The overall objective is testing long term functional abilities of primitive hemopoietic stem cells (PHSC) in newborn mouse blood, which models human umbilical cord blood at birth. This project focuses on long term clinical effectiveness of PHSC, from which all myeloid and lymphoid cells are continuously descended. PHSC are essential to clinical use of cord blood, and will be analyzed using newly available congenic mice with distinguishable markers. Direct analyses of long term function in vivo define PHSC numbers and functional abilities, avoiding enrichment, tissue culture, or retroviral marking, which might affect PHSC. Functions per PHSC are determined using Poisson statistics in a new competitive dilution assay. B6 and BALB strains will be compared since they show major differences in regulation of PHSC faction with age. Such genetic differences may also exist in human beings. The hypotheses to be tested are: (1) that PHSC in cord blood, adult marrow and fetal liver function equally well. Initial results suggest that PHSC from BALB but not B6 show large functional changes with age. Competitive repopulating abilities per PHSC will be compared using various donor types, including B6 and BALB. (2) that the same chromosomal regions affect PHSC in cord blood as fetal liver. RI and bilineal congenic lines will be used to test whether genes at similar genetic loci affect cord blood PHSC and cause loss of PHSC functional capacity from fetal to adult life in BALB but not in B6. (3)that in vitro assays predict cord blood PHSC function. Large differences in functional abilities of BALB fetal and adult PHSC, provide standards to evaluate whether the LTC-IC or cobblestone assays can estimate long term function of cord blood PHSC. (4) that effects of freezing on long term repopulating abilities can be minimized. Clinical use of cord blood often requires freezing. Effects of various freezing methods on PHSC function will be tested using genetically disparate cell types.
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