BackgroundDevelopment of clinical-grade cell preparations is central to meeting the regulatory requirements for cellular therapies under good manufacturing practice-compliant (cGMP) conditions. Since addition of animal serum in culture media may compromise safe and efficient expansion of mesenchymal stem cells (MSCs) for clinical use, this study aimed to investigate the potential of two serum/xeno-free, cGMP culture systems to maintain long-term “stemness” of oral MSCs (dental pulp stem cells (DPSCs) and alveolar bone marrow MSCs (aBMMSCs)), compared to conventional serum-based expansion.MethodsDPSC and aBMMSC cultures (n = 6/cell type) were established from pulp and alveolar osseous biopsies respectively. Three culture systems were used: StemPro_MSC/SFM_XenoFree (Life Technologies); StemMacs_MSC/XF (Miltenyi Biotek); and α-MEM (Life Technologies) with 15% fetal bovine serum. Growth (population doublings (PDs)), immunophenotypic (flow cytometric analysis of MSC markers) and senescence (β-galactosidase (SA-β-gal) activity; telomere length) characteristics were determined during prolonged expansion. Gene expression patterns of osteogenic (ALP, BMP-2), adipogenic (LPL, PPAR-γ) and chondrogenic (ACAN, SOX-9) markers and maintenance of multilineage differentiation potential were determined by real-time PCR.ResultsSimilar isolation efficiency and stable growth dynamics up to passage 10 were observed for DPSCs under all expansion conditions. aBMMSCs showed lower cumulative PDs compared to DPSCs, and when StemMacs was used substantial delays in cell proliferation were noted after passages 6–7. Serum/xeno-free expansion produced cultures with homogeneous spindle-shaped phenotypes, while serum-based expansion preserved differential heterogeneous characteristics of each MSC population. Prolonged expansion of both MSC types but in particular the serum/xeno-free-expanded aBMMSCs was associated with downregulation of CD146, CD105, Stro-1, SSEA-1 and SSEA-4, but not CD90, CD73 and CD49f, in parallel with an increase of SA-gal-positive cells, cell size and granularity and a decrease in telomere length. Expansion under both serum-free systems resulted in “osteogenic pre-disposition”, evidenced by upregulation of osteogenic markers and elimination of chondrogenic and adipogenic markers, while serum-based expansion produced only minor changes. DPSCs retained a diminishing (CCM, StemPro) or increasing (StemMacs) mineralization potential with passaging, while aBMMSCs lost this potential after passages 6–7 under all expansion conditions.ConclusionsThese findings indicate there is still a vacant role for development of qualified protocols for clinical-grade expansion of oral MSCs; a key milestone achievement for translation of research from the bench to clinics.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-017-0705-0) contains supplementary material, which is available to authorized users.
Objectives: Peri-implantitis (PI) is an inflammatory disease associated with periimplant bone loss and impaired healing potential. There is limited evidence about the presence of mesenchymal stromal cells (MSCs) and their regenerative properties within the granulation tissue (GT) of infrabony peri-implantitis defects. The aim of the present study was to characterize the cells derived from the GT of infrabony PI lesions (peri-implantitis derived mesenchymal stromal cells-PIMSCs).Material and Methods: PIMSC cultures were established from GT harvested from PI lesions with a pocket probing depth ≥6 mm, bleeding on probing/suppuration, and radiographic evidence of an infrabony component from four systemically healthy individuals. Cultures were analyzed for embryonic (SSEA4, NANOG, SOX2, OCT4A), mesenchymal (CD90, CD73, CD105, CD146, STRO1) and hematopoietic (CD34, CD45) stem cell markers using flow cytometry. PIMSC cultures were induced for neurogenic, angiogenic and osteogenic differentiation by respective media. Cultures were analyzed for morphological changes and mineralization potential (Alizarin Red S method). Gene expression of neurogenic (NEFL, NCAM1, TUBB3, ENO2), angiogenic (VEGFR1, VEGFR2, PECAM1) and osteogenic (ALPL, BGLAP, BMP2, RUNX2) markers was determined by quantitative RT-PCR.Results: PIMSC cultures demonstrated high expression of embryonic and mesenchymal stem cell markers with inter-individual variability. After exposure to neurogenic, angiogenic and osteogenic conditions, PIMSCs showed pronounced tri-lineage differentiation potential, as evidenced by their morphology and expression of respective markers. High mineralization potential was observed.Conclusions: This study provides evidence that MSC-like populations reside within the GT of PI lesions and exhibit a multilineage differentiation potential. Further studies are needed to specify the biological role of these cells in the healing processes of inflamed PI tissues and to provide indications for their potential use in regenerative therapies.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.