Controlling Expansion and Cardiomyogenic Differentiation of Human Pluripotent Stem Cells in Scalable Suspension Culture

Abstract

SummaryTo harness the potential of human pluripotent stem cells (hPSCs), an abundant supply of their progenies is required. Here, hPSC expansion as matrix-independent aggregates in suspension culture was combined with cardiomyogenic differentiation using chemical Wnt pathway modulators. A multiwell screen was scaled up to stirred Erlenmeyer flasks and subsequently to tank bioreactors, applying controlled feeding strategies (batch and cyclic perfusion). Cardiomyogenesis was sensitive to the GSK3 inhibitor CHIR99021 concentration, whereas the aggregate size was no prevailing factor across culture platforms. However, in bioreactors, the pattern of aggregate formation in the expansion phase dominated subsequent differentiation. Global profiling revealed a culture-dependent expression of BMP agonists/antagonists, suggesting their decisive role in cell-fate determination. Furthermore, metallothionein was discovered as a potentially stress-related marker in hPSCs. In 100 ml bioreactors, the production of 40 million predominantly ventricular-like cardiomyocytes (up to 85% purity) was enabled that were directly applicable to bioartificial cardiac tissue formation.

Cardiac differentiation of human pluripotent stem cells in scalable suspension culture

“…Within 10 d of differentiation, the protocol results in >60% NKX2.5-GFP-positive cells, indicating CM formation (Fig. 4c,d); we have previously demonstrated equivalent results for other cell lines, using numerous other CM-specific markers and functional assays to confirm CM functionality 31 . Furthermore, as indicated in the protocol (Step 16A(ix)), we advise that those using this method closely monitor cell numbers (in independent wells of a specific experiment and between independent experiments as well) and strictly stick to the timing of the respective process steps.…”

“…By overcoming previous limitations, we recently demonstrated hPSC expansion and cardiomyogenic differentiation via a 'onestep' suspension culture protocol in a 100-ml-scale bioreactor platform, which allowed the induction of >80% CM purity in defined medium without further lineage enrichment 31 . To facilitate straightforward application of this method, this protocol provides a detailed description of the expansion and differentiation strategy (illustrated in Fig.…”

A Universal and Robust Integrated Platform for the Scalable Production of Human Cardiomyocytes From Pluripotent Stem Cells

“…However, these techniques have limited differentiation potential, scalability (microwell-mediated control, hanging drop, and microprinting technologies), universality, and/or reproducibility because of the differentiation protocol itself or the low throughput of the methods used such as forced aggregation techniques before transferring cells to dynamic culture conditions. In addition, most of the protocols depend on using expensive and complex media or reagents (mTeSR1; StemCell Technologies, Vancouver, BC, Canada, http://www.stemcell.com; or StemPro-34; Thermo Fisher Scientific, Waltham, MA, http:// www.thermofisher.com) or microcarriers for expansion of hPSCs and their directed differentiation to cardiomyocytes [24,25].…”

Stem cell derived in vivo-like human cardiac bodies in a microfluidic device for toxicity testing by beating frequency imaging

“…35 Antibodies specific to cardiac troponin T (1 : 200; clone 13-11; Thermo Scientific, MA, USA), sarcomeric α-actinin (1 : 800; clone EA-53; Sigma-Aldrich), α-myosin heavy chain (1 : 25; Hybridoma Bank, IA, USA), and respective isotype controls (Dako, Denmark) were applied in combination with appropriate Cy3-/Cy5-conjugated secondary antibodies (1 : 200; Jackson ImmunoResearch Laboratories, PA, USA) to analyze the cardiomyocyte content, which typically ranged at 80-90% in CBs as previously published. 33 …”