Human dental pulp stem cells (hDPSCs) are easily obtained multipotent cells, however, their potential value in regenerative medicine is hindered by the phenotypic and functional changes after conventional monolayer expansion. Here, we employed single-cell RNA sequencing (scRNA-seq) to comprehensively study the transcriptional difference between the freshly isolated and monolayer cultured DPSCs. The cell cluster analysis based on our scRNA-seq data showed that monolayer culture resulted in a significant cellular composition switch compared to the freshly isolated DPSCs. However, one subpopulation, characterized as MCAM(+)JAG(+)PDGFRA(−), maintained the most transcriptional characteristics compared to their freshly isolated counterparts. Notably, immunofluorescent staining revealed that the MCAM(+)JAG(+)PDGFRA(−) hDPSCs uniquely located in the perivascular region of human dental pulp tissue. Flow-cytometry analysis confirmed that their proportion remained relatively stable (~2%) regardless of physiological senescence or dental caries. Consistent with the annotation of scRNA-seq data, MCAM(+)JAG(+)PDGFRA(−) hDPSCs showed higher proliferation capacity and enhanced in vitro multilineage differentiation potentials (osteogenic, chondrogenic and adipogenic) compared with their counterparts PDGFRA(+) subpopulation. Furthermore, the MCAM(+)JAG(+)PDGFRA(−) hDPSCs showed enhanced bone tissue formation and adipose tissue formation after 4-week subcutaneous implantation in nude mice. Taken together, our study for the first time revealed the cellular composition switch of monolayer cultured hDPSCs compared to the freshly isolated hDPSCs. After in vitro expansion, the MCAM(+)JAG(+)PDGFRA(−) subpopulation resembled the most transcriptional characteristics of fresh hDPSCs which may be beneficial for further tissue regeneration applications.

人牙髓干细胞（hDPSC）是容易获得的多能细胞，然而，其在再生医学中的潜在价值受到常规单层扩增后表型和功能变化的阻碍。在这里，我们采用单细胞RNA测序（scRNA-seq）来全面研究新鲜分离的DPSC和单层培养的DPSC之间的转录差异。基于我们的 scRNA-seq 数据的细胞簇分析表明，与新鲜分离的 DPSC 相比，单层培养导致显着的细胞组成变化。然而，与新鲜分离的亚群相比，一个被称为 MCAM(+)JAG(+)PDGFRA(−) 的亚群保持了大部分转录特征。值得注意的是，免疫荧光染色显示 MCAM(+)JAG(+)PDGFRA(−) hDPSC 独特地位于人牙髓组织的血管周围区域。流式细胞术分析证实，无论生理衰老还是龋齿，它们的比例都保持相对稳定（~2%）。与 scRNA-seq 数据的注释一致，与对应的 PDGFRA(+) 亚群相比，MCAM(+)JAG(+)PDGFRA(−) hDPSC 表现出更高的增殖能力和增强的体外多谱系分化潜力（成骨、软骨形成和脂肪形成） 。此外，MCAM(+)JAG(+)PDGFRA(−) hDPSCs在裸鼠皮下植入4周后显示出骨组织形成和脂肪组织形成增强。总而言之，我们的研究首次揭示了单层培养的 hDPSC 与新鲜分离的 hDPSC 相比的细胞组成变化。 体外扩增后，MCAM(+)JAG(+)PDGFRA(−) 亚群与新鲜 hDPSC 的转录特征最为相似，这可能有利于进一步的组织再生应用。