Cardiomyocyte (CM) implantation is a promising treatment for heart failure through myocardial remuscularization. Clinical research is still ongoing, with studies involving large animal models and tissue-engineered grafts with increasing experimental data. Thorough predictions for larger animal results and human outcomes remain obstructed because small animal evidence proves insufficient in accounting for fundamental physiological and immunological differences.
Long-term CM engraftment has not been achieved in immunocompetent models under clinically compatible immunosuppression conditions despite the strong immune responses that continue challenging xenograft studies.
This research was approved by multiple institutional authorities and ethics committees. The Stanford Animal Research Committee authorized the use of nude rats in animal studies with approval from Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit for the rhesus macaque research. BioVAT-HF-DZHK20 Phase 1/2 clinical trial received authorization from both Paul-Ehrlich-Institut and the Ethics Committee of the University Medical Center Göttingen. All patients provided informed consent.
Four lines of rhesus macaque iPS cells generated through either Sendai virus transduction or episomal plasmid reprogramming were used experimental subjects. The researchers included human iPS cells as well as the TC1133 cell line. The researchers authenticated their cell lines and conducted mycoplasma testing to ensure integrity.
The ABCF+I protocol was used for cardiomyocyte differentiation, whereas the ABCF-CRAB-VCF protocol generated stromal cells (StCs). Cell biologists used growth factor-enriched media to combine CMs, StCs, and medical-grade bovine collagen to construct engineered heart muscle (EHM).
The assessment of contractile properties in EHM loops used isometric force measurements during electrical field stimulation. Single-nucleus RNA sequencing (snRNA-seq) examined cell composition in both flash-frozen iPS-derived cells together with EHMs. To analyze EHM composition, Flow cytometry used detection methods for the proteins sarcomeric actinin (ACTN2) and vimentin (VIM).
The established protocols successfully differentiated iPS cells into CMs and StCs. The flow cytometry results indicated high quantities of ACTN2-positive cardiomyocytes and VIM-positive stromal cells in EHMs. The isometric force testing revealed that EHMs exhibited muscular strength, which responded well when subjected to mechanical force based on Frank-Starling principles.
RNA sequencing of small nuclear RNA produced outcomes that revealed different cell populations located inside EHMs; through principal component analysis and clustering, the research group identified separate gene expression patterns that corresponded to cardiomyocytes, stromal cells, and endothelial-like cells. The Louvain algorithm detected all anticipated cellular clusters, thus proving the accurate differentiation process. The CMs showed expression patterns consistent with mature cells, thus demonstrating their functionality.
The research successfully used iPS cells to create functional CMs and StCs before integrating these cells into EHMs. This characteristic of EHMs to contract serves as supportive evidence for their potential application in cardiac tissue engineering. Specific information on the cell composition and gene expression patterns of EHMs that showed consistent and predicted differentiation processes was discovered using snRNA-seq research. Â Â Â Â
The ABCF-CRAB-VCF and ABCF+I techniques performed better than earlier approaches in producing cardiac cell types efficiently and at scale. The observed heart muscle contractions prove consistent with studies that have examined engineered cardiac tissues, thus enabling applications within regenerative medicine. The research needs more development to understand the translation possibilities, emphasizing how generated cardiac tissues function during long periods inside living organisms.
This study successfully developed EHMs using cells from iPS-derived cardiomyocytes and stromal cells, which exhibited excellent differentiation and functional connections. The research team performed contractility assays along with snRNA-seq testing to establish both the structural and molecular accuracy of their constructs. The research enables the advancement of cardiac tissue engineering by offering new possibilities for heart failure therapy and regenerative medicine. Further research involves the living subjects is necessary to evaluate the long-term treatment efficacy and therapeutic viability.
References: Jebran AF, Seidler T, Tiburcy M, et al. Engineered heart muscle allografts for heart repair in primates and humans. Nature. 2025. doi:10.1038/s41586-024-08463-0
