Characterization of a Stemness-Optimized Purification Method for Human Dental-Pulp Stem Cells: An Approach to Standardization

Abstract

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 p-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 COL5A1 and COL6A1, while osteogenic marker genes were detected at varying levels. (c/+) were the only population missing BDNF 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.

Biomechanical Modulation of Dental Pulp Stem Cell (DPSC) Properties for Soft Tissue Engineering

“…In this study, pulp cells were enriched via STRO-1-dependent magnetic-activated cell sorting. STRO-1+ dental pulp cells have been shown to exhibit excellent SC properties, including a high proliferative capacity, multilineage differentiation potential, and a low tendency toward cellular senescence [ 49 ]. Standardization of DPSCs isolation and enrichment protocols is a central precondition for reproducible and safe application of this cell type in regenerative treatments in the clinic.…”

“…The medium was exchanged every 2–3 days until cells reached a confluence of 80–90%. DPSCs were characterized as described previously [ 49 ].…”