脱落乳牙牙髓干细胞在口腔颌面部组织再生及免疫调节中应用的研究进展

乳牙牙髓干细胞是一种成体干细胞,提取自脱落的乳牙,易获取、无创伤,符合伦理要求,同时具有强增殖能力及多分化潜能;其低免疫原性的特点使得移植后宿主无发生免疫反应或排斥反应的风险,也不需要接受免疫抑制治疗.这些特性使乳牙牙髓干细胞成为再生医学中细胞治疗所需的理想种子细胞之一.文章就脱落乳牙牙髓干细胞(stem cells ...     

SHED repair critical-size calvarial defects in mice

Objective:  Stem cells from human exfoliated deciduous teeth (SHED) are a population of highly proliferative postnatal stem cells capable of differentiating into odontoblasts, adipocytes, neural cells, and osteo-inductive cells. To examine whether SHED-mediated bone regeneration can be utilized for therapeutic purposes, we used SHED to repair critical-size calvarial defects in immunocompromised mice.
Materials and methods:  We generated calvarial defects and transplanted SHED with hydroxyapatite/tricalcium phosphate as a carrier into the defect areas.
Results:  SHED were able to repair the defects with substantial bone formation. Interestingly, SHED-mediated osteogenesis failed to recruit hematopoietic marrow elements that are commonly seen in bone marrow mesenchymal stem cell-generated bone. Furthermore, SHED were found to co-express mesenchymal stem cell marker, CC9/MUC18/CD146, with an array of growth factor receptors such as transforming growth factor β receptor I and II, fibroblast growth factor receptor I and III, and vascular endothelial growth factor receptor I, implying their comprehensive differentiation potential.
Conclusions:  Our data indicate that SHED, derived from neural crest cells, may select unique mechanisms to exert osteogenesis. SHED might be a suitable resource for orofacial bone regeneration.     

人乳牙牙髓干细胞在干细胞治疗中的应用

乳牙牙髓干细胞（SHED）是牙源性干细胞的一种,属外胚间充质干细胞。作为一种理想的干细胞来源,SHED在干细胞治疗中有良好的应用前景。本文阐述了SHED的生物学特征及其在干细胞治疗中的优势,探讨了SHED在组织再生和修复中发挥的多向分化潜能、细胞分泌功能和免疫调节功能等方面的功能作用。此外,本文还介绍了SHED在各系统、器官疾病治疗中的临床应用,重点阐述了用SHED进行干细胞移植在牙髓—牙本质再生、颌骨再生、神经系统疾病治疗和免疫系统疾病治疗方面的研究进展。     

Dental pulp tissue engineering with stem cells from exfoliated deciduous teeth

Stem cells from human exfoliated deciduous teeth (SHED) have been isolated and characterized as multipotent cells. However, it is not known whether SHED can generate a dental pulp-like tissue in vivo. The purpose of this study was to evaluate morphologic characteristics of the tissue formed when SHED seeded in biodegradable scaffolds prepared within human tooth slices are transplanted into immunodeficient mice. We observed that the resulting tissue presented architecture and cellularity that closely resemble those of a physiologic dental pulp. Ultrastructural analysis with transmission electron microscopy and immunohistochemistry for dentin sialoprotein suggested that SHED differentiated into odontoblast-like cells in vivo. Notably, SHED also differentiated into endothelial-like cells, as demonstrated by B-galactosidase staining of cells lining the walls of blood-containing vessels in tissues engineered with SHED stably transduced with LacZ. This work suggests that exfoliated deciduous teeth constitute a viable source of stem cells for dental pulp tissue engineering.     

Stem Cell Proliferation Pathways Comparison between Human Exfoliated Deciduous Teeth and Dental Pulp Stem Cells by Gene Expression Profile from Promising Dental Pulp

IntroductionMesenchymal stem cells (MSCs) have been used for clinical application in tissue engineering and regenerative medicine (TERM). To date, the most common source of MSCs has been bone marrow. However, the bone marrow aspirate is an invasive and painful procedure for the donor. Thus, the identification and characterization of alternative sources of MSCs are of great importance. This study focused on the characterization of stem cells from human exfoliated deciduous teeth (SHED) compared with dental pulp stem cells (DPSCs) and bone marrow–derived mesenchymal stem cells (BMMSCs).MethodsWe have compared “stemness” such as the proliferation rate and the expression of stem cell marker of DPSCs, SHED, and BMMSCs. In addition, gene expression profile of DPSCs and SHED were analyzed by using DNA microarray.ResultsAll cells isolated from the three sources exhibited MSC characteristics including a fibroblastic morphology, and the expression of mesenchymal stem-cell markers. The proliferation rate of SHED was significantly higher than that of DPSCs and BMMSCs (P < 0.05). The comparison of the gene expression profiles indicated 4386 genes with a changed expression between DPSCs and SHED by 2.0-fold or more. Higher expression in SHED was observed for genes that participate in pathways related to cell proliferation and extracellular matrix, including several cytokines such as fibroblast growth factor and tumor growth factor beta.ConclusionsBecause of its advantages of a higher proliferation capability, abundant cell supply, and painless stem cell collection with minimal invasion, SHED could be a desirable option as a cell source for potential therapeutic applications.     

人乳牙牙髓干细胞和成人牙髓干细胞研究进展

近年来,成体干细胞不断地从不同的组织中被分离出来,该类细胞具有多向分化潜能、较强的增殖能力和持久的自我更新能力,具备充当组织工程种子细胞的天然优势。2000年和2003年,研究者先后从成人牙髓组织和人乳牙牙髓组织中分离出具有干细胞特征的细胞,这两种细胞的发现对牙组织工程将产生重要的意义。现就这两种成体干细胞的研究进展做一综述,并展望其应用前景。     

Differentiation of stem cells from human deciduous and permanent teeth into spiral ganglion neuron-like cells

ObjectiveStem cells from pulp tissue are a promising cell-based therapy for neurodegenerative patients based on their origin in the neural crest. The aim of this study was to differentiate and evaluate the ability of human dental pulp stem cells from permanent teeth (DPSC) and stem cells from human exfoliated deciduous teeth (SHED) to differentiate into spiral ganglion neurons.DesignAfter isolation and characterization of mesenchymal stem cell properties, DPSC and SHED were treated with the neurotrophins brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and glial cell-derived neurotrophic factor (GDNF). The differentiation was identified by immunostaining and qRT-PCR analysis of neuronal markers and measuring intracellular calcium activity.ResultsAfter 2 weeks of induction, morphological changes were observed in both DPSC and SHED. The differentiated cells expressed neuron-specific class III beta-tubulin, GATA binding protein 3 (GATA3) and tropomyosin receptor kinase B, protein markers of spiral ganglion neurons. These cells also showed upregulation of the genes encoding these proteins, namely GATA3 and neurotrophic receptor tyrosine kinase 2. Intracellular calcium dynamics that reflect neurotransmitter release were observed in differentiated DPSC and SHED.ConclusionThese results demonstrate that dental pulp stem cells from permanent and deciduous teeth can differentiate into spiral ganglion neuron-like cells.     

Comparative characterization of stem cells from human exfoliated deciduous teeth and dental pulp stem cells

ObjectiveThis study focused on the characterization of stem cells from human exfoliated deciduous teeth (SHED) in comparison with dental pulp stem cells (DPSCs) to certify SHED as a key element in tissue engineering.MethodsIn the present study, SHED and DPSCs were assayed for their cell surface antigens and proliferation by measuring the cell cycles, growth rates, Ki67-positive efficiencies, and colony-forming units (CFUs). The evaluation of multi-differentiation was performed using alizarin red and oil red O and real-time PCR in vitro. The mineralization capability of the cells was examined in vivo by implanting with ceramic bovine bone (CBB) into subcutaneous of immunocompromised mice for 8 weeks. A three-dimensional pellet cultivation system is proposed for SHED and DPSCs to recreate the biological microenvironment that is similar to that of a regenerative milieu.ResultsSHED showed a higher proliferation rate and differentiation capability in comparison with DPSCs in vitro, and the results of the in vivo transplantation suggest that SHED have a higher capability of mineralization than the DPSCs. The mRNA expression levels of inflammatory cytokines, including matrix metalloproteinase-1 (MMP1), tissue inhibitors of metalloproteinase-1 (TIMP1), matrix metalloproteinase-2 (MMP2), tissue inhibitors of metalloproteinase-2 (TIMP2) and interleukin-6 (IL-6) were higher in SHED than that in DPSCs. In addition, the expression levels of Col I and proliferating cell nuclear antigen (PCNA) in SHED sheets were significantly higher than those in DPSCs sheets.ConclusionsThis study systematically demonstrated the differences in the growth and differentiation characteristics between SHED and DPSCs. Consequently, SHED may represent a suitable, accessible and potential alternative source for regenerative medicine and therapeutic applications.     

牙本质细胞外基质蛋白对乳牙牙髓干细胞体外增殖、分化能力的影响

目的:提取牙本质细胞外基质蛋白(Dentinextracellularmatrixproteins,DEMPs)研究其对人脱落的乳牙牙髓干细胞(Stemcellfromhumanexfoliateddeciduousteeth,SHED)体外增殖分化能力的影响。方法:采用组织块联合酶消化法获得SHED并进行体外培养。成骨诱导液诱导细胞,鉴定其多向分化能力;拔取健康牛牙,用4mol/L盐酸胍和0.5mol/LEDTA提取DEMPs,四唑盐比色法(MTT)检测不同浓度DEMPs对SHED增殖能力的影响,同时测定DEMPs对SHED碱性磷酸酶(ALP)活性的影响;实时荧光定量PCR(RT-PCR)检测牙本质磷蛋白与牙本质基质蛋白1的mRNA表达情况。结果:DEMPs诱导细胞培养3d、5d可促进细胞增殖。细胞培养5、7d上调ALP活性,RT-PCR结果显示,DEMPs诱导后可促进DSPP与DMP-1基因的表达。结论:牙本质细胞外基质蛋白可以促进SHED的增殖与牙向分化能力。     

The efficacy of mesenchymal stem cells to regenerate and repair dental structures

OBJECTIVES: Identification, characterization, and potential application of mesenchymal stem cells (MSC) derived from human dental tissues. METHODS: Dental pulp and periodontal ligament were obtained from normal human impacted third molars. The tissues were digested in collagenase/dispase to generate single cell suspensions. Cells were cultured in alpha-MEM supplemented with 20% fetal bovine serum, 2 mM l-glutamine, 100 microM l-ascorbate-2-phosphate. Magnetic and fluorescence activated cell sorting were employed to characterize the phenotype of freshly isolated and ex vivo expanded cell populations. The developmental potential of cultured cells was assessed following co-transplantation with hydroxyapetite/tricalcium phosphate (HA/TCP) particles into immunocompromised mice for 8 weeks. RESULTS: MSC were identified in adult human dental pulp (dental pulp stem cells, DPSC), human primary teeth (stem cells from human exfoliated deciduous teeth, SHED), and periodontal ligament (periodontal ligament stem cells, PDLSC) by their capacity to generate clongenic cell clusters in culture. Ex vivo expanded DPSC, SHED, and PDLSC populations expressed a heterogeneous assortment of makers associated with MSC, dentin, bone, smooth muscle, neural tissue, and endothelium. PDLSC were also found to express the tendon specific marker, Scleraxis. Xenogeneic transplants containing HA/TCP with either DPSC or SHED generated donor-derived dentin-pulp-like tissues with distinct odontoblast layers lining the mineralized dentin-matrix. In parallel studies, PDLSC generated cementum-like structures associated with PDL-like connective tissue when transplanted with HA/TCP into immunocompromised mice. CONCLUSION: Collectively, these data revealed the presence of distinct MSC populations associated with dental structures with the potential of stem cells to regenerate living human dental tissues in vivo.     

釉质基质蛋白对乳牙牙髓干细胞体外增殖、分化能力的影响

目的：研究釉质基质蛋白（enamel matrix proteins,EMPs）对人脱落乳牙牙髓干细胞（stem cells from hu—man exfoliated dediduous teeth,SHED）体外增殖分化能力的影响。方法：利用酶消化法联合组织块法获得脱落乳牙牙髓干细胞,并进行形态学观察。三氯乙酸法制备EMPs,用不同浓度的EMPs对SHED进行诱导,利用四唑盐比色法（MTT）检测并分析诱导后的SHED增殖活性的变化,检测经诱导后的培养液中碱性磷酸酶（ALP）。RT—PCR检测牙本质涎磷蛋白（dentin sialophosphoprotein,DSPP）及牙本质基质蛋白1（dentin matrix protein1,DMP-1）的mRNA表达。结果：人脱落乳牙牙髓干细胞呈集落生长,并且在体外具有一定的自我增殖能力。EMPs对乳牙牙髓干细胞的增殖无明显影响,而能够显著提高ALP的活性,并呈现一定的剂量依赖性。经EMPs诱导后,细胞相对高表达DSPP、DMP-1mRNA。结论：EMPs对于SHED向成牙本质样分化具有积极作用。     

乳牙牙髓干细胞的研究进展

乳牙牙髓干细胞（SHED）来源于脱落的乳牙牙髓,具有较强的增殖能力、自我更新能力、多向分化潜能,而且乳牙为生物废弃物,符合伦理要求,所以渐渐成为干细胞领域研究的新热点。本文就SHED的生物学特征、培养方法、鉴定方法、多向分化潜能及其在疾病治疗中应用的研究进展作一综述。     

人乳牙牙髓干细胞体外表达和分泌胶质细胞源性神经营养因子的研究

目的 研究人乳牙牙髓干细胞(SHED)表达和分泌胶质细胞源性神经营养因子(GDNF)的能力及规律,为进一步探讨SHED治疗帕金森病的作用机制提供依据.方法 通过酶消化法体外分离培养人乳牙牙髓中的干细胞,当传代至第3代时,加入神经干细胞的特殊培养基Neurobasal A和细胞因子进行神经球诱导培养,通过Real-Ti...     

牙源性干细胞及牙髓再生的研究进展

牙髓组织中含有细胞、血管、神经和纤维等,是一个复杂的3D结构系统。随着干细胞生物学和组织工程学的相互结合和促进,牙髓再生逐渐成为可能。牙体组织中分离出的多种干细胞,如牙髓干细胞、脱落乳牙牙髓干细胞、根尖牙乳头干细胞、牙囊干细胞等,都具有再生牙髓的潜能。文章就牙源性干细胞及以牙源性干细胞为基础的牙髓再生的研究进展做一综述。     

Comparison of human dental follicle cells (DFCs) and stem cells from human exfoliated deciduous teeth (SHED) after neural differentiation in vitro

Dental stem cells from human exfoliated deciduous teeth (SHED) and dental follicle cells (DFCs) are neural crest-derived stem cells from human dental tissues. Interestingly, SHED and DFCs can successfully differentiate into neuron-like cells. We hypothesized that SHED and DFCs have the same neural cell differentiation potentials. To evaluate neural cell differentiation, we cultivated SHED and DFCs in four different serum-replacement media (SRMs) and analyzed cell morphology, cell proliferation, and gene expression patterns before and after differentiation. In a standard cell culture medium, SHED and DFCs have not only similar cell morphologies, but they also have similar gene expression patterns for known stem cell markers. However, only SHED expressed the neural stem cell marker Pax6. After cultivation in SRMs, cell proliferations of DFCs and SHED were reduced and the cell morphology was spindle-like with long processes. However, differentiated DFCs and SHED had different neural cell marker expression patterns. For example, gene expression of the late neural cell marker microtubule-associated protein 2 was upregulated in DFCs and downregulated in SHED in SRM with the B27 supplement. In contrast, SHED formed neurosphere-like cell clusters in SRM with the B27 supplement, epidermal growth factor, and fibroblast growth factor-2. Moreover, SHED differentially expressed the glial cell marker glial fibrillary acidic protein, which in contrast was weakly or not expressed in DFCs. In conclusion, SHED and DFCs have different neural differentiation potentials under the same cell culture conditions.     

人乳牙牙髓干细胞和恒牙牙髓干细胞的蛋白质组学比较

目的 采用蛋白质组学方法研究人乳牙牙髓干细胞（SHED）和恒牙牙髓干细胞（DPSC）中的蛋白表达差异.方法 应用双向凝胶电泳技术分离SHED和DPSC的细胞总蛋白.通过比较两种细胞的蛋白组学图谱,确定差异表达的蛋白点,而后对差异点进行基质辅助激光解析电离飞行时间质谱分析和蛋白数据库信息检索,对差异蛋白进行功能分类.结果 建立了SHED和DPSC的蛋白质组图谱,经软件分析出45个差异蛋白点,其中26个表达上调,19个表达下调,再经质谱鉴定出48种蛋白,其生物学功能涉及细胞周期、代谢等.结论 SHED与DPSC中蛋白的差异表达体现了两种细胞在结构和功能上的异同性,为进一步研究SHED和DPSC在增殖、分化中的差异,以及牙齿相关干细胞在组织工程和再生医学研究中的应用提供参考.     

牙源性间充质干细胞诱导iPS细胞的效率与时间的研究

目的研究比较不同种牙源性间充质干细胞诱导iPS细胞的效率与时间。方法分离牙髓干细胞（DPSCs）、脱落乳牙干细胞（SHED）、牙乳头干细胞（SCAP）。应用慢病毒介导Lin28、Nanog、Oct4和Sox2因子重编程获得iPS细胞。比较在同等条件下三种细胞获得iPS细胞的克隆数与平均诱导时间。结果DPSC诱导iPS细胞的效率是0.167%,高于SHED细胞和SCAP细胞的诱导效率（0.125%,0.033%）;DPSC诱导iPS细胞的平均重编程时间是20.1d,均少于SHED细胞和SCAP细胞（23.73d,25.25d）,差异均有统计学意义。结论三种不同牙源性细胞有不同的iPS细胞诱导效率与重编程时间,牙髓干细胞有较好的诱导iPS细胞的应用潜能。     

Synergistic effects of chitosan scaffold and TGFβ1 on the proliferation and osteogenic differentiation of dental pulp stem cells derived from human exfoliated deciduous teeth

ObjectiveMultipotent stem cells derived from human exfoliated deciduous teeth (SHED) represent a promising cell source for tissue regeneration. In the present study we decided to test the inductive effect of chitosan and transforming growth factor-β₁ (TGFβ₁) as a scaffold/factor combination on SHED proliferation and osteogenic differentiation.DesignCell proliferation was quantitatively assessed by PrestoBlue, live/dead assay was performed and cell attachment to chitosan scaffold was examined by scanning electron microscopy (SEM). For osteogenic differentiation analysis, alkaline phosphatase activity was quantified, cells were stained with Alizarin Red, and the lineage specific genes/proteins ALP, COL I, BSP, and OCN were analysed by real-time PCR and Western blot.ResultsSHED remained viable and attached well to the chitosan structure. Moreover, TGFβ₁ significantly enhanced the proliferative activity of SHED on the chitosan scaffold. Our data further revealed that chitosan and TGFβ₁ enhanced the osteogenic differentiation of SHED, as evidenced by high ALP activity, strong mineral deposition, and the up-regulation of ALP, COL I, BSP, and OCN gene/protein expression.ConclusionTogether, data from our study indicate that the combination of chitosan scaffolds and TGFβ₁ enhanced proliferation and osteogenic differentiation of SHED. These findings suggest that the combined application of chitosan scaffold and TGFβ₁ in conjunction with SHED might be beneficial for in vivo bone regeneration.