VII. Differences in adult stem cells (ASCs)
We are planning to define adult HSC functions in vivo to test other adult stem cells (ASCs), and to determine whether the BALB/B6 differences are general for all ASCs. Two requirements are essential: ASCs must be unambiguously identified by genetic markers, and ASCs must function normally in vivo. Ideal models will be recipients with genetically deficient endogenous ASCs that we will replace with ASCs from anemic mice. Using RNAi inserted by viral vectors, we will try to produce recipients with targeted SC defects, to get double-stranded expression in specific tissues, inhibiting specific genes, receptors, or nuclear factors (oct-8, c-Kit). We will look for correlated defects in various central nervous system (CNS) cells descended from the same precursors.
We will use our expertise in defining adult hematopoietic stem cells (AHSCs) in vivo to test how well bone marrow stem cells (BMSCs) repopulate the brain, and how well neural stem cells (NSCs) repopulate the myeloid and lymphoid systems, relative to each other. We will compare relative repopulating abilities of mixtures of NSC and BMSC subsets in both brain and in the myeloid/lymphoid systems. We will have 2 essential rules: 1) All SCs must be unambiguously identified by genetic markers. 2) The precursor must function normaly in vivo.
A major problem in plasticity studies has been the very limited degree of repopulation. This may have resulted from the use of recipients with normal precursor cells, which greatly limit donor cell repopulation. For testing HSC functions, we will use W-anemic recipients known to have effective HSCs. For testing NSC functions, we will use recipients with genetic defects that may have parallel effects on NSCs. Using competitive repopulation, in vivo, we will test the hypothesis that the same precursor can produce progeny that repopulate both hematopoietic and brain cells.
We will compare correlations between brain and hematopoietic cells after transplanting mixtures of 2 marked cell types into groups of 30 recipients. If a single precursor repopulates both brain and blood, we will see its function in both types of tissues in the same recipients, and not in the others. Cloning or simple limiting dilution will not be used, as too few donor cells would be present to maintain normal health in the recipient. Retroviral insertion markers will verify results from competitive dilution.
