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.

Figure 6. Hypothetical model for vascular niche JAG1/DLL4-mediated induction of Notch activation of hematopoietic specification.

Hemogenic cells (CD34⁺PECAM1⁺VEC⁺VEGFR⁺ CD45^lo/–) placed in a vascular niche that produces JAG1/DLL4 lead to Notch activation and upregulation of RUNX1 and GATA2, which, in combination with additional unknown factors (indicated by ??), support the endothelial-to-hematopoietic transition. Blockade of either JAG1 or DLL4 expression by vascular cells (shRNA to the membrane-bound Notch ligands) altered competing Notch-1, -2 ligand stoichiometry, reduced Notch activation in hemogenic precursors, lowered RUNX1 and GATA2 expression, and resulted in fewer CD34⁺CD45⁺ cells.

Figure 5. Gene-modified MniPSC-MPP from primary recipient mouse BM reconstitute secondary recipient mice.

(A) Representative PCR analysis of mouse BM from EC-MniPSC-MPP mouse for data shown in Figure 4D showing detection of primate β-actin and lentivirus LTR. (B) PCR of GFP transgene (top gel) and macaque (Mn) BSG (lower gel) in gDNA from GFP⁺ sorted monkey PBMCs, unmodified, negative control human and mouse hematopoietic cells, and splenic gDNA from representative engrafted mice. (C) Subsets in second-degree recipients. (D) CFUs from BM of second-degree recipient mice. Secondary transplantation studies were conducted in 3 mice/group over 2 independent experiments.

Figure 4. In vivo selection increases long-term engraftment of EC-induced MniPSC-MPP.

(A) Experimental schematic. (B) In vivo selection of EC-expanded MniPSC-MPP (n = 10) versus cytokine-expanded MniPSC-MPP (n = 7). Arrows indicate O⁶BG/BCNU. Primate CD45⁺ cells in EC-induced MniPSC-MPP–transplanted mouse before and after in vivo selection (4 and 16 weeks, respectively). (C) CFU-GEMM from BM of EC-induced MniPSC-MPP–transplanted mice. Original magnification, ×20. Scale bars: 100 μm. Bottom panels: CFU frequencies in primary recipient BM. (D) Summary of gene marking by PCR in organs. Values are mean of biological replicates (mean ± SD for all mice/group). Transplantation studies were conducted in 8 to 12 mice/group over 2 independent experiments. **P < 0.005, Student’s t test.

Figure 3. hESCs differentiated in a vascular niche give rise to engrafting MPP with in vivo myeloid and lymphoid potential.

(A) Kinetics of EC-induced hes2-MPP engraftment compared with mice transplanted with ECs alone. (B) Distinction between the human and mouse CD45. (C) Th1 cytokine production by human CD3⁺ cells and CD14⁺ cells. For cytokine assays, cells were isolated from organs of 3 mice/group (3 biological replicates) and 3 technical replicates were run for each test.

Figure 2. Long-term multilineage engraftment of vascular niche–induced MniPSC-MPP.

Detection of primate CD45⁺ cells in blood of mice transplanted with EC-induced MniPSC-MPP, cytokine-induced MniPSC-MPP, or Mn BM CD34⁺ cells. (A) Kinetics of primate CD45⁺ cells in blood. *P < 0.05; **P < 0.005, Student’s t test. (B) Distinction between primate and mouse CD45⁺ cells by flow cytometry analysis. Middle panels: lymphoid and myeloid subset analysis. Right panels: flow cytometry plots showing CD3, CD20 single-positive and CD13, CD14 double-positive cells. (C) Primate CD45⁺ cells in BM. Dots indicate individual mice. Lines show mean/group. (D) Top panels: frequency of BM CFUs. M, macrophage; GM, granulocyte-macrophage; E, erythroid; GEMM, mixed. Bottom panels: colonies from BM of EC MniPSC-MPP mouse. Original magnification, ×4. Middle panels: Wright-stained macrophage and erythroid cells. Original magnification, ×20. Right panels: qRT-PCR for primate γ- and β-hemoglobin from erythroid (BFU-E) cells. Transcripts normalized to β-actin and calibrated to macaque blood (line indicates calibrator level). Transplantation studies were conducted in 8 to 12 mice/group over 3 independent experiments.

Figure 1. Endothelial Notch ligands regulate emergence and expansion of hematopoietic progenitor cells from PSCs.

(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.