Abstract
Background. In vitro methods for hematopoietic differentiation of human pluripotent stem cells (hPSC) are a matter of priority for the in-depth research into the mechanisms of early embryogenesis. So-far, published results regarding the generation of hematopoietic cells come from studies using either 2D or 3D culture formats, hence, it is difficult to discern their particular contribution to the development of the concept of a unique in vitro model in close resemblance to in vivo hematopoiesis. Aim of the study. To assess using the same culture conditions and the same time course, the potential of each of these two formats to support differentiation of human pluripotent stem cells to primitive hematopoiesis without exogenous activation of Wnt signaling. Methods. We used in parallel 2D and 3D formats, the same culture environment and assay methods (flow cytometry, IF, qPCR) to investigate stages of commitment and specification of mesodermal, and hemogenic endothelial cells to CD34 hematopoietic cells and evaluated their clonogenic capacity in a CFU system. Results. We show an adequate formation of mesoderm, an efficient commitment to hemogenic endothelium, a higher number of CD34 hematopoietic cells, and colony-forming capacity potential only in the 3D format-supported differentiation. Conclusions. This study shows that the 3D but not the 2D format ensures the induction and realization by endogenous mechanisms of human pluripotent stem cells’ intrinsic differentiation program to primitive hematopoietic cells. We propose that the 3D format provides an adequate level of upregulation of the endogenous Wnt/β-catenin signaling.
Highlights
In vitro generation of human hematopoietic stem cells from embryonic and induced pluripotent stem cells serves multiple beneficial purposes: mechanistic studies of hematopoiesis, development of cell therapy for hematological diseases, induced transplant tolerance, disease modeling, and drug screening, among others [1] [2]
Our main objective was to compare the capacity of these formats to support the cell commitment events during differentiation of human pluripotent stem cells (hPSC) to primitive hematopoiesis in the lack of exogenous stimulation of Wnt/β-catenin shown previously to direct differentiation to definitive hematopoietic lineages [37,38,39,40,41,42]
The Immunofluorescence analysis with antibodies to Tra160, Oct4 and NANOG showed no significant differences between the two cultures, evidencing that hPSCs used in the study were similar from the monolayer at a density of 1 × 104(2D) or as spheres (3D) and culturing for 5 days
Summary
In vitro generation of human hematopoietic stem cells from embryonic and induced pluripotent stem cells (hPSC) serves multiple beneficial purposes: mechanistic studies of hematopoiesis, development of cell therapy for hematological diseases, induced transplant tolerance, disease modeling, and drug screening, among others [1] [2]. Much effort has been applied to defining the appropriate differentiation conditions required to obtain hematopoietic stem cells in vitro; for example signal gradients [6], cytokines in suitable combinations with the appropriate timing and specific microenvironments, such as feeder cells or artificial niches and with different culture systems, such as embryoid bodies (EBs) [7]. -far, published results regarding the generation of hematopoietic cells come from studies using either 2D or 3D culture formats, it is difficult to discern their particular contribution to the development of the concept of a unique in vitro model in close resemblance to in vivo hematopoiesis. To assess using the same culture conditions and the same time course, the potential of each of these two formats to support differentiation of human pluripotent stem cells to primitive hematopoiesis without exogenous activation of Wnt signaling
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