The broad objective is to determine how to regulate differentiation and self renewal of the most primitive hemopoietic stem cell (PHSC), as success of clinical marrow transplantation depends on these cells. This project explores a recent finding that BMC (bone marrow cells) from the CXB-12 RI strain have more than 10 times the long term repopulating ability expected from inbred strains, including 11 other CXB RI strains.
The specific aims define the biology and genetics of this hyper PHSC phenotype, using recipients and donors homozygous for I1-2d to remove hybrid resistance.
Aim 1 defines CXB-12 strain PHSC. Whether BMC have increased numbers of PHSC, increased function per PHSC, or both, will be tested by competitive limiting dilution. T and B cells will be removed, to test if they affect the assay. Whether CXB-12 PHSC have improved homing after grafting or migration will be tested in shielded mice. Effects of development and aging on PHSC, of 5FU on precursor assays, of transplantation, of cell cycle-specific drugs and of enrichment using stem cell markers, will be tested.
Aim 2 defines the genetics. Whether the CXB 12 phenotype is caused by a single co-dominant mutation, or a co-dominant locus in a unique recombination, will be tested. Analysis of phenotypes and genetic markers from 200 back-crossed donors will locate the chromosome locus and candidate genes. 5FU may reveal the phenotype in other assays. The parent strains will be made congenic for the locs.
Aim 3 tests if hyper PHSC function is intrinsic to the PHSC, by comparing CXB-12 strain PHSC with parent or Fl hybrid PHSC in the same allophenic donor. Allophenics will be produced by fusing early CXB-12 embryos with F1 embryos, genetically marked to distinguish PHSC progeny. Identity, number and function of individual PHSC will be defined by competitive dilution.
