Modeling of hematopoiesis using ES and patients-derived iPS cells

Abstract

In vitro differentiation of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) into hematopoietic cells may help to find new strategies for the treatment of incurable hematopoiesis disorders and to improve (hematopoietic stem cells) HSCs transplantation. In this study we mainly focus on the mechanisms of hematopoietic differentiation of ESCs and iPSCs at early and late stages, using our knowledge from iPSCs of cyclic neutropenia (CyN) and congenital neutropenia (CN) patients. We found that TPO and bone morphogenetic protein 4 (BMP4) signaling pathway are directly connected. This could elevate early hematopoiesis by TPO/c-mpl signaling. TPO-mediated binding of the HIF-1α transcription factor to the BMP4 gene promoter mediates early hematopoiesis and activation of BMP4 target genes in ESCs. We also used an embryoid body (EB)-based protocol of granulocytic differentiation of human iPSCs to evaluate the in vitro myeloid differentiation of iPSCs derived from one CyN patient harboring a sporadic heterozygous ELANE mutation (p.W241L), and one CN patient with an inherited familial ELANE mutation (p.C151Y) in comparison with myeloid differentiation of iPSCs derived from a healthy donor. CyN is a hematologic disorder show cycles at approximately 21-day intervals in blood cell counts particularly granulocytic neutrophils, monocytes, platelets, and reticulocyte numbers. The majority of CyN patients (approximately 90%) show inherited mutations in the ELANE gene and CN patients also have inherited ELANE mutations. It is unclear how a mutation in the same gene causes CN or CyN. In addition, the pathomechanism of cycling in hematopoiesis pathway downstream of ELANE mutations is still unclear. Using the embryoid body (EB)-based protocol of granulocytic differentiation of hiPSCs, we found that the combination of IL-3 with G-CSF is the best condition to improve our neutrophil maturation protocol for further experiments applicable for CN patients derived from iPSCs. We detected diminished absolute numbers of CD15+CD16+ that were derived from iPSCs of a CyN patient, as compared to that of a healthy individual on day 28 of culture. We also found a remarkable reduction of absolute numbers of myeloid and granulocytic cells that were generated from iPSCs of a CN patient, as compared to cells of healthy individuals on day 28 of culture.