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. 2013 Oct;35(5):1545-57.
doi: 10.1007/s11357-012-9457-z. Epub 2012 Jul 28.

Anti-aging Effects of Vitamin C on Human Pluripotent Stem Cell-Derived Cardiomyocytes

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Free PMC article

Anti-aging Effects of Vitamin C on Human Pluripotent Stem Cell-Derived Cardiomyocytes

Yoon Young Kim et al. Age (Dordr). .
Free PMC article

Abstract

Human pluripotent stem cells (hPSCs) have arisen as a source of cells for biomedical research due to their developmental potential. Stem cells possess the promise of providing clinicians with novel treatments for disease as well as allowing researchers to generate human-specific cellular metabolism models. Aging is a natural process of living organisms, yet aging in human heart cells is difficult to study due to the ethical considerations regarding human experimentation as well as a current lack of alternative experimental models. hPSC-derived cardiomyocytes (CMs) bear a resemblance to human cardiac cells and thus hPSC-derived CMs are considered to be a viable alternative model to study human heart cell aging. In this study, we used hPSC-derived CMs as an in vitro aging model. We generated cardiomyocytes from hPSCs and demonstrated the process of aging in both human embryonic stem cell (hESC)- and induced pluripotent stem cell (hiPSC)-derived CMs. Aging in hESC-derived CMs correlated with reduced membrane potential in mitochondria, the accumulation of lipofuscin, a slower beating pattern, and the downregulation of human telomerase RNA (hTR) and cell cycle regulating genes. Interestingly, the expression of hTR in hiPSC-derived CMs was not significantly downregulated, unlike in hESC-derived CMs. In order to delay aging, vitamin C was added to the cultured CMs. When cells were treated with 100 μM of vitamin C for 48 h, anti-aging effects, specifically on the expression of telomere-related genes and their functionality in aging cells, were observed. Taken together, these results suggest that hPSC-derived CMs can be used as a unique human cardiomyocyte aging model in vitro and that vitamin C shows anti-aging effects in this model.

Figures

Fig. 1
Fig. 1
Schematic representation of experiments. The definition and features of CMs derived from hPSCs, which include hESCs and hiPSCs, are indicated for each temporal stage. Representative pictures of beating and aged CMs are included for illustration. a A representative image of beating CMs. The contractile region is indicated by the red arrow. b The morphology of aged CMs. As the in vitro culture period lengthened, the differentiated cells showed a darker color in the center region and the beating rate decreased. c Sequential moving images of beating CMs are temporally arranged from left to right, and the time is indicated at the bottom-left of each image in red
Fig. 2
Fig. 2
Evaluation of cardiac characteristics in hPSC-derived CMs. The expression of cardiac-specific genes was evaluated at the mRNA and protein level. Each stage of differentiation in hPSCs demonstrated expression of critical cardiac cascade genes. A Immunostaining of hPSC-derived CMs. Expression of cardiac specific genes in hESC-derived (a–e) and hiPSC-derived CMs (f–j) at each stage. Cardiac transcription factor Nkx2.5, protein for contraction cardiac troponin I (cTnI), structural protein αMHC, and the cardiomyocyte secreting protein ANF were immunolabeled and evaluated using laser scanning microscopy. Images were captured at 200× magnification (insets) and enlarged images at 800× magnification are represented. a, f Stage 1(day 12), Nkx2.5 (green), cTn I (red); b, g stage 2 (day 18), Nkx2.5 (green), αMHC (red); d, i stage 3 (day 24), Nkx2.5 (green), ANF (red). Images of replated hESC-derived (c, e) and hiPSC-derived CMs (h, j) were captured at 200× magnification (insets) and enlarged images at 1,200× magnification are represented. c, h Stage 2 (day 18), Nkx2.5 (green), αMHC (red); e, j stage 3 (day 24), Nkx2.5 (green), ANF (red). B Expression of cardiac specific genes in hPSC-derived CMs using qRT-PCR. Expression level was normalized to GAPDH and run in triplicate. C Positive populations for Nkx2.5 and αMHC were evaluated. More than 60 % of differentiated cells were positive for Nkx2.5 and αMHC as measured by FACS analysis
Fig. 3
Fig. 3
Aging phenomenon in hPSC-derived CMs. Various assays were used for the evaluation of aging-related phenomena. A SA-β-gal staining was performed in each stage of hPSC-derived CMs. Beta-gal-stained cells were observed under the microscope, and small (40× magnification, inset) and enlarged images (100× magnification) are represented: a–d hESC-derived CMs; e–h hiPSC-derived CMs. a, e Beta-gal-stained CMs in a whole plate; b, f stage 1 (day 12); c, g stage 2 (day 18); d, h stage 3 (day 24); i the number of SA-β-gal-stained cells was counted under a microscope. As indicated by the drawn bars, the number of positively stained cells increased as in vitro differentiation progressed and this number increased significantly through stage 2 (day 18) and stage 3 (day 24). B Ultrastructural analysis of aged CMs using transmission electron microscopy. Observation of lipofuscin, an aging pigment, within aged cells at each stage was performed. The yellow asterisk indicates the prescence of pigment. a hESC-derived CM at day 18 (stage 2) showed faint precipitation of aging pigment; b hESC-derived CM at day 24 (stage 3) showed accumulated lipofuscin pigment spots; c hiPSC-derived CM at day 18 (stage 2) showed relatively abundant small spots of lipofuscin; d hiPSC-derived CM at day 24 (stage 3) showed larger, accumulated lipofuscin pigmentation. C Expression of aging-related genes in hPSC-derived CMs measured by qRT-PCR. The expression of hTR, a gene encoding the RNA components of telomerase, decreased in days 18 and 24 hESC-derived CMs. The expression of TRF2 also decreased in days 18 and 24 CMs. The expression pattern of hTR and TRF2 in hiPSC-derived CMs differed from hESC-derived CMs, as they did not demonstrate significantly decreased expression in stages 2 and 3. D Expression of cell cycle-related genes in hPSC-derived CMs measured by qRT-PCR. The expression of cyclin D1, cyclin D2, cyclin D3, and Cdk2 decreased with each progressing stage of differentiation. The expression of cyclin D3 and Cdk2 decreased as differentiation proceeded in both CMs
Fig. 3
Fig. 3
Aging phenomenon in hPSC-derived CMs. Various assays were used for the evaluation of aging-related phenomena. A SA-β-gal staining was performed in each stage of hPSC-derived CMs. Beta-gal-stained cells were observed under the microscope, and small (40× magnification, inset) and enlarged images (100× magnification) are represented: a–d hESC-derived CMs; e–h hiPSC-derived CMs. a, e Beta-gal-stained CMs in a whole plate; b, f stage 1 (day 12); c, g stage 2 (day 18); d, h stage 3 (day 24); i the number of SA-β-gal-stained cells was counted under a microscope. As indicated by the drawn bars, the number of positively stained cells increased as in vitro differentiation progressed and this number increased significantly through stage 2 (day 18) and stage 3 (day 24). B Ultrastructural analysis of aged CMs using transmission electron microscopy. Observation of lipofuscin, an aging pigment, within aged cells at each stage was performed. The yellow asterisk indicates the prescence of pigment. a hESC-derived CM at day 18 (stage 2) showed faint precipitation of aging pigment; b hESC-derived CM at day 24 (stage 3) showed accumulated lipofuscin pigment spots; c hiPSC-derived CM at day 18 (stage 2) showed relatively abundant small spots of lipofuscin; d hiPSC-derived CM at day 24 (stage 3) showed larger, accumulated lipofuscin pigmentation. C Expression of aging-related genes in hPSC-derived CMs measured by qRT-PCR. The expression of hTR, a gene encoding the RNA components of telomerase, decreased in days 18 and 24 hESC-derived CMs. The expression of TRF2 also decreased in days 18 and 24 CMs. The expression pattern of hTR and TRF2 in hiPSC-derived CMs differed from hESC-derived CMs, as they did not demonstrate significantly decreased expression in stages 2 and 3. D Expression of cell cycle-related genes in hPSC-derived CMs measured by qRT-PCR. The expression of cyclin D1, cyclin D2, cyclin D3, and Cdk2 decreased with each progressing stage of differentiation. The expression of cyclin D3 and Cdk2 decreased as differentiation proceeded in both CMs
Fig. 4
Fig. 4
The anti-aging effect of vitamin C on hESC-derived CMs. To reverse the aging phenomenon of hESC-derived CMs, vitamin C at 100 and 250 μM was added for 24 and 48 h. A SA-β-gal staining of day 24 hESC-derived CMs after treatment of vitamin C (100 μM). The number of SA-β-gal-stained cells and the intensity of staining were both significantly reduced. a, b β-gal-stained, hESC-derived CMs (150× magnification); c, d β-gal-stained, hESC-derived CMs following 24 h vitamin C treatment (150× magnification); e according to FACS measurement, the number of stained cells significantly decreased after vitamin C treatment. B Telomerase activity and expression of telomere-related genes hTR and hTERT at each stage of hESC-derived CM differentiation. a Telomerase activity in hESC-derived CMs. The product of each group was sequentially loaded. The left lane holds non-treated control and the right lane holds the respective vitamin C 100 μM 48 h-treated groups. b Expressions of telomerase-related genes, hTR and hTERT, and TRF2 were analyzed for each stage in hESC-derived CMs. Gene expression between the control group and vitamin C-treated group is compared
Fig. 5
Fig. 5
Alterations of functionality in hESC-derived CMs. The addition of vitamin C enhanced the mitochondrial function and beating frequency of hESC-derived aged CMs. A JC-1, which exhibits potential-dependent accumulation in mitochondria, was used as an indicator of mitochondrial potential. Vitamin C treatment significantly enhanced JC-1 red fluorescence (polarization, high membrane potential). Conversely, JC-1 green fluorescence (depolarization, low membrane potential) was reduced in hESC-derived CMs at day 24 when treated with vitamin C. a Control; b vitamin C 100 μM, 24 h; c vitamin C 100 μM, 48 h. B Population of JC-1 positive cells. JC-1 stained, hESC-derived CMs at day 24 were dissociated into single cells then immediately analyzed using flow cytometry. The red fluorescence-positive population increased when treated with vitamin C. a Control; b vitamin C 100 μM, 24 h; c vitamin C 100 μM, 48 h. C Effect of vitamin C on beating frequency of hESC-derived CMs. Treated CMs showed a smaller reduction in beating frequency compared to control

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