Cargando…

Practical adoption of state-of-the-art hiPSC-cardiomyocyte differentiation techniques

Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes are a valuable resource for cardiac therapeutic development; however, generation of these cells in large numbers and high purity is a limitation in widespread adoption. Here, design of experiments (DOE) is used to investigate the car...

Descripción completa

Detalles Bibliográficos
Autores principales: Rupert, Cassady E., Irofuala, Chinedu, Coulombe, Kareen L. K.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7064240/
https://www.ncbi.nlm.nih.gov/pubmed/32155214
http://dx.doi.org/10.1371/journal.pone.0230001
_version_ 1783504843823906816
author Rupert, Cassady E.
Irofuala, Chinedu
Coulombe, Kareen L. K.
author_facet Rupert, Cassady E.
Irofuala, Chinedu
Coulombe, Kareen L. K.
author_sort Rupert, Cassady E.
collection PubMed
description Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes are a valuable resource for cardiac therapeutic development; however, generation of these cells in large numbers and high purity is a limitation in widespread adoption. Here, design of experiments (DOE) is used to investigate the cardiac differentiation space of three hiPSC lines when varying CHIR99027 concentration and cell seeding density, and a novel image analysis is developed to evaluate plate coverage when initiating differentiation. Metabolic selection via lactate purifies hiPSC-cardiomyocyte populations, and the bioenergetic phenotype and engineered tissue mechanics of purified and unpurified hiPSC-cardiomyocytes are compared. Findings demonstrate that when initiating differentiation one day after hiPSC plating, low (3 μM) Chiron and 72 x 10(3) cells/cm(2) seeding density result in peak cardiac purity (50–90%) for all three hiPSC lines. Our results confirm that metabolic selection with lactate shifts hiPSC-cardiomyocyte metabolism towards oxidative phosphorylation, but this more “mature” metabolic phenotype does not by itself result in a more mature contractile phenotype in engineered cardiac tissues at one week of culture in 3D tissues. This study provides widely adaptable methods including novel image analysis code and parameters for refining hiPSC-cardiomyocyte differentiation and describes the practical implications of metabolic selection of cardiomyocytes for downstream tissue engineering applications.
format Online
Article
Text
id pubmed-7064240
institution National Center for Biotechnology Information
language English
publishDate 2020
publisher Public Library of Science
record_format MEDLINE/PubMed
spelling pubmed-70642402020-03-23 Practical adoption of state-of-the-art hiPSC-cardiomyocyte differentiation techniques Rupert, Cassady E. Irofuala, Chinedu Coulombe, Kareen L. K. PLoS One Research Article Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes are a valuable resource for cardiac therapeutic development; however, generation of these cells in large numbers and high purity is a limitation in widespread adoption. Here, design of experiments (DOE) is used to investigate the cardiac differentiation space of three hiPSC lines when varying CHIR99027 concentration and cell seeding density, and a novel image analysis is developed to evaluate plate coverage when initiating differentiation. Metabolic selection via lactate purifies hiPSC-cardiomyocyte populations, and the bioenergetic phenotype and engineered tissue mechanics of purified and unpurified hiPSC-cardiomyocytes are compared. Findings demonstrate that when initiating differentiation one day after hiPSC plating, low (3 μM) Chiron and 72 x 10(3) cells/cm(2) seeding density result in peak cardiac purity (50–90%) for all three hiPSC lines. Our results confirm that metabolic selection with lactate shifts hiPSC-cardiomyocyte metabolism towards oxidative phosphorylation, but this more “mature” metabolic phenotype does not by itself result in a more mature contractile phenotype in engineered cardiac tissues at one week of culture in 3D tissues. This study provides widely adaptable methods including novel image analysis code and parameters for refining hiPSC-cardiomyocyte differentiation and describes the practical implications of metabolic selection of cardiomyocytes for downstream tissue engineering applications. Public Library of Science 2020-03-10 /pmc/articles/PMC7064240/ /pubmed/32155214 http://dx.doi.org/10.1371/journal.pone.0230001 Text en © 2020 Rupert et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Rupert, Cassady E.
Irofuala, Chinedu
Coulombe, Kareen L. K.
Practical adoption of state-of-the-art hiPSC-cardiomyocyte differentiation techniques
title Practical adoption of state-of-the-art hiPSC-cardiomyocyte differentiation techniques
title_full Practical adoption of state-of-the-art hiPSC-cardiomyocyte differentiation techniques
title_fullStr Practical adoption of state-of-the-art hiPSC-cardiomyocyte differentiation techniques
title_full_unstemmed Practical adoption of state-of-the-art hiPSC-cardiomyocyte differentiation techniques
title_short Practical adoption of state-of-the-art hiPSC-cardiomyocyte differentiation techniques
title_sort practical adoption of state-of-the-art hipsc-cardiomyocyte differentiation techniques
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7064240/
https://www.ncbi.nlm.nih.gov/pubmed/32155214
http://dx.doi.org/10.1371/journal.pone.0230001
work_keys_str_mv AT rupertcassadye practicaladoptionofstateofthearthipsccardiomyocytedifferentiationtechniques
AT irofualachinedu practicaladoptionofstateofthearthipsccardiomyocytedifferentiationtechniques
AT coulombekareenlk practicaladoptionofstateofthearthipsccardiomyocytedifferentiationtechniques