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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.


Enrichment of cardiomyocytes with sodium L-lactate. a Summary of cardiac differentiation of different human iPS lines using efficient cardiac differentiation followed by lactate enrichment. b Flow cytometry analysis of cardiac-specific troponin T staining at day 16 of cardiac differentiation of line iLB-C1-30 m-r12 showed about 63 % cTNT positive cardiomyocytes without lactate enrichment and 96 % cTNT positive cells after lactate enrichment
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Fig2: Enrichment of cardiomyocytes with sodium L-lactate. a Summary of cardiac differentiation of different human iPS lines using efficient cardiac differentiation followed by lactate enrichment. b Flow cytometry analysis of cardiac-specific troponin T staining at day 16 of cardiac differentiation of line iLB-C1-30 m-r12 showed about 63 % cTNT positive cardiomyocytes without lactate enrichment and 96 % cTNT positive cells after lactate enrichment

Mentions: After optimization of cardiac differentiation using a standard iPS line, we checked the efficacy of the devised protocol on multiple iPS lines representing different origins of cells (fibroblasts, keratinocyte and cord blood cells) as well as methods of reprogramming (Retrovirus, Lentivirus and Sendai virus) to cover the full spectrum of state-of-the-art iPS technology (see details on iPS lines used in the materials section). Although our optimized protocol gave rise to a highly enriched population of beating cells with the standard iPS cell line (del-AR1034ZIMA 001), the outcome with the other iPS lines indeed varied substantially. In fact, we obtained yields of cTNT-positive cells ranging from 33 to 92 % (Fig. 2a) demonstrating the high line-to-line variability using the basic standard protocol. In order to maximize purity of cardiomyocytes from different iPS lines to the same level, we decided to apply lactate based cardiac enrichment in the late phase of our protocol. As has been recently reported glucose-depleted, lactate-supplemented culture medium strongly selects for cardiomyocytes [32]. Since only cardiomyocytes can metabolize lactate for energy supply, other non-cardiac cells were expected to die out during this 4 days treatment resulting in higher purity of cardiomyocytes. In order to achieve this, we switched the medium at day 12 of cardiac differentiation to basal medium without glucose but supplemented with lactate. In fact when we applied lactate enrichment, we could obtain 95 % pure cTNT-positive cells from the iPS line iLB-C-30-r12 which otherwise gave about 63 % positive cardiomyocytes (Fig. 2a and b). Even the iPS line fl-AR1034ZIMA, carrying loxP-flanked reprogramming transgenes [35] and being strongly resistant towards cardiac differentiation, showed efficient enrichment from 34 to 74 % cTNT-positive cells (Fig. 2a).Fig. 2


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)

Enrichment of cardiomyocytes with sodium L-lactate. a Summary of cardiac differentiation of different human iPS lines using efficient cardiac differentiation followed by lactate enrichment. b Flow cytometry analysis of cardiac-specific troponin T staining at day 16 of cardiac differentiation of line iLB-C1-30 m-r12 showed about 63 % cTNT positive cardiomyocytes without lactate enrichment and 96 % cTNT positive cells after lactate enrichment
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4493626&req=5

Fig2: Enrichment of cardiomyocytes with sodium L-lactate. a Summary of cardiac differentiation of different human iPS lines using efficient cardiac differentiation followed by lactate enrichment. b Flow cytometry analysis of cardiac-specific troponin T staining at day 16 of cardiac differentiation of line iLB-C1-30 m-r12 showed about 63 % cTNT positive cardiomyocytes without lactate enrichment and 96 % cTNT positive cells after lactate enrichment
Mentions: After optimization of cardiac differentiation using a standard iPS line, we checked the efficacy of the devised protocol on multiple iPS lines representing different origins of cells (fibroblasts, keratinocyte and cord blood cells) as well as methods of reprogramming (Retrovirus, Lentivirus and Sendai virus) to cover the full spectrum of state-of-the-art iPS technology (see details on iPS lines used in the materials section). Although our optimized protocol gave rise to a highly enriched population of beating cells with the standard iPS cell line (del-AR1034ZIMA 001), the outcome with the other iPS lines indeed varied substantially. In fact, we obtained yields of cTNT-positive cells ranging from 33 to 92 % (Fig. 2a) demonstrating the high line-to-line variability using the basic standard protocol. In order to maximize purity of cardiomyocytes from different iPS lines to the same level, we decided to apply lactate based cardiac enrichment in the late phase of our protocol. As has been recently reported glucose-depleted, lactate-supplemented culture medium strongly selects for cardiomyocytes [32]. Since only cardiomyocytes can metabolize lactate for energy supply, other non-cardiac cells were expected to die out during this 4 days treatment resulting in higher purity of cardiomyocytes. In order to achieve this, we switched the medium at day 12 of cardiac differentiation to basal medium without glucose but supplemented with lactate. In fact when we applied lactate enrichment, we could obtain 95 % pure cTNT-positive cells from the iPS line iLB-C-30-r12 which otherwise gave about 63 % positive cardiomyocytes (Fig. 2a and b). Even the iPS line fl-AR1034ZIMA, carrying loxP-flanked reprogramming transgenes [35] and being strongly resistant towards cardiac differentiation, showed efficient enrichment from 34 to 74 % cTNT-positive cells (Fig. 2a).Fig. 2

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.