Expression of Abcg2 in Murine Skeletal Muscle Cells Identifies Two Populations with Different Myogenic Potential.

Stem cells can be identified by a side population (SP) phenotype in a variety of adult and embryonic tissues. We have previously shown that expression of the Abcg2 serves as a prospective marker for isolating HSCs suggesting that Abcg2 expression may also serve as a marker for stem cell activity in other non-hematopoetic tissues. In particular, skeletal muscle SP cells have been shown to have stem cell activity in muscle reconstitution experiments and the SP population in skeletal muscle is significantly reduced in Abcg2 null mice. To investigate the possibility that Abcg2 can serve as a muscle stem cell marker, we used our mouse strain in which a GFP reporter gene was inserted into the Abcg2 locus. Skeletal muscle cells from adult Abcg2/GFP knock-in mice were isolated based on GFP expression and tested for stem cell activity. To exclude contamination by hematopoetic cells, all experiments were performed on cells gated for the CD45 ⁻/Ter119⁻ phenotype. Flow cytometric analysis showed that 11.6 ± 4.2 % of these muscle cells expressed the Abcg2/GFP allele. Since myogenic progenitor cells have the CD34⁺/ Sca-1⁻phenotype, GFP positive and negative cell populations were further analyzed for CD34 and Sca-1 expression. This analysis showed that 15.6 ± 5.3 % of Abcg2/GFP⁺ cells were CD34⁺/ Sca-1⁻. In contrast, 51.3 ±18.3 % of Abcg2/GFP⁻ cells were CD34⁺/ Sca-1⁻. These results indicated that Abcg2/GFP⁻ cell population may have a higher frequency of myogenic progenitor cells when compared to Abcg2/GFP⁺ cells. Analysis of skeletal muscle SP cells for GFP expression showed that 57.5±12 % of the SP and 10.8±0.9 % of non-SP or main population (MP) cells expressed the Abcg2/GFP allele. When SP and MP cell populations were analyzed for CD34 and Sca-1 expression, the highest percentage of CD34⁺/Sca-1⁻ cells were found in MP/GFP⁻ cell population (33.8±5.3%). Since 61.7 % of total cells were MP/GFP⁻ cells, the greatest absolute number of cells with the myogenic phenotype were found to be located in MP/GFP⁻ population. The growth characteristics and differentiation potential of Abcg2/GFP⁺ and Abcg2/GFP⁻ cells were then assessed in a myogenic clonal culture assay. Sorted Abcg2/GFP⁺ and Abcg2/GFP⁻ cells were plated in collagen-coated plates in proliferation medium. Both cell populations increased in number and formed large colonies after 7 days in culture. When these cells were then cultured in myogenic differentiation medium for 4 days, only GFP⁻ cells differentiated into contracting myofibers. In contrast, GFP⁺ cells differentiated mostly into adherent fibroblast like cells. This data was further validated by DNA micro-arrays analysis of GFP⁺ and GFP⁻ cell populations. We found that GFP⁻ cells expressed skeletal muscle-specific genes such as MyoD, myf-5, myogenin and troponin whereas GFP⁺ population did not express any of these genes. Based on these data, we conclude that myogenic progenitor cells did not express the Abcg2/GFP allele. We are currently characterizing the Abcg2/GFP⁺ population for potential mesenchymal stem cell activity. Transplantation assays to determine myogenic activity of GFP⁺ and GFP⁻ populations in vivo are in progress.