In Vivo Selection and Long-Term Engraftment Of Hematopoietic Stem Cells Generated Via Vascular Niche Induction Of Nonhuman Primate Induced Pluripotent Stem Cells
Authors: Gori Jennifer L , Butler Jason M , Chandrasekaran Devikha , Beard Brian C , Nolan Daniel J , Ginsberg Michael , Adair Jennifer E , Rafii Shahin , Kiem Hans-Peter 
Published: October 2019

Clinical use of human pluripotent stem cell (PSC)-hematopoietic stem cells (HSCs) is impeded by low engraftment potential. This block suggests that additional vascular derived angiocrine signals and hematopoietic cues must be provided to produce authentic HSCs. In addition, gene modification of induced (i)PSCs with a chemotherapy resistance transgene would provide a selective mechanism to stabilize or increase engraftment of HSCs. We therefore hypothesized that modifying iPSCs to express the O6-benzylguanine (O6BG)-resistant P140K variant of methylguanine methyltransferase (MGMT), would support in vivo selection of early-engrafted iPSC-HSCs. We further postulated that Akt-activated human endothelial cells afforded by transduction of the E4ORF1 gene (E4ORF1+ECs) through angiocrine upregulation of Notch and IGF ligands would provide the necessary signals under xenobiotic-free conditions to promote definitive hematopoiesis. This vascular induction platform could drive the emergence of true HSCs. We focused on pigtail macaque (Mn)iPSCs, as a scalable, clinically relevant nonhuman primate model. MniPSCs modified to express P140K had 15-fold higher MGMT levels compared to levels in human peripheral blood mononuclear cells. P140K-MniPSCs differentiated into chemoresistant CD34+ hematopoietic progenitors (50% CD34+) with a predominant long-term (LT)-HSC-like phenotype (CD34+CD38-Thy1+CD45RA-CD49f+). Hematopoietic progenitors maintained colony forming potential after O6BG and bis-chloroethylnitrosourea (BCNU) treatment. HSCs expanded on E4ORF1+ECs maintained colony forming potential, in contrast to cells cultured with cytokines alone, with a 22-fold increase in CD34+ cell content and 10-fold increase in LT-HSC-like cells. Importantly, MniPSC-HSCs expanded with the E4ORF1+ECs had long-term engraftment in NSG mice at levels comparable to Mn bone marrow HSC engrafted mice. O6BG/BCNU treatment increased engraftment to 35% CD45+ cells the blood of mice transplanted with E4ORF1+EC expanded P140K-MniPSC-HSCs, which was maintained 16 weeks post transplantation. Primate CD45+ cell levels in the blood after selection were significantly higher for this cohort compared to mice transplanted with P140K-MniPSC-HSCs expanded in the “cytokines alone” condition (18% vs. 3% CD45+, P<0.05). On average, 15% CD34+ and 37% CD45+ cells were detected in the bone marrow of mice transplanted with E4ORF1+EC-expanded P140K-MniPSC HSCs, which is significantly higher than levels detected in the other cohorts (Table 1). CD45+ cells in the marrow were predominantly myeloid but lymphoid subsets were also present (10-25% CD3+ cells). Remarkably, the level of gene marking in CFCs and number of gene marked CFCs from mouse bone marrow was substantially higher for mice transplanted with E4ORF1+EC expanded compared to cytokine expanded P140K-MniPSC-HSCs (Table 1). Finally, to confirm engraftment of authentic HSCs, secondary transplants were established. Although engraftment was achieved in all secondary transplanted cohorts, the level of nonhuman primate cells detected was significantly higher in animals transplanted with E4ORF1+EC expanded P140K-MniPSC-HSCs. Significantly more lymphocytes (CD45+CD3+ and CD45+CD56+) and monocytes (CD45+CD14+) were detected in the blood of these secondary transplant recipients. These findings confirm generation of bona fide HSCs derived from nonhuman primate iPSCs and demonstrate that O6BG/BCNU chemotherapy supports in vivo selection of P140K-MniPSC-HSCs generated by co-culture with the E4ORF1+EC vascular platform. Our studies mark a significant advance toward clinical translation of PSC-based blood therapeutics and the development of a nonhuman primate preclinical model.
Table 1 CD34+ and CD45+ engraftment and gene marking in the bone marrow of mice transplanted with nonhuman primate HPSCs from MniPSCs and bone marrow. HSCs E4ORF1+ECs O6BG/BCNU Mean %CD34+ Mean %CD45+ % gene marking in CFCs (lentivirus+) total lentivirus+ CFCs per 105 cells GFP-MniPSC + - 3 16 9 ± 2 13 ± 2 P140K-MniPSC + - 4 19 12 ± 5 17 ± 7 P140K-MniPSC - + 0.4 24 3 ± 2 2 ± 1 P140K-MniPSC + + 15 37 27 ± 24 111 ± 96 Mn BM CD34+ - - 2 21 0 0

Disclosures:
Nolan: Angiocrine Bioscience: Employment. Ginsberg:Angiocrine Bioscience: Employment. Rafii:Angiocrine Bioscience: Founder Other.