Human pluripotent stem cells (hPSCs) provide a powerful platform for generating functional hematopoietic cells for blood disease modeling and therapeutic testing. However, the quantity and quality of hPSC-derived blood cells remain to be improved. Here, by performing extensive single-cell transcriptomic analyses to map fate choices and gene expression programs during hematopoietic differentiation of hPSCs, we construct the first hematopoietic landscape of hPSCs at the single-cell level and identify strategies to promote hematopoietic progenitor (HP) generation from hPSCs with functional T cell potential.
By focusing specifically on cell populations and molecular events involved in endothelial-to-hematopoietic transition (EHT), we compared the difference of early hematopoiesis between hPSCs and human embryos (Yang Zeng et al. Cell Research. 2019) and found aerobic metabolism was dysregulated during in-vitro-directed differentiation. The decreased oxygen metabolism program was further deciphered as a key molecular event occurred during the EHT. Providing hypoxia at the stage of EHT enhanced hematopoietic differentiation of hPSCs via specifying arterial programs, including arterial hemogenic endothelium (AHE) and arterial endothelium cells (AE).
To further determine the effect of AE on hematopoietic development, we isolated AE, venous endothelium and mesenchymal cells identified in our single-cell transcriptomic analyses and cocultured them with AHE respectively for HP generation. AE were finally validated as a critical regulator of definitive HP specification with more T cell potential. T cells generated from AE-primed HPs (AE-T) were highly functional and exhibited polyfunctional production of interferon (IFN)-γ, tumor necrosis factor alpha (TNF-α), and IL-2 in response to phorbol 12-myristate 13-acetate (PMA) and ionomycin. To further evaluate the function of AE-T, we engineered T cells with CD19-CAR. The in vitro cytotoxicity of CAR-engineered AE-T was performed both in CD19+ cell lines (Nalm-6 and Raji) and human primary B-ALL samples. The efficacy of CAR-engineered AE-T in vivo was evaluated in a mouse xenograft model inoculated intravenously with luciferase-expressing Nalm-6 cells. Similar to CD19 CAR-transduced peripheral blood T cells, the AE-T potently inhibited tumor growth both in vitro and in vivo.
Collectively, our study provides benchmark datasets to understand the origins of human hematopoiesis and presents an advance for guiding the generation of functional T cells in vitro for clinical applications.
Disclosures
No relevant conflicts of interest to declare.
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