Genetic regulation of hematopoietic stem cell (HSC) functions
We define strain differences in differentiation, self-renewal, and aging in hematopoietic stem cells (HSCs). Modern genetic tools (recombinant inbred [RI] and bilineal congenic lines analyzed with DNA markers) identify chromosome regions that regulate strain differences. Candidate genes must have DNA and expression patterns consistent with biological effects. For example, we test whether telomeres are shorter in cells descended from the old stem cells, whose function becomes less effective with age. (Results suggest that telomeres are not involved.)
Candidate genes are further defined in congenic strains recombined with the parent strain for increased precision, and ultimately by induced mutagenesis. The major problem in using genetically segregating donors is that hybrid resistance can affect stem cell functions. To solve this, we focus on congenics so that donors and recipients are matched for major histocompatibility genes, and we segregate only at a specific small chromosome region. This eliminates hybrid resistance. Studies using stem cell phenotype markers are important because they offer quick results, but they must be confirmed in vivo.
By far, the largest set of bilineal congenics is available with linked alleles from the 129 strain that were moved to the C57BL/6J (B6) background in the induced mutation program at The Jackson Laboratory. These alleles were genetically linked to homologous recombinants from ES cell lines of the 129 strain that have been back-crossed to the B6 background. Now we are defining effects on stem cell development, self-renewal, and aging in the 129 strain, comparing results with those from the standard B6 strain.
We are analyzing strain differences in differentiation, self-renewal, and aging in stem cells using haplotype mapping (phenome mapping), which allows us to predict the existence of quantitative trait loci (QTLs) by testing for association between the haplotypes and phenotypes using computerized databases. We will be testing HSC function in both short- and long-term assays, using many strains, to identify minute chromosome regions, identical by descent, that contain regulatory genes. We will confirm these regions using transgenics, and ultimately through induced mutagenesis.
As a first step, we screened 26 mutant genotypes of mice with single gene deletions, all on the B6 background. We tested whether each deletion alters the frequency of bone marrow cells (BMCs) carrying Lin-Sca1+KIT+CD34- markers, because this phenotype includes HSCs. Among the 26 mutants tested, 20 showed no significant change. Lin-Sca1+KIT+CD34- cell frequency was reduced in 5 gene-deletion mutants: ATP-binding cassette, sub-family Bmember 2 (Abcb2); cyclin-dependent kinase inhibitor 1a (Cdkn1a or p21); Lymphotoxin A (Lta); Nitric oxide synthase 1 (Nos1); and Nitric oxide synthase 2 (Nos2) (TeKippe et al., 2003).
Only the deletion of transformation-related protein 53 (Trp53, originally called p53) caused an increase in Lin-Sca1+KIT+CD34- cell frequency. Therefore, we further defined other aspects of hematopoiesis in Trp53-targeted mutant (Trp53-/-) mice. In comparison to wild-type controls, Trp53-/- mice had normal blood and bone marrow cell counts, increased ITGAM+ and decreased PTPRC+ cell proportions in blood and bone marrow, twice as many Lin-Sca1+KIT+CD34- BMCs, and 37% more day 9 colony forming units, spleen (CFUs-S). In the competitive repopulation assay, 5 months after transplantation, BMCs from Trp53-/- donors engrafted lethally irradiated recipients 2- to 4-times better than BMCs from Trp53+/+ donors. Only 44% of recipients of BMCs from Trp53-/- donors survived 5 months after transplantation, compared with 92% of recipients of BMCs from Trp53+/+ donors.