Ex Vivo expansion Of Umbilical Cord Blood CD34+ Cells Under Hypoxic Conditions Using Novel Compound#999 With Cytokines
Authors: Khan Saadiya , Stewart Alison L. , Mukherjee Siddhartha , Scheiber Stuart L. , Ebert Benjamin L. , Golub Todd R. , Scadden David T. , Moore Malcolm A.S. 
Published: October 2019

Introduction

Umbilical cord blood is an increasingly utilized source for hematopoietic stem transplantation. However the limitation is inadequate hematopoietic stem and progenitor cell (HSPC) dose leading to poor engraftment and prolonged neutropenia. Umbilical cord blood transplants (UCBT) were initially restricted to small sized children and adults. The advent of double umbilical cord blood transplants (DUCBT) led to both children and adults transplants with sufficient numbers of HSPCs. However there continue to be issues with insufficient engraftment, extended duration of cytopenia, risk of infections and prolonged duration of hospital stay.

There is ongoing research to investigate optimal ex vivo umbilical cord blood (UCB) HSPC expansion with the intention to ensure sustained engraftment, reduce the prolonged periods of neutropenia and curtail the high risk of infectious complications in the immediate post-transplant period. HSPC expansion with cytokines alone produces about 7-fold increase of HSPC over 12-14days. However most IRB approved protocols require that a significant percentage of these cord blood cells be transplanted without manipulation and then the expanded cells be transplanted later. To detect a significant advantage to this expanded fraction we have calculated that HSPCs need to be expanded 8-10 fold. To achieve this we have combined an optimal cytokine combination with hypoxia and the additive of Aryl hydrocarbon Receptor (AhR) antagonist Stem Reginin1 (SR1); previously reported to facilitate HSPC expansion (Boitano et al 2010 Science).

Objectives

Here we evaluated if there was any potential synergistic effect of combining AhR antagonist SR1 with hypoxia for ex vivo HSPC expansion. Additionally we looked at the effect of adding #999; a small molecule identified using high-throughput screening that selectively expands murine hematopoietic stem cells.

Methods

UCB derived phenotypic CD34+ cells were cultured in the presence of stem cell factor (SCF), Flt3 ligand (Flt3L) and thrombopoietin (TPO) on a feeder layer of OP9 cells transduced with lentiviral vector expressing red fluorescent protein in both normoxia and hypoxia (3% oxygen). Total cell numbers (TNC) were counted, CD34+ cells were measured through flowcytometry and the self-renewal and multi-lineage differentiation was measured through week-5 cobblestone area forming (CAFC) and colony forming (CFC) assays respectively.

Results

CD34+ cells cultured in the presence of SCF, Flt3L and TPO (50ng/ml each) resulted in a 100fold expansion of CD34+ cells compared to input cells at 2 weeks. SR1 when added to the above cytokine cocktail led to a 200-fold expansion while #999 used with cytokines resulted in 118-fold expansion at 2weeks. Using both small molecules together in the presence of cytokines did not show an additive effect (207fold increase). Repeating the above experiments in hypoxia (3% oxygen) showed 196-fold increase with cytokines alone, 289-fold increase with SR1, 211-fold increase with #999 and again no additive effect of SR1 and #999 together.

CD34+ cells cultured with SR1 or #999 with cytokines produced approximately 1.9 and 1.2 times more CFC than those with cytokines alone respectively. SR1 treated cells on week-5 CAFC showed 3-fold and #999 treated cells 1.3-fold more cobblestones compared to cytokines alone. In hypoxia CD34+ cells cultured with #999 gave rise to more colonies as compared to both SR1 (2-fold more) and cytokines (3-fold more). CAFC data for these are pending.

The degree of HSPC expansion with SR1 in addition to cytokines can be increased in hypoxic conditions. # 999 when used with cytokines in hypoxia can also lead to the same degree of HSPC expansion as SR1 in normoxia. The combination of SR1 and #999 showed no additive effect in either normoxia or hypoxia.

Conclusion

Compound 999 when used in hypoxia leads to a significant expansion of HSPCs compared to cytokines alone or SR1 plus cytokines in normoxia. In vivo xenograft murine studies are been conducted so as to compare and evaluate the engraftment potential of these ex vivo expanded CD34+ cells in irradiated NSG mice.

Disclosures:

Mukherjee: Onconova Therapeutics: Research Funding. Ebert:Genoptix: Consultancy; Celgene: Consultancy.