14 research outputs found
Contribution of stem cells to skeletal muscle regeneration.
Stem cells for skeletal muscle originate from dermomyotome of the embryo. The early marker of these cells is expression of both transcription factors Pax3 and Pax7 (Pax3+/Pax7+ cells). The skeletal muscles in the adult organism have a remarkable ability to regenerate. Skeletal muscle damage induces degenerative phase, followed by activation of inflammatory and satellite cells. The satellite cells are quiescent myogenic precursor cells located between the basal membrane and the sarcolemma of myofiber and they are characterized by Pax7 expression. Activation of the satellite cells is regulated by muscle growth and chemokines. Apart from the satellite cells, a population of adult stem cells (muscle side population--mSP) exists in the skeletal muscles. Moreover, the cells trafficking from different tissues may be involved in the regeneration of damaged muscle. Trafficking of cells in the process of damaged muscle regeneration may be traced in the SCID mice
Myogenic Differentiation of Mouse Embryonic Stem Cells That Lack a Functional Pax7 Gene
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
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
Contribution of stem cells to skeletal muscle regeneration.
Stem cells for skeletal muscle originate from dermomyotome of the embryo. The early marker of these cells is expression of both transcription factors Pax3 and Pax7 (Pax3+/Pax7+ cells). The skeletal muscles in the adult organism have a remarkable ability to regenerate. Skeletal muscle damage induces degenerative phase, followed by activation of inflammatory and satellite cells. The satellite cells are quiescent myogenic precursor cells located between the basal membrane and the sarcolemma of myofiber and they are characterized by Pax7 expression. Activation of the satellite cells is regulated by muscle growth and chemokines. Apart from the satellite cells, a population of adult stem cells (muscle side population--mSP) exists in the skeletal muscles. Moreover, the cells trafficking from different tissues may be involved in the regeneration of damaged muscle. Trafficking of cells in the process of damaged muscle regeneration may be traced in the SCID mice
Restricted myogenic potential of mesenchymal stromal cells isolated from umbilical cord
Nonhematopoietic cord blood cells and mesenchymal cells of umbilical cord Wharton's jelly have been shown to be able to differentiate into various cell types. Thus, as they are readily available and do not raise any ethical issues, these cells are considered to be a potential source of material that can be used in regenerative medicine. In our previous study, we tested the potential of whole mononucleated fraction of human umbilical cord blood cells and showed that they are able to participate in the regeneration of injured mouse skeletal muscle. In the current study, we focused at the umbilical cord mesenchymal stromal cells isolated from Wharton's jelly. We documented that limited fraction of these cells express markers of pluripotent and myogenic cells. Moreover, they are able to undergo myogenic differentiation in vitro, as proved by coculture with C2C12 myoblasts. They also colonize injured skeletal muscle and, with low frequency, participate in the formation of new muscle fibers. Pretreatment of Wharton's jelly mesenchymal stromal cells with SDF-1 has no impact on their incorporation into regenerating muscle fibers but significantly increased muscle mass. As a result, transplantation of mesenchymal stromal cells enhances the skeletal muscle regeneration
Adhesion proteins--an impact on skeletal myoblast differentiation.
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
Interleukin 4 Moderately Affects Competence of Pluripotent Stem Cells for Myogenic Conversion
Changes in expression and localization of adhesion proteins during <i>Soleus</i> muscle regeneration.
<p>A - level of mRNAs encoding adhesion proteins analyzed by sqRT-PCR. Optical densities of representative bands are shown as a percentage of GAPDH band density taken as a 100%. B – localization of integrin alpha3 (red) in intact and in regenerating muscle at day 3, 5, 7, 14. Nuclei – blue. White arrows show degenerating myofibers. Scale bars 50 µm.C –colocalization of Pax7, MyoD, myogenin (MG) (red) with integrin alpha3 (green) in intact muscle and at day 1, 3 and 5 of regeneration (intact, d1, d3, d5 respectively). White arrows - myoblasts, pink – muscle fiber nuclei, yellow - MyoD, myogenin and integrin alpha3 negative cells. D – control staing with secondary antibodies only. Nuclei – blue. Scale bars 50 µm. E - localization of integrin beta1, ADAM12, CD9, or M-cadherin (green) at day 3 of regeneration. Nuclei – blue. Scale bars 50 µm. F – immunoblotting analysis of M-cadherin (M-cad), integrin beta1, ADAM12 (AD12), CD9 and integrin alpha3 level during muscle regeneration (days 1, 3, 7, 14).</p
Downregulation of alpha3 integrin expression reduces fusion of MPCs-derived myoblasts.
<p>A - Pappenheim's staining of fusing myoblasts. B – total number of nuclei calculated at day 12 of myoblast culture. C –index of fusion analyzed at day 12 of culture in control and experimental myoblasts shown as a percentage of myotube nuclei per number of all nuclei. D - proportion of 2–4, 5–7, 8–10 and >11 nuclear myotubes per number of all myotubes, respectively. NT – control, not transfected myoblasts, TR – control myoblasts cultured in medium supplemented with transfection reagent, siRNA-cont - control myoblasts transfected with scramble siRNA, siRNA-alpha3 – myoblasts transfected with siRNA downregulating the expression of integrin alpha3. Error bars indicate SEM, results were analyzed by Kruskal-Wallis test. Kruskal-Wallis One Way Analysis of Variance showed differences between experimental groups which were considered statistically significant when p<0.05 (marked with asterisks). * p≤0.05.</p