Atsushi Asakura

Myogenic specification of side population cells in skeletal muscle

Skeletal muscle contains myogenic progenitors called satellite cells and muscle-derived stem cells that have been suggested to be pluripotent. We further investigated the differentiation potential of muscle-derived stem cells and satellite cells to elucidate relationships between these two populations of cells. FACS® analysis of muscle side population (SP) cells, a fraction of muscle-derived stem cells, revealed expression of hematopoietic stem cell marker Sca-1 but did not reveal expression of any satellite cell markers. Muscle SP cells were greatly enriched for cells competent to form hematopoietic colonies. Moreover, muscle SP cells with hematopoietic potential were CD45 positive. However, muscle SP cells did not differentiate into myocytes in vitro. By contrast, satellite cells gave rise to myocytes but did not express Sca-1 or CD45 and never formed hematopoietic colonies. Importantly, muscle SP cells exhibited the potential to give rise to both myocytes and satellite cells after intramuscular transplantation. In addition, muscle SP cells underwent myogenic specification after co-culture with myoblasts. Co-culture with myoblasts or forced expression of MyoD also induced muscle differentiation of muscle SP cells prepared from mice lacking Pax7 gene, an essential gene for satellite cell development. Therefore, these data document that satellite cells and muscle-derived stem cells represent distinct populations and demonstrate that muscle-derived stem cells have the potential to give rise to myogenic cells via a myocyte-mediated inductive interaction.

The Potential of Muscle Stem Cells

Skeletal muscle contains two types of stem cells: satellite cells, which function as myogenic precursors, and a population of multipotent adult stem cells. Satellite cells are believed to form a stable, self-renewing pool of stem cells in adult muscle where they function in tissue growth and repair. An additional stem cell population in adult muscle displays a remarkable capacity to differentiate into hematopoietic cells as well as muscle following transplantation. This article discusses the characteristics and properties of these cell populations, the relationship between them, and the potential for stem cell-based muscle therapeutics.

NeuroD2 and neuroD3: distinct expression patterns and transcriptional activation potentials within the neuroD gene family.

We have identified two new genes, neuroD2 and neuroD3, on the basis of their similarity to the neurogenic basic-helix-loop-helix (bHLH) gene neuroD. The predicted amino acid sequence of neuroD2 shows a high degree of homology to neuroD and MATH-2/NEX-1 in the bHLH region, whereas neuroD3 is a more distantly related family member. neuroD3 is expressed transiently during embryonic development, with the highest levels of expression between days 10 and 12. neuroD2 is initially expressed at embryonic day 11, with persistent expression in the adult nervous system. In situ and Northern (RNA) analyses demonstrate that different regions of the adult nervous system have different relative amounts of neuroD and neuroD2 RNA. Similar to neuroD, expression of neuroD2 in developing Xenopus laevis embryos results in ectopic neurogenesis, indicating that neuroD2 mediates neuronal differentiation. Transfection of vectors expressing neuroD and neuroD2 into P19 cells shows that both can activate expression through simple E-box-driven reporter constructs and can activate a reporter driven by the neuroD2 promoter region, but the GAP-43 promoter is preferentially activated by neuroD2. The noncongruent expression pattern and target gene specificity of these highly related neurogenic bHLH proteins make them candidates for conferring specific aspects of the neuronal phenotype.

Constitutive Notch Activation Upregulates Pax7 and Promotes the Self-Renewal of Skeletal Muscle Satellite Cells

ABSTRACT Notch signaling is a conserved cell fate regulator during development and postnatal tissue regeneration. Using skeletal muscle satellite cells as a model and through myogenic cell lineage-specific NICDOE (overexpression of constitutively activated Notch 1 intracellular domain), here we investigate how Notch signaling regulates the cell fate choice of muscle stem cells. We show that in addition to inhibiting MyoD and myogenic differentiation, NICDOE upregulates Pax7 and promotes the self-renewal of satellite cell-derived primary myoblasts in culture. Using MyoD−/− myoblasts, we further show that NICDOE upregulates Pax7 independently of MyoD inhibition. In striking contrast to previous observations, NICDOE also inhibits S-phase entry and Ki67 expression and thus reduces the proliferation of primary myoblasts. Overexpression of canonical Notch target genes mimics the inhibitory effects of NICDOE on MyoD and Ki67 but not the stimulatory effect on Pax7. Instead, NICD regulates Pax7 through interaction with RBP-Jκ, which binds to two consensus sites upstream of the Pax7 gene. Importantly, satellite cell-specific NICDOE results in impaired regeneration of skeletal muscles along with increased Pax7+ mononuclear cells. Our results establish a role of Notch signaling in actively promoting the self-renewal of muscle stem cells through direct regulation of Pax7.

MyoD induces myogenic differentiation through cooperation of its NH2- and COOH-terminal regions

MyoD and Myf5 are basic helix-loop-helix transcription factors that play key but redundant roles in specifying myogenic progenitors during embryogenesis. However, there are functional differences between the two transcription factors that impact myoblast proliferation and differentiation. Target gene activation could be one such difference. We have used microarray and polymerase chain reaction approaches to measure the induction of muscle gene expression by MyoD and Myf5 in an in vitro model. In proliferating cells, MyoD and Myf5 function very similarly to activate the expression of likely growth phase target genes such as L-myc, m-cadherin, Mcpt8, Runx1, Spp1, Six1, IGFBP5, and Chrnβ1. MyoD, however, is strikingly more effective than Myf5 at inducing differentiation-phase target genes. This distinction between MyoD and Myf5 results from a novel and unanticipated cooperation between the MyoD NH2- and COOH-terminal regions. Together, these results support the notion that Myf5 functions toward myoblast proliferation, whereas MyoD prepares myoblasts for efficient differentiation.

MyoD regulates apoptosis of myoblasts through microRNA-mediated down-regulation of Pax3

Suppression of the myogenic transcription factor MyoD is required for maintenance of muscle stem cells.

Stem Cells in Adult Skeletal Muscle

Muscle satellite cells are a self-renewing pool of stem cells that give rise to daughter myogenic precursor cells in adult skeletal muscle, where they function in postnatal tissue growth and regeneration. Adult skeletal muscle also contains a novel stem cell population purified as a side population (SP), which actively excludes Hoechst 33342 dye. Muscle SP cells that express the hematopoietic stem cell marker Sca-1 possess the ability to differentiate into hematopoietic cells, skeletal muscle, and satellite cells following transplantation. The muscle SP fraction also contains cells expressing the hematopoietic marker CD45 that are capable of differentiation into hematopoietic cells and muscle cells. Thus, these novel muscle stem cells appear to have characteristics similar to those of hematopoietic stem cells, and can participate in muscle regeneration. This review outlines recent findings regarding different stem cell populations in skeletal muscle, and discusses their involvement in muscle regeneration.

Increased survival of muscle stem cells lacking the MyoD gene after transplantation into regenerating skeletal muscle

MyoD is a myogenic master transcription factor that plays an essential role in muscle satellite cell (muscle stem cell) differentiation. To further investigate the function of MyoD in satellite cells, we examined the transplantation of satellite cell-derived myoblasts lacking the MyoD gene into regenerating skeletal muscle. After injection into injured muscle, MyoD−/− myoblasts engrafted with significantly higher efficiency compared with wild-type myoblasts. In addition, MyoD−/− myoblast-derived satellite cells were detected underneath the basal lamina of muscle fibers, indicating the self-renewal property of MyoD−/− myoblasts. To gain insights into MyoD gene deficiency in muscle stem cells, we investigated the pathways regulated by MyoD by GeneChip microarray analysis of gene expression in wild-type and MyoD−/− myoblasts. MyoD deficiency led to down-regulation of many muscle-specific genes and up-regulation of some stem cell markers. Importantly, in MyoD−/− myoblasts, many antiapoptotic genes were up-regulated, whereas genes known to execute apoptosis were down-regulated. Consistent with these gene expression profiles, MyoD−/− myoblasts were revealed to possess remarkable resistance to apoptosis and increased survival compared with wild-type myoblasts. Forced expression of MyoD or the proapoptotic protein Puma increased cell death in MyoD−/− myoblasts. Therefore, MyoD−/− myoblasts may preserve stem cell characteristics, including their resistance to apoptosis, expression of stem cell markers, and efficient engraftment and contribution to satellite cells after transplantation. Furthermore, our data offer evidence for improved therapeutic stem cell transplantation for muscular dystrophy, in which suppression of MyoD in myogenic progenitors would be beneficial to therapy by providing a selective advantage for the expansion of stem cells.

Muscle satellite cell heterogeneity and self-renewal.

Adult skeletal muscle possesses extraordinary regeneration capacities. After muscle injury or exercise, large numbers of newly formed muscle fibers are generated within a week as a result of expansion and differentiation of a self-renewing pool of muscle stem cells termed muscle satellite cells. Normally, satellite cells are mitotically quiescent and reside beneath the basal lamina of muscle fibers. Upon regeneration, satellite cells are activated, and give rise to daughter myogenic precursor cells. After several rounds of proliferation, these myogenic precursor cells contribute to the formation of new muscle fibers. During cell division, a minor population of myogenic precursor cells returns to quiescent satellite cells as a self-renewal process. Currently, accumulating evidence has revealed the essential roles of satellite cells in muscle regeneration and the regulatory mechanisms, while it still remains to be elucidated how satellite cell self-renewal is molecularly regulated and how satellite cells are important in aging and diseased muscle. The number of satellite cells is decreased due to the changing niche during ageing, resulting in attenuation of muscle regeneration capacity. Additionally, in Duchenne muscular dystrophy (DMD) patients, the loss of satellite cell regenerative capacity and decreased satellite cell number due to continuous needs for satellite cells lead to progressive muscle weakness with chronic degeneration. Thus, it is necessary to replenish muscle satellite cells continuously. This review outlines recent findings regarding satellite cell heterogeneity, asymmetric division and molecular mechanisms in satellite cell self-renewal which is crucial for maintenance of satellite cells as a muscle stem cell pool throughout life. In addition, we discuss roles in the stem cell niche for satellite cell maintenance, as well as related cell therapies for approaching treatment of DMD.

Resident Endothelial Precursors in Muscle, Adipose, and Dermis Contribute to Postnatal Vasculogenesis

A novel population of tissue‐resident endothelial precursors (TEPs) was isolated from small blood vessels in dermal, adipose, and skeletal muscle of mouse based on their ability to be grown as spheres. Cellular and molecular analyses of these cells revealed that they were highly related regardless of the tissue of origin and distinct from embryonic neural stem cells. Notably, TEPs did not express hematopoietic markers, but they expressed numerous characteristics of angiogenic precursors and their differentiated progeny, such as CD34, Flk‐1, Tie‐1, CD31, and vascular endothelial cadherin (VE‐cadherin). TEPs readily differentiated into endothelial cells in newly formed vascular networks following transplantation into regenerating skeletal muscle. Taken together, these experiments suggest that TEPs represent a novel class of endothelial precursors that are closely associated with small blood vessels in muscle, adipose, and dermal tissue. This finding is of particular interest since it could bring new insight in cancer angiogenesis and collateral blood vessels developed following ischemia.