Autonomous regulation of stem cell frequency

Lineage restricted growth factors regulate peripheral blood cell homeostasis. However, mechanisms that affect the frequency of primitive stem cells with long term proliferative potential remain largely unknown. We have shown that stem cell numbers in DBA/2 (D2) mice are higher than in C57BL/6 (B6) mice. To assess whether this is a cell-extrinsic (e.g. due to variation of growth factor concentrations) or cell-autonomous trait we have developed a model in which D2 and B6 stem cells co-exist is a single animal. Chimeric mice were produced by transplanting irradiated B6D2F1 recipients with mixtures of D2 and B6 day-14 fetal liver cells. Donor leukocyte contribution was assessed using anti-H2Kb/d antibodies. In agreement with our previous findings, repopulating ability of D2 fetal liver cells was 10 fold higher than B6 cells. In order to determine the actual frequency of D2 and B6 stem and progenitor cells in these chimeric animals, we performed several experiments. Firstly, mice in which D2 and B6 leukocyte contributions were equal, were injected with G-CSF. Peripheral blood CFU-GM were cultured and the genotype of each colony was assessed using SSLP-PCR. D2 CFU-GM were 4-8 fold more numerous than B6 progenitors. Marrow was harvested from these chimeric mice and D2 and B6 cell populations were separated by flow cytometry. CAFC analysis on sorted samples showed that the frequency of D2 late appearing CAFC subsets was 10 fold higher, indicating that chimerism of mobilized progenitors reflected marrow stem cell chimerism. To confirm these in vitro data we performed a secondary transplantation using unfractionated chimeric marrow, transplanted in limiting doses to F1 recipients. Comparison of the proportion of animals showing D2 and B6 leukocyte contributions revealed that the frequency of D2 LTRA was 10 fold higher than B6 LTRA numbers. Our data demonstrate that two genetically distinct stem cell populations, coexisting in individual animals, independently maintain their parental phenotype/frequency. This indicates that stem cells maintain their population size autonomously.