Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Jun 1;18(6):1173.
doi: 10.3390/ijms18061173.

Differential Sarcomere and Electrophysiological Maturation of Human iPSC-Derived Cardiac Myocytes in Monolayer vs. Aggregation-Based Differentiation Protocols

Dorota Jeziorowska  1   2 , Vincent Fontaine  3   4 , Charlène Jouve  5   6 , Eric Villard  7 , Sébastien Dussaud  8 , David Akbar  9 , Valérie Letang  10 , Pauline Cervello  11 , Jean-Michiel Itier  12 , Marie-Pierre Pruniaux  13 , Jean-Sébastien Hulot  14   15
Affiliations

Differential Sarcomere and Electrophysiological Maturation of Human iPSC-Derived Cardiac Myocytes in Monolayer vs. Aggregation-Based Differentiation Protocols

Dorota Jeziorowska et al. Int J Mol Sci. .

Abstract

Human induced pluripotent stem cells (iPSCs) represent a powerful human model to study cardiac disease in vitro, notably channelopathies and sarcomeric cardiomyopathies. Different protocols for cardiac differentiation of iPSCs have been proposed either based on embroid body formation (3D) or, more recently, on monolayer culture (2D). We performed a direct comparison of the characteristics of the derived cardiomyocytes (iPSC-CMs) on day 27 ± 2 of differentiation between 3D and 2D differentiation protocols with two different Wnt-inhibitors were compared: IWR1 (inhibitor of Wnt response) or IWP2 (inhibitor of Wnt production). We firstly found that the level of Troponin T (TNNT2) expression measured by FACS was significantly higher for both 2D protocols as compared to the 3D protocol. In the three methods, iPSC-CM show sarcomeric structures. However, iPSC-CM generated in 2D protocols constantly displayed larger sarcomere lengths as compared to the 3D protocol. In addition, mRNA and protein analyses reveal higher cTNi to ssTNi ratios in the 2D protocol using IWP2 as compared to both other protocols, indicating a higher sarcomeric maturation. Differentiation of cardiac myocytes with 2D monolayer-based protocols and the use of IWP2 allows the production of higher yield of cardiac myocytes that have more suitable characteristics to study sarcomeric cardiomyopathies.

Keywords: cardiomyocytes; cardiomyopathies; differentiation; induced pluripotent stem cells; sarcomere.

PubMed Disclaimer

Conflict of interest statement

Jean-Sébastien Hulot and Eric Villard declare a research grant to the institution from Sanofi. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

Figures

Figure 1
Figure 1
Comparison of protocols. (A) Schematic representation of differentiation protocols; blebbistin = blebbistatin (B) Schematic overview of the canonical Wnt pathway and the targets of the Wnt inhibitors used; (C) Aspect of differentiated iPSCs on day 30 in transmitted light; (D) Troponin expression at day 27 ± 2 analyzed by flow cytometry. Typical flow cytometry plots show negative IgG control in blue and cTnT+ cells in red (E) Quantification (box-whisker plots showing minimum, first quartile, median, third quartile and maximum) of cardiac troponin T expression measured by flow cytometry in the three compared protocols from 11 to 16 differentiations with 3 different clones. *** p < 0.001.
Figure 1
Figure 1
Comparison of protocols. (A) Schematic representation of differentiation protocols; blebbistin = blebbistatin (B) Schematic overview of the canonical Wnt pathway and the targets of the Wnt inhibitors used; (C) Aspect of differentiated iPSCs on day 30 in transmitted light; (D) Troponin expression at day 27 ± 2 analyzed by flow cytometry. Typical flow cytometry plots show negative IgG control in blue and cTnT+ cells in red (E) Quantification (box-whisker plots showing minimum, first quartile, median, third quartile and maximum) of cardiac troponin T expression measured by flow cytometry in the three compared protocols from 11 to 16 differentiations with 3 different clones. *** p < 0.001.
Figure 2
Figure 2
High content cell imaging to analyze the cell size, nucleation and morphology using the cTnT and Dapi staining. (A) Design of the automated cellular imaging high content analysis; (B) Composite image of the detection of cTnT+ and Dapi+ cells. Detection setting for total, round and long cells (C) Cell surface comparison at day 27 ± 2. N > 1500 cells from at least three different differentiations with three different iPSC clones, **** p < 0.0001; (D) Distribution of cell surface area in generated cTNT+ cells from the 2D-IWR1 (green) and 2D-IWP2 (blue); (E) Frequency of number of nuclei in the total cells for each protocol (F) Distribution of round and long cells obtained with each protocol. ** p < 0.01.
Figure 3
Figure 3
Measurement of the sarcomere length. (A) Deconvolution microscopy imaging of hiPSC-CM in the three differentiation protocols. α-sarcomeric actinin is stained in red and nucleus in blue. The sarcomere was measured by tracing a line of 20 µm across the sarcomeres using the Fiji software. The intensity of fluorescence across the line was translated into longitudinal plots; (B) Distribution of the sarcomere sizes. n = 45 cells for each protocol, from three different differentiations and three different iPSC clones. ** p < 0.01.
Figure 4
Figure 4
Quantification of sarcomere mRNA and proteins. (A) The mRNA expression of cTni and ssTni was quantified by quantitative RT PCR. (n = 8 to 12 from four iPSC clones, p < 0.05 Kruskal-Wallis and Dunn’s Multiple Comparison Test, hVentriCM was excluded from statistical analysis); (B) Typical western blot of cTNi and ssTNi proteins; (C) Quantification of the ratio of cTNi/GAPDH on ssTNi/GAPDH proteins in hiPSC-CM generated with the 2D-IWR1 vs. 2D-IWP2 protocols. Quantifications from 4 differentiations with 4 different iPS clones, * p < 0.05, Mann–Whitney test.
See this image and copyright information in PMC

References

    1. Lahti A.L., Kujala V.J., Chapman H., Koivisto A.P., Pekkanen-Mattila M., Kerkela E., Hyttinen J., Kontula K., Swan H., Conklin B.R., et al. Model for long QT syndrome type 2 using human iPS cells demonstrates arrhythmogenic characteristics in cell culture. Dis. Model. Mech. 2012;5:220–230. doi: 10.1242/dmm.008409. - DOI - PMC - PubMed
    1. Ma D., Wei H., Zhao Y., Lu J., Li G., Sahib N.B., Tan T.H., Wong K.Y., Shim W., Wong P., et al. Modeling type 3 long QT syndrome with cardiomyocytes derived from patient-specific induced pluripotent stem cells. Int. J. Cardiol. 2013;168:5277–5286. doi: 10.1016/j.ijcard.2013.08.015. - DOI - PubMed
    1. Stillitano F., Hansen J., Kong C.W., Karakikes I., Funck-Brentano C., Geng L., Scott S., Reynier S., Wu M., Valogne Y., et al. Modeling susceptibility to drug-induced long QT with a panel of subject-specific induced pluripotent stem cells. Elife. 2017;6:e19406. doi: 10.7554/eLife.19406. - DOI - PMC - PubMed
    1. Itzhaki I., Maizels L., Huber I., Gepstein A., Arbel G., Caspi O., Miller L., Belhassen B., Nof E., Glikson M., et al. Modeling of catecholaminergic polymorphic ventricular tachycardia with patient-specific human-induced pluripotent stem cells. J. Am. Coll. Cardiol. 2012;60:990–1000. doi: 10.1016/j.jacc.2012.02.066. - DOI - PubMed
    1. Hick A., Wattenhofer-Donze M., Chintawar S., Tropel P., Simard J.P., Vaucamps N., Gall D., Lambot L., Andre C., Reutenauer L., et al. Neurons and cardiomyocytes derived from induced pluripotent stem cells as a model for mitochondrial defects in friedreich’s ataxia. Dis. Model. Mech. 2013;6:608–621. doi: 10.1242/dmm.010900. - DOI - PMC - PubMed