Session: 501. Hematopoietic Stem and Progenitor Cells and Hematopoiesis: Basic and Translational: Basic and Developmental
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Translational Research, genomics, hematopoiesis, cell expansion, Therapies, Biological Processes, molecular biology, Technology and Procedures, omics technologies
The objective of the present study was to characterize the molecular programs supporting the strong expansion of HSCs in X2A cultures. RNA-Seq data from SCACs expanded CD34+CD45RA- cells revealed the upregulation of several receptor tyrosine kinases (RTKs) including AXL, but also its ligand GAS6 preferentially with X2A. AXL was recently shown to be essential for the self-renewal of leukemia initiating cells. Single cell RNA-seq analyses of CD34+ cells was done to characterize the graft composition of SCACs expanded HSPCs. These analyses revealed that AXL expression is enriched within CD34+CD45RA-CD90+CD49f+ HSC-enriched clusters. Image-flowcytometry analyses confirmed that HSC-enriched cell populations respond to GAS6-activation, resulting in the activation of ERK1/2 downstream of AXL activation. Epigenetic analysis revealed that GAS6/AXL upregulation is regulated by AA2P through DNA-demethylation in a TET dependent manner. Using genetic knockdown, chemical inhibition and GAS6 neutralizing antibody approaches we confirmed that GAS6 activation of AXL drives human HSPC expansion within X2A. In addition, supplementation of SCAC or single SCA-based cultures with GAS6 significantly enhanced the expansion of HSPC subsets with little induction of differentiation (e.g., 640- Vs. 1975-fold for CD34+CD45RA- HSPC in X2A and X2A+GAS6 cultures, respectively (n=3, p<0.05)). Transplant assays with day-14 expanded HSPC (progeny of 850 CD34+ starting cells/mouse) revealed that GAS6 supplementation in X2A cultures increased ensuing engraftment by 10-fold in both primary and secondary NSG mice (n=2, p<0.0001). For instance, the % of human CD45+ BM cells in secondary mice were of 2.3±0.1% and 25.7±1.3% in X2A and X2A+GAS6 groups. Remarkably, the net numbers of human colony-forming unit and CD34+ BM cells were increased by 7- and 15-fold (p<0.0001) in primary, and by 20- and 26-fold (p<0.0001) in secondary transplants by GAS6, respectively. In contrast, GAS6 neutralization in cultures impaired ensuing serial engraftment by 8- and 30-fold respectively Vs. X2A control (p<0.01). Lastly, SCAC and GAS6 were also shown to improve the expansion of adult G-CSF mobilized peripheral blood stem cells.
In conclusion, we demonstrate that AA2P-mediated HSPC expansion is driven through DNA demethylation leading to enhanced expression of AXL and its ligand GAS6. We also show for the first time that GAS6 is a potent growth factor that promote robust expansion of human HSPCs including serial engrafting HSCs resulting in unexpectedly high engraftment.
Disclosures: No relevant conflicts of interest to declare.
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