Hypoxia drives hematopoiesis with the enhancement of T lineage through eliciting arterial specification of hematopoietic endothelial progenitors from hESC

Abstract

Background: Hematopoietic stem cells are able to self-renew and differentiate into all blood cell lineages. Hematopoietic stem cell transplantation is a mainstay of life-saving therapy for hematopoietic malignancies and hypoproliferative disorders. In vitro hematopoietic differentiation of human pluripotent stem cells (hPSCs) is a promising approach for modeling hematopoietic development and cell replacement therapies. Although using hPSCs to derive hematopoietic progenitor cells has achieved some successes in the past, differentiation from hPSCs to produce all hematopoietic cells which can provide robust long-term multilineage engraftment is still very difficult. Here, we reported a novel culture system for hematopoietic differentiation from human embryonic stem cells (hESCs) with optimal cytokines combinations under hypoxia condition.

Methods: In vitro production of T lineage hematopoietic stem/progenitor cells from hESCs by using hypoxia differentiation system, the effects and the potential mechanism of hypoxia promoting T lineage hematopoiesis were investigated by RT-qPCR validation, cell cycle assay and flow cytometry analysis.

Results: Using our differentiation system, almost 80% CD45⁺ cells generated from hESCs were hematopoietic cells and particularly could be further induced into CD3⁺TCRαβ⁺ T cells in vitro. We detected more CD34⁺CD144⁺ hematopoietic endothelial progenitors (HEPs) induced from hESCs than those in normoxia conditions, and the early HEPs-related gene DLL4 was upregulated by enhancing the hypoxia signaling via potential HIF-1α/NOTCH1/DLL4 axis to enhance arterial feature, thus drove T lineage during the hematopoiesis. Strikingly, hematopoietic cells generated in our system exhibited the potential for all multilineage reconstruction including lymphoid, myeloid and erythroid lineages in vivo by transplantation assay.

Conclusion: Our results demonstrated that hypoxia plays an important role in T lineage hematopoiesis by promoting the expression of arterial endothelial gene DLL4 and upregulation of NOTCH1 through the activation of the HIF-1α signaling pathway. These results provide a significant approach for in vitro and in vivo production of fully functional hematopoietic stem/progenitor cells from hESCs.

Keywords: Endothelial-to-hematopoietic transition; Hematopoietic; Hemogenic endothelium; Human embryonic stem cells; Hypoxia.