Unlocking the potential of human-induced pluripotent stem cells: Cellular responses and secretome profiles in peptide hydrogel 3D culture

Authors: CUI, MUYUN - Kansas State University; WU, WEI - Kansas State University; LI, QUAN - Kansas State University; QI, GUANGYAN - Kansas State University; Liu, Xuming; BAI, JIANFA - Kansas State University; Chen, Ming Shun; LI, PING - Kansas State University; SUN, XIUZHI SUSAN - Kansas State University

Submitted to: Cell
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/3/2023
Publication Date: 1/12/2024
Citation: Cui, M., Wu, W., Li, Q., Qi, G., Liu, X., Bai, J., Chen, M., Li, P., Sun, X. 2024. Unlocking the potential of human-induced pluripotent stem cells: Cellular responses and secretome profiles in peptide hydrogel 3D culture. Cell. https://doi.org/10.3390/cells13020143.
DOI: https://doi.org/10.3390/cells13020143

Interpretive Summary: Traditional cell culture has been carried out in petri-dishes or flasks in two dimensions. As our human society moves towards cell therapy and in vitro tissue regeneration and transplantation, three dimensional cell culture holds great promise. This research is developing and improving three dimensional culturing of human stem cells using a peptide hydrogel as matrix. The research demonstrated superior cell viability, enhanced protein secretion with significantly higher quantity of unique proteins, increased numbers of extracellular vesicles and increased abundance of growth factors compared to those in 2D culture. Our results also offer insights into how to optimize three dimensional cell culturing, which shall be useful for using three dimensional cell culturing in translational biomedical research towards clinical applications.

Technical Abstract: Human-induced pluripotent stem cells (hiPSCs) have shown great potential for human health, but their growth and properties have been significantly limited by the traditional monolayer (2D) cell culture method for more than 15 years. Three-dimensional (3D) culture technology has demonstrated tremendous advantages over 2D, particularly, the 3D PGmatrix-hiPSC derived from a peptide hydrogel offers a breakthrough pathway for the maintenance and expansion of physiologically relevant hiPSC 3D colonies (spheroids). In this study, the impact of 3D culture conditions in PGmatrix on hiPSC performance, integrity, and secretome profiles was determined across two commonly used hiPSC cell lines derived from fibroblast cells (hiPSC-F) and peripheral blood mononuclear cells (hiPSC-P) in two most popular hiPSC culture media (mTeSR1 and essential eight (E8)). The 3D culture conditions varied in hydrogel strength, 3D embedded matrix, and 3D suspension matrix. Results showed that hiPSC cultured in 3D PGmatrix-hiPSC demonstrated the ability to maintain consistently high cell viability that was above 95% across all the 3D conditions with cell expansion rates of 10-20 folds depending on 3D conditions and cell lines. RT-qPCR analysis suggested pluripotent gene markers are stable and not significantly affected by cell lines or 3D PGmatrix conditions tested in this study. Mass spectrometry-based analysis of secretome from hiPSCs cultured in 3D PGmatrix-hiPSC revealed significantly higher quantity of unique proteins, including extracellular vesicles (EVs) related and growth factors, compared to those in 2D culture. Moreover, it is the first time to identify that hiPSC in the medium with rich supplement (i.e., mTeSR1) released more growth-regulating factors, while in the medium with fewer supplements (i.e., E8) secreted more survival growth factors and extracellular proteins. These findings offer insights into how these differences may impact hiPSC behavior and deepen our understanding of hiPSC responses to 3D culture conditions, aiding the optimization of hiPSC properties in translational biomedical research towards clinical applications.