Ascorbic acid enhances the cardiac differentiation of induced pluripotent stem cells through promoting the proliferation of cardiac progenitor cells

Abstract

Generation of induced pluripotent stem cells (iPSCs) has opened new avenues for the investigation of heart diseases, drug screening and potential autologous cardiac regeneration. However, their application is hampered by inefficient cardiac differentiation, high interline variability, and poor maturation of iPSC-derived cardiomyocytes (iPS-CMs). To identify efficient inducers for cardiac differentiation and maturation of iPSCs and elucidate the mechanisms, we systematically screened sixteen cardiomyocyte inducers on various murine (m) iPSCs and found that only ascorbic acid (AA) consistently and robustly enhanced the cardiac differentiation of eleven lines including eight without spontaneous cardiogenic potential. We then optimized the treatment conditions and demonstrated that differentiation day 2-6, a period for the specification of cardiac progenitor cells (CPCs), was a critical time for AA to take effect. This was further confirmed by the fact that AA increased the expression of cardiovascular but not mesodermal markers. Noteworthily, AA treatment led to approximately 7.3-fold (miPSCs) and 30.2-fold (human iPSCs) augment in the yield of iPS-CMs. Such effect was attributed to a specific increase in the proliferation of CPCs via the MEK-ERK1/2 pathway by through promoting collagen synthesis. In addition, AA-induced cardiomyocytes showed better sarcomeric organization and enhanced responses of action potentials and calcium transients to β-adrenergic and muscarinic stimulations. These findings demonstrate that AA is a suitable cardiomyocyte inducer for iPSCs to improve cardiac differentiation and maturation simply, universally, and efficiently. These findings also highlight the importance of stimulating CPC proliferation by manipulating extracellular microenvironment in guiding cardiac differentiation of the pluripotent stem cells.

Figure 1

AA robustly enhances cardiogenesis of 3F and 4F miPSCs. (A) Concentration-dependent relationships of AA. Data were collected at day 10. (B) Time windows for AA-promoted cardiac differentiation. Left panel, schematic diagram of the differentiation protocols; middle and right panels, corresponding efficiency in 3F and 4F lines. (C) Differentiation profile of cardiomyocytes in both iPSC lines. (D) Percentages of cTnT⁺ cardiomyocytes at day 15 in the total population derived from both iPSC lines with or without AA treatment. n = 3 each. Data are expressed as means ± SEM. ^*P < 0.05, ^**P < 0.01 vs control.

Figure 2

AA increases the content and improves the sarcomeric organization of iPS-CMs. (A) Representative images showing increased beating areas (a-d) and the content of α-actinin⁺ (e-h) or cTnT⁺ (i-l) cardiomyocytes in day-10 EBs treated with AA. Scale bars = 100 μm. (B) Sarcomeric structure analysis of the day-18 iPS-CMs by α-actinin and cTnT staining. The insets were magnifications of the framed areas showing more organized cross-striation alignment of sarcomeres in AA-induced iPS-CMs. Scale bars = 25 μm. Nuclei were counterstained with Hoechst33258 (blue).

Figure 3

AA increases the expression of cardiac genes. (A) RT-PCR analysis showing the particular upregulation of cardiac genes in AA-stimulated EBs compared with the untreated ones. (B) Quantitative RT-PCR analysis indicated the increased expression of cardiac progenitor but not mesodermal transcripts (n = 3). Data are expressed as means ± SEM. ^*P < 0.05, ^**P < 0.01 vs control.

Figure 4

Rescue of innate cardiac deficiency by AA treatment. (A) AA induced the development of contracting EBs in other 5 iPSC lines without spontaneous cardiogenic potential. Data were collected at day 10. (B) Differentiation profile of cardiomyocytes in three representative iPSC lines. (C) Quantitative RT-PCR analysis of the expression level of cardiac genes. (D) Immunostaining showing the only emergence of α-actinin⁺ and cTnT⁺ cardiomyocytes in AA-treated EBs at day 10. Scale bars = 100 μm. Nuclei were counterstained with Hoechst33258 (blue). Data are expressed as means ± SEM. ^*P < 0.05, ^**P < 0.01 vs control.

Figure 5

AA enhances the response of day 16-18 iPS-CMs to β-adrenergic and muscarinic stimulations. (A) Representative APs of AA-induced iPS-CMs showing characteristics of nodal-like, atrial-like, and ventricular-like APs. Dotted lines indicate 0 mV. (B) Comparison of Iso (10 nmol/l)-stimulated increases (left panel) and Cch (1 μmol/l)-induced decreases (right panel) in characteristics of APs between control and AA-applied iPS-CMs (n = 10-13). BF, beating frequency; DD, diastolic depolarization; APA, AP amplitude; V_max, maximum rise rate. (C) Representative tracings of spontaneous rhythmic Ca²⁺ transients with (grey) and without (black) Iso-simulation (10 nmol/l). (D) Iso-induced increases in basal [Ca²⁺]_i, APA, upstroke V_max, and decay rate of Ca²⁺ transients in control or AA-applied iPS-CMs (n = 12-13). Data are expressed as means ± SEM. ^*P < 0.05, ^**P < 0.01 vs control.

Figure 6

AA-enhanced collagen synthesis is required for its cardiomyocyte-promoting effect. (A) Immunostaining of Col IV at day-6 EBs treated with or without AA and collagen synthesis inhibitor AzC and CIS. (B and C) Cardiomyocyte-promoting effect of AA was abolished by AzC and CIS, and could be partially rescued by Col IV. Scale bars = 100 μm. Nuclei were counterstained with Hoechst33258 (blue). Data are expressed as means ± SEM. ^*P < 0.01 vs corresponding values.

Figure 7

AA specifically enhances the proliferation of CPCs in a collagen synthesis-dependent manner. (A) Immunostaining of cTnT and BrdU in day-10 iPS-CMs. Data were quantified from 6-8 random fields in two assays. Scale bars = 25 μm. Nuclei were counterstained with PI (red). (B) Double staining of Nkx2-5 and BrdU at day-5 (n = 3). (C) Immunostaining of Mef2c and BrdU in day-8 CPCs (3 days after sorting). Data were quantified from five random fields in two assays. Scale bars = 100 μm. (D) Effects of signal inhibitors on proliferation of Nkx2-5⁺ CPCs and percentage of contracting EBs (n = 3). Data are expressed as means ± SEM. ^*P < 0.01 vs corresponding values. ns, no significant difference.

Figure 8

AA promotes cardiogenesis of human iPSCs (hiPSCs). (A) Morphology (a) and alkaline phosphatase staining (b) of undifferentiated hiPSCs colonies. (B) Percentages of contracting EBs during differentiation. (C) Percentages of cTnT⁺ cardiomyocytes in hiPSC-derived EBs at day 30. (D, E) Quantitative RT-PCR (D) and immunostaining (E) analysis showed the remarkable increases in expression of cardiac markers following AA treatment (n = 3). (F) Time windows for AA-promoted cardiogenesis. Scale bars = 100 μm. Nuclei were counterstained with Hoechst33258 (blue). ^*P < 0.05, ^**P < 0.01 vs control.