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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.


Ultrastructural analysis of 21-day old human iPS (del-AR1034ZIMA 001) cell-derived cardiomyocytes. a Two cells in close contact displaying sarcomer-like organization of contractile filaments. Scale bar: 1,000 nm (b–c) Higher magnification showing the presence of fascia adherens-like and gap-junctions like cellular contacts and initial sarcomeric organisation of actin and myosin filaments. Scale bar: b 1,000 nm, c 250 nm. d iPS cell-derived cardiomyocyte-like cells show sarcomer organization of contractile filaments with already identifiable A- and I-bands together with Z-lines as well as H-zones. Abbrevations: m mitochondria, N Nucleus, FA-lS Fascia adherens-like structure, GJ-lS Gap junctions-like structure, Z Z-line, H H-zone. Scale Bar: 250 nm
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Fig4: Ultrastructural analysis of 21-day old human iPS (del-AR1034ZIMA 001) cell-derived cardiomyocytes. a Two cells in close contact displaying sarcomer-like organization of contractile filaments. Scale bar: 1,000 nm (b–c) Higher magnification showing the presence of fascia adherens-like and gap-junctions like cellular contacts and initial sarcomeric organisation of actin and myosin filaments. Scale bar: b 1,000 nm, c 250 nm. d iPS cell-derived cardiomyocyte-like cells show sarcomer organization of contractile filaments with already identifiable A- and I-bands together with Z-lines as well as H-zones. Abbrevations: m mitochondria, N Nucleus, FA-lS Fascia adherens-like structure, GJ-lS Gap junctions-like structure, Z Z-line, H H-zone. Scale Bar: 250 nm

Mentions: In order to study the maturation state of iPS-derived cardiomyocyte-like cells at ultra-structural level we performed transmission electron microscopy analysis of 21 day old cardiomyocytes. Many cells show nascent parallel arrays of myofilament bundles anchored at Z-line like electron dense structures (Suppl. Figure 1). They show different spatial orientation within the same cell as well as branching. Moreover, we observed sarcomer-like organization of contractile filaments (Fig. 4a). Additionally, fascia adherens-like and gap-junctions-like cellular contacts as well as initial sarcomeric organisation of actin and myosin filaments were detected. The sarcomeric structures of contractile filaments exhibited already identifiable A- and I-bands together with Z-lines as well as H-zones (Fig. 4b-d, Suppl. Figure 1).Fig. 4


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)

Ultrastructural analysis of 21-day old human iPS (del-AR1034ZIMA 001) cell-derived cardiomyocytes. a Two cells in close contact displaying sarcomer-like organization of contractile filaments. Scale bar: 1,000 nm (b–c) Higher magnification showing the presence of fascia adherens-like and gap-junctions like cellular contacts and initial sarcomeric organisation of actin and myosin filaments. Scale bar: b 1,000 nm, c 250 nm. d iPS cell-derived cardiomyocyte-like cells show sarcomer organization of contractile filaments with already identifiable A- and I-bands together with Z-lines as well as H-zones. Abbrevations: m mitochondria, N Nucleus, FA-lS Fascia adherens-like structure, GJ-lS Gap junctions-like structure, Z Z-line, H H-zone. Scale Bar: 250 nm
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4493626&req=5

Fig4: Ultrastructural analysis of 21-day old human iPS (del-AR1034ZIMA 001) cell-derived cardiomyocytes. a Two cells in close contact displaying sarcomer-like organization of contractile filaments. Scale bar: 1,000 nm (b–c) Higher magnification showing the presence of fascia adherens-like and gap-junctions like cellular contacts and initial sarcomeric organisation of actin and myosin filaments. Scale bar: b 1,000 nm, c 250 nm. d iPS cell-derived cardiomyocyte-like cells show sarcomer organization of contractile filaments with already identifiable A- and I-bands together with Z-lines as well as H-zones. Abbrevations: m mitochondria, N Nucleus, FA-lS Fascia adherens-like structure, GJ-lS Gap junctions-like structure, Z Z-line, H H-zone. Scale Bar: 250 nm
Mentions: In order to study the maturation state of iPS-derived cardiomyocyte-like cells at ultra-structural level we performed transmission electron microscopy analysis of 21 day old cardiomyocytes. Many cells show nascent parallel arrays of myofilament bundles anchored at Z-line like electron dense structures (Suppl. Figure 1). They show different spatial orientation within the same cell as well as branching. Moreover, we observed sarcomer-like organization of contractile filaments (Fig. 4a). Additionally, fascia adherens-like and gap-junctions-like cellular contacts as well as initial sarcomeric organisation of actin and myosin filaments were detected. The sarcomeric structures of contractile filaments exhibited already identifiable A- and I-bands together with Z-lines as well as H-zones (Fig. 4b-d, Suppl. Figure 1).Fig. 4

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.