Proteomic profiling of various human dental stem cells-a systematic review

BACKGROUND The proteomic signature or profile best describes the functional component of a cell during its routine metabolic and survival activities.Additional complexity in differentiation and maturation is observed in stem/progenitor cells.The role of functional proteins at the cellular level has long been attributed to anatomical niches,and stem cells do not deflect from this attribution.Human dental stem cells(hDSCs),on the whole,are a combination of mesenchymal and epithelial coordinates observed throughout craniofacial bones to pulp.AIM To specify the proteomic profile and compare each type of hDSC with other mesenchymal stem cells(MSCs)of various niches.Furthermore,we analyzed the characteristics of the microenvironment and preconditioning changes associated with the proteomic profile of hDSCs and their influence on committed lineage differentiation.METHODS Literature searches were performed in PubMed,EMBASE,Scopus,and Web of Science databases,from January 1990 to December 2018.An extra inquiry of the grey literature was completed on Google Scholar,ProQuest,and OpenGrey.Relevant MeSH terms(PubMed)and keywords related to dental stem cells were used independently and in combination.RESULTS The initial search resulted in 134 articles.Of the 134 full-texts assessed,96 articles were excluded and 38 articles that met the eligibility criteria were reviewed.The overall assessment of hDSCs and other MSCs suggests that differences in the proteomic profile can be due to stem cellular complexity acquired from varied tissue sources during embryonic development.However,our comparison of the proteomic profile suffered inconsistencies due to the heterogeneity of various hDSCs.We believe that the existence of a heterogeneous population of stem cells at a given niche determines the modalities of regeneration or tissue repair.Added prominences to the differences present between various hDSCs have been reasoned out.CONCLUSION Systematic review on proteomic studies of various hDSCs are promising as an eye-opener for revisiting the proteomic profile and in-depth analysis to elucidate more refined mechanisms of hDSC functionalities.

Application of dental stem cells in three-dimensional tissue regeneration

Dental stem cells can differentiate into different types of cells.Dental pulp stem cells,stem cells from human exfoliated deciduous teeth,periodontal ligament stem cells,stem cells from apical papilla,and dental follicle progenitor cells are five different types of dental stem cells that have been identified during different stages of tooth development.The availability of dental stem cells from discarded or removed teeth makes them promising candidates for tissue engineering.In recent years,three-dimensional(3D)tissue scaffolds have been used to reconstruct and restore different anatomical defects.With rapid advances in 3D tissue engineering,dental stem cells have been used in the regeneration of 3D engineered tissue.This review presents an overview of different types of dental stem cells used in 3D tissue regeneration,which are currently the most common type of stem cells used to treat human tissue conditions.

NOTCH3 is expressed in human apical papilla and in subpopulations of stem cells isolated from the tissue

NOTCH plays a role in regulating stem cell function and fate decision.It is involved in tooth development and injury repair.Information regarding NOTCH expression in human dental root apical papilla(AP)and its residing stem cells(SCAP)is limited.Here we investigated the expression of NOTCH3,its ligand JAG1,and mesenchymal stem cell markers CD146 and STRO-1 in the AP or in the primary cultures of SCAP isolated from AP.Our in situ immunostaining showed that in the AP NOTCH3 and CD146 were co-expressed and associated with blood vessels having NOTCH3 located more peripherally.In cultured SCAP,NOTCH3 and JAG1 were co-expressed.Flow cytometry analysis showed that 7%,16%and 98%of the isolated SCAP were positive for NOTCH3,STRO-1 and CD146,respectively with a rare 1.5%subpopulation of SCAP co-expressing all three markers.The expression level of NOTCH3 reduced when SCAP underwent osteogenic differentiation.Our findings are the first step towards defining the regulatory role of NOTCH3 in SCAP fate decision.

新型生物陶瓷材料对人根尖牙乳头干细胞增殖及成骨分化的影响

目的评估新型生物陶瓷类材料c-Root SP对人根尖牙乳头干细胞(hSCAPs)的细胞增殖及骨诱导能力。方法制备不同浓度c-Root SP浸提液,采用CCK-8法检测其对hSCAPs的细胞增殖情况,使用碱性磷酸酶(ALP)活性测定法测定ALP酶水平和茜素红染色法观察钙化结节形成情况,数据采用单因素方差分析,与iRootSP对比,评价c-Root SP促进细胞增殖及骨诱导能力。结果c-Root SP对hSCAPs的细胞增殖具有浓度依赖性,1/10浓度浸提液增殖水平最好,c-Root SP1/5、1/10、1/1000浓度浸提液对hSCAPs的细胞增殖的影响显著高于同浓度iRoot SP浸提液(P<0.05)。成骨方面,矿化诱导7天,iRoot SP ALP活性显著高于c-Root SP(P<0.05),矿化诱导14天c-Root SP矿化结节显著多于iRoot SP。结论c-Root SP具有对hSCAPs的良好细胞增殖及骨诱导能力,与iRoot SP相似。

根尖牙乳头干细胞来源凋亡囊泡对巨噬细胞糖酵解相关酶的表达及炎症表型影响的初探研究

目的:探究根尖牙乳头干细胞(SCAP)来源的凋亡囊泡(apoVs)是否通过影响糖酵解相关酶调控巨噬细胞(BMDMs)炎症表型,进而对炎症反应产生调节作用。方法:体外培养鉴定人源SCAP,经星形孢菌素诱导凋亡后提取apoVs,利用扫描电镜、流式细胞术等对其进行表征鉴定。分别空白处理、脂多糖(LPS)处理、LPS同apoVs共处理大鼠骨髓来源的BMDMs,利用流式细胞术检测促炎表型BMDMs表面标志分子CD80、CD86的表达,利用Western blot检测BMDMs糖酵解相关酶的表达。结果:根尖牙乳头干细胞来源的apoVs能被BMDMs摄取,促炎表型标志分子CD80、CD86的表达下调,糖酵解相关酶葡萄糖转运蛋白(GLUT)1、GLUT3的表达显著下调(P<0.05)。结论:SCAP来源apoVs能降低促炎型BMDMs的分布,并对其糖酵解相关酶的表达产生影响。

根尖牙乳头干细胞外泌体通过AHR调节CDC42转录促进血管内皮细胞迁移的机制研究

目的:探究根尖牙乳头干细胞来源的外泌体(SCAP-Exo)调控细胞分裂周期蛋白42(CDC42)转录介导血管内皮细胞(VECs)迁移的分子机制。方法:超速离心法提取SCAP-Exo并应用透射电镜和纳米粒子追踪实验鉴定。构建CDC42低表达的VECs,应用SCAP-Exo处理VECs,实时荧光定量PCR与Western blot实验检测SCAP-Exo处理后CDC42的基因和蛋白表达水平,通过划痕实验和Transwell迁移实验检测VECs迁移能力。通过Western blot和免疫荧光染色实验,检测SCAP-Exo对VECs中芳香烃受体(AHR)核转位以及CDC42表达的影响。结果:SCAP-Exo可以上调CDC42基因和蛋白表达水平,增强VECs的迁移能力。与SCAP-Exo处理的正常VECs相比,VECs低表达CDC42后加入SCAP-Exo,迁移能力明显降低。SCAP-Exo可以促进VECs内AHR转位至细胞核内,当抑制VECs中AHR核转位后,VECs内CDC42的基因和蛋白表达水平下降。结论:SCAP-Exo通过激活VECs内AHR入核促进CDC42转录,进而上调CDC42的蛋白表达水平,从而增强VECs的迁移能力。

WDR63促进人根尖牙乳头干细胞成神经分化功能

目的:研究WD重复结构域63(WDR63)对人根尖牙乳头干细胞(SCAPs)体外成神经分化能力的作用。方法:通过实时荧光定量PCR检测未转染慢病毒SCAPs中WDR63基因在成神经分化诱导3、6、9 d的表达变化,利用慢病毒转染分别获得WDR63稳定过表达和敲低的SCAPs,通过类神经球形成实验对各组SCAPs进行神经分化诱导,对类神经球的直径进行定量分析,免疫荧光染色实验检测SCAPs中巢蛋白(Nestin)和βⅢ-微管蛋白(βⅢ-tubulin)的表达,实时荧光定量PCR实验检测神经分化相关指标βⅢ-tubulin、神经源性分化蛋白(NeuroD)、神经细胞黏附分子(NCAM)和Nestin基因的表达。结果:未转染慢病毒SCAPs中WDR63的表达量在成神经分化后的3、6、9 d逐渐增加(P<0.05);过表达WDR63后,WDR63基因(P<0.001)和蛋白表达增加,神经球直径增加(P<0.01),Nestin和βⅢ-tubulin神经球样细胞荧光强度增加(P<0.01),βⅢ-tubulin、NeuroD、NCAM和Nestin基因表达升高(P<0.05);敲低WDR63后,WDR63基因(P<0.01)和蛋白的表达降低,神经球直径降低(P<0.05),Nestin和βⅢ-tubulin神经球样细胞荧光强度减少(P<0.05),βⅢ-tubulin、NeuroD、NCAM和Nestin基因表达降低(P<0.05)。结论:WDR63可促进SCAPs的成神经分化功能。

根尖牙乳头干细胞成骨分化的研究进展

根尖牙乳头干细胞(stem cells from apical papilla,SCAP)具有很强的多系分化潜能,其中成骨分化可以应用于骨组织再生,为口腔颌骨缺损治疗提供新思路。成骨分化是个复杂的网络调控过程,诸如各种细胞因子、表观遗传物质、各种信号分子和信号通路等内源性物质均可产生不同程度的影响。这些因素相互作用可以促进SCAP的增殖、迁移和成骨分化,但其在SCAP成骨分化的不同进程中的具体机制和内在联系各不相同。对近年来有关促进SCAP成骨分化的各种因素及其可能的调控机制研究文献进行综述,以期为其进一步的应用研究提供新信息。

IL-34介导的NF-кB信号通路对根尖牙乳头干细胞成牙成骨定向分化的调节作用

目的:探讨白细胞介素-34(IL-34)介导的核因子кB(NF-кB)对根尖牙乳头干细胞(SCAPs)成牙/成骨定向分化调节作用。方法:使用酶消化法从小鼠根尖牙乳头组织传代培养SCAPs;流式细胞术鉴定SCAPs中CD44、CD90、CD45及集落刺激因子1受体(CSF-1R)的表达;实验分为4组:空白对照组、IL-34组(100 ng/mL IL-34)、CSF-1R组(100 ng/mL IL-34+25 ng/mL anti-CSF-1R)、NF-кB组(100 ng/mL IL-34+1umol/L BMS-345541)。通过蛋白质印迹法(Western blot)分析各组30、60、120 min时SCAPs胞浆中NF-кB关键蛋白p-IкBα、IкBα、p-P65及P65蛋白的表达情况;矿化诱导4组SCAPs 3、7、14 d时,茜素红染色和半定量分析比较各组细胞的矿化结节形成能力;通过RT-qPCR检测各组SCAPs中成骨相关基因Runt相关转录因子-2(RUNX2)、牙本质涎磷蛋白(DSPP)、骨钙素(OCN)mRNA水平。结果:流式细胞术显示,SCAPs中CD44(90.4%)、CD90(93.5%)阳性表达,造血干细胞CD45(3.1%)阴性表达,SCAPs表达CSF-1R(23.2%)阳性。与对照组比较,IL-34组SCAPs胞浆蛋白内p-IкBα、p-P65表达水平上调,矿化诱导3、7、14 d矿化结节形成量增多,细胞中RUNX2、DSPP、OCN mRNA相对表达量升高,差异均有统计学意义(P<0.05)。与IL-34组比较,CSF-1R组和NF-кB组SCAPs胞浆蛋白内p-IкBα、p-P65表达水平下调(P<0.05),矿化诱导3、7、14 d矿化结节形成量减少,细胞中RUNX2、DSPP、OCN mRNA相对表达量降低,差异均有统计学意义(P<0.05)。结论:IL-34可通过IL-34/CSF-1R调节SCAPs中NF-кB信号通路,并通过IL-34/CSF-1R/NF-кB信号调节SCAPs的成牙/成骨定向分化能力。

Oral stem cells,decoding and mapping the resident cells populations

The teeth and their supporting tissues provide an easily accessible source of oral stem cells.These different stem cell populations have been extensively studied for their properties,such as high plasticity and clonogenicity,expressing stem cell markers and potency for multilineage differentiation in vitro.Such cells with stem cell properties have been derived and characterised from the dental pulp tissue,the apical papilla region of roots in development,as well as the supporting tissue of periodontal ligament that anchors the tooth within the alveolar socket and the soft gingival tissue.Studying the dental pulp stem cell populations in a continuously growing mouse incisor model,as a traceable in vivo model,enables the researchers to study the properties,origin and behaviour of mesenchymal stem cells.On the other side,the oral mucosa with its remarkable scarless wound healing phenotype,offers a model to study a well-coordinated system of healing because of coordinated actions between epithelial,mesenchymal and immune cells populations.Although described as homogeneous cell populations following their in vitro expansion,the increasing application of approaches that allow tracing of individual cells over time,along with single-cell RNA-sequencing,reveal that different oral stem cells are indeed diverse populations and there is a highly organised map of cell populations according to their location in resident tissues,elucidating diverse stem cell niches within the oral tissues.This review covers the current knowledge of diverse oral stem cells,focusing on the new approaches in studying these cells.These approaches“decode”and“map”the resident cells populations of diverse oral tissues and contribute to a better understanding of the“stem cells niche architecture and interactions.Considering the high accessibility and simplicity in obtaining these diverse stem cells,the new findings offer potential in development of translational tissue engineering approaches and innovative therapeutic solutions.

Extracellular vesicles derived from human dental mesenchymal stem cells stimulated with low-intensity pulsed ultrasound alleviate inflammation-induced bone loss in a mouse model of periodontitis

Extracellular vesicles(EVs)derived from mesenchymal stem cells(MSCs)have emerged as a new mode of intercellular crosstalk and are responsible for many of the thera-peutic effects of MSCs.To promote the application of MSC-EVs,recent studies have focused on the manipulation of MSCs to improve the production of EVs and EV-mediated activities.The current paper details an optimization method using non-invasive low-intensity pulsed ul-trasound(LIPUS)as the stimulation for improving oral MSC-EV production and effectiveness.Stem cells from apical papilla(SCAP),a type of oral mesenchymal stem cell,displayed inten-sity-dependent pro-osteogenic and anti-inflammatory responses to LIPUS without significant cytotoxicity or apoptosis.The stimuli increased the secretion of EVs by promoting the expres-sion of neutral sphingomyelinases in SCAP.In addition,EVs from LIPUS-induced SCAP exhibited stronger efficacy in promoting the osteogenic differentiation and anti-inflammation of peri-odontal ligament cells in vitro and alleviating oral inflammatory bone loss in vivo.In addition,LIPUS stimulation affected the physical characteristics and miRNA cargo of SCAP-EVs.Further investigations indicated that miR-935 is an important mediator of the pro-osteogenic and anti-inflammatory capabilities of LIPUS-induced SCAP-EVs.Taken together,these findings demonstrate that LIPUS is a simple and effective physical method to optimize SCAP-EV produc-tion and efficacy.

TNF-α对人根尖乳头干细胞增殖及分化能力的影响

目的:研究肿瘤坏死因子α(TNF-α)对人根尖乳头干细胞增殖及分化能力的影响。方法:采用组织块法获得人根尖乳头干细胞,免疫荧光法鉴定干细胞表面标志物;经TNF-α刺激后检测对干细胞增殖、克隆形成率及分化能力的影响。结果:免疫荧光显示人波形丝蛋白、STRO-1阳性,角蛋白阴性;10μg/L TNF-α在4天时促进根尖乳头干细胞的增殖,提高克隆形成率;抑制碱性磷酸酶活性及矿化结节形成。结论:炎性因子TNF-α对人根尖乳头干细胞的生物学特性具有一定的影响。

犬根尖牙乳头干细胞的分离培养和生物学特性

目的:分离培养比格犬(Beagle犬)根尖牙乳头干细胞并研究其生物学特性。方法:拔除Beagle犬上颌年轻恒前牙,切取根尖牙乳头,采用酶消化法分离培养Beagle犬根尖牙乳头细胞,进行成纤维细胞克隆形成实验、生长曲线测定、多向分化能力研究及间充质干细胞相关表面标记物STRO-1、CD146和CK的表达分析,并将其移植至免疫缺陷鼠皮下观察矿化组织的形成。结果:Beagle犬根尖牙乳头组织中存在一群具有高度增殖能力的细胞,在相同培养条件下,其克隆形成率明显高于人根尖牙乳头干细胞,差异具有统计学意义(P<0.001);所获得Beagle犬根尖牙乳头干细胞具有成骨、成脂和成软骨等多向分化能力;在体外经成骨诱导后可形成矿化结节,成脂诱导后可形成脂滴,成软骨诱导后免疫组织化学染色Ⅱ型胶原阳性表达;同时该群细胞STRO-1和CD146阳性表达,而CK阴性表达;与羟基磷灰石混合移植于免疫缺陷鼠皮下可形成矿化组织和牙髓-牙本质复合体样结构。结论:Beagle犬根尖牙乳头中存在具有高增殖能力和多向分化能力的根尖牙乳头干细胞。

TGF-β1对人根尖牙乳头干细胞增殖和分化的影响

目的:研究转化生长因子(TGF-β1)在体外对根尖牙乳头干细胞(SCAP)增殖和分化的影响。方法:采用酶消化法获得原代培养人SCAP,并对获得克隆化的SCAP进行免疫组化染色和多向分化能力的初步鉴定。用MTT比色法观察不同浓度的TGF-β1对细胞增殖情况,并通过检测ALP活性和矿化诱导后COL1、DSPP、OCN mRNA表达,分析TGF-β1对SCAP分化能力的影响。结果:5、10、20、40 ng/mL组的TGF-β1在SCAP培养1周内可显著促进细胞增殖(P<0.05);在TGF-β1作用8 d后,细胞ALP活性增强(P<0.05);SCAP矿化诱导2周后,TGF-β1组中COL1、OCN、DSPP的mRNA表达上调(P<0.05)。结论:TGF-β1促进SCAP细胞增殖,并通过提高ALP活性和上调COL1,OCN、DSPP mRNA的表达促进SCAP分化。

根尖牙乳头干细胞细胞膜片构建及其成骨/成牙本质分化性能研究

目的:构建根尖牙乳头干细胞(stem cells from apical papilla,SCAP)细胞膜片,并对其组织学形态和成骨/成牙本质分化性能进行研究,为SCAP细胞膜片应用于年轻恒牙牙髓再生提供实验基础。方法:分离年轻恒牙第三磨牙牙乳头,进行SCAP原代培养。采用流式细胞术检测SCAP表面标志物,茜素红S染色检测其成骨/成牙本质分化。取第3代SCAP,膜片诱导液连续培养14d,形成细胞膜片。对SCAP细胞膜片进行H-E染色,组织学观察;流式细胞术检测SCAP细胞膜片的细胞周期变化;实时定量PCR(qRT-PCR)检测细胞膜片中Runx2、ALP、Col-I基因表达;Western印迹法检测细胞膜片Runx2、ALP蛋白表达水平。采用SPSS17.0软件包对实验结果进行统计学分析,两样本细胞周期检测和q RT-PCR基因检测均数的比较采用t检验。结果:组织学H-E染色显示,SCAP细胞膜片由5~6层细胞组成,细胞量大,细胞之间排列紧密,且富含细胞外基质。SCAP细胞膜片细胞周期G2+S期所占比例较低,而G1期比例较高,提示SCAP细胞膜片增殖性能受到显著抑制(P<0.05)。同时,qRT-PCR和Western印迹法结果显示,与SCAP相比,SCAP细胞膜片成骨/成牙本质分化能力显著升高(P<0.05)。结论:SCAP细胞膜片富含细胞外基质,且成骨/成牙本质分化能力高于单纯SCAP,SCAP细胞膜片应用于牙髓再生更具有优势。

根尖牙乳头干细胞的研究现状

根尖周牙乳头干细胞(Stem Cells from the Apical Papilla,SCAP)存在于未完全发育成形的恒牙根尖周组织中,是具有高度增生、自我更新能力和多向分化潜能的成体干细胞。SCAP是牙根发育时成牙本质细胞的重要来源,在牙根的形成和发育中起着重要的作用,SCAP的研究将对牙髓修复,牙本质再生以及生物学牙根组织工程产生重要作用。该文就其研究现状作一综述。

TNF-α对根尖乳头干细胞与牙周膜干细胞增殖活性影响的比较研究

目的:体外培养鼠根尖乳头干细胞和牙周膜干细胞,比较研究肿瘤坏死因子α(TNF-α)对两者增殖活性的影响,以期得出在炎症状态下两者用于牙本质再生的可能性。方法:取幼鼠根尖尚未发育完全的健康下颌牙,用酶消化法获得根尖乳头干细胞和牙周膜干细胞;免疫荧光法鉴定干细胞表面标志物;将含有TNF-α浓度为0、5、10、50 ng/ml的培养液加入两者的第3代细胞中,使用MTT法检测以比较其对两种细胞增殖活性的影响;采用SPSS 11.0软件包对实验组和对照组数据进行单因素方差分析。结果:SCAP与PDLSCs均呈长梭形,形似成纤维细胞;SCAP与PDLSCs均表达干细胞特性;SCAP较PDLSCs具有更强的增殖活性;在10ng/ml和50ng/ml浓度时,TNF-α能促进2种细胞的增殖。结论:与PDLSCs比较,SCAP增殖活性更强,在炎性环境下亦是如此,根尖乳头干细胞可能在年轻恒牙根尖周炎症经适当治疗后牙根继续发育中起到重要作用。

人牙根尖乳头干细胞及其在组织工程中的研究进展

最近,国外研究人员采用酶消化法,在人根尖尚未形成的第三磨牙牙根部牙乳头组织中分离出成体干细胞,并定义为牙乳头干细胞。研究表明,与牙髓干细胞相比较,牙乳头干细胞有着更高的溴脱氧尿苷吸收率、更多的倍增数和更强的组织再生能力,其可能是一种比牙髓干细胞更早期、更优越的牙源性干细胞的资源,在牙髓再生和组织工程运用中具有广阔的应用前景。本文就其生物学特性、在牙根形成方面的潜在作用和在生物牙根工程方面的应用等作一综述。

血小板裂解液对人根尖牙乳头干细胞和牙周膜干细胞体内外增殖、矿化能力的影响

目的:研究血小板裂解液(Platelet Lysate,PL)在体内外对人根尖牙乳头干细胞(SCAP)和牙周膜干细胞(PDLSCs)增殖、矿化的干预效应,以期找到PL应用于这两种细胞的最佳方式。方法:在矿化诱导培养体系中按一定体积比加入PL,分别作用于体内、外复合HA-TCP支架材料三维生长的SCAP和PDLSCs,扫描电镜(SEM)以及组织学观察细胞生长、分化情况。结果:体外三维培养模式下,PL能够促进SCAP和PDLSCs的增殖以及矿化细胞外基质的形成,并利于在支架材料上形成完整的细胞膜片(cell sheet)样结构,以上效应对于PDLSCs作用更为显著。而在体内移植模式下,经50 mL/L、10 mL/L PL培养体系预培养的SCAP能形成包含成牙本质细胞样细胞、牙本质样基质和牙髓组织的牙本质牙髓复合体样结构;而PDLSCs经50 mL/LPL干预后,可分化为成牙骨质细胞样细胞,并生成牙骨质样基质、牙周膜样组织。结论:一定体积浓度比的PL能促进SCAP和PDLSCs在体内外的增殖以及成骨、成牙本质分化。

bFGF对人根尖牙乳头干细胞成脂分化影响的研究

目的:研究碱性成纤维细胞生长因子(bFGF)在体外对人根尖牙乳头干细胞(stem cellsfrom apical papilla,SCAP)成脂分化的影响。方法:采用酶消化法获得原代SCAP,用含有5 ng/mL bFGF的成脂诱导液对其诱导培养不同时间,显微镜观察细胞形态变化、油红0染色观察SCAP成脂分化能力、RT-PCR检测PPAR-γ2 mRNA表达情况。结果:SCAP在含有5 ng/mL bFGF成脂诱导液中培养3周,细胞中脂滴形成数量多于对照组,而且细胞密度也相对较大,细胞体积小,未见拉长、变大等现象。RT-PCR检测显示,诱导培养1周时,实验组与对照组PPAR-γ2 mRNA表达无显著性差异(P>0.05);培养3周时,实验组PPAR-γ2 mRNA表达量明显高于对照组(P<0.05)。结论:bFGF具有促进SCAP成脂分化的潜能。