Abstract
Functional heart cells and tissues sourced from human pluripotent stem cells (hPSCs) have great potential for substantially advancing treatments of cardiovascular maladies. Realization of this potential will require the development of cost-effective and tunable bioprocesses for manufacturing hPSC-based cell therapeutics. Here, we report the development of a xeno-free platform for guiding the cardiogenic commitment of hPSCs. The system is based on a fully defined, open-source formulation without complex supplements, which have varied and often undetermined effects on stem cell physiology. The formulation was used to systematically investigate factors inducing the efficient commitment to cardiac mesoderm of three hPSC lines. Contractile clusters of cells appeared within a week of differentiation in planar cultures and by day 13 over 80% of the cells expressed cardiac progeny markers such as TNNT2. In conjunction with expansion, this differentiation strategy was employed in stirred-suspension cultures of hPSCs. Scalable differentiation resulted in 0.4–2 million CMs/ml or ∼5–20 TNNT2-positive cells per seeded hPSC without further enrichment. Our findings will contribute to the engineering of bioprocesses advancing the manufacturing of stem cell-based therapeutics for heart diseases.
Highlights
Heart disease, which is a major cause of morbidity and mortality (Benjamin et al, 2018), is associated with loss of cardiac myocytes and declining function eventually leading to organ failure
In formulations with DMEM (Supplementary Figure 1A) different human pluripotent stem cells (hPSCs) lines exhibited doubling times that followed the same trend as those for cells maintained in the medium used for routine maintenance of self-renewing hPSCs
The proliferation of hPSCs cultured in medium with Recombinant human serum albumin (rHA) was similar as in the same base medium containing the more complex free fatty acid mix (FFA; linoleic acid, linolenic acid, and tocopherol acetate), a component of the B27 supplement, or FFA and rHA together (Supplementary Figure 1B)
Summary
Heart disease, which is a major cause of morbidity and mortality (Benjamin et al, 2018), is associated with loss of cardiac myocytes and declining function eventually leading to organ failure. The shortage of donor organs and the required life-long immunosuppression are limiting factors for heart transplantation. To this end, human pluripotent stem cell (hPSC)-based cellular therapies hold promise for reconstituting the damaged cardiac tissue and improving its function. Many utilize non-physiological components [e.g., polyvinyl alcohol (Burridge et al, 2014)], chemically defined lipids with animal-sourced components (e.g., cholesterol), and complex supplements such as B27 (Lian et al, 2012) The latter, which was originally developed for neuronal cell cultivation (Brewer et al, 1993), contains an assortment of components including antioxidants, lipids, steroids, and bovine serum albumin (BSA), with potentially multifarious but largely unexplored roles in the specification of hPSCs to heart cells
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