Prevention of Tumor Formation after Allogeneic In Utero Transplantation of Cynomolgus Monkey Embryonic Stem Cell-Derived Hematopoietic Precursors.
Authors: Hanazono Yutaka , Shibata Hiroaki , Ageyama Naohide , Tanaka Yujiro , Kishi Yukiko , Nakamura Shinichiro , Sasaki Kyoko , Hayashi Satoshi , Kitano Yoshihiro , Terao Keiji 
Published: October 2019

Although human ES cell-based transplantation therapies would hold a great potential in the treatment of a variety of diseases and injuries, there is a concern for teratoma formation after transplantation. In this study, we assessed the risk of tumor formation during the hematopoietic engraftment derived from cynomolgus ES cells (cyESCs) in an allogeneic transplantation model. cyESCs expressing GFP were cultured on OP9 stromal cells and induced to differentiate into the putative hematopoietic precursors. According to the flow cytometric analysis, CD34 was up-regulated on day 6 but decreased thereafter. Notably, CD31, CD144 (VE-cadherin) and VEGFR-2 (Flk-1) were all up-regulated on day 6. These are key markers of hemangioblasts (which generate endothelial and hematopoietic lineages). Despite the hemangioblast marker expression on day 6, the SCL gene was up-regulated at this time point as assessed by RNA-PCR, implying that the hematopoietic commitment might have already occurred on day 6. CD45, however, was not detected until day 12. The day-6 cells were transplanted in utero into allogeneic (cynomolgus) preimmune fetuses (n = 3) in the liver under ultrasound guidance around the end of first trimester (49–66 days/full term 165 days). We transplanted day-6 cells because the CD34 expression was the highest at this time point. We transplanted the cells into the liver because the liver is the major hematopoietic organ at these gestation days.

Fetuses were delivered at 3 months after transplantation (almost at full term). The transplanted cell-derived, GFP-positive hematopoietic colony-forming cells were successfully detected in the newborns (4–5%). Hematopoietic engraftment from cyESCs was thus achieved albeit at low levels. However, teratomas formed in all the three newborns. They were derived from transplanted cells, because they expressed GFP. The risk of tumor formation was unexpectedly high, given that we had seldom observed tumor formation in immunodeficient mice or fetal sheep that had been transplanted with the same day-6 cyESC-derivatives. Innate immune responses against cynomolgus-derived tumors might be more rigorous in xeno-transplanted mice and sheep than in allo-transplanted monkeys, resulting in failure to detect tumorigenesis in the xeno-transplantation models. Our monkey allogeneic transplantation setting would therefore allow the strict evaluation of in vivo safety of transplantation therapies using ES cells.

It turned out that day-6 cyESC-derivatives included residual SSEA-4-positive pluripotent cells (38.2 ± 10.3%) despite the rigorous differentiation culture. Presumably those cells were responsible for the teratoma formation. We purged an SSEA-4-positive fraction of day-6 cyESC-derivatives using a cell sorter and transplanted the negative fraction into the fetal liver (n = 6). At delivery, tumors were no longer observed in all the six animals, while the cyESC-derived hematopoietic engraftment was unperturbed (2–5%). SSEA-4 is therefore a clinically-relevant pluripotency marker of primate ES cells. Purging cells with this surface marker would be a promising method for clinical progenitor cell preparations using human ES cells.