The Unexpected G0/G1 Cell Cycle Status of Mobilized Hematopoietic Stem Cells From Peripheral Blood

Abstract

Treatment with a combination of cytokines and chemotherapy can effectively stimulate the release of hematopoietic stem cells (HSC) into the peripheral blood (PB), which can then be harvested for transplantation. The cell cycle status of the harvested HSC from mobilized PB (MPB) is of interest because of the impact that cell cycling may have on optimizing the conditions for ex vivo expansion, retrovirus-mediated gene transfer, and the engraftment of transplanted tissues. Therefore, we characterized the cell cycling status of mobilized HSC from mice and humans. The murine HSC, which express the phenotype c-kit+ Thy-1.1lo Lin−/lo Sca-1+, were purified from PB, bone marrow (BM), and spleen after the mice were treated with the mobilizing regimen of granulocyte colony-stimulating factor (G-CSF ) or a combination of cyclophosphamide (CTX) and G-CSF. Human HSC (CD34+ Thy-1+ Lin−) and progenitor cells (CD34+ Thy-1− Lin−) were isolated from the BM of untreated healthy volunteers and from MPB of healthy volunteers and patients treated with G-CSF or a combination of CTX and GM-CSF. Cell cycle status was determined by quantitating the amount of DNA in the purified cells after staining with the dye Hoechst 33342. Fluorescence-activated cell sorting analysis of the progenitor cells from the murine and human samples showed an unexpected finding, ie, virtually none of the cells from the MPB was cycling. The G0/G1 status of HSC from MPB was surprising, because a significant proportion of HSC from BM are actively proliferating and, after mobilization, the HSC in the spleen and BM were also actively cycling.

Dynamic cell cycle kinetics of normal CD34+ cells and CD38+/− subsets of haemopoietic progenitor cells in G‐CSF‐mobilized peripheral blood

“…In this study we have employed a recently developed FCM assay, which simultaneously measures the content of BrdUrd and DNA on leucocyte subsets defined by LDAs, to elucidate the progression of normal CD34 þ cells through cell cycle by calculating the LI, Ts and Tpot. The data presented here corroborate and extend recent studies on mobilized HSC which have shown that virtually no CD34 þ cells from healthy donors or patients are in S/G 2 M phase (Leitner et al, 1996;Roberts & Metcalf, 1995;Siegert & Serke, 1996;Uchida et al, 1997), and that HGF are required to induce cell cycling in these cells (Knaän-Shanzer et al, 1996;Leitner et al, 1996;Strauss et al, 1991;von Kalle et al, 1994). Moreover, mobilized HSC are thought to be in a prolonged G 1 phase rather than deeply quiescent (G 0 ) (Lemoli et al, 1997;Roberts & Metcalf, 1995).…”

Highly Efficient Lentiviral Gene Transfer in CD34 + and CD34 + /38 − /lin − Cells from Mobilized Peripheral Blood after Cytokine Prestimulation

“…The reason for these differences is not yet known, but several reports suggest that stem cells derived from mPB may be in a deeper state of dormancy than cells from CB or BM [21]. Thus, prolonged stimulation of these cells might be required to allow retroviral integration in mPB HSCs with, in counterpart, a detrimental effect on the maintenance of the long-term reconstituting ability of these cells [22,23]. Despite these limitations, and in the particular context of a selective advantage of the transduced cells, gene therapy with retroviral vectors was achieved recently in X-linked severe combined immunodeficiency (SCID) by γc gene transfer into CD34 + cells [24,25].…”

Cyclical mobilization and gated importation of thymocyte progenitors in the adult mouse: evidence for a thymus‐bone marrow feedback loop

“…2, right panel). Regarding the question of the cell-cycle kinetics of circulating prothymocytes, it would not be surprising to find that those that home directly to the thymus, like mobilized HSCs and myeloid and erythroid progenitor cells (129,130), are mostly nonproliferating. However, it is quite possible that the prothymocytes that return to the BM for further differentiation (13) may be cycling.…”