Limits...
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

Fibroblasts can be directly reprogrammed to spontaneously contracting patches with chemical cocktails. (A) The scheme of direct cardiac reprogramming with small molecule cocktails. Fibroblasts were plated in fibroblast growth medium for one day and then the medium was changed into cardiac reprogramming medium (CRM) containing the small molecule cocktails (first stage). At day 16, the medium was changed into cardiomyocyte maintaining medium (CMM, second stage). The first beating clusters could be observed on day 6–8. (B) Representative morphologies of various MEF-derived beating clusters induced by the small molecule cocktail CRFVPT. See also Supplementary information, Movie S2. (C) Number of the beating clusters induced from MEFs with CRFVPT at various time points. Results are presented as means ± SEM, n = 3. (D) Screening for compounds essential for cardiomyocyte induction. Numbers of beating clusters at day 20 are shown. (E) Morphology of TTF-derived beating cells by small molecule cocktail CRFVPT at day 14. See also Supplementary information, Movie S3. (F) Induction of TTF-derived beating cells with CMM at the second stage supplemented with various growth factors (NRG1, 100 ng/ml; G-CSF, 20 ng/ml; Tβ−4, 100 ng/ml; GDF11, 100 ng/ml). (G) Induction of TTF-derived beating cells with CRFVPT plus Rolipram (3 μM) in the first stage, and the growth factors (100 ng/ml NRG-1 and 20 ng/ml G-CSF) in the second stage. Data are means ± SEM, n = 3. *P < 0.05; **P < 0.01; ***P < 0.001. (H) Immunostaining of cardiac markers Mef2c, Gata4, Nkx2.5, α-MHC, α-actinin, cTnT, cTnI, N-cad, and Cx43 in beating clusters generated from MEFs on day 24. Nuclei were stained with Hoechst. Scale bars represent 50 μm in B and E, 20 μm in H.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: Fibroblasts can be directly reprogrammed to spontaneously contracting patches with chemical cocktails. (A) The scheme of direct cardiac reprogramming with small molecule cocktails. Fibroblasts were plated in fibroblast growth medium for one day and then the medium was changed into cardiac reprogramming medium (CRM) containing the small molecule cocktails (first stage). At day 16, the medium was changed into cardiomyocyte maintaining medium (CMM, second stage). The first beating clusters could be observed on day 6–8. (B) Representative morphologies of various MEF-derived beating clusters induced by the small molecule cocktail CRFVPT. See also Supplementary information, Movie S2. (C) Number of the beating clusters induced from MEFs with CRFVPT at various time points. Results are presented as means ± SEM, n = 3. (D) Screening for compounds essential for cardiomyocyte induction. Numbers of beating clusters at day 20 are shown. (E) Morphology of TTF-derived beating cells by small molecule cocktail CRFVPT at day 14. See also Supplementary information, Movie S3. (F) Induction of TTF-derived beating cells with CMM at the second stage supplemented with various growth factors (NRG1, 100 ng/ml; G-CSF, 20 ng/ml; Tβ−4, 100 ng/ml; GDF11, 100 ng/ml). (G) Induction of TTF-derived beating cells with CRFVPT plus Rolipram (3 μM) in the first stage, and the growth factors (100 ng/ml NRG-1 and 20 ng/ml G-CSF) in the second stage. Data are means ± SEM, n = 3. *P < 0.05; **P < 0.01; ***P < 0.001. (H) Immunostaining of cardiac markers Mef2c, Gata4, Nkx2.5, α-MHC, α-actinin, cTnT, cTnI, N-cad, and Cx43 in beating clusters generated from MEFs on day 24. Nuclei were stained with Hoechst. Scale bars represent 50 μm in B and E, 20 μm in H.

Mentions: A two-stage optimization strategy was carried out to improve the induction efficiency and stabilize the cardiomyocyte-like spontaneously contracting cells (Figure 1A). We first optimized the cardiac reprogramming medium (CRM) used in the first stage of the induction with CRFVPT. We found bFGF, which is essential in CiPSC induction, was dispensable for the generation of contracting cells. In fact, bFGF even reduced vitamin C-stimulated cardiac transdifferentiation (Supplementary information, Figure S1B). The combination of 15% fetal bovine serum (FBS) and 5% knockout serum replacement (KSR) was found to be more effective in inducing beating cells than the original 10% FBS and 10% KSR combination used in CiPSC induction. The addition of N2 and B27 further increased the generation of beating clusters (Supplementary information, Figure S1C). The matrix microstructures have also been reported to play a role in cardiac reprogramming15. We tested different extracellular matrix gels and found that BD Matrigel-coated dishes were better than gelatin-coated or uncoated dishes (Supplementary information, Figure S1D). More beating colonies instead of single beating cells could be found after these optimizations. We then optimized the cardiomyocyte-maintaining medium (CMM) used in the second stage (after day 16) of the induction. Exposure to the cocktail CRFVPT beyond day 16 did not further improve the efficiency (Supplementary information, Figure S1E and S1F); we thus removed CRFVPT from the CMM. In contrast, 2i (CHIR99021 and PD0325901), LIF, and insulin, which have been reported to benefit the maintenance of cardiomyocytes in vitro16,17,18, were found to be very effective in increasing the number of beating clusters (Supplementary information, Figure S1F). BMP4, which has been reported to be beneficial for cardiomyocyte induction19, did not further enhance the effect of 2i and LIF (Supplementary information, Figure S1F).


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)

Fibroblasts can be directly reprogrammed to spontaneously contracting patches with chemical cocktails. (A) The scheme of direct cardiac reprogramming with small molecule cocktails. Fibroblasts were plated in fibroblast growth medium for one day and then the medium was changed into cardiac reprogramming medium (CRM) containing the small molecule cocktails (first stage). At day 16, the medium was changed into cardiomyocyte maintaining medium (CMM, second stage). The first beating clusters could be observed on day 6–8. (B) Representative morphologies of various MEF-derived beating clusters induced by the small molecule cocktail CRFVPT. See also Supplementary information, Movie S2. (C) Number of the beating clusters induced from MEFs with CRFVPT at various time points. Results are presented as means ± SEM, n = 3. (D) Screening for compounds essential for cardiomyocyte induction. Numbers of beating clusters at day 20 are shown. (E) Morphology of TTF-derived beating cells by small molecule cocktail CRFVPT at day 14. See also Supplementary information, Movie S3. (F) Induction of TTF-derived beating cells with CMM at the second stage supplemented with various growth factors (NRG1, 100 ng/ml; G-CSF, 20 ng/ml; Tβ−4, 100 ng/ml; GDF11, 100 ng/ml). (G) Induction of TTF-derived beating cells with CRFVPT plus Rolipram (3 μM) in the first stage, and the growth factors (100 ng/ml NRG-1 and 20 ng/ml G-CSF) in the second stage. Data are means ± SEM, n = 3. *P < 0.05; **P < 0.01; ***P < 0.001. (H) Immunostaining of cardiac markers Mef2c, Gata4, Nkx2.5, α-MHC, α-actinin, cTnT, cTnI, N-cad, and Cx43 in beating clusters generated from MEFs on day 24. Nuclei were stained with Hoechst. Scale bars represent 50 μm in B and E, 20 μm in H.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: Fibroblasts can be directly reprogrammed to spontaneously contracting patches with chemical cocktails. (A) The scheme of direct cardiac reprogramming with small molecule cocktails. Fibroblasts were plated in fibroblast growth medium for one day and then the medium was changed into cardiac reprogramming medium (CRM) containing the small molecule cocktails (first stage). At day 16, the medium was changed into cardiomyocyte maintaining medium (CMM, second stage). The first beating clusters could be observed on day 6–8. (B) Representative morphologies of various MEF-derived beating clusters induced by the small molecule cocktail CRFVPT. See also Supplementary information, Movie S2. (C) Number of the beating clusters induced from MEFs with CRFVPT at various time points. Results are presented as means ± SEM, n = 3. (D) Screening for compounds essential for cardiomyocyte induction. Numbers of beating clusters at day 20 are shown. (E) Morphology of TTF-derived beating cells by small molecule cocktail CRFVPT at day 14. See also Supplementary information, Movie S3. (F) Induction of TTF-derived beating cells with CMM at the second stage supplemented with various growth factors (NRG1, 100 ng/ml; G-CSF, 20 ng/ml; Tβ−4, 100 ng/ml; GDF11, 100 ng/ml). (G) Induction of TTF-derived beating cells with CRFVPT plus Rolipram (3 μM) in the first stage, and the growth factors (100 ng/ml NRG-1 and 20 ng/ml G-CSF) in the second stage. Data are means ± SEM, n = 3. *P < 0.05; **P < 0.01; ***P < 0.001. (H) Immunostaining of cardiac markers Mef2c, Gata4, Nkx2.5, α-MHC, α-actinin, cTnT, cTnI, N-cad, and Cx43 in beating clusters generated from MEFs on day 24. Nuclei were stained with Hoechst. Scale bars represent 50 μm in B and E, 20 μm in H.
Mentions: A two-stage optimization strategy was carried out to improve the induction efficiency and stabilize the cardiomyocyte-like spontaneously contracting cells (Figure 1A). We first optimized the cardiac reprogramming medium (CRM) used in the first stage of the induction with CRFVPT. We found bFGF, which is essential in CiPSC induction, was dispensable for the generation of contracting cells. In fact, bFGF even reduced vitamin C-stimulated cardiac transdifferentiation (Supplementary information, Figure S1B). The combination of 15% fetal bovine serum (FBS) and 5% knockout serum replacement (KSR) was found to be more effective in inducing beating cells than the original 10% FBS and 10% KSR combination used in CiPSC induction. The addition of N2 and B27 further increased the generation of beating clusters (Supplementary information, Figure S1C). The matrix microstructures have also been reported to play a role in cardiac reprogramming15. We tested different extracellular matrix gels and found that BD Matrigel-coated dishes were better than gelatin-coated or uncoated dishes (Supplementary information, Figure S1D). More beating colonies instead of single beating cells could be found after these optimizations. We then optimized the cardiomyocyte-maintaining medium (CMM) used in the second stage (after day 16) of the induction. Exposure to the cocktail CRFVPT beyond day 16 did not further improve the efficiency (Supplementary information, Figure S1E and S1F); we thus removed CRFVPT from the CMM. In contrast, 2i (CHIR99021 and PD0325901), LIF, and insulin, which have been reported to benefit the maintenance of cardiomyocytes in vitro16,17,18, were found to be very effective in increasing the number of beating clusters (Supplementary information, Figure S1F). BMP4, which has been reported to be beneficial for cardiomyocyte induction19, did not further enhance the effect of 2i and LIF (Supplementary information, Figure S1F).

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