SPECIAL ISSUE: RESEARCH ARTICLE Exploring peptide‐functionalized alginate scaffolds for engineering cardiac tissue from human embryonic stem cell‐ derived cardiomyocytes in serum‐free medium Dana Hayoun‐Neeman 1 | Nataly Korover 1 | Sharon Etzion 2 | Rivka Ofir 2 | Rachel G. Lichtenstein 1,2 | Smadar Cohen 1,2,3 1 Avram and Stella Goren‐Goldstein Department of Biotechnology Engineering, Ben‐Gurion University of the Negev, Beer Sheva, Israel 2 Regenerative Medicine and Stem Cell (RMSC) Research Center 3 The Else Katz Institute for Nanoscale Science and Technology, Ben‐Gurion University of the Negev, Beer‐Sheva 84105, Israel Correspondence Smadar Cohen and Rachel G. Lichtenstein, Avram and Stella Goren‐Goldstein Department of Biotechnology Engineering, Ben‐Gurion University of the Negev, Beer Sheva, Israel. Email: scohen@bgu.ac.il; ruha@bgu.ac.il Funding information Jordan Baruch Stem Cell Fund Engineering human cardiac tissue is a promising solution for myocardial repair of injured hearts and for drug screening. Herein, we examined the capability of chemi- cally defined alginate scaffolds to promote cardiac tissue regeneration from human embryonic stem cell‐derived cardiomyocytes (hESC‐CMs) in serum‐free, chemically defined medium. The cells were single seeded or coseeded with human dermal fibro- blasts (HFs) in macroporous scaffolds made from pristine alginate or alginate modified with arginine‐glycine‐aspartate (RGD) peptide and heparin‐binding peptide (HBP). Our results show that the addition of fibroblasts to the 3‐D culture is indispensable for the formation of functional cardiac tissues and that the presence of RGD/HBP attached to the alginate matrix further improves its functionality. The engineered tis- sue displayed the typical fiber morphology with massive striation. An increase in con- traction amplitude and calcium transients with time, together with a decrease in excitation threshold, indicated advancement toward tissue maturation. Our results thus point to the importance of co‐cultivating fibroblasts with hESCs‐CMs in chemi- cally defined peptide‐functionalized alginate scaffolds and culture medium for regenerating functional cardiac tissue in vitro. KEYWORDS cardiomyocytes, fibroblasts, human embryonic stem cells, RGD/HBP‐functionalized scaffold, tissue engineering 1 | INTRODUCTION The ability of adult mammalian heart tissue to regenerate after an extensive injury is limited, and it cannot repair sizeable heart defects, such as after myocardial infarction (MI). Patients suffering from MI can progress to heart failure as the injured ventricles dilate and remodel. The recent development of differentiation protocols for human pluripotent stem cells (hPSCs) and the generation of large num- bers of human cardiomyocytes (CMs) 1 have raised the prospect of using these cells in various therapeutic strategies to regenerate dam- aged heart tissue 2 and for their use in cell‐based assays to screen for drug cardiotoxicity in drug discovery programs. 3 To realize their potential in these applications, the strategy of tissue engineering has been applied to promote the functional maturity of the hPSC‐derived CMs. 4 The accumulated experience with in vitro cardiac tissue engineer- ing using cardiac cells, isolated from animal or human heart tissues, points to the applicability of this strategy in yielding functional cardiac tissue. 5 Much of this acquired information has been implemented in the recent transition to using hPSC‐derived CMs (hPSC‐CMs) as the Dana Hayoun‐Neeman and Nataly Korover have equally contributed to this work. Received: 28 February 2019 Accepted: 15 March 2019 DOI: 10.1002/pat.4602 Polym Adv Technol. 2019;1–13. © 2019 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/pat 1