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OSR Journal of Student Research

Abstract

Human pluripotent stem cells (hPSC) provide a valuable model for studying organogenesis and recapitulating human diseases. Notably, genetic modification techniques can be harnessed to realize potential of these cells. hPSC display the features of indefinite self-renewal and the potential to differentiate into three germ layers, which holds great promise for regenerative medicine and human disease research. Genetic manipulation continues to be a powerful to study the properties of hPSC. Despite current advances in optimization of transfection procedures, genetic manipulation of hPSC remains a capricious process. On the other hand, controlled differentiation also yielded a variable and unpredictable efficiency in different hPSC lines. Recent studies have shown that serum starvation-induced cell cycle synchronization significantly improved reprogramming efficiency in human fibroblast and increased transient gene delivery into mouse embryonic stem cells. Nevertheless, hPSC lines are routinely cultured in KnockOut Serum Replacement (KO-SR), a serum-free formulation that directly replaces serum in culture. In this regard, we hypothesized that reduction of KO-SR exerts a profound effect in transfection and differentiation efficiency of hPSC. Our procedure mirrors effect of serum starvation by synchronizing hPSC cultures in G1 phase. We showed that transient transfection efficiency could be increased by culturing hPSC in 5% KO-SR for 2 days. Furthermore, our procedure resulted in six-fold increment in the derivation of stably eGFP-expressing transfectants. Interestingly, KO-SR synchronized hPSC also differentiated in a more homogenous manner, as evidenced by a significantly stronger induction of lineage-specific progenitors and an absence of OCT4-expressing residual undifferentiated cells. These results demonstrate that cell cycle synchronization is a key milepost on the path to reducing heterogeneity of hPSC and should be useful in exploring the use of these cells in a predictable manner.

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