Characteristics of tumors developed in mdx mice after transplantation of GFP-positive mesenchymal stem cells isolated from bone marrow of transgenic C57BL/6 mice

The purpose of the work was morphological and histochemical examination of tissue differentiation in tumors developed in mdx mice after the intramuscular transplantation of GFP-positive mesenchymal bone-marrow stem cells (MSC-GFP) derived from C576BL/6 transgenic mice and cultivated for 43–45 passages. These cells did not generate tumors in syngeneic adult C57BL/6 mice. The tumors were classified as mesenhymomas, fibrosarcomas, and sarcoma. Adipocyte and chondrocyte clusters, as well as bone areas with erythroid, myeloid, and thrombocyte hematopoiesis and neural tissue with glia cells were observed inside of tumors. Types of tissue tumor differentiation were similar to those described in the literature for MSCs induced to differente in vitro by specific treatment. However, the differentiation spectrum in MSC-GFP-produced tumors was broader than the differentiation of tumors derived from adult mouse MSCs spontaneously transformed or transfected in vitro. The results presented here, along with our previous data, demonstrate that the transfection of stem cells, including totipotent stem cells, with genetic constructs is accompanied by the destabilization of the cell genome, even if the activity of inserted gene (GFP) does not affect general cell functioning.     

Participation of bone-marrow stem cells in the differentiation of mdx mice striated muscle

Two sets of experiments were carried out. The first one involved chimeric mice, obtained by intravenously injections of bone marrow derived cells taken from transgenic C57BL/6 mice, expressing GFP, to 5 Gy X-ray irradiated mdx or C57BL/6 mice. In 2 months M. quadriceps femoris of chimeric mice were destroyed by surgical clamp. Following the next 4-5 weeks, the same muscles were studied for the presence of GFP-positive striated muscle fibres. In the case of chimeric C57BL/6 mice GFP-positive striated muscle fibres were observed in 0.3 +/- 0.5 and in 0.2 +/- 0.3 % of destroyed muscle, and in lateral (control) muscle, consequently. In the case of chimeric mdx mice, positive results were observed in 1.7 +/- 0.4 and in 0.5 +/- 0.3 % of destroyed and control muscles, respectively. In the second set of experiments, the GFP-positive bone marrow cells were used for multiple intramuscular injections to M. quadriceps femoris of C57BL/6 or mdx mice in a dose of 2 x 10(5)-5 x 10(5) cells per mouse. Before injection, GFP-positive bone marrow cells were fractionated in a 63 % Percoll solution and then were exhausted from differentiated cells by magnetic manner using CD4, CD8, CD38, CD45R, CD119, Ly-6G, and F4/80 antibodies. After 2-3 weeks, as many as 0.15 +/- 0.40 and 0.1 +/- 0.2 % of GFP-positive muscle fibres were found in injected and control muscles of C57BL/6 mice, respectively. In the case of mdx mice, the frequency of GFP-positive striated muscle fibres was 2.0 +/- 0.8 and 1.2 +/- 0.6 % for injected and control muscles, respectively. A conclusion is made that bone marrow stem cells can take part in differentiation of mdx mouse muscles after their delivery by needle injections.     

Structure of neuromuscular junctions and differentiation of striated muscle fibers in mdx mice after bone-marrow stem cell therapy

Mdx mice are an experimental model of Duchenne muscular dystrophy caused by mutations in the dystrophin gene. Repeated cycles of muscle degeneration-regeneration are common for mdx mice. Disrupted neuromuscular junctions also characterize mdx mice. The structure of mdx mice neuromuscular junctions and the differentiation of striated muscle fibers were investigated 4, 8, 16, and 24 weeks after transplantation of C57BL/6 Lin(−) bone-marrow stem cells. We found that the death of striated muscle fibers decreased 4 weeks after the transplantation of bone-marrow stem cells. Accumulation of muscle fibers without centrally located nuclei began in 8 weeks and dystrophin synthesis increased in 16–24 weeks after the bone-marrow stem cells transplantation. On the longitudinal sections of quadriceps muscle of mdx mice 4 weeks after transplantation, we observed a reduced quantity of acetylcholine receptor clusters and an increase in their area in neuromuscular junctions. Sixteen weeks after the transplantation, the total area of neuromuscular junctions increased due to an enlarged number of acethylcholine receptors and their extended area. The single intramuscular transplantation of C57BL/6 Lin(−) bone-marrow stem cells induces the differentiation of mdx mice striated muscle fibers and improves the structure of neuromuscular junctions.     

Persistence of a chimerical phenotype after hepatocyte differentiation of human bone marrow mesenchymal stem cells

Abstract.  Objectives : Recent studies have suggested the potential of mesenchymal stem cells (MSCs) to differentiate into a hepatocyte-like lineage. Here, we evaluate the efficacy of hepatocyte differentiation of MSCs by studying acquisition of hepatocyte-like features together with alteration of the native mesenchymal phenotype. Material and methods : In vitro , we have investigated protein and mRNA level expression of hepatocyte and mesenchymal markers of mesenchymal-derived hepatocyte-like cells (MDHLCs) and we have evaluated their functionality using metabolic assays. In vivo , we investigated co-expression of hepatocyte (albumin, α-foetoprotein, cytokeratin 18) and mesenchymal (fibronectin, vimentin) markers after transplantation of MSCs or MDHLCs into severe combined immune deficiency mice. Results : We observed that while in vitro these cells acquired some phenotypic and functional features of mature hepatocytes, they partially preserved their mesenchymal phenotype. After intrasplenic transplantation, engrafted MSCs with isolated expression of fibronectin and α-foetoprotein were observed. When these cells were injected into the liver, they expressed all analysed markers, confirming the chimaeric co-expression observed in vitro . Conversely, liver-engrafted MDHLCs conserved their hepatocyte-lineage markers but lost their chimaeric phenotype. Conclusions : Hepatocyte differentiation of MSCs predominantly allows the acquisition of phenotypic hallmarks and provides chimaeric cells that maintain expression of initial lineage markers. However, advanced maturation to the hepatocyte-like phenotype could be obtained in vivo by conditioning MSCs prior to transplantation or by infusing cells into the liver micro-environment.     

Microdystrophin基因重组腺病毒的构建及转染mdx骨髓间充质干细胞的表达

构建含有人microdystrophin基因的重组腺病毒,来感染dystrophin基因敲除小鼠mdx的骨髓间充质干细胞(ＭＳＣ)进行基因修饰,为同种异体基因修饰的干细胞移植治疗ＤＭＤ疾病奠定基础。用NotⅠ酶切含microdystrophin基因的pBSK-MICRO质粒,获得microdystrophin基因。片段回收后定向插入腺病毒穿梭质粒pShuttle-CMV,获得重组质粒pShuttle-CMV-MICRO。PmeⅠ线性化重组质粒pShuttle-CMV-MICRO,去磷酸化后回收后与腺病毒骨架质粒pAdeasy-1共电转化BJ5183感受态细胞。同源重组后用选择性培养基筛选阳性克隆,提取质粒,用脂质体介导转染293细胞,通过观察293细胞病变及PCR扩增目的基因等方法鉴定重组的腺病毒。然后将病毒上清转染DMD模型鼠mdx小鼠的骨髓间充质干细胞,通过RT-PCR以及间接免疫荧光检测microdystrophin的转录及蛋白表达。成功构建了含有microdystrophin基因的重组腺病毒,病毒滴度为5.58×10¹²vp/mL。间接免疫荧光检测可见microdystrophin蛋白在mdx小鼠MSCs中高效表达。该重组腺病毒载体的构建及成功转染到mdx MSCs内表达为下一步用microdystrophin基因修饰的mdx MSCs进行同种异体移植治疗ＤＭＤ疾病奠定了基础。     

Effects of FGF2 and FGF9 on osteogenic differentiation of bone marrow-derived progenitors

Bone repair is a major concern in reconstructive surgery. Transplants containing osteogenically committed mesenchymal stem cells (MSCs) provide an alternative source to the currently used autologous bone transplants which have limited supply and require additional surgery to the patient. A major drawback, however is the lack of a critical mass of cells needed for successful transplantation. The purpose of the present study was to test the effects of FGF2 and FGF9 on expansion and differentiation of MSCs in order to establish an optimal culture protocol resulting in sufficient committed osteogenic cells required for successful in vivo transplantation. Bone marrow-derived MSCs cultured in αMEM medium supplemented with osteogenic supplements for up to three passages (control medium), were additionally treated with FGF2 and FGF9 in various combinations. Cultures were evaluated for viability, calcium deposition and in vivo osteogenic capacity by testing subcutaneous transplants in nude mice. FGF2 had a positive effect on the proliferative capacity of cultured MSCs compared to FGF9 and control medium treated cultures. Cultures treated with FGF2 followed by FGF9 showed an increased amount of extracted Alizarin red indicating greater osteogenic differentiation. Moreover, the osteogenic capacity of cultured cells transplanted in immunodeficient mice revealed that cells that were subjected to treatment with FGF2 in the first two passages and subsequently to FGF9 in the last passage only, were more successful in forming new bone. It is concluded that the protocol using FGF2 prior to FGF9 is beneficial to cell expansion and commitment, resulting in higher in vivo bone formation for successful bone tissue engineering.     

Bioimaging for the monitoring of the in vivo distribution of infused mesenchymal stem cells in a mouse model of the graft-versus-host reaction

Cell therapy using MSCs (mesenchymal stem cells) might be effective treatment for refractory GVHD (graft-versus-host disease). However, the fate and distribution of MSCs after transplantation remains unclear. In this study, an animal model was developed to monitor the dynamic distribution of MSCs in mice with GVHD. A GVHD mouse model was established by transplanting C57BL/6 donor bone marrow cells and C57BL/6 EGFP (enhanced green fluorescent protein) splenocytes into lethally irradiated BALB/c nude recipient mice. Donor MSCs were obtained from MHC-identical C57BL/6 RFP (red fluorescent protein) mice and infused into the recipient mice on the same transplantation day. In vivo movement of the donor splenocytes (EGFP) and MSCs (RFP) were evaluated by measuring the biofluorescence (IVIS-Xenogen system). Donor splenocytes and MSCs reached the lungs first, and then the gastrointestinal tract, lymph nodes and skin, in that order; the transit time and localization site of these cells were very similar. In the recipient mouse with GVHD, the number of detectable cells declined with time, as assessed by biofluorescence imaging and confirmed by RT (real-time)-PCR. This bioimaging system might be useful for preclinical testing and the design of therapeutic strategies for monitoring the dynamic distribution of MSCs with GVHD.     

Mesenchymal stem cells are recruited into wounded skin and contribute to wound repair by transdifferentiation into multiple skin cell type

Mesenchymal stem cells (MSCs) can differentiate not only into mesenchymal lineage cells but also into various other cell lineages. As MSCs can easily be isolated from bone marrow, they can be used in various tissue engineering strategies. In this study, we assessed whether MSCs can differentiate into multiple skin cell types including keratinocytes and contribute to wound repair. First, we found keratin 14-positive cells, presumed to be keratinocytes that transdifferentiated from MSCs in vitro. Next, we assessed whether MSCs can transdifferentiate into multiple skin cell types in vivo. At sites of mouse wounds that had been i.v. injected with MSCs derived from GFP transgenic mice, we detected GFP-positive cells associated with specific markers for keratinocytes, endothelial cells, and pericytes. Because MSCs are predominantly located in bone marrow, we investigated the main MSC recruitment mechanism. MSCs expressed several chemokine receptors; especially CCR7, which is a receptor of SLC/CCL21, that enhanced MSC migration. Finally, MSC-injected mice underwent rapid wound repaired. Furthermore, intradermal injection of SLC/CCL21 increased the migration of MSCs, which resulted in an even greater acceleration of wound repair. Taken together, we have demonstrated that MSCs contribute to wound repair via processes involving MSCs differentiation various cell components of the skin.     

小鼠间充质干细胞诱导分化成脂肪细胞miRNA表达的变化

目的：探讨小鼠间充质干细胞（MSCs）定向诱导分化成脂肪细胞微小RNA（miRNA）表达的变化,为进一步研究miRNA调控MSCs向脂肪细胞分化的分子机制奠定基础。方法：采用全骨髓体外分离结合差速贴壁法纯化扩增C57BL／6小鼠MSCs,形态学观察细胞生长情况,并用免疫组化方法鉴定细胞表面抗原CD29、CIM4和CD34的表达。脂肪细胞分化诱导剂诱导MSCs分化为脂肪细胞,利用油红O染色,判断MSCs成脂分化情况。运用rrfiRNA芯片技术检测MSC8和脂肪细胞中差异表达的miRNA。结果：①倒置显微镜下观察,传5代后可获得均一性较高的MSCs;免疫组化显示90％以上的骨髓间质干细胞CD29、CD44阳性,CD34阴性。MSCs经脂肪诱导剂诱导后,胞内大量脂滴形成,油红O染色阳性;②基因微阵列分析表明,小鼠MSCs分化成脂肪细胞差异表达的miRNA共75个,其中20个表达上调、55个表达下调。结论：MSCs分化成脂肪细胞存在miRNA表达的变化,某些miRNA很可能具有重要的调控MSCs成脂分化的作用。     

BM stem cell transplantation rescues pathophysiologic features of aged dystrophic mdx muscle

BACKGROUND: The value of transplantation of BM stem cells in aged (12-month-old) mdx was evaluated because it is thought to be a more ideal model for studying the praxiology of Duchenne muscular dystrophy (DMD). The possible mechanisms of stem cell differentiation were then discussed. METHODS: BM was isolated from 8-10-week-old male C57 BL/10 mice. After injecting BM cells into 12-month-old female mdx mice through the tail vein, the expression of dystrophin and MyoD was detected at different time points by immunofluorescence staining, RT-PCR and Western blot. RESULTS: The C57 male mice donor-specific and Y-chromosome-specific sequence could be detected in all female aged mdx mice, implying the success of the transplantation. Expression of dystrophin and MyoD was detected and increased over time. DISCUSSION: BM cells were recruited to the muscle and partially restored specific pathophysiologic features of the dystrophic muscle in aged mdx mice. Muscle differentiation of BM cells recapitulated embryonic myogenesis.     

Establishment of a preadipocyte cell line derived from mature adipocytes of GFP transgenic mice and formation of adipose tissue

We established a preadipocyte cell line from mature adipocytes obtained from subcutaneous fat tissue of green fluorescent protein (GFP) transgenic mice. The floating top layer, containing mature adipocytes, was isolated from subcutaneous fat tissue by collagenase digestion and filtration. Fluorescence-activated cell sorting and microscopic analysis revealed that the floating cell fraction comprised a highly homogeneous adipocyte population with no adipose stromal-vascular cells. Isolated mature adipocytes dedifferentiated into fibroblast-like cells and actively proliferated in ceiling culture. In vitro studies showed that the cells could redifferentiate into mature adipocytes in an identical way to 3T3-L1 preadipocytes. No changes in the differentiation pattern were observed during the propagation of our cells. They were successfully maintained and differentiated for at least 22 passages. We named these cells dedifferentiated fat (DFAT-GFP) cells. When DFAT-GFP cells were implanted subcutaneously into C57BL/6N mice, they developed highly vascularized fat pads that morphologically resembled normal subcutaneous adipose tissue and consisted of GFP-positive cells; however, implanted 3T3-L1 cells did not have such an effect on the mice. We conclude that DFAT-GFP cells provide a model that should enable us to study the mechanisms of adipocyte differentiation and adipose tissue formation in vivo and in vitro. This work was supported by grants from the Japan Ministry of Education, Science, Sports, and Culture (no. 19580348) and from MEXT. HAITEKU (2007–2011).     

Stem cells from umbilical cord blood differentiate into myotubes and express dystrophin in vitro only after exposure to in vivo muscle environment

BACKGROUND INFORMATION: Duchenne muscular dystrophy is a disease characterized by progressive and irreversible muscle degeneration for which there is no therapy. HUCB (human umbilical cord blood) has been considered as an important source of haematopoietic and mesenchymal stem cells, each having been shown to differentiate into distinct cell types. However, it remains unclear if these cells are able to differentiate into muscle cells. RESULTS: We have showed that stem cells from HUCB did not differentiate into myotubes or express dystrophin when cultured in muscle-conditioned medium or with human muscle cells. However, delivery of GFP (green fluorescent protein)-transduced mononucleated cells from HUCB, which comprises both haematopoietic and mesenchymal populations, into quadriceps muscle of mdx (mouse dystrophy X-chromosome linked) mice resulted in the expression of human myogenic markers. After recovery of these cells from mdx muscle and in vitro cultivation, they were able to fuse and form GFP-positive myotubes that expressed dystrophin. CONCLUSIONS: These results indicate that chemical factors and cell-to-cell contact provided by in vitro conditions were not enough to trigger the differentiation of stem cells into muscle cells. Nevertheless, we showed that the HUCB-derived stem cells were capable of acquiring a muscle phenotype after exposure to an in vivo muscle environment, which was required to activate the differentiation programme.     

Bone-derived mesenchymal stromal cells from HIV transgenic mice exhibit altered proliferation,differentiation capacity and paracrine functions along with impaired therapeutic potential in kidney injury

Mesenchymal stem cells (MSCs) secrete paracrine factors that could be cytoprotective and serve roles in immunoregulation during tissue injury. Although MSCs express HIV receptors, and co-receptors, and are susceptible to HIV infection, whether HIV-1 may affect biological properties of MSCs needs more study. We evaluated cellular proliferation, differentiation and paracrine functions of MSCs isolated from compact bones of healthy control mice and Tg26 HIV-1 transgenic mice. The ability of MSCs to protect against cisplatin toxicity was studied in cultured renal tubular cells as well as in intact mice. We successfully isolated MSCs from healthy mice and Tg26 HIV-1 transgenic mice and found the latter expressed viral Nef, Vpu, NL4-3 and Vif genes. The proliferation and differentiation of Tg26 HIV-1 MSCs was inferior to MSCs from healthy mice. Moreover, transplantation of Tg26 HIV-1 MSCs less effectively improved outcomes compared with healthy MSCs in mice with acute kidney injury. Also, Tg26 HIV-1 MSCs secreted multiple cytokines, but at significantly lower levels than healthy MSCs, which resulted in failure of conditioned medium from these MSCs to protect cultured renal tubular cells from cisplatin toxicity. Therefore, HIV-1 had adverse biological effects on MSCs extending to their proliferation, differentiation, function, and therapeutic potential. These findings will help in advancing mechanistical insight in renal injury and repair in the setting of HIV-1 infection.     

Transgenic mice produced by retroviral transduction of male germ line stem cells in vivo

Spermatogonial stem cells are the only stem cells in the postnatal body that can transmit parental genetic information to the offspring, making them an attractive target cell population for animal transgenesis. Although transgenic mice and rats were recently produced by retrovirus transduction of these cells in vitro, with transplantation of the transduced cells into infertile recipients, the difficulty of restoring fertility and preparing recipients using spermatogonial transplantation limits practical application of the technique. In this article, we describe a novel approach for producing transgenic animals by transducing spermatogonial stem cells in vivo using a retrovirus vector. Microinjection of retrovirus into immature seminiferous tubules resulted in the direct transduction of spermatogonial stem cells in situ, and the animals produced transgenic offspring after mating with females. Transgenic mice were produced in C57BL/6, BALB/C, A, and C3H backgrounds, with an average efficiency of 2.8%. The transgene was transmitted stably and expressed in the next generation. The technique overcomes the drawback of the in vitro-transduction approach, and will be useful as a novel method for producing transgenic animals as well as providing a means for analyzing the self-renewal and differentiation processes of spermatogonial stem cells in vivo.     

Treatment with chicken-egg-white or whole-egg extracts maintains and enhances the survival and differentiation of spleen cells

The identification of egg extracts with the ability to maintain and enhance the survival and differentiation of cells would be widely useful in cellular biology research. In this study, we compared the different abilities of spleen cells to survive and differentiate in vivo after permeabilization by five different types of egg extracts. Five types of egg extracts were prepared. The spleen cells from male GFP-transgenic mice were permeabilized by the extracts for 30 min, cultured for 12 days, and then transfused into irradiated female mice. At varying days after transplantation, the percentage of GFP-expressing surviving spleen cells was detected in the peripheral blood by flow cytometry. At 120 days after transplantation, bone marrow cells from the female mice were analyzed for the presence of cells containing the Y chromosome. Surviving GFP-positive spleen cells that had been permeabilized with either chicken-egg-white or whole-egg extracts could be detected in the female mice after transplantation. A lower percentage of GFP-positive cells was also detected after permeabilization by the other extracts tested, and no GFP-positive cells were found in the female mouse transfused with spleen cells permeabilized with Hank’s Buffered Salt Solution (HBSS) as a control. At 120 days after transplantation, the percentage of cells containing a Y chromosome in the bone marrow positively correlated with the percentage of GFP-positive cells in the peripheral blood. After permeabilization by chicken-egg-white or whole-egg extracts, spleen cells demonstrated significantly enhanced survival and differentiation functions compared with the spleen cells treated with the other egg extracts tested. These results show that chicken-egg-white and whole-egg extracts have roles in maintaining and enhancing the survival and differentiation of spleen cells. Therefore, these two types of extracts may be of future use in maintaining the function of stem cells.     

Human intronless genes: functional groups, associated diseases, evolution, and mRNA processing in absence of splicing

Duchenne muscular dystrophy is the most prevalent inheritable muscle disease. Transplantation of autologous stem cells with gene direction is an ideal therapeutic approach for the disease. The current study aimed to investigate the restoration of myofibers in mdx mice after mdx bone marrow-derived mesenchymal stem cell (mMSC) transplantation with human microdystrophin delivery. Possible mechanisms of action were also studied. In our research, mMSCs were successfully transduced by retrovirus carrying a functional human microdystrophin gene. Transplantation of transduced mMSCs enabled persistent dystrophin restoration in the skeletal muscle of mdx mice up to the 12th week after transplantation. Simultaneous coexpression of human microdystrophin and desmin showed that implanted mMSCs are capable of long-term survival as muscle satellite cells.     

Telomerase deficiency impairs differentiation of mesenchymal stem cells

Expression of telomerase activity presumably is involved in maintaining self-replication and the undifferentiated state of stem cells. Adult mouse bone marrow mesenchymal stem cells (mMSCs) are multipotential cells capable of differentiating into a variety of lineage cell types, including adipocytes and chondrocytes. Here we show that the lacking telomerase of mMSC lose multipotency and the capacity to differentiate. Primary cultures of mMSCs were obtained from both telomerase knockout (mTR(-/-)) and wild-type (WT) mice. The MSCs isolated from mTR(-/-) mice failed to differentiate into adipocytes and chondrocytes, even at early passages, whereas WT MSCs were capable of differentiation. Consistent with other cell types, late passages mTR(-/-)MSCs underwent senescence and were accompanied by telomere loss and chromosomal end-to-end fusions. These results suggest that in addition to its known role in cell replication, telomerase is required for differentiation of mMSCs in vitro. This work may be significant for further potentiating adult stem cells for use in tissue engineering and gene therapy and for understanding the significance of telomerase expression in the process of cell differentiation.     

Mesenchymal stromal cells promote tumor growth through the enhancement of neovascularization

Mesenchymal stromal cells (MSCs), also called mesenchymal stem cells, migrate and function as stromal cells in tumor tissues. The effects of MSCs on tumor growth are controversial. In this study, we showed that MSCs increase proliferation of tumor cells in vitro and promote tumor growth in vivo. We also further analyzed the mechanisms that underlie these effects. For use in in vitro and in vivo experiments, we established a bone marrow-derived mesenchymal stromal cell line from cells isolated in C57BL/6 mice. Effects of murine MSCs on tumor cell proliferation in vitro were analyzed in a coculture model with B16-LacZ cells. Both coculture with MSCs and treatment with MSC-conditioned media led to enhanced growth of B16-LacZ cells, although the magnitude of growth stimulation in cocultured cells was greater than that of cells treated with conditioned media. Co-injection of B16-LacZ cells and MSCs into syngeneic mice led to increased tumor size compared with injection of B16-LacZ cells alone. Identical experiments using Lewis lung carcinoma (LLC) cells instead of B16-LacZ cells yielded similar results. Consistent with a role for neovascularization in MSC-mediated tumor growth, tumor vessel area was greater in tumors resulting from co-injection of B16-LacZ cells or LLCs with MSCs than in tumors induced by injection of cancer cells alone. Co-injected MSCs directly supported the tumor vasculature by localizing close to vascular walls and by expressing an endothelial marker. Furthermore, secretion of leukemia inhibitory factor, macrophage colony-stimulating factor, macrophage inflammatory protein-2 and vascular endothelial growth factor was increased in cocultures of MSCs and B16-LacZ cells compared with B16-LacZ cells alone. Together, these results indicate that MSCs promote tumor growth both in vitro and in vivo and suggest that tumor promotion in vivo may be attributable in part to enhanced angiogenesis.     

Engraftment of mesenchymal stem cells into dystrophin-deficient mice is not accompanied by functional recovery

Mesenchymal stem cell preparations have been proposed for muscle regeneration in musculoskeletal disorders. Although MSCs have great in vitro expansion potential and possess the ability to differentiate into several mesenchymal lineages, myogenesis has proven to be much more difficult to induce. We have recently demonstrated that Pax3, the master regulator of the embryonic myogenic program, enables the in vitro differentiation of a murine mesenchymal stem cell line (MSCB9-Pax3) into myogenic progenitors. Here we show that injection of these cells into cardiotoxin-injured muscles of immunodeficient mice leads to the development of muscle tumors, resembling rhabdomyosarcomas. We then extended these studies to primary human mesenchymal stem cells (hMSCs) isolated from bone marrow. Upon genetic modification with a lentiviral vector encoding PAX3, hMSCs activated the myogenic program as demonstrated by expression of myogenic regulatory factors. Upon transplantation, the PAX3-modified MSCs did not generate rhabdomyosarcomas but rather, resulted in donor-derived myofibers. These were found at higher frequency in PAX3-transduced hMSCs than in mock-transduced MSCs. Nonetheless, neither engraftment of PAX3-modified or unmodified MSCs resulted in improved contractility. Thus these findings suggest that limitations remain to be overcome before MSC preparations result in effective treatment for muscular dystrophies.