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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

Lineage tracing of chemical induced transdifferentiation of MEFs toward cardiomyocytes. (A) Schematic diagram of the genetic fate mapping method used to trace the origin of CiCMs reprogrammed from Fsp1-Cre:R26RtdTomato MEFs. (B) Co-localization of tdTomato (red) with various cardiac markers, including Mef2c, NKX2.5, α-actinin, cTnT, cTnI and α-MHC in beating clusters generated from Fsp1-Cre:R26RtdTomato MEFs on day 24 with chemical cocktail CRFVPT. Nuclei are stained with Hoechst. (C) Spontaneous calcium transients in the tdTomato-CiCMs at day 25 of induction. Left, linescan images; right, traces of calcium signals. (D) A representative tdTomato-CiCM used for electrophysiological characterization. (E) Representative action potentials (AP) of tdTomato-CiCM. (F) AP parameters of tdTomato-CiCMs. Data are means ± SEM. Scale bars in B and D represent 20 μm.
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fig3: Lineage tracing of chemical induced transdifferentiation of MEFs toward cardiomyocytes. (A) Schematic diagram of the genetic fate mapping method used to trace the origin of CiCMs reprogrammed from Fsp1-Cre:R26RtdTomato MEFs. (B) Co-localization of tdTomato (red) with various cardiac markers, including Mef2c, NKX2.5, α-actinin, cTnT, cTnI and α-MHC in beating clusters generated from Fsp1-Cre:R26RtdTomato MEFs on day 24 with chemical cocktail CRFVPT. Nuclei are stained with Hoechst. (C) Spontaneous calcium transients in the tdTomato-CiCMs at day 25 of induction. Left, linescan images; right, traces of calcium signals. (D) A representative tdTomato-CiCM used for electrophysiological characterization. (E) Representative action potentials (AP) of tdTomato-CiCM. (F) AP parameters of tdTomato-CiCMs. Data are means ± SEM. Scale bars in B and D represent 20 μm.

Mentions: To ultimately confirm that the CiCMs were indeed transdifferentiated from fibroblasts but not the possible contaminating progenitor cells, we used a lineage-tracing experiment to track the origin of the CiCMs. Transgenic mice that express Cre recombinase under the control of the fibroblast specific protein-1 (Fsp1 or S100A4) promoter were crossed with the R26RtdTomato mice, in which the expression of tdTomato is prevented by a loxP-flanked STOP cassette. Since Fsp1 is specifically expressed in fibroblasts29, the progeny of these mice (Fsp1-Cre:R26RtdTomato) would have the red fluorescent protein tdTomato expressed specifically in the fibroblasts (Figure 3A).


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)

Lineage tracing of chemical induced transdifferentiation of MEFs toward cardiomyocytes. (A) Schematic diagram of the genetic fate mapping method used to trace the origin of CiCMs reprogrammed from Fsp1-Cre:R26RtdTomato MEFs. (B) Co-localization of tdTomato (red) with various cardiac markers, including Mef2c, NKX2.5, α-actinin, cTnT, cTnI and α-MHC in beating clusters generated from Fsp1-Cre:R26RtdTomato MEFs on day 24 with chemical cocktail CRFVPT. Nuclei are stained with Hoechst. (C) Spontaneous calcium transients in the tdTomato-CiCMs at day 25 of induction. Left, linescan images; right, traces of calcium signals. (D) A representative tdTomato-CiCM used for electrophysiological characterization. (E) Representative action potentials (AP) of tdTomato-CiCM. (F) AP parameters of tdTomato-CiCMs. Data are means ± SEM. Scale bars in B and D represent 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: Lineage tracing of chemical induced transdifferentiation of MEFs toward cardiomyocytes. (A) Schematic diagram of the genetic fate mapping method used to trace the origin of CiCMs reprogrammed from Fsp1-Cre:R26RtdTomato MEFs. (B) Co-localization of tdTomato (red) with various cardiac markers, including Mef2c, NKX2.5, α-actinin, cTnT, cTnI and α-MHC in beating clusters generated from Fsp1-Cre:R26RtdTomato MEFs on day 24 with chemical cocktail CRFVPT. Nuclei are stained with Hoechst. (C) Spontaneous calcium transients in the tdTomato-CiCMs at day 25 of induction. Left, linescan images; right, traces of calcium signals. (D) A representative tdTomato-CiCM used for electrophysiological characterization. (E) Representative action potentials (AP) of tdTomato-CiCM. (F) AP parameters of tdTomato-CiCMs. Data are means ± SEM. Scale bars in B and D represent 20 μm.
Mentions: To ultimately confirm that the CiCMs were indeed transdifferentiated from fibroblasts but not the possible contaminating progenitor cells, we used a lineage-tracing experiment to track the origin of the CiCMs. Transgenic mice that express Cre recombinase under the control of the fibroblast specific protein-1 (Fsp1 or S100A4) promoter were crossed with the R26RtdTomato mice, in which the expression of tdTomato is prevented by a loxP-flanked STOP cassette. Since Fsp1 is specifically expressed in fibroblasts29, the progeny of these mice (Fsp1-Cre:R26RtdTomato) would have the red fluorescent protein tdTomato expressed specifically in the fibroblasts (Figure 3A).

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