PS1224 DOWNREGULATION OF ENDOTHELIAL GENE MYCT1 IN HUMAN HSCS DURING EX VIVO CULTURE COMPROMISES THEIR FUNCTION

Abstract

Background:Hematopoietic stem cells (HSCs) are rare undifferentiated cells that, due to their ability to engraft, self‐renew and generate all blood lineages, represent a life‐saving therapy for hematopoietic malignancies and hereditary blood disorders. However, expansion of HSCs in vitro has had limited success thus far, and represents a bottleneck for their wider clinical use. Our previous studies showed that OP9 MSC stromal cells can expand immunophenotypic human HSPCs that are multipotent; however, the culture‐expanded HSPCs were functionally compromised when transplanted in immunodeficient mice. It is therefore necessary to understand the molecular mechanisms that regulate human HSC properties, and how they can be preserved in culture. To this end, we identified MYCT1 (Myc target 1) as a gene that is dysregulated in cultured HSPC, and may be linked to their compromised function in culture.Aims:Our aim is to understand how downregulation of MYCT1 in cultured HSC impacts HSC function.Methods:MYCT1 gene expression was assessed in sorted human HSPC before and after culture on OP9 stroma (0, 12 h, 2 and 5 weeks by microarray, and at 0 and 4 weeks by RNA‐seq), and in distinct hematopoietic subsets in various tissues from early human development to cord blood and bone marrow. To assess the impact of MYCT1 levels on HSPC expansion, highly self‐renewing human fetal liver HSPCs were isolated and transduced with lentiviral vectors for MYCT1 overexpression (OE) or shRNA knockdown (KD). In vitro expansion of HSPCs was assessed by quantifying CD34+ CD38‐ CD90+ cells at 2, 4, 6, and 8 weeks (OE) or at 5, 10, 15 and 30 days (KD). ChIP‐seq experiments for H3K4me3, H3K9ac, H3K79me2, and H3K6me3 to assess epigenetic regulation of MYCT1 gene were performed on sorted HSPCs at day 0 and 4 weeks in culture. MYCT1 co‐regulated genes were identified from the CO‐Regulation Database (Fahrenbach et al., 2014).Results:Analysis of our RNAseq datasets from early human development to post‐natal hematopoietic tissues revealed that MYCT1 is expressed highly in hemogenic endothelium and subsequently maintained in HSCs in all developmental and post‐natal hematopoietic tissues, but drastically downregulated during differentiation. Strikingly, MYCT1 was one of the most downregulated genes in cultured human HSCs. Analysis of Chip‐seq data revealed a drastic loss of histone marks of active TSS (H3K4me3, H3K9ac) and active gene body (H3K79me2, H3K36me3) surrounding the MYCT1 promoter and gene body in cultured human HSPC. Restoring MYCT1 expression in human HSPCs promoted their expansion in culture, whereas MYCT1 knockdown caused premature exhaustion of the undifferentiated HSPCs. However, the specific mechanisms through which MYCT1 expression is maintained and how it regulates human HSC function are unknown. Analysis of publicly available databases of gene co‐regulation and KEGG pathway analysis revealed a significant association of MYCT1 with genes involved in cell‐cell and cell‐ECM adhesion and trans‐endothelial migration. About 70% of MYCT1 co‐regulated genes were also downregulated in human HSPC during culture. These data suggest that MYCT1 may be an important node in a network of genes responsible for HSC‐niche interactions.Summary/Conclusion:We hypothesize that the HSC‐enriched gene MYCT1 governs human HSC function by regulating their adhesion and migratory capacity, allowing proper HSC homing and interaction with the niche, and that restoring proper MYCT1 levels in cultured HSC will improve the ability to expand transplantable human HSCs in culture.