Joint regulation of Wnt and bone morphogenetic protein signaling pathways to promote differentiation of human induced pluripotent stem cells into cardiomyocytes_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

YAN Ying ^1,2 , LIU Feng ³ , HOU Xiaojie ³ , WAN Juyi ³ , XIONG Qi ³ ,  ZHOU Rui ⁴ ,  LIAO Bin ³

1. Clinical Medicine College of Chengdu University of Traditional Chinese Medicine, Chengdu Sichuan, 610075, P.R.China;
2. College of Integrative Chinese and Western Medicine, Southwest Medical University, Luzhou Sichuan, 646000, P.R.China;
3. Department of Cardiac Macrovascular Surgery, Affiliated Hospital of Southwest Medical University, Luzhou Sichuan, 646000, P.R.China;
4. Institute of Cardiovascular Medicine, Southwest Medical University, Luzhou Sichuan, 646000, P.R.China;

Corresponding author：

ZHOU Rui, Email: zhouhuaxizhu@126.com; LIAO Bin, Email: 1875153677@qq.com

Keywords：

Human induced pluripotent stem cells; Wnt signaling pathway; bone morphogenetic protein signaling pathway; cardiomyocytes

DOI：

10.7507/1002-1892.201912087

Video：

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Objective To explore the role of joint regulation of Wnt and bone morphogenetic protein (BMP) signaling pathways in the differentiation of human induced pluripotent stem cells (hiPSCs) into cardiomyocytes.Methods HiPSCs were cultured and observed under inverted phase contrast microscope. Immunofluorescence staining was used to observe the expressions of hiPSCs pluripotent markers (OCT3/4, NANOG, and TRA-1-60). HiPSCs were passaged which were taken for subsequent experiments within the 35th passage. When the fusion degree of hiPSCs was close to 100%, the CHIR99021 (Wnt pathway activator) was added on the 0th day of differentiation. Different concentrations of IWP4 (inhibitor of Wnt production) were added on the 3rd day of differentiation, and the best concentration of IWP4 was added at different time points. The optimal concentration and the best effective period of IWP4 were obtained by detecting the expression of troponin T (TNNT2) mRNA by real-time fluorescence quantitative PCR. Then, on the basis of adding CHIR99021 and IWP4, different concentrations of BMP-4 were added on the 5th day of differentiation, and the best concentration of BMP-4 was added at different time points. The optimal concentration and best effective period of BMP-4 were obtained by detecting the expression of TNNT2 mRNA. Finally, hiPSCs were divided into three groups: Wnt group, BMP group, and Wnt+BMP group. On the basis of adding CHIR99021 on the 0th day of differentiation, IWP4, BMP-4, and IWP4+BMP-4 were added into Wnt group, BMP group, and Wnt+BMP group respectively according to the screening results. Cells were collected on the 7th and the 15th days of differentiation. The expressions of myocardial precursor cell markers [ISL LIM homeobox 1 (ISL1), NK2 homeobox 5 (NKX2-5)] and cardiomyocyte specific markers [myocyte enhancer factor 2C (MEF2C), myosin light chain 2 (MYL2), MYL7, and TNNT2] were detected by real-time fluorescent quantitative PCR. Cells were collected on the 28th day of differentiation, and the expression of cardiac troponin T (cTnT) was detected by flow cytometry and immunofluorescence staining.Results The results of cell mophology and immunoflurescence staining showed that the OCT3/4, NANOG, and TRA-1-60 were highly expressed in hiPSCs, which suggested that hiPSCs had characteristics of pluripotency. The optimal concentration of IWP4 was 10.0 μmol/L (P<0.05) and the best effective period was the 3rd day (P<0.05) in inducing hiPSCs to differentiate into cardiomyocytes. The optimal concentration of BMP-4 was 20.0 ng/mL (P<0.05) and the best effective period was the 3rd day (P<0.05). The relative expressions of ISL1, NKX2-5, MEF2C, MYL2, MYL7, and TNNT2 mRNAs, the positive expression ratio of cTnT detected by flow cytometry, and sarcomere structure detected by immunofluorescence staining of Wnt+BMP group were superior to those of Wnt group (P<0.05).Conclusion Joint regulation of Wnt and BMP signaling pathways can improve the differentiation efficiency of hiPSCs into cardiomyocytes.

Citation： YAN Ying, LIU Feng, HOU Xiaojie, WAN Juyi, XIONG Qi, ZHOU Rui, LIAO Bin. Joint regulation of Wnt and bone morphogenetic protein signaling pathways to promote differentiation of human induced pluripotent stem cells into cardiomyocytes. Chinese Journal of Reparative and Reconstructive Surgery, 2020, 34(10): 1313-1321. doi: 10.7507/1002-1892.201912087 Copy

1.	Bedada FB, Wheelwright M, Metzger JM. Maturation status of sarcomere structure and function in human iPSC-derived cardiac myocytes. Biochim Biophys Acta, 2016, 1863(7 Pt B): 1829-1838.
2.	Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 2006, 126(4): 663-676.
3.	Takahashi K, Tanabe K, Ohnuki M, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell, 2007, 131(5): 861-872.
4.	Fujiwara M, Yan P, Otsuji TG, et al. Induction and enhancement of cardiac cell differentiation from mouse and human induced pluripotent stem cells with cyclosporin-A. PLoS One, 2011, 6(2): e16734.
5.	穆军升, 许士俊, 张健群, 等. 维生素 C 明显提高小鼠诱导多能干细胞分化为心肌细胞的诱导效率. 心肺血管病杂志, 2016, 35(1): 59-64.
6.	王记, 陆玉琴, 徐鑫, 等. 抑瘤素 M 促进小鼠诱导多能干细胞向心肌分化. 基础医学与临床, 2017, 37(10): 1401-1406.
7.	陈焱, 李晓莉, 曾迪, 等. 静电纺丝聚己内酯纳米纤维支架支持小鼠诱导多能干细胞表达心肌细胞标志物. 基础医学与临床, 2016, 36(4): 486-491.
8.	Zhao M, Tang YW, Zhou Y, et al. Deciphering role of Wnt signalling in cardiac mesoderm and cardiomyocyte differentiation from human iPSCs: Four-dimensional control of Wnt pathway for hiPSC CMs differentiation. Sci Rep, 2019, 9(1): 19389.
9.	Kattman SJ, Witty AD, Gagliardi M, et al. Stage-specific optimization of activin/nodal and BMP signaling promotes cardiac differentiation of mouse and human pluripotent stem cell lines. Cell Stem Cell, 2011, 8(2): 228-240.
10.	Klaus A, Saga Y, Taketo MM, et al. Distinct roles of Wnt/beta-catenin and Bmp signaling during early cardiogenesis. Proc Natl Acad Sci U S A, 2007, 104(47): 18531-18536.
11.	Lian X, Hsiao C, Wilson G, et al. Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling. Proc Natl Acad Sci U S A, 2012, 109(27): E1848-1857.
12.	Garside VC, Chang AC, Karsan A, et al. Co-ordinating Notch, BMP, and TGF-β signaling during heart valve development. Cell Mol Life Sci, 2013, 70(16): 2899-2917.
13.	Abdelwahid E, Rice D, Pelliniemi LJ, et al. Overlapping and differential localization of Bmp-2, Bmp-4, Msx-2 and apoptosis in the endocardial cushion and adjacent tissues of the developing mouse heart. Cell Tissue Res, 2001, 305(1): 67-78.
14.	Monzen K, Nagai R, Komuro I. A role for bone morphogenetic protein signaling in cardiomyocyte differentiation. Trends Cardiovasc Med, 2002, 12(6): 263-269.
15.	刘锋, 方易冰, 熊琪, 等. TBX3 及 TBX18 在人源诱导多能干细胞向窦房结样细胞富集分化中的作用. 中国修复重建外科杂志, 2019, 33(4): 497-506.
16.	Hubert F, Payan SM, Rochais F. FGF10 signaling in heart development, homeostasis, disease and repair. Front Genet, 2018, 9: 599.
17.	Andersen P, Tampakakis E, Jimenez DV, et al. Precardiac organoids form two heart fifields via Bmp/Wnt signaling. Nat Commun, 2018, 9(1): 1-13.
18.	张友明, 朱红涛. Wnt 信号通路与心血管疾病的研究进展. 国际心血管病杂志, 2016, 43(5): 269-272.
19.	Mazzotta S, Neves C, Bonner RJ, et al. Distinctive roles of canonical and noncanonical Wnt signaling in human embryonic cardiomyocyte development. Stem Cell Reports, 2016, 7(4): 764-776.
20.	Marvin MJ, Di Rocco G, Gardiner A, et al. Inhibition of Wnt activity induces heart formation from posterior mesoderm. Genes Dev, 2001, 15(3): 316-327.
21.	Schneider VA, Mercola M. Wnt antagonism initiates cardiogenesis in xenopus laevis. Genes Dev, 2001, 15(3): 304-315.
22.	Arkell RM, Fossat N, Tam PP. Wnt signalling in mouse gastrulation and anterior development: new players in the pathway and signal output. Curr Opin Genet Dev, 2013, 23(4): 454-460.
23.	Ueno S, Weidinger G, Osugi T, et al. Biphasic role for Wnt/beta-catenin signaling in cardiac specification in zebrafish and embryonic stem cells. Proc Natl Acad Sci U S A, 2007, 104(23): 9685-9690.
24.	Barron M, Gao M, Lough J. Requirement for BMP and FGF signaling during cardiogenic induction in non-precardiac mesoderm is specific, transient, and cooperative. Dev Dyn, 2000, 218(2): 383-393.
25.	Shi Y, Katsev S, Cai C, et al. BMP signaling is required for heart formation in vertebrates. Dev Biol, 2000, 224(2): 226-237.
26.	Uchimura T, Komatsu Y, Tanaka M, et al. Bmp2 and Bmp4 genetically interact to support multiple aspects of mouse development including functional heart development. Genesis, 2009, 47(6): 374-384.

1. Bedada FB, Wheelwright M, Metzger JM. Maturation status of sarcomere structure and function in human iPSC-derived cardiac myocytes. Biochim Biophys Acta, 2016, 1863(7 Pt B): 1829-1838.
2. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 2006, 126(4): 663-676.
3. Takahashi K, Tanabe K, Ohnuki M, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell, 2007, 131(5): 861-872.
4. Fujiwara M, Yan P, Otsuji TG, et al. Induction and enhancement of cardiac cell differentiation from mouse and human induced pluripotent stem cells with cyclosporin-A. PLoS One, 2011, 6(2): e16734.
5. 穆军升, 许士俊, 张健群, 等. 维生素 C 明显提高小鼠诱导多能干细胞分化为心肌细胞的诱导效率. 心肺血管病杂志, 2016, 35(1): 59-64.
6. 王记, 陆玉琴, 徐鑫, 等. 抑瘤素 M 促进小鼠诱导多能干细胞向心肌分化. 基础医学与临床, 2017, 37(10): 1401-1406.
7. 陈焱, 李晓莉, 曾迪, 等. 静电纺丝聚己内酯纳米纤维支架支持小鼠诱导多能干细胞表达心肌细胞标志物. 基础医学与临床, 2016, 36(4): 486-491.
8. Zhao M, Tang YW, Zhou Y, et al. Deciphering role of Wnt signalling in cardiac mesoderm and cardiomyocyte differentiation from human iPSCs: Four-dimensional control of Wnt pathway for hiPSC CMs differentiation. Sci Rep, 2019, 9(1): 19389.
9. Kattman SJ, Witty AD, Gagliardi M, et al. Stage-specific optimization of activin/nodal and BMP signaling promotes cardiac differentiation of mouse and human pluripotent stem cell lines. Cell Stem Cell, 2011, 8(2): 228-240.
10. Klaus A, Saga Y, Taketo MM, et al. Distinct roles of Wnt/beta-catenin and Bmp signaling during early cardiogenesis. Proc Natl Acad Sci U S A, 2007, 104(47): 18531-18536.
11. Lian X, Hsiao C, Wilson G, et al. Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling. Proc Natl Acad Sci U S A, 2012, 109(27): E1848-1857.
12. Garside VC, Chang AC, Karsan A, et al. Co-ordinating Notch, BMP, and TGF-β signaling during heart valve development. Cell Mol Life Sci, 2013, 70(16): 2899-2917.
13. Abdelwahid E, Rice D, Pelliniemi LJ, et al. Overlapping and differential localization of Bmp-2, Bmp-4, Msx-2 and apoptosis in the endocardial cushion and adjacent tissues of the developing mouse heart. Cell Tissue Res, 2001, 305(1): 67-78.
14. Monzen K, Nagai R, Komuro I. A role for bone morphogenetic protein signaling in cardiomyocyte differentiation. Trends Cardiovasc Med, 2002, 12(6): 263-269.
15. 刘锋, 方易冰, 熊琪, 等. TBX3 及 TBX18 在人源诱导多能干细胞向窦房结样细胞富集分化中的作用. 中国修复重建外科杂志, 2019, 33(4): 497-506.
16. Hubert F, Payan SM, Rochais F. FGF10 signaling in heart development, homeostasis, disease and repair. Front Genet, 2018, 9: 599.
17. Andersen P, Tampakakis E, Jimenez DV, et al. Precardiac organoids form two heart fifields via Bmp/Wnt signaling. Nat Commun, 2018, 9(1): 1-13.
18. 张友明, 朱红涛. Wnt 信号通路与心血管疾病的研究进展. 国际心血管病杂志, 2016, 43(5): 269-272.
19. Mazzotta S, Neves C, Bonner RJ, et al. Distinctive roles of canonical and noncanonical Wnt signaling in human embryonic cardiomyocyte development. Stem Cell Reports, 2016, 7(4): 764-776.
20. Marvin MJ, Di Rocco G, Gardiner A, et al. Inhibition of Wnt activity induces heart formation from posterior mesoderm. Genes Dev, 2001, 15(3): 316-327.
21. Schneider VA, Mercola M. Wnt antagonism initiates cardiogenesis in xenopus laevis. Genes Dev, 2001, 15(3): 304-315.
22. Arkell RM, Fossat N, Tam PP. Wnt signalling in mouse gastrulation and anterior development: new players in the pathway and signal output. Curr Opin Genet Dev, 2013, 23(4): 454-460.
23. Ueno S, Weidinger G, Osugi T, et al. Biphasic role for Wnt/beta-catenin signaling in cardiac specification in zebrafish and embryonic stem cells. Proc Natl Acad Sci U S A, 2007, 104(23): 9685-9690.
24. Barron M, Gao M, Lough J. Requirement for BMP and FGF signaling during cardiogenic induction in non-precardiac mesoderm is specific, transient, and cooperative. Dev Dyn, 2000, 218(2): 383-393.
25. Shi Y, Katsev S, Cai C, et al. BMP signaling is required for heart formation in vertebrates. Dev Biol, 2000, 224(2): 226-237.
26. Uchimura T, Komatsu Y, Tanaka M, et al. Bmp2 and Bmp4 genetically interact to support multiple aspects of mouse development including functional heart development. Genesis, 2009, 47(6): 374-384.

Chinese Journal of Reparative and Reconstructive Surgery

Joint regulation of Wnt and bone morphogenetic protein signaling pathways to promote differentiation of human induced pluripotent stem cells into cardiomyocytes

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