Int J Stem Cells.  2021 Nov;14(4):410-422. 10.15283/ijsc21051.

p53 Promotes Differentiation of Cardiomyocytes from hiPSC through Wnt Signaling-Mediated Mesendodermal Differentiation

• Liu Y
• Zhang P
• Huang W
• Liu F
• Long D
• Peng W
• Dang X
• Zeng X
• Zhou R

• KMID: 2522560
• DOI: http://doi.org/10.15283/ijsc21051

Abstract

Background and Objectives
Manipulating different signaling pathways via small molecules could efficiently induce cardiomyocytes from human induced pluripotent stem cells (hiPSC). However, the effect of transcription factors on the hiPSC-directed cardiomyocytes differentiation remains unclear. Transcription factor, p53 has been demonstrated indispensable for the early embryonic development and mesendodermal differentiation of embryonic stem cells (ESC). We tested the hypothesis that p53 promotes cardiomyocytes differentiation from human hiPSC.
Methods and Results
Using the well-characterized GiWi protocol that cardiomyocytes are generated from hiPSC via temporal modulation of Wnt signaling pathway by small molecules, we demonstrated that forced expression of p53 in hiPSC remarkably improved the differentiation efficiency of cardiomyocytes from hiPSC, whereas knockdown endogenous p53 decreased the yield of cardiomyocytes. This p53-mediated increased cardiomyocyte differentiation was mediated through WNT3, as evidenced by that overexpression of p53 upregulated the expression of WNT3, and knockdown of p53 decreased the WNT3 expression. Mechanistic analysis showed that the increased cardiomyocyte differentiation partially depended on the amplified mesendodermal specification resulted from p53-mediated activation of WNT3-mediated Wnt signaling. Consistently, endogenous WNT3 knockdown significantly ameliorated mesendodermal specification and subsequent cardiomyocyte differentiation.
Conclusions
These results provide a novel insight into the potential effect of p53 on the development and differentiation of cardiomyocyte during embryogenesis.

Keyword

hiPSC; Cardiomyocytes; p53; Wnt signaling

Figure

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  Fig. 1 Identification of the hiPSC. (A) Representative images displayed the typical morphology of hiPSC. (B) qRT-PCR results showed hiPSC has similar mRNA levels to the human embryonic stem cells (hESC) regarding the pluripotency marker genes (OCT4, SOX2, NANOG and KLF4). (C) Pluripotency markers of hiPSC (OCT4, NANOG, SSEA4 and TRA-1-60) were confirmed by immunofluorescence (IF) assay. (D) qRT-PCR results showed that hiPSC has similar mRNA levels to the hESC regarding the proliferation marker genes, MKI67 and AURKB. (E) MKI67 was further identified using IF method. Expression values of the PCR analysis were normalized to GAPDH. Data are presented as mean±SD (t test, NS not significant; n=3).

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  Fig. 2 Identification of the hiPSC derived cardiomyocytes. (A) Schematic illustration of an experimental protocol for the cardiomyocytes differentiation from hiPSC via sequential transient modulation of canonical Wnt signaling pathway using GiWi method. Gi, GSK3 inhibitor; Wi, Wnt inhibitor; iCM, induced cardiomyocytes; IF, immunofluorescence; FC, flow cytometry. (B) Time course analysis of the expression of cardiac progenitor marker (NKX2-5) and cardiomyocytes marker (TNNT2) was performed using qRT-PCR assay. (C) The protein of cardiac progenitor marker (NKX2-5) and cardiomyo-cytes marker (cTNT and α-ACTININ) was further evaluated by IF method. Data are presented as mean±SD.

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  Fig. 3 Overexpression of P53 gene in hiPSC by lentivirus methods. (A) Representative images of GFP fluorescence displayed highly efficient infection of hiPSC cells in both P53 and control group. (B) The qRT-PCR results showed that hiPSC with lenti-P53 caused about 8-fold increase of P53 mRNA compared with control group. (C) Representative image of Western Blot showed significantly higher p53 protein level of lenti-P53 group than control, and quantification was done by densitometry (D). (E) The qRT-PCR results showed that overexpressed P53 had no significant effects on the mRNA expression of the pluripotency marker (OCT4, SOX2, NANOG and KLF4) of hiPSC. (F, G) The Western Blot results showed that overexpressed P53 did not change OCT4 protein level but slightly decreased Nanog with statistical significance. Representative image was shown (F) and quantified by densitometry (G). (H) hiPSC with lenti-P53 displayed similar mRNA levels of proliferation markers (MKI67 and AURKB). (I, J) Representative image of cell cycle analysis using flow cytometry showed cell cycle was not influenced by lenti-P53 (I) and quantification by densitometry (J). Expression values of the PCR analysis were normalized to GAPDH. Data are presented as mean±SD (t test, *p<0.05, NS not significant; n=3).

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  Fig. 4 p53 increases the differentiation efficiency of cardiomyocytes from hiPSC. (A, B) The qRT-PCR results showed that overexpression of P53 significantly increased mRNA levels of the cardiomyocytes markers (TNNT2, TNNI3 and α-ACTNIN) (A) and also that of cardiac progenitor marker (NKX2-5) (B) at day 21. (C, D) Representative image of flow cytometry (C) displayed more yields of cardiomyocytes derived from hiPSC indicated by cTNT＋ population by lenti-P53 and quantification was performed (D). (E, F) It was showed using qRT-PCR method that p53 resulted in remarkably increased mRNA levels of mesendodermal markers (BRACHYURY and EOMES) at day 3 from differentiation (E) and cardiac mesodermal makers (MESP1 and KDR) at day 5 (F), respectively. Expression values of the PCR analysis were normalized to GAPDH. Data are presented as mean±SD (t test, *p<0.05; n=3, performed in 3 independent experiments).

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  Fig. 5 Knockdown of P53 gene decreases the cardiomyocytes generation from hiPSC. (A) The qRT-PCR results showing successful knockdown of P53 in HiPSC. (B) Representative image of Western Blot displayed significantly decreased p53 protein level of lenti-Sh-P53 group (B) and quantification was done by densitometry (C). (D, E) Knockdown of P53 significantly declined mRNA levels of makers of cardiomyocytes (TNNT2) (D) and cardiac progenitor (NKX2-5) (E). (F, G) Representative image of flow cytometry (F) displayed notably decreased cTNT＋ cardiomyocytes differentiated by hiPSC by knockdown of P53 in contrast with control，and quantification was done (G). (H, I) The qRT-PCR results showed that knockdown of P53 markedly reduced the mRNA levels of mesendodermal markers (BRACHYURY and EOMES) at day 3 from differentiation (E) and cardiac mesodermal makers (MESP1 and KDR) at day 5 (F), respectively. Expression values of the PCR analysis were normalized to GAPDH. Data are presented as mean±SD (t test, *p<0.05; n=3, performed in 3 independent experiments).

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  Fig. 6 Enhanced mesendodermal differentiation mediated by Wnt signaling pathway partially accounted for the effects of p53 on the cardiac differentiation from hiPSC. (A) The qRT-PCR results showing hiPSC with lenti-P53 had raised mRNA level of WNT3. It was further confirmed by representative image of Western Blot (B) and quantification by densitometry (C). (D) The mRNA level of WNT3 in hiPSC was decreased by knockdown of P53 as shown by qRT-PCR results. (E) Representative image of Western Blot displayed notably reduced protein level of WNT3 caused by knockdown of P53, and quantification was done by densitometry (G∼I). WNT3 gene was successfully downregulated by Sh-WNT3 using qRT-PCR (G) and Western Blot analysis (H, I). (J, K) The qRT-PCR results showed that Sh-WNT3 notably reversed P53-caused upregulation of mesendodermal markers (BRACHYURY and EOMES) (J) and cardiac mesodermal makers (MESP1 and KDR) (K) at day 3 and 5 from differentiation respectively. (L, M) The qRT-PCR results showed that knockdown of WNT3 significantly abrogated P53-dependent increased mRNA levels of mesendodermal markers (BRACHYURY and EOMES) at day 3 from differentiation (L) and cardiac mesodermal makers (MESP1 and KDR) at day 5 (M). Expression values of the PCR analysis were normalized to GAPDH. Data are presented as mean±SD (t test, *p<0.05; n=3, performed in 3 independent experiments).

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  Fig. 7 Upregulated WNT3 by p53 could further amplify the activation of Wnt signaling pathway caused by GSK inhibition (Gi). However, the amplified Wnt pathway by p53 was completely blocked by Wnt inhibitor (Wi) at late stage. (A, B) The Western Blot result showing at day 1 from differentiation, overexpressing P53 or WNT3 alone could increase the localization of β-catenin in the nuclear fraction indicating activation of Wnt pathway. Moreover, P53 overexpression together with GSK inhibitor (Gi) could further amplify the activation of Gi-caused activation of Wnt signaling as shown by the representative image of Western Blot (A) and quantification by densitometry (B). (C, D) The representative image of Western Blot showed that synergically increased Wnt signaling could remarkably abrogated by WNT inhibitor (Wi) at day 5, indicated by significantly decreased nuclear β-catenin level (C) and quantification was done by densitometry (D). Expression values of the Western Blot analysis of nuclear and total protein were normalized to Histone H3 and GAPDH respectively. Data are presented as mean±SD (t test, *p<0.05, **p<0.01; n=3, performed in 3 independent experiments).

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  Fig. 8 Graphic abstract. Working model of how p53 enhances more cardiomyocytes generation from hiPSC. During the period from HiPSC to mesendoderm, p53 activates the Wnt signaling pathway through transactivating WNT3 expression which boosts mesendodermal differentiation, and finally yields more cardiomyocytes generation.

Reference

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