Efficient Differentiation of Human Pluripotent Stem Cells into Dendritic Cells by Chemical Approach

Background: Since patient condition affects the function of dendritic cells (DCs), developing fully functional DCs from human pluripotent stem cells (hPSCs) has been promising for DC-based immunotherapy. Here we established a new protocol for efficient production of hPSCs-derived DCs.Materials and Methods: Using 3D culture condition and small molecules, hPSCs were committed and differentiated into hae-matopoietic progenitor cells (HPCs). Then, HPCs were induced to generate DCs and analyzed for morphological and functional properties.Results: In order to efficiently derive DCs from hPSCs, we first generated HPCs. To this, hPSCs were induced to differentiate as aggregates in the presence of CHIR99021 and ascorbic acid small molecules. In this protocol, we observed that most of the aggregates tend to cavitate and form cystic body. Second, the cystic bodies were plated onto a matrigel-coated dish and cul-tured in expansion media in order to release non-adherent hae-matopoietic-like cells. The released cells possessed robust my-eloid colony forming activity. Third, we collected non-adherent cells and transferred them into the ultra-low attachment plates containing DC differentiation media and observed efficient DC-like cells formation. Flowcytometry assay, scanning elec-tron microscopy, Wright-Giemsa staining demonstrated that hPSC-derived DCs produced by this protocol were comparable with human peripheral blood monocyte-derived DCs in terms of cell surface receptors and morphology. In total, we were able to generate 1.5×106 DCs from 105 PSCs. Furthermore, inde-pendent hPSC lines were used to show reproducibility of the protocol.Conclusion: We assumed that 3D culture condition more close-ly imitates natural tissue development than 2D culture systems. To corroborate this hypothesis, we established a protocol in 3D culture condition with the presence of small molecules. Our re-sults suggest that this approach can be effective in production of DCs and can be useful in many fields including human cancer immunotherapy.

Keywords:

Pluripotent Stem Cell , Haematopoietic Stem Cell , Small Molecules , Dendritic Cells