Induction of Human iPSC-Derived Cardiomyocyte Proliferation Revealed by Combinatorial Screening in High Density Microbioreactor Arrays.

Drew M Titmarsh,Richard J Mills,Alejandro Hidalgo,Nick R Glass,Ernst J Wolvetang,Enzo R Porrello,James E Hudson,Justin J Cooper-White

doi:10.1038/srep24637

Abstract

Inducing cardiomyocyte proliferation in post-mitotic adult heart tissue is attracting significant attention as a therapeutic strategy to regenerate the heart after injury. Model animal screens have identified several candidate signalling pathways, however, it remains unclear as to what extent these pathways can be exploited, either individually or in combination, in the human system. The advent of human cardiac cells from directed differentiation of human pluripotent stem cells (hPSCs) now provides the ability to interrogate human cardiac biology in vitro, but it remains difficult with existing culture formats to simply and rapidly elucidate signalling pathway penetrance and interplay. To facilitate high-throughput combinatorial screening of candidate biologicals or factors driving relevant molecular pathways, we developed a high-density microbioreactor array (HDMA) – a microfluidic cell culture array containing 8100 culture chambers. We used HDMAs to combinatorially screen Wnt, Hedgehog, IGF and FGF pathway agonists. The Wnt activator CHIR99021 was identified as the most potent molecular inducer of human cardiomyocyte proliferation, inducing cell cycle activity marked by Ki67, and an increase in cardiomyocyte numbers compared to controls. The combination of human cardiomyocytes with the HDMA provides a versatile and rapid tool for stratifying combinations of factors for heart regeneration.

Highlights

A central pursuit for researchers pursuing cardiac regeneration is the induction and control of proliferation in differentiated cardiomyocytes, adult cardiomyocytes, which otherwise exhibit very low levels of proliferation[6]
The new design developments were the expansion to four factors, extension to 50 serial culture chambers, parallel replication of device columns, and downsizing of the culture chamber dimensions
The High Density Microbioreactor Array (HDMA)’s microfluidic architecture can accommodate an array of culture chambers comprising a parallel replicates, b serial replicates, c factor concentration levels and d factors resulting in n = abcd total array elements

Summary

Introduction

A central pursuit for researchers pursuing cardiac regeneration is the induction and control of proliferation in differentiated cardiomyocytes, adult cardiomyocytes, which otherwise exhibit very low levels of proliferation[6]. The HDMA platform enables two essential functions: firstly, the ability to access miniaturised cultures of representative cell populations, at a high level of integration, which is vital for high-throughput experimentation; and secondly, the ability to autonomously generate a detailed spectrum of culture environments from a selection of input media. This in turn streamlines the combinatorial investigation of a set of factors of interest, which is relevant to probing signalling pathways that induce cardiomyocyte proliferation. We present and validate the HDMA platform, confirming its capability to culture hPSCs and thereafter employ it in a combinatorial screen of putative proliferation factors in hPSC-derived cardiomyocytes

Methods

Results

Conclusion