Human induced pluripotent stem cell derived erythroblasts can undergo definitive erythropoiesis and co‐express gamma and beta globins

Abstract

Human induced pluripotent stem cells (hiPSCs), like embryonic stem cells, are under intense investigation for novel approaches to model disease and for regenerative therapies. Here, we describe the derivation and characterization of hiPSCs from a variety of sources and show that, irrespective of origin or method of reprogramming, hiPSCs can be differentiated on OP9 stroma towards a multi-lineage haemo-endothelial progenitor that can contribute to CD144+ endothelium, CD235a+ erythrocytes (myeloid lineage) and CD19+ B lymphocytes (lymphoid lineage). Within the erythroblast lineage, we were able to demonstrate by single cell analysis (flow cytometry), that hiPSC-derived erythroblasts express alpha globin as previously described, and that a sub-population of these erythroblasts also express haemoglobin F (HbF), indicative of fetal definitive erythropoiesis. More notably however, we were able to demonstrate that a small sub-fraction of HbF positive erythroblasts co-expressed HbA in a highly heterogeneous manner, but analogous to cord blood-derived erythroblasts when cultured using similar methods. Moreover, the HbA expressing erythroblast population could be greatly enhanced (44·0 ± 6·04%) when a defined serum-free approach was employed to isolate a CD31+ CD45+ erythro-myeloid progenitor. These findings demonstrate that hiPSCs may represent a useful alternative to standard sources of erythrocytes (RBCs) for future applications in transfusion medicine.

A molecular roadmap of definitive erythropoiesis from human induced pluripotent stem cells

“…Our previous work has highlighted the ability of hiPSC‐derived erythroblasts to express adult haemoglobin (HbA) (Yang et al , ), suggesting a definitive lineage. To conduct a comparative molecular analysis of these with cord‐derived (UCB CD34 + ) erythroblasts and their primitive counterparts, we sought initially to isolate and characterize EryPs, which are the first erythroblasts to develop from pluripotent sources (Sturgeon et al , ) and mirror those emerging from the yolk sac blood islands in the embryo (Palis et al , ; Baron & Fraser, ; Fraser et al , ; Baron et al , ; Palis, ).…”

“…The c19 hiPSC line was derived in‐house from Normal Human Dermal Fibroblasts (NHDFs) (Lonza Biologics, Slough, UK) using lentiviral vectors, as described elsewhere (Carpenter et al , ). Additional lines include OC1 and CE1 hiPSC lines, which were generated from UCB mononuclear cells and erythroblasts respectively, and OPM1 from adult peripheral blood, as described previously (Yang et al , ). Cells were maintained in mTeSR1 (Stem Cell Technologies, Vancouver, Canada;) media with active ingredients that include bovine serum albumin, recombinant human (rh) fibroblast growth factor (rh‐bFGF), rh‐transforming growth factor β (rh‐TGFβ), Lithium Chloride, Pipecolic acid, γ‐aminobutyric acid.…”

“…hiPSC and cord‐derived erythroblasts were assessed at various time points by flow cytometry, with live cell staining that included red cell surface antigens, and fixed staining for haemoglobin expression, with methods that have been described previously (Yang et al , ). Analysis was performed on an LSR II flow cytometer (BD Biosciences) with gating for live cells.…”

Human haematopoietic stem cell development: from the embryo to the dish

“…Erythroid cells differentiated from human PSCs usually express ε-and γ-globins (Chang et al, 2006;Lu et al, 2008;Hatzistavrou et al, 2009;Dias et al, 2011), although prolonged culture in human plasma or on stromal layers has yielded predominantly γ-or γ-and β-globin-expressing cells (Qiu et al, 2008;Lapillonne et al, 2010;Yang et al, 2014;Fujita et al, 2016). Importantly, the switch to β-globin synthesis occurs perinatally, and thus is not expected at the AGM or foetal liver stages of haematopoiesis.…”

Umbilical Cord Blood Hematopoietic Stem and Progenitor Cell Expansion for Therapeutic Use

“…Alternatively, and although initial studies have used ibroblasts [61][62][63], UCB may be reprogrammed to induced pluripotent stem (iPS) cells [64], which can then be diferentiated into diferent cell lineages, e.g., to generate red blood cells and platelets. As these end cells lack nuclei, they may allay certain safety concerns with respect to iPS cells and tumorigenicity (provided that enucleated cells can survive transplantation).…”