Analysis of Highly Self-Renewing GPI-80+ Human Fetal Hematopoietic Stem Cells Identifies Novel Regulators of Stemness

Achievements in pluripotent stem cell and reprogramming strategies provide hope for generating hematopoietic stem cells (HSC) in culture and for obtaining unlimited sources of transplantable cells. To reach this goal, deeper understanding of the regulatory mechanisms that distinguish the self-renewing HSC from non-self-renewing progenitors during human development is required. We analyzed the molecular signature of GPI-80 (VNN-2) expressing human second trimester fetal liver HSPC, the only population that harbors the truly self-renewing fetal HSC. Microarray and RNAseq analysis comparing CD34⁺CD38^-CD90⁺GPI-80⁺ HSC to CD34⁺CD38^-CD90⁺GPI80^- HPC demonstrated remarkable molecular similarity of these two functionally distinct populations, including comparable expression of many key transcription factors involved in HSC development and maintenance (e.g. SCL, RUNX1, MLL1, HOXA9, BMI1, GFI1, ETV6 etc.). Nevertheless, this analysis identified a subset of transcriptional regulators uniquely up-regulated in GPI80⁺ HSC, such as MYCT1, HLF, MLLT3 and HIF3A. These factors were down-regulated in human fetal liver HSPC upon expansion on MSC stroma culture, during which they become compromised in in vivo engraftment ability despite maintaining HSC surface immunophenotype. multipotency and expression of most known HSC regulators. Moreover, these factors were absent or expressed at low levels in human ES cell derived HPC, which can acquire HSC surface phenotype but are unable to self-renew. The expression of MYCT1, MLLT3 and HLF was also enriched in undifferentiated HSPC subset as compared to progenitors in the adult bone marrow, while HIF3a expression was low post-natally. Altogether, these analyses implied strong correlation of the expression of these factors with HSC self-renewal properties. Strikingly, knockdown of MYCT1, HLF, MLLT3 and HIF3A in human fetal liver HSPC using lentiviral shRNA impaired maintenance of undifferentiated HSPC in MSC stroma co-culture, while their inducible overexpression augmented HSPC expansion and prevented their premature exhaustion. Uncovering how these novel HSC regulators protect the highly self-renewing fetal HSC will help define the pre-requisites for establishing and maintaining stemness in developing human HSC, and for generating HSC for therapeutic use.