Combining Hypoxia and Bioreactor Hydrodynamics Boosts Induced Pluripotent Stem Cell Differentiation Towards Cardiomyocytes

Abstract

Cardiomyocytes (CMs) derived from induced pluripotent stem cells (iPSCs) hold great promise for patient-specific disease modeling, drug screening and cell therapy. However, existing protocols for CM differentiation of iPSCs besides being highly dependent on the application of expensive growth factors show low reproducibility and scalability. The aim of this work was to develop a robust and scalable strategy for mass production of iPSC-derived CMs by designing a bioreactor protocol that ensures a hypoxic and mechanical environment. Murine iPSCs were cultivated as aggregates in either stirred tank or WAVE bioreactors. The effect of dissolved oxygen and mechanical forces, promoted by different hydrodynamic environments, on CM differentiation was evaluated. Combining a hypoxia culture (4 % O2 tension) with an intermittent agitation profile in stirred tank bioreactors resulted in an improvement of about 1000-fold in CM yields when compared to normoxic (20 % O2 tension) and continuously agitated cultures. Additionally, we showed for the first time that wave-induced agitation enables the differentiation of iPSCs towards CMs at faster kinetics and with higher yields (60 CMs/input iPSC). In an 11-day differentiation protocol, clinically relevant numbers of CMs (2.3 × 109 CMs/1 L) were produced, and CMs exhibited typical cardiac sarcomeric structures, calcium transients, electrophysiological profiles and drug responsiveness. This work describes significant advances towards scalable cardiomyocyte differentiation of murine iPSC, paving the way for the implementation of this strategy for mass production of their human counterparts and their use for cardiac repair and cardiovascular research.Electronic supplementary materialThe online version of this article (doi:10.1007/s12015-014-9533-0) contains supplementary material, which is available to authorized users.

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

“…These parameters include oxygen tension, hydrodynamic culture conditions, feeding and media refreshment strategy, and the development of innovative SM delivery technologies to increase the differentiation efficacy and homogeneity and minimize hPSC cell loss after differentiation induction. More recently, it has been demonstrated that optimizing the bioprocess parameters, including oxygen tension (hypoxia), and bioreactor hydrodynamics can boost mouse iPSC differentiation toward CMs [43]. However, these finding should also be validated for differentiation of hPSC cell lines.…”

Effective Hypothermic Storage of Human Pluripotent Stem Cell-Derived Cardiomyocytes Compatible With Global Distribution of Cells for Clinical Applications and Toxicology Testing

“…This cell line was genetically modified to integrate and stably express a transgene containing puromycin‐ N ‐acetyl transferase and enhanced green fluorescent protein (eGFP) genes, both under control of the cardiac‐restricted promoter α‐myosin heavy chain (α‐MHC) [33]. miPSCs were expanded and differentiated as aggregates in fully controlled bioreactors as previously described by our group [8]. Briefly, iPSCs were inoculated in the WAVE Cellbag (GE Healthcare, Piscataway, NJ, http://www.gelifesciences.com) as single cells, at a concentration of 7 × 10 4 cell per ml, and were cultivated under defined conditions (temperature, 37°C; pO 2 , 4% O 2 ; CO 2 , 5%; surface aeration rate, 0.1 air volume per working volume per minute (vvm); rocking angle, 4°; agitation rate, 10–26 rpm; working volume, 1 liter) in differentiation medium (Iscove's modified Dulbecco's medium with GlutaMAX, supplemented with 20% [vol/vol] fetal bovine serum, 1% [vol/vol] nonessential amino acids, 1% [vol/vol] penicillin/streptomycin, and 50 μM β‐mercaptoethanol [all from Invitrogen, Carlsbad, CA, http://www.invitrogen.com]).…”

Human pluripotent stem cells: Prospects and challenges as a source of cardiomyocytes for in vitro modeling and cell-based cardiac repair

“…However, the efficiency of differentiation of human PSCs in suspension systems has been variable [201–204]. Correia and colleagues differentiated human iPSCs to cardiomyocytes using the Cellbag™-WAVE bioreactor (GE Healthcare) in 4% oxygen, rather than continuous or intermittent agitation in a stirred-tank bioreactor [205]. They reported obtaining a purity of ~75% and an impressive yield of 60 cardiomyocytes per input undifferentiated cell.…”