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Skeletal muscle stem cells Development of an in vitro assay and validation of multi-lineage hematopoietic potential in vivo

Skeletal muscle stem cells Development of an in vitro assay and validation of multi-lineage hematopoietic potential in vivo

Blood 96(11 Part 1): 279a, November 16

An emerging theme in stem cell biology is the flexibility retained by stem cells to express different developmental programs appropriate to specific tissue microenvironments. To investigate the developmental potential and plasticity of stem cells in skeletal muscle, we have prepared single cell suspensions by mincing and enzymatic digestion of muscles obtained from perfused mice. To determine the numbers of progenitor and stem cells in this cell preparation, we adapted the cobblestone area-forming cell (CAFC) assay originally described for hematopoietic cells. Seeding on a stromal layer to support stem cell differentiation, the CAFC assay permits the determination of CAFC frequencies at limiting dilution and provides temporal data indicating the hierarchy of stem and progenitor cells (late developing colonies arising from more primitive cells). Beginning at 7 days of culture, and extending through at least 5 weeks, small colonies of 5-20 cells developed from the skeletal muscle cell preparations. These muscle-derived CAFC formed at a frequency (2-4 colonies per 105 cells) comparable to the frequency of hematopoietic stem cells found in adult marrow. Furthermore, as seen in the hematopoietic system, different mouse strains showed different frequencies and kinetics of muscle CAFC. To determine the in vivo potential of the skeletal muscle cell preparations to differentiate into hematopoietic cells, we injected single cell suspensions into irradiated (8Gy) C57BL/6 mice. Six weeks following transplant of 4 X 106 cells prepared but not otherwise selected from muscle, donor cells com-prised apprx30% of the blood leukocytes in several lineages. Donor cells contributed to engraftment in a dose-dependent manner. Assuming a seeding efficiency of 10% for the in vivo assay, muscle cell preparations contained roughly similar numbers of primitive cells as judged by the in vivo and in vitro assays, despite the fact that differentiation into another cell lineage was required in vivo. The results suggests that the muscle. CAFC assay can detect a muscle stem cell that is capable of altering its developmental program and will be useful for further investigation of these stem cells and their potential use for disease treatment.

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