Dataset Information

Non-Human Primate iPSC Generation, Cultivation, and Cardiac Differentiation under Chemically Defined Conditions.

ABSTRACT: Non-human primates (NHP) are important surrogate models for late preclinical development of advanced therapy medicinal products (ATMPs), including induced pluripotent stem cell (iPSC)-based therapies, which are also under development for heart failure repair. For effective heart repair by remuscularization, large numbers of cardiomyocytes are required, which can be obtained by efficient differentiation of iPSCs. However, NHP-iPSC generation and long-term culture in an undifferentiated state under feeder cell-free conditions turned out to be problematic. Here we describe the reproducible development of rhesus macaque (Macaca mulatta) iPSC lines. Postnatal rhesus skin fibroblasts were reprogrammed under chemically defined conditions using non-integrating vectors. The robustness of the protocol was confirmed using another NHP species, the olive baboon (Papio anubis). Feeder-free maintenance of NHP-iPSCs was essentially dependent on concurrent Wnt-activation by GSK-inhibition (Gi) and Wnt-inhibition (Wi). Generated NHP-iPSCs were successfully differentiated into cardiomyocytes using a combined growth factor/GiWi protocol. The capacity of the iPSC-derived cardiomyocytes to self-organize into contractile engineered heart muscle (EHM) was demonstrated. Collectively, this study establishes a reproducible protocol for the robust generation and culture of NHP-iPSCs, which are useful for preclinical testing of strategies for cell replacement therapies in NHP.

SUBMITTER: Stauske M

PROVIDER: S-EPMC7349583 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Non-Human Primate iPSC Generation, Cultivation, and Cardiac Differentiation under Chemically Defined Conditions.

Stauske Michael M Rodriguez Polo Ignacio I Haas Wadim W Knorr Debbra Yasemin DY Borchert Thomas T Streckfuss-Bömeke Katrin K Dressel Ralf R Bartels Iris I Tiburcy Malte M Zimmermann Wolfram-Hubertus WH Behr Rüdiger R

Cells 20200529 6

Non-human primates (NHP) are important surrogate models for late preclinical development of advanced therapy medicinal products (ATMPs), including induced pluripotent stem cell (iPSC)-based therapies, which are also under development for heart failure repair. For effective heart repair by remuscularization, large numbers of cardiomyocytes are required, which can be obtained by efficient differentiation of iPSCs. However, NHP-iPSC generation and long-term culture in an undifferentiated state unde ...[more]

PMID: 32485910

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Project description:Cardiovascular-related disorders are a significant worldwide health problem. Cardiovascular disease (CVD) is the leading cause of death in developed countries, making up a third of the mortality rate in the US1. Congenital heart defects (CHD) affect ~1% of all live births2, making it the common birth defect in humans. Current technologies provide some insight into how these disorders originate but are limited in their ability to provide a complete overview of disease pathogenesis and progression due to their lack of physiological complexity. There is a pressing need to develop more faithful organ-like platforms recapitulating complex in vivo phenotypes to study human development and disease in vitro. Here, we report the most faithful in vitro organoid model of human cardiovascular development to date using human pluripotent stem cells (hPSCs). Our protocol is highly efficient, scalable, shows high reproducibility and is compatible with high-throughput approaches. Furthermore, our hHOs showed very high similarity to human fetal hearts, both morphologically and in cell-type complexity. hHOs were differentiated using a two-step manipulation of Wnt signaling using chemical inhibitors and growth factors in completely defined media and culture conditions. Organoids were successfully derived from multiple independent hPSCs lines with very similar efficiency. hHOs started beating at ~6 days, were mostly spherical and grew up to ~1 mm in diameter by day 15 of differentiation. hHOs developed sophisticated, interconnected internal chambers and confocal analysis for cardiac markers revealed the presence of all major cardiac lineages, including cardiomyocytes (TNNT2+), epicardial cells (WT1+, TJP+), cardiac fibroblasts (THY1+, VIM+), endothelial cells (PECAM1+), and endocardial cells (NFATC1+). Morphologically, hHOs developed well-defined epicardial and adjacent myocardial regions and presented a distinct vascular plexus as well as endocardial-lined microchambers. RNA-seq time-course analysis of hHOs, monolayer differentiated iPSCs and fetal human hearts revealed that hHOs recapitulate human fetal heart tissue development better than previously described differentiation protocols. hHOs allow higher-order interaction of distinct heart tissues for the first time, and display biologically relevant physical and topographical 3D cues that closely resemble the human fetal heart. Our model constitutes a powerful novel tool for discovery and translational studies in human cardiac development and disease.

Dataset Information

Non-Human Primate iPSC Generation, Cultivation, and Cardiac Differentiation under Chemically Defined Conditions.

Publications

Non-Human Primate iPSC Generation, Cultivation, and Cardiac Differentiation under Chemically Defined Conditions.

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