Epigenetic memory in induced pluripotent stem cells which is related to the somatic cell type of origin of the stem cells might lead to variations in the differentiation capacities of the pluripotent stem cells. of the stem cells we found a similar hematopoietic induction potential and erythroid differentiation pattern of induced pluripotent stem cells of different somatic cell origin. All human induced pluripotent stem cell lines showed terminal maturation into normoblasts and enucleated reticulocytes generating predominantly fetal hemoglobin. Differences were only observed in the growth rate of erythroid cells which was slightly higher in the induced pluripotent stem cells derived from CD34+ hematopoietic stem cells. More detailed methylation analysis of the hematopoietic and erythroid promoters recognized comparable CpG methylation levels in the induced pluripotent stem cell lines derived from CD34+ cells and those derived from neural stem cells which confirms their comparable erythroid differentiation potential. Introduction During the last years enormous progress has been made in the manufacture of human red blood cells (RBC). Using human hematopoietic stem cells (HSC) from cord blood (CB) or bone marrow as the primary source growth rates higher than 105-fold 1 accompanied by fully terminal maturation into enucleated reticulocytes 1 have been achieved. Recently the first proof-of-principle experiment was performed by transfusing a small sample of manufactured RBC into a human recipient.7 However despite this achievement the large-scale expansion of RBC for transfusion purposes (1 RBC unit contains 1012 RBC) remains problematic as human HSC are a limited source. Up to now protocols for the growth of multipotent HSC are not available. One promising option might be the generation of RBC from human pluripotent stem cells a theoretically unlimited source characterized by properties of self-renewal. Until recently the generation of RBC from human embryonic stem cells (hESC) was limited by ethical issues. Furthermore it is B-Raf-inhibitor 1 unknown whether any of the hESC lines approved in the USA and produced under good developing practice conditions have the universal O Rhesus unfavorable phenotype.8 These limitations were overcome by the discovery of induced pluripotent stem cells (iPSC). Human iPSC which resemble hESC and recapitulation of physiological erythropoiesis in its entirety which includes mesoderm induction generation of HSC erythroid maturation hemoglobin switching and enucleation remains a challenge. Compared to the established protocols for Prox1 the adult system RBC generation from iPSC is usually less efficient. In addition to a poor growth rate of erythroid cells the terminal differentiation of cells generated from iPSC fails particularly with regards to enucleation and switching from embryonic to fetal and finally to adult hemoglobin. Increasing evidence from murine23 24 and human systems25 26 B-Raf-inhibitor 1 indicates that iPSC exhibit an epigenetic memory related to their donor cell type of origin. Although iPSC show characteristics and behaviors of ESC incomplete removal of tissue-specific methylation or aberrant methylation has been observed which might influence their differentiation behavior. Due to this potential epigenetic memory and its influence on hematopoietic differentiation iPSC from CD34+ HSC may be more suitable for erythroid differentiation than the commonly used fibroblast-derived iPSC. To investigate the influence of an epigenetic memory on the growth of iPSC into hematopoietic B-Raf-inhibitor 1 and erythroid cells we generated iPSC lines from human CB-derived CD34+ HSC and human NSC.15 We evaluated their global gene methylation status and their potential to differentiate into hematopoietic progenitors and mature RBC under conditions. Whereas CD34+ HSC are the physiological source for RBC in humans and are of mesodermal origin NSC are derived from the ectodermal germ layer. For the sake of completeness fibroblast-derived iPSC27 and hESC H1 were included in our study as “controls”. Methods Generation of human cord blood CD34+ induced pluripotent stem cells CD34+ HSC were isolated from human CB using MACS sorting (Miltenyi Biotec Germany). Informed consent was obtained from the donating mothers and the investigation was approved by the B-Raf-inhibitor 1 Ethics Committee of Heinrich-Heine-University Düsseldorf Medical School. CD34+ cells were stimulated with stem cell factor (SCF) thrombopoietin (TPO) fms-related tyrosine kinase 3 ligand (FLT3-L) and interleukin 6 (IL-6) as explained elsewhere28 and reprogrammed with either OCT4 SOX2 KLF4 and c-MYC or only OCT4 and SOX2. Lentiviral vectors encoding the human.