Neuronal differentiation of stem cells isolated from adult muscle

Abstract

Lineage uncommitted pluripotent stem cells reside in the connective tissue of skeletal muscle. The present study was carried out with pluripotent stem cells (PPSCs) isolated from 6‐month old rat muscle. Before differentiation, these cells were vimentin+, CD90+, CD45−, and varied in their expression of CD34. The PPSCs were expanded as non‐adherent aggregates under similar conditions to those used to generate neurospheres from embryonic or neural stem cells. The PPSC‐derived neurospheres were positive for nestin, an early marker present in neuronal precursors, and expressed the two alternative mRNA forms of the neuroectodermal marker Pax‐6, as well as mRNA for Oct‐4, a gene related to the pluripotentiality of stem cells. To confirm their neural potential, PPSC‐derived neurospheres were plated on coated coverslips under varying conditions: Neurobasal medium with N2 or B27, and either NT3 or BDNF. After 4–6 days the cells expressed neuronal (Tuj1+, NF68), astrocytic (GFAP) and oligodendrocytic (MOSP+, MBP+) markers, both by immunocytochemistry and RT‐PCR. In addition, PPSCs were cultured as monolayers under adherent conditions, exposed to growth factors and defined differentiating conditions for 5 hr, and subsequently kept for 2 days in a maturation medium. At this point they gave rise to a mixed population of early neural progenitors (Nestin+ or NG2+), immature and mature neurons (Tuj1+ and NF145+) and myelin producing oligodendrocytes (CNPase + and MOSP+). Our study shows that PPSCs present in adult muscle can overcome germ lineage restrictions and express the molecular characteristics of brain cells. Therefore, PPSCs isolated from adult muscle could provide a novel source for autologous cell replacement in neurodegenerative and demyelinating diseases. © 2002 Wiley‐Liss, Inc.

Dedifferentiation of Adult Human Myoblasts Induced by Ciliary Neurotrophic Factor In Vitro

“…The myoblasts in these clones can divide for more than 25 passages with preserving their myogenic specificity because the myoblasts randomly selected from 3 to 25 passages were uniformly positive for MyoD, the key marker of myogenic lineagecommitted myoblasts (Kablar et al, 1999;Parker et al, 2003;Chen et al, 2004); while grown in DM to induce myogenic differentiation, they can form multinucleated myotubes stained positive for Myosin, a marker of myogenic terminal differentiation myotubes (Gal-Levi et al, 1998;Odelberg et al, 2000;Langley et al, 2002). The data shown in Figure 1 let us exclude the possibility that multiple distinct stem/progenitor cells identified in adult skeletal muscle present in the myoblast clones: first, although muscle satellite cells, the precursor cells of myoblasts, may express Myf5, the expression of MyoD is not detectable in the cells (Jennifer et al, 2003); second, because the myoblast clones are uniformly negative for Nestin, CD34, and CD45, there are no such heterogeneous contamination cells as previously identified Nestin ϩ NSCs (Romero-Ramos et al, 2002), CD34 ϩ /CD45 ϩ HSCs (Jackson et al, 1999), CD45 ϩ SP cells (Asakura et al, 2002), and CD34 ϩ MDSCs (Qu-Petersen et al, 2002) in adult skeletal muscle; in addition, there is also not the presence of fibroblasts and MSCs because they do not differentiate to form multinucleated myotubes expressing Myosin when grown under the same DM conditions to the myoblasts; finally, these possible heterogeneous cells in the adult skeletal muscle never express myogenic specific protein MyoD. Taken together, these results provide strong evidence against the interpretation that the myoblast culture system contains multiple distinct cells.…”

“…To exclude the presence of fibroblasts and mesenchymal stem cells (MSCs) in the myoblast clones, NIH3T3 and human MSCs were also cultured in the same myoblast GM or DM to determine the myogenic specificity of these cells. Furthermore, the Nestin ϩ NSCs (neural stem cells) and CD45 ϩ /CD34 ϩ HSCs (hemopoietic stem cells), which were used as the positive control of the immunocytochemical analysis with anti-Nestin, anti-CD34, and anti-CD45 antibodies to exclude the presence of previously reported progenitor/stem cells isolated from adult skeletal muscle, such as Nestin ϩ neuronal differentiation of stem cells (Romero-Ramos et al, 2002), and CD45 ϩ /CD34 ϩ SP cells/MDSCs (Asakura et al, 2002;Jankowski et al, 2002;Qu-Petersen et al, 2002;Tamaki et al, 2003) were plated onto the collagen-coated glass coverslips for 12 h to adhere. The control human MSCs and HSCs were gifts from Professor XiaoDan Guo and Xiaoxia Jiang, Ph.D., whereas NSCs of adult rat were isolated as previously described (Xu et al, 2003).…”

“…The Desmin ϩ clones were dispersed and plated on collagencoated glass coverslips in GM for 24 h to adhere and proliferate, or in differentiation medium (DM; see Materials and Methods) for 72 h to induce myogenic differentiation. Immunocytochemistry revealed that, of the five clones analyzed, all of them grown for 12 h in GM were positive for MyoD ( Figure 1A), the markers of myogenic lineage-committed myoblasts (Kablar et al, 1999;Jankowski et al, 2002;Tamaki et al, 2003), but were negative for Nestin ( Figure 1B), a muscle-derived NSC marker (Romero-Ramos et al, 2002). They also were negative for CD34 ( Figure 1B) and CD45 ( Figure 1B), the markers of muscle-derived CD45 ϩ /CD34 ϩ SP cells/MDSCs (Asakura et al, 2002;Jankowski et al, 2002;Qu-Petersen et al, 2002;Tamaki et al, 2003).…”

“…These clones were subjected to immunocytochemical analysis by staining with Desmin, a marker of myogenic lineage-committed myoblasts. Only the Desmin ϩ clones were allowed to further propagated and five of these clones were analyzed.To characterize the Desmin ϩ clones better, we analyzed them for production of markers associated with other progenitor/stem cells which have been isolated and identified from adult skeletal muscle, such as muscle satellite cells (Jankowski et al, 2002), the precursor cells of myoblasts, SP (side population) cells (Asakura et al, 2002;Jankowski et al, 2002;Tamaki et al, 2003), MDSCs (muscle-derived stem) cells (Jankowski et al, 2002;Qu-Petersen et al, 2002), and NSCs (neural stem cells; Romero-Ramos et al, 2002). The Desmin ϩ clones were dispersed and plated on collagencoated glass coverslips in GM for 24 h to adhere and proliferate, or in differentiation medium (DM; see Materials and Methods) for 72 h to induce myogenic differentiation.…”

Epigenetics and the plasticity of differentiation in normal and cancer stem cells

“…Another distinct population of bone marrow-derived stem cells can differentiate to cells of various lineages including muscle, endothelial, epithelial and neural cells (Yoon et al, 2005). Other adult stem cells that can be induced to differentiate to a different cell type, include skeletal muscle stem cells to blood cells (Jackson et al, 1999;Cao et al, 2003) and neural cells (Romero-Ramos et al, 2002); cardiac muscle stem cells to endothelial cells (Mohri et al, 2006); neural stem cells to blood and endothelial cells (Bjornson et al, 1999;Wurmser et al, 2004); hair follicle stem cells to neural lineage cells (Amoh et al, 2005a, b); and testis-derived stem cells to a wide variety of cell types (KanatsuShinohara et al, 2004;Guan et al, 2006). The use of adult stem cells has thus considerable therapeutic potential for the regeneration of damaged tissues.…”