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Updated: Nov 28, 2025
Hemogenic Endothelium Differentiation from Human Pluripotent Stem Cells in A Feeder- and Xeno-free Defined Condition
Published on: June 16, 2019
Definitive hematopoietic stem/progenitor cells from human embryonic stem cells through serum/feeder-free
Selami Demirci1, Juan J Haro-Mora1, Alexis Leonard1
1Sickle 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.
