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

Abstract

Recent advances in the generation of cardiomyocytes (CMs) from human pluripotent stem cells (hPSCs), in conjunction with the promising outcomes from preclinical and clinical studies, have raised new hopes for cardiac cell therapy. We report the development of a scalable, robust, and integrated differentiation platform for large-scale production of hPSC-CM aggregates in a stirred suspension bioreactor as a single-unit operation. Precise modulation of the differentiation process by small molecule activation of WNT signaling, followed by inactivation of transforming growth factor-b and WNT signaling and activation of sonic hedgehog signaling in hPSCs as size-controlled aggregates led to the generation of approximately 100% beating CM spheroids containing virtually pure (∼90%) CMs in 10 days. Moreover, the developed differentiation strategy was universal, as demonstrated by testing multiple hPSC lines (5 human embryonic stem cell and 4 human inducible PSC lines) without cell sorting or selection. The produced hPSC-CMs successfully expressed canonical lineage-specific markers and showed high functionality, as demonstrated by microelectrode array and electrophysiology tests. This robust and universal platform could become a valuable tool for the mass production of functional hPSC-CMs as a prerequisite for realizing their promising potential for therapeutic and industrial applications, including drug discovery and toxicity assays. STEM CELLS

Exosomes secreted by hypoxic cardiosphere‐derived cells enhance tube formation and increase pro‐angiogenic miRNA

“…Labeled exosomes were washed with PBS and ultracentrifuged at 100 000 g for 100 min (Type 45 Ti rotor, 30 000 rpm, k‐factor 133, Beckman coulter, L‐100XP ultracentrifuge). PKH26‐labeled exosomes (10 µg/mL) were co‐cultured with human embryonic stem cell derived cardiomyocytes (hESC‐CMs) for 2 h. hESC‐CMs were obtained according to a previously described protocol . Exosome uptake by the hESC‐CMs was stopped by washing with cold PBS.…”

Scaling up a chemically‐defined aggregate‐based suspension culture system for neural commitment of human pluripotent stem cells

“…Although the expansion and cardiac differentiation of hPSCs as suspension aggregates in scalable platforms are thoroughly studied , examples of neural induction under these conditions are rare, and derivation of NP cells from hPSCs is usually performed under static conditions . Recent work has demonstrated that inhibition of Activin/Nodal and bone morphogenetic protein (BMP) signaling directs hPSCs into neuroectoderm, while blocking signals for mesoderm and endoderm .…”

“…Successful expansion of hPSCs in spinner flasks with a working volume up to 100 mL using serum-free media, such as mTeSR1, has been extensively performed [7,9,[12][13][14]. Moreover, scalable 3D aggregate culture systems have also been used for the controlled differentiation of hPSCs although, to date, the majority of protocols has been designed to direct the differentiation towards the cardiac lineage [15][16][17][18]. Still, there is a lack of scalable systems for the controlled neural differentiation of hPSCs.…”

Stem Cell Therapy of Myocardial Infarction: A Promising Opportunity in Bioengineering

“…After 5 days of culture, the hPSC aggregates are 175 ± 25 µm and CM differentiation is then initiated. At 30 days later, 100% of the EBs are beating and up to 90% cTnT+ CM are produced . By optimizing the PSC aggregate size, small molecule concentrations, induction timing, and agitation rate, 1.5–2 × 10 9 hPSC‐CMs are produced in a 1 L spinner flask with a high purity (>90%) .…”