Superior Induced Pluripotent Stem Cell Generation through Phactr3-Driven Mechanomodulation of Both Early and Late Phases of Cell Reprogramming.

Autor:	Chowdhury MM; Australian Institute of Bioengineering and Nanotechnology (AIBN), The University of Queensland, St. Lucia, QLD 4072, Australia., Zimmerman S; Albert Einstein College of Medicine, Bronx, NY 10461, USA., Leeson H; Australian Institute of Bioengineering and Nanotechnology (AIBN), The University of Queensland, St. Lucia, QLD 4072, Australia., Nefzger CM; Institute of Molecular Bioscience, The University of Queensland, St. Lucia, QLD 4072, Australia., Mar JC; Australian Institute of Bioengineering and Nanotechnology (AIBN), The University of Queensland, St. Lucia, QLD 4072, Australia.; Albert Einstein College of Medicine, Bronx, NY 10461, USA., Laslett A; Australian Regenerative Medicine Institute, Monash University, Clayton, VIC 3800, Australia., Polo JM; Department of Anatomy and Developmental Biology, Monash Biomedicine Discovery Institute and the Australian Regenerative Medicine Institute, Monash University, Clayton, VIC 3800, Australia.; Adelaide Centre for Epigenetics and the South Australian Immunogenomics Cancer Institute, Faculty of Health and Medical Sciences, The University of Adelaide, Adelaide, SA 5005, Australia., Wolvetang E; Australian Institute of Bioengineering and Nanotechnology (AIBN), The University of Queensland, St. Lucia, QLD 4072, Australia., Cooper-White JJ; Australian Institute of Bioengineering and Nanotechnology (AIBN), The University of Queensland, St. Lucia, QLD 4072, Australia.; School of Chemical Engineering, Andrew N. Liveris Building, The University of Queensland, St. Lucia, QLD 4072, Australia.
Jazyk:	angličtina
Zdroj:	Biomaterials research [Biomater Res] 2024 May 21; Vol. 28, pp. 0025. Date of Electronic Publication: 2024 May 21 (Print Publication: 2024).
DOI:	10.34133/bmr.0025
Abstrakt:	Human cell reprogramming traditionally involves time-intensive, multistage, costly tissue culture polystyrene-based cell culture practices that ultimately produce low numbers of reprogrammed cells of variable quality. Previous studies have shown that very soft 2- and 3-dimensional hydrogel substrates/matrices (of stiffnesses ≤ 1 kPa) can drive ~2× improvements in human cell reprogramming outcomes. Unfortunately, these similarly complex multistage protocols lack intrinsic scalability, and, furthermore, the associated underlying molecular mechanisms remain to be fully elucidated, limiting the potential to further maximize reprogramming outcomes. In screening the largest range of polyacrylamide (pAAm) hydrogels of varying stiffness to date (1 kPa to 1.3 MPa), we have found that a medium stiffness gel (~100 kPa) increased the overall number of reprogrammed cells by up to 10-fold (10×), accelerated reprogramming kinetics, improved both early and late phases of reprogramming, and produced induced pluripotent stem cells (iPSCs) having more naïve characteristics and lower remnant transgene expression, compared to the gold standard tissue culture polystyrene practice. Functionalization of these pAAm hydrogels with poly-l-dopamine enabled, for the first-time, continuous, single-step reprogramming of fibroblasts to iPSCs on hydrogel substrates (noting that even the tissue culture polystyrene practice is a 2-stage process). Comparative RNA sequencing analyses coupled with experimental validation revealed that a novel reprogramming regulator, protein phosphatase and actin regulator 3, up-regulated under the gel condition at a very early time point, was responsible for the observed enhanced reprogramming outcomes. 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. Competing Interests: Competing interests: The authors declare that they have no competing interests. (Copyright © 2024 Mohammad Mahfuz et al.)
Databáze:	MEDLINE
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