Single-Cell-State Culture of Human Pluripotent Stem Cells Increases Transfection Efficiency
Authors: Nii Takenobu , Kohara Hiroshi , Marumoto Tomotoshi , Sakuma Tetsushi , Yamamoto Takashi , Tani Kenzaburo 
Published: October 2019

Abstract
Human pluripotent stem cells (hPSCs), such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), have the potential to self-renew indefinitely and differentiate into various cell types. hPSCs can differentiate into various stem or progenitor cell populations used for regenerative medicine and drug development. Newly developed genome editing technology has advanced the use of hPSCs for such purposes. However, to fully utilize hPSCs to achieve this goal, more efficient gene transfer methods under defined conditions are required.
Development of efficient genome editing methods, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9), for use in hPSCs holds great promise in the fields of basic and clinical research. Among these methods, TALENs are more efficient and safer for use in hPSCs to achieve specific gene editing, as ZFNs had a low gene editing efficiency and CRISPR/Cas9 was accompanied by more severe off-target effects than TALENs. Electroporation is a widely used transfection method for hPSC genome editing; however, this method results in reduced cell viability and gene editing efficiency.
In the past decade, various methods were developed for gene transfer into hPSCs; however, hPSCs form tightly packed colonies, making gene transfer difficult. In this study, we established a culture method of hPSCs at a single-cell-state to reduce cell density, and investigated gene transfection efficiency followed by gene editing efficiency. hPSCs cultured in a single-cell-state were transfected using non-liposomal transfection reagents with plasmid DNA driven by the human elongation factor 1-alpha 1 (EF1α) promoter or mRNA encoding enhanced green fluorescent protein (eGFP). The proportion of eGFP+ cells considerably increased in single-cell-state cultures (DNA: 95.80 ± 2.51%, mRNA: 99.70 ± 0.10%). Moreover, most of the cells were viable (control: 93.10 ± 0.40%, DNA: 83.40 ± 2.03%, mRNA: 86.71 ± 0.19%). The mean fluorescence intensity (MFI) was approximately three-fold higher than that in cells transfected by electroporation (electroporation (EPN): 6631 ± 992; transfection (TFN): 17933 ± 1595). eGFP expression was detected by fluorescence microscopy until day seven post-transfection. Our results also demonstrate an inverse correlation between cell density and transfection efficiency. To test whether transfection using this method affected the "stemness" of hPSCs, we examined SSEA4 and NANOG expression in eGFP-transfected cells by flow cytometry analysis. The percentage of both SSEA4+ and NANOG+ cells was greater than 90%. Moreover, transplantation of eGFP-transfected cells into immunodeficient mice led to the formation of teratomas. These results strongly suggested that single-cell-state hPSC culture improved transfection efficiency without inducing differentiation or loss of pluripotency.
Moreover, we used our efficient transfection method to edit the hPSC genome using TALENs. We constructed a Platinum TALEN driven by the EF1α promoter targeting the adenomatous polyposis coli (APC) gene and analyzed the efficiency of gene editing using the Cel-1 assay. Our efficient transfection method induced mutations more efficiently than electroporation (Transfection: 11.1 ± 1.38%, Electroporation: 3.2 ± 0.89). These results showed that TALENs increased gene editing efficiency in single-cell-state hPSC cultures.
Overall, our efficient hPSC transfection method using single-cell-state culture provides an excellent experimental system to investigate the full potential of hPSCs. We expect that this method may contribute to the fields of hPSC-based regenerative medicine and drug discovery.

Disclosures
No relevant conflicts of interest to declare.