Timely inhibition of Notch signaling by DAPT promotes cardiac differentiation of murine pluripotent stem cells

Abstract

The Notch signaling pathway plays versatile roles during heart development. However, there is contradictory evidence that Notch pathway either facilitates or impairs cardiomyogenesis in vitro. In this study, we developed iPSCs by reprogramming of murine fibroblasts with GFP expression governed by Oct4 promoter, and identified an effective strategy to enhance cardiac differentiation through timely modulation of Notch signaling. The Notch inhibitor DAPT (N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester) alone drove the iPSCs to a neuronal fate. After mesoderm induction of embryoid bodies initiated by ascorbic acid (AA), the subsequent treatment of DAPT accelerated the generation of spontaneously beating cardiomyocytes. The timed synergy of AA and DAPT yielded an optimal efficiency of cardiac differentiation. Mechanistic studies showed that Notch pathway plays a biphasic role in cardiomyogenesis. It favors the early-stage cardiac differentiation, but exerts negative effects on the late-stage differentiation. Therefore, DAPT administration at the late stage enforced the inhibition of endogenous Notch activity, thereby enhancing cardiomyogenesis. In parallel, DAPT dramatically augmented the expression of Wnt3a, Wnt11, BMP2, and BMP4. In conclusion, our results highlight a practicable approach to generate cardiomyocytes from iPSCs based on the stage-specific biphasic roles of Notch signaling in cardiomyogenesis.

Figure 1. Derivation of iPSCs using an inducible lentiviral system.

(A) Schematic representation of iPSC generation. (B) RT-PCR analysis of exogenous Yamanaka factors, ES pluripotent markers in fully and partially reprogrammed iPSCs (iPS-Dox6d, iPS-Dox12d). Parental MEFs and ESCs were used as negative and positive controls. (C) Immunofluorescence analysis of pluripotent markers, Nanog and SSEA-1 (Red). Oct4-GFP⁺ cells (Green) represented fully reprogramming iPSCs. Scale bar 50 µm. (D) Western blot analysis confirmed the pluripotent properties of iPSCs expressing Oct4, Nanog and Sox2. Parental MEF and ES cells were used as negative and positive controls. (E) Teratoma formation of iPSCs transplanted into immunodeficient mice. After 4 weeks, iPSC-derived tumors were sectioned and stained with hematoxylin and eosin. Shown were neural (ectoderm), muscle (mesoderm) and glandular (endoderm) tissues from left to right. Scale bar 100 µm. (F) Karyotyping analysis showing normal karyotyping of iPSCs after passage 10.

Figure 2. AA induces cardiac differentiation of iPSCs.

(A) iPSCs were cultured in hanging drops and induced with AA. With the disappearance of Oct4-drived GFP expression, beating cardiomyocytes were observed at day 12 and increased until day 16 of iPSC differentiation, as indicated by closed dashed lines. Scale bar 50 µm. (B) Immunofluorescence analysis of iPSC-derived cardiomyocytes at day 16 with a monoclonal antibody against sarcomeric α-actinin (Red). Undifferentiated iPSCs were identified by GFP expression droven by Oct4-promoter (Green). Scale bar 50 µm. (C) Cardiac-specific gene expression profiles were analyzed by RT-PCR during iPSC differentiation at indicated time points. 18S rRNA expression was used as an internal control. (D) Relative expression levels of several Notch pathway members were analyzed by real-time RT-PCR. Data are shown as relative gene expression with means ± s.d (n = 3) (*p<0.05 vs control group at day 0).

Figure 3. DAPT alone instructs neuronal differentiation of iPSCs.

(A) Schematic diagram of differentiation protocol with DAPT. (B) Differentiated iPSCs induced by DAPT alone exhibited neural phenotypes with a more spindle-shaped morphology from day 12 to day 16. Scale bar 50 µm. (C) Immunofluorescence analysis of DAPT-treated iPSCs at day16 with neural (Nestin and Pax6) or cardiac markers (α-actinin). Nuclei were counterstained with Hoechst33342 (blue). Scale bars 50 µm. (D) RT-PCR analysis for cardiac markers (GATA4, Nkx2.5, α-MHC, β-MHC) as well as neural markers (Nestin, Pax6) during DAPT induction at indicated time points. 18S rRNA expression was used as an internal control. Representative results were from three independent experiments.

Figure 4. DAPT promotes cardiac differentiation from intermediate mesoderm.

(A) Experimental strategies to optimize the induction procedure of four groups designated as G1 to G4. (B) The percentage of contracting EBs was counted at day 8, day 12 and day 16 among these groups. Data obtained from three independent experiments were shown as means ± s.d (*p<0.05, **p<0.01). (C) Immunofluorescence staining of α-actinin (Red) in AA (G3) and AA plus DAPT (G4) groups at day 16. GFP-positive expression indicated the existence of endogenous Oct4, representing undifferentiated iPSCs. Nuclei were counterstained with Hoechst33342 (Blue). Scale bar 50 µm. (D) Immunofluorescence staining of Tropnin T (Red) in AA (G3) and AA plus DAPT (G4) groups at day 16. Nuclei were counterstained with Hoechst33342 (Blue). Scale bar 50 µm. (E) Percentage of Troponin T-positive cells was calculated from at least five randomly selected fields at day 16. Data obtained from three independent experiments were shown as means ± s.d (*p<0.05). (F) The expression of α-actinin, Oct4, and Nanog was determined by Western blot analysis. Results are representative of three independent experiments.

Figure 5. DAPT elevates the expression of cardiac transcriptional factors.

(A) After AA induction for 4 days, iPSCs were subsequently treated by AA or AA plus DAPT. Real-time PCR analysis was performed to estimate the relative level of cardiac-specific and pluripotency-associated genes. (B) Real-time PCR analysis of mesoderm, cardiac progenitor and cardiac-specific transcription factor markers. Data are shown as relative gene expression with means ± s.d (n = 3) (*p<0.05, **p<0.01).

Figure 6. Notch1 promotes the expression of marker genes for the early-stage cardiac differentiation.

(A) Immunoblot analysis showed the protein levels of Notch1 during iPSCs differentiation into cardiomyocytes. β-actin was used as an internal control (upper panel). The band intensity for Notch1 was normalized with that of β-actin and presented as relative intensity (lower panel) (*p<0.05 vs day 0). (B) Overexpression of NICD1 promoted the expression of Isl1 and GATA4. After 24 h of transfection, the iPSCs were cultured in hanging drops with AA induction for 4 days. The mRNA level of Isl1 and GATA4 was measured and normalized to 18S rRNA gene. (*p<0.05 vs mock group). (C) Real-time PCR was performed to determine RNAi efficiency mediated by three pairs of Notch1 RNAi duplexes (upper panel). The expression of Isl1 and GATA4 was evaluated under the condition of Notch1 knock-down by RNAi-2 duplexes (lower panel). The iPSCs were treated as the same method in B. (D) DAPT treatment during the early stage of cardiac differentiation inhibited the expression of Isl1 and GATA4. The iPSCs were cultured with AA and DAPT in hanging drops for 4 days. Notch1 protein and the expression of Isl1 and GATA4 mRNA were measured by immunoblot (upper panel) or real-time PCR (lower panel), respectively. All data are shown as means ± s.d (n = 3) (*p<0.05, **p<0.01).

Figure 7. DAPT promotes the late-stage cardiac differentiation by Notch inhibition.

(A) Immunoblot analysis demonstrated the protein amount of Notch1. After AA induction for 4 days, iPSCs were subsequently treated by AA in the presence or absence of DAPT during the cardiomyocyte differentiation. Cell lysates were analyzed for Notch1 or β-actin (upper panel). Normalized densitometric quantification of Notch1 bands was performed with images of three independent experiments (lower panel). (B) Real-time PCR analysis was performed to determine the expression of Notch family genes expression at indicated time points. (C) and (D) Real-time PCR analysis of Wnt3a, Wnt11, BMP2, and BMP4 expression. (E) Immunoblot analysis demonstrated the protein amount of β-catenin. β-actin was used as the internal control. All data are shown as means ± s.d (n = 3) (*p<0.05, **p<0.01).