Division of Oral Biotechnology, Center for Dental Medicine, Medical Center-University of Freiburg, Faculty of Medicine, Albert-Ludwigs-University of Freiburg, Hugstetter Str. 55, 79106 Freiburg, Germany.
Department of Operative Dentistry and Periodontology, Centre for Dental Medicine Medical Center-University of Freiburg, Faculty of Medicine, Albert-Ludwigs-University of Freiburg, 79106 Freiburg, Germany.
Cells. 2022 Oct 12;11(20):3204. doi: 10.3390/cells11203204.
Human dental pulp stem cells (hDPSCs) are promising for oral/craniofacial regeneration, but their purification and characterization is not yet standardized. hDPSCs from three donors were purified by magnetic activated cell sorting (MACS)-assisted STRO-1-positive cell enrichment (+), colony derivation (c), or a combination of both (c/+). Immunophenotype, clonogenicity, stemness marker expression, senescence, and proliferation were analyzed. Multilineage differentiation was assessed by qPCR, immunohistochemistry, and extracellular matrix mineralization. To confirm the credibility of the results, repeated measures analysis and post hoc -value adjustment were applied. All hDPSC fractions expressed STRO-1 and were similar for several surface markers, while their clonogenicity and expression of CD10/44/105/146, and 166 varied with the purification method. (+) cells proliferated significantly faster than (c/+), while (c) showed the highest increase in metabolic activity. Colony formation was most efficient in (+) cells, which also exhibited the lowest cellular senescence. All hDPSCs produced mineralized extracellular matrix. Regarding osteogenic induction, (c/+) revealed a significant increase in mRNA expression of and , while osteogenic marker genes were detected at varying levels. (c/+) were the only population missing gene transcription increase during neurogenic induction. All hDPSCs were able to differentiate into chondrocytes. In summary, the three hDPSCs populations showed differences in phenotype, stemness, proliferation, and differentiation capacity. The data suggest that STRO-1-positive cell enrichment is the optimal choice for hDPSCs purification to maintain hDPSCs stemness. Furthermore, an (immuno) phenotypic characterization is the minimum requirement for quality control in hDPSCs studies.
人牙髓间充质干细胞（hDPSCs）在口腔/颅面再生中具有广阔的应用前景，但目前其分离和鉴定方法尚未标准化。本研究采用磁激活细胞分选（MACS）辅助 STRO-1 阳性细胞富集（+）、集落衍生（c）或两者联合（c/+）的方法，从 3 名供者中分离纯化 hDPSCs。分析细胞免疫表型、克隆形成能力、干细胞标志物表达、衰老和增殖情况。通过 qPCR、免疫组化和细胞外基质矿化评估多能分化。采用重复测量分析和事后 - 值调整对结果进行验证。所有 hDPSC 亚群均表达 STRO-1，几种表面标志物相似，但其克隆形成能力和 CD10/44/105/146、166 的表达随纯化方法而变化。（+）细胞的增殖速度明显快于（c/+）细胞，而（c）细胞的代谢活性增加最高。集落形成效率以（+）细胞最高，细胞衰老程度最低。所有 hDPSCs 均能产生矿化细胞外基质。在成骨诱导方面，（c/+）细胞 和 的 mRNA 表达显著增加，而在成骨标志物基因的检测中则呈现出不同的表达水平。（c/+)在神经诱导中是唯一未检测到 基因转录增加的细胞群。所有 hDPSCs 均能分化为软骨细胞。综上所述，三种 hDPSC 亚群在表型、干性、增殖和分化能力方面存在差异。数据表明，STRO-1 阳性细胞富集是 hDPSCs 分离纯化中维持 hDPSCs 干性的最佳选择。此外，（免疫）表型特征分析是 hDPSCs 研究质量控制的最低要求。
