In vitro clonal analysis of murine pluripotent stem cells isolated from skeletal muscle and adipose stromal cells.

Objective: Possible clinical utility of pluripotent stem cells (PSCs) with multilineage differentiation capacity depends on their ability to adapt to tissue-specific differentiation conditions. Previous data from our laboratory suggest that putative PSCs exhibiting an immunophenotype of CD45(-)Sca-1+CD117(-)CD90+ can be isolated from multiple tissues. In the present study, the clonal in vitro differentiation potential of two isolates of PSCs was examined.

Materials And Methods: Clonal analysis of the differentiation potential of skeletal muscle- (SM) and adipose stromal cell (ASC)-derived PSCs into myogenic, adipogenic, and neurogenic cells was investigated by expanding single PSCs prior to specification under three separate differentiation conditions.

Results: Differentiation of SM- and ASC-derived PSCs into myotubes, adipocytes, and neuronal-like cells was evident in clonal cultures promoting differentiation along these lineages. A total of 2.0%, 1.0%, and 0.33% of SM-derived clones demonstrated unipotent, bipotent, and tripotent differentiation, respectively, into combinations of myocytes, adipocytes, and neuronal cells. As a percentage of SM-derived PSCs, tripotent clones comprised 0.016% of total muscle cells. Similar results were obtained with ASC-derived PSCs, suggesting phenotypic and functional similarities between PSCs from both tissues. Following differentiation of single PSCs into three lineages, a clear and complete commitment to tissue-specific gene expression accompanied by inactivation of lineage-unrelated genes could not be demonstrated in several SM- and ASC-derived clones.

Conclusions: These data demonstrate that phenotypically defined PSCs remain functionally heterogeneous at the single-cell level and illustrate that morphologic lineage commitment may be independent of exclusive expression and/or loss of associated lineage specific genes.