Numerous protocols of cardiac differentiation have been established by essentially focusing on specific growth factors on human pluripotent stem cell (hPSC) differentiation efficiency. However, the optimal environmental factors to obtain cardiac myocytes in network are still unclear. The mesoderm germ layer differentiation is known to be enhanced by low oxygen exposure. Here, we hypothesized that low oxygen exposure enhances the molecular and functional maturity of the cardiomyocytes. We aimed at comparing the molecular and functional consequences of low (5% O2 or LOE) and high oxygen exposure (21% O2 or HOE) on cardiac differentiation of hPSCs in 2D- and 3D-based protocols. hPSC-CMs were differentiated through both the 2D (monolayer) and 3D (embryoid body) protocols using several lines. Cardiac marker expression and cell morphology were assessed. The mitochondrial localization and metabolic properties were evaluated. The intracellular Ca2+ handling and contractile properties were also monitored. The 2D cardiac monolayer can only be differentiated in HOE. The 3D cardiac spheroids containing hPSC-CMs in LOE further exhibited cardiac markers, hypertrophy, steadier SR Ca2+ release properties revealing a better SR Ca2+ handling, and enhanced contractile force. Preserved distribution of mitochondria and similar oxygen consumption by the mitochondrial respiratory chain complexes were also observed. Our results brought evidences that LOE is moderately beneficial for the 3D cardiac spheroids with hPSC-CMs exhibiting further maturity. In contrast, the 2D cardiac monolayers strictly require HOE.

1st Department of Internal Medicine Cardioangiology St Anne's Hospital Masaryk University 65691 Brno Czech Republic

CEITEC Masaryk University 62500 Brno Czech Republic

Department of Biology Faculty of Medicine Masaryk University 62500 Brno Czech Republic

International Clinical Research Center St Anne's University Hospital Brno 65691 Brno Czech Republic

Montpellier Ressources Imagerie BioCampus Montpellier CNRS INSERM University of Montpellier 34000 Montpellier France

PhyMedExp INSERM University of Montpellier CNRS 34000 Montpellier France

Zobrazit více v PubMed

Acimovic I., Vilotic A., Pesl M., Lacampagne A., Dvorak P., Rotrekl V., Meli A.C. Human pluripotent stem cell-derived cardiomyocytes as research and therapeutic tools. BioMed Res. Int. 2014 doi: 10.1155/2014/512831. PubMed DOI PMC

Simon M.C., Keith B. The role of oxygen availability in embryonic development and stem cell function. Nat. Rev. Mol. Cell Biol. 2008;9:285–296. doi: 10.1038/nrm2354. PubMed DOI PMC

Horton R.E., Auguste D.T. Synergistic effects of hypoxia and extracellular matrix cues in cardiomyogenesis. Biomaterials. 2012;33:6313–6319. doi: 10.1016/j.biomaterials.2012.05.063. PubMed DOI

Leri A., Rota M., Hosoda T., Goichberg P., Anversa P. Cardiac stem cell niches. Stem Cell Res. 2014;13:631–646. doi: 10.1016/j.scr.2014.09.001. PubMed DOI PMC

Korski K.I., Kubli D.A., Wang B.J., Khalafalla F.G., Monsanto M.M., Firouzi F., Echeagaray O.H., Kim T., Adamson R.M., Dembitsky W.P., et al. Hypoxia prevents mitochondrial dysfunction and senescence in human c-kit+ cardiac progenitor cells. Stem Cells. 2019;37:555–567. doi: 10.1002/stem.2970. PubMed DOI PMC

Mummery Christine L., Zhang J., Elizabeth S.N., David A.E., Andrew G.E., Timothy J.K. Differentiation of human embryonic stem cells and induced pluripotent stem cells to cardiomyocytes. Circ. Res. 2012;111:344–358. doi: 10.1161/CIRCRESAHA.110.227512. PubMed DOI PMC

Niebruegge S., Bauwens C.L., Peerani R., Thavandiran N., Masse S., Sevaptisidis E., Nanthakumar K., Woodhouse K., Husain M., Kumacheva E., et al. Generation of human embryonic stem cell-derived mesoderm and cardiac cells using size-specified aggregates in an oxygen-controlled bioreactor. Biotechnol. Bioeng. 2009;102:493–507. doi: 10.1002/bit.22065. PubMed DOI

Kudova J., Prochazkova J., Vasicek O., Perecko T., Sedlackova M., Pesl M., Pachernik J., Kubala L. Hif-1alpha deficiency attenuates the cardiomyogenesis of mouse embryonic stem cells. PLoS ONE. 2016;11:e0158358. doi: 10.1371/journal.pone.0158358. PubMed DOI PMC

Krishnan J., Ahuja P., Bodenmann S., Knapik D., Perriard E., Krek W., Perriard J.-C. Essential role of developmentally activated hypoxia-inducible factor 1alpha for cardiac morphogenesis and function. Circ. Res. 2008;103:1139–1146. doi: 10.1161/01.RES.0000338613.89841.c1. PubMed DOI

Iyer N.V., Kotch L.E., Agani F., Leung S.W., Laughner E., Wenger R.H., Gassmann M., Gearhart J.D., Lawler A.M., Yu A.Y., et al. Cellular and developmental control of o2 homeostasis by hypoxia-inducible factor 1 alpha. Genes Dev. 1998;12:149–162. doi: 10.1101/gad.12.2.149. PubMed DOI PMC

Medley T.L., Furtado M., Lam N.T., Idrizi R., Williams D., Verma P.J., Costa M., Kaye D.M. Effect of oxygen on cardiac differentiation in mouse ips cells: Role of hypoxia inducible factor-1 and wnt/beta-catenin signaling. PLoS ONE. 2013;8:e80280. doi: 10.1371/journal.pone.0080280. PubMed DOI PMC

Acimovic I., Refaat M.M., Moreau A., Salykin A., Reiken S., Sleiman Y., Souidi M., Pribyl J., Kajava A.V., Richard S., et al. Post-translational modifications and diastolic calcium leak associated to the novel ryr2-d3638a mutation lead to cpvt in patient-specific hipsc-derived cardiomyocytes. J. Clin. Med. 2018;7:423. doi: 10.3390/jcm7110423. PubMed DOI PMC

Pesl M., Acimovic I., Pribyl J., Hezova R., Vilotic A., Fauconnier J., Vrbsky J., Kruzliak P., Skladal P., Kara T., et al. Forced aggregation and defined factors allow highly uniform-sized embryoid bodies and functional cardiomyocytes from human embryonic and induced pluripotent stem cells. Heart Vessel. 2014;29:834–846. doi: 10.1007/s00380-013-0436-9. PubMed DOI

Dubois N.C., Craft A.M., Sharma P., Elliott D.A., Stanley E.G., Elefanty A.G., Gramolini A., Keller G. Sirpa is a specific cell-surface marker for isolating cardiomyocytes derived from human pluripotent stem cells. Nat. Biotechnol. 2011;29:1011–1018. doi: 10.1038/nbt.2005. PubMed DOI PMC

Kattman S.J., Witty A.D., Gagliardi M., Dubois N.C., Niapour M., Hotta A., Ellis J., Keller G. Stage-specific optimization of activin/nodal and bmp signaling promotes cardiac differentiation of mouse and human pluripotent stem cell lines. Cell Stem. Cell. 2011;8:228–240. doi: 10.1016/j.stem.2010.12.008. PubMed DOI

Burridge P.W., Thompson S., Millrod M.A., Weinberg S., Yuan X., Peters A., Mahairaki V., Koliatsos V.E., Tung L., Zambidis E.T. A universal system for highly efficient cardiac differentiation of human induced pluripotent stem cells that eliminates interline variability. PLoS ONE. 2011;6:e18293. doi: 10.1371/journal.pone.0018293. PubMed DOI PMC

Palmer J.W., Tandler B., Hoppel C.L. Biochemical differences between subsarcolemmal and interfibrillar mitochondria from rat cardiac muscle: Effects of procedural manipulations. Arch. Biochem. Biophys. 1985;236:691–702. doi: 10.1016/0003-9861(85)90675-7. PubMed DOI

Chen Y., Amende I., Hampton T.G., Yang Y., Ke Q., Min J.-Y., Xiao Y.-F., Morgan J.P. Vascular endothelial growth factor promotes cardiomyocyte differentiation of embryonic stem cells. Am. J. Physiol. Heart Circ. Physiol. 2006;291:H1653–H1658. doi: 10.1152/ajpheart.00363.2005. PubMed DOI

Millauer B., Wizigmann-Voos S., Schnurch H., Martinez R., Moller N.P., Risau W., Ullrich A. High affinity vegf binding and developmental expression suggest flk-1 as a major regulator of vasculogenesis and angiogenesis. Cell. 1993;72:835–846. doi: 10.1016/0092-8674(93)90573-9. PubMed DOI

Louzier V., Raffestin B., Leroux A., Branellec D., Caillaud J.M., Levame M., Eddahibi S., Adnot S. Role of vegf-b in the lung during development of chronic hypoxic pulmonary hypertension. Am. J. Physiol. Lung Cell. Mol. Physiol. 2003;284:L926–L937. doi: 10.1152/ajplung.00247.2002. PubMed DOI

Bellomo D., John P.H., Ginters U.S., Carol A.P., Penny S.T., Gartside M., Mould A., Marian M.C., Ian D.T., Sean M.G., et al. Mice lacking the vascular endothelial growth factor-b gene (vegfb) have smaller hearts, dysfunctional coronary vasculature, and impaired recovery from cardiac ischemia. Circ. Res. 2000;86:e29–e35. doi: 10.1161/01.RES.86.2.e29. PubMed DOI

Cameron C.M., Harding F., Hu W.-S., Kaufman D.S. Activation of hypoxic response in human embryonic stem cell–derived embryoid bodies. Exp. Biol. Med. 2008;233:1044–1057. doi: 10.3181/0709-RM-263. PubMed DOI

Hu D., Linders A., Yamak A., Correia C., Kijlstra J.D., Garakani A., Xiao L., Milan D.J., van der Meer P., Serra M., et al. Metabolic maturation of human pluripotent stem cell-derived cardiomyocytes by inhibition of hif1α and ldha. Circ. Res. 2018;123:1066–1079. doi: 10.1161/CIRCRESAHA.118.313249. PubMed DOI PMC

Eschenhagen T., Mummery C., Knollmann B.C. Modelling sarcomeric cardiomyopathies in the dish: From human heart samples to ipsc cardiomyocytes. Cardiovasc. Res. 2015;105:424–438. doi: 10.1093/cvr/cvv017. PubMed DOI PMC

Ronaldson-Bouchard K., Yeager K., Teles D., Chen T., Ma S., Song L., Morikawa K., Wobma H.M., Vasciaveo A., Ruiz E.C., et al. Engineering of human cardiac muscle electromechanically matured to an adult-like phenotype. Nat. Protoc. 2019;14:2781–2817. doi: 10.1038/s41596-019-0189-8. PubMed DOI PMC

Kim D.H., Lipke E.A., Kim P., Cheong R., Thompson S., Delannoy M., Suh K.Y., Tung L., Levchenko A. Nanoscale cues regulate the structure and function of macroscopic cardiac tissue constructs. Proc. Natl. Acad. Sci. USA. 2010;107:565–570. doi: 10.1073/pnas.0906504107. PubMed DOI PMC

Ribeiro M.C., Tertoolen L.G., Guadix J.A., Bellin M., Kosmidis G., D’Aniello C., Monshouwer-Kloots J., Goumans M.J., Wang Y.L., Feinberg A.W., et al. Functional maturation of human pluripotent stem cell derived cardiomyocytes in vitro--correlation between contraction force and electrophysiology. Biomaterials. 2015;51:138–150. doi: 10.1016/j.biomaterials.2015.01.067. PubMed DOI

Parikh S.S., Blackwell D.J., Gomez-Hurtado N., Frisk M., Wang L., Kim K., Dahl C.P., Fiane A., Tonnessen T., Kryshtal D.O., et al. Thyroid and glucocorticoid hormones promote functional t-tubule development in human-induced pluripotent stem cell-derived cardiomyocytes. Circ. Res. 2017;121:1323–1330. doi: 10.1161/CIRCRESAHA.117.311920. PubMed DOI PMC

Jeziorowska D., Fontaine V., Jouve C., Villard E., Dussaud S., Akbar D., Letang V., Cervello P., Itier J.-M., Pruniaux M.-P., et al. Differential sarcomere and electrophysiological maturation of human ipsc-derived cardiac myocytes in monolayer vs. Aggregation-based differentiation protocols. Int. J. Mol. Sci. 2017;18:1173. doi: 10.3390/ijms18061173. PubMed DOI PMC

Bedada F.B., Chan S.S.K., Metzger S.K., Zhang L., Zhang J., Garry D.J., Kamp T.J., Kyba M., Metzger J.M. Acquisition of a quantitative, stoichiometrically conserved ratiometric marker of maturation status in stem cell-derived cardiac myocytes. Stem. Cell Rep. 2014;3:594–605. doi: 10.1016/j.stemcr.2014.07.012. PubMed DOI PMC

Qu C., Puttonen K.A., Lindeberg H., Ruponen M., Hovatta O., Koistinaho J., Lammi M.J. Chondrogenic differentiation of human pluripotent stem cells in chondrocyte co-culture. Int. J. Biochem. Cell Biol. 2013;45:1802–1812. doi: 10.1016/j.biocel.2013.05.029. PubMed DOI

Pesl M., Pribyl J., Acimovic I., Vilotic A., Jelinkova S., Salykin A., Lacampagne A., Dvorak P., Meli A.C., Skladal P., et al. Atomic force microscopy combined with human pluripotent stem cell derived cardiomyocytes for biomechanical sensing. Biosens. Bioelectron. 2016;85:751–757. doi: 10.1016/j.bios.2016.05.073. PubMed DOI

Pribyl J., Pešl M., Caluori G., Acimovic I., Jelinkova S., Dvorak P., Skladal P., Rotrekl V. Biomechanical characterization of human pluripotent stem cell-derived cardiomyocytes by use of atomic force microscopy. In: Santos N.C., Carvalho F.A., editors. Atomic Force Microscopy: Methods and Protocols. Springer; New York, NY, USA: 2019. pp. 343–353. PubMed

Sleiman Y., Souidi M., Kumar R., Yang E., Jaffré F., Zhou T., Bernardin A., Reiken S., Cazorla O., Kajava A.V., et al. Modeling polymorphic ventricular tachycardia at rest using patient-specific induced pluripotent stem cell-derived cardiomyocytes. EBioMedicine. 2020;60:103024. doi: 10.1016/j.ebiom.2020.103024. PubMed DOI PMC

Jelinkova S., Vilotic A., Pribyl J., Aimond F., Salykin A., Acimovic I., Pesl M., Caluori G., Klimovic S., Urban T., et al. Dmd pluripotent stem cell derived cardiac cells recapitulate in vitro human cardiac pathophysiology. Front. Bioeng. Biotechnol. 2020;8:535. doi: 10.3389/fbioe.2020.00535. PubMed DOI PMC

Aminophylline Induces Two Types of Arrhythmic Events in Human Pluripotent Stem Cell-Derived Cardiomyocytes