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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 were generated through cardiac precursor stage but not via iPSCs. (A) Time-lapse images of CiCMs generated from OG2 MEFs carrying an Oct4::GFP reporter (upper panels, phase and GFP). α-MHC (Red) was stained at day 26. Lower panel show iPSCs cells (GFP+) at day 12 after induction with Yamanaka factors (Oct4, Sox2, cMyc, and Klf4). (B) Expression of cardiac precursor markers: Sca-1, Abcg2, Wt1, Flk1, and Mesp1 by quantitative RT-PCR during CiCMs induction. Data are means ± SEM, n = 3. *P < 0.05 *P < 0.01 **P < 0.001, versus MEFs (day 0). (C) Sca-1+ cells at day 20 of induction (left panel). Continuous culture of Sca-1+ cells in either smooth muscle cell differentiation medium or endothelial cell differentiation medium for another two weeks produced α-SMA and Cnn2 positive cells (middle panel), or PECAM and VE-cadherin positive cells (right panel), respectively. Scale bars represent 200 μm in A and 20 μm in C. (D) A model for direct reprogramming of MEFs into cardiomyocytes with chemicals. Chemical combination might induce the generation of a mixed progenitor (or unstable intermediate) population. With favorable culture conditions, these progenitor/intermediate cells might be induced to become pluripotent or develop into various functional cells.
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fig4: CiCMs were generated through cardiac precursor stage but not via iPSCs. (A) Time-lapse images of CiCMs generated from OG2 MEFs carrying an Oct4::GFP reporter (upper panels, phase and GFP). α-MHC (Red) was stained at day 26. Lower panel show iPSCs cells (GFP+) at day 12 after induction with Yamanaka factors (Oct4, Sox2, cMyc, and Klf4). (B) Expression of cardiac precursor markers: Sca-1, Abcg2, Wt1, Flk1, and Mesp1 by quantitative RT-PCR during CiCMs induction. Data are means ± SEM, n = 3. *P < 0.05 *P < 0.01 **P < 0.001, versus MEFs (day 0). (C) Sca-1+ cells at day 20 of induction (left panel). Continuous culture of Sca-1+ cells in either smooth muscle cell differentiation medium or endothelial cell differentiation medium for another two weeks produced α-SMA and Cnn2 positive cells (middle panel), or PECAM and VE-cadherin positive cells (right panel), respectively. Scale bars represent 200 μm in A and 20 μm in C. (D) A model for direct reprogramming of MEFs into cardiomyocytes with chemicals. Chemical combination might induce the generation of a mixed progenitor (or unstable intermediate) population. With favorable culture conditions, these progenitor/intermediate cells might be induced to become pluripotent or develop into various functional cells.

Mentions: We were intrigued by an observation that many colonies or cell patches formed before the initiation of beating, and examined if the generation of CiCMs went through an iPSC or precursor stage as previously reported in transcription factor-mediated cardiac reprogramming10. Quantitative RT-PCR analysis revealed that the expression of pluripotency genes such as Oct4, Sox2, Nanog, and Rex1 remained at a very low level during the CRFVPT-mediated cardiac reprogramming (Supplementary information, Figure S2D). In contrast, cardiac markers were gradually upregulated (Supplementary information, Figure S2C). We also performed time-lapse imaging of OG2 MEFs (MEFs containing an Oct4:GFP reporter) undergoing CRFVPT-induced cardiac reprogramming (Figure 4A). Beating clusters began to appear at day 12 in this experiment, and immunocytochemical staining revealed that these beating clusters expressed α-MHC at day 26 (Figure 4A). However, GFP+ cells were never detected throughout the entire reprogramming process. This is in clear contrast to Yamanaka factor (Oct4, Klf4, Sox2, and cMyc)-mediated reprogramming, in which GFP+ iPSC colonies can be observed at day 12 (Figure 4A). These data indicate that chemical-mediated cardiac reprogramming does not transverse an iPSC 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 were generated through cardiac precursor stage but not via iPSCs. (A) Time-lapse images of CiCMs generated from OG2 MEFs carrying an Oct4::GFP reporter (upper panels, phase and GFP). α-MHC (Red) was stained at day 26. Lower panel show iPSCs cells (GFP+) at day 12 after induction with Yamanaka factors (Oct4, Sox2, cMyc, and Klf4). (B) Expression of cardiac precursor markers: Sca-1, Abcg2, Wt1, Flk1, and Mesp1 by quantitative RT-PCR during CiCMs induction. Data are means ± SEM, n = 3. *P < 0.05 *P < 0.01 **P < 0.001, versus MEFs (day 0). (C) Sca-1+ cells at day 20 of induction (left panel). Continuous culture of Sca-1+ cells in either smooth muscle cell differentiation medium or endothelial cell differentiation medium for another two weeks produced α-SMA and Cnn2 positive cells (middle panel), or PECAM and VE-cadherin positive cells (right panel), respectively. Scale bars represent 200 μm in A and 20 μm in C. (D) A model for direct reprogramming of MEFs into cardiomyocytes with chemicals. Chemical combination might induce the generation of a mixed progenitor (or unstable intermediate) population. With favorable culture conditions, these progenitor/intermediate cells might be induced to become pluripotent or develop into various functional cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig4: CiCMs were generated through cardiac precursor stage but not via iPSCs. (A) Time-lapse images of CiCMs generated from OG2 MEFs carrying an Oct4::GFP reporter (upper panels, phase and GFP). α-MHC (Red) was stained at day 26. Lower panel show iPSCs cells (GFP+) at day 12 after induction with Yamanaka factors (Oct4, Sox2, cMyc, and Klf4). (B) Expression of cardiac precursor markers: Sca-1, Abcg2, Wt1, Flk1, and Mesp1 by quantitative RT-PCR during CiCMs induction. Data are means ± SEM, n = 3. *P < 0.05 *P < 0.01 **P < 0.001, versus MEFs (day 0). (C) Sca-1+ cells at day 20 of induction (left panel). Continuous culture of Sca-1+ cells in either smooth muscle cell differentiation medium or endothelial cell differentiation medium for another two weeks produced α-SMA and Cnn2 positive cells (middle panel), or PECAM and VE-cadherin positive cells (right panel), respectively. Scale bars represent 200 μm in A and 20 μm in C. (D) A model for direct reprogramming of MEFs into cardiomyocytes with chemicals. Chemical combination might induce the generation of a mixed progenitor (or unstable intermediate) population. With favorable culture conditions, these progenitor/intermediate cells might be induced to become pluripotent or develop into various functional cells.
Mentions: We were intrigued by an observation that many colonies or cell patches formed before the initiation of beating, and examined if the generation of CiCMs went through an iPSC or precursor stage as previously reported in transcription factor-mediated cardiac reprogramming10. Quantitative RT-PCR analysis revealed that the expression of pluripotency genes such as Oct4, Sox2, Nanog, and Rex1 remained at a very low level during the CRFVPT-mediated cardiac reprogramming (Supplementary information, Figure S2D). In contrast, cardiac markers were gradually upregulated (Supplementary information, Figure S2C). We also performed time-lapse imaging of OG2 MEFs (MEFs containing an Oct4:GFP reporter) undergoing CRFVPT-induced cardiac reprogramming (Figure 4A). Beating clusters began to appear at day 12 in this experiment, and immunocytochemical staining revealed that these beating clusters expressed α-MHC at day 26 (Figure 4A). However, GFP+ cells were never detected throughout the entire reprogramming process. This is in clear contrast to Yamanaka factor (Oct4, Klf4, Sox2, and cMyc)-mediated reprogramming, in which GFP+ iPSC colonies can be observed at day 12 (Figure 4A). These data indicate that chemical-mediated cardiac reprogramming does not transverse an iPSC 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