Advantages of Cell Proliferation and Immune Regulation in CD146+NESTIN+ HUMSCs Obtained from Extremely Premature Infants: Insights from Single-Cell RNA Sequencing

Background In our prior study, we discovered that human umbilical cord Wharton’s Jelly derived mesenchymal stem cells (HUMSCs) obtained from extremely preterm infants demonstrated superior characteristics compared to term infants, particularly regarding cell proliferation, pluripotency, and cell damage repair ability. To explore the underlying heterogeneity between these cells further, we utilized single-cell RNA sequencing (scRNA-seq) to examine their transcriptional differences and potential molecular pathways involved in this heterogeneity. Methods We conducted scRNA-seq on HUMSCs obtained from three distinct gestational ages- 22+5 weeks, 28 weeks, and 39 weeks, respectively. To assist in the analysis, we employed the scRNA-seq data from two bone marrow mesenchymal stem cells (BMSCs) samples available in existing literature as reference datasets. Subsequently, we undertook bioinformatics analysis on the obtained transcriptomic data using the R programming language. Results Upon merging the five samples, we were able to identify a total of 17 cell subpopulations with high expression of fibroblast and MSC markers. The expression of CD146 was found to be significantly higher in HUMSCs as compared to BMSCs. Moreover, we observed higher expression of Nestin+ cells in premature HUMSCs. Cell cycle analysis revealed that the majority of HUMSCs were in the G2M phase, while BMSCs were mainly in the G1 phase. Pseudotime analysis showed that HUMSCs had a lower degree of differentiation compared to BMSCs, and this decreased with increasing gestational age. Custom gene set scoring analysis revealed that the cells expressed genes related to osteogenesis, chondrogenesis, adipogenesis, stemness, immunology, and vasculogenesis; with preterm HUMSCs displaying an immunological edge. Differential gene analysis and gene enrichment analysis indicated that CD146+Nestin+ HUMSC subpopulations displayed upregulation in immune regulation, cell proliferation-related gene expression, and gene regulatory pathways. Conclusion scRNA-seq analysis revealed differences between BMSCs and HUMSCs at both preterm and term infant. Specifically, the expression of CD146+ and Nestin+ cells was significantly higher in preterm HUMSCs, which may contribute to their advantages in immune regulation, cell proliferation-related gene expression, and regulatory pathways. These findings hold great significance in advancing our understanding of the molecular mechanisms of HUMSCs and their potential applications in disease treatment, transplantation, and regenerative medicine.