Molecular detection of stem cell plasticity

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Definitive evidence of adult stem cell plasticity would be of outstanding significance for understanding organ development and would promise novel curative cell replacement therapies. Rigorous proof of bone marrow stem cell plasticity requires the detection of molecular identity between cells in hematopoiesis and organ regeneration. Retro- and lentivirus vector integration generates unique, stable integration sites and is an ideal marker for clonal derivation. We have developed a PCR-based technique for the direct genomic sequencing of unique retro- and lentiviral integration sites at the single cell level. This proposal aims to study the immigration and organ-specific differentiation of MGMT/GFP-marked oligoclonal bone marrow stem cells after transplantation into murine models of human hepatitis B virus large antigen transgenic mice and cathepsin L knockout mice. Hepatoregeneration and differentiation into cardiomyocytes will be studied in these transgenic models with and without radiation damage. GFP-positive cells will be identified together with specific markers of differentiation by double or triple color immunofluorescence. Non-contact laser optical microdissection of GFP-marked, organ-specific cells followed by single cell integration site analysis will definitively prove or disprove a common clonal origin of such cells with hematopoiesis. The quantification of the number and frequency of pluripotent stem cells presents in murine marrow will define the extent of plasticity and help to establish a better understanding of multipotent stem cell physiology.