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Amniotic membrane as novel scaffold for human iPSC-derived cardiomyogenesis

  • Shagufta ParveenEmail author
  • Shishu Pal Singh
  • M. M. Panicker
  • Pawan Kumar Gupta
Article

Abstract

Recent approaches of using decellularized organ matrices for cardiac tissue engineering prompted us to culture human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs) on the human amniotic membrane (hAM). Since hAM has been used lately to patch diseased hearts in patients and has shown anti-inflammatory and anti-fibrotic benefits, it qualifies as a cardiac compatible and clinically relevant heart tissue scaffold. The aim of this study was to test the ability of the hAM to support attachment, differentiation, and maturation of hiPSC-derived CMs in vitro. hAMs were prepared from term placenta. An in-house generated hiPSC line was used for CM derivation. hiPSC-derived cardiac progenitors were cultured on the surface of cryopreserved hAMs and in the presence of cytokines promoting cardiac differentiation. CMs grown on hAM and popular basement membrane matrix (BMM) Matrigel™ were compared for the following aspects of cardiac development: the morphology of cardiomyocytes with respect to shape and cellular alignments, levels of cardiac-related gene transcript expression, functionality in terms of spontaneous calcium fluxes and mitochondrial densities and distributions. hAM is biocompatible with hiPSC-derived CMs. hAM increased cardiac transcription regulator and myofibril protein transcript expression, accelerated intracellular calcium transients, and enhanced cellular mitochondrial complexity of its cardiomyocytes in comparison to cardiomyocytes differentiated on Matrigel™. Our data suggests that hAM supports differentiation and improves cardiomyogenesis in comparison to Matrigel™. hAMs are natural, easily and largely available. The method of preparing hAM cardiac sheets described here is simple with potential for clinical transplantation.

Graphical abstract

A An outline of the differentiation protocol with stage-specific growth factors and culture media used. B Cell fates from pluripotent stem cells to cardiomyocytes during differentiation on the amniotic membrane. C-FPhotomicrographs of cells at various stages of differentiation. Scale bars represent 100 μm.

Keywords

Placental induced pluripotent stem cells Human amniotic membrane hiPSC-derived cardiomyocytes Improved cardiomyogenesis hAM cardiac sheets 

Notes

Acknowledgements

We would like to thank Dr. Jyothi Prasanna (SORM) for contributing amniotic membranes used in the study. MMP acknowledges the support of NCBS (TIFR). The study was supported by a CSIR grant (no. 27(293)/13 EMR II) and funds from MAHE. SPS and MMP were supported by funds from NCBS (TIFR).

Author contributions

SP designed and carried out the entire study. SPS performed confocal microscopy, helped to develop and carry out the calcium imaging, MMP provided the microscopy facilities, experimental input, and comments on the manuscript, and PKG provided comments on the manuscript and discussion.

Compliance with ethical standards

Conflict of interest

None.

Supplementary material

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Supplementary Fig. 1 Characterization of hiPSCs. hiPSCs expressing pluripotency markers. (Lipid Droplet associated blue fluorescence, alkaline phosphatase, SSEA4, OCT4, TRA 1-60, NANOG and SOX2. Differentiation of hiPSCs into cells of ectoderm, mesoderm and endoderm lineages expressing lineage specific markers (NESTIN, DESMIN and SOX17). (PNG 6.40 mb)
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Supplementary Fig. 2

Confirmation of non-viability of the cryopreserved amniotic membrane and its role as extracellular matrix. A. Viability of cells on fresh and cryopreserved amniotic membrane shown by dye exclusion test with propidium iodide. B. Hematoxylin & Eosin staining of sectioned fresh and cryopreserved amniotic membrane showing the layer of epithelial cells and basement membrane. C. Fresh Amniotic membrane contain intact RNA (Lane 1), cryopreserved amniotic membrane show no intact RNA (Lane 2), RNA extracted from an equivalent amniotic membrane cardiac sheet (Lane 3) and molecular ladder (Lane 4). D. Immunostaining of the amniotic membrane with ECM marker Collagen Type II. Scale bars represent 100 μm. (PNG 872 kb)

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References

  1. Adamowicz J, Pokrywczyńska M, Tworkiewicz J, Kowalczyk T, van Breda SV, Tyloch D, Kloskowski T, Bodnar M, Skopinska-Wisniewska J, Marszałek A, Frontczak-Baniewicz M, Kowalewski TA, Drewa T (2016) New amniotic membrane based biocomposite for future application in reconstructive urology. Li Z, ed. PLoS One 11:e0146012CrossRefGoogle Scholar
  2. Barski D, Gerullis H, Ecke T, Yang J, Varga G, Boros M, Pintelon I, Timmermans J-P, Otto T (2017) Bladder reconstruction with human amniotic membrane in a xenograft rat model: a preclinical study. Int J Med Sci 14:310–318CrossRefGoogle Scholar
  3. Bedada FB, Wheelwright M, Metzger JM (1863) Maturation status of sarcomere structure and function in human iPSC-derived cardiac myocytes. Biochim Biophys Acta - Mol Cell Res The Authors 2016:1829–1838Google Scholar
  4. Birket MJ, Ribeiro MC, Verkerk AO, Ward D, Leitoguinho AR, den Hartogh SC, Orlova VV, Devalla HD, Schwach V, Bellin M, Passier R, Mummery CL (2015) Expansion and patterning of cardiovascular progenitors derived from human pluripotent stem cells. Nat Biotechnol Nat Publ Group 33:1–12CrossRefGoogle Scholar
  5. Buccini S, Haider KH, Ahmed RPH, Jiang S, Ashraf M (2012) Cardiac progenitors derived from reprogrammed mesenchymal stem cells contribute to angiomyogenic repair of the infarcted heart. Basic Res Cardiol 107:301CrossRefGoogle Scholar
  6. Cargnoni A, Di MM, Campagnol M, Nassuato C, Albertini A, Parolini O (2009) Amniotic membrane patching promotes ischemic rat heart repair. Cell Transplant 18:1147–1159CrossRefGoogle Scholar
  7. Carvajal-Vergara X, Sevilla A, D’Souza SL, Ang Y-S, Schaniel C, Lee D-F, Yang L, Kaplan AD, Adler ED, Rozov R, Ge Y, Cohen N, Edelmann LJ, Chang B, Waghray A, Su J, Pardo S, Lichtenbelt KD, Tartaglia M, Gelb BD, Lemischka IR (2010) Patient-specific induced pluripotent stem-cell-derived models of LEOPARD syndrome. Nature 465:808–812CrossRefGoogle Scholar
  8. Chong JJH, Yang X, Don CW, Minami E, Liu Y-W, Weyers JJ, Mahoney WM, Van BB, Cook SM, Palpant NJ, Gantz JA, Fugate JA, Muskheli V, Gough GM, Vogel KW, Astley CA, Hotchkiss CE, Baldessari A, Pabon L, Reinecke H, Gill EA, Nelson V, Kiem H-P, Laflamme MA, Murry CE (2014) Human embryonic-stem-cell-derived cardiomyocytes regenerate non-human primate hearts. Nature 510:273–277CrossRefGoogle Scholar
  9. Eschenhagen T, Bolli R, Braun T, Field LJ, Fleischmann BK, Frisén J, Giacca M, Hare JM, Houser S, Lee RT, Marbán E, Martin JF, Molkentin JD, Murry CE, Riley PR, Ruiz-Lozano P, Sadek HA, Sussman MA, Hill JA (2017) Cardiomyocyte regeneration. Circulation 136:680–686CrossRefGoogle Scholar
  10. Feaster TK, Cadar AG, Wang L, Williams CH, Chun YW, Hempel J, Bloodworth N, Merryman WD, Lim CC, Wu JC, Knollmann BC, Hong CC (2015) Matrigel mattress: a method for the generation of single contracting human-induced pluripotent stem cell-derived cardiomyocytes. Circ Res.  https://doi.org/10.1161/CIRCRESAHA.115.307580
  11. Francisco JC, Cunha RC, Simeoni RB, Guarita-souza LC, Ferreira RJ, Irioda AC, Maria C, Souza CO, Venkata G, Srikanth N, Nityanand S, Chachques JC, Athayde K, De Carvalho T. (2013) Amniotic membrane as a potent source of stem cells and a matrix for engineering heart tissue. Journal of Biomedical Science and Engineering 06(12):1178–1185Google Scholar
  12. Freund C, Ward-van Oostwaard D, Monshouwer-Kloots J, van den BS, van RM, Xu X, Zweigerdt R,Mummery C, Passier R (2008) Insulin redirects differentiation from cardiogenic mesoderm and endoderm to neuroectoderm in differentiating human embryonic stem cells. Stem Cells 26(3):724–733Google Scholar
  13. Gilbert SH, Benoist D, Benson AP, White E, Tanner SF, Holden AV, Dobrzynski H, Bernus O, Radjenovic A (2012) Visualization and quantification of whole rat heart laminar structure using high-spatial resolution contrast-enhanced MRI. AJP Hear Circ Physiol 302:H287–H298CrossRefGoogle Scholar
  14. Gomes JA, Romano A, Santos MS, Dua HS (2005) Amniotic membrane use in ophthalmology. Curr Opin Ophthalmol 16:233–240CrossRefGoogle Scholar
  15. Guyette JP, Charest JM, Mills RW, Jank BJ, Moser PT, Gilpin SE, Gershlak JR, Okamoto T, Gonzalez G, Milan DJ, Gaudette GR, Ott HC (2016) Bioengineering human myocardium on native extracellular matrix. Circ Res 118:56–72CrossRefGoogle Scholar
  16. Ilic D, Vicovac L, Nikolic M, Lazic Ilic E (2016) Human amniotic membrane grafts in therapy of chronic non-healing wounds: Table 1. Br Med Bull 117:59–67CrossRefGoogle Scholar
  17. Itzhaki I, Maizels L, Huber I, Zwi-Dantsis L, Caspi O, Winterstern A, Feldman O, Gepstein A, Arbel G, Hammerman H, Boulos M, Gepstein L (2011) Modelling the long QT syndrome with induced pluripotent stem cells. Nature 471(7337):225–229Google Scholar
  18. Jung CB, Moretti A, Mederos y Schnitzler M, Iop L, Storch U, Bellin M, Dorn T, Ruppenthal S, Pfeiffer S, Goedel A, Dirschinger RJ, Seyfarth M, Lam JT, Sinnecker D, Gudermann T, Lipp P, Laugwitz KL (2012) Dantrolene rescues arrhythmogenic RYR2 defect in a patient-specific stem cell model of catecholaminergic polymorphic ventricular tachycardia. EMBO Mol Med 4:180–191CrossRefGoogle Scholar
  19. Kawamura M, Miyagawa S, Fukushima S, Saito A, Miki K, Ito E, Sougawa N, Kawamura T, Daimon T, Shimizu T, Okano T, Toda K, Sawa Y (2013) Enhanced survival of transplanted human induced pluripotent stem cell-derived cardiomyocytes by the combination of cell sheets with the pedicled omental flap technique in a porcine heart. Circulation 128:87–95CrossRefGoogle Scholar
  20. Khalpey Z, Marsh KM, Ferng A, Bin RI, Friedman M, Indik J, Avery R, Jokerst C, Oliva I (2015) First in man: amniotic patch reduces postoperative inflammation. Am J Med Elsevier Inc 128:e5–e6CrossRefGoogle Scholar
  21. Li S, Cheng H, Tomaselli GF, Li RA (2014) Mechanistic basis of excitation-contraction coupling in human pluripotent stem cell-derived ventricular cardiomyocytes revealed by Ca2+ spark characteristics: direct evidence of functional Ca2+-induced Ca2+ release. Hear Rhythm Elsevier 11:133–140CrossRefGoogle Scholar
  22. Lim JJ, Koob TJ, Fonger J, Koob TJ (2017) Dehydrated human amnion/chorion membrane allograft promotes cardiac repair following myocardial infarction. 2:2–7Google Scholar
  23. Liu Z, Zhou J, Du Z, Duan C (2013) The tumourigenicity of iPS cells and their differentiated derivates. J Cell Mol Med 17:782–791CrossRefGoogle Scholar
  24. Lu T-Y, Lin B, Kim J, Sullivan M, Tobita K, Salama G, Yang L (2013) Repopulation of decellularized mouse heart with human induced pluripotent stem cell-derived cardiovascular progenitor cells. Nat Commun Nature Publishing Group 4:2307CrossRefGoogle Scholar
  25. Marsh KM, Ferng AS, Pilikian T, Desai AA, Avery R, Friedman M, Oliva I, Jokerst C, Schipper D, Khalpey Z (2017) Anti-inflammatory properties of amniotic membrane patch following pericardiectomy for constrictive pericarditis. J Cardiothorac Surg 12:6CrossRefGoogle Scholar
  26. Masumoto H, Ikuno T, Takeda M, Fukushima H, Marui A, Katayama S, Shimizu T, Ikeda T, Okano T, Sakata R, Yamashita JK (2015) Human iPS cell-engineered cardiac tissue sheets with cardiomyocytes and vascular cells for cardiac regeneration. Sci Rep 4:6716CrossRefGoogle Scholar
  27. Masumoto H, Matsuo T, Yamamizu K, Uosaki H, Narazaki G, Katayama S, Marui A, Shimizu T, Ikeda T, Okano T, Sakata R, Yamashita JK (2012) Pluripotent stem cell-engineered cell sheets reassembled with defined cardiovascular populations ameliorate reduction in infarct heart function through cardiomyocyte-mediated neovascularization. Stem Cells 30:1196–1205CrossRefGoogle Scholar
  28. Miyagawa S, Domae K, Yoshikawa Y, Fukushima S, Nakamura T, Saito A, Sakata Y, Hamada S, Toda K, Pak K, Takeuchi M, Sawa Y (2017) Phase I clinical trial of autologous stem cell–sheet transplantation therapy for treating cardiomyopathy. J Am Heart Assoc 6:e003918CrossRefGoogle Scholar
  29. Mohseni R (2014) Safe Transplantation Of Pluripotent Stem Cell By Preventing Teratoma Formation. Journal of Stem Cell Research & Therapy 04(06)Google Scholar
  30. Mrugala A, Sui A, Plummer M, Altman I, Papineau E, Frandsen D, Hill D, Ennis WJ (2016) Amniotic membrane is a potential regenerative option for chronic non-healing wounds: a report of five cases receiving dehydrated human amnion/chorion membrane allograft. Int Wound J 13:485–492CrossRefGoogle Scholar
  31. Niknejad H, Peirovi H, Jorjani M, Ahmadiani A, Ghanavi J, Seifalian AM (2008) Properties of the amniotic membrane for potential use in tissue engineering. Eur Cell Mater 15:88–99CrossRefGoogle Scholar
  32. Okita K, Matsumura Y, Sato Y, Okada A, Morizane A, Okamoto S, Hong H, Nakagawa M, Tanabe K, Tezuka K, Shibata T, Kunisada T, Takahashi M, Takahashi J, Saji H, Yamanaka S (2011) A more efficient method to generate integration-free human iPS cells. Nat Methods 8:409–412CrossRefGoogle Scholar
  33. Ong S-B, Hausenloy DJ (2010) Mitochondrial morphology and cardiovascular disease. Cardiovasc Res 88:16–29CrossRefGoogle Scholar
  34. Ott HC, Matthiesen TS, Goh S-K, Black LD, Kren SM, Netoff TI, Taylor DA (2008) Perfusion-decellularized matrix: using nature’s platform to engineer a bioartificial heart. Nat Med 14:213–221CrossRefGoogle Scholar
  35. Palpant NJ, Pabon L, Friedman CE, Roberts M, Hadland B, Zaunbrecher RJ, Bernstein I, Zheng Y, Murry CE (2016) Generating high-purity cardiac and endothelial derivatives from patterned mesoderm using human pluripotent stem cells. Nat Protoc Nature Publishing Group 12:15–31CrossRefGoogle Scholar
  36. Parolini O, Caruso M (2011) Review: preclinical studies on placenta-derived cells and amniotic membrane: an update. Placenta 32:S186–S195CrossRefGoogle Scholar
  37. Rahman I, Said DG, Maharajan VS, Dua HS (2009) Amniotic membrane in ophthalmology: indications and limitations. Eye 23:1954–1961CrossRefGoogle Scholar
  38. Riboh JC, Saltzman BM, Yanke AB, Cole BJ (2016) Human amniotic membrane-derived products in sports medicine. Am J Sports Med 44:2425–2434CrossRefGoogle Scholar
  39. Riegler J, Tiburcy M, Ebert A, Tzatzalos E, Raaz U, Abilez OJ, Shen Q, Kooreman NG, Neofytou E, Chen VC, Wang M, Meyer T, Tsao PS, Connolly AJ, Couture LA, Gold JD, Zimmermann WH, Human Engineered WJC (2015) Heart muscles engraft and survive long term in a rodent myocardial infarction ModelNovelty and significance. Circ Res 117:720–730CrossRefGoogle Scholar
  40. Robertson C, Tran DD, George SC (2013) Concise review: maturation phases of human pluripotent stem cell-derived cardiomyocytes. Stem Cells 31:829–837CrossRefGoogle Scholar
  41. Rocchetti M, Sala L, Dreizehnter L, Crotti L, Sinnecker D, Mura M, Pane LS, Altomare C, Torre E, Mostacciuolo G, Severi S, Porta A, De FGM, George AL, Schwartz PJ, Gnecchi M, Moretti A, Zaza A (2017) Elucidating arrhythmogenic mechanisms of long-QT syndrome CALM1-F142L mutation in patient-specific induced pluripotent stem cell-derived cardiomyocytes. Cardiovasc Res 113:531–541CrossRefGoogle Scholar
  42. Roy R, Haase T, Ma N, Bader A, Becker M, Seifert M, Choi Y-H, Falk V, Stamm C (2016) Decellularized amniotic membrane attenuates postinfarct left ventricular remodeling. J Surg Res 200:409–419CrossRefGoogle Scholar
  43. Segers VFM, Lee RT (2008) Stem-cell therapy for cardiac disease. Nature 451:937–942CrossRefGoogle Scholar
  44. Sekine H, Shimizu T, Dobashi I, Matsuura K, Hagiwara N, Takahashi M, Kobayashi E, Yamato M, Okano T (2011) Cardiac cell sheet transplantation improves damaged heart function via superior cell survival in comparison with dissociated cell injection. Tissue Eng Part A 17:2973–2980CrossRefGoogle Scholar
  45. Shiba Y, Fernandes S, Zhu W-Z, Filice D, Muskheli V, Kim J, Palpant NJ, Gantz J, Moyes KW, Reinecke H, Van BB, Dardas T, Mignone JL, Izawa A, Hanna R, Viswanathan M, Gold JD, Kotlikoff MI, Sarvazyan N, Kay MW, Murry CE, Laflamme MA (2012) Human ES-cell-derived cardiomyocytes electrically couple and suppress arrhythmias in injured hearts. Nature 489:322–325CrossRefGoogle Scholar
  46. Shinozawa T, Nakamura K, Shoji M, Morita M, Kimura M, Furukawa H, Ueda H, Shiramoto M, Matsuguma K, Kaji Y, Ikushima I, Yono M, Liou S-Y, Nagai H, Nakanishi A, Yamamoto K, Izumo S (2017) Recapitulation of clinical individual susceptibility to drug-induced QT prolongation in healthy subjects using iPSC-derived cardiomyocytes. Stem Cell Reports ElsevierCompany 8:226–234CrossRefGoogle Scholar
  47. Streckfuss-Bomeke K, Wolf F, Azizian A, Stauske M, Tiburcy M, Wagner S, Hubscher D, Dressel R, Chen S, Jende J, Wulf G, Lorenz V, Schon MP, Maier LS, Zimmermann WH, Hasenfuss G, Guan K (2013) Comparative study of human-induced pluripotent stem cells derived from bone marrow cells, hair keratinocytes, and skin fibroblasts. Eur Heart J 34:2618–2629CrossRefGoogle Scholar
  48. Sun N, Yazawa M, Liu J, Han L, Sanchez-Freire V, Abilez OJ, Navarrete EG, Hu S, Wang L, Lee A, Pavlovic A, Lin S, Chen R, Hajjar RJ (2012) Patient-specific induced pluripotent stem cells as model for familial dilated cardiomyopathy. Sci Transl Med 4(130):130ra47CrossRefGoogle Scholar
  49. Veerman CC, Kosmidis G, Mummery CL, Casini S, Verkerk AO, Bellin M (2015) Immaturity of human stem-cell-derived cardiomyocytes in culture: fatal flaw or soluble problem? Stem Cells Dev 24:1035–1052CrossRefGoogle Scholar
  50. Yang L, Soonpaa MH, Adler ED, Roepke TK, Kattman SJ, Kennedy M, Henckaerts E, Bonham K, Abbott GW, Linden RM, Field LJ, Keller GM (2008) Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population. Nature 453:524–528CrossRefGoogle Scholar
  51. Yang X, Pabon L, Murry CE (2014) Engineering adolescence: maturation of human pluripotent stem cell-derived cardiomyocytes. Circ Res 114:511–523CrossRefGoogle Scholar
  52. Yazawa M, Hsueh B, Jia X, Pasca AM, Bernstein JA, Hallmayer J, Dolmetsch RE (2011) Using induced pluripotent stem cells to investigate cardiac phenotypes in Timothy syndrome. Nature 471:230–234CrossRefGoogle Scholar
  53. Zhang J, Wilson GF, Soerens AG, Koonce CH, Yu J, Palecek SP, Thomson JA, Kamp TJ (2009) Functional cardiomyocytes derived from human induced pluripotent stem cells. Circ Res 104:e30–e41Google Scholar
  54. Zhang L, Zou D, Li S, Wang J, Qu Y, Ou S, Jia C, Li J, He H, Liu T, Yang J, Chen Y, Liu Z, Li W (2016) An ultra-thin amniotic membrane as carrier in corneal epithelium tissue-engineering. Sci Rep 6:21021CrossRefGoogle Scholar
  55. Zhao Q, Wang X, Wang S, Song Z, Wang J, Ma J (2017) Cardiotoxicity evaluation using human embryonic stem cells and induced pluripotent stem cell-derived cardiomyocytes. Stem Cell Res Ther 8:54CrossRefGoogle Scholar
  56. Zimmermann WH (2016) Strip and dress the human heart. Circ Res 118:12–13CrossRefGoogle Scholar

Copyright information

© The Society for In Vitro Biology 2019

Authors and Affiliations

  • Shagufta Parveen
    • 1
    Email author
  • Shishu Pal Singh
    • 2
  • M. M. Panicker
    • 2
  • Pawan Kumar Gupta
    • 3
  1. 1.Manipal Academy of Higher EducationSchool of Regenerative MedicineBengaluruIndia
  2. 2.National Centre for Biological Sciences, TIFRBangaloreIndia
  3. 3.Stempeutics Research Pvt. Ltd.BangaloreIndia

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