Scaling-Up of Dental Pulp Stem Cells Isolated from Multiple Niches

Abstract

Dental pulp (DP) can be extracted from child’s primary teeth (deciduous), whose loss occurs spontaneously by about 5 to 12 years. Thus, DP presents an easy accessible source of stem cells without ethical concerns. Substantial quantities of stem cells of an excellent quality and at early (2–5) passages are necessary for clinical use, which currently is a problem for use of adult stem cells. Herein, DPs were cultured generating stem cells at least during six months through multiple mechanical transfers into a new culture dish every 3–4 days. We compared stem cells isolated from the same DP before (early population, EP) and six months after several mechanical transfers (late population, LP). No changes, in both EP and LP, were observed in morphology, expression of stem cells markers (nestin, vimentin, fibronectin, SH2, SH3 and Oct3/4), chondrogenic and myogenic differentiation potential, even after cryopreservation. Six hours after DP extraction and in vitro plating, rare 5-bromo-2′-deoxyuridine (BrdU) positive cells were observed in pulp central part. After 72 hours, BrdU positive cells increased in number and were found in DP periphery, thus originating a multicellular population of stem cells of high purity. Multiple stem cell niches were identified in different zones of DP, because abundant expression of nestin, vimentin and Oct3/4 proteins was observed, while STRO-1 protein localization was restricted to perivascular niche. Our finding is of importance for the future of stem cell therapies, providing scaling-up of stem cells at early passages with minimum risk of losing their “stemness”.

Insulin-Like Growth Factor 1 Receptor and p38 Mitogen-Activated Protein Kinase Signals Inversely Regulate Signal Transducer and Activator of Transcription 3 Activity to Control Human Dental Pulp Stem Cell Quiescence, Propagation, and Differentiation

“…Inactivation of IGF-1R and p38 MAPK by their respective inhibitors, sc204008 and PD169316, induced Ki67 in DPCs ex vivo and decreased the quiescence of PY low and G0-arrested human DPSCs cells in vitro, demonstrating that p38 MAPK and IGF-1R are responsible for the mitotic quiescence of these cells. The identity of DPSCs was corroborated by their phenotype, stem cell activity, and localization of Ki67-positive cells perfectly matching the position of BrdU-positive DPSCs reported by Téclès et al [4] and Ishikawa et al [19] and the position of Oct4-positive pluripotent DPSCs reported in another study [74]. Taken together, our data establish that IGF-1R and p38 MAPK signaling pathways control human DPSC quiescence and demonstrate that these pathways must be inactivated for mitotic division to occur in DPSCs.…”

How has dental pulp stem cells isolation been conducted? A scoping review

“…The DPSCs isolation process has evolved overtime and currently it can be done using two main techniques: explant 18,19,20 and enzymatic; 19,20 the Table 4. Distribution according to the supplementation used in DPSCs culture medium preparation (some studies used more than one culture medium).…”

“…Lizier and collaborators developed a cell culture method that preserves DPSCs viability for longer periods, minimizing the slow proliferation rate. 18 The methodology is based on the explant technique, and it consists in transferring tissue fragments to a different plate every time the desired cell density is achieved. It allows the de novo cell migration to a new plate, enabling the achievement of larger amounts of pulp stem cells.…”

“…It allows the de novo cell migration to a new plate, enabling the achievement of larger amounts of pulp stem cells. 18 Few studies have compared the enzymatic and explant techniques for DPSCs isolation. 23,29,31 In a well-designed study, Hilkens and collaborators showed that cells isolated by the enzymatic or the explant techniques had the same proliferating rate and colony formation capacity, and could successfully differentiate into adipogenic, chrondrogenic and osteogenic cell types, indicating that both isolation methodologies could be applied to obtain suitable autologous DPSCs.…”

Human adult dental pulp CD117/c-kit-positive networks of stromal cells

“…Dental pulp is a complex microenvironment where mesenchymal stem cells and progenitor cells cohabit [18], usually in perivascular stem cell niches [7,17,26].…”