Thus, positive impact of Sdf-1 on muscle regeneration is related to the mobilisation of endogenous cells, that is satellite cells and myoblasts, as well as non-muscle stem cells, expressing Cxcr4 and CD34.

The transcription factor Pax7 plays a key role during embryonic myogenesis and sustains the proper function of satellite cells, which serve as adult skeletal muscle stem cells. Overexpression of Pax7 has been shown to promote the myogenic differentiation of pluripotent stem cells. However, the effects of the absence of functional Pax7 in differentiating embryonic stem cells (ESCs) have not yet been directly tested. Herein, we studied mouse stem cells that lacked a functional Pax7 gene and characterized the differentiation of these stem cells under conditions that promoted the derivation of myoblasts in vitro. We analyzed the expression of myogenic factors, such as myogenic regulatory factors and muscle-specific microRNAs, in wild-type and mutant cells. Finally, we compared the transcriptome of both types of cells and did not find substantial differences in the expression of genes related to the regulation of myogenesis. As a result, we showed that the absence of functional Pax7 does not prevent the in vitro myogenic differentiation of ESCs.

Formation of mammalian skeletal muscle myofibers, that takes place during embryogenesis, muscle growth or regeneration, requires precise regulation of myoblast adhesion and fusion. There are few evidences showing that adhesion proteins play important role in both processes. To follow the function of these molecules in myoblast differentiation we analysed integrin alpha3, integrin beta1, ADAM12, CD9, CD81, M-cadherin, and VCAM-1 during muscle regeneration. We showed that increase in the expression of these proteins accompanies myoblast fusion and myotube formation in vivo. We also showed that during myoblast fusion in vitro integrin alpha3 associates with integrin beta1 and ADAM12, and also CD9 and CD81, but not with M-cadherin or VCAM-1. Moreover, we documented that experimental modification in the expression of integrin alpha3 lead to the modification of myoblast fusion in vitro. Underexpression of integrin alpha3 decreased myoblasts' ability to fuse. This phenomenon was not related to the modifications in the expression of other adhesion proteins, i.e. integrin beta1, CD9, CD81, ADAM12, M-cadherin, or VCAM-1. Apparently, aberrant expression only of one partner of multiprotein adhesion complexes necessary for myoblast fusion, in this case integrin alpha3, prevents its proper function. Summarizing, we demonstrated the importance of analysed adhesion proteins in myoblast fusion both in vivo and in vitro.

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.

Abstract:The goal of our study was to compare the skeletal muscle regeneration induced by two types of injury: either crushing, that causes muscle degeneration as a result of mechanical devastation of myofibers, or the injection of a cardiotoxin that is a myotoxic agent causing myolysis of myofibers leading to muscle degeneration. Regenerating muscles were analyzed at selected intervals, until the 14 th day following the injury. We analyzed their weight and morphology. We also studied the expression of different myosin heavy chain isoforms as a molecular marker of the regeneration progress. Histological analysis revealed that inflammatory response and myotube formation in crushed muscles was delayed compared to cardiotoxin-injected ones. Moreover, the expression of myosin heavy chain isoforms was observed earlier in cardiotoxin-injured versus crushed muscles. We conclude that the dynamics of skeletal muscle regeneration depends on the method of injury.

The transcription factor Pax7 plays a key role during embryonic myogenesis and in adult organisms in that it sustains the proper function of satellite cells, which serve as adult skeletal muscle stem cells. Recently we have shown that lack of Pax7 does not prevent the myogenic differentiation of pluripotent stem cells. In the current work we show that the absence of functional Pax7 in differentiating embryonic stem cells modulates cell cycle facilitating their proliferation. Surprisingly, deregulation of Pax7 function also positively impacts at the proliferation of mouse embryonic fibroblasts. Such phenotypes seem to be executed by modulating the expression of positive cell cycle regulators, such as cyclin E.

Pluripotent stem cells, such as embryonic stem cells and induced pluripotent stem cells, are an important tool in the studies focusing at the differentiation of various cell types, including skeletal myoblasts. They are also considered as a source of the cells that due to their pluripotent character and availability could be turned into any required tissue and then used in future in regenerative medicine. However, the methods of the derivation of some of cell types from pluripotent cells still need to be perfected. This chapter summarizes the history and current advancements in the derivation and testing of pluripotent stem cells-derived skeletal myoblasts. It focuses at the in vitro methods allowing the differentiation of stem cells grown in monolayer or propagated as embryoid bodies, and also at in vivo tests allowing the verification of the functionality of obtained skeletal myoblasts.

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 SIGNIFICANCE STATEMENTIn the current study, characterization of microRNomes of mouse embryonic stem cells (ESCs) was performed and the potential use of transient microRNA (miRNA) expression as a tool to induce differentiation of these cells was tested. Analyses of microRNomes of two ESC lines revealed that miRNA expression patterns reflect differentiation abilities of each of the analyzed cell line. Also, it was documented that transient overexpression of selected miRNA is sufficient to affect the phenotype of differentiating ESCs. Using this technique and appropriately chosen miRNAs, we were able to improve myogenic differentiation.