Original Article | Published:

Cardiac differentiation of induced pluripotent stem cells on elastin-like protein-based hydrogels presenting a single-cell adhesion sequence

Polymer Journal (2018) | Download Citation


Substrate-dependent cardiac differentiation of induced pluripotent stem cells (iPSCs) has been studied on various extracellular matrix (ECM)-derived substrates, such as collagen type I (Col-I). However, ECM-derived substrates have multiple cell-adhesive amino acid sequences and stimulate various signaling pathways in cells, making it difficult to clarify the mechanism of substrate-dependent stem cell differentiation. A substrate presenting one of these sequences is a powerful tool for elucidating the mechanism. We designed elastin-like proteins (ELPs) composed of repetitive VPGIG sequences with or without the RGD cell adhesion motif (ELP-RGD/ELP-Ctrl) and used a chemical crosslinker to generate hydrogels. By adjusting the ELP and crosslinker concentrations, we obtained ELP-Ctrl and ELP-RGD hydrogels with a Young’s modulus of 0.3 kPa. The ELP-Ctrl and ELP-RGD gels were used as a substrate for the cardiac differentiation of cultured murine iPSCs. Cells on the ELP-RGD gel showed four times higher gene expression of the contractile protein troponin T type 2 than those on a Col-I gel, which is an effective substrate for iPSC cardiac differentiation. The ELP-RGD gel might stimulate integrin-derived signaling pathways in the cells to promote cardiac differentiation. This study showed the potential of ELP hydrogels for studying substrate-dependent iPSC cardiac differentiation by enabling the control of cell-adhesive sequence presentation.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


  1. 1.

    Miskon A, Ehashi T, Mahara A, Uyama H, Yamaoka T. Beating behavior of primary neonatal cardiomyocytes and cardiac-differentiated P19CL6 cells on different extracellular matrix components. J Artif Organs. 2009;12:111–7.

  2. 2.

    Miskon A, Mahara A, Uyama H, Yamaoka T. A suspension induction for myocardial differentiation of rat mesenchymal stem cells on various extracellular matrix proteins. Tissue Eng Part C. 2010;16:979–87.

  3. 3.

    Yamaoka T, Hirata M, Dan T, Yamashita A, Otaka A, Nakaoki T, et al. Individual evaluation of cardiac marker expression and self-beating during cardiac differentiation of P19CL6 cells on different culture substrates. J Biomed Mater Res A. 2017;105:1166–74.

  4. 4.

    Hansson EM, Lindsay ME, Chien KR. Regeneration next: toward heart stem cell therapeutics. Cell Stem Cell. 2009;5:364–77.

  5. 5.

    Seo JH, Hirata M, Kakinoki S, Yamaoka T, Yui N. Dynamic polyrotaxane-coated surfaces for effective differentiation of mouse induced pluripotent stem cells into cardiomyocytes. RSC Adv. 2016;6:35668–76.

  6. 6.

    Hirata M, Yamaoka T. Effect of stem cell niche elasticity/ECM protein on the self-beating cardiomyocyte differentiation of induced pluripotent stem (iPS) cells at different stages. Acta Biomater. 2018;65:44–52.

  7. 7.

    Gai H, Leung EL, Costantino PD, Aguila JR, Nguyen DM, Fink LM, et al. Generation and characterization of functional cardiomyocytes using induced pluripotent stem cells derived from human fibroblasts. Cell Biol Int. 2009;33:1184–93.

  8. 8.

    Kaichi S, Hasegawa K, Takaya T, Yokoo N, Mima T, Kawamura T, et al. Cell line-dependent differentiation of induced pluripotent stem cells into cardiomyocytes in mice. Cardiovasc Res. 2010;88:314–23.

  9. 9.

    Minami I, Yamada K, Otsuji TG, Yamamoto T, Shen Y, Otsuka S, et al. A small molecular that promotes cardiac differentiation of human pluripotent stem cells under defined, cytokine- and xeno-free conditions. Cell Rep. 2012;2:1448–60.

  10. 10.

    Burridge PW, Matsa E, Shukla P, Lin ZC, Churko JM, Ebert AD, et al. Chemically defined generation of human cardiomyocytes. Nat Methods. 2014;11:855–60.

  11. 11.

    Jung JP, Hu D, Domian IJ, Ogle BM. An integrated statistical model for enhanced murine cardiomyocyte differentiation via optimized engagement of 3D extracellular matrices. Sci Rep. 2015;5:18705. https://doi.org/10.1038/srep18705

  12. 12.

    Macrí-Pellizzeri L, Pelacho B, Sancho A, Lglesias-García O, Simón-Yarza AM, Soriano-Navarro M, et al. Substrate stiffness and composition specifically direct differentiation of induced pluripotent stem cells. Tissue Eng Part A. 2015;21:1633–41.

  13. 13.

    Higuchi A, Kumar SS, Ling Q, Alarfaj AA, Munusamy MA, Murugan K, et al. Polymeric design of cell culture materials that guide the differentiation of human pluripotent stem cell. Prog Polym Sci. 2017;65:83–126.

  14. 14.

    Zhang S. Beyond the petri dish. Nat Biotechnol. 2004;22:151–2.

  15. 15.

    Meyer DE, Chilkoti A. Genetically encoded synthesis of protein-based polymers with precisely specified molecular weight and sequence by recursive directional ligation: examples from the elastin-like polypeptide system. Biomacromolecules. 2002;3:357–67.

  16. 16.

    Trabbic-Carlson K, Liu L, Kim B, Chilkoti A. Expression and purification of recombinant proteins from Escherichia coli: Comparison of an elastin-like polypeptide fusion with an oligohistidine fusion. Protein Sci. 2004;13:3274–84.

  17. 17.

    Engler AJ, Sen S, Sweeney HL, Discher DE. Matrix elasticity directs stem cell lineage specification. Cell. 2006;126:677–89.

  18. 18.

    Zio KD, Tirrell DA. Mechanical properties of artificial protein matrices engineered for control of cell and tissue behavior. Macromolecules. 2003;36:1553–8.

  19. 19.

    Lim DW, Nettles DL, Setton LA, Chilkoti A. Rapid crosslinking of elastin-like polypeptides with hydroxylmethylphosphines in aqueous solution. Biomacromolecules. 2007;8:1463–70.

  20. 20.

    Chung C, Lampe KJ, Heilshorn SC. Tetrakis(hydroxylmethyl) phosphonium chloride as a covalent crosslinking agent for cell encapsulation within protein-based hydrogels. Biomacromolecules. 2012;13:3912–6.

  21. 21.

    Davis GE. Affinity of integrins for damaged extracellular matrix: αVβ3 binds to denatured collagen type I through RGD sites. Biochem Biophys Res Commun. 1992;182:1025–31.

  22. 22.

    Kraehenbuehl TP, Zammaretti P, Van der Vlies AJ, Schoenmakers RG, Lutolf MP, Hubbell JA. Three-dimensional extracellular matrix-directed cardioprogenitor differentiation: systematic modulation of a synthetic cell-responsive PEG-hydrogel. Biomaterials. 2008;29:2757–66.

  23. 23.

    Mahara A, Kiicj KL, Yamaoka T. In vivo guided vascular regeneration with a non-porous elastin-like polypeptide hydrogel tubular scaffold. J Biomed Mater Res A. 2017;105:1746–55.

  24. 24.

    Kambe Y, Murakoshi A, Urakawa H, Kimura Y, Yamaoka T. Vascular induction and cell infiltration into peptide-modified bioactive silk fibroin hydrogels. J Mater Chem B. 2017;5:7557–71.

  25. 25.

    Kraehenbuehl TP, Zammaretti P, Van der Vlies AJ, Schoenmakers RG, Lutolf MP, Jaconi ME, et al. Three-dimensional extracellular matrix-derived cardioprogenitor differentiation: systematic modulation of a synthetic cell-responsive PEG-hydrogel. Biomaterials. 2008;29:2757–66.

  26. 26.

    Hirata M, Yamaoka T. Hepatocytic differentiation of iPS cells on decellularized liver tissue. J Artif Organs. 2017;20:318–25.

  27. 27.

    Livak JJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and 2−ΔΔCT method. Methods. 2001;25:402–8.

  28. 28.

    Yamaoka T, Tamura T, Seto Y, Tada T, Kunugi S, Tirrell DA. Mechanism for the phase transition of a genetically engineered elastin model peptide (VPGIG)40 in aqueous solution. Biomacromolecules. 2003;4:1680–5.

  29. 29.

    Trabbic-Carlson K, Setton LA, Chilkoti A. Swelling and mechanical behaviors of chemically cross-linked hydrogels of elastin-like polypeptides. Biomacromolecules. 2003;4:572–80.

  30. 30.

    Fässler R, Rohwedel J, Maltsev V, Bloch W, Lentini S, Guan K, et al. Differentiation and integrity of cardiac muscle cells are impaired in the absence of β1 integrin. J Cell Sci. 1996;109:2989–99.

  31. 31.

    Hodgkinson CP, Gomez JA, Payne AJ, Zhang L, Wang X, Dal-Pra S, et al. Abi3bp regulates cardiac progenitor cell proliferation and differentiation. Circ Res. 2014;115:1007–17.

  32. 32.

    Konstandin MH, Toko H, Gastelum GM, Quijada P, De La Torre A, Quintana M, et al. Fibronectin is essential for regenerative cardiac progenitor cell response following myocardial infarction. Circ Res. 2013;113:115–25.

Download references


This work was supported financially in part by the Intramural Research Funds from the NCVC (25-2-2 and 29-6-2) and by the Japan Society for the Promotion of Science Grant-in-Aid for Challenging Exploratory Research (26560251).

Author information


  1. Department of Biomedical Engineering, National Cerebral and Cardiovascular Center (NCVC) Research Institute, 5-7-1 Fujishirodai, Suita, Osaka, 565-8565, Japan

    • Yusuke Kambe
    • , Takayuki Tokushige
    • , Atsushi Mahara
    •  & Tetsuji Yamaoka
  2. Faculty of Chemistry, Materials and Bioengineering, Kansai University, 3-3-35 Yamatecho, Suita, Osaka, 565-8680, Japan

    • Takayuki Tokushige
    •  & Yasuhiko Iwasaki


  1. Search for Yusuke Kambe in:

  2. Search for Takayuki Tokushige in:

  3. Search for Atsushi Mahara in:

  4. Search for Yasuhiko Iwasaki in:

  5. Search for Tetsuji Yamaoka in:

Conflict of interest

The authors declare that they have no conflict of interest.

Corresponding author

Correspondence to Tetsuji Yamaoka.

Electronic supplementary material

About this article

Publication history