Efficient reprogramming of human fibroblasts using RNA reprogramming with DAPT and iDOT1L under normoxia conditions

Kazuki Morita; Akihiro Nakamura; Masakazu Machida; Tomoyuki Kawasaki; Rina Nakanishi; Justin Ichida; Takanori Iwata; Akihiro Umezawa; Hidenori Akutsu

doi:10.1016/j.reth.2022.09.002

Efficient reprogramming of human fibroblasts using RNA reprogramming with DAPT and iDOT1L under normoxia conditions

Regen Ther. 2022 Sep 19:21:389-397. doi: 10.1016/j.reth.2022.09.002. eCollection 2022 Dec.

Authors

Kazuki Morita^{1

2}, Akihiro Nakamura³, Masakazu Machida², Tomoyuki Kawasaki², Rina Nakanishi², Justin Ichida⁴, Takanori Iwata¹, Akihiro Umezawa², Hidenori Akutsu²

Affiliations

¹ Department of Periodontology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), Tokyo 113-8510, Japan.
² Centre for Regenerative Medicine, National Research Institute for Child Health and Development, Tokyo, Japan.
³ Department of Microbiology, Saitama Medical University, Saitama, Japan.
⁴ Department of Stem Cell Biology and Regenerative Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, 90033, USA.

Abstract

Introduction: Human induced pluripotent stem cells (hiPSCs) are generated through the reprogramming of somatic cells expressing a defined set of transcription factors. The advent of autologous iPSCs has enabled the generation of patient-specific iPSC lines and is expected to contribute to the exploration of cures and causes of diseases, drug screening, and tailor-made regenerative medicines. Efficient control of hiPSC derivation is beneficial for industrial applications. However, the mechanisms underlying somatic cell reprogramming remain unknown, while reprogramming efficiency remains extremely low, especially in human cells.

Methods and results: We previously reported that chemical inhibition of the NOTCH signaling pathway and DOT1L promoted the generation of hiPSCs from keratinocytes, but the mechanisms and effect of this double inhibition on other types of cells remain to be investigated. Here, we found that the NOTCH/DOT1L inhibition markedly increased iPSC colony generation from human fibroblast cells via mRNA reprogramming, and mesenchymal to epithelial transition (MET)-related genes are significantly expressed in the early phase of the reprogramming. We successfully derived hiPSC lines using a single-cell sorting system under efficient reprogramming conditions.

Conclusions: This user-friendly reprogramming approach paves the way for the development of hiPSC derivations in industrial applications of disease modeling and drug screening.

Keywords: ACTB, actin beta; ALP, alkaline phosphatase; ALPL, alkaline phosphatase, liver/bone/kidney; CDH1, cadherin 1; CDH2, cadherin 2; CDKN1A, cyclin dependent kinase inhibitor 1A; DSC2, desmocollin 2; EGFR, epidermal growth factor receptor; ERBB3, erb-B2 receptor tyrosine kinase 3; FOXH1, forkhead box H1; KLF4, Krüppel-like factor 4; KRT14, keratin 14; LIN28A, Lin-28 homolog A; MET; MMP2, matrix metallopeptidase 2; NANOG, nanog homeobox; NODAL, nodal growth differentiation factor; PDGFRA, platelet derived growth factor receptor alpha; POU5F1, POU class 5 homeobox 1; Reprogramming; SOX2, sex determining region Y-box transcription factor 2; TMEFF1, transmembrane protein with epidermal growth factor like and two follistatin like domains 1; TP53, tumor protein p53; TWIST1, twist family basic helix-loop-helix transcription factor 1; VIM, vimentin; iPS cells.