Human Fibroblast Reprogramming to Naïve Induced Pluripotent Stem Cells in Microfluidics

Abstract

Induced pluripotent stem cells (iPSCs) are generated by expression of transcription factors OCT4,SOX2, KLF4 and cMYC (OSKM) in somatic cells. In contrast to murine naïve iPSCs, conventional humaniPSCs are in a more developmentally advanced state called primed pluripotency. Here we report adetailed protocol for the generation of human naïve iPSCs (niPSCs) from less than 1000 primaryhuman somatic cells without stable genetic manipulation by delivery of modified messenger RNAswith microfluidics. Expression of OSKM in combination with NANOG for 12 days generates niPSCs thatare free of transgenes, karyotypically normal, and display transcriptional, epigenetic and metabolicfeatures indicative of the naïve state. Specifically, we describe the production of microfluidics chips,the reprogramming of somatic cells into niPSCs and their expansion out of the microfluidics chips. Ourmicrofluidics approach will allow robust and cost-effective production of patient-specific niPSCs forregenerative medicine applications, including disease modelling and drug screening