Hematopoietic Microchimerism in Sheep After In Utero Transplantation of Cultured Cynomolgus Embryonic Stem Cells

Abstract

Although directed differentiation of human embryonic stem (ES) cells would enable a ready supply of cells and tissues required for transplantation therapy, the methodology is limited. We have developed a novel method for hematopoietic development from primate ES cells. We first cultured cynomolgus monkey ES cells in vitro and transplanted the cells in vivo into fetal sheep liver, generating sheep with cynomolgus hematopoiesis. Cynomolgus ES cells were induced to mesodermal cells on murine stromal OP9 cells with multiple cytokines for 6 days. The cells (average 4.8 x 10 cells) were transplanted into fetal sheep in the liver (n=4) after the first trimester (day 55-73, full term 147 days). The animals were delivered at full term, and two of them were intraperitoneally administered with human stem-cell factor (SCF). Cynomolgus hematopoietic progenitor cells were detected in bone marrow at a level of 1% to 2% in all four sheep up to 17 months posttransplant. No teratoma was found in the lambs. After SCF administration, the fractions of cynomolgus hematopoiesis increased by several-fold (up to 13%). Cynomolgus cells were also detected in the circulation, albeit at low levels (<0.1%). Long-term hematopoietic microchimerism from primate ES cells was observed after in vitro differentiation to mesodermal cells, followed by in vivo introduction into the fetal liver microenvironment. The mechanism of such directed differentiation of ES cells remains to be elucidated, but this procedure should allow further investigation.