Differential Maintenance of Cell Cycle Status of Human Bone Marrow- and Umbilical Cord Blood-Derived CD34+ Cells after Homing to the Bone Marrow Microenvironment.
Authors: Poteat Bradley A. , Chitteti Brahmananda Reddy , Pollok Karen E. , Suvannasankha Attaya , Srour Edward F. 
Published: April 2020

Abstract
Transplanted human bone marrow (BM) CD34+ cells remain mitotically quiescent up to 72h following their homing to the BM of irradiated NOD/SCID mice (Blood2002;99(5):1585). To investigate whether umbilical cord blood (UCB)-derived CD34+ cells behave similarly and to assess the impact of the BM microenvironment on this observed temporary cell cycle arrest, we examined the cell cycle status of BM- and UCB-derived CD34+ cells recovered from the BM of irradiated (325 cGy) and non-irradiated NOD/SCID IL2Rγnull (NS2) mice 20h after transplantation (AT). To understand the molecular control of this sustained, but short-lived induced quiescence, expression of cell cycle-related proteins among BM-homed cells was examined by real-time quantitative PCR. Freshly isolated CD34+ cells were stained with CFSE-1 and transplanted into normal or conditioned NS2 mice or cultured in vitro with and without SCF, Flt3, GM-CSF, and IL-3. BM was harvested from NS2 recipients 20h AT and CFSE+ cells were recovered by cell sorting. Cell cycle status was assessed by PI staining in a fraction of recovered CFSE+ and cells cultured in vitro and mRNA was isolated from the remaining cells in both groups. The proportion of cells in G0/G1 phase of cell cycle among BM CD34+ cells was maintained in recipient BM 20h AT regardless of whether the microenvironment was irradiated or not (fresh: 83.7% ± 4.4%, n = 14; irradiated: 82.7% ± 5.3%, n = 6; non-irradiated: 84.4% ± 4.1%, n = 10), suggesting that exit of BM-homed cells from G0/G1 is either a strictly cell intrinsic property or is a phenomenon mediated by microenvironmental modulators present in both intact and injured BM. However, cultured BM CD34+ cells cycled efficiently such that after 20h, only 75.5% ± 5.7%, n = 12 remained in G0/G1. Surprisingly, BM-homed UCB CD34+ cells did not remain quiescent and at 20h AT, only 84.85% ± 13.1% (n = 5) were in G0/G1 compared to 97.0% ± 1.8% (n = 14) for freshly isolated cells, suggesting that different cell cycle regulatory mechanisms control BM versus UCB CD34+ cells. UCB CD34+ cells recovered from the BM of irradiated recipient mice 20h AT had increased levels of Bcl-2 and CDKN1B (p27) while those of CDKN1C (p57) and p53 were decreased relative to those isolated from non-irradiated recipients. These findings suggest that at least in the case of UCB CD34+ cells, different cell cycle regulatory networks may impact cell cycle progression of these cells in an irradiated versus intact BM microenvironment. Compared to freshly isolated BM CD34+ cells, those recovered from the marrow of non-irradiated recipients 20h AT had significantly elevated levels of mRNA for CDKN1a (p21), CDKN1B (p27), p53, and N-cadherin, while the mRNA levels for these molecules in comparable cells cultured in vitro for 20h was relatively unchanged. Taken together, these data illustrate that human CD34+ cells from different tissues behave differently during the first few hours following their homing to the BM and that this behavior may be partially regulated by the status of the microenvironment. Furthermore, data from BM-derived CD34+ cells suggest that following homing, active repression of cell cycle progression may be mediated by induced upregulation of p21 and p27. Mechanisms leading to the upregulation of these cell cycle regulatory molecules remain to be investigated.