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Robust Generation of Cardiomyocytes from Human iPS Cells Requires Precise Modulation of BMP and WNT Signaling.

Kadari A, Mekala S, Wagner N, Malan D, Köth J, Doll K, Stappert L, Eckert D, Peitz M, Matthes J, Sasse P, Herzig S, Brüstle O, Ergün S, Edenhofer F - Stem Cell Rev (2015)

Bottom Line: In particular we demonstrate cardiomyocyte differentiation within 15 days with an efficiency of up to 95 % as judged by flow cytometry staining against cardiac troponin T.Cardiomyocytes derived were functionally validated by alpha-actinin staining, transmission electron microscopy as well as electrophysiological analysis.We expect our protocol to provide a robust basis for scale-up production of functional iPS cell-derived cardiomyocytes that can be used for cell replacement therapy and disease modeling.

View Article: PubMed Central - PubMed

Affiliation: Stem Cell and Regenerative Medicine Group, Institute of Anatomy and Cell Biology, University of Würzburg, 97070, Würzburg, Germany.

ABSTRACT
Various strategies have been published enabling cardiomyocyte differentiation of human induced pluripotent stem (iPS) cells. However the complex nature of signaling pathways involved as well as line-to-line variability compromises the application of a particular protocol to robustly obtain cardiomyocytes from multiple iPS lines. Hence it is necessary to identify optimized protocols with alternative combinations of specific growth factors and small molecules to enhance the robustness of cardiac differentiation. Here we focus on systematic modulation of BMP and WNT signaling to enhance cardiac differentiation. Moreover, we improve the efficacy of cardiac differentiation by enrichment via lactate. Using our protocol we show efficient derivation of cardiomyocytes from multiple human iPS lines. In particular we demonstrate cardiomyocyte differentiation within 15 days with an efficiency of up to 95 % as judged by flow cytometry staining against cardiac troponin T. Cardiomyocytes derived were functionally validated by alpha-actinin staining, transmission electron microscopy as well as electrophysiological analysis. We expect our protocol to provide a robust basis for scale-up production of functional iPS cell-derived cardiomyocytes that can be used for cell replacement therapy and disease modeling.

No MeSH data available.


Related in: MedlinePlus

Optimization of myocardial induction of human iPS line (iLB-C-50-s9). a RT PCR analysis to assess the expression of T brachyury at day 2 of cardiac induction using different conditions, namely Ch, B + Ch and A + B + Ch b Immunostaining using cardiac precursors maker ISL1 at day 5 of cardiac differentiation using small molecule combination (B + Ch). Scale bar: 100 μM c Flow cytometry analysis of cardiac-specific troponin T staining at day 15 of cardiac differentiation showed 21.4 and 92.5 % of cTNT positive cells in the case of only CHIR99021/XAV939, and a combination of BMP4 with CHIR99021/XAV939, respectively. Abbreviations: T T-brachyury, BA beta-actin, Ch CHIR99021, B BMP4, A Activin A, NC negative control; cTNT cardiac troponin T
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Fig1: Optimization of myocardial induction of human iPS line (iLB-C-50-s9). a RT PCR analysis to assess the expression of T brachyury at day 2 of cardiac induction using different conditions, namely Ch, B + Ch and A + B + Ch b Immunostaining using cardiac precursors maker ISL1 at day 5 of cardiac differentiation using small molecule combination (B + Ch). Scale bar: 100 μM c Flow cytometry analysis of cardiac-specific troponin T staining at day 15 of cardiac differentiation showed 21.4 and 92.5 % of cTNT positive cells in the case of only CHIR99021/XAV939, and a combination of BMP4 with CHIR99021/XAV939, respectively. Abbreviations: T T-brachyury, BA beta-actin, Ch CHIR99021, B BMP4, A Activin A, NC negative control; cTNT cardiac troponin T

Mentions: Since cardiomyocyte differentiation critically depends on WNT signaling, our first step was to screen molecules with which one can tightly control WNT signaling in iPS cells and their progeny, both in an agonistic and antagonistic manner, respectively. In order to quantitatively assess the level of WNT signaling, we employed a WNT reporter cell line and assessed the functionality of candidate molecules to modulate WNT signaling. In order to identify potent WNT activator, we used CHIR99021 (designated CHIR hereafter) and BIO. We found that 5 μM CHIR99021 (designated CHIR hereafter) strongly activates WNT signaling (data not shown) while BIO appeared toxic to the cells. To identify potent molecules with potent WNT inhibition, we screened previously described molecules XAV939, IWR1, KY02111 and WNT-C59. According to this analysis XAV939 and IWR1 showed strongest WNT inhibition without causing excessive cell death. Hence we decided to use CHIR as WNT activator and XAV939 or IWR1 as WNT inhibitor during the subsequent experiments. In order to achieve efficient cardiovascular induction we decided to formulate different combinations of growth factors and small molecules modulating important signaling pathways such as TGFβ (Activin A, BMP4) and FGF (FGF2) as well as WNT (CHIR, XAV939, IWR1) [15]. As a quick read out for cardiovascular induction we decided to check the expression of T-brachyury at day two (Fig. 1a) and Isl1 at day 5 (Fig. 1b) of differentiation. As an end-point analysis we monitored the capability of cultures to exhibit spontaneously beating patches in a semi-quantitative manner (Suppl. Tabl. 1-3). Comprehensive quantification of cardiac differentiation was carried out by flow cytometry analysis using cTNT specific antibodies (Fig. 1c). According to these analyses we found that the combination of BMP4 (25 ng/ml) and CHIR (5 μM) strongly enhanced expression of T-brachyury whereas additional application of Activin A had no effect (Fig. 1a). Neither increasing the CHIR concentration nor extending the incubation period beyond 48 h had a beneficial effect (Suppl. Tabl. 1). Applying 25 ng/ml BMP4 turned out to be the optimal concentration since higher and lower concentrations, respectively, reduced the number of beating patches (Suppl. Tabl. 2). After an initial phase of WNT activiation the precise timing of WNT inhibition is critical. Our data shows that application of both WNT inhibitors, XAV939 or IWR1, between day 3 and 8 results in optimal cardiac differentiation (Suppl. Tabl. 3). After day 5 of differentiation cells showed strong expression of the early cardiac marker ISL1 in almost every cell (Fig. 1b). Using these optimized conditions we generated cardiomyocyte-like cells of up to 95 % purity from human iPS line (iLB-C-50-s9) at day 12 of differentiation as judged by flow cytometry analysis using cTNT specific antibodies (Fig. 1c).Fig. 1


Robust Generation of Cardiomyocytes from Human iPS Cells Requires Precise Modulation of BMP and WNT Signaling.

Kadari A, Mekala S, Wagner N, Malan D, Köth J, Doll K, Stappert L, Eckert D, Peitz M, Matthes J, Sasse P, Herzig S, Brüstle O, Ergün S, Edenhofer F - Stem Cell Rev (2015)

Optimization of myocardial induction of human iPS line (iLB-C-50-s9). a RT PCR analysis to assess the expression of T brachyury at day 2 of cardiac induction using different conditions, namely Ch, B + Ch and A + B + Ch b Immunostaining using cardiac precursors maker ISL1 at day 5 of cardiac differentiation using small molecule combination (B + Ch). Scale bar: 100 μM c Flow cytometry analysis of cardiac-specific troponin T staining at day 15 of cardiac differentiation showed 21.4 and 92.5 % of cTNT positive cells in the case of only CHIR99021/XAV939, and a combination of BMP4 with CHIR99021/XAV939, respectively. Abbreviations: T T-brachyury, BA beta-actin, Ch CHIR99021, B BMP4, A Activin A, NC negative control; cTNT cardiac troponin T
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4493626&req=5

Fig1: Optimization of myocardial induction of human iPS line (iLB-C-50-s9). a RT PCR analysis to assess the expression of T brachyury at day 2 of cardiac induction using different conditions, namely Ch, B + Ch and A + B + Ch b Immunostaining using cardiac precursors maker ISL1 at day 5 of cardiac differentiation using small molecule combination (B + Ch). Scale bar: 100 μM c Flow cytometry analysis of cardiac-specific troponin T staining at day 15 of cardiac differentiation showed 21.4 and 92.5 % of cTNT positive cells in the case of only CHIR99021/XAV939, and a combination of BMP4 with CHIR99021/XAV939, respectively. Abbreviations: T T-brachyury, BA beta-actin, Ch CHIR99021, B BMP4, A Activin A, NC negative control; cTNT cardiac troponin T
Mentions: Since cardiomyocyte differentiation critically depends on WNT signaling, our first step was to screen molecules with which one can tightly control WNT signaling in iPS cells and their progeny, both in an agonistic and antagonistic manner, respectively. In order to quantitatively assess the level of WNT signaling, we employed a WNT reporter cell line and assessed the functionality of candidate molecules to modulate WNT signaling. In order to identify potent WNT activator, we used CHIR99021 (designated CHIR hereafter) and BIO. We found that 5 μM CHIR99021 (designated CHIR hereafter) strongly activates WNT signaling (data not shown) while BIO appeared toxic to the cells. To identify potent molecules with potent WNT inhibition, we screened previously described molecules XAV939, IWR1, KY02111 and WNT-C59. According to this analysis XAV939 and IWR1 showed strongest WNT inhibition without causing excessive cell death. Hence we decided to use CHIR as WNT activator and XAV939 or IWR1 as WNT inhibitor during the subsequent experiments. In order to achieve efficient cardiovascular induction we decided to formulate different combinations of growth factors and small molecules modulating important signaling pathways such as TGFβ (Activin A, BMP4) and FGF (FGF2) as well as WNT (CHIR, XAV939, IWR1) [15]. As a quick read out for cardiovascular induction we decided to check the expression of T-brachyury at day two (Fig. 1a) and Isl1 at day 5 (Fig. 1b) of differentiation. As an end-point analysis we monitored the capability of cultures to exhibit spontaneously beating patches in a semi-quantitative manner (Suppl. Tabl. 1-3). Comprehensive quantification of cardiac differentiation was carried out by flow cytometry analysis using cTNT specific antibodies (Fig. 1c). According to these analyses we found that the combination of BMP4 (25 ng/ml) and CHIR (5 μM) strongly enhanced expression of T-brachyury whereas additional application of Activin A had no effect (Fig. 1a). Neither increasing the CHIR concentration nor extending the incubation period beyond 48 h had a beneficial effect (Suppl. Tabl. 1). Applying 25 ng/ml BMP4 turned out to be the optimal concentration since higher and lower concentrations, respectively, reduced the number of beating patches (Suppl. Tabl. 2). After an initial phase of WNT activiation the precise timing of WNT inhibition is critical. Our data shows that application of both WNT inhibitors, XAV939 or IWR1, between day 3 and 8 results in optimal cardiac differentiation (Suppl. Tabl. 3). After day 5 of differentiation cells showed strong expression of the early cardiac marker ISL1 in almost every cell (Fig. 1b). Using these optimized conditions we generated cardiomyocyte-like cells of up to 95 % purity from human iPS line (iLB-C-50-s9) at day 12 of differentiation as judged by flow cytometry analysis using cTNT specific antibodies (Fig. 1c).Fig. 1

Bottom Line: In particular we demonstrate cardiomyocyte differentiation within 15 days with an efficiency of up to 95 % as judged by flow cytometry staining against cardiac troponin T.Cardiomyocytes derived were functionally validated by alpha-actinin staining, transmission electron microscopy as well as electrophysiological analysis.We expect our protocol to provide a robust basis for scale-up production of functional iPS cell-derived cardiomyocytes that can be used for cell replacement therapy and disease modeling.

View Article: PubMed Central - PubMed

Affiliation: Stem Cell and Regenerative Medicine Group, Institute of Anatomy and Cell Biology, University of Würzburg, 97070, Würzburg, Germany.

ABSTRACT
Various strategies have been published enabling cardiomyocyte differentiation of human induced pluripotent stem (iPS) cells. However the complex nature of signaling pathways involved as well as line-to-line variability compromises the application of a particular protocol to robustly obtain cardiomyocytes from multiple iPS lines. Hence it is necessary to identify optimized protocols with alternative combinations of specific growth factors and small molecules to enhance the robustness of cardiac differentiation. Here we focus on systematic modulation of BMP and WNT signaling to enhance cardiac differentiation. Moreover, we improve the efficacy of cardiac differentiation by enrichment via lactate. Using our protocol we show efficient derivation of cardiomyocytes from multiple human iPS lines. In particular we demonstrate cardiomyocyte differentiation within 15 days with an efficiency of up to 95 % as judged by flow cytometry staining against cardiac troponin T. Cardiomyocytes derived were functionally validated by alpha-actinin staining, transmission electron microscopy as well as electrophysiological analysis. We expect our protocol to provide a robust basis for scale-up production of functional iPS cell-derived cardiomyocytes that can be used for cell replacement therapy and disease modeling.

No MeSH data available.


Related in: MedlinePlus