Generation of CD34⁺ cells from human embryonic stem cells using a clinically applicable methodology and engraftment in the fetal sheep model

Until now, exvivo generation of CD34⁺ hematopoietic stem cells (HSCs) from human embryonic stem cells (hESCs) mostly involved use of feeder cells of nonhuman origin. Although they provided invaluable models to study hematopoiesis, invivo engraftment of hESC-derived HSCs remains a challenging task. In this study, we used a novel coculture system composed of human bone marrow-derived mesenchymal stromal/stem cells (MSCs) and peripheral blood CD14⁺ monocyte-derived macrophages to generate CD34⁺ cells from hESCs invitro. Human ESC-derived CD34⁺ cells generated using this method expressed surface makers associated with adult human HSCs and upregulated hematopoietic stem cell genes comparable to human bone marrow-derived CD34⁺ cells. Finally, transplantation of purified hESC-derived CD34⁺ cells into the preimmune fetal sheep, primed with transplantation of MSCs derived from the same hESC line, demonstrated multilineage hematopoietic activity with graft presence up to 16 weeks after transplantation. This invivo demonstration of engraftment and robust multilineage hematopoietic activity by hESC-derived CD34⁺ cells lends credence to the translational value and potential clinical utility of this novel differentiation and transplantation protocol.