Assessing Cell Competition in Human Pluripotent Stem Cell (hPSC) Cultures

Cell-cell interactions are essential for development and tissue maintenance in multicellular organisms. These interactions regulate processes like cell competition, which helps maintain tissue fitness by eliminating less-fit cells. Prior research has shown that cell competition occurs in various organisms, including mammals. However, the role of cell competition in human pluripotent stem cells (hPSCs) was unclear. hPSCs are widely used in developmental and regenerative medicine studies. Recent evidence suggests that cell competition can occur in hPSC cultures and during reprogramming. This raises questions about how cell competition might influence cell fate and culture composition. Understanding this process could improve hPSC-based modeling and therapeutic applications. Despite these insights, the mechanisms and protocols for studying cell competition in hPSCs remain limited. This gap motivated the development of new methods to assess cell competition in hPSC cultures.

The aim of this study is to develop and optimize protocols for investigating cell competition in hPSC cultures. Cell competition is a biological process that eliminates less-fit cells in favor of fitter ones. In hPSCs, this process may influence culture composition and cell fate. The researchers sought to establish a reliable system to study this phenomenon. They focused on protocols that allow for the assessment of competitive interactions and elimination of less-fit cells. The study also aimed to determine how cell competition manifests in hPSCs. By using fluorescent labeling and co-culture assays, the team could track competitive dynamics. The goal was to provide a framework for future studies on cell-cell interactions in hPSCs. This work may help clarify how cell competition contributes to tissue homeostasis and development.

The study outlines a series of protocols to investigate cell competition in hPSCs. The first step involves electroporation to establish a fluorescent reference cell line. This allows for tracking of specific cell populations during experiments. The researchers also used single-cell dissociation and cloning to generate labeled hPSCs. They then performed proliferation assays in separate and co-culture conditions to compare growth dynamics. Apoptosis levels were assessed using flow cytometry to detect cell death. Transwell assays were used to study cell interactions without direct contact. Immunohistochemistry and image quantification of cleaved caspase-3 helped identify apoptosis markers. Additional assays, such as cell confrontation and compression, were used to observe mechanical extrusion. These methods together provide a comprehensive approach to studying cell competition in hPSCs.

The protocols successfully enabled the assessment of cell competition in hPSC cultures. Fluorescent labeling allowed for clear identification of competitive interactions. Proliferation assays showed differences in growth rates between fitter and less-fit cells. Apoptosis levels were higher in less-fit cells, suggesting elimination through programmed cell death. Transwell assays indicated that direct cell contact is necessary for competition to occur. Immunohistochemistry confirmed increased cleaved caspase-3 in less-fit cells. Cell confrontation and compression assays revealed mechanical extrusion as a possible elimination mechanism. Time-lapse imaging provided visual evidence of cell dynamics during competition. These findings suggest that hPSCs can serve as a model for studying cell competition.

The authors propose that hPSCs can be used to study cell competition during development and disease. The protocols described allow for tracking and analyzing competitive interactions in hPSC cultures. The findings suggest that less-fit cells are eliminated through apoptosis and mechanical extrusion. These results support the idea that cell competition functions in hPSCs as it does in other organisms. The study does not assign essentiality to any specific mechanism but highlights multiple possible pathways. The researchers suggest that these protocols can help model cell-cell interactions in various contexts. The work provides a framework for future studies on cell competition in hPSCs. The authors emphasize the potential of hPSCs as a tool for understanding developmental and regenerative processes.