Clinical Grade Ex-Vivo Expansion of Granulomonocytic Cells and Progenitors Using High-Dose SCF and G-CSF without Exhaustion of Stem Cell Activity.
Authors: Bonnet Marie-Laure , Guillier Martine , Greissinger Natacha , Marracho Marie-Catherine , Bedouel Muriel , Cailliot Christian , Vainchenker William , Boccaccio Catherine , Turhan Ali G. 
Published: April 2020

Abstract
Ex-vivo expansion of mature hematopoietic cells and progenitors is a major cell therapy technology aiming to shorten chemotherapy-induced cytopenias. The goal of amplifying the most primitive hematopoietic stem cell populations in clinically acceptable conditions is currently difficult, due to the absence of clinical grade early acting-cytokines and lack of adequate stem cell assays. Stem cell factor (SCF) and granulocyte colony stimulating factor (G-CSF) are both clinically applicable and they have been shown to induce a dose-response effect with regard to the expansion of mature cell populations when used in combination with early- acting cytokines. The combined effects of high concentrations of G-CSF and SCF on hematopoietic cells and progenitors and the effects of this procedure on the more primitive stem cell subsets have not been studied. We have developed a preclinical protocol to evaluate the feasibility of expansion of granulomonocytic cells using SCF and G-CSF with the goal of translating this protocol to the clinical application. In preliminary assays, the effects of low and high concentrations of both cytokines on total nucleated cell numbers and progenitors from purifed CD34+ cells (50 104cells/ml) have been tested. As compared to low concentrations (G-CSF 10 ng/ml and SCF 50 ng/ml) high concentrations of G-CSF and SCF (100 ng/ml and 300 ng/ml respectively) induced a two-fold increase of the nucleated cells and progenitors after 9 days of culture (n=7 experiments). High concentrations were therefore chosen for further experiments. CD34+ cells purified from mobilized peripheral blood grafts (purity 99%) have been cultured in a clinically acceptable medium containing clinical grade fetal calf serum ( 10 %), G-CSF (100 ng/ml) and SCF (300 ng/ml) for 9 days. Cells were analyzed using apoptosis tests, immunophenotyping and clonogenic assays at day 0 and day+9. In several experiments ( n= 9) a mean 21-fold expansion of total viable cell numbers was obtained, with a mean 4-fold expansion of clonogenic progenitors. Expanded cells had consistently CD11b+, CD13+, CD15+ phenotype and 2–4% of them remained CD34+. In a scale-up experiment started with 23.106 CD34+ cells, the total cell numbers expanded 16-fold at day+9, with generation of high numbers of CD11b + (18%), CD13+(98%) and CD15+ cells (91%) demonstrating the feasibility of the protocol in clinical scale. To determine if ex-vivo expansion would lead to a significant exhaustion of more primitive stem cells, we have evaluated the NOD/SCID-repopulating cell (RC) contents of the cultures before (day 0) and after ex-vivo expansion (day 9), in mice transplanted with day 0 and day+9 cells. In two experiments, the numbers of NOD/SCID mice engrafted with CD45+ human cells have been found increase from 30% ( 3/10) at day 0 to 100 % ( 10/10 ) at day 9 (Exp1 ) and from 50 % (3/6) at day 0 to 90 % ( 7/8 ) at day9 (Exp 2), demonstrating the persistence of NOD/SCID-RC potential after ex-vivo expansion. Overall the large-scale granulomonocytic cell production protocol that we developed could be of major interest in order to maintain the dose-intensity in high dose chemotherapy regimens and to shorten neutropenia after autologous PBSC transplantation while maintaing the stem cell potential of the graft.