title: 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 × 10(9) 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.
authors.affiliation: Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, Oeiras, 2780-157, Portugal.
authors.forename: Cláudia
authors.initials: C
authors.lastname: Correia
authors.forename: Margarida
authors.initials: M
authors.lastname: Serra
authors.forename: Nuno
authors.initials: N
authors.lastname: Espinha
authors.forename: Marcos
authors.initials: M
authors.lastname: Sousa
authors.forename: Catarina
authors.initials: C
authors.lastname: Brito
authors.forename: Karsten
authors.initials: K
authors.lastname: Burkert
authors.forename: Yunjie
authors.initials: Y
authors.lastname: Zheng
authors.forename: Jürgen
authors.initials: J
authors.lastname: Hescheler
authors.forename: Manuel J T
authors.initials: MJ
authors.lastname: Carrondo
authors.forename: Tomo
authors.initials: T
authors.lastname: Sarić
authors.forename: Paula M
authors.initials: PM
authors.lastname: Alves
mesh_terms: D000818:Animals; D019149:Bioreactors; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation / Q000502:physiology*; D002460:Cell Line; D057446:Hydrodynamics; D000860:Hypoxia / Q000503:physiopathology*; D057026:Induced Pluripotent Stem Cells / Q000502:physiology*; D051379:Mice; D032383:Myocytes, Cardiac / Q000502:physiology*