Authors:

Nathan Petrucci

Date Created:

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About Paper:

Current immunotherapies, including CAR T cell therapies, rely on the time-consuming and costly procedures of harvesting and culturing patient or donor cells. Pluripotent stem cells provide a potential alternative source of cells which could alleviate the time and expense involved in immunotherapies. However, the lack of robust and efficient protocols to differentiate pluripotent stem cells into hemogenic endothelium is a current barrier preventing the implementation of stem cell-derived cells in immunotherapy. In this study, building off existing methods to produce hemogenic endothelium, we attempt to develop a new protocol with higher yield and less complexity. Modified mRNA encoding the ETV2 gene was synthesized from plasmid DNA. The successful production of modified mRNA was verified by gel electrophoresis and NanoDrop. The modified mRNA was lipofected into human pluripotent stem cells at different times in the differentiation process. The transient expression of ETV2 in the cells effectively differentiated them into hemogenic endothelium. The cells were characterized by using fluorescence immunostaining and flow cytometry analysis. All treatments successfully produced hemogenic endothelium, but further analysis is required to definitively determine the time of ETV2 expression which maximizes yield. Additionally, in future studies the ETV2-directed hemogenic endothelium will be further differentiated into hematopoietic stem cells. Ultimately, this established protocol could help streamline the process of producing immune cells from human pluripotent stem cells, improving their feasibility of use in clinical immunotherapy.

Source:

Purdue University / 2023

Topics:

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Co-authors:

Nathan Petrucci