Induced pluripotent stem cell-based mapping of ß-globin expression throughout human erythropoietic development.

Robust ß-globin expression in erythroid cells derived from induced pluripotent stem cells (iPSCs) increases the resolution with which red blood cell disorders such as sickle cell disease and ß thalassemia can be modeled in vitro. To better quantify efforts in augmenting ß-globin expression, we report the creation of a ß-globin reporter iPSC line that allows for the mapping of ß-globin expression throughout human erythropoietic development in real time at single-cell resolution. Coupling this tool with single-cell RNA sequencing (scRNAseq) identified features that distinguish ß-globin-expressing cells and allowed for the dissection of the developmental and maturational statuses of iPSC-derived erythroid lineage cells. Coexpression of embryonic, fetal, and adult globins in individual cells indicated that these cells correspond to a yolk sac erythromyeloid progenitor program of hematopoietic development, representing the onset of definitive erythropoiesis. Within this developmental program, scRNAseq analysis identified a gradient of erythroid maturation, with ß-globin-expressing cells showing increased maturation. Compared with other cells, ß-globin-expressing cells showed a reduction in transcripts coding for ribosomal proteins, increased expression of members of the ubiquitin-proteasome system recently identified to be involved in remodeling of the erythroid proteome, and upregulation of genes involved in the dynamic translational control of red blood cell maturation. These findings emphasize that definitively patterned iPSC-derived erythroblasts resemble their postnatal counterparts in terms of gene expression and essential biological processes, confirming their potential for disease modeling and regenerative medicine applications.

Notch and Aryl Hydrocarbon Receptor Signaling Impact Definitive Hematopoiesis from Human Pluripotent Stem Cells.

Induced pluripotent stem cells (iPSCs) stand to revolutionize the way we study human development, model disease, and eventually, treat patients. However, these cell sources produce progeny that retain embryonic and/or fetal characteristics. The failure to mature to definitive, adult-type cells is a major barrier for iPSC-based disease modeling and drug discovery. To directly address these concerns, we have developed a chemically defined, serum and feeder-free-directed differentiation platform to generate hematopoietic stem-progenitor cells (HSPCs) and resultant adult-type progeny from iPSCs. This system allows for strict control of signaling pathways over time through growth factor and/or small molecule modulation. Through direct comparison with our previously described protocol for the production of primitive wave hematopoietic cells, we demonstrate that induced HSPCs are enhanced for erythroid and myeloid colony forming potential, and strikingly, resultant erythroid-lineage cells display enhanced expression of adult ß globin indicating definitive pathway patterning. Using this system, we demonstrate the stage-specific roles of two key signaling pathways, Notch and the aryl hydrocarbon receptor (AHR), in the derivation of definitive hematopoietic cells. We illustrate the stage-specific necessity of Notch signaling in the emergence of hematopoietic progenitors and downstream definitive, adult-type erythroblasts. We also show that genetic or small molecule inhibition of the AHR results in the increased production of CD34+ CD45+ HSPCs while conversely, activation of the same receptor results in a block of hematopoietic cell emergence. Results presented here should have broad implications for hematopoietic stem cell transplantation and future clinical translation of iPSC-derived blood cells. Stem Cells 2018;36:1004-1019.