Cell Biology International ISSN 1065-6995 doi: 10.1002/cbin.10302
RESEARCH ARTICLE
Comparative study of directional differentiation of human and mouse embryonic stem cells into cardiomyocytes Junsheng Mu1*, Xianshuai Li1, Shumin Yuan2, Jianqun Zhang1 and Ping Bo1 1 Department of Cardiac Surgery, Capital Medical University Affiliated Beijing Anzhen Hospital, Beijin Institute of Heart, Lung and Blood Vessel Diseases, Beijing 100029, P.R. China 2 State Key Laboratory of Animal Research Institute of the Chinese Academy of Sciences Biological Membrane and Membrane Biotechnology, Beijing 100101, P.R. China
Abstract This comparative study investigates the method, efficiency, and anti-hypoxic ability of cardiomyocytes, directionally induced from human (h) and mouse (m) embryonic stem cells (ESCs). hESCs were induced into cardiomyocytes by suspension culture, without inducers, or adherent culture using the inducers activin A and BMP4. mESCs were induced into cardiomyocytes by hanging-drop method, without inducers or induced with vitamin C. All four methods successfully induced ESCs to differentiate into cardiomyocytes. There was a significant difference between groups with and without inducers. A significant difference was found between mESC and hESC groups with inducers. The average beating frequency of cardiomyocytes differentiated from hESC was lower than cardiomyocytes differentiated from mESC, while the average beating frequency of cardiomyocytes differentiated from the same cell line, despite different culture methods, did not differ. Beating cardiomyocytes of each group were positive for cTnT staining. Spontaneous action potentials of beating cardiomyocytes were detected by patch-clamp experiments in each group. Different apoptotic ratios were detected in beating cardiomyocytes in each group and the difference between cardiomyocytes induced from mESCs and hESCs was statistically significant. The differentiation efficiencies in the groups without inducers were significantly higher than those without inducers. The induction of mESCs was more simple and efficient compared with hESCs. Without the presence of other protective factors, the anti-hypoxic ability of cardiomyocytes induced from hESCs was stronger and the beating times were longer in vitro compared with mESCs. Keywords: cardiomyocytes; differentiation; embryoid body; human embryonic stem cells; mouse embryonic stem cells
Introduction Myocardial infarction is a serious and common disease in clinics, with a poor prognosis and a high mortality in the long-term. The mature mammalian heart has limited regenerative capacity. If damage occurs to a significant number of cardiomyocytes it can be irreparable, which can seriously impair ventricular function and eventually lead to heart failure. Although there has been noticeable progress in pharmacology, interventional therapy and surgical treatment, the prognosis of patients with heart failure remains poor. At present, organ transplantation is the only effective treatment for chronic heart failure. The morbidity of myocardial infarction increased obviously all over the world
in recent years and better treatments are in urgent need in clinic. In view of the important medical issues, reconstructing the damaged heart with new cardiomyogenic cells is an attractive method. Recent research in stem cells gives hope to patients with heart disease. Embryonic stem cells (ESCs) have the greatest developmental totipotency and the highest research value among stem cell types. The experimental methods of isolating and culturing ESCs in vitro have been well-established, and ESCs can be greatly expanded in culture and differentiated into definitive cardiomyocytes (Kehat et al., 2001; Xu et al., 2002a; Mummery et al., 2003). Cardiomyocytes induced from ESCs have powerful proliferative capacity both in vitro and in vivo following implantation (Snir et al., 2003; Laflamme et al., 2005;
Corresponding author: e-mail: [email protected] Junsheng Mu and Xianshuai Li contributed equally to this study.
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McDevitt et al., 2005), implying that delivery of an initially sub-therapeutic cell dose may suffice to obtain a functionally meaningful cardiac implant over time. Although the technology of inducing differentiation of hESCs and mESCs into cardiomyocytes are already available, there has been no comparative study of their directional differentiation into cardiomyocytes. We have compared four different strategies of inducing hESCs and mESCs differentiation into cardiomyocytes to provide an experimental basis for study of inducing differentiation of ESCs into cardiomyocytes in vitro. Materials and methods
Propagation of hESC and mESC lines The X-01 hESC line was obtained from Shanghai Si Dan Sai Biotechnology Co Ltd, and the R1 mESC line was obtained from Shanghai Institutes for Biological Sciences. Continuous cultures of the X-01 cell line was grown on 0.2% gelatincoated, mitomycin C (Sigma; 10 mg/mL) inactivated mouse embryonic fibroblast (MEF) feeders in standard hESC culture medium consisting of 80% KO-DMEM (Gibco), 20% serum replacement (SR, Gibco), 1% non-essential amino acid solution (Gibco), 1 mM L-glutamine (Gibco), 0.1% bmercaptoethanol (Sigma), 4 ng/mL human basic fibroblast growth factor (bFGF, Gibco). Media was replenished every day with media pre-equilibrated in an incubator for 2 h at 37 C and 5% CO2 and cells were passaged every fifth/sixth day by incubation in 200 units/mL collagenase IV for 5– 10 min at 37 C and then dissociated. The cultures were maintained at 37 C and 5% CO2 in air and used for characterization and differentiation studies. MEF feeders were cultured in DMEM High Glucose (Gibco) supplemented with 10% FBS (Gibco), 1% non-essential amino acid solution, 1 mM L-glutamine. Similarly, continuous cultures of the R1 cell line was grown on 0.2% gelatin-coated, mitomycin C inactivated MEF feeders in a standard mESC culture medium consisting of 85% DMEM (Gibco), 15% EScell qualified FBS (Gibco), 1% non-essential amino acid solution, 1 mM L-glutamine, 0.1% b-mercaptoethanol and leukemia inhibitory factor (LIF). Media was replenished every day with media pre-equilibrated in an incubator for 2 h at 37 C and air plus 5% CO2 and cells were passaged every second/third day by incubation in 0.05% trypsin for 3–4 min at 37 C and then dissociated. Protocol 1: Differentiation of cardiomyocytes via EB formation (hESC without inducers group) Undifferentiated hES cells were dissociated into clumps using 200 U/mL collagenase IV at 37 C for 5–10 min and cultured in suspension using low attachment plates (Corning) to form EBs. The differentiation medium
Directional differentiation of human and mouse ESCs
contained 80% KO-DMEM, 20% FBS, 1% non-essential amino acid solution, 1 mM L-glutamine, 0.1% b-mercaptoethanol. After 4 days in suspension, EBs were plated on 0.2 Matrigel (BD) coated plates to score the contracting cardiac clusters and record the beating rate. The number of days of differentiation includes the days in which the cells were maintained in suspension. For example, differentiation day 10 relates to cells that were maintained in suspension for 4 days, plated and cultured for an additional 6 days. Protocol 2: Differentiation of cardiomyocytes via adherent culture (hESC with inducers group) Undifferentiated hES cells were dissociated into clumps using 200 U/mL collagenase IV at 37 C for 5–10 min and seeded onto Matrigel-coated plates at 100,000 cells/cm2. Medium were refreshed daily with MEF-CM plus 8 ng/mL bFGF for 6 days. To induce cardiac differentiation, we replaced MEF-CM with RPMI-B27 medium (Gibco) supplemented with the following cytokines: 100 ng/mL human recombinant activin A (Gibco) for 24 h, followed by 10 ng/mL human recombinant BMP4 (Gibco) for 4 days. The medium was exchanged for RPMI-B27 without supplementary cytokines; culture medium was refreshed every 2–3 days for 2–3 additional weeks. Widespread spontaneous beating activity was typically observed by day 12 after addition of activin A. Protocol 3: Differentiation of cardiomyocytes via EB formation (mESC without inducers group) Undifferentiated mES cells were dissociated into clumps using 0.05% trypsin for 3–4 min at 37 C and seeded at 7000 cells/cm2 in 0.2% gelatin-coated cell culture plates with medium without LIF, and for inducing EB formation, EBs were formed in hanging drops of 400 cells in 20 mL of medium without LIF. After 2 days, EBs were plated on gelatin-coated dishes in standard mESC culture medium without LIF and vitamin C. Protocol 4: Differentiation of cardiomyocytes via EB formation (group of mESC with inducers) This protocol was the same as protocol 3 except the last step. After 2 days, EBs were plated on gelatin-coated dishes in standard mESC culture medium without LIF but with the addition of vitamin C.
Immunostaining For immunostaining, ESC were induced to differentiate into beating colonies through the four protocols. Following this, beating colonies were fixed in 4% paraformaldehyde for 10 min, washed three times with PBS (Gibco), and permeabilized with 0.4% triton X-100 at RT for 10 min. Colonies were then washed three times with PBS, blocked
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with 5% bovine serum albumin (BSA) for 30 min, followed by incubation with primary antibody against cTnT at 4 C overnight. After washing, the colonies were exposed to the corresponding secondary antibody at RT for 45 min. The colonies were then washed again, and mounted with Vectashield medium for photomicroscopy.
of 330–380 nm. Apoptotic cells were identified on the basis of morphologic changes in their nuclear assembly by observing chromatin condensation and fragmentation by Hoechst 33258 staining. In each group, 10 microscopic fields were randomly selected and counted.
Statistical analysis Electrophysiological recordings Beating colonies were disassociated into isolated cardiomyocytes by collagenase or trypsin, and after culturing for 36 h, beating cardiomyocytes were selected for patch-clamp experiments. Spontaneous action potentials of cardiomyocytes were recorded by current-clamp technique with an Axon-200A amplifier. The glass microelectrodes were filled with a solution containing: 50 mM KCl, 80 mM Kasp, 1 mM MgCl2, 3 mM MgATP, 10 mM HEPES, 10 mM EGTA (pH 7.4 with KOH), the electrode resistance was between 2 and 4 MV. Cells were superfused with a bathing solution that contained: 140 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM D-glucose (pH 7.4 with NaOH). Experiments were conducted at 37 C.
Hoechst 33258 staining A Hoechst 33258 cell apoptosis staining kit (Beyotime) was used to confirm morphological changes in the nuclei. Cells were fixed with 4% paraformaldehyde for 10 min at RT, washed three times with cold PBS and treated with 10 mg/L Hoechst 33258 in the dark for 5 min. The cells were washed with PBS and immediately photographed under a fluorescence microscope at an excitation wavelength
All experiments were performed at least three times, and data were expressed as mean standard deviation and analyzed by Student’s t-test or one-way ANOVA with post hoc analysis. P < 0.05 was considered statistically significant. Results
Generation of beating cardiomyocytes All four methods successfully induced differentiation of ESCs into cardiomyocytes. hESC clones had clear boundaries, flat nest-like shapes, and high nuclear:cytoplasmic ratios (Figure 1A). hESCs grew relatively slowly, with a passage time of 5–6 days. While the mESC clone showed morphological diversity, relative density, large nuclei, scant cytoplasm, and a small number of differentiated cells could sometimes be seen in the marginal zone whose morphology was like that of epithelial cells or spindle-shaped fibroblasts (Figure 1B). mESCs grew relatively fast, and the passage time was 2–3 days. Embryoid bodies (EBs) began to adhere at 2– 3 h after plating and completely adhered within 24 h. Day 2 EBs had fully expanded into flat shapes that extended outward, with the middle part slightly raised (Figures 1C and 1D). After 7 days differentiation, beating colonies began
Figure 1 (A) hESC clone; (B) mESC clone; (C) the second day of differentiating of hESC clone; (D) the second day of differentiating of mESC clone; (E) beating colony induced from hESC (video has been taken); (F) beating colony induced from mESC (video has been taken).
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to appear, and most EBs were very widely spread. The thick inner section of the EBs were unequal and there were sometimes multiple beating areas with different sizes, frequencies, and amplitudes (Figures 1E and 1F). Videos have also been recorded.
Immunostaining and electrophysiological recordings After ESCs were induced to differentiate into beating colonies, they were immunofluorescently stained for the cardiomyocyte-specific marker cTnT. Almost the entire beating colony was positive for cTnT (Figures 2A and Figure 2). Beating colonies were disassociated into isolated cardiomyocytes for patch-clamp experiments, and spontaneous action potentials were recorded from atria-like cardiomyocytes (Figures 2C and 2D). Action potentials were recorded from two clusters. Figure 2C is a spontaneous action potential of atria-like cardiomyocytes induced from mESC, while in Figure 2D it is from atria-like cardiomyocytes induced from hESC. However, the morphology of action potentials varied from different cell lines and differentiation conditions. Action potentials here show that the cardiomyocytes induced from both cell lines have electrophysiological function.
Directional differentiation of human and mouse ESCs
Comparison of the differentiation efficiency human and mouse ESCs into cardiomyocytes One EB was inoculated per well, and a total of 96 EBs were inoculated for each group. From day 5 following adherent differentiation, we measured the efficiency of differentiation. In the hESC without inducers group, the mean time to appearance of beating cardiomyocytes was 13.9 0.9 days, the percentage was 20.8% and the average beating frequency was 63.8 5.6 times/min. In the hESC with inducers group, the mean time to appearance of beating cardiomyocytes was 13.0 1.1 days, the percentage was 66.7% and the average beating frequency was 63.0 7.0 times/min; in the mESC without inducers group, the mean time to appearance of beating cardiomyocytes was 14.3 1.0 days, the percentage was 12.5% and the average beating frequency was 80.2 3.9 times/min; in the mESC with inducers group, the mean time to appearance of beating cardiomyocytes was 12.2 1.2 days, the percentage was 81.25% and the average beating frequency was 79.9 7.7 times/min (Figure 3). The statistics show that the groups with inducers improved the differentiation efficiency more significantly than the groups without inducers, as the mean time to the
Figure 2 (A and B) Light field of beating colony induced from hESC and its immunofluorescence staining of cTnT; (C) spontaneous action potential of atria-like cardiomyocytes induced from mESC; (D) spontaneous action potential of atria-like cardiomyocytes induced from hESC.
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Figure 3 (A) Comparison of the average time of appearing beating colonies in each group, *P-values are
RESEARCH ARTICLE
Comparative study of directional differentiation of human and mouse embryonic stem cells into cardiomyocytes Junsheng Mu1*, Xianshuai Li1, Shumin Yuan2, Jianqun Zhang1 and Ping Bo1 1 Department of Cardiac Surgery, Capital Medical University Affiliated Beijing Anzhen Hospital, Beijin Institute of Heart, Lung and Blood Vessel Diseases, Beijing 100029, P.R. China 2 State Key Laboratory of Animal Research Institute of the Chinese Academy of Sciences Biological Membrane and Membrane Biotechnology, Beijing 100101, P.R. China
Abstract This comparative study investigates the method, efficiency, and anti-hypoxic ability of cardiomyocytes, directionally induced from human (h) and mouse (m) embryonic stem cells (ESCs). hESCs were induced into cardiomyocytes by suspension culture, without inducers, or adherent culture using the inducers activin A and BMP4. mESCs were induced into cardiomyocytes by hanging-drop method, without inducers or induced with vitamin C. All four methods successfully induced ESCs to differentiate into cardiomyocytes. There was a significant difference between groups with and without inducers. A significant difference was found between mESC and hESC groups with inducers. The average beating frequency of cardiomyocytes differentiated from hESC was lower than cardiomyocytes differentiated from mESC, while the average beating frequency of cardiomyocytes differentiated from the same cell line, despite different culture methods, did not differ. Beating cardiomyocytes of each group were positive for cTnT staining. Spontaneous action potentials of beating cardiomyocytes were detected by patch-clamp experiments in each group. Different apoptotic ratios were detected in beating cardiomyocytes in each group and the difference between cardiomyocytes induced from mESCs and hESCs was statistically significant. The differentiation efficiencies in the groups without inducers were significantly higher than those without inducers. The induction of mESCs was more simple and efficient compared with hESCs. Without the presence of other protective factors, the anti-hypoxic ability of cardiomyocytes induced from hESCs was stronger and the beating times were longer in vitro compared with mESCs. Keywords: cardiomyocytes; differentiation; embryoid body; human embryonic stem cells; mouse embryonic stem cells
Introduction Myocardial infarction is a serious and common disease in clinics, with a poor prognosis and a high mortality in the long-term. The mature mammalian heart has limited regenerative capacity. If damage occurs to a significant number of cardiomyocytes it can be irreparable, which can seriously impair ventricular function and eventually lead to heart failure. Although there has been noticeable progress in pharmacology, interventional therapy and surgical treatment, the prognosis of patients with heart failure remains poor. At present, organ transplantation is the only effective treatment for chronic heart failure. The morbidity of myocardial infarction increased obviously all over the world
in recent years and better treatments are in urgent need in clinic. In view of the important medical issues, reconstructing the damaged heart with new cardiomyogenic cells is an attractive method. Recent research in stem cells gives hope to patients with heart disease. Embryonic stem cells (ESCs) have the greatest developmental totipotency and the highest research value among stem cell types. The experimental methods of isolating and culturing ESCs in vitro have been well-established, and ESCs can be greatly expanded in culture and differentiated into definitive cardiomyocytes (Kehat et al., 2001; Xu et al., 2002a; Mummery et al., 2003). Cardiomyocytes induced from ESCs have powerful proliferative capacity both in vitro and in vivo following implantation (Snir et al., 2003; Laflamme et al., 2005;
Corresponding author: e-mail: [email protected] Junsheng Mu and Xianshuai Li contributed equally to this study.
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Cell Biol Int 38 (2014) 1098–1105 © 2014 International Federation for Cell Biology
J. Mu et al.
McDevitt et al., 2005), implying that delivery of an initially sub-therapeutic cell dose may suffice to obtain a functionally meaningful cardiac implant over time. Although the technology of inducing differentiation of hESCs and mESCs into cardiomyocytes are already available, there has been no comparative study of their directional differentiation into cardiomyocytes. We have compared four different strategies of inducing hESCs and mESCs differentiation into cardiomyocytes to provide an experimental basis for study of inducing differentiation of ESCs into cardiomyocytes in vitro. Materials and methods
Propagation of hESC and mESC lines The X-01 hESC line was obtained from Shanghai Si Dan Sai Biotechnology Co Ltd, and the R1 mESC line was obtained from Shanghai Institutes for Biological Sciences. Continuous cultures of the X-01 cell line was grown on 0.2% gelatincoated, mitomycin C (Sigma; 10 mg/mL) inactivated mouse embryonic fibroblast (MEF) feeders in standard hESC culture medium consisting of 80% KO-DMEM (Gibco), 20% serum replacement (SR, Gibco), 1% non-essential amino acid solution (Gibco), 1 mM L-glutamine (Gibco), 0.1% bmercaptoethanol (Sigma), 4 ng/mL human basic fibroblast growth factor (bFGF, Gibco). Media was replenished every day with media pre-equilibrated in an incubator for 2 h at 37 C and 5% CO2 and cells were passaged every fifth/sixth day by incubation in 200 units/mL collagenase IV for 5– 10 min at 37 C and then dissociated. The cultures were maintained at 37 C and 5% CO2 in air and used for characterization and differentiation studies. MEF feeders were cultured in DMEM High Glucose (Gibco) supplemented with 10% FBS (Gibco), 1% non-essential amino acid solution, 1 mM L-glutamine. Similarly, continuous cultures of the R1 cell line was grown on 0.2% gelatin-coated, mitomycin C inactivated MEF feeders in a standard mESC culture medium consisting of 85% DMEM (Gibco), 15% EScell qualified FBS (Gibco), 1% non-essential amino acid solution, 1 mM L-glutamine, 0.1% b-mercaptoethanol and leukemia inhibitory factor (LIF). Media was replenished every day with media pre-equilibrated in an incubator for 2 h at 37 C and air plus 5% CO2 and cells were passaged every second/third day by incubation in 0.05% trypsin for 3–4 min at 37 C and then dissociated. Protocol 1: Differentiation of cardiomyocytes via EB formation (hESC without inducers group) Undifferentiated hES cells were dissociated into clumps using 200 U/mL collagenase IV at 37 C for 5–10 min and cultured in suspension using low attachment plates (Corning) to form EBs. The differentiation medium
Directional differentiation of human and mouse ESCs
contained 80% KO-DMEM, 20% FBS, 1% non-essential amino acid solution, 1 mM L-glutamine, 0.1% b-mercaptoethanol. After 4 days in suspension, EBs were plated on 0.2 Matrigel (BD) coated plates to score the contracting cardiac clusters and record the beating rate. The number of days of differentiation includes the days in which the cells were maintained in suspension. For example, differentiation day 10 relates to cells that were maintained in suspension for 4 days, plated and cultured for an additional 6 days. Protocol 2: Differentiation of cardiomyocytes via adherent culture (hESC with inducers group) Undifferentiated hES cells were dissociated into clumps using 200 U/mL collagenase IV at 37 C for 5–10 min and seeded onto Matrigel-coated plates at 100,000 cells/cm2. Medium were refreshed daily with MEF-CM plus 8 ng/mL bFGF for 6 days. To induce cardiac differentiation, we replaced MEF-CM with RPMI-B27 medium (Gibco) supplemented with the following cytokines: 100 ng/mL human recombinant activin A (Gibco) for 24 h, followed by 10 ng/mL human recombinant BMP4 (Gibco) for 4 days. The medium was exchanged for RPMI-B27 without supplementary cytokines; culture medium was refreshed every 2–3 days for 2–3 additional weeks. Widespread spontaneous beating activity was typically observed by day 12 after addition of activin A. Protocol 3: Differentiation of cardiomyocytes via EB formation (mESC without inducers group) Undifferentiated mES cells were dissociated into clumps using 0.05% trypsin for 3–4 min at 37 C and seeded at 7000 cells/cm2 in 0.2% gelatin-coated cell culture plates with medium without LIF, and for inducing EB formation, EBs were formed in hanging drops of 400 cells in 20 mL of medium without LIF. After 2 days, EBs were plated on gelatin-coated dishes in standard mESC culture medium without LIF and vitamin C. Protocol 4: Differentiation of cardiomyocytes via EB formation (group of mESC with inducers) This protocol was the same as protocol 3 except the last step. After 2 days, EBs were plated on gelatin-coated dishes in standard mESC culture medium without LIF but with the addition of vitamin C.
Immunostaining For immunostaining, ESC were induced to differentiate into beating colonies through the four protocols. Following this, beating colonies were fixed in 4% paraformaldehyde for 10 min, washed three times with PBS (Gibco), and permeabilized with 0.4% triton X-100 at RT for 10 min. Colonies were then washed three times with PBS, blocked
Cell Biol Int 38 (2014) 1098–1105 © 2014 International Federation for Cell Biology
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with 5% bovine serum albumin (BSA) for 30 min, followed by incubation with primary antibody against cTnT at 4 C overnight. After washing, the colonies were exposed to the corresponding secondary antibody at RT for 45 min. The colonies were then washed again, and mounted with Vectashield medium for photomicroscopy.
of 330–380 nm. Apoptotic cells were identified on the basis of morphologic changes in their nuclear assembly by observing chromatin condensation and fragmentation by Hoechst 33258 staining. In each group, 10 microscopic fields were randomly selected and counted.
Statistical analysis Electrophysiological recordings Beating colonies were disassociated into isolated cardiomyocytes by collagenase or trypsin, and after culturing for 36 h, beating cardiomyocytes were selected for patch-clamp experiments. Spontaneous action potentials of cardiomyocytes were recorded by current-clamp technique with an Axon-200A amplifier. The glass microelectrodes were filled with a solution containing: 50 mM KCl, 80 mM Kasp, 1 mM MgCl2, 3 mM MgATP, 10 mM HEPES, 10 mM EGTA (pH 7.4 with KOH), the electrode resistance was between 2 and 4 MV. Cells were superfused with a bathing solution that contained: 140 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM D-glucose (pH 7.4 with NaOH). Experiments were conducted at 37 C.
Hoechst 33258 staining A Hoechst 33258 cell apoptosis staining kit (Beyotime) was used to confirm morphological changes in the nuclei. Cells were fixed with 4% paraformaldehyde for 10 min at RT, washed three times with cold PBS and treated with 10 mg/L Hoechst 33258 in the dark for 5 min. The cells were washed with PBS and immediately photographed under a fluorescence microscope at an excitation wavelength
All experiments were performed at least three times, and data were expressed as mean standard deviation and analyzed by Student’s t-test or one-way ANOVA with post hoc analysis. P < 0.05 was considered statistically significant. Results
Generation of beating cardiomyocytes All four methods successfully induced differentiation of ESCs into cardiomyocytes. hESC clones had clear boundaries, flat nest-like shapes, and high nuclear:cytoplasmic ratios (Figure 1A). hESCs grew relatively slowly, with a passage time of 5–6 days. While the mESC clone showed morphological diversity, relative density, large nuclei, scant cytoplasm, and a small number of differentiated cells could sometimes be seen in the marginal zone whose morphology was like that of epithelial cells or spindle-shaped fibroblasts (Figure 1B). mESCs grew relatively fast, and the passage time was 2–3 days. Embryoid bodies (EBs) began to adhere at 2– 3 h after plating and completely adhered within 24 h. Day 2 EBs had fully expanded into flat shapes that extended outward, with the middle part slightly raised (Figures 1C and 1D). After 7 days differentiation, beating colonies began
Figure 1 (A) hESC clone; (B) mESC clone; (C) the second day of differentiating of hESC clone; (D) the second day of differentiating of mESC clone; (E) beating colony induced from hESC (video has been taken); (F) beating colony induced from mESC (video has been taken).
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to appear, and most EBs were very widely spread. The thick inner section of the EBs were unequal and there were sometimes multiple beating areas with different sizes, frequencies, and amplitudes (Figures 1E and 1F). Videos have also been recorded.
Immunostaining and electrophysiological recordings After ESCs were induced to differentiate into beating colonies, they were immunofluorescently stained for the cardiomyocyte-specific marker cTnT. Almost the entire beating colony was positive for cTnT (Figures 2A and Figure 2). Beating colonies were disassociated into isolated cardiomyocytes for patch-clamp experiments, and spontaneous action potentials were recorded from atria-like cardiomyocytes (Figures 2C and 2D). Action potentials were recorded from two clusters. Figure 2C is a spontaneous action potential of atria-like cardiomyocytes induced from mESC, while in Figure 2D it is from atria-like cardiomyocytes induced from hESC. However, the morphology of action potentials varied from different cell lines and differentiation conditions. Action potentials here show that the cardiomyocytes induced from both cell lines have electrophysiological function.
Directional differentiation of human and mouse ESCs
Comparison of the differentiation efficiency human and mouse ESCs into cardiomyocytes One EB was inoculated per well, and a total of 96 EBs were inoculated for each group. From day 5 following adherent differentiation, we measured the efficiency of differentiation. In the hESC without inducers group, the mean time to appearance of beating cardiomyocytes was 13.9 0.9 days, the percentage was 20.8% and the average beating frequency was 63.8 5.6 times/min. In the hESC with inducers group, the mean time to appearance of beating cardiomyocytes was 13.0 1.1 days, the percentage was 66.7% and the average beating frequency was 63.0 7.0 times/min; in the mESC without inducers group, the mean time to appearance of beating cardiomyocytes was 14.3 1.0 days, the percentage was 12.5% and the average beating frequency was 80.2 3.9 times/min; in the mESC with inducers group, the mean time to appearance of beating cardiomyocytes was 12.2 1.2 days, the percentage was 81.25% and the average beating frequency was 79.9 7.7 times/min (Figure 3). The statistics show that the groups with inducers improved the differentiation efficiency more significantly than the groups without inducers, as the mean time to the
Figure 2 (A and B) Light field of beating colony induced from hESC and its immunofluorescence staining of cTnT; (C) spontaneous action potential of atria-like cardiomyocytes induced from mESC; (D) spontaneous action potential of atria-like cardiomyocytes induced from hESC.
Cell Biol Int 38 (2014) 1098–1105 © 2014 International Federation for Cell Biology
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Directional differentiation of human and mouse ESCs
Figure 3 (A) Comparison of the average time of appearing beating colonies in each group, *P-values are
