Aggregate Size Optimization in Microwells for Suspension-based Cardiac Differentiation of Human Pluripotent Stem Cells

Cardiac differentiation of human pluripotent stems cells (hPSCs) is typically carried out in suspension cell aggregates. Conventional aggregate formation of hPSCs involves dissociating cell colonies into smaller clumps, with size control of the clumps crudely controlled by pipetting the cell suspens...

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Main Authors:	Bauwens, Celine L., Toms, Derek, Ungrin, Mark
Format:	Online
Language:	English
Published:	MyJove Corporation 2016
Online Access:	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5092056/

id	pubmed-5092056
recordtype	oai_dc
spelling	pubmed-50920562016-11-15 Aggregate Size Optimization in Microwells for Suspension-based Cardiac Differentiation of Human Pluripotent Stem Cells Bauwens, Celine L. Toms, Derek Ungrin, Mark Developmental Biology Cardiac differentiation of human pluripotent stems cells (hPSCs) is typically carried out in suspension cell aggregates. Conventional aggregate formation of hPSCs involves dissociating cell colonies into smaller clumps, with size control of the clumps crudely controlled by pipetting the cell suspension until the desired clump size is achieved. One of the main challenges of conventional aggregate-based cardiac differentiation of hPSCs is that culture heterogeneity and spatial disorganization lead to variable and inefficient cardiomyocyte yield. We and others have previously reported that human embryonic stem cell (hESC) aggregate size can be modulated to optimize cardiac induction efficiency. We have addressed this challenge by employing a scalable, microwell-based approach to control physical parameters of aggregate formation, specifically aggregate size and shape. The method we describe here consists of forced aggregation of defined hPSC numbers in microwells, and the subsequent culture of these aggregates in conditions that direct cardiac induction. This protocol can be readily scaled depending on the size and number of wells used. Using this method, we can consistently achieve culture outputs with cardiomyocyte frequencies greater than 70%. MyJove Corporation 2016-09-25 /pmc/articles/PMC5092056/ /pubmed/27768032 http://dx.doi.org/10.3791/54308 Text en Copyright © 2016, Journal of Visualized Experiments http://creativecommons.org/licenses/by-nc-nd/3.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. To view a copy of this license, visithttp://creativecommons.org/licenses/by-nc-nd/3.0/
repository_type	Open Access Journal
institution_category	Foreign Institution
institution	US National Center for Biotechnology Information
building	NCBI PubMed
collection	Online Access
language	English
format	Online
author	Bauwens, Celine L. Toms, Derek Ungrin, Mark
spellingShingle	Bauwens, Celine L. Toms, Derek Ungrin, Mark Aggregate Size Optimization in Microwells for Suspension-based Cardiac Differentiation of Human Pluripotent Stem Cells
author_facet	Bauwens, Celine L. Toms, Derek Ungrin, Mark
author_sort	Bauwens, Celine L.
title	Aggregate Size Optimization in Microwells for Suspension-based Cardiac Differentiation of Human Pluripotent Stem Cells
title_short	Aggregate Size Optimization in Microwells for Suspension-based Cardiac Differentiation of Human Pluripotent Stem Cells
title_full	Aggregate Size Optimization in Microwells for Suspension-based Cardiac Differentiation of Human Pluripotent Stem Cells
title_fullStr	Aggregate Size Optimization in Microwells for Suspension-based Cardiac Differentiation of Human Pluripotent Stem Cells
title_full_unstemmed	Aggregate Size Optimization in Microwells for Suspension-based Cardiac Differentiation of Human Pluripotent Stem Cells
title_sort	aggregate size optimization in microwells for suspension-based cardiac differentiation of human pluripotent stem cells
description	Cardiac differentiation of human pluripotent stems cells (hPSCs) is typically carried out in suspension cell aggregates. Conventional aggregate formation of hPSCs involves dissociating cell colonies into smaller clumps, with size control of the clumps crudely controlled by pipetting the cell suspension until the desired clump size is achieved. One of the main challenges of conventional aggregate-based cardiac differentiation of hPSCs is that culture heterogeneity and spatial disorganization lead to variable and inefficient cardiomyocyte yield. We and others have previously reported that human embryonic stem cell (hESC) aggregate size can be modulated to optimize cardiac induction efficiency. We have addressed this challenge by employing a scalable, microwell-based approach to control physical parameters of aggregate formation, specifically aggregate size and shape. The method we describe here consists of forced aggregation of defined hPSC numbers in microwells, and the subsequent culture of these aggregates in conditions that direct cardiac induction. This protocol can be readily scaled depending on the size and number of wells used. Using this method, we can consistently achieve culture outputs with cardiomyocyte frequencies greater than 70%.
publisher	MyJove Corporation
publishDate	2016
url	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5092056/
_version_	1613709364788461568

Aggregate Size Optimization in Microwells for Suspension-based Cardiac Differentiation of Human Pluripotent Stem Cells

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