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Direct reprogramming of mouse fibroblasts into cardiomyocytes with chemical cocktails.

Fu Y, Huang C, Xu X, Gu H, Ye Y, Jiang C, Qiu Z, Xie X - Cell Res. (2015)

Bottom Line: These chemical-induced cardiomyocyte-like cells (CiCMs) express cardiomyocyte-specific markers, exhibit sarcomeric organization, and possess typical cardiac calcium flux and electrophysiological features.Genetic lineage tracing confirms the fibroblast origin of these CiCMs. Further studies show the generation of CiCMs passes through a cardiac progenitor stage instead of a pluripotent stage.Bypassing the use of viral-derived factors, this proof of concept study lays a foundation for in vivo cardiac transdifferentiation with pharmacological agents and possibly safer treatment of heart failure.

View Article: PubMed Central - PubMed

Affiliation: Shanghai Key Laboratory of Signaling and Disease Research, Laboratory of Receptor-based Bio-medicine, School of Life Sciences and Technology, Tongji University, Shanghai 200092, China.

ABSTRACT
The direct conversion, or transdifferentiation, of non-cardiac cells into cardiomyocytes by forced expression of transcription factors and microRNAs provides promising approaches for cardiac regeneration. However, genetic manipulations raise safety concerns and are thus not desirable in most clinical applications. The discovery of full chemically induced pluripotent stem cells suggest the possibility of replacing transcription factors with chemical cocktails. Here, we report the generation of automatically beating cardiomyocyte-like cells from mouse fibroblasts using only chemical cocktails. These chemical-induced cardiomyocyte-like cells (CiCMs) express cardiomyocyte-specific markers, exhibit sarcomeric organization, and possess typical cardiac calcium flux and electrophysiological features. Genetic lineage tracing confirms the fibroblast origin of these CiCMs. Further studies show the generation of CiCMs passes through a cardiac progenitor stage instead of a pluripotent stage. Bypassing the use of viral-derived factors, this proof of concept study lays a foundation for in vivo cardiac transdifferentiation with pharmacological agents and possibly safer treatment of heart failure.

No MeSH data available.


Related in: MedlinePlus

CiCMs exhibit typical cardiac calcium flux, electrophysiological features, and gene expression profile. (A) Heatmap illustration of microarray data from MEFs, MEF-CiCMs (beating clusters picked at day 24) and cardiomyocytes. Groups 1 and 2 contain genes that are upregulated or downregulated for > 5 fold in CiCMs compared with MEFs. (B) GO term enrichment analysis of genes that display > 5 fold change in expression in CiCMs and cardiomyocytes compared with MEFs. Left, upregulated genes; right, downregulated genes. (C) Calcium flux in MEF-derived CiCMs at day 25 of the induction. Calcium transients were recorded at basal condition, or after 5 μM carbachol (Cch) or 1 μM isoproterenol (Iso) treatment. Left, linescan images of calcium transients; right, traces of calcium transients. See also Supplementary information, Movie S4. (D) Calcium transient frequency and (E) decay rate at basal state (n = 18), and after treatment of Cch (n = 8) or Iso (n = 13). Data are presented as means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001. (F) Representative action potentials (AP) of CiCMs induced with CRFVPT cocktail. Em, membrane potential in millivolts. (G) Immunostaining of MLC2a, MLC2v, α-actinin and HCN4 in MEF-derived CiCMs. Scale bars represent 20 μm. (H) AP parameters of CiCMs including maximum upstroke velocity (dv/dtMax), overshoot potential (OSP), minimum diastolic potential (MDP), AP amplitude (APA), beating frequency (Freq), AP durations (APDs) at the level of 50% (APD50) and 90% repolarization (APD90). Data are means ± SEM.
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fig2: CiCMs exhibit typical cardiac calcium flux, electrophysiological features, and gene expression profile. (A) Heatmap illustration of microarray data from MEFs, MEF-CiCMs (beating clusters picked at day 24) and cardiomyocytes. Groups 1 and 2 contain genes that are upregulated or downregulated for > 5 fold in CiCMs compared with MEFs. (B) GO term enrichment analysis of genes that display > 5 fold change in expression in CiCMs and cardiomyocytes compared with MEFs. Left, upregulated genes; right, downregulated genes. (C) Calcium flux in MEF-derived CiCMs at day 25 of the induction. Calcium transients were recorded at basal condition, or after 5 μM carbachol (Cch) or 1 μM isoproterenol (Iso) treatment. Left, linescan images of calcium transients; right, traces of calcium transients. See also Supplementary information, Movie S4. (D) Calcium transient frequency and (E) decay rate at basal state (n = 18), and after treatment of Cch (n = 8) or Iso (n = 13). Data are presented as means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001. (F) Representative action potentials (AP) of CiCMs induced with CRFVPT cocktail. Em, membrane potential in millivolts. (G) Immunostaining of MLC2a, MLC2v, α-actinin and HCN4 in MEF-derived CiCMs. Scale bars represent 20 μm. (H) AP parameters of CiCMs including maximum upstroke velocity (dv/dtMax), overshoot potential (OSP), minimum diastolic potential (MDP), AP amplitude (APA), beating frequency (Freq), AP durations (APDs) at the level of 50% (APD50) and 90% repolarization (APD90). Data are means ± SEM.

Mentions: Quantitative RT-PCR also confirmed the time-dependent increase in the expression of cardiac-specific genes, including Nkx2.5, Mef2c, Gata4, β-MHC, cTnT, and Ryr2, during the induction (Supplementary information, Figure S2C). The global gene expression patterns of MEFs, MEF-CiCMs, and cardiomyocytes were analyzed with microarrays. Compared with MEFs, 477 genes were upregulated and 276 were downregulated for more than 5 folds in MEF-CiCMs. CiCMs and cardiomyocytes showed very similar expression patterns of these genes and were thus clustered into one group (Figure 2A). Gene ontology (GO) term enrichment analysis of these genes indicated that genes involved in muscle development, myofibril assembly, muscle contraction, and especially cardiac muscle development were significantly upregulated in CiCM samples, while genes involved in cell cycle and mitosis control were significantly downregulated (Figure 2B), indicating a clear transition from dividing MEFs to differentiated cardiomyocyte-like state.


Direct reprogramming of mouse fibroblasts into cardiomyocytes with chemical cocktails.

Fu Y, Huang C, Xu X, Gu H, Ye Y, Jiang C, Qiu Z, Xie X - Cell Res. (2015)

CiCMs exhibit typical cardiac calcium flux, electrophysiological features, and gene expression profile. (A) Heatmap illustration of microarray data from MEFs, MEF-CiCMs (beating clusters picked at day 24) and cardiomyocytes. Groups 1 and 2 contain genes that are upregulated or downregulated for > 5 fold in CiCMs compared with MEFs. (B) GO term enrichment analysis of genes that display > 5 fold change in expression in CiCMs and cardiomyocytes compared with MEFs. Left, upregulated genes; right, downregulated genes. (C) Calcium flux in MEF-derived CiCMs at day 25 of the induction. Calcium transients were recorded at basal condition, or after 5 μM carbachol (Cch) or 1 μM isoproterenol (Iso) treatment. Left, linescan images of calcium transients; right, traces of calcium transients. See also Supplementary information, Movie S4. (D) Calcium transient frequency and (E) decay rate at basal state (n = 18), and after treatment of Cch (n = 8) or Iso (n = 13). Data are presented as means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001. (F) Representative action potentials (AP) of CiCMs induced with CRFVPT cocktail. Em, membrane potential in millivolts. (G) Immunostaining of MLC2a, MLC2v, α-actinin and HCN4 in MEF-derived CiCMs. Scale bars represent 20 μm. (H) AP parameters of CiCMs including maximum upstroke velocity (dv/dtMax), overshoot potential (OSP), minimum diastolic potential (MDP), AP amplitude (APA), beating frequency (Freq), AP durations (APDs) at the level of 50% (APD50) and 90% repolarization (APD90). Data are means ± SEM.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4559819&req=5

fig2: CiCMs exhibit typical cardiac calcium flux, electrophysiological features, and gene expression profile. (A) Heatmap illustration of microarray data from MEFs, MEF-CiCMs (beating clusters picked at day 24) and cardiomyocytes. Groups 1 and 2 contain genes that are upregulated or downregulated for > 5 fold in CiCMs compared with MEFs. (B) GO term enrichment analysis of genes that display > 5 fold change in expression in CiCMs and cardiomyocytes compared with MEFs. Left, upregulated genes; right, downregulated genes. (C) Calcium flux in MEF-derived CiCMs at day 25 of the induction. Calcium transients were recorded at basal condition, or after 5 μM carbachol (Cch) or 1 μM isoproterenol (Iso) treatment. Left, linescan images of calcium transients; right, traces of calcium transients. See also Supplementary information, Movie S4. (D) Calcium transient frequency and (E) decay rate at basal state (n = 18), and after treatment of Cch (n = 8) or Iso (n = 13). Data are presented as means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001. (F) Representative action potentials (AP) of CiCMs induced with CRFVPT cocktail. Em, membrane potential in millivolts. (G) Immunostaining of MLC2a, MLC2v, α-actinin and HCN4 in MEF-derived CiCMs. Scale bars represent 20 μm. (H) AP parameters of CiCMs including maximum upstroke velocity (dv/dtMax), overshoot potential (OSP), minimum diastolic potential (MDP), AP amplitude (APA), beating frequency (Freq), AP durations (APDs) at the level of 50% (APD50) and 90% repolarization (APD90). Data are means ± SEM.
Mentions: Quantitative RT-PCR also confirmed the time-dependent increase in the expression of cardiac-specific genes, including Nkx2.5, Mef2c, Gata4, β-MHC, cTnT, and Ryr2, during the induction (Supplementary information, Figure S2C). The global gene expression patterns of MEFs, MEF-CiCMs, and cardiomyocytes were analyzed with microarrays. Compared with MEFs, 477 genes were upregulated and 276 were downregulated for more than 5 folds in MEF-CiCMs. CiCMs and cardiomyocytes showed very similar expression patterns of these genes and were thus clustered into one group (Figure 2A). Gene ontology (GO) term enrichment analysis of these genes indicated that genes involved in muscle development, myofibril assembly, muscle contraction, and especially cardiac muscle development were significantly upregulated in CiCM samples, while genes involved in cell cycle and mitosis control were significantly downregulated (Figure 2B), indicating a clear transition from dividing MEFs to differentiated cardiomyocyte-like state.

Bottom Line: These chemical-induced cardiomyocyte-like cells (CiCMs) express cardiomyocyte-specific markers, exhibit sarcomeric organization, and possess typical cardiac calcium flux and electrophysiological features.Genetic lineage tracing confirms the fibroblast origin of these CiCMs. Further studies show the generation of CiCMs passes through a cardiac progenitor stage instead of a pluripotent stage.Bypassing the use of viral-derived factors, this proof of concept study lays a foundation for in vivo cardiac transdifferentiation with pharmacological agents and possibly safer treatment of heart failure.

View Article: PubMed Central - PubMed

Affiliation: Shanghai Key Laboratory of Signaling and Disease Research, Laboratory of Receptor-based Bio-medicine, School of Life Sciences and Technology, Tongji University, Shanghai 200092, China.

ABSTRACT
The direct conversion, or transdifferentiation, of non-cardiac cells into cardiomyocytes by forced expression of transcription factors and microRNAs provides promising approaches for cardiac regeneration. However, genetic manipulations raise safety concerns and are thus not desirable in most clinical applications. The discovery of full chemically induced pluripotent stem cells suggest the possibility of replacing transcription factors with chemical cocktails. Here, we report the generation of automatically beating cardiomyocyte-like cells from mouse fibroblasts using only chemical cocktails. These chemical-induced cardiomyocyte-like cells (CiCMs) express cardiomyocyte-specific markers, exhibit sarcomeric organization, and possess typical cardiac calcium flux and electrophysiological features. Genetic lineage tracing confirms the fibroblast origin of these CiCMs. Further studies show the generation of CiCMs passes through a cardiac progenitor stage instead of a pluripotent stage. Bypassing the use of viral-derived factors, this proof of concept study lays a foundation for in vivo cardiac transdifferentiation with pharmacological agents and possibly safer treatment of heart failure.

No MeSH data available.


Related in: MedlinePlus