Exogenous expression of human apoA-I enhances cardiac differentiation of pluripotent stem cells - PubMed (original) (raw)
Exogenous expression of human apoA-I enhances cardiac differentiation of pluripotent stem cells
Kwong-Man Ng et al. PLoS One. 2011.
Abstract
The cardioprotective effects of high-density lipoprotein cholesterol (HDL-C) and apolipoprotein A1 (apoA-I) are well documented, but their effects in the direction of the cardiac differentiation of embryonic stem cells are unknown. We evaluated the effects of exogenous apoA-I expression on cardiac differentiation of ESCs and maturation of ESC-derived cardiomyocytes. We stably over-expressed full-length human apoA-I cDNA with lentivirus (LV)-mediated gene transfer in undifferentiated mouse ESCs and human induced pluripotent stem cells. Upon cardiac differentiation, we observed a significantly higher percentage of beating embryoid bodies, an increased number of cardiomyocytes as determined by flow cytometry, and expression of cardiac markers including α-myosin heavy chain, β-myosin heavy chain and myosin light chain 2 ventricular transcripts in LV-apoA-I transduced ESCs compared with control (LV-GFP). In the presence of noggin, a BMP4 antagonist, activation of BMP4-SMAD signaling cascade in apoA-I transduced ESCs completely abolished the apoA-I stimulated cardiac differentiation. Furthermore, co-application of recombinant apoA-I and BMP4 synergistically increased the percentage of beating EBs derived from untransduced D3 ESCs. These together suggests that that pro-cardiogenic apoA-I is mediated via the BMP4-SMAD signaling pathway. Functionally, cardiomyocytes derived from the apoA-I-transduced cells exhibited improved calcium handling properties in both non-caffeine and caffeine-induced calcium transient, suggesting that apoA-I plays a role in enhancing cardiac maturation. This increased cardiac differentiation and maturation has also been observed in human iPSCs, providing further evidence of the beneficial effects of apoA-I in promoting cardiac differentiation. In Conclusion, we present novel experimental evidence that apoA-I enhances cardiac differentiation of ESCs and iPSCs and promotes maturation of the calcium handling property of ESC-derived cardiomyocytes via the BMP4/SMAD signaling pathway.
Conflict of interest statement
Competing Interests: The authors have declared that no competing interests exist.
Figures
Figure 1. Expression of human apoA-I in D3 mouse ESCs.
Undifferentiated ESCs were cultured in the absence of a feeder layer and transduced with lentiviral particles containing the full-length wild type apoA-I cDNA under the control of CMV promoter. This expression cassette was linked to the IRES-GFP reported cassette for the identification of transduced cells, while the lentiviral particles containing the empty construct served as a control. To evaluate the success of gene transfer, undifferentiated empty construct- and apoA-I-transduced cells were examined for expression of GFP (A). The secretion of apoA-I in the concentrated spent medium was determined by Western blot analysis using an antibody specific to apoA-I.
Figure 2. Effect of apoA-I gene transfer on generation of beating embryoid body and cardiac cells.
(A) Empty construct and apoA-I-transduced mouse ESCs were differentiated using the conventional “hanging-drop” method, and the resultant embryoid bodies (EBs) were plated onto gelatin coated plates. The occurrence of beating areas within the EBs was observed and counted for 8 days starting from the day of plating. (B) Percentage of ESC-derived cardiomyocytes (troponin-T positive cells) on day 8 as determined by flow cytometry. (C) Individual cardiomyocytes were isolated from the beating area of the EBs and identified with immunnohistochemistry using antibody specific to the cardiac troponin-T. (D) Cardiac maker gene expression in the embryoid bodies derived from empty construct- and apoA-I-transduced ESCs as revealed by real-time quantitative PCR analysis. MHCA: α-myosin heavy chain; MHCB: β-myosin heavy chain; MLC2V: myosin light chain 2 ventricular transcripts. Data shown as mean ± SEM from at least 3 independent experiments, n = 3–5, *p<0.05; **p<0.005.
Figure 3. apoA-I gene transfer activates the BMP4-SMAD1/5 signaling cascade in undifferentiated ESCs.
(A) The mRNA levels of bone morphogenic protein 4 (BMP4), activin receptor-like kinase 1 (ALK1) and activin receptor-like kinase 2 (ALK2) in the undifferentiated ESCs transduced with empty construct or apoA-I were evaluated by real-time quantitative PCR analysis using ribosomal protein S16 as internal control. (B) The phosphorylation status and total protein levels of the SMAD proteins were evaluated with Western blot analysis using β-actin as loading control. (C) The densitometry quantification of the Western blot results. Data shown as mean ± SEM from 3 independent experiments,* p<0.05; ** p<0.005.
Figure 4. Inhibition of the BMP4 signaling pathway abolished the pro-cardiogenic effects of apoA-I gene transfer.
The BMP4 signaling pathway was inhibited by the application of noggin (1 µ/ml) to the apoA-I- transduced ESCs upon plating. (A) The phosphorylation status of the differentiating cells was evaluated 24 hr after noggin treatment. (B) The appearance of beating clusters during the 8-day period of differentiation. (C) The percentage of troponin-T positive cells as determined by flow cytometry analysis. Data shown as mean ± SEM from 3 independent experiments, # p<0.05 comparing to the LV-apoA-I group; *p<0.05 and ** p<0.005 comparing to the LV-GFP group.
Figure 5. Synergistic effect of recombinant apoA-I and BMP4 on the cardiac differentiation of D3 ESCs.
Untransduced D3 ESCs were subjected to cardiac differentiation in the presences or absence of recombinant apoA-1 (100 nM) and/or BMP4 (0.5 ng/ml). The appearance of beating clusters during the 8-day period of differentiation were recorded. Data shown as mean ± SEM from 3 independent experiments, ** p<0.005 comparing to the control group.
Figure 6. Calcium handling property of the cardiomyocytes derived from empty construct- and apoA-I-transduced ESCs.
(A) Representative tracings of rhythmic spontaneous Ca2+ transients in cardiomyocytes derived from empty construct- and apoA-I- transduced cells. (B): Amplitude, (C) Maximal upstroke velocity (Vmax upstroke), (D) Maximal decay velocity (Vmax decay) of Ca2+ transients in the mESC-derived cardiomyocytes. (E) Representative tracings of caffeine-induced Ca2+ release from sarcoplasmic reticulum in cardiomyocytes derived from wild type, empty construct and apoA-I-1α transduced cells (right), demonstrating caffeine-sensitive Ca2+ stores and fractional release of total sarcoplasmic reticulum Ca2+ load during spontaneous activation. (F): Amplitude, (G) Maximal upstroke velocity (Vmax upstroke), (H) Maximal decay velocity (Vmax decay) of Ca2+ transients in the ESC-derived cardiomyocytes. Data shown as mean ± SEM from the recordings of 20–30 cells from 3–5 independent experiments, * p<0.05; ** p<0.005.
Figure 7. The expression of calcium handling components in cardiomyocytes.
The mRNA levels of sodium/calcium exchanger (NCX1), sarcoplasmic reticulum Ca2+ ATPase (SERCA2A) and ryanodine receptor 2 (RYR2) of embryoid bodies (at d8 after plating) were evaluated by real-time quantitative PCR analysis using ribosomal protein S16 as internal control. Data shown as mean ± SEM from 3 independent experiments, ** p<0.005.
Figure 8. Effect of apoA-I gene transfer on the cardiac differentiation of IMR90 human IPSCs.
(A) Percentage of ESC-derived cardiomyocytes (α-actinin (sarcomeric)-positive cells) 30 days after plating as determined by flow cytometry. (B) The beating EBs were digested into monolayers and the cardiomyocytes were identified with immunnohistochemistry using antibody specific to the cardiac troponin-T. Data shown as mean ± SEM from at least 3 independent experiments, *p<0.05; **p<0.005. Scale bar = 50 µm.
Figure 9. Calcium homeostasis in the cardiomyocytes derived from IMR90 (LV-GFP and LV-apoA-I).
(A) representative tracings of the calcium transient in the absence (control) or presence of ryanodine. (B) statistical analysis of the amplitude, calcium release rate and calcium re-uptake rates of the IMR90-derived cardiomyocytes in the absence of Ryanodine. (C) amplitudes and calcium release rates after ryanodine application normalized to values recorded under control ryanodine-free conditions. Data shown as mean ± SEM from the recordings of 15 cells from 3–5 independent experiments, ** p<0.005.
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