Superior induced pluripotent stem cell generation through phactr-3-driven mechano-modulation of both early and late phases of cell reprogramming

Abstract

Background:Human cell reprogramming traditionally involves time-intensive, multi-stage, costly tissue culture polystyrene (TCPS)-based cell culture practices that ultimately produce low numbers of reprogrammed cells of variable quality. Previous studies have shown that very soft two-and three-dimensional hydrogel substrates/matrices (of stiffnesses </= 1 kPa) can drive ~2X improvements in human cell reprogramming outcomes. Unfortunately, these similarly complex multi-stage protocols lack intrinsic scalability, and furthermore, the associated underlying molecular mechanisms remain to be fully elucidated, limiting the potential to further maximise reprogramming outcomes. Methods:Polyacrylamide hydrogels of varying stiffness (1 kPa – 1.3 MPa) were surface activated with either Sulfo-SAMPAH or poly-L-dopamine and thereafter gelatin functionalised. Mouse and human fibroblast cells were reprogrammed on these substrates using endogenous (mouse) or exogenous (human) transcription factors for 18 days. Cells were phenotyped during the each of the reprogramming phases. RNA sequencing and bioinformatic analysis elucidated critical molecular drivers of reprogramming upon exposure to the hydrogels, confirmed through gene knockdown experiments. Results:In screening the largest range of polyacrylamide hydrogels of varying stiffness to date, we found that a medium stiffness gel (~100 kPa) significantly increased the overall number of reprogrammed cells by up to ten-fold (10X), accelerated reprogramming kinetics, improved both early and late phases of reprogramming, and produced iPSCs having more naïve characteristics and lower remnant transgene expression, compared to the gold standard tissue culture polystyrene practice. Functionalisation of these pAAm hydrogels with poly-L-dopamine (PDA) enabled, for the first-time, continuous, single-step reprogramming of fibroblasts to iPSCs on hydrogel substrates (noting that even the TCPS practice is a two-stage process). Comparative RNA-Seq analyses coupled with experimental validation revealed that a novel reprogramming regulator, Phactr3, upregulated in the gel condition at a very early time-point, was responsible for the observed enhanced reprogramming outcomes. Conclusions:This study provides a novel culture protocol and substrate for continuous hydrogel-based cell reprogramming and previously unattained clarity of the underlying mechanisms via which substrate stiffness modulates reprogramming kinetics and iPSC quality outcomes, opening new avenues for producing higher numbers of quality iPSCs or other reprogrammed cells at shorter timescales.