Kinetics of Self-Renewal and Differentiation Divisions of Transplanted Hematopoietic Stem and Progenitor Cells Early After Transplantation Into Lethally-Irradiated Recipients.

Abstract 2443

Poster Board II-420

Transplanted hematopoietic progenitor cells (HPC) and stem cells (HSC) provide short- and long-term hematopoietic support, respectively, in myeloablated recipients after transplantation. Despite the reliance on these cells for successful clinical engraftment and reconstitution of transplant recipients, little is known regarding their proliferation kinetics in vivo during the period of engraftment, or how this relates to the vast literature describing steady state hematopoiesis. We have previously established methodology that can track donor HSC and HPC in mice after transplantation using retention and loss of CFSE fluorescence to identify CFSEbright and CFSEdim cells, respectively. Cells identified as CFSEbright on d5 post-transplantation were shown to be exclusively enriched for donor long-term repopulating potential, thus comprising all the HSC within the donor cell population. In the current study, we used this methodology to examine the long-term repopulating potential and progenitor activity of CFSEbright, CFSEmid, and CFSEdim cells isolated from primary recipients on days 3, 5, 7, and 10 after transplantation of low density bone marrow (LDBM) cells. We aimed to determine when HSC activity moved from CFSEbright cells into the CFSEmid, as a means of estimating the time point at which donor HSC undergo self-renewal divisions in recipient BM. Likewise, using clonogenic assays, HPC content of the three CFSE fractions was followed to determine the kinetics and nature of proliferation of donor progenitor cells. As expected, the percentage and absolute number of CFSEbright and CFSEmid cells decreased by day 10 to approximately 1-10% of day 3 values, while CFSEdim cells increased ∼200-fold to comprise >95% of donor cells by day 10 (n=14-16 mice/time point). Interestingly, when the HPC content of the various CFSE populations was examined, all HPC activity at day 3 post-transplant resided in the CFSEmid cells, suggesting that HPC divide rapidly upon transplantation and leave the CFSEbright pool within 1-2 days. Progenitor activity began to appear in the CFSEdim population by d5 post-transplant, increasing 5- to 20-fold in absolute number by day 10, roughly paralleling the increase in absolute number of CFSEdim cells during this same time frame. While the frequency of total HPC in CFSEmid and CFSEdim populations was similar to that of steady state LDBM, 5- to 15-fold more of these progenitors were identified as CFU-GEMM and HPP-CFC compared to steady state BM, suggesting that engrafting cells expand their primitive HPC content at a faster rate than steady state BM. In contrast to the rapid proliferation kinetics of engrafting HPC, results of competitive transplantation studies of the various CFSE fractions suggest that long-term multi-lineage engraftment potential moves from the CFSE-bright to the CFSE-mid population around day 7-8 post-transplantation. Using the number of transplanted CFSE graft cells and their 6mo chimerism values to determine an enrichment factor for HSC potential, we estimate that d5 CFSEbright cells are 28-fold more enriched for HSC activity than steady state LDBM, while d7 CFSEmid cells are 8-fold more enriched. These data suggest that within the first 7 days post-transplant, 1 in 3.5 cell divisions of CFSEbright cells are self-renewal in nature. In contrast, using the same formula, CFSEdim cells were found to possess ∼1% of the HSC activity of steady state LDBM when analyzed up to 1 month post-transplantation, suggesting that CFSEdim cells are functionally weakened at these early time points post-transplant, and thus unable to provide significant chimerism in secondary recipients. Taken together, these data suggest that donor HSC undergo self-renewal divisions at approximately 1 week post-transplant and at a much higher rate than during steady state hematopoiesis. In addition, transplanted HPC were found to proliferate between 1-2 days post-transplant, and appear to give rise to a pool of progenitors 5 to 15-fold more enriched for primitive HPC than that present in steady state LDBM. These results add to our understanding of HSC/HPC engraftment and the kinetics of self-renewal and differentiation divisions in vivo, and may have clinical implications in designing methodologies to optimize hematopoietic engraftment and reconstitution.