Porcine cells restrict human cell proliferation via cellular competition in a human-porcine mesenchymal stem cells co-culture model

The xenotransplantation of human cells into porcine hosts holds immense potential in the fields of regenerative medicine and organ transplantation. However, the low survival rate of human-derived cells within porcine remains a critical bottleneck constraining the application of xenotransplantation. Whether porcine cells exert negative effect on human cell growth is not studied. Here, we established an <i>in vitro</i> direct co-culture model of human and porcine mesenchymal stem cells (hMSCs and pMSCs) to investigate the competitive relationship between human and porcine-derived cells. The results demonstrated that the proliferation capability of hMSCs in the co-culture system was significantly suppressed compared to those cultured in isolation. Moreover, an increasing number of pMSCs exhibited enhanced inhibition of hMSC proliferation. Notably, results from transwell assays and treatment with porcine-conditioned medium indicated that the inhibition of hMSCs by pMSCs was not mediated through soluble cytokines. To elucidate the underlying molecular mechanisms, RNA sequencing analysis was performed and the result revealed that direct co-culture significantly downregulated the expression of proliferation-related genes in hMSCs, including <i>CYP1B1</i>, <i>SLC7A11</i>, <i>TFAP2C</i>, and <i>PSAT1</i>. Concurrently, the co-culture paradigm disrupted endoplasmic reticulum function and multiple amino acid transport processes within hMSCs, while activating the NF-κB signaling pathway, thereby achieving negative regulation of hMSC proliferation. Collectively, our primary study characterized the competitive interactions between hMSCs and pMSCs and uncovered possible underlying mechanisms which provided new experimental foundations for improving human cell survival in porcine hosts to advance xenotransplantation application.