Isolation, characterization, and transplantation of stem

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Hematopoietic stem cells reside within the bone marrow post-natally, providing all hematopoietic lineages for the life of the host, and their extensive characterization over the last several decades has led to clinical application including bone marrow transplantation for benign and malignant disorders affecting the hematopoietic compartment. Isolation of bone marrow cells based upon expression of the CD34 antigen enriches for the primitive compartment, and techniques to isolate the CD34-positive population are commonly used in clinical practice. Recently, a method for the isolation of murine bone marrow cells capable of reconstituting hematopoiesis after lethal irradiation based solely upon dual wavelength flow cytometry after staining with the vital dye, Hoechst 33342, was described by Goodell et. al. The molecular basis of these side population cells was recently attributed to expression of the ABC transporter, ABCG2. Side population (SP) cells are highly enriched for hematopoietic repopulating ability, with one cell capable of reconstituting hematopoiesis in irradiated recipient mice. We reasoned that this phenotype is conserved among organs with the capacity for post-natal regeneration. We have recently isolated SP cells from the non-parynchymal portion of human cadaveric liver and have cultured these cells in hepatic culture media. Hepatic SP cells generated colonies of cells demonstrating granule rich cytoplasm and dense, often double nuclei consistent with hepatocytes. These hepatocyte-like cells expressed markers of human hepatocytes including HepPar, cytokeratin-8, and human albumin. RT-PCR confirmed the expression of hepatocyte markers including albumin, cytokeratin-18, along with the more specific markers, ?1 antitrypsin and the human P450 gene, CYP2B6. We have now developed an in vivo rescue model in NOD/SCID mice treated with sublethal dosing of carbon-tetra-chloride, and initial results suggest that SP cells are capable of affecting a normalization of liver function test similar to mature hepatocytes when compared to controls. Further in vivo characterization of these SP cells is ongoing. Additionally, SP cells have been isolated from the adult pancreas, and microarray analysis demonstrates gene expression profile similar to that of bone marrow derived SP cells. Initial attempts to differentiate pancreatic SP cells toward a mature beta-cell phenotype have met with only limited success and these studies are ongoing. Finally, we have cloned the rhesus ABCG2 gene to determine the effects of forced expression of this transporter in the primitive hematopoietic compartment. Our results confirm the existence of rhesus-ABCG2, establish its importance in conferring the SP phenotype in large animals, suggest no detrimental effect of its overexpression upon differentiation in vivo, and imply a potential role for its overexpression as an in vivo selection strategy for gene therapy applications1.