Vascular niche promotes hematopoietic multipotent progenitor formation from pluripotent stem cells
Jennifer L. Gori, … , Shahin Rafii, Hans-Peter Kiem
Jennifer L. Gori, … , Shahin Rafii, Hans-Peter Kiem
Published February 9, 2015
Citation Information: J Clin Invest. 2015;125(3):1243-1254. https://doi.org/10.1172/JCI79328.
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Technical Advance Stem cells

Vascular niche promotes hematopoietic multipotent progenitor formation from pluripotent stem cells

Abstract

Pluripotent stem cells (PSCs) represent an alternative hematopoietic stem cell (HSC) source for treating hematopoietic disease. The limited engraftment of human PSC–derived (hPSC-derived) multipotent progenitor cells (MPP) has hampered the clinical application of these cells and suggests that MPP require additional cues for definitive hematopoiesis. We hypothesized that the presence of a vascular niche that produces Notch ligands jagged-1 (JAG1) and delta-like ligand-4 (DLL4) drives definitive hematopoiesis. We differentiated hes2 human embryonic stem cells (hESC) and Macaca nemestrina–induced PSC (iPSC) line-7 with cytokines in the presence or absence of endothelial cells (ECs) that express JAG1 and DLL4. Cells cocultured with ECs generated substantially more CD34+CD45+ hematopoietic progenitors compared with cells cocultured without ECs or with ECs lacking JAG1 or DLL4. EC-induced cells exhibited Notch activation and expressed HSC-specific Notch targets RUNX1 and GATA2. EC-induced PSC-MPP engrafted at a markedly higher level in NOD/SCID/IL-2 receptor γ chain–null (NSG) mice compared with cytokine-induced cells, and low-dose chemotherapy-based selection further increased engraftment. Long-term engraftment and the myeloid-to-lymphoid ratio achieved with vascular niche induction were similar to levels achieved for cord blood–derived MPP and up to 20-fold higher than those achieved with hPSC-derived MPP engraftment. Our findings indicate that endothelial Notch ligands promote PSC-definitive hematopoiesis and production of long-term engrafting CD34+ cells, suggesting these ligands are critical for HSC emergence.

Authors

Jennifer L. Gori, Jason M. Butler, Yan-Yi Chan, Devikha Chandrasekaran, Michael G. Poulos, Michael Ginsberg, Daniel J. Nolan, Olivier Elemento, Brent L. Wood, Jennifer E. Adair, Shahin Rafii, Hans-Peter Kiem

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Figure 1

Endothelial Notch ligands regulate emergence and expansion of hematopoietic progenitor cells from PSCs.

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Endothelial Notch ligands regulate emergence and expansion of hematopoie...
(A) Left: CD34+CD45+ cell yield per million input of cells ± ECs and Notch ligand– replete and –depleted conditions. Right: fold change in CD34+CD45+ cells relative to MPP generated in cytokines. JAG1-KD, ECs transduced with shRNA to JAG1; DLL4-KD, ECs transduced with shRNA to DLL4, –, cytokines alone. (B) Notch signaling target expression on day 15. Left: MniPSC-7–MPP; right: day hes2-MPP after coculture in indicated conditions. Levels calibrated to expression in PSC-MPP cocultured with WT ECs and normalized to β-actin. (C) Number of hematopoietic CFUs from day-15 MniPSC-7–MPP after expansion ± WT ECs. Right: Mn BM CD34+ activity (control). CFUs per 100,000 cells plated. Plating efficiency noted in parentheses. Far right: CFUs from day 15 MniPSC-7–MPP and BM CD34+ cells. Original magnification, ×20. (D) Primate CD45+ cells in blood 12 weeks after transplantation (1 transplant experiment, n = 3 mice/group, bars represent mean/group). **P < 0.005; ***P < 0.0005, Student’s t test. Differentiation studies ± Notch ligand–depleted ECs were conducted in 2 MniPSC lines and 1 hESC line (hes2) in 3 independent experiments per cell line. Differentiation studies comparing induction with cytokines alone and WT ECs were conducted in 2 MniPSC lines and 1 hESC line in 6 independent experiments per cell line.