Abstract
The hemangioblast, the progenitor common to hematopoetic and endothelial lineages, expresses the vascular endothelial growth factor (VEGF) receptor flk-1 on its surface. Based on lineage-tracking studies indicating that skeletal muscle cells are derived from flk-1-expressing precursors (Sato et al. Genesis35:153–159, 2003), we tested the hypothesis that the hemangioblast is also a progenitor to the muscle lineage. We studied the development of skeletal muscle progentiors from mouse embryonic stem cells cultured in suspension to form embryoid bodies (EB). These EB were harvested at different time points, disaggregated into single cells and analyzed by flow cytometry. After 10–18 days of culture, the EB contained abundant cells that expressed α7-integrin, a skeletal muscle-specific cell surface antigen. Immunofluorescence microscopic study of sorted and cytospun α7-integrin-positive cells from EB revealed positive staining with MyoD, sarcomeric α-actinin and desmin antibodies, indicating that they were bona fide muscle cells. To test if these muscle cells were derived from hemangioblasts, green fluorescent protein (GFP)-expressing EB were collected at day 4 of development, disaggregated into single cells and replated in methylcellulose medium with the addition of VEGF, stem cell factor and thrombopoietin. After 5 days in methylcellulose culture, clusters of loosely aggregated cells appeared. These cells have features characteristic of hemangioblasts and exhibit large nuclear-to-cytoplasmic ratios. The hemangioblast colonies were individually picked, diaggregated into single cells and plated with a myoblastic cell line (C2C12 cells) in media containing 10% fetal calf serum. Co-culturing of the hemangioblasts with a myoblastic feeder layer was designed to provide the environmental signals necessary for myogenic development. After 48 hours, the co-culture medium was changed to 2% serum to promote muscle differentiation. Three days later, the plates were fixed and analyzed. A small number of GFP-expressing cells assumed an elongated, multinucleated, myofiber-like appearance and stained positiviely for skeletal muscle markers α7-integrin and desmin. We next tested the myogenic potential of hemangioblasts in vivo. The tibialis anterior muscles of C57BL/6 mice were injected with cardiotoxin, a protein kinase inhibitor that depolarizes muscle fibers and induces muscle regeneration. After two days, hemangioblast colonies were picked from methylcellulose culture and injected into the regenerating muscles. Occasional GFP-expressing fibers that stained positive for desmin were seen when analyzed several weeks post-injection. These results indicate that the hemangioblast may be a progenitor not only to hematopoetic and endothelial lineages, but also to skeletal muscle. Future experiments include investigating the significance and mechanism of the hemangioblast’s development into muscle and explore the role of cell-cell fusion in this phenomenon.
Epigenetic Reprogramming of Human Embryonic Stem Cells into Skeletal Muscle Cells and Generation of Contractile Myospheres