Odontogenic capability: bone marrow stromal stem cells versus dental pulp stem cells

Background information. Although adult bone‐marrow‐derived cell populations have been used to make teeth when recombined with embryonic oral epithelium, the differences between dental and non‐dental stem‐cell‐mediated odontogenesis remain an open question. Results. STRO‐1⁺ (stromal precursor cell marker) DPSCs (dental pulp stem cells) and BMSSCs (bone marrow stromal stem cells) were isolated from rat dental pulp and bone marrow respectively by magnetic‐activated cell‐sorting techniques. Their odontogenic capacity was compared under the same inductive microenvironment produced by ABCs (apical bud cells) from 2‐day‐old rat incisors. Co‐cultured DPSCs/ABCs in vitro showed more active odontogenic differentiation ability than mixed BMSSCs/ABCs, as indicated by the accelerated matrix mineralization, up‐regulated alkaline phosphatase activity, cell‐cycle modification, and the expression of tooth‐specific proteins and genes. After cultured for 14 days in the renal capsules of rat hosts, recombined DPSC/ABC pellets formed typical tooth‐shaped tissues with balanced amelogenesis and dentinogenesis, whereas BMSSC/ABC recombinants developed into atypical dentin—pulp complexes without enamel formation. DPSC and BMSSC pellets in vivo produced osteodentin‐like structures and fibrous connective tissues respectively. Conclusions. DPSCs presented more striking odontogenic capability than BMSSCs under the induction of postnatal ABCs. This report provides critical insights into the selection of candidate cells for tooth regeneration between dental and non‐dental stem cell populations.     

Allogenic tooth transplantation inhibits the maintenance of dental pulp stem/progenitor cells in mice

Our recent study suggested that allogenic tooth transplantation may affect the maintenance of dental pulp stem/progenitor cells. This study aims to elucidate the influence of allograft on the maintenance of dental pulp stem/progenitor cells following tooth replantation and allo- or auto-genic tooth transplantation in mice using BrdU chasing, immunohistochemistry for BrdU, nestin and Ki67, in situ hybridization for Dspp, transmission electron microscopy and TUNEL assay. Following extraction of the maxillary first molar in BrdU-labeled animals, the tooth was immediately repositioned in the original socket, or the roots were resected and immediately allo- or auto-grafted into the sublingual region in non-labeled or the same animals. In the control group, two types of BrdU label-retaining cells (LRCs) were distributed throughout the dental pulp: those with dense or those with granular reaction for BrdU. In the replants and autogenic transplants, dense LRCs remained in the center of dental pulp associating with the perivascular environment throughout the experimental period and possessed a proliferative capacity and maintained the differentiation capacity into the odontoblast-like cells or fibroblasts. In contrast, LRCs disappeared in the center of the pulp tissue by postoperative week 4 in the allografts. The disappearance of LRCs was attributed to the extensive apoptosis occurring significantly in LRCs except for the newly-differentiated odontoblast-like cells even in cases without immunological rejection. The results suggest that the host and recipient interaction in the allografts disturbs the maintenance of dense LRCs, presumably stem/progenitor cells, resulting in the disappearance of these cell types.     

牙髓干细胞的研究进展

牙髓干细胞是来源于牙髓组织中的一种成体干细胞,该种细胞具有高度增殖、自我更新的能力和多向分化潜能。牙髓干细胞的研究对牙髓再生、牙体修复等牙组织工程将产生重要的意义。该文就牙髓干细胞的研究现状作一综述,并对其应用前景进行探讨。     

人体牙髓干细胞的分离与鉴定

目的探讨牙髓干细胞（DPSC）对牙周病,外伤及肿瘤等造成下颌骨缺损、口腔软组织与神经损伤的修复治疗作用。方法本研究利用组织块培养法分离出人体DPSC,用流式细胞仪进行了鉴定,并进行DPSC成骨、成脂、成神经的分化研究。结果分离出3株DPSC,流式细胞分析表明DPSC表达CD73和CD90标志物,但不表达生血干细胞标志物CD34。用茜素红染色表明DPSC能分化成骨细胞,油红O染色表明DPSC能分化成脂肪细胞,免疫免疫荧光染色表明DPSC分化的细胞表达神经细胞特异标志物TUJ1。结论组织块培养能够高效快速分离表达CD73和CD90的DPSC,在体外诱导条件下DPSC能分化为成骨细胞、脂肪细胞和神经细胞,此研究为DPSC在治疗和修复骨组织缺损和神经损伤中的临床应用提供了实验依据。     

Postnatal stem/progenitor cells derived from the dental pulp of adult chimpanzee

Background  

Chimpanzee dental pulp stem/stromal cells (ChDPSCs) are very similar to human bone marrow derived mesenchymal stem/stromal cells (hBMSCs) as demonstrated by the expression pattern of cell surface markers and their multipotent differentiation capability.     

Wnt signaling reprograms metabolism in dental pulp stem cells

Human dental pulp stem cells (DPSCs) can differentiate to a wide range of different cell lineages, and share some gene expression and functional similarities with pluripotent stem cells. The stemness of DPSCs can also be pharmacologically enhanced by the activation of canonical Wnt signaling. Here, we examined the metabolic profile of DPSCs during reprogramming linked to Wnt activation, by a short (48 hr) exposure to either the GSK3-β inhibitor BIO (6-bromoindirubin-3´-oxine) or human recombinant protein WNT-3A. Both treatments largely increased glucose consumption, and induced a gene overexpression of pyruvate and mitochondrial acetyl-coA producing enzymes, thus activating mitochondrial tricarboxylic acid cycle (TCA) metabolism in DPSCs. This ultimately led to an accumulation of reducing power and a mitochondrial hyperpolarization in DPSCs. Interestingly, Nile Red staining showed that lipid fuel reserves were being stored in Wnt-activated DPSCs. We associate this metabolic reprogramming with an energy-priming state allowing DPSCs to better respond to subsequent high demands of energy and biosynthesis metabolites for cellular growth. These results show that enhancement of the stemness of DPSCs by Wnt activation comes along with a profound metabolic remodeling, which is distinctly characterized by a crucial participation of mitochondrial metabolism.     

Allogenic banking of dental pulp stem cells for innovative therapeutics

Medical research in regenerative medicine and cell-based therapy has brought encouraging perspectives for the use of stem cells in clinical trials. Multiple types of stem cells, from progenitors to pluripotent stem cells, have been investigated. Among these, dental pulp stem cells (DPSCs) are mesenchymal multipotent cells coming from the dental pulp, which is the soft tissue within teeth. They represent an interesting adult stem cell source because they are recovered in large amount in dental pulps with non-invasive techniques compared to other adult stem cell sources. DPSCs can be obtained from discarded teeth, especially wisdom teeth extracted for orthodontic reasons. To shift from promising preclinical results to therapeutic applications to human, DPSCs must be prepared in clinical grade lots and transformed into advanced therapy medicinal products (ATMP). As the production of patient-specific stem cells is costly and time-consuming, allogenic biobanking of clinical grade human leukocyte antigen (HLA)-typed DPSC lines provides efficient innovative therapeutic products. DPSC biobanks represent industrial and therapeutic innovations by using discarded biological tissues (dental pulps) as a source of mesenchymal stem cells to produce and store, in good manufacturing practice (GMP) conditions, DPSC therapeutic batches. In this review, we discuss about the challenges to transfer biological samples from a donor to HLA-typed DPSC therapeutic lots, following regulations, GMP guidelines and ethical principles. We also present some clinical applications, for which there is no efficient therapeutics so far, but that DPSCs-based ATMP could potentially treat.     

Scaling-up of dental pulp stem cells isolated from multiple niches

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".     

Capacity of dental pulp differentiation after tooth transplantation

Under pathological conditions, dental pulp elaborates both bone and dentin matrix in which the contribution of periodontal tissue cannot be excluded. This study has aimed to clarify the capability of dental pulp to deposit bone matrix in an auto-graft experiment by using (1) immunohistochemistry for 5-bromo-2′-deoxyuridine (BrdU) and nestin and (2) histochemistry for tartrate-resistant acid phosphatase (TRAP). Following the extraction of the molars of 3-week-old mice, the roots and pulp floor were resected and immediately transplanted into the sublingual region. On Days 5–7, tubular dentin formation commenced next to the pre-existing dentin at the pulp horn in which nestin-positive odontoblast-like cells were arranged. Up until Day 14, bone-like tissue formation occurred in the pulp chamber in which intense TRAP-positive cells appeared. These results suggest that odontoblast- and osteoblast-lineage cells reside in the dental pulp. Overall, specific dental pulp regeneration should provide fundamental knowledge for the realization of human tooth regeneration in the near future.This work was supported in part by a grant from MEXT to promote the 2001-multidisciplinary research project (in 2001–2005), KAKENHI (B) (no. 16390523 to H.O.) from MEXT, and the Japan-Korea Joint Research Project from JSPS and KOSEF (no. F01-2005-000-10212-0).     

Stage-specific embryonic antigen-4 identifies human dental pulp stem cells

Embryonic stem cell-associated antigens are expressed in a variety of adult stem cells as well as embryonic stem cells. In the present study, we investigated whether stage-specific embryonic antigen (SSEA)-4 can be used to isolate dental pulp (DP) stem cells. DP cells showed plastic adherence, specific surface antigen expression, and multipotent differentiation potential, similar to mesenchymal stem cells (MSC). SSEA-4+ cells were found in cultured DP cells in vitro as well as in DP tissue in vivo. Flow cytometric analysis demonstrated that 45.5% of the DP cells were SSEA-4+. When the DP cells were cultured in the presence of all-trans-retinoic acid, marked downregulation of SSEA-3 and SSEA-4 and the upregulation of SSEA-1 were observed. SSEA-4+ DP cells showed a greater telomere length and a higher growth rate compared to ungated and SSEA-4- cells. A clonal assay demonstrated that 65.5% of the SSEA-4+ DP cells had osteogenic potential, and the SSEA-4+ clonal DP cells showed multilineage differentiation potential toward osteoblasts, chondrocytes, and neurons in vitro. In addition, the SSEA-4+ DP cells had the capacity to form ectopic bone in vivo. Thus, our results suggest that SSEA-4 is a specific cell surface antigen that can be used to identify DP stem cells.     

Dynamic hydrostatic pressure promotes differentiation of human dental pulp stem cells

The masticatory apparatus absorbs high occlusal forces, but uncontrolled parafunctional or orthodontic forces damage periodontal ligament (PDL), cause pulpal calcification, pulp necrosis and tooth loss. Morphology and functional differentiation of connective tissue cells can be controlled by mechanical stimuli but effects of uncontrolled forces on intra-pulpal homeostasis and ability of dental pulp stem cells (DPSCs) to withstand direct external forces are unclear. Using dynamic hydrostatic pressure (HSP), we tested the hypothesis that direct HSP disrupts DPSC survival and odontogenic differentiation. DPSCs from four teenage patients were subjected to HSP followed by assessment of cell adhesion, survival and recovery capacity based on odontogenic differentiation, mineralization and responsiveness to bone morphogenetic protein-2 (BMP-2). HSP down-regulated DPSC adhesion and survival but promoted differentiation by increasing mineralization, in vivo hard tissue regeneration and BMP-2 responsiveness despite reduced cell numbers. HSP-treated DPSCs displayed enhanced odontogenic differentiation, an indication of favorable recovery from HSP-induced cellular stress.     

Isolation of pluripotent stem cells from human third molar dental pulp

Potent stem/progenitor cells have been isolated from normal human dental pulps, termed dental pulp stem cells (DPSCs). However, no study has described the presence of stem cell populations in human dental pulp from the third molar with embryonic phenotypes. The dental pulp tissue was cultured in media with the presence of LIF, EGF, and PDGF. In the present study, we describe a new population of pluripotent stem cells that were isolated from dental pulp (DPPSC). These cells are SSEA-4(+), Oct4(+), Nanog(+), FLK-1(+), HNF3beta(+), Nestin(+), Sox2(+), Lin28(+), c-Myc(+), CD13(+), CD105(+), CD3(-), CD45(-), CD90(low), CD29(+), CD73(low), STRO-1(low) and CD146(-). We have investigated by SEM analysis and q-RT-PCR the capacity of DPPSCs to 3D differentiate in vitro using the Cell Carrier 3D glass scaffold into tissues that have similar characteristics to embryonic mesoderm and endoderm layers. These data would support the use of these cells, which are derived from an easily accessible source and can be used in future regeneration protocols for many tissue types that differentiate from the three embryonic layers.