The influence of physiological matrix conditions on permanent culture of induced pluripotent stem cell-derived cardiomyocytes

Heras-Bautista, Carlos O; Katsen-Globa, Alisa; Schloerer, Nils; Dieluweit, Sabine; Aziz, Osama M. Abd El; Peinkofer, Gabriel; Attia, Walid A.; Khalil, Markus; Brockmeier, Konrad; Hescheler, Jürgen; Pfannkuche, Kurt

doi:10.1016/j.biomaterials.2014.05.027

Cited by 38 publications

(38 citation statements)

References 34 publications

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“…Sarcomere lengths in trained ES-CMs (6 weeks), were comparable to the typical sarcomere lengths of adult mouse CMs described in the literature [36,37] (Figure 2i). Meanwhile, similar to results reported for neonatal rat CMs, after 6 weeks of culture on PS, staining for α-actinin was very weak, consistent with loss of sarcomere integrity [38]. Finally, we assayed trained ES-CMs for the presence of t-tubules.…”

Section: Further Characterization Of Mouse Es-cms Undergoing 2d Auxotsupporting

confidence: 82%

Sub-Confluent Culture of Mouse Embryonic Stem Cell-derived Ventricular Cardiomyocytes On & In Gels - Enhancement of Maturation Phenotype Relative to Tissue Culture Polystyrene via Enabling of Auxotonic Contraction

Mittal

Atmanli

et al. 2017

Preprint

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Cardiac myocytes (CMs) obtained by differentiating embryonic stem cells (ES-CMs) have an immature phenotype and promoting the maturation of such PSC-derived cardiomyocytes remains a major limitation in the development of stem cell models of human cardiovascular disease. We cultured murine ES-CMs in a collagen gel (3D) at a low density, or on collagen-coated polystyrene (2D) and found that 3D culture results in dramatic improvement of the maturation rate and end-state gene expression of ES-CMs. There are two main differences between CMs cultured in 3D versus 2D; in 3D the mechanical stiffness of the environment is lower, enabling auxotonic instead of isometric contraction; and, in 3D the amount of cell-cell interaction is higher. To isolate the contributions, we first cultured ES-CMs on gels (2D substrates) that are softer than tissue culture plastic, enabling auxotonic contraction, while controlling for dimensionality and cell interaction. This indeed promoted a mature gene expression profile, while also enabling the maintenance of sarcomeres. Next, we determined that increased cell-cell interaction inhibits the mature gene expression of ES-CMs. Thus, auxotonic contraction is the likely mechanism for improved gene expression in sub-confluent 3D culture. However, 2D auxotonic contraction may offer a suitable compromise between obtaining enhanced gene expression and morphology. After 6 weeks of culture on gels, via Di-8-ANEPPs and WGA staining we also detected CMs forming a t-tubule network. Collectively these results demonstrate that 3D and 2D cultures that enable auxotonic contraction enhance aspects of the maturation of ES-CMs.

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Section: Further Characterization Of Mouse Es-cms Undergoing 2d Auxotsupporting

confidence: 82%

Sub-Confluent Culture of Mouse Embryonic Stem Cell-derived Ventricular Cardiomyocytes On & In Gels - Enhancement of Maturation Phenotype Relative to Tissue Culture Polystyrene via Enabling of Auxotonic Contraction

Mittal

Atmanli

et al. 2017

Preprint

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“…Adopting the current 2D CellDrum technology to a 3D model may bring it closer to the three-dimensional environment in ventricular tissue. In addition, the CellDrum benefits from the fact that myocytes are not grown on a stiff substrate but on highly flexible membranes mimicking to some extend the natural compliance of soft tissue [70]. Yet, the highly anisotropic three dimensional environment and several other factors still rely on full organ tests suffering from the drawback of a non-scientific experimental setup -the heart of an animal.…”

Section: Discussionmentioning

confidence: 99%

Mechano-Pharmacological Characterization of Cardiomyocytes Derived from Human Induced Pluripotent Stem Cells

Goßmann

Frotscher

Linder³

et al. 2016

Cell Physiol Biochem

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Background/Aims: Common systems for the quantification of cellular contraction rely on animal-based models, complex experimental setups or indirect approaches. The herein presented CellDrum technology for testing mechanical tension of cellular monolayers and thin tissue constructs has the potential to scale-up mechanical testing towards medium-throughput analyses. Using hiPS-Cardiac Myocytes (hiPS-CMs) it represents a new perspective of drug testing and brings us closer to personalized drug medication. Methods: In the present study, monolayers of self-beating hiPS-CMs were grown on ultra-thin circular silicone membranes and deflect under the weight of the culture medium. Rhythmic contractions of the hiPS-CMs induced variations of the membrane deflection. The recorded contraction-relaxation-cycles were analyzed with respect to their amplitudes, durations, time integrals and frequencies. Besides unstimulated force and tensile stress, we investigated the effects of agonists and antagonists acting on Ca²⁺ channels (S-Bay K8644/verapamil) and Na⁺ channels (veratridine/lidocaine). Results: The measured data and simulations for pharmacologically unstimulated contraction resembled findings in native human heart tissue, while the pharmacological dose-response curves were highly accurate and consistent with reference data. Conclusion: We conclude that the combination of the CellDrum with hiPS-CMs offers a fast, facile and precise system for pharmacological, toxicological studies and offers new preclinical basic research potential.

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“…Cardiac differentiation of miPS and mES cells was performed in mass culture as previously described [2] with the addition of ascorbic acid (3 mg/ml) from day 0 to day 3 of differentiation [21]. Colonies of undifferentiated cells (Fig.…”

Section: Methodsmentioning

confidence: 99%

Functional Expression and Regulation of Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels (HCN) in Mouse iPS Cell-derived Cardiomyocytes afterUTF1-Neo Selection

Semmler

Lehmann

Pfannkuche

et al. 2014

Cell Physiol Biochem

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Background/Aims: In vitro reprogramming of somatic cells holds great potential to serve as an autologous source of cells for tissue repair. However, major difficulties in achieving this potential include obtaining homogeneous and stable cells for transplantation. High electrical activity of cells such as cardiomyocytes (CMs) is crucial for both, safety and efficiency of cell replacement therapy. Moreover, the function of the cardiac pacemaker is controlled by the activities of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. Here we have examined changes in HCN gene expression and function during cardiomyogenesis. Methods: We differentiated murine iPS cells selected by an undifferentiated transcription factor 1 (UTF1) -promoter-driven G418 resistance to CMs in vitro and characterized them by RT-PCR, immunocytochemistry, and electrophysiology. Results: As key cardiac markers alpha-actinin and cardiac troponin T could be identified in derived CMs. Immunocytochemical staining of CMs showed the presence of all HCN subunits (HCN1-4). Electrophysiology experiments revealed developmental changes of action potentials and I_f currents as well as functional hormonal regulation and sensitivity to I_f channel blockers. Conclusion: We conclude that iPS cells derived from UTF-selection give rise to functional CMs in vitro, with established hormonal regulation pathways and functionally expressed I_f current in a development-dependent manner; and have all phenotypes with the pacemaker as predominant subtype. This might be of great importance for transplantation purposes.

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The influence of physiological matrix conditions on permanent culture of induced pluripotent stem cell-derived cardiomyocytes

Cited by 38 publications

References 34 publications

Sub-Confluent Culture of Mouse Embryonic Stem Cell-derived Ventricular Cardiomyocytes On & In Gels - Enhancement of Maturation Phenotype Relative to Tissue Culture Polystyrene via Enabling of Auxotonic Contraction

Sub-Confluent Culture of Mouse Embryonic Stem Cell-derived Ventricular Cardiomyocytes On & In Gels - Enhancement of Maturation Phenotype Relative to Tissue Culture Polystyrene via Enabling of Auxotonic Contraction

Mechano-Pharmacological Characterization of Cardiomyocytes Derived from Human Induced Pluripotent Stem Cells

Functional Expression and Regulation of Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels (HCN) in Mouse iPS Cell-derived Cardiomyocytes afterUTF1-Neo Selection

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