Scalable Cardiac Differentiation of Pluripotent Stem Cells Using Specific Growth Factors and Small Molecules

Abstract

The envisioned routine application of human pluripotent stem cell (hPSC)-derived cardiomyocytes (CMs) for therapies and industry-compliant screening approaches will require efficient and highly reproducible processes for the mass production of well-characterized CM batches.On their way toward beating CMs, hPSCs initially undergo an epithelial-to-mesenchymal transition into a primitive-streak (PS)-like population that later gives rise to all endodermal and mesodermal lineages, including cardiovascular progenies (CVPs). CVPs are multipotent and possess the capability to give rise to all major cell types of the heart, including CMs, endothelial cells, cardiac fibroblasts, and smooth muscle cells. This article provides an historical overview and describes the stepwise development of protocols that typically result in the appearance of beating CMs within 7-12 days of hPSC differentiation.We describe the development of directed and closely controlled cardiomyogenic differentiation, which now enables the induction of >90% CM purity without further lineage enrichment. Although secreted lineage specifiers (revealed from developmental biology) were initially used, we outline the advantages of chemical pathway modulators, as defined by more recent screening approaches. Subsequently, we discuss the use of defined culture media for upscaling the production of hPSC-CMs in controlled bioreactors and how this, in principle, unlimited source of human CMs can be used to progress heart regeneration and stimulate the drug discovery pipeline. Graphical Abstract.

Selection of human induced pluripotent stem cells lines optimization of cardiomyocytes differentiation in an integrated suspension microcarrier bioreactor

“…Overall, our process yielded > 40 CMs per seeded hiPSC (Table 2D), which is at least eightfold more than other reports. They ranged from 1 to 5 CMs/hPSC [10,13,14,27,28,38,57,60], and 35% more than our previous report [58]. This difference in CM yields is mainly attributed to the high proliferative capacity in the initial expansion phase (~16fold) and differentiation phase (~3-fold), as well as > 96% of Troponin T obtained after the recovery phase.…”

Cryopreservation of human pluripotent stem cell-derived cardiomyocytes is not detrimental to their molecular and functional properties

“…Advances in hPSC-CM generation and phenotyping have made them valuable as in vitro models for studying human heart development and cardiovascular disease, as well as for cardiac safety pharmacology, drug screening, and drug discovery (Brandão et al, 2017;Hartman et al, 2016;Magdy et al, 2018). While it is now possible to efficiently generate large quantities of relatively pure hPSC-CMs (Kempf and Zweigerdt, 2018), these procedures are timeconsuming, laborious, and are still subject to variability in reproducibility between successive differentiations (Burridge et al, 2015;Kempf et al, 2014). Efforts to cryopreserve hPSC-CMs have led to improved consistency in functional assays with comparable results obtained when the same batch of cells are used even between different laboratories (Hwang et al, 2015;Millard et al, 2018).…”

Human Induced Pluripotent Stem-Cell-Derived Cardiomyocytes as Models for Genetic Cardiomyopathies

“…Although cardiac differentiation protocols for iPSCs have been improved significantly over recent years [345,470], it should be kept in mind that iPSC-derived cardiomyocytes are still a heterogeneous cell population. Especially for bulk down-stream applications like proteomics, genomics, or metabolomics, this might have a significant impact.…”