A strategy to replace the high loss of cells, especially cardiomyocytes, in injury is to deliver engineered cardiac cells or tissues (graft) into the damaged host tissue. Embryonic stem cells (ESCs) and Human-induced Pluripotent Stem Cells (hiPSC) are the most readily available sources of human-lineage to derive cardiomyocytes, because they can proliferate indefinitely and be differentiated into cells of different lineages. The key objective in cardiac tissue/cell engineering include: 1) reaching high proliferative efficacy, 2) high rate of cardiomyocyte differentiation, and 3) reaching efficient engraftment, also reducing host rejection to graft.

A–C) hiPSCs were differentiated into CMs via the Sandwich method, and the lineage of the differentiated hiPSC-CMs was confirmed via the expression of (A) slow myosin heavy chain (SMHC) and α-sarcomeric actin (α-SA); (B) cardiac troponin T (cTnT) and the ventricular- specific cardiomyocyte protein myosin light chain 2v (MLC2v); and (C) cTnT and the gap-junction protein connexin-43 (Con-43); nuclei were counterstained with DAPI. The boxed region in the second panel of (C) is shown at higher magnification. (G–L) The lineage of the differentiated hiPSC-ECs was confirmed via the expression of (G) CD31, (H) CD144, and (I) vWF-8; and (J–L) the lineage of the differentiated hiPSC-SMCs was confirmed via the expression of (J) smooth-muscle actin (SMA), (K) SM22, and (L) calponin. Nuclei were counterstained with DAPI. Magnification (G)–(L) = 200×

     Zhang lab has mastered the technique to create hiPSC-derived cardiac cells for a long time. Thus, we focus on strategies to increase proliferate and engraftment rate of the engineered cells and tissues. In 2014, we, led by Dr. Ye Lei, created a new hiPSC-derived cardiac tissue, which consisted of cardiomyocytes, endothelial cells, and smooth muscle cells differentiated from the same hiPSC origin. This engineered tissue was successfully delivered into a porcine injured-heart model; the tissue survived in the host four weeks after delivery, demonstrated better engraftment rate and improve the host cardiac function compared to delivering hiPSC-derived cardiomyocytes alone. Then, in 2021, the work led by Dr. Meng Zhao showed that by overexpressing Cyclin D2 (CCND2), the hiPSC-derived cardiomyocytes not only significantly increased the engraftment rate but also promoted the host cardiomyocyte proliferation in the porcine model. Furthermore, exosomes, which contain microRNAs, released by CCND2-overexpressing hiPSC-derived cardiomyocytes interact with the host cardiomyocytes and support their proliferation.

Cyclin D2 (CCND2)–overexpressing cardiomyocytes (CCND2OECMs) proliferated after transplantation into infarcted pig hearts. A, Gross anatomy of a single cardiac slice from a porcine heart at 4 weeks after the surgery: each slice was divided into different subregions for Y chromosome quantification and other analyses. B and C, Immunofluorescence images of tissue sections of the porcine hearts subjected to myocardial infarction and transplantation of CCND2OECMs 4 weeks after the treatment. The transplanted human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) were identified by coexpression of cardiac troponin T (cTnT; red) and human nuclear antigen (HNA; green). The infarct zone, the border zone, the graft, and the native (recipient) cardiomyocytes in the infarcted porcine hearts are marked inside the white dashed ovals or simply marked (native cardiomyocytes) on the image panels (B and C). Scale bars=100 µm and 20 µm. D through F, Sections from the infarct border zone were stained for HNA to identify cardiomyocytes of the human origin (hiPSC-CMs), for cTnT to verify cardiomyocyte identity, and for Ki67 (D) and phosphorylated histone 3 (PH3; E) to verify proliferating activity of the cardiomyocytes. TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) staining was performed to assess the level of apoptosis in the cardiomyocytes (F). Nuclei were counterstained with DAPI (4′,6-diamidino-2-phenylindole) and hiPSC-CM proliferation was quantified and presented as a percentage of HNA-expressing cells that also expressed Ki67 or PH3; hiPSC-CM apoptosis was quantified as a percentage of the HNA-expressing cells positive for TUNEL staining. Scale bar=20 µm. *P<0.05 relative to MI+CCND2WTCM.

Read more:

– Ye L, Zhang P, Duval S, Su L, Xiong Q, Zhang J. Thymosin β4 increases the Potency of Transplanted Mesenchymal Stem Cells for Myocardial Repair. Circulation. 2013;128(11 Suppl 1):S32-S41. PMID: 24030419; PMCID: PMC3886821

– Ye L, Chang YH, Xiong Q, Zhang P, Zhang L, Somasundaram P, Lepley M, Swingen C, Su L, Wendel JS, Guo J, Jang A, Rosenbush D, Greder L, Dutton JR, Zhang J, Kamp TJ, Kaufman DS, Ge Y, Zhang J. Cardiac Repair in a Porcine Model of Acute Myocardial Infarction with Human Induced Pluripotent Stem Cell-Derived Cardiovascular Cells. Cell Stem Cell. 2014;15(6):750-761. PMID: 25479750; PMCID: PMC4275050

– Zhu W, Zhao M, Mattapally S, Chen S, Zhang J. CCND2 Overexpression Enhances the Regenerative Potency of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes: Remuscularization of Injured Ventricle. Circulation Research. 2018 Jan 5;122(1):88-96. Epub 2017 Oct 10. PMID: 29018036; PMCID: PMC5756126

– Zhao M, Nakada Y, Wei Y, Bian W,  Chu Y,  Borovjagin AV, Xie M, Zhu W, Nguyen T, Zhou Y, Serpooshan V, Walcott GP, Zhang J.  Cyclin D2 overexpression enhances the efficacy of human induced pluripotent stem cell-derived cardiomyocytes for myocardial recovery in a swine model of myocardial infarction.  Circulation May 2021

– Kahn-Krell AM, Pretorius D,  Ou J, Fast V, Litovsky S, Joel Berry J, Liu M, Zhang J.  Bioreactor Suspension Culture: Differentiation and Production of Cardiomyocyte Spheroids From Human Induced Pluripotent Stem Cells. Frontiers in bioengineering-and-biotechnology