Int J Stem Cells.  2022 Aug;15(3):247-257. 10.15283/ijsc21148.

The Biphasic Effect of Retinoic Acid Signaling Pathway on the Biased Differentiation of Atrial-like and Sinoatrial Node-like Cells from hiPSC

Affiliations
  • 1National Regional Children’s Medical Center (Northwest), Key Laboratory of Precision Medicine to Pediatric Diseases of Shaanxi Province, Xi’an Key Laboratory of Children’s Health and Diseases, Shaanxi Institute for Pediatric Diseases, Xi’an Children’s Hospital, Affiliated Children’s Hospital of Xi’an Jiaotong University, Xi’an, China
  • 2The Key Laboratory of Medical Electrophysiology of Ministry of Education and Medical Electrophysiological Key Laboratory of Sichuan Province, Collaborative Innovation Center for Prevention and Treatment of Cardiovascular Disease of Sichuan Province, Institute of Cardiovascular Research, Southwest Medical University, Luzhou, China

Abstract

Background and Objectives
Although human-induced pluripotent stem cells (hiPSC) can be efficiently differentiated into cardiomyocytes (CMs), the heterogeneity of the hiPSC-CMs hampers their applications in research and regenerative medicine. Retinoic acid (RA)-mediated signaling pathway has been proved indispensable in cardiac development and differentiation of hiPSC toward atrial CMs. This study was aimed to test whether RA signaling pathway can be manipulated to direct the differentiation into sinoatrial node (SAN) CMs.
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, RA signaling pathway was manipulated during the differentiation of hiPSC-CMs on day 5 post-differentiation, a crucial time point equivalent to the transition from cardiac mesoderm to cardiac progenitor cells in cardiac development. The resultant CMs were characterized at mRNA, protein and electrophysiology levels by a combination of qPCR, immunofluorescence, flow cytometry, and whole-cell patch clamp. The results showed that activation of the RA signaling pathway biased the differentiation of atrial CMs, whereas inhibition of the signaling pathway biased the differentiation of sinoatrial node-like cells (SANLCs).
Conclusions
Our study not only provides a novel and simple strategy to enrich SANLCs but also improves our under-standing of the importance of RA signaling in the differentiation of hiPSC-CMs.

Keyword

Retinoic acid (RA) signaling; Human-induced pluripotent stem cell (hiPSC); Atrial-like cells; Sinoatrial node-like cells (SANLCs); Biased differentiation

Figure

  • Fig. 1 Characterization of the hiPSC. hiPSC expresses high levels of OCT4 (A, E, I), NANOG (B, F, J), TRA-1-60 (C, K), and Ki67 (D, H, L) by IF, qPCR and flow cytometry respectively. In addition, pluripotent marker SOX2 was also highly expressed in hiPSC using the qPCR data (G). For qPCR analysis (E∼G), the terminal differentiated human fibroblast (hF) cell line (BJ) served as negative control and human embryonic stem cell (hESC) line (HN4) as positive control. The expression was normalized to that of GAPDH. Data are presented as ‘Mean±SD’ from at least 3 independent experiments with duplicate on each sample, with ns denoting non-significant and nd, not detectable. Scale bars=100 μm (400× magnification).

  • Fig. 2 Characterization of the cardiomyocytes derived from hiPSC. Representative images of immunofluorescence and FACS plots showing that cardiomyocytes derived from hiPSC on day 35 after differentiation express α-actinin by IF (A) and cTNT by IF (B) and flow cytometry (C). Scale bars=100 μm (400× magnification).

  • Fig. 3 Activation of RA signaling pathway by RA biases the differentiation of hiPSC toward atrial cardiomyocytes. (A) RA was introduced on day 5 at the concentrations indicated, the effect on the expression of NPPA, MYL7, COUPTFII, KCNJ5, and CX40 was analyzed by qPCR. (B) 2 μM RA was introduced on day 5 after the differentiation, and the expression of SHOX2, TBX18, TBX3, HCN4, ISL1, CX30.2, CACNB1, CACNA1A, KCNN4, KCNK2, KCND2, and SCN3B was quantitated by qPCR on day 21. The expression was normalized to that of GAPDH. Data are presented as ‘Mean±SD’ from at least 3 indepen-dent experiments with duplicate on each sample, with ns denoting non -significant, *denoting p<0.05, and **denoting p<0.01.

  • Fig. 4 Inhibition of RA signaling pathway by BMS biases the differentiation of hiPSC toward Sinoatrial node-like cells. (A) BMS was introduced on day 5 at the concentrations indicated, the effect on the expression of NPPA, MYL7, COUPTFII, KCNJ5, and CX40 was analyzed by qPCR. (B) 5 μM BMS was introduced on day 5 after the differentiation, and the expression of SHOX2, TBX18, TBX3, HCN4, ISL1, CX30.2, CACNB1, CACNA1A, KCNN4, KCNK2, KCND2, and SCN3B was quantitated by qPCR on day 21. The expression was normalized to that of GAPDH. Data are presented as ‘Mean±SD’ from at least 3 independent experiments with duplicate on each sample, with ns denoting non-significant, *denoting p<0.05, **denoting p<0.01, and ***denoting p<0.001.

  • Fig. 5 Inhibition of RA signaling pathway by BMS biases the differentiation of hiPSC to Sinoatrial node-like cells. BMS at 5 μM was introduced on day 5 after the differentiation, and the expression of COUPTFII (A, B), TBX18 (C, D), and TBX3 (E, F) was evaluated by IF. Representative images and corresponding quantitations showing that inhibition of RA signaling pathway by BMS decreases the percentage of COUPTFII (A, B) positive cardio-myocytes and increases the percentage of TBX18-(C, D) and TBX3-positive (E, F) cardiomyocytes. Scale bars=100 μm (400× magnification).

  • Fig. 6 Inhibition of RA signaling pathway by BMS biases the differentiation of hiPSC to Sinoatrial node-like cells. BMS at 5 μM was introduced on day 5 after the differentiation, and the expression of COUPTFII, cTNT+/SHOX2+, and cTNT+/NKX2.5− was evaluated by flow cytometry. Representative FACS plots showing that inhibition of RA signaling pathway by BMS decreases the percentage of COUPTFII positive cardiomyocytes (A) and increases the percentage of SHOX2+/cTNT+ (B) and cTNT+/NKX2.5− (C) cardio-myocytes.

  • Fig. 7 Inhibition of RA signaling pathway by BMS biases the differentiation of cardiomyocytes possessing typical electrophysiological features of Sinoatrial node-like cells. BMS at 5 μM was introduced on day 5 after the differentiation, and the beating frequency and AP were analyzed after the differentiation on day 21 and 60 respectively. BMS significantly increased the beating frequency of cardiomyocytes (A), sensitized cardiomyocytes to TCN ion channel inhibitor (B), decreased the ratio of cardiomyocytes displaying typical atrial-like AP (C, middle panel; D, blue bar, 5/15), and increased the ratio of cardiomyocytes displaying typical pacemaker-like AP (C, bottom panel; D, green bar, 4/15), compared to RA group (10/15 and 2/15 respectively). Data in (A) and (B) are presented as ‘Mean±SD’ from recording of 7 cardiomyocytes, with *denoting p<0.05 and ****denoting p<0.0001.

  • Fig. 8 Graphic abstract. Working model of biphasic effect of modulating RA signaling on the enrichment differentiation of atrial-like and sinoatrial node-like cells based on the GiWi method. During the cardiac mesoderm stage of pan cardio-myocytes differentiation from hiPSCS, activation of RA signaling pathway promotes biased differentiation of atrial-like cells. In contrast, enrichment differentiation of sinoatrial node-like cells could be enabled by RA inhibition.


Reference

References

1. Takahashi K, Yamanaka S. 2006; Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 126:663–676. DOI: 10.1016/j.cell.2006.07.024. PMID: 16904174. PMID: https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=33747195353&origin=inward.
2. Sung TC, Liu CH, Huang WL, Lee YC, Kumar SS, Chang Y, Ling QD, Hsu ST, Higuchi A. 2019; Efficient differentiation of human ES and iPS cells into cardiomyocytes on biomaterials under xeno-free conditions. Biomater Sci. 7:5467–5481. DOI: 10.1039/C9BM00817A. PMID: 31656967. PMID: https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85075271004&origin=inward.
3. Kishino Y, Fujita J, Tohyama S, Okada M, Tanosaki S, Someya S, Fukuda K. 2020; Toward the realization of cardiac regenerative medicine using pluripotent stem cells. Inflamm Regen. 40:1. DOI: 10.1186/s41232-019-0110-4. PMID: 31938077. PMCID: PMC6956487. PMID: 3d846dad7b4c4d6d8f29f58d98f60373. PMID: https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85083666553&origin=inward.
4. Hartman ME, Dai DF, Laflamme MA. 2016; Human pluripotent stem cells: prospects and challenges as a source of cardiomyocytes for in vitro modeling and cell-based cardiac repair. Adv Drug Deliv Rev. 96:3–17. DOI: 10.1016/j.addr.2015.05.004. PMID: 25980938. PMCID: PMC4644514. PMID: https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=84951860090&origin=inward.
5. Burridge PW, Zambidis ET. 2013; Highly efficient directed differentiation of human induced pluripotent stem cells into cardiomyocytes. Methods Mol Biol. 997:149–161. DOI: 10.1007/978-1-62703-348-0_12. PMID: 23546754. PMID: https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=84877078624&origin=inward.
6. He JQ, Ma Y, Lee Y, Thomson JA, Kamp TJ. 2003; Human embryonic stem cells develop into multiple types of cardiac myocytes: action potential characterization. Circ Res. 93:32–39. DOI: 10.1161/01.RES.0000080317.92718.99. PMID: 12791707. PMID: https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=0038300672&origin=inward.
7. Park M, Yoon YS. 2018; Cardiac regeneration with human pluripotent stem cell-derived cardiomyocytes. Korean Circ J. 48:974–988. DOI: 10.4070/kcj.2018.0312. PMID: 30334384. PMCID: PMC6196153. PMID: https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85056373666&origin=inward.
8. Sugiura T, Hibino N, Breuer CK, Shinoka T. 2016; Tissue-engineered cardiac patch seeded with human induced pluripotent stem cell derived cardiomyocytes promoted the regeneration of host cardiomyocytes in a rat model. J Cardio-thorac Surg. 11:163. DOI: 10.1186/s13019-016-0559-z. PMID: 27906085. PMCID: PMC5131419. PMID: https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=84999863221&origin=inward.
9. Miao S, Zhao D, Wang X, Ni X, Fang X, Yu M, Ye L, Yang J, Wu H, Han X, Qu L, Li L, Lan F, Shen Z, Lei W, Zhao ZA, Hu S. 2020; Retinoic acid promotes metabolic maturation of human embryonic stem cell-derived cardio-myocytes. Theranostics. 10:9686–9701. DOI: 10.7150/thno.44146. PMID: 32863954. PMCID: PMC7449904. PMID: https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85090108048&origin=inward.
10. Devalla HD, Schwach V, Ford JW, Milnes JT, El-Haou S, Jackson C, Gkatzis K, Elliott DA, Chuva de Sousa Lopes SM, Mummery CL, Verkerk AO, Passier R. 2015; Atrial-like cardiomyocytes from human pluripotent stem cells are a robust preclinical model for assessing atrial-selective pharm-acology. EMBO Mol Med. 7:394–410. DOI: 10.15252/emmm.201404757. PMID: 25700171. PMCID: PMC4403042. PMID: https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=84926165142&origin=inward.
11. Lee JH, Protze SI, Laksman Z, Backx PH, Keller GM. 2017; Human pluripotent stem cell-derived atrial and ventricular cardiomyocytes develop from distinct mesoderm popula-tions. Cell Stem Cell. 21:179–194.e4. DOI: 10.1016/j.stem.2017.07.003. PMID: 28777944. PMID: https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85026505552&origin=inward.
12. Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, Slukvin II, Thomson JA. 2007; Induced pluripotent stem cell lines derived from human somatic cells. Science. 318:1917–1920. DOI: 10.1126/science.1151526. PMID: 18029452. PMID: https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=36749043230&origin=inward.
13. Burridge PW, Matsa E, Shukla P, Lin ZC, Churko JM, Ebert AD, Lan F, Diecke S, Huber B, Mordwinkin NM, Plews JR, Abilez OJ, Cui B, Gold JD, Wu JC. 2014; Chemically defined generation of human cardiomyocytes. Nat Methods. 11:855–860. DOI: 10.1038/nmeth.2999. PMID: 24930130. PMCID: PMC4169698. PMID: https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=84905242471&origin=inward.
14. Paige SL, Plonowska K, Xu A, Wu SM. 2015; Molecular regulation of cardiomyocyte differentiation. Circ Res. 116:341–353. DOI: 10.1161/CIRCRESAHA.116.302752. PMID: 25593278. PMCID: PMC4299877. PMID: https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=84921954526&origin=inward.
15. Zhang Q, Jiang J, Han P, Yuan Q, Zhang J, Zhang X, Xu Y, Cao H, Meng Q, Chen L, Tian T, Wang X, Li P, Hescheler J, Ji G, Ma Y. 2011; Direct differentiation of atrial and ventricular myocytes from human embryonic stem cells by alternating retinoid signals. Cell Res. 21:579–587. DOI: 10.1038/cr.2010.163. PMID: 21102549. PMCID: PMC3203651. PMID: https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=79954418253&origin=inward.
16. Lian X, Zhang J, Azarin SM, Zhu K, Hazeltine LB, Bao X, Hsiao C, Kamp TJ, Palecek SP. 2013; Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt/β-catenin signaling under fully defined conditions. Nat Protoc. 8:162–175. DOI: 10.1038/nprot.2012.150. PMID: 23257984. PMCID: PMC3612968. PMID: https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=84872016692&origin=inward.
17. El-Battrawy I, Zhao Z, Lan H, Cyganek L, Tombers C, Li X, Buljubasic F, Lang S, Tiburcy M, Zimmermann WH, Utikal J, Wieland T, Borggrefe M, Zhou XB, Akin I. 2018; Electrical dysfunctions in human-induced pluripotent stem cell-derived cardiomyocytes from a patient with an arrhythmogenic right ventricular cardiomyopathy. Europace. 20:f46–f56. DOI: 10.1093/europace/euy042. PMID: 29566126. PMID: https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85048631276&origin=inward.
18. Liu F, Fang Y, Hou X, Yan Y, Xiao H, Zuo D, Wen J, Wang L, Zhou Z, Dang X, Zhou R, Liao B. 2020; Enrichment differentiation of human induced pluripotent stem cells into sinoatrial node-like cells by combined modulation of BMP, FGF, and RA signaling pathways. Stem Cell Res Ther. 11:284. DOI: 10.1186/s13287-020-01794-5. PMID: 32678003. PMCID: PMC7364513. PMID: 78f108ffbfa84397ae25d6418e0cb953. PMID: https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85088212795&origin=inward.
19. Kapoor N, Liang W, Marbán E, Cho HC. 2013; Direct conversion of quiescent cardiomyocytes to pacemaker cells by expression of Tbx18. Nat Biotechnol. 31:54–62. DOI: 10.1038/nbt.2465. PMID: 23242162. PMCID: PMC3775583. PMID: https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=84872192083&origin=inward.
20. Gorabi AM, Hajighasemi S, Tafti HA, Atashi A, Soleimani M, Aghdami N, Saeid AK, Khori V, Panahi Y, Sahebkar A. 2019; TBX18 transcription factor overexpression in human-induced pluripotent stem cells increases their differentiation into pacemaker-like cells. J Cell Physiol. 234:1534–1546. DOI: 10.1002/jcp.27018. PMID: 30078203. PMID: https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85052653407&origin=inward.
21. Zhao H, Wang F, Zhang W, Yang M, Tang Y, Wang X, Zhao Q, Huang C. 2020; Overexpression of TBX3 in human induced pluripotent stem cells (hiPSCs) increases their differentiation into cardiac pacemaker-like cells. Biomed Ph-armacother. 130:110612. DOI: 10.1016/j.biopha.2020.110612. PMID: 32771895. PMID: https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85089030426&origin=inward.
22. Protze SI, Liu J, Nussinovitch U, Ohana L, Backx PH, Gepstein L, Keller GM. 2017; Sinoatrial node cardiomyocytes derived from human pluripotent cells function as a biological pacemaker. Nat Biotechnol. 35:56–68. DOI: 10.1038/nbt.3745. PMID: 27941801. PMID: https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85011032595&origin=inward.
23. Yechikov S, Kao HKJ, Chang CW, Pretto D, Zhang XD, Sun YH, Smithers R, Sirish P, Nolta JA, Chan JW, Chiamvimonvat N, Lieu DK. 2020; NODAL inhibition promotes differentiation of pacemaker-like cardiomyocytes from human induced pluripotent stem cells. Stem Cell Res. 49:102043. DOI: 10.1016/j.scr.2020.102043. PMID: 33128951. PMCID: PMC7814970. PMID: https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85093081098&origin=inward.
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