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

Abstract

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. 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. 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. 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.