Definitive hematopoietic stem/progenitor cells from human embryonic stem cells through serum/feeder-free organoid-induced differentiation.
Selami Demirci, Juan J Haro-Mora, Alexis Leonard, Claire Drysdale, Daniela Malide, Keyvan Keyvanfar, Khaled Essawi, Raul Vizcardo, Naritaka Tamaoki, Nicholas P Restifo, John F Tisdale, Naoya Uchida
Author Information
Selami Demirci: 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.
Juan J Haro-Mora: 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.
Alexis Leonard: 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.
Claire Drysdale: 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.
Khaled Essawi: 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.
Raul Vizcardo: National Cancer Institute, Center for Cancer Research, NIH, Bethesda, MD, USA.
Naritaka Tamaoki: National Cancer Institute, Center for Cancer Research, NIH, Bethesda, MD, USA.
Nicholas P Restifo: National Cancer Institute, Center for Cancer Research, NIH, Bethesda, MD, USA.
John F Tisdale: 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. johntis@mail.nih.gov. ORCID
Naoya Uchida: 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.
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.
