Isolation and prolonged expansion of oral mesenchymal stem cells under clinical-grade, GMP-compliant conditions differentially affects “stemness” properties

Abstract

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.

Mesenchymal stem cells: Cell therapy and regeneration potential

“…B. Liu, Chen, et al, 2012;Moya et al, 2018;J. Zhang, Jia, et al, 2013) Xenofree isolation and culturing (Bakopoulou et al, 2017;Gottipamula et al, 2013;Laitinen et al, 2016;Swamynathan et al, 2014) Abbreviation: MSC, mesenchymal stromal/stem cell.…”

Evaluation of growth, stemness, and angiogenic properties of dental pulp stem cells cultured in cGMP xeno-/serum-free medium

“…Previously, we showed that bone marrow-derived MSCs (BM-MSCs) and adipose tissue-derived MSCs (ASCs) cultured in a serum-free medium (MesenCult-XF) proliferated faster compared with FBS containing medium but had significantly reduced expression of the MSC marker, CD105 (Brohlin et al 2017). However, to be able to use DPSCs clinically, the cells should be preferably cultivated in a defined medium produced under conditions of current good manufacturing practice (cGMP) (Bakopoulou et al 2017). Previous studies have also shown that human serum was a suitable alternative to FBS for expansion of DPSCs while keeping their angiogenic potential and regenerative capacity (Khanna-Jian et al 2012;Piva et al 2017).…”

Characterisation of proliferation, differentiation potential, and gene expression among clonal cultures of human dental pulp cells

“…Furthermore, we found that the ratio of clonogenic cells expressing both STRO-1 and CD146 decreased at > 40 PDL (Table 1; Additional file 2: Table S2). These reductions in the expression ratio of STRO-1 and CD146 were due to the increased number of passages during longterm culture [36,37]. Somoza et al analysed 38 human bone marrow-derived cell clones and found that 10 (26%) were both osteogenic and adipogenic, two (5%) were osteogenic only, 21 (55%) were adipogenic only, and five (13%) did not demonstrate either differentiation potential [15]; notably, these results in bone marrow-derived cell clones were similar to our results in dental pulp-derived cell clones ( Table 2 at 24.1 PDL).…”