Edyta Brzoska

Distinct patterns of MMP-9 and MMP-2 activity in slow and fast twitch skeletal muscle regeneration in vivo

Skeletal muscles exhibit great plasticity and an ability to reconstruct in response to injury. However, the repair process is often inefficient and hindered by the development of fibrosis. We explored the possibility that during muscle repair, the different regeneration ability of the fast (extensor digitorum longus; EDL) and slow twitch (Soleus) muscles depends on the differential expression of metalloproteinases (MMP-9 and MMP-2) involved in the remodeling of the extracellular matrix. Our results show that MMP-9 and MMP-2 are present in the intact muscle and are up-regulated after crush-induced muscle injury. The expression and the activity of these two enzymes depend on the type of muscle and the phase of muscle regeneration. In the regenerating Soleus muscle, elevated levels of MMP-9 occurred during the myolysis and reconstruction phase. In contrast, regenerating EDL muscles exhibited decreased MMP-9 levels during myolysis and increased MMP-2 activity at the reconstruction phase. Moreover, satellite cells (mononuclear myoblasts) derived from Soleus and EDL muscles showed no differences in localization or activity of MMP-9 and MMP-2 during proliferation and differentiation in vitro. MMP-9 activity was present during all stages of myoblast differentiation, whereas MMP-2 activity reached its highest level during myoblast fusion. We conclude that MMPs are involved in muscle repair, and that fast and slow twitch muscles exhibit different patterns of MMP-9 and MMP-2 activity.

Sdf-1 (CXCL12) improves skeletal muscle regeneration via the mobilisation of Cxcr4 and CD34 expressing cells

The regeneration of skeletal muscles involves satellite cells, which are muscle‐specific precursor cells. In muscles, injured either mechanically or as a consequence of a disease, such as muscular dystrophy, local release of the growth factors and cytokines leads to satellite cells activation, proliferation and differentiation of the resulting myoblasts, followed by the formation of new myofibres. Various cell types, such as stem and progenitor cells, originating from other tissues different than the muscle, are also able to follow a myogenic program. Participation of these cells in the repair process depends on their precise mobilisation to the site of the injury.

Comparison of satellite cell-derived myoblasts and C2C12 differentiation in two- and three-dimensional cultures: changes in adhesion protein expression.

Changes in the expression of adhesion proteins involved in myoblast differentiation were investigated in monolayer (two‐dimensional) and 3D (three‐dimensional) cell cultures. The expression of integrin alpha3 subunit, integrin beta1 subunit, ADAM12 (a disintegrin and metalloproteinase 12), tetraspanins CD9 and CD81 and M‐cadherin were examined in the murine myoblast cell line C2C12 and in a primary culture of rat satellite cells. Myoblasts in monolayer and 3D cultures showed significant differences in their morphology and cytoskeletal organization. All of the studied proteins participated in myoblast fusion in each culture examined, but differences in their levels of expression were observed. Satellite cell‐derived myoblasts exhibited higher expression of adhesion protein mRNAs than C2C12 cells. Also, C2C12 cells from a 3D culture showed slightly higher expression of adhesion protein transcripts than the same cells cultured as a monolayer. Significantly, the levels of adhesion protein mRNAs were found to change in parallel in all cell culture types. Despite this finding, it is important that differences between satellite cell‐derived myoblasts and cell line C2C12 grown in monolayer and 3D cultures are taken into account when studying processes of myoblast differentiation in vitro.

Regulation of muscle stem cells activation: the role of growth factors and extracellular matrix.

Vertebrate skeletal muscle is composed of organized multinucleate muscle fibers and also various subpopulations of cells localized in between. Some of them can be considered as the stem cells, however, few of them are able to follow myogenic program. First and most extensively studied so far, are the satellite cells that serve as tissue-specific precursors for muscle growth and repair. They are located between the basal membrane and the sarcolemma of adult muscle myofibers. They remain quiescent but can be activated in response to muscle damage resulted from mechanical injury, stretching, exercise, denervation, or progressing muscle dystrophy. Except the satellite cells also other stem cells could participate in muscle fibers reconstruction. Such cells as pericytes and mesangioblasts, muscle-derived stem cells, including so-called muscle side population, or CD133 expressing cells, were proved to be able to undergo myogenic differentiation in experiments involving their in vitro coculture with myoblasts or transplantation to injured skeletal muscle. In the current review, we will summarize stimuli influencing skeletal muscle stem cells activation, that is, growth factors which are secreted by muscle fibers, satellite cells, inflammatory cells, or released from basal lamina. We will also describe factors present within the skeletal muscle niche which interactions with stem cells lead to their activation, proliferation, asymmetric divisions, migration, and finally differentiation into myotubes, and then terminally differentiated myofibers.

Restricted Myogenic Potential of Mesenchymal Stromal Cells Isolated From Umbilical Cord

Nonhematopoietic cord blood cells and mesenchymal cells of umbilical cord Whartons 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 Whartons 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 Whartons 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.

Regulation of Muscle Stem Cells Activation

Vertebrate skeletal muscle is composed of organized multinucleate muscle fibers and also various subpopulations of cells localized in between. Some of them can be considered as the stem cells, however, few of them are able to follow myogenic program. First and most extensively studied so far, are the satellite cells that serve as tissue-specific precursors for muscle growth and repair. They are located between the basal membrane and the sarcolemma of adult muscle myofibers. They remain quiescent but can be activated in response to muscle damage resulted from mechanical injury, stretching, exercise, denervation, or progressing muscle dystrophy. Except the satellite cells also other stem cells could participate in muscle fibers reconstruction. Such cells as pericytes and mesangioblasts, muscle-derived stem cells, including so-called muscle side population, or CD133 expressing cells, were proved to be able to undergo myogenic differentiation in experiments involving their in vitro coculture with myoblasts or transplantation to injured skeletal muscle. In the current review, we will summarize stimuli influencing skeletal muscle stem cells activation, that is, growth factors which are secreted by muscle fibers, satellite cells, inflammatory cells, or released from basal lamina. We will also describe factors present within the skeletal muscle niche which interactions with stem cells lead to their activation, proliferation, asymmetric divisions, migration, and finally differentiation into myotubes, and then terminally differentiated myofibers.

Pax3 and Pax7 expression during myoblast differentiation in vitro and fast and slow muscle regeneration in vivo

In this report, we focused on Pax3 and Pax7 expression in vitro during myoblast differentiation and in vivo during skeletal muscle regeneration. We showed that Pax3 and Pax7 were present in EDL (extensor digitorum longus) and Soleus muscle derived cells. These cells express in vitro a similar level of Pax3 mRNA, however, differ in the levels of mRNA encoding Pax7. Analysis of Pax3 and Pax7 proteins showed that Soleus and EDL satellite cells differ in the level of Pax3/7 proteins and also in the number of Pax3/7 positive cells. Moreover, Pax3/7 expression was restricted to undifferentiated cells, and both proteins were absent at further stages of myoblast differentiation, indicating that Pax3 and Pax7 are down‐regulated during myoblast differentiation. However, we noted that the population of undifferentiated Pax3/7 positive cells was constantly present in both in vitro cultured satellite cells of EDL and Soleus. In contrast, there was no significant difference in Pax3 and Pax7 during in vivo differentiation accompanying regeneration of EDL and Soleus muscle. We demonstrated that Pax3 and Pax7, both in vitro and in vivo, participated in the differentiation and regeneration events of muscle and detected differences in the Pax7 expression pattern during in vitro differentiation of myoblasts isolated from fast and slow muscles.

From Planarians to Mammals - the many faces of regeneration

This report presents the history of the involvement of the Department of Cytology in studies of different aspects of regeneration. It can be divided into two major phases; the first focused on the regeneration of Turbellarians and the second on the regeneration of rat skeletal muscles including the differentiation of satellite cells in vitro. Regeneration of Turbellarians was investigated both at the cellular and molecular levels including the role of the protein kinase C (PKC) in this process. Studies on skeletal muscle regeneration initially focused on factors involved in regulation of signal transduction pathways. Next, we explored the influence of growth factors on the modulation of the regeneration process. Another important aspect of our studies was investigating of the distribution and function of different proteins involved in adhesion and fusion of myoblasts. Finally, we are also conducting research on the role of stem cells from other tissues in the regeneration of skeletal muscle.

Stromal derived factor-1 and granulocyte-colony stimulating factor treatment improves regeneration of Pax7−/− mice skeletal muscles

The skeletal muscle has the ability to regenerate after injury. This process is mediated mainly by the muscle specific stem cells, that is, satellite cells. In case of extensive damage or under pathological conditions, such as muscular dystrophy, the process of muscle reconstruction does not occur properly. The aim of our study was to test whether mobilized stem cells, other than satellite cells, could participate in skeletal muscle reconstruction.

Sdf-1 (CXCL12) induces CD9 expression in stem cells engaged in muscle regeneration

IntroductionUnderstanding the mechanism of stem cell mobilization into injured skeletal muscles is a prerequisite step for the development of muscle disease therapies. Many of the currently studied stem cell types present myogenic potential; however, when introduced either into the blood stream or directly into the tissue, they are not able to efficiently engraft injured muscle. For this reason their use in therapy is still limited. Previously, we have shown that stromal-derived factor-1 (Sdf-1) caused the mobilization of endogenous (not transplanted) stem cells into injured skeletal muscle improving regeneration. Here, we demonstrate that the beneficial effect of Sdf-1 relies on the upregulation of the tetraspanin CD9 expression in stem cells.MethodsThe expression pattern of adhesion proteins, including CD9, was analysed after Sdf-1 treatment during regeneration of rat skeletal muscles and mouse Pax7-/- skeletal muscles, that are characterized by the decreased number of satellite cells. Next, we examined the changes in CD9 level in satellite cells-derived myoblasts, bone marrow-derived mesenchymal stem cells, and embryonic stem cells after Sdf-1 treatment or silencing expression of CXCR4 and CXCR7. Finally, we examined the potential of stem cells to fuse with myoblasts after Sdf-1 treatment.ResultsIn vivo analyses of Pax7-/- mice strongly suggest that Sdf-1-mediates increase in CD9 levels also in mobilized stem cells. In the absence of CXCR4 receptor the effect of Sdf-1 on CD9 expression is blocked. Next, in vitro studies show that Sdf-1 increases the level of CD9 not only in satellite cell-derived myoblasts but also in bone marrow derived mesenchymal stem cells, as well as embryonic stem cells. Importantly, the Sdf-1 treated cells migrate and fuse with myoblasts more effectively.ConclusionsWe suggest that Sdf-1 binding CXCR4 receptor improves skeletal muscle regeneration by upregulating expression of CD9 and thus, impacting at stem cells mobilization to the injured muscles.