Abstract Body: For the goal of regenerative cardiology, cellular therapy may be used to restore injured heart muscle. The molecular mechanisms behind the engraftment and maturation of these transplanted cells, however, remain poorly understood. Thus, to obtain a comprehensive understanding of the complex transcriptional landscapes within their transplanted spatial context, we utilized spatial transcriptomics in this study.

We have previously demonstrated the safety and effectiveness of transplanting our cardiovascular progenitors (CVPs) produced from laminin-221 into pig hearts that had suffered myocardial infarction (MI). Here, we undertake 10x spatial transcriptomics in chronic MI pig models at 1-, 4-, and 12 weeks post-transplantation to further our understanding of these cells in vivo.

With confidence, we can distinguish between the transcripts from the pigs and the humans in the infarcted region. By using human-specific anti-Ku80 antibodies for immunohistochemical labeling of the pig tissue samples, this in silico prediction was verified. The CVPs' engraftment and longevity in the infracted region were shown by the long-term identification and staining of human cells at various time points. It is interesting to note that after 12 weeks, ribosomal and mitochondrial activity were higher than at 1 and 2 weeks. These results are consistent with the hallmarks of cardiac maturation, in which cells transit from immature fetal-like cells to mature cardiomyocytes (CMs) by switching their metabolic energy source from glucose to fatty acids. Also, by comparing the heart-specific genes, including MYH6 and MYH7, we were able to quantify the myosin isoform switches in the xenograft over time. Furthermore, to delineate the fate of the transplanted cells, we performed a ligand-receptor interaction analysis. Intriguing, we identified the midkine protein as a possible secreted human paracrine factor that enhances angiogenesis in the graft.

In conclusion, we have shown the successful engraftment and maturation of human CVPs toward CMs using spatial transcriptomic technology. These data will be used to understand other aspects of regenerative cardiology such as the mechanism of VTs and in vivo cellular repair mechanisms. A shiny app atlas has been generated to share our transcriptomics data for all to explore gene expression of human transplanted cells in myocardial infarcted pig hearts.