Definitive hematopoietic stem/progenitor cells from human embryonic stem cells through serum/feeder-free organoid-induce
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(2020) 11:493
RESEARCH
Open Access
Definitive hematopoietic stem/progenitor cells from human embryonic stem cells through serum/feeder-free organoidinduced differentiation Selami Demirci1, Juan J. Haro-Mora1, Alexis Leonard1, Claire Drysdale1, Daniela Malide2, Keyvan Keyvanfar3, Khaled Essawi1, Raul Vizcardo4, Naritaka Tamaoki4, Nicholas P. Restifo4, John F. Tisdale1* and Naoya Uchida1
Abstract Background: Ex vivo production of hematopoietic stem/precursor cells (HSPCs) represents a promising versatile approach for blood disorders. Methods: To derive definitive HSPCs from human embryonic stem cells (ESCs), we differentiated mesodermally specified embryoid bodies (EBs) on gelatin-coated plates in serum/feeder-free conditions. Results: Seven-day EB maturation followed by an 8-day differentiation period on OP9 cells provided the highest number of definitive (CD34+ CD235a−, 69%, p < 0.01) and lowest number of primitive (CD34− CD235a+, 1.55%, p < 0.01) precursor cells along with the highest colony-forming units (149.8 ± 11.6, p < 0.01) in feeder-free conditions. Maximal HSPC fraction (CD34+ CD38− CD45RA− CD49f+ CD90+) was 7.6–8.9% after 10 days of hematopoietic differentiation with 14.5% adult β-globin expression following RBC differentiation. Myeloid and erythroid colonies were restricted strictly to the CD34+ CD43+ fraction (370.5 ± 65.7, p < 0.001), while the CD34− CD43+ fraction produced only a small number of colonies (21.6 ± 11.9). In addition, we differentiated the CD34+ CD43+ cells towards T-lymphocytes using the OP9/DLL1 co-culture system demonstrating double-positive T cells (CD4+ CD8+) with CD3+ expression displaying a broad T cell receptor (TCR) repertoire. Confocal imaging of organoid-like structures revealed a close association of CD31+ cells with CD34+ and CD43+ cells, suggesting a potential emergence of HSPCs through endothelial to hematopoietic transition. Furthermore, fluorescently labeled organoids exhibited the emergence of spherical non-attached cells from rare progenitors at the border of the organoid center. Conclusions: In summary, definitive HSPCs can be derived from ESCs through a dynamic cellular process from an organoid-like structure, where erythroid progeny are capable of producing adult hemoglobin and lymphoid progeny shows a diverse TCR repertoire. Keywords: iPSCs, Hemogenic endothelium, HSCs, T cell differentiation, β-Globin, Definitive hematopoiesis
* Correspondence: [email protected] 1 Sickle Cell Branch, National Heart Lung and Blood Institute (NHLBI)/National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (NIH), 9000 Rockville Pike, Bldg. 10, 9N112, Bethesda, MD 20892, USA Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source,
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