Identification of a Common Pluripotent Stem Cell from Murine Post-Natal Skeletal Muscle and Adipose Tissue

Jonathan C. Howell¹, Mervin C. Yoder², Jalees Rehman³, Keith L. March³, Wei-Hua Lee², Jin Zhong² and Edward F. Srour^{1, 2, 3}

Pluripotential stem cells (PSC) with transdifferentiation capacity have a remarkable potential utility as a treatment modality for a large number of malignant and non-malignant diseases. A number of cells possessing characteristics of PSC have been identified and isolated from several adult murine tissues. We therefore hypothesized that murine tissues may contain a common PSC and that these cells may either reside permanently in specific sites or continue to circulate and colonize tissues as needed.

Previous data from our laboratory suggest that cells exhibiting an immunophenotype of CD45-Sca-1+c-kit- can be isolated from several murine tissues and may represent putative common pluripotent stem cells. Phenotypic examination of cell isolates documented that CD45-Sca-1+c-kit- cells can be identified within skeletal muscle cells, bone marrow cells, peripheral blood mononuclear cells, brain cells, small intestinal mucosa and adipose stromal cells (ASC). In order to expand the phenotypic definition and further evaluate the differentiation potential of these cells, we sorted both murine neonatal muscle and adult adipose stromal cells (ASC) based on CD34 and Thy-1 expression within the CD45-Sca-1+c-kit- phenotype. Utilization of CD34 and Thy-1 identified four distinct subgroups of CD45-Sca-1+c-kit- cells and CD45-Sca-1+c-kit-CD34+Thy-1+ cells were very prominent. The pluripotent differentiation capacity of these and their three complimentary subgroups was investigated in comparative in vitro differentiation assays. Only CD45-Sca-1+c-kit-CD34+Thy-1+ muscle and adipose-derived common pluripotent stem cells (CoPSC) differentiated in response to IGF-1 into Oil Red O staining adipocytes, or into osteoblasts that stained positively with Alizarin Red S in response to stimulation with BMP-2, or into spontaneously twitching myotubes when cultured with horse serum and 5-azacytidine. Additionally, muscle-derived CoPSC demonstrated neurogenic differentiation in response to bFGF and PDGF and expressed Tau and NF-200 proteins, as assessed by confocal microscopy. When CD45-Sca-1+c-kit- cells from muscle or bone marrow were transplanted intracerebellarly into Purkinje Cell Degenerative (pcd) mice, the behavior of these mice improved 28 days after transplantation relative to mice injected with vehicle alone suggesting that these cells contributed to the appearance of functional neuronal cells that may have improved the ataxic condition characteristic of these mice.

Whether candidate stem cells truly posses pluripotential differentiation capabilities can only be assessed by clonal analysis. Single muscle-derived CoPSC cells were used in a two-step assay to examine whether progeny cells can be exogenously directed into multiple differentiation pathways. Our preliminary results demonstrated that 0.02%, 0.008%, and 0.003% of muscle-derived CoPSC were unipotent, bipotent and tripotent in their ability to differentiate along adipogenic, myogenic, and neurogenic pathways. Taken together, these results suggest that a rare cell population identified as CD45-Sca-1+c-kit- may represent a common PSC present in multiple murine tissues and that these cells may be capable of clonal pluripotential differentiation.

Departments of ¹Microbiology/Immunology, ²Pediatrics, Herman B Wells Center for Pediatric Research and Medicine, ³Indiana University School of Medicine, Indianapolis, IN