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Whether cytokines can modulate the fate of primitive hematopoietic progenitor cells (HPCs) through successive in vitro cell divisions has not been established. Single human marrow CD34+CD38–/lo cells in the G0 phase of cell cycle were cultured under 7 different cytokine combinations, monitored for proliferation on days 3, 5, and 7, then assayed for long-term culture-initiating cell (LTC-IC) function on day 7. LTC-IC function was then retrospectively correlated with prior number of in vitro cell divisions to determine whether maintenance of LTC-IC function after in vitro cell division is dependent on cytokine exposure. In the presence of proliferation progression signals, initial cell division was independent of cytokine stimulation, suggesting that entry of primitive HPCs into the cell cycle is a stochastic property. However, kinetics of proliferation beyond day 3 and maintenance of LTC-IC function were sensitive to cytokine stimulation, such that LTC-IC underwent an initial long cell cycle, followed by more synchronized shorter cycles varying in length depending on the cytokine combination. Nonobese diabetic/severe combined immunodeficiency (NOD/SCID) transplantation studies revealed analogous results to those obtained with LTC-ICs. These data suggest that although exit from quiescence and commitment to proliferation might be stochastic, kinetics of proliferation, and possibly fate of primitive HPCs, might be modulated by extrinsic factors.

Differences in engraftment potential of hematopoietic stem cells (HSCs) in distinct phases of cell cycle may result from the inability of cycling cells to home to the bone marrow (BM) and may be influenced by the rate of entry of BM-homed HSCs into cell cycle. Alternatively, preferential apoptosis of cycling cells may contribute to their low engraftment potential. This study examined homing, cell cycle progression, and survival of human hematopoietic cells transplanted into nonobese diabetic severe combined immunodeficient (NOD/SCID) recipients. At 40 hours after transplantation (AT), only 1% of CD34(+) cells, or their G(0) (G(0)CD34(+)) or G(1) (G(1)CD34(+)) subfractions, was detected in the BM of recipient mice, suggesting that homing of engrafting cells to the BM was not specific. BM of NOD/SCID mice receiving grafts containing approximately 50% CD34(+) cells harbored similar numbers of CD34(+) and CD34(-) cells, indicating that CD34(+) cells did not preferentially traffic to the BM. Although more than 64% of human hematopoietic cells cycled in culture at 40 hours, more than 92% of cells recovered from NOD/SCID marrow were quiescent. Interestingly, more apoptotic human cells were detected at 40 hours AT in the BM of mice that received xenografts of expanded cells in S/G(2)+M than in recipients of G(0)/G(1) cells (34.6% +/- 5.9% and 17.1% +/- 6.3%, respectively; P

The bone marrow (BM), in addition to its role in providing hematopoietic stem cell support, is becoming increasingly recognized as a significant reservoir for T cells. Recent data have shown the BM to be the predominant site for homeostatic peripheral expansion (HPE). Constitutive HPE is a mechanism which allows maintenance of T cell numbers, while lymphopenia-induced HPE restores T cell numbers following T cell-depleting measures such as myelosuppression. While the role of BM in constitutive HPE in non-irradiated mice has been well documented, little is known of the role of BM in HPE following myeloablation. In the present study, we examined the contribution of BM to lymphopenia-induced HPE following myeloablative radiation conditioning and HSC transplantation, and documented the kinetics of expansion of donor-derived T cell subsets in the irradiated marrow and spleen. To this end, C57BL/6 and BoyJ congenic mice, differing in CD45 allelic expression, were used to track donor-derived T cell subsets using 8 color flow cytometry 5 days after transplantation of 10–20 × 10e6 low density BM cells (non-T cell depleted) into lethally-irradiated recipients (950cGy). At the time of analysis (d5 post transplant) the percentage of total donor cells in BM was approximately 20%, and 10% in the spleen. Of interest, an enhanced migration of CD8+ cells to BM was observed, increasing from 3±0.6% in steady state marrow to 8±3.3% of donor cells in transplanted mice. While only 10.1±1.0% of CD8+ cells in steady state BM exhibited a central memory phenotype (TCM, CD44hi, CD45RBhi, CD62Lhi), this percentage increased to 90±2.7% of donor CD8+ cells in day 5 transplanted BM (p

We have previously shown that in vitro migration and NOD/SCID engraftment potential of mobilized peripheral blood (MPB) CD34+ cells can be significantly increased 2–3 fold by preincubation of graft cells with 100 ng/ml of SDF-1α (CXCL-12) for 30mins at 4°C. However, bone marrow (BM) and cord blood (CB) CD34+ cells did not exhibit similar increases in migration or engraftment after SDF-1α preincubation and, in the case of CB, preincubation somewhat reduced in vitro migration. We believe that these biological differences in SDF-1α responses may begin to explain some of the reported differences in hematopoietic potential among these three sources of CD34+ cells, and may be related to rate and degree of CXCR4 receptor recycling after SDF-1α exposure. In the present work, we examined patterns of CXCR4 expression both before (part 1 studies) and after (part 2 studies) SDF-1α pretreatment, and found that, in general, MPB CD34+ cells appear to possess an inherent ability to mobilize internal CXCR4 stores with greater efficiency than BM and CB CD34+ cells, while CB appears most sensitive to SDF-1α-induced CXCR4 downmodulation. This conclusion is supported by the following results: In part 1 studies, when untreated CD34+ cells were incubated with SDF-1α (100 ng/ml, 37°C), CXCR4 expression initially decreased in all tissues, but returned to basal levels in BM and MPB cells, while re-expression on CB cells remained below baseline for the duration of the 2hr assay. In parallel cultures where SDF-1α was washed out after 60mins, CXCR4 re-expression was most rapid in MPB CD34+ cells, reaching 4.5±2.7-fold higher levels of expression after 30mins, compared to 1.8±1.1-fold for BM (p