Single-cell transcriptome of early hematopoiesis guides arterial endothelial-enhanced functional T cell generation from human PSCs

Abstract

Hematopoietic differentiation of human pluripotent stem cells (hPSCs) requires orchestration of dynamic cell and gene regulatory networks but often generates blood cells that lack natural function. Here, we performed extensive single-cell transcriptomic analyses to map fate choices and gene expression patterns during hematopoietic differentiation of hPSCs and showed that oxidative metabolism was dysregulated during in vitro directed differentiation. Applying hypoxic conditions at the stage of endothelial-to-hematopoietic transition in vitro effectively promoted the development of arterial specification programs that governed the generation of hematopoietic progenitor cells (HPCs) with functional T cell potential. Following engineered expression of the anti-CD19 chimeric antigen receptor, the T cells generated from arterial endothelium-primed HPCs inhibited tumor growth both in vitro and in vivo. Collectively, our study provides benchmark datasets as a resource to further understand the origins of human hematopoiesis and represents an advance in guiding in vitro generation of functional T cells for clinical applications.

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

“…Previous studies have shown that the hypoxia could drive the hematopoietic differentiation [ 15 ]. The occurrence of the EHT in vitro closely mimics how hematopoietic cells develop in vivo, although this process is accompanied by gradual downregulation of aerobic metabolism; however, it still remains in highly active aerobic metabolism when compared to those in vivo [ 15 ].…”

“…Previous studies have shown that the hypoxia could drive the hematopoietic differentiation [ 15 ]. The occurrence of the EHT in vitro closely mimics how hematopoietic cells develop in vivo, although this process is accompanied by gradual downregulation of aerobic metabolism; however, it still remains in highly active aerobic metabolism when compared to those in vivo [ 15 ]. To experimentally demonstrate the acquisition of the hematopoiesis under the hypoxia conditions, hESCs were therefore induced to differentiate into hematopoietic lineage under the hypoxia (5%) or the normoxia (21%) conditions.…”

“…For T cell differentiation [ 15 ], 5 × 10 5 hESC-derived CD45 + cells were centrifuged with 1 × 10 6 OP9 cells (derived from neonatal OP/OP mouse cranium for supporting the production of T cell) to form small 3D aggregates, which were then plated onto a 0.4-mm Millicell transwell insert (EMD Millipore) placed in a six-well plate containing 1 mL of T cell differentiation medium consisting of RPMI 1640 (Gibco), 4% B27 supplement (Thermo Fisher Scientific), 30 mM l-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma-Aldrich), 1% penicillin/streptomycin (Thermo Fisher Scientific), 20 ng/mL stem cell factor (SCF), 5 ng/mL FLT3L, 5 ng/mL IL-7 and 10 ng/mL DLL4 (all from PeproTech). Differentiation medium was changed every 2 days and cultured for 8 weeks.…”

“…Based on this, we aimed to optimize the in vitro differentiation system by regulating the role of exogenous cytokines to improve the yield of hematopoietic cells derived from hPSCs. Previous study has also provided compelling evidence that despite the gradual downregulation of oxygen metabolism, the endothelial-to-hematopoietic transition (EHT) in vitro is still in an active state of aerobic metabolism compared with the in vivo data of the fetal liver and bone marrow, which identified as an important molecular feature during the EHT stage [ 15 ].…”

“…Even brief exposure could have a significant negative effect on HSCs, resulting in a decrease in the number and function of HSCs and ultimately a decrease in the efficacy of transplantation [ 19 , 20 ]. HIF-1α, a key transcription factor of cells in the response to hypoxia [ 21 , 22 ], not only is an important molecular feature during the EHT stage [ 15 ], but also interferes with mitochondrial aerobic metabolism and decreases ROS production [ 23 ], thereby repressing HSC apoptosis [ 15 , 19 ]. Studies of HSCs with multi-lineages observed in mouse embryos have shown that HIF-1α effectively binds to a common sequence at promoters or enhancers of a series of downstream target genes whose function is usually associated with HSC maintenance in bone marrow niches, such as EPO, VEGFA, and SDF-1/CXCL12 [ 24 ].…”