Comparison of Muscle Regeneration after BMSC-Conditioned Medium, Syngeneic, or Allogeneic BMSC Injection.

For many years optimal treatment for dysfunctional skeletal muscle characterized, for example, by impaired or limited regeneration, has been searched. Among the crucial factors enabling its development is finding the appropriate source of cells, which could participate in tissue reconstruction or serve as an immunomodulating agent (limiting immune response as well as fibrosis, that is, connective tissue formation), after transplantation to regenerating muscles. MSCs, including those derived from bone marrow, are considered for such applications in terms of their immunomodulatory properties, as their naive myogenic potential is rather limited. Injection of autologous (syngeneic) or allogeneic BMSCs has been or is currently being tested and compared in many potential clinical treatments. In the present study, we verified which approach, that is, the transplantation of either syngeneic or allogeneic BMSCs or the injection of BMSC-conditioned medium, would be the most beneficial for skeletal muscle regeneration. To properly assess the influence of the tested treatments on the inflammation, the experiments were carried out using immunocompetent mice, which allowed us to observe immune response. Combined analysis of muscle histology, immune cell infiltration, and levels of selected chemokines, cytokines, and growth factors important for muscle regeneration, showed that muscle injection with BMSC-conditioned medium is the most beneficial strategy, as it resulted in reduced inflammation and fibrosis development, together with enhanced new fiber formation, which may be related to, i.e., elevated level of IGF-1. In contrast, transplantation of allogeneic BMSCs to injured muscles resulted in a visible increase in the immune response, which hindered regeneration by promoting connective tissue formation. In comparison, syngeneic BMSC injection, although not detrimental to muscle regeneration, did not result in such significant improvement as CM injection.

Non-Coding RNAs as Regulators of Myogenesis and Postexercise Muscle Regeneration.

miRNAs and lncRNAs do not encode proteins, but they play an important role in the regulation of gene expression. They differ in length, biogenesis, and mode of action. In this work, we focus on the selected miRNAs and lncRNAs involved in the regulation of myogenesis and muscle regeneration. We present selected miRNAs and lncRNAs that have been shown to control myogenic differentiation and show that manipulation of their levels could be used to improve myogenic differentiation of various types of stem and progenitor cells. Finally, we discuss how physical activity affects miRNA and lncRNA expression and how it affects muscle well-being.

Comparison of Differentiation Pattern and WNT/SHH Signaling in Pluripotent Stem Cells Cultured under Different Conditions.

Pluripotent stem cells (PSCs) are characterized by the ability to self-renew as well as undergo multidirectional differentiation. Culture conditions have a pivotal influence on differentiation pattern. In the current study, we compared the fate of mouse PSCs using two culture media: (1) chemically defined, free of animal reagents, and (2) standard one relying on the serum supplementation. Moreover, we assessed the influence of selected regulators (WNTs, SHH) on PSC differentiation. We showed that the differentiation pattern of PSCs cultured in both systems differed significantly: cells cultured in chemically defined medium preferentially underwent ectodermal conversion while their endo- and mesodermal differentiation was limited, contrary to cells cultured in serum-supplemented medium. More efficient ectodermal differentiation of PSCs cultured in chemically defined medium correlated with higher activity of SHH pathway while endodermal and mesodermal conversion of cells cultured in serum-supplemented medium with higher activity of WNT/JNK pathway. However, inhibition of either canonical or noncanonical WNT pathway resulted in the limitation of endo- and mesodermal conversion of PSCs. In addition, blocking WNT secretion led to the inhibition of PSC mesodermal differentiation, confirming the pivotal role of WNT signaling in this process. In contrast, SHH turned out to be an inducer of PSC ectodermal, not mesodermal differentiation.

PAX7 Balances the Cell Cycle Progression via Regulating Expression of Dnmt3b and Apobec2 in Differentiating PSCs.

PAX7 transcription factor plays a crucial role in embryonic myogenesis and in adult muscles in which it secures proper function of satellite cells, including regulation of their self renewal. PAX7 downregulation is necessary for the myogenic differentiation of satellite cells induced after muscle damage, what is prerequisite step for regeneration. Using differentiating pluripotent stem cells we documented that the absence of functional PAX7 facilitates proliferation. Such action is executed by the modulation of the expression of two proteins involved in the DNA methylation, i.e., Dnmt3b and Apobec2. Increase in Dnmt3b expression led to the downregulation of the CDK inhibitors and facilitated cell cycle progression. Changes in Apobec2 expression, on the other hand, differently impacted proliferation/differentiation balance, depending on the experimental model used.

Hypoxia preconditioned bone marrow-derived mesenchymal stromal/stem cells enhance myoblast fusion and skeletal muscle regeneration.

BACKGROUND: The skeletal muscle reconstruction occurs thanks to unipotent stem cells, i.e., satellite cells. The satellite cells remain quiescent and localized between myofiber sarcolemma and basal lamina. They are activated in response to muscle injury, proliferate, differentiate into myoblasts, and recreate myofibers. The stem and progenitor cells support skeletal muscle regeneration, which could be disturbed by extensive damage, sarcopenia, cachexia, or genetic diseases like dystrophy. Many lines of evidence showed that the level of oxygen regulates the course of cell proliferation and differentiation. METHODS: In the present study, we analyzed hypoxia impact on human and pig bone marrow-derived mesenchymal stromal cell (MSC) and mouse myoblast proliferation, differentiation, and fusion. Moreover, the influence of the transplantation of human bone marrow-derived MSCs cultured under hypoxic conditions on skeletal muscle regeneration was studied. RESULTS: We showed that bone marrow-derived MSCs increased VEGF expression and improved myogenesis under hypoxic conditions in vitro. Transplantation of hypoxia preconditioned bone marrow-derived MSCs into injured muscles resulted in the improved cell engraftment and formation of new vessels. CONCLUSIONS: We suggested that SDF-1 and VEGF secreted by hypoxia preconditioned bone marrow-derived MSCs played an essential role in cell engraftment and angiogenesis. Importantly, hypoxia preconditioned bone marrow-derived MSCs more efficiently engrafted injured muscles; however, they did not undergo myogenic differentiation.

Beneficial Effect of IL-4 and SDF-1 on Myogenic Potential of Mouse and Human Adipose Tissue-Derived Stromal Cells.

Under physiological conditions skeletal muscle regeneration depends on the satellite cells. After injury these cells become activated, proliferate, and differentiate into myofibers reconstructing damaged tissue. Under pathological conditions satellite cells are not sufficient to support regeneration. For this reason, other cells are sought to be used in cell therapies, and different factors are tested as a tool to improve the regenerative potential of such cells. Many studies are conducted using animal cells, omitting the necessity to learn about human cells and compare them to animal ones. Here, we analyze and compare the impact of IL-4 and SDF-1, factors chosen by us on the basis of their ability to support myogenic differentiation and cell migration, at mouse and human adipose tissue-derived stromal cells (ADSCs). Importantly, we documented that mouse and human ADSCs differ in certain reactions to IL-4 and SDF-1. In general, the selected factors impacted transcriptome of ADSCs and improved migration and fusion ability of cells in vitro. In vivo, after transplantation into injured muscles, mouse ADSCs more eagerly participated in new myofiber formation than the human ones. However, regardless of the origin, ADSCs alleviated immune response and supported muscle reconstruction, and cytokine treatment enhanced these effects. Thus, we documented that the presence of ADSCs improves skeletal muscle regeneration and this influence could be increased by cell pretreatment with IL-4 and SDF-1.

Pax7 as molecular switch regulating early and advanced stages of myogenic mouse ESC differentiation in teratomas.

BACKGROUND: Pluripotent stem cells present the ability to self-renew and undergo differentiation into any cell type building an organism. Importantly, a lot of evidence on embryonic stem cell (ESC) differentiation comes from in vitro studies. However, ESCs cultured in vitro do not necessarily behave as cells differentiating in vivo. For this reason, we used teratomas to study early and advanced stages of in vivo ESC myogenic differentiation and the role of Pax7 in this process. Pax7 transcription factor plays a crucial role in the formation and differentiation of skeletal muscle precursor cells during embryonic development. It controls the expression of other myogenic regulators and also acts as an anti-apoptotic factor. It is also involved in the formation and maintenance of satellite cell population. METHODS: In vivo approach we used involved generation and analysis of pluripotent stem cell-derived teratomas. Such model allows to analyze early and also terminal stages of tissue differentiation, for example, terminal stages of myogenesis, including the formation of innervated and vascularized mature myofibers. RESULTS: We determined how the lack of Pax7 function affects the generation of different myofiber types. In Pax7-/- teratomas, the skeletal muscle tissue occupied significantly smaller area, as compared to Pax7+/+ ones. The proportion of myofibers expressing Myh3 and Myh2b did not differ between Pax7+/+ and Pax7-/- teratomas. However, the area of Myh7 and Myh2a myofibers was significantly lower in Pax7-/- ones. Molecular characteristic of skeletal muscles revealed that the levels of mRNAs coding Myh isoforms were significantly lower in Pax7-/- teratomas. The level of mRNAs encoding Pax3 was significantly higher, while the expression of Nfix, Eno3, Mck, Mef2a, and Itga7 was significantly lower in Pax7-/- teratomas, as compared to Pax7+/+ ones. We proved that the number of satellite cells in Pax7-/- teratomas was significantly reduced. Finally, analysis of neuromuscular junction localization in samples prepared with the iDISCO method confirmed that the organization of neuromuscular junctions in Pax7-/- teratomas was impaired. CONCLUSIONS: Pax7-/- ESCs differentiate in vivo to embryonic myoblasts more readily than Pax7+/+ cells. In the absence of functional Pax7, initiation of myogenic differentiation is facilitated, and as a result, the expression of mesoderm embryonic myoblast markers is upregulated. However, in the absence of functional Pax7 neuromuscular junctions, formation is abnormal, what results in lower differentiation potential of Pax7-/- ESCs during advanced stages of myogenesis.

The Survey of Cells Responsible for Heterotopic Ossification Development in Skeletal Muscles-Human and Mouse Models.

Heterotopic ossification (HO) manifests as bone development in the skeletal muscles and surrounding soft tissues. It can be caused by injury, surgery, or may have a genetic background. In each case, its development might differ, and depending on the age, sex, and patient's conditions, it could lead to a more or a less severe outcome. In the case of the injury or surgery provoked ossification development, it could be, to some extent, prevented by treatments. As far as genetic disorders are concerned, such prevention approaches are highly limited. Many lines of evidence point to the inflammatory process and abnormalities in the bone morphogenetic factor signaling pathway as the molecular and cellular backgrounds for HO development. However, the clear targets allowing the design of treatments preventing or lowering HO have not been identified yet. In this review, we summarize current knowledge on HO types, its symptoms, and possible ways of prevention and treatment. We also describe the molecules and cells in which abnormal function could lead to HO development. We emphasize the studies involving animal models of HO as being of great importance for understanding and future designing of the tools to counteract this pathology.

IL-4 and SDF-1 Increase Adipose Tissue-Derived Stromal Cell Ability to Improve Rat Skeletal Muscle Regeneration.

Skeletal muscle regeneration depends on the satellite cells, which, in response to injury, activate, proliferate, and reconstruct damaged tissue. However, under certain conditions, such as large injuries or myopathies, these cells might not sufficiently support repair. Thus, other cell populations, among them adipose tissue-derived stromal cells (ADSCs), are tested as a tool to improve regeneration. Importantly, the pro-regenerative action of such cells could be improved by various factors. In the current study, we tested whether IL-4 and SDF-1 could improve the ability of ADSCs to support the regeneration of rat skeletal muscles. We compared their effect at properly regenerating fast-twitch EDL and poorly regenerating slow-twitch soleus. To this end, ADSCs subjected to IL-4 and SDF-1 were analyzed in vitro and also in vivo after their transplantation into injured muscles. We tested their proliferation rate, migration, expression of stem cell markers and myogenic factors, their ability to fuse with myoblasts, as well as their impact on the mass, structure and function of regenerating muscles. As a result, we showed that cytokine-pretreated ADSCs had a beneficial effect in the regeneration process. Their presence resulted in improved muscle structure and function, as well as decreased fibrosis development and a modulated immune response.

The factors present in regenerating muscles impact bone marrow-derived mesenchymal stromal/stem cell fusion with myoblasts.

BACKGROUND: Satellite cells, a population of unipotent stem cells attached to muscle fibers, determine the excellent regenerative capability of injured skeletal muscles. Myogenic potential is also exhibited by other cell populations, which exist in the skeletal muscles or come from other niches. Mesenchymal stromal/stem cells inhabiting the bone marrow do not spontaneously differentiate into muscle cells, but there is some evidence that they are capable to follow the myogenic program and/or fuse with myoblasts. METHODS: In the present study we analyzed whether IGF-1, IL-4, IL-6, and SDF-1 could impact human and porcine bone marrow-derived mesenchymal stromal/stem cells (hBM-MSCs and pBM-MSCs) and induce expression of myogenic regulatory factors, skeletal muscle-specific structural, and adhesion proteins. Moreover, we investigated whether these factors could induce both types of BM-MSCs to fuse with myoblasts. IGF-1, IL-4, IL-6, and SDF-1 were selected on the basis of their role in embryonic myogenesis as well as skeletal muscle regeneration. RESULTS: We found that hBM-MSCs and pBM-MSCs cultured in vitro in the presence of IGF-1, IL-4, IL-6, or SDF-1 did not upregulate myogenic regulatory factors. Consequently, we confirmed the lack of their naïve myogenic potential. However, we noticed that IL-4 and IL-6 impacted proliferation and IL-4, IL-6, and SDF-1 improved migration of hBM-MSCs. IL-4 treatment resulted in the significant increase in the level of mRNA encoding CD9, NCAM, VCAM, and m-cadherin, i.e., proteins engaged in cell fusion during myotube formation. Additionally, the CD9 expression level was also driven by IGF-1 treatment. Furthermore, the pre-treatment of hBM-MSCs either with IGF-1, IL-4, or SDF-1 and treatment of pBM-MSCs either with IGF-1 or IL-4 increased the efficacy of hybrid myotube formation between these cells and C2C12 myoblasts. CONCLUSIONS: To conclude, our study revealed that treatment with IGF-1, IL-4, IL-6, or SDF-1 affects BM-MSC interaction with myoblasts; however, it does not directly promote myogenic differentiation of these cells.

The role of CXC receptors signaling in early stages of mouse embryonic stem cell differentiation.

Interplay between CXCR7 and other CXC receptors, namely CXCR4 or CXCR3, binding such ligands as SDF-1 or ITAC, was shown to regulate multiple cellular processes. The developmental role of signaling pathways mediated by these receptors was proven by the phenotypes of mice lacking either functional CXCR4, or CXCR7, or SDF-1, showing that formation of certain lineages relies on these factors. In this study, using in vitro differentiating mouse embryonic stem cells that lacked the function of CXCR7, we asked the question about the role of CXCR mediated signaling during early steps of differentiation. Our analysis showed that interaction of SDF-1 or ITAC with CXC receptors is necessary for the regulation of crucial developmental regulators expression and that CXCR7 is involved in the control of ESC pluripotency and differentiation into mesodermal lineages.

Interleukin 4 Moderately Affects Competence of Pluripotent Stem Cells for Myogenic Conversion.

Pluripotent stem cells convert into skeletal muscle tissue during teratoma formation or chimeric animal development. Thus, they are characterized by naive myogenic potential. Numerous attempts have been made to develop protocols enabling efficient and safe conversion of pluripotent stem cells into functional myogenic cells in vitro. Despite significant progress in the field, generation of myogenic cells from pluripotent stem cells is still challenging-i.e., currently available methods require genetic modifications, animal-derived reagents, or are long lasting-and, therefore, should be further improved. In the current study, we investigated the influence of interleukin 4, a factor regulating inter alia migration and fusion of myogenic cells and necessary for proper skeletal muscle development and maintenance, on pluripotent stem cells. We assessed the impact of interleukin 4 on proliferation, selected gene expression, and ability to fuse in case of both undifferentiated and differentiating mouse embryonic stem cells. Our results revealed that interleukin 4 slightly improves fusion of pluripotent stem cells with myoblasts leading to the formation of hybrid myotubes. Moreover, it increases the level of early myogenic genes such as Mesogenin1, Pax3, and Pax7 in differentiating embryonic stem cells. Thus, interleukin 4 moderately enhances competence of mouse pluripotent stem cells for myogenic conversion.

Adipose Tissue-Derived Stromal Cells in Matrigel Impacts the Regeneration of Severely Damaged Skeletal Muscles.

In case of large injuries of skeletal muscles the pool of endogenous stem cells, i.e., satellite cells, might be not sufficient to secure proper regeneration. Such failure in reconstruction is often associated with loss of muscle mass and excessive formation of connective tissue. Therapies aiming to improve skeletal muscle regeneration and prevent fibrosis may rely on the transplantation of different types of stem cell. Among such cells are adipose tissue-derived stromal cells (ADSCs) which are relatively easy to isolate, culture, and manipulate. Our study aimed to verify applicability of ADSCs in the therapies of severely injured skeletal muscles. We tested whether 3D structures obtained from Matrigel populated with ADSCs and transplanted to regenerating mouse gastrocnemius muscles could improve the regeneration. In addition, ADSCs used in this study were pretreated with myoblasts-conditioned medium or anti-TGFß antibody, i.e., the factors modifying their ability to proliferate, migrate, or differentiate. Analyses performed one week after injury allowed us to show the impact of 3D cultured control and pretreated ADSCs at muscle mass and structure, as well as fibrosis development immune response of the injured muscle.

Muscular Contribution to Adolescent Idiopathic Scoliosis from the Perspective of Stem Cell-Based Regenerative Medicine.

Adolescent idiopathic scoliosis (AIS) is a relatively frequent disease within a range 0.5%-5.0% of population, with higher frequency in females. While a resultant spinal deformity is usually medically benign condition, it produces far going psychosocial consequences, which warrant attention. The etiology of AIS is unknown and current therapeutic approaches are symptomatic only, and frequently inconvenient or invasive. Muscular contribution to AIS is widely recognized, although it did not translate to clinical routine as yet. Muscle asymmetry has been documented by pathological examinations as well as systemic muscle disorders frequently leading to scoliosis. It has been also reported numerous genetic, metabolic and radiological alterations in patients with AIS, which are linked to muscular and neuromuscular aspects. Therefore, muscles might be considered an attractive and still insufficiently exploited therapeutic target for AIS. Stem cell-based regenerative medicine is rapidly gaining momentum based on the tremendous progress in understanding of developmental biology. It comes also with a toolbox of various stem cells such as satellite cells or mesenchymal stem cells, which could be transplanted; also, the knowledge acquired in research on regenerative medicine can be applied to manipulation of endogenous stem cells to obtain desired therapeutic goals. Importantly, paravertebral muscles are located relatively superficially; therefore, they can be an easy target for minimally invasive approaches to treatment of AIS. It comes in pair with a fast progress in image guidance, which allows for precise delivery of therapeutic agents, including stem cells to various organs such as brain, muscles, and others. Summing up, it seems that there is a link between AIS, muscles, and stem cells, which might be worth of further investigations with a long-term goal of setting foundations for eventual bench-to-bedside translation.

Novel insights and innovations in biotechnology towards improved quality of life.

Resolution of old problems with new tools seems to be a new way of challenging and finding the keys to innovation. A holistic view of different branches of science and industry, including services for society, is critically important for the development of modern science. The International Congress Eurobiotech 2017 was a special opportunity for such a universal view. In this paper, we discuss the application of different small RNAs, stem cells, epigenetics and sequencing, as well as art and bioeconomy. Our most significant message is not a new one but is very universal: biotechnology is vital for society.

Induction of bone marrow-derived cells myogenic identity by their interactions with the satellite cell niche.

BACKGROUND: Skeletal muscle regeneration is possible thanks to unipotent stem cells, which are satellite cells connected to the myofibers. Populations of stem cells other than muscle-specific satellite cells are considered as sources of cells able to support skeletal muscle reconstruction. Among these are bone marrow-derived mesenchymal stem cells (BM-MSCs), which are multipotent, self-renewing stem cells present in the bone marrow stroma. Available data documenting the ability of BM-MSCs to undergo myogenic differentiation are not definitive. In the current work, we aimed to check if the satellite cell niche could impact the ability of bone marrow-derived cells to follow a myogenic program. METHODS: We established a new in-vitro method for the coculture of bone marrow-derived cells (BMCs) that express CXCR4 (CXCR4+BMCs; the stromal-derived factor-1 (Sdf-1) receptor) with myofibers. Using various tests, we analyzed the myogenic identity of BMCs and their ability to fuse with myoblasts in vitro and in vivo. RESULTS: We showed that Sdf-1 treatment increased the number of CXCR4+BMCs able to bind the myofiber and occupy the satellite cell niche. Moreover, interaction with myofibers induced the expression of myogenic regulatory factors (MRFs) in CXCR4+BMCs. CXCR4+BMCs, pretreated by the coculture with myofibers and Sdf-1, participated in myotube formation in vitro and also myofiber reconstruction in vivo. We also showed that Sdf-1 overexpression in vivo (in injured and regenerating muscles) supported the participation of CXCR4+BMCs in new myofiber formation. CONCLUSION: We showed that CXCR4+BMC interaction with myofibers (that is, within the satellite cell niche) induced CXCR4+BMC myogenic commitment. CXCR4+BMCs, pretreated using such a method of culture, were able to participate in skeletal muscle regeneration.

Transient MicroRNA Expression Enhances Myogenic Potential of Mouse Embryonic Stem Cells.

MicroRNAs (miRNAs) are known regulators of various cellular processes, including pluripotency and differentiation of embryonic stem cells (ESCs). We analyzed differentiation of two ESC lines-D3 and B8, and observed significant differences in the expression of miRNAs and genes involved in pluripotency and differentiation. We also examined if transient miRNA overexpression could serve as a sufficient impulse modulating differentiation of mouse ESCs. ESCs were transfected with miRNA Mimics and differentiated in embryoid bodies and embryoid body outgrowths. miRNAs involved in differentiation of mesodermal lineages, such as miR145 and miR181, as well as miRNAs regulating myogenesis (MyomiRs)-miR1, miR133a, miR133b, and miR206 were tested. Using such approach, we proved that transient overexpression of molecules selected by us modulated differentiation of mouse ESCs. Increase in miR145 levels upregulated Pax3, Pax7, Myod1, Myog, and MyHC2, while miR181 triggered the expression of such crucial myogenic factors as Myf5 and MyHC2. As a result, the ability of ESCs to initiate myogenic differentiation and form myotubes was enhanced. Premature expression of MyomiRs had, however, an adverse effect on myogenic differentiation of ESCs. Stem Cells 2018;36:655-670.

In Memoriam - Prof. Andrzej Krzysztof Tarkowski (1933-2016).

Professor Andrzej Krzysztof Tarkowski passed away last September (2016) at the age of 83. His findings, have become indispensable tools for immunological, genetic, and oncological studies, as well as for generating transgenic animals which are instrumental for studying gene function in living animals. His work and discoveries provided a tremendous input to the contemporary developmental biology of mammals.

Stem cells migration during skeletal muscle regeneration - the role of Sdf-1/Cxcr4 and Sdf-1/Cxcr7 axis.

The skeletal muscle regeneration occurs due to the presence of tissue specific stem cells - satellite cells. These cells, localized between sarcolemma and basal lamina, are bound to muscle fibers and remain quiescent until their activation upon muscle injury. Due to pathological conditions, such as extensive injury or dystrophy, skeletal muscle regeneration is diminished. Among the therapies aiming to ameliorate skeletal muscle diseases are transplantations of the stem cells. In our previous studies we showed that Sdf-1 (stromal derived factor -1) increased migration of stem cells and their fusion with myoblasts in vitro. Importantly, we identified that Sdf-1 caused an increase in the expression of tetraspanin CD9 - adhesion protein involved in myoblasts fusion. In the current study we aimed to uncover the details of molecular mechanism of Sdf-1 action. We focused at the Sdf-1 receptors - Cxcr4 and Cxcr7, as well as signaling pathways induced by these molecules in primary myoblasts, as well as various stem cells - mesenchymal stem cells and embryonic stem cells, i.e. the cells of different migration and myogenic potential. We showed that Sdf-1 altered actin organization via FAK (focal adhesion kinase), Cdc42 (cell division control protein 42), and Rac-1 (Ras-Related C3 Botulinum Toxin Substrate 1). Moreover, we showed that Sdf-1 modified the transcription profile of genes encoding factors engaged in cells adhesion and migration. As the result, cells such as primary myoblasts or embryonic stem cells, became characterized by more effective migration when transplanted into regenerating muscle.

Myogenic potential of mouse embryonic stem cells lacking functional Pax7 tested in vitro by 5-azacitidine treatment and in vivo in regenerating skeletal muscle.

Regeneration of skeletal muscle relies on the presence of satellite cells. Satellite cells deficiency accompanying some degenerative diseases is the reason for the search for the "replacement cells" that can be used in the muscle therapies. Due to their unique properties embryonic stem cells (ESCs), as well as myogenic cells derived from them, are considered as a promising source of therapeutic cells. Among the factors crucial for the specification of myogenic precursor cells is Pax7 that sustains proper function of satellite cells. In our previous studies we showed that ESCs lacking functional Pax7 are able to form myoblasts in vitro when differentiated within embryoid bodies and their outgrowths. In the current study we showed that ESCs lacking functional Pax7, cultured in vitro in monolayer in the medium supplemented with horse serum and 5azaC, expressed higher levels of factors associated with myogenesis, such as Pdgfra, Pax3, Myf5, and MyoD. Importantly, skeletal myosin immunolocalization confirmed that myogenic differentiation of ESCs was more effective in case of cells lacking Pax7. Our in vivo studies showed that ESCs transplanted into regenerating skeletal muscles were detectable at day 7 of regeneration and the number of Pax7-/- ESCs detected was significantly higher than of control cells. Our results support the concept that lack of functional Pax7 promotes proliferation of differentiating ESCs and for this reason more of them can turn into myogenic lineage.