Reprogrammed Adult Human Endothelium into Hematopoietic Stem Cells Yields Functional T Cells In Vivo

• Published in: Blood 2018-11, Vol.132 (Supplement 1), p.169-169
• Main Authors: Barcia Durán, José Gabriel, Lu, Tyler M., Lis, Raphael, Rafii, Shahin
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2018-169

During development, the hematopoietic stem cells that go on to populate the bone marrow and give rise to all blood cell lineages emerge from a specialized endothelial subpopulation. We have previously harnessed this vestigial identity to achieve the direct conversion of adult endothelial cells (ECs) into long-term engraftable hematopoietic stem and progenitor cells (rEC-HSPCs); however, to date, we had only detected and characterized functional T cells that result from the transplantation, engraftment, and differentiation of mouse rEC-HSPCs (Lis, R. et al., Conversion of adult endothelium to immunocompetent haematopoietic stem cells. Nature, 545:439-445. 2017). We first reprogrammed adult human endothelium using lentiviral vectors encoding FOSB, GFI1, SPI1, and RUNX1 (FGRS) under constitutive promoters and an inductive vascular niche (Sandler V. et al., Reprogramming of human endothelium into hematopoietic cells requires vascular niche induction. Nature, 511:312-8. 2014). The resulting rEC-HSPCs maintained exogenous expression of the four transcription factors for over 20 weeks post-transplantation into immuno-compromised NSG mice. Constitutive expression of Spi1, however, has been shown to hinder lymphoid differentiation in vivo by blocking T lymphopoiesis (Anderson, M. et al. Constitutive expression of PU.1 in fetal hematopoietic progenitors blocks T cell development at the pro-T cell stage. Immunity 16:285-296. 2002). In addition, mice of the NSG strain cannot educate native B or T cells to maturity. Our system of constitutive exogenous FGRS expression was therefore unable to confer transplanted immuno-compromised mice the ability to generate an adaptive immune response. Here, we obtained human rEC-HSPCs making use of (i) doxycycline-inducible vectors to temporarily overexpress FGRS and (ii) transgenic substrains of NSG mice (one carrying human stem cell factor, granulocyte/macrophage colony-stimulating factor, and interleukin 3; the other, human major histocompatibility complex class I as well as beta-2 microglobulin) for transplantation assays. We show that human rEC-HSPCs engraft primary- and secondary-transplanted mice for over a year at levels of up to 20% in the spleen or bone marrow. Engrafted cells differentiate into all blood lineages including phenotypically and functionally mature T cells in the absence of exogenous FGRS expression in vivo. Notably, the resulting T cells undergo TCR rearrangement and are able to clear viral particles one week