Definitive hematopoietic stem/progenitor cells from human embryonic stem cells through serum/feeder-free organoid-induced differentiation

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.

The Role of SCL Isoforms in Embryonic Hematopoiesis

“…In comparison with the other types of vascular endothelial cells, CD34 is a quite specific marker for hemogenic endothelium and CD31 is a general marker for all [ 30 ]. In vitro differentiation experiments concluded that CD43 defined definitive hematopoietic stem/progenitor cells from human embryonic stem cells [ 31 , 32 ]. Unlike the SCL-S, all the CD31, CD34, and CD43 markers were still not observed on the endothelial layer of E9.5 aorta ( Figure 4 A), instead, dispersed CD31 + /CD34 + /CD43 + cells were found in the vessel cavities ( Figure 4 A,B) and weak but significant c-Kit and CD45 markers were revealed on most of these cells ( Figure 4 C).…”

Establishment of human hematopoietic organoids for evaluation of hematopoietic injury and regeneration effect

“…hPSCs, such as hESCs, are ideal seed cells for obtaining large numbers of HSPCs and for scalable manufacturing of human hematopoietic or BM organoids. Accumulating evidence demonstrates that hESCs can differentiate into HSPCs and multilineage blood cells [ 36 , 37 ]. Therefore, we used a stepwise induction protocol to generate human CD34 + HSPCs from hESCs.…”

Research Trends in Lung Organoids: Bibliometric Analysis and Visualization

“…Branchways are key structures for air conduction in the lungs. However, lung organoids are typically spherical and cannot fully simulate this morphological structure [75]. In addition to epithelial cells, many other cell types are involved in the pulmonary microenvironment.…”