Specification of Hemato-Endothelial-Like Structures and Generation of Hematopoietic Progenitor Cells from Human Pluripotent Stem Cells

Abstract

Background and purpose: Human pluripotent stem cells (hPSCs) with the ability to differentiate into adult cells have provided a new perspective for treatment of some diseases. But, the efficiency of differentiation methods to generate hematopoietic progenitor cells (HPCs) is faced with multiple challenges. In the present study, we investigated the formation of hemato-endothelial-like structures and HPCs from hPSCs. Materials and methods: To generate hemato-endothelial structures and HPCs, in first method, three-dimensional aggregations of hPSCs were co-cultured on OP9 cells. In second and third methods, embryoid bodies (EBs) were differentiated spontaneously or directly in the culture medium containing BMP4, bFGF, SCF, and VEGF. In differentiation process, cell morphology was evaluated by microscopic observation and the expression of CD34 and CD45 markers were analyzed by flow cytometry. In addition, the ability of HPCs to differentiate into various types of blood cells was evaluated using colony formation assay, Wright-Giemsa staining, and CD86 immunofluorescence staining. Results: Findings showed that all three methods generated cells with endothelial-like morphology and HPCs. These cells were able to proliferate, form cell clusters with different efficiency and differentiate into erythroid cells, macrophages, and dendritic cells. Conclusion: The use of cultured cells in defined conditions without a feeder layer is preferred in cell therapy. Therefore, despite the ability of all these methods to generate HPCs, direct differentiation of EBs under defined conditions is a better option for generatingHPCs from hPSCs.

Keywords

• pluripotent stem cells
• hematopoietic progenitor cells
• op9 stromal cell
• embryoid body