Transplanted CD34+ Cells Stimulated with Flt3 Ligand and Granulocyte-Colony Stimulating Factor Display An Impaired Early Reconstitution Capacity
Authors: Kaya Fati ma SF Aerts , Hartong Simone CC , Visser Trudi P , Verstegen Monique MA , Wognum Albertus W. , Wagemaker Gerard 
Published: April 2020

Abstract
Flt3/flk2 belongs to the class III hematopoietic growth factor receptors with intrinsic kinase activity, ligand-activated tyrosine phosphorylation and a central role in early stages of hematopoiesis. Flt3-ligand (FL) has been shown to enhance the effects of several growth factors, including Granulocyte-colony stimulating factor (G-CSF). In this study the reconstitution ability of FL+G-CSF primed CD34+ cells was evaluated in a nonhuman primate model of radiation-induced myeloablative treatment followed by transplantation of stimulated autologous bone marrow (BM) and/or peripheral blood (PB) cells. Rhesus monkeys (n=9) were subjected to subcutaneous injections with FL (100 μg/kg/day) and G-CSF (100 μg/kg/day) for 7 to 14 days and subsequently irradiated with a lethal dose of total body irradiation (TBI) followed by transplantation with primed stem cells or with a midlethal dose of TBI (n=2) without transplantation. White blood cell counts (WBC) in PB showed a steep increase and reached a mean level of 98.9×109/L (71.5–131.7×109/L) after 7 days (P&lt;0.01). BM cellularity increased from 35.2×109/L before treatment to 193.8×109/L after priming (95.0–348.2×109/L) (P&lt;0.01). CD34+ cells displayed a 1-log increase (P&lt;0.01) in BM and a 2-log increase in PB (P&lt;0.01). Both total nucleated and CD34+ cell numbers reached after 7 days a steady state level, which was maintained until cessation of growth factor treatment. Treatment with FL+G-CSF resulted in a moderate increase (2-fold) of CFU-GM in BM after 7 days (P&lt;0.05) but did not affect BFU-E, consistent with the predilection of FL for myeloid and lymphoid cells, in contrast to SCF, which predominantly influences the erythroid and thrombopoietic lineage. The effect of treatment with FL+G-CSF on WBC was significantly greater than treatment with either G-CSF at day 7 (P&lt;0.05) or FL at day 7 and 14 (P&lt;0.01). Lethally irradiated monkeys, transplanted with FL+G-CSF stimulated cells displayed a much slower regeneration of WBC, platelets and reticulocytes relative to the cell number transplanted and were consequently in need of prolonged antimicrobial prophylaxis and transfusions. Furthermore, these monkeys displayed regeneration patterns consistent with a 1-log depletion of repopulating cells relative to non-stimulated BM cells. Mid-lethal irradiated FL+G-CSF treated monkeys were not transplanted to study the intrinsic regeneration capacity of primed BM. These monkeys displayed normal regeneration of reticulocytes, platelets and WBC. Thus, stimulation with FL+G-CSF leads to a steep and synergistic increase of nucleated and CD34+ cells in both BM and PB. Priming with FL+G-CSF does not affect the intrinsic regeneration capacity of BM after radiation; but when transplanted, primed CD34+ cells do not per se contribute to accelerated short-term engraftment after transplantation, rather the reconstitution patterns were consistent with on average one order of magnitude stem cell depletion relative to the numbers of CD34+ cells transplanted.