstained with Dapi.
response they provoke [27]. We had to overcome this obstacle by transient immunosuppression with cyclophosphamide administration during the course of the study. In our in vivo experiments, we seldom observed the fusion of adipocytes (Figs. 5l). There might be a few possible explanations to this: (i) There is a rich stromal environment and a strong connective tissue support of the fat tissue that protects it from external physical trauma. The development of fusion requires strong intercellular interactions and this strong stromal support may not permit to favor the transdifferentiating adipocytes to communicate each other adequately to develop fusion. (ii) While there are a number of studies in literature that document the favorable effect of cyclophosphamide on fusion in vitro [28,29], the direct inhibitory role has also been documented in an in vivo study [30] which might be the case. (iii) Lastly, MyoD overexpression could over-differentiate transduced cells without giving any time to fuse [31].
Conclusion It was reported that MyoD could induce myogenic conversion in adipocyte precursors [12]; our data provide evidence that it is also capable of initiating the myogenic program in mature adipocytes in vivo. Both adipocytes and myotubes are terminally differentiated cells. In all the previous reported examples of transdifferentiation via transcription factors, tissue specific commitment could only be demonstrated in cells bearing proliferative capacity isolated from the mature tissue environment of cell lines [2,3]. In this study, we present evidence in three independent systems that a terminally differentiated mature adipocyte can be converted into myogenic program without prior dedifferentiation or cell cycle progression. Yet, this is the only example of transdifferentiation induction without any prior cell divisions. Apart from this, the expression of this alien transcription factor have been relatively well tolerated in these adipocytes. As the future direction of this study, we are currently working on a tissue-specific promoter to achieve MyoD expression uniquely in adipocytes. We have the intention to test this tissue-specific expression vector on the denervation-induced skeletal muscle fatty degeneration model [32]. An in vivo model is required to understand if the conversion of the adipocytes would result in organized myofibers with basement membrane.
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Fatty infiltration is the hallmark of the tissue deterioration in a wide variety of muscle disorders such as immobility, tendon release, denervation, and majorly the dystrophies. Once the underlying pathological condition is reversed, MyoD gene transfer might help to augment the contractile function of the muscle. We believe that the myogenic conversion of the adipocytes may provide therapeutic benefits to the dystrophic muscle only if the underlying genetic defect might be concomitantly corrected. We are currently investigating this approach as a potential therapeutical avenue for a number of myopathies where fatty infiltration is the hallmark of degeneration.
Acknowledgments The authors are grateful to Thibaut Marais, Bernard Gjata, and Isabelle Adamski for technical assistance. This work was supported by grants from the Association Franc¸aise Contre les Myopathies and the Centre National de la Recherche Scientifique.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.yexcr.2005. 03.038.
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Research Article
Myogenic program induction in mature fat tissue (with MyoD expression) Y.C. Kocaefea,b,*, D. Israelia, M. Ozgucb, O. Danosa, L. Garciaa,* a
Genethon CNRS UMR8115 1 bis rue de l’Internationale 91002 Evry cedex, France Hacettepe University, Faculty of Medicine, Department of Medical Biology, Ankara, Turkey
b
Received 5 July 2004, revised version received 9 March 2005 Available online 25 May 2005
Abstract MyoD exerts a master transcriptional control over the myogenic differentiation cascade. Here, we study different approaches to induce myogenic transdifferentiation in mature adipocytes utilizing MyoD gene transfer. Organotypic cultures of fat tissue and a long-term culture of in vitro differentiated adipocytes deduced that MyoD provoked morphological changes in mature adipocytes that can be summarized as loss of fat content, acquisition of a fusiform shape and eventual fusion with committed neighbor cells. In vivo, MyoD gene transfer into rat interscapular and inguinal fat pads demonstrated that while structural proteins of muscle lineage were expressed, they co-existed with specific adipocyte proteins. Expression of these proteins diminished over time likewise the fat content. The transdifferentiation process initiated by MyoD did not require cell cycle progression and was well tolerated by the fully differentiated and mature adipocytes. D 2005 Elsevier Inc. All rights reserved. Keywords: MyoD; Myogenic conversion; Transdifferentiation; Muscle dystrophy; Myogenic fusion
Introduction The switching of a differentiated cell type from one lineage to another is called transdifferentiation. Although this phenomenon is a common process in embryonic development, it is a rare event in adulthood, and is referred as metaplasia under pathologic conditions. In addition to various examples in drosophila [1], induction of transdifferentiation in tissues and organs through experimental intervention in mammals has also been demonstrated [2– 4]. Master transcription factors of specific transcription cascades are the key factors for ignition of differentiation processes. MyoD, a helix –loop –helix DNA binding transcription factor [5], is regarded as the master control over the myogenic differentiation cascade [6,7]. It binds to the enhancers of muscle specific genes through a homodimer or heterodimers
* Corresponding authors. Y.C. Kocaefe is to be contacted at Hacettepe University, Faculty of Medicine, Department of Medical Biology 06100, Ankara, Turkey. Fax: +90 312 3096060. L. Garcia, Genethon 1 bis rue de l’Internationale 91002 Evry, France. Fax: +33 1 60 778698. E-mail addresses: [email protected] (Y.C. Kocaefe), [email protected] (L. Garcia). 0014-4827/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2005.03.038
of positive acting transcriptional activators [8,9]. The downstream effectors of MyoD include other myogenic regulation factors (MRFs) such as Myf5, Myogenin, and MRF-4 [10,11]. The forced expression of MyoD triggers myogenesis in a wide range of mesenchymal cells and cell lines, a phenomenon referred to as ‘‘myogenic conversion’’ [12 – 14]. Although this phenomenon was initially shown on adipocyte cell line [12], transdifferentiation of differentiated mature adipocytes into muscle lineage has not yet been challenged. The adipogenic transcription program is initiated by PPARg and C/EBPa factors. Ectopic expression of either of these factors or upregulation of PPARg by activators can induce the adipogenic program in committed myoblasts [15] or fibroblasts [16]. While the fatty infiltration is the hallmark of the end point of many muscle diseases such as Duchenne muscular dystrophy, the origin of the adipocytes infiltrating the degenerating muscle is still unknown. Gene replacement therapy for the treatment of muscle dystrophies would apply to early-diagnosed cases before muscle deterioration. Once the fatty degeneration is established, there is no more contractile tissue left in the muscle to be cured by approaches of gene replacement therapy. However, if the conversion of the adipocytes
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residing in the degenerated muscle into myogenic program may be achieved under the frame of an organized muscle, it is conceivable that this could possibly maintain the contractile function. In this study, we have tested the possibility to induce the transdifferentiation of mature fat tissue into muscle utilizing MyoD gene transfer. We have challenged myogenic conversion in adipocytes in three different setups. Firstly, we have designed a system to obtain mature differentiated adipocytes from primary preadipocytes. Yet, to this day, no method has been described to isolate and cultivate pure primary mature adipocytes for long term. To overcome this, we have secondly utilized cultures of organ explants of fat pads, which could be maintained up to several weeks. This allowed us to compare various viral vectors to assess targeting of mature adipocytes. Lastly, we investigated the consequences of MyoD gene transfer in fat pads of rats in vivo.
Materials and methods Gene transfer vectors (i) 1st-generation serotype 5 adenovirus vector, (ii) serotype 2 adeno-associated virus (AAV), and (iii) secondgeneration lentiviral vector pseudotyped with VSVg (vesicular stomatitis virus g protein) envelope were employed to express EGFP protein driven by CMV promoter except the last, where the PGK promoter was used instead. The production methods have been described previously elsewhere [17 – 19]. We also used the same adenoviral gene expression vector for the delivery of mouse MyoD cDNA [genebank accession: 6996931] under the control of the CMV promoter. Downstream to the MyoD cDNA, the expression cassette also included an IRES (Internal Ribosomal Entry Site) and an EGFP reporter to follow the transgene expression (Clontech Laboratories, Paolo Alto, CA). Organ culture The interscapular and inguinal fat tissues of 6-month-old Sprague –Dawley rats were resected with special care to avoid contamination by surrounding superficial muscles. Tissues were then grossly dissected with a scalpel into small pieces with a diameter of a few millimeters. Afterward, samples were placed in culture flasks with adipogenic induction medium for 24 h. This medium consisted of 1:1 F12:DMEM (Gibco, France) supplemented with 10% fetal calf serum, 0.5 mM IBMX, 10 Ag/ml insulin, 1 AM dexamethasone, and 0.2 nM T3 (Sigma, France) [20]. The induction medium was removed gradually starting from the 1st day by replacing partially with an adipogenic maintenance medium in which fetal calf serum was reduced to 4% and did not contain IBMX, dexamethasone, and T3.
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Cultures were kept in 5% CO2 in 37-C incubator. The medium was partially refreshed every 2 days. The tissues were transducted at day 1 with either adenoviral or adenoassociated viral and lentiviral expression vectors harboring EGFP or MyoD. Tissues were investigated on a daily basis with fluorescent microscopy and fixated at different time points with 4% paraformaldehyde for further confocal microscopy analysis. Differentiating preadipocytes and long-term culture The inguinal and interscapular fat pads were dissected as above and were minced with scalpel into smaller pieces under dissection microscope while special care was taken to remove the vascular structures and connective tissue. The minced adipose tissue is digested with 0.5 v/w of collagenase B and dispase II (Roche Diagnostics, Mannheim, Germany) at 37-C for 1 h with gentle agitation. After washing twice and filtering through 40-Am filters, the cells were plated at near confluency in pre-treated (described in the Results and discussion section) Lab-Tek glass chambers (Nunc, Roskilde, Denmark) with 105 cells for each chamber of 1 cm2 surface area with adipogenic induction medium. Every 2 days, the medium was replaced partially with adipogenic maintenance medium. It was possible to observe the first inclusions of lipid accumulation after the first week. The preadipocytes acquire a multilocular view by their 4th week and only after the 10th to 12th week, the multilocular lipid vesicles fuse to give the cell the look of a mature monolocular adipocyte, gaining a stability in their shape and size. Animals and surgical procedures Six- to nine-month-old (mean weight 240 T 50 g) male Sprague – Dawley rats were used in the study. All animal procedures were performed according to an institutionapproved protocol and under strict biological containment. The animals were placed on a regimen of 50 Ag of cyclophosphamide delivered intraperitoneally (IP) every 72 h during the course of the experiment. The interscapular or the inguinal skin incised following 70 Ag of IP ketamine anesthesia and fat pads were revealed. A total of 50 Al (7 1010 physical particles) viral vector preparation was injected in an infiltrating manner at 18 to 20 different locations on the target fat tissues. The skin was sutured back after the intervention. At 2-, 3-, 4-, and 5-week time points, injected rats were sacrificed via pentobarbital overdose. Transducted fat tissues were resected, mounted, and fresh frozen for further analysis. Immunohistochemistry The explanted tissues from rats at different time points were investigated for the expression of muscle-specific proteins after cryosectioning. The tissue-specific antibodies used were monoclonal anti-desmin (Sigma, France), mono-
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clonal myosin heavy chain (MHC) (Novocastra, France), monoclonal anti-a-actinin (Abcam, Cambridge, UK), and guinea pig polyclonal; anti-perilipin and anti-adipophilin (Progen, Heidelberg, Germany). Transgene expression has been confirmed via polyclonal MyoD antibody (Santacruz Biotech, France). Immunostaining procedure was conducted according to antibody producers’ directions and analyzed by Bio-Rad multiphoton confocal microscopy (Bio-Rad, Hercules, USA).
Results and discussion Organotypic fat tissue explants and long-term culture of differentiated preadipocytes The main obstacle working with adipocytes is to isolate them alive and to maintain them for a sufficient period of time. Organ culture of adipose tissue has long been accepted as the model of choice for biochemistry experiments where mature adipocytes and their activities can be traced in an isolated environment for a short time [20,21]. This setup is adequate to observe the impact of a short-term modulator or the outcome of environmental changes on the adipocyte metabolism. In our hands, we could keep the explanted mature adipocytes alive for up to 3 weeks. We observed no significant difference in maintaining adipocytes from either interscapular or inguinal fat pads. During the first week, adipocytes tended to lose their fat content rapidly. This is a well-known phenomenon which has been inaccurately named ‘‘dedifferentiation’’ [22]. There is a significant number of studies using preadipocytes and/or adipofibroblasts to investigate adipogenic commitment and differentiation [23]. Although these cells could easily be handled, they naturally harbor a significant plasticity unless they are fully differentiated. We utilized an alternative approach of differentiating the primary preadipocytes to obtain mature fully differentiated adipocytes. Once the adipogenic differentiation was induced, the precursors underwent commitment and started accumulating fat vesicles (Figs. 1a– d). On the long course of incubation, the average time for these adipocytes to achieve the monolocular mature phenotype took at least 10 to 12 weeks. After the 12th week, the adipocytes were stable in size and shape in culture (Figs. 1e, f). As the adipocytes grow and acquire more fat, they tend to lose their adhesion capabilities and eventually break loose and start floating. This was the main obstacle of this method and generally occurred as the cells achieved the monolocular shape. One approach to overcome this problem was to grow the preadipocytes to confluency before differentiation, fix them with ethanol, wash repeatedly with PBS, and achieve a surface for a second round of plating and differentiation. In our hands, this approach enhanced the adhesion properties and gave better results compared to collagen-coated tissue culture chambers.
Gene transfer into mature adipocytes In the course of this study, one of the primary questions was which mode of gene transfer has to be employed to target mature adipocytes. As very little information was available in the literature, we conducted a triplicate set of experiments to assess the tropism of adenoviral (1stgeneration serotype 5), adeno-associated (serotype 2) and lentiviral (self-inactivating 2nd generation) EGFP expression vectors for the mature adipocytes. Special care was taken to observe adipocytes via epifluorescence microscopy, as they tend to diffract the light by their nature and can easily cause misinterpretation of the GFP signal. The organotypic tissue culture provided a stable short-term system to follow the reporter expression. Indeed, the organ culture of adipose tissue revealed that primary mature adipocytes could be kept alive up to 20 days maximally. Starting from the third week, it was possible to observe cells undergoing apoptosis by their morphology, as well as necrosis in the center of the tissue piece. As it was not possible to assess the exact number of cells in pieces of explanted fat tissue, viral vectors were added into medium arbitrarily at various amounts ranging from 1 108 to 7 1010 physical particles/ml. Although this approach did not achieve a meticulous titration assay, it was sufficient to sort out the best vector system for our application that was the adenoviral vectors. During the 3-week follow-up of tissues transducted with adenovirus, the GFP expression could be visualized starting from the 24th hour within the mature adipocytes (Fig. 2a). As the promoter we used had no tissue specificity, GFP was also observed in a wide variety of stromal cells, vascular structures, and endothelia within the tissue. The observations on the lentiviral-transduced tissues revealed that numerous small cells residing between the adipocytes did uptake the vector whereas no expression could be observed in mature adipocytes. Starting from the 1st week of observation, these small cells began to accumulate fat deposits (Fig. 2b). Taking into account that these organotypic cultures were carried out in the presence of adipogenic induction factors (IBMX, dexamethasone, Insulin), it is likely that lentiviral-targeted cells were the preadipocytes, which differentiated later in the course of the culture. For the adeno-associated viral vector, we observed GFP expression limited to stromal cells in between the adipocytes. Contrary to cells transduced with lentiviral vector, these cells did not differentiate into adipocytes (Figs. 2c). We concluded that in our system, adenoviral vectors were the most appropriate. The in vitro differentiated mature adipocytes also enabled us to make a further assessment of the transduction efficiency with an EGFP expression vector. The differentiation was initiated with approximately 105 cells and some of the adipocytes detached and lost on the course of the long process. The 2 109 physical particles yields 100% transduction of the non-adipocyte cells in the longterm culture but only 10 to 15% of the mature adipocytes
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Fig. 1. Differentiation and maintenance of preadipocytes in long-term tissue cultures. Panels a to f show primary preadipocytes from rat interscapular fat pads at 0, 1, 4, 8, 12, and 15 weeks, respectively. Note that the adipocytes are rather flat until the 8th week, and then acquire a round shape with increasing volume and transition to monolocular morphology. After week 12, adipocytes were stable in their size and shape. Scale bar represents 20 Am.
were observed to be GFP (+). Increasing the mount of viral vector did not enhance the ratio further. It has been previously demonstrated that stable expression of the adenovirus receptor CAR in 3T3-L1 cell line provided a 100-fold increase in the transfection efficiency via adenoviral vectors [24]. This observation suggests that the limited expression of CAR on mature adipocytes might be the obstacle restricting transfection. Myogenic conversion of mature adipocytes using Adenoviral MyoD vectors MyoD-transduced adipocytes in organotypic cultures were fixed in 4% PFA and traced under confocal microscopy. Multiple z-stack acquisition also enabled to achieve 3-D reconstitution of the images. The GFP reporter in the MyoD expression cassette permitted to trace the transduced adipocytes in living organotypic cultures. While the signal was generally weak due to the thin cytoplasm, it was
possible to observe the GFP in living mature adipocytes starting from 48 h after Adeno-MyoD transduction. Beginning from day 15, mature adipocytes underwent a number of significant morphologic changes; they developed polarity while acquiring a lemon-like shape with protruding tentaclelike extensions from poles (Fig. 3a). The control adipocytes transduced with GFP-only expressing adenoviral vectors did not demonstrate such changes. Neither the virus nor the promoter we have utilized in this study have any specificity for adipose tissue. As a consequence of that, some mesenchymal cells, fibroblasts, and perivascular pluripotent cells residing in the tissue, which were transduced, also committed to myogenic differentiation. As expected, these non-adipocyte cells harbored more plasticity and started to fuse rapidly from the 7th day onward as thin myotubes. The discrimination between these newly forming myotubes and the adipocytes were made by one simple criterion, which is ‘‘cytoplasmic monoloculated fat droplet’’. As a highly hydrophilic globular protein, the GFP was excluded from
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Fig. 2. Explanted adipocytes in organotypic cultures transduced with various EGFP expression vectors. GFP expression in mature adipocytes could be observed 2 days after transduction with recombinant adenoviral vectors (a). Lentiviral vectors only transduced some small cells surrounding mature adipocytes, which started accumulating fat droplets and presented a multilocular preadipocyte phenotype on the course of organotypic culture. Thirteen days after transduction, these preadipocytes are clearly distinguishable with their fat droplets as indicated by arrowheads (b). The adeno-associated viral vector targeted small stromal cells that did not demonstrate any fat deposits at the end of the second week (c). Scale bar represents 20 AM.
this fat droplet and concentrate in the thin cytoplasm of the adipocyte. Starting from 15th day after transduction, it was possible to observe the adipocytes that were undergoing morphological changes beginning to fuse with other committed cells in their neighborhood (Fig. 3b). It is clear
Fig. 3. Morphologic changes of mature adipocytes in organotypic culture conditions after transduction with 2 109 physical particles/ml of AdenoMyoD expression vector. By 15 days after transduction, adipocytes (TA) acquired polarity and developed tendril-like extensions from poles. With time, these extensions stretched out (arrow) to nearby myotubes (mt) (a). Further in the course of these changes, with the advancement of the loss of fat content, eventual fusion to a neighbor myotube could be observed (b). Immunostaining of MyoD appears in yellow, stars indicate fat droplets.
that, in order for an adipocyte to achieve this, it needs a remarkable time period and some loss of cytoplasmic fat content. The lack of vector specificity allowed us to observe the fusion of adipocytes with other plastic stromal cells in the neighborhood. Please refer to online supplementary documentation for animated 3-D images of these structures. Since explants of primary mature adipocytes couldn’t be kept further than 3 weeks, we assessed the effect of MyoD on mature adipocytes derived from in vitro differentiated primary preadipocytes. After the 12th week of differentiation, upcoming mature adipocytes were transduced with Adenoviral MyoD expression vector as described before. Consistent with the organotypic observations, transduced adipocytes developed polarity after the first week, which progressed to tendril-like extensions. They eventually started to fuse to neighbor cells expressing MyoD (Fig. 4) after the second week. It was also possible to observe that although keeping their round shape, some adipocytes harbored multiple nuclei showing that this fusiform morphological change is not necessary for their fusion (please refer to online Supplementary material in Appendix A). This approach of differentiating preadipocytes in long-term tissue culture conditions provided mature monolocular adipocytes with stability in tissue culture, amenable to modification and stable observation on the long run. The abovementioned morphological changes were observed in about 50% of the transduced mature adipocytes but subsequent fusion could be observed in roughly 10% of them. It was difficult to perform immunostaining on these
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Fig. 4. Adipocytes differentiated and grown to maturity in long-term tissue culture displaying morphological changes subsequent to transduction with Adenoviral MyoD expression vector (2 109 physical particles/ml). Transduced adipocytes (T) acquired a fusiform shape and polarity in 10 days. Note the morphological difference to the non-transduced neighbor (nT) (a). This alteration in morphology is followed by subsequent progress to fusion with transduced neighbor cells (b) in 19 days. Scale bar represents 20 Am.
cells due to their extremely fragile nature. Even very mild fixation or permeabilization deformed or detached them. Desmin expression could be observed in adipocytes demonstrating morphological changes and fusion at 7 days after the transduction (data not shown). In vivo MyoD gene transfer to mature adipocytes in fat pads The interscapular and inguinal fat pads of the rats have been revealed and observed after sacrificing. The viral vector injected tissue appeared harder on the gross examination. Beginning from the 2-week time points, musclespecific proteins could be demonstrated within the transduced adipocytes. The expression of the transgene and muscle-specific proteins has been studied via immunostaining (Fig. 5). The adipocytes were discriminated specifically by immunostaining for perilipin and adipophilin which are structural proteins surrounding the fat lobule. Desmin, which is expressed as an early marker on the myogenic differentiation program, was the first muscle-specific protein observed within the adipocytes. It could be detected in the thin cytoplasm of some adipocytes within 7 to 10 days of transduction (Figs. 5a, d– g). To assess the later stages of the
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differentiation, we have checked for the presence of myosin heavy chain (Figs. 5b, h) and sarcomeric a-actinin (Figs. 5c, i –k). The presence of these two structural muscle proteins further supports the progression of the myogenic program in mature adipocytes. The lace-like staining patterns of MHC and sarcomeric a-actinin proteins suggest a nascent sarcomeric organization. a-Actinin and MHC proteins were visible by immunostaining starting about 1 week after the onset of desmin expression in adipocytes. Although the exact time-course observations could not be made due to variations between samples, it was evident that the intensity of the staining patterns of MHC and a-actinin increased over time. The mature monoloculated adipocytes transduced with MyoD vectors seemed to lose their fat content with time, acquiring a thicker cytoplasm. Likewise, on the course of advancement of the myogenic transdifferentiation, the perilipin staining of transduced adipocytes was much fainter than their untransduced neighbors (Figs. 5i, j). In addition, adipophilin staining was never visible in any adipocyte undergoing myogenic conversion, indicating a regression from adipogenic differentiation program (Fig. 5k). It has been previously demonstrated that adipocytes undergoing lipolysis under the effect of TNFa downregulated their perilipin content [25]. This phenomenon is explained by perilipin’s providing more accessibility to lipases functioning in the breakdown of the fat content [26]. None of the above observations were made with adenoviral-EGFP expression vectors injected to time-matched control animals and the myogenic conversion of the adipocytes was observed in every animal that was injected with the MyoD expression vector. We have conducted our experiments on both interscapular and inguinal fat pads as these pads can easily be reached and manipulated. Although there is a consensus in literature that while the inguinal fat pads represent more of white adipose tissue (WAT) and interscapular fat is referred as brown adipose tissue (BAT), the latter is composed of mixed lobules of multiloculated brown adipocytes surrounded by large (80 – 100 Am diameter) monoloculated white adipocytes. As brown fat tends to decrease and be replaced by white fat with age, we especially choose to use older rats to achieve a more heterogeneous adipocyte population. We observed no difference in both time points and patterns of expression of muscle proteins between these two tissues. The diameter of the fat deposit in a MyoDtransducted adipocyte seemed to decrease over time while the cytoplasm thickness increased. This is concordant with our experiments proposing that while the myogenic transcription program is ongoing, the adipogenic transcription program diminishes. This may be regarded as a dedifferentiation process where adipocytes are digressing from their original transcription program and adopting a new fate (Fig. 6). Consequently, the MyoD-induced program might be regarded as dominant to the adipogenic program. Unfortunately, adenoviral vectors are not the best choice for a long-term in vivo experiment due to the inflammatory
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Fig. 5. In vivo myogenic conversion in fat pads via Adenoviral MyoD gene delivery. Confocal analysis of muscle-specific proteins (green staining) in adipocytes from inguinal fat pads 3 weeks after viral vector injection: desmin (a), MHC (b) and a-actinin (c). The adipocyte-specific protein perilipin is shown in red. Myogenic conversion could also be achieved in interscapular adipocytes after vector injections (d – l). Ten days after vector delivery, desmin expression could be detected in the thin cytoplasm of large adipocytes (asterisk) (d). A higher magnification of a single adipocyte is provided in panel e. At a much later time point (5 weeks), cells expressing desmin had little fat content and much thicker cytoplasm indicating a massive loss of fat (f—arrowheads) compared to an earlier time point (d, e) and untransduced neighbors (f—stars). A higher magnification is provided in panel g. MHC can be detected at about 2 weeks after transduction (h). At 3 weeks, transduced adipocytes expressing MHC displayed a mixed population with varying amount of fat deposits (stars). a-Actinin staining in 4-week samples reveals a lace-like pattern resembling sarcomeric organization (green). The perilipin staining of these adipocytes (stars) were much fainter compared to their untransduced neighbors (arrowheads) (i, j). Interestingly, these adipocytes undergoing myogenic conversion were also almost negative for adipophilin (red staining) compared to untransduced neighbor adipocytes (stars) (k). Even though seldom, it was possible to observe MyoD-transduced adipocytes undergoing fusion (arrowhead) (l), nuclear staining of MyoD in red and MHC in green. Nuclei were counterstained with Dapi. Scale bars represent 10 Am in panels e, i, j; 50 Am in panel f and 20 Am elsewhere.
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Fig. 6. Selected adipocytes representing different time points (from left to right; 10, 12, 15, 21, 22, 28, 35, 42 days, respectively) illustrating the putative course of the myogenic transdifferentiation process. MHC staining in green, nuclei are counterstained with Dapi.
response they provoke [27]. We had to overcome this obstacle by transient immunosuppression with cyclophosphamide administration during the course of the study. In our in vivo experiments, we seldom observed the fusion of adipocytes (Figs. 5l). There might be a few possible explanations to this: (i) There is a rich stromal environment and a strong connective tissue support of the fat tissue that protects it from external physical trauma. The development of fusion requires strong intercellular interactions and this strong stromal support may not permit to favor the transdifferentiating adipocytes to communicate each other adequately to develop fusion. (ii) While there are a number of studies in literature that document the favorable effect of cyclophosphamide on fusion in vitro [28,29], the direct inhibitory role has also been documented in an in vivo study [30] which might be the case. (iii) Lastly, MyoD overexpression could over-differentiate transduced cells without giving any time to fuse [31].
Conclusion It was reported that MyoD could induce myogenic conversion in adipocyte precursors [12]; our data provide evidence that it is also capable of initiating the myogenic program in mature adipocytes in vivo. Both adipocytes and myotubes are terminally differentiated cells. In all the previous reported examples of transdifferentiation via transcription factors, tissue specific commitment could only be demonstrated in cells bearing proliferative capacity isolated from the mature tissue environment of cell lines [2,3]. In this study, we present evidence in three independent systems that a terminally differentiated mature adipocyte can be converted into myogenic program without prior dedifferentiation or cell cycle progression. Yet, this is the only example of transdifferentiation induction without any prior cell divisions. Apart from this, the expression of this alien transcription factor have been relatively well tolerated in these adipocytes. As the future direction of this study, we are currently working on a tissue-specific promoter to achieve MyoD expression uniquely in adipocytes. We have the intention to test this tissue-specific expression vector on the denervation-induced skeletal muscle fatty degeneration model [32]. An in vivo model is required to understand if the conversion of the adipocytes would result in organized myofibers with basement membrane.
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Fatty infiltration is the hallmark of the tissue deterioration in a wide variety of muscle disorders such as immobility, tendon release, denervation, and majorly the dystrophies. Once the underlying pathological condition is reversed, MyoD gene transfer might help to augment the contractile function of the muscle. We believe that the myogenic conversion of the adipocytes may provide therapeutic benefits to the dystrophic muscle only if the underlying genetic defect might be concomitantly corrected. We are currently investigating this approach as a potential therapeutical avenue for a number of myopathies where fatty infiltration is the hallmark of degeneration.
Acknowledgments The authors are grateful to Thibaut Marais, Bernard Gjata, and Isabelle Adamski for technical assistance. This work was supported by grants from the Association Franc¸aise Contre les Myopathies and the Centre National de la Recherche Scientifique.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.yexcr.2005. 03.038.
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