title: Direct reprogramming of mouse fibroblasts to cardiomyocyte-like cells using Yamanaka factors on engineered poly(ethylene glycol) (PEG) hydrogels.
abstract: Direct reprogramming strategies enable rapid conversion of somatic cells to cardiomyocytes or cardiomyocyte-like cells without going through the pluripotent state. A recently described protocol couples Yamanaka factor induction with pluripotency inhibition followed by BMP4 treatment to achieve rapid reprogramming of mouse fibroblasts to beating cardiomyocyte-like cells. The original study was performed using Matrigel-coated tissue culture polystyrene (TCPS), a stiff material that also non-specifically adsorbs serum proteins. Protein adsorption-resistant poly(ethylene glycol) (PEG) materials can be covalently modified to present precise concentrations of adhesion proteins or peptides without the unintended effects of non-specifically adsorbed proteins. Here, we describe an improved protocol that incorporates custom-engineered materials. We first reproduced the Efe et al. protocol on Matrigel-coated TCPS (the original material), reprogramming adult mouse tail-tip mouse fibroblasts (TTF) and mouse embryonic fibroblasts (MEF) to cardiomyocyte-like cells that demonstrated striated sarcomeric α-actinin staining, spontaneous calcium transients, and visible beating. We then designed poly(ethylene glycol) culture substrates to promote MEF adhesion via laminin and RGD-binding integrins. PEG hydrogels improved proliferation and reprogramming efficiency (evidenced by beating patch number and area, gene expression, and flow cytometry), yielding almost twice the number of sarcomeric α-actinin positive cardiomyocyte-like cells as the originally described substrate. These results illustrate that cellular reprogramming may be enhanced using custom-engineered materials.
authors.affiliation: Department of Biomedical Engineering and Center for Materials Innovation, Washington University, Campus Box 1097, One Brookings Dr., St. Louis, MO 63130, USA.
authors.forename: Amanda W
authors.initials: AW
authors.lastname: Smith
authors.forename: Jake D
authors.initials: JD
authors.lastname: Hoyne
authors.forename: Peter K
authors.initials: PK
authors.lastname: Nguyen
authors.forename: Dylan A
authors.initials: DA
authors.lastname: McCreedy
authors.forename: Haytham
authors.initials: H
authors.lastname: Aly
authors.forename: Igor R
authors.initials: IR
authors.lastname: Efimov
authors.forename: Stacey
authors.initials: S
authors.lastname: Rentschler
authors.forename: Donald L
authors.initials: DL
authors.lastname: Elbert
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D065150:Cellular Reprogramming / Q000502:physiology; D005347:Fibroblasts / Q000473:pathology*; D005434:Flow Cytometry; D020100:Hydrogels / Q000737:chemistry*; D007150:Immunohistochemistry; D051379:Mice; D008858:Microscopy, Phase-Contrast; D032383:Myocytes, Cardiac / Q000378:metabolism; D011092:Polyethylene Glycols / Q000737:chemistry*; D055153:Stem Cell Niche / Q000502:physiology
chemical_list: D020100:Hydrogels; D011092:Polyethylene Glycols