毛囊干细胞及其隆突微环境

毛囊隆突(bulge)是毛囊干细胞特定的微环境,它维持并调节干细胞的特性,使干细胞在静息态、自我更新和分化上保持平衡。现重点从毛囊隆突的物理结构上来简要揭示微环境对干细胞的调控。     

小鼠胚胎干细胞定向血管内皮和造血细胞分化体系的建立

目的:建立小鼠胚胎干细胞体外定向分化为血管内皮细胞和造血细胞的体系,并验证诱导后2种细胞的表面分子特征。方法:以小鼠胚胎成纤维细胞为饲养层,首先在无血清培养基StemPro中加入骨形态发生蛋白4(BMP4)、激活素A、碱性成纤维细胞生长因子(FGF-Basic)和血管内皮细胞生长因子(VEGF),诱导小鼠胚胎干细胞系R1/E 4 d后形成拟胚体;再将拟胚体消化后与OP9-DL1基质细胞共孵育,分别用干细胞因子(SCF)、VEGF和SCF、FLt3、白细胞介素3(IL-3)诱导向内皮和造血2个方向分化,并以CD31、CD45、CD144、Kit、CD201作为表面标志,流式检测诱导后细胞的表面分子特征和诱导效率;诱导10 d后免疫组化染色,进行内皮细胞的形态学鉴定。结果:诱导分化10 d后,免疫组化染色观察到多个内皮管状结构,流式检测CD31^+的内皮细胞比例为1.35%±0.05%,进一步分析CD31^+CD144^+CD45^-群体,有3.0%±0.2%的细胞表型为Kit^+CD201^+,提示该部分细胞可能是处于分化上游的内皮干祖细胞;CD45^+的造血细胞比例为35.0%±0.5%,其中0.35%±0.05%的细胞表达Kit和CD201,提示该部分细胞可能是处于分化上游的造血干祖细胞。结论:本研究将胚胎干细胞诱导为内皮细胞和造血细胞,并且能诱导出具有内皮、造血干祖细胞分子特征的细胞,可作为理想的体外诱导分化体系。     

Epidermal stem cells: the cradle of epidermal determination, differentiation and wound healing

The field of epidermal stem cells has dramatically advanced in the last decade, leading to a better understanding of the molecular factors, signalling pathways and cellular events that identify and characterize stem cells, thus revealing their immense potential for therapeutic use. Furthermore, multipotent epidermal stem cells present the major advantage of easy accessibility with the discovery of their specific location within the bulge of the hair follicle. This review focuses on the most recent findings on epidermal stem cells, and their potential role in initial epidermal commitment, differentiation and wound healing processes in the skin.     

脂肪间充质干细胞的分离培养、多向分化及其应用前景

脂肪组织几乎遍布于动物体全身,在整个生命过程中有极强的可塑性. 近年研究表明,运用相似的分离方法,可从人、小鼠、大鼠、兔和猪等物种脂肪组织中分离获得脂肪间充质干细胞. 与骨髓来源的间充质干细胞相比,它具有相似的表面标记和分化潜能;在合适的诱导条件下,这种细胞能分别向3个胚层的细胞分化,如成肌细胞、心肌细胞、软骨细胞、成骨细胞、脂肪细胞、神经细胞、血管内皮细胞和肝细胞等;脂肪间充质干细胞具有来源丰富,取材安全方便和扩增速率高的特点,使其在细胞治疗和组织工程方面具有更广阔的应用前景.     

一种体外快速分离脂肪来源干细胞的方法

目的:以小鼠为模型,建立一种基于流式细胞仪为检测手段的快速分离脂肪来源干细胞的方法,解决间充质干细胞进入实际应用过程中遇到的难题。方法:取BALB/c小鼠腹股沟内侧的皮下脂肪组织,采用Ⅰ型胶原酶消化等系列措施,获取脂肪干细胞(adipose-derived stem cells,ADSCs)。所得细胞分离培养4代后,流式细胞仪分选出CD73+CD45-ADSCs后成骨诱导分化。碱性磷酸酶染色和实时荧光定量PCR检测其分化情况。结果:刚分离培养的ADSCs细胞普遍呈圆形或椭圆形,传至第三代的细胞,非MSCs细胞逐渐被淘汰,剩余的细胞形态逐渐变得一致,细胞形态呈梭形。流式细胞仪检测发现ADSCs细胞表面抗原标记CD73+CD45-为20.7%。所得的ADSCs成骨诱导分化后碱性磷酸酶(alkaline phosphatase,ALP)染色呈阳性,实时荧光定量PCR检测成骨标志基因发现它们表达上调,其中ALP的表达高达22倍。结论:本方法可以获得纯度较高的ADSCs,且耗时少成本低;且提示可采用该方法来获得大量的人源ADSCs用于组织工程修复。     

从人胚胎干细胞畸胎瘤中有效获取间充质干细胞和神经前体细胞

通过人胚胎干细胞(human embryonic stem cells,hESC)体外分化方法和畸胎瘤形成可以分化获得多种成体细胞．但目前尚不清楚是否可以从hESCs畸胎瘤中分离某些特异性细胞．通过体外筛选方法,有效地从hESCs畸胎瘤中分离出神经前体细胞(neural progenitor cells,NPCs)和间充质干细胞(mesenchymal stem cells,MSCs)．这种hESCs畸胎瘤来源的NPCs和MSCs与体内神经前体细胞和间充质干细胞有着相似的分子标记和特性,并具有进一步的分化潜能——分别可以诱导成为神经元、神经胶质细胞、脂肪细胞和骨骼细胞等．根据人胚胎干细胞畸胎瘤中含有不同分化阶段的外胚层、中胚层和内胚层的组织或细胞,认为人胚胎干细胞畸胎瘤可以作为另一个细胞来源以获取多种(包括人胚胎干细胞体外分化难以得到的)各种前体/干细胞和终末分化细胞．     

STX17在不同毛色小鼠皮肤中差异表达与在毛囊中的定位

突触融合蛋白17 (STX17)是一种囊泡蛋白,参与细胞中物质的运输.为研究Stx17在不同毛色皮肤中是否存在差异表达及明确它在毛囊中的定位,进行了普通PCR、real-time PCR、免疫组化和蛋白免疫印迹实验对小鼠皮肤组织和体外培养黑素细胞的Stx17基因及蛋白的检测.普通PCR检测得出小鼠皮肤和黑色素细胞总RNA有Stx17 CDS区序列的表达;荧光定量检测显示,在白、灰、黑3种组织中Stx17均有表达,在灰色腹部表达量最高,是黑色皮肤的1.682倍,昆明鼠白色皮肤中表达量最低,是黑色皮肤的0.115倍;皮肤组织免疫组化结果显示,STX17表达于毛囊的上皮根鞘,且毛囊上段和中段表达量高于下段,黑色素细胞的免疫组化分析得出,STX17在黑色素细胞的细胞质和细胞膜上均有表达;蛋白免疫印迹结果显示,在白色、灰色和黑色皮肤均有STX17蛋白阳性条带且灰色皮肤中表达量最高,黑色皮肤次之,白色皮肤中表达量是最低的,这与荧光定量检测结果一致,体外培养的小鼠黑色素细胞中也有STX17蛋白阳性条带.实验结果表明,小鼠Stx17基因在皮肤组织、毛囊角化细胞以及黑色素细胞中均有表达,Stx17可能参与毛色的形成,且在小鼠腹部毛色变浅中起到了一定的作用.     

VEGF通过调节黏着斑促进间充质干细胞的黏附及铺展

间充质干细胞（mesenchymalstemcells,MSCs）具有多向分化潜能并能在体外趋化剂或细胞因子的作用下进行定向迁移,体内移植后可趋向迁移至脑瘤病灶区。细胞黏附是细胞迁移的首要条件,了解细胞黏附及其调控有助于细胞迁移机制的研究。细胞黏附及铺展涉及到黏着斑（f0－caladhesions,FAs）的动态变化以及细胞骨架的重排。细胞铺展面积在黏附过程中逐渐增大,黏附初期形成的小的黏着复合物逐渐成熟,聚集在一起形成较大的FAs。肌动蛋白（F—actin）聚集形成的螺线圈样微丝结构逐渐被应力纤维代替,细胞也由圆形变为具有极性的梭形或多角形。黏着斑激酶（focal adhesion kinase,FAK）和桩蛋白（paxillin）具有调节FAs聚合及骨架重排的作用,其中,Y397－FAK和Y31／Y118－paxillin的磷酸化活性在细胞铺展过程中不断变化。FAs组装时,Y397－FAK的磷酸化活性升高;FAs成熟后,Y397．FAK的磷酸化活性下降。活化的FAK能够磷酸4LY31／Y118-paxillin,激活paxillin参与调节细胞骨架的形成和排列。血管内皮生长因子（vascular endothelial growthfactor,VEGF）诱导~SMSCs黏附过程中,细胞面积变大,完全铺展的时间缩短,黏着斑及细胞骨架的形成均提前。另外,VEGF诱导的细胞铺展过程中形成的FAs形态细长,数量较多。该研究表明,VEGF通过调节黏着斑和细胞骨架促L~MSCs的黏附与铺展,提示vEGF可以通过调节黏着斑进而调控MSCs的定向迁移,为细胞迁移行为的研究提供理论基础。     

Activity of mesenchymal stem cells in therapies for chronic skin wound healing

Chronic or non-healing skin wounds present an ongoing challenge in advanced wound care, particularly as the number of patients increases while technology aimed at stimulating wound healing in these cases remains inefficient. Mesenchymal stem cells (MSCs) have proved to be an attractive cell type for various cell therapies due to their ability to differentiate into various cell lineages, multiple donor tissue types, and relative resilience in ex-vivo expansion, as well as immunomodulatory effects during transplants. More recently, these cells have been targeted for use in strategies to improve chronic wound healing in patients with diabetic ulcers or other stasis wounds. Here, we outline several mechanisms by which MSCs can improve healing outcomes in these cases, including reducing tissue inflammation, inducing angiogenesis in the wound bed, and reducing scarring following the repair process. Approaches to extend MSC life span in implant sites are also examined.     

胶质细胞的神经干细胞/祖细胞特性

近年来,关于胶质细胞有许多令人惊奇的发现。其中最令人感兴趣的是部分胶质细胞在体内外都表现出神经千细胞／祖细胞的特性,在适当条件下能分化成神经元、星形胶质细胞和／或少突胶质细胞。不仅存在于非哺乳类脊椎动物整个生命周期的放射胶质显示出这一特性,存在于成年哺乳动物脑室下区和颗粒下层的星形胶质细胞也是如此。在体外培养中,部分胶质细胞具有形成多潜能神经球的能力。在体内,胶质细胞充当前驱细胞时的命运受到细胞间相互作用、细胞因子、血脉系统、胞外基质以及基膜等所构建的微环境的影响。胶质细胞的这些特性将对神经修复产生深远影响。     

Recent highlights on bone stem cells: a report from Bone Stem Cells 2009, and not only…

The use of stem cells has opened new prospects for the treatment of orthopaedic conditions characterized by large bone defects. However, many issues still exist to which answers are needed before routine, large-scale application becomes possible. Bone marrow stromal cells (MSC), which are clonogenic, multipotential precursors present in the bone marrow stroma, are generally employed for bone regeneration. Stem cells with multilineage differentiation similar to MSC have also been demonstrated in adipose tissue, peripheral blood, umbilical cord and amniotic fluid. Each source presents its own advantages and drawbacks. Unfortunately, no unique surface antigen is expressed by MSC, and this hampers simple MSC enrichment from heterogeneous populations. MSC are identified through a combination of physical, morphological and functional assays. Different in vitro and in vivo models have been described for the research on bone stem cells. These models should predict the in vivo bone healing capacity of MSC and if the induced osteogenesis is similar to the physiological one. Although stem cells offer an exciting possibility of a renewable source of cells and tissues for replacement, orthopaedic applications often represent case reports whereas controlled randomized trials are still lacking. Further biological aspects of bone stem cells should be elucidated and a general consensus on the best models, protocols and proper use of scaffolds and growth factors should be achieved.     

mESC衍生内皮祖细胞定向分化为血管内皮细胞促伤口愈合

缺血性功能障碍是重要的全球健康问题。血管内皮细胞 (vascular endothelial cell, VEC) 在血管生成和创面修复中发挥关键作用,血管重建不足可导致慢性不愈合伤口。因此,了解有效的血管内皮细胞生成策略有助于受损组织中的血管再生。胚胎干细胞 (embryonic stem cell, ESC) 在组织的内皮化研究中应用广泛。内皮祖细胞 (endothelial progenitor cell, EPC) 是血管内皮细胞发育中不可或缺的部分。本研究目的在于找到一种小鼠胚胎干细胞 (mouse embryonic stem cell, mESC) 衍生为内皮祖细胞的快速、易筛选且高重复性的方法,并从内皮祖细胞定向分化中获得存活率高和功能性好的血管内皮细胞。结果表明,胚胎干细胞通过10 ng/mL VEGF和5 ng/mL bFGF定向诱导分化为增殖能力强的“铺路石”样祖细胞。同时,差异贴壁法有助于EPC的筛选。而EPC可诱导3 d的祖细胞高表达CD133和CD34(相对表达量分别为0.88 ± 0.04和2.12 ± 0.02);采用acctuse酶消化祖细胞,并在50 ng/mL VEGF和25 ng/mL bFGF的条件下诱导7 d分化为血管内皮样细胞,该细胞不仅高表达内皮细胞标志基因CD31、CD144、LAMA5、Tek、KDR和vWF,高表达标志蛋白CD31、CD144、LAMA5(相对表达量分别为1.07 ± 0.03、0.60 ± 0.02和0.70 ± 0.02),而且具有良好的迁移、成管和Weibel Palade (W-P) 小体形成能力。随后,将PBS、EPC和VEC分别应用于大小相同的创面治疗,EPC和VEC均能加快组织愈合程度(相对愈合率分别为78.93 ± 75.35%、95.57 ± 83.73%和100.00 ± 0.00%),VEC明显增强了伤口的血管生成能力和炎症反应。该研究初步证实,mESC衍生的EPC定向诱导7 d后可分化为血管内皮细胞。此内皮细胞具有较好的组织修复功能,干细胞促进血管生成的生理途径有望成为组织重塑的新靶点。     

PRC1 Fine-tunes Gene Repression and Activation to Safeguard Skin Development and Stem Cell Specification

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Propyl Gallate Inhibits Adipogenesis by Stimulating Extracellular Signal-Related Kinases in Human Adipose Tissue-Derived Mesenchymal Stem Cells

Propyl gallate (PG) used as an additive in various foods has antioxidant and anti-inflammatory effects. Although the functional roles of PG in various cell types are well characterized, it is unknown whether PG has effect on stem cell differentiation. In this study, we demonstrated that PG could inhibit adipogenic differentiation in human adipose tissue-derived mesenchymal stem cells (hAMSCs) by decreasing the accumulation of intracellular lipid droplets. In addition, PG significantly reduced the expression of adipocyte-specific markers including peroxisome proliferator-activated receptor-γ (PPAR-γ), CCAAT enhancer binding protein-α (C/EBP-α), lipoprotein lipase (LPL), and adipocyte fatty acid-binding protein 2 (aP2). PG inhibited adipogenesis in hAMSCs through extracellular regulated kinase (ERK) pathway. Decreased adipogenesis following PG treatment was recovered in response to ERK blocking. Taken together, these results suggest a novel effect of PG on adipocyte differentiation in hAMSCs, supporting a negative role of ERK1/2 pathway in adipogenic differentiation.     

心脏干/祖细胞与心肌损伤修复

细胞移植是一种有希望的组织再生的治疗手段.多种类型的细胞已经用于动物心 肌损伤的修复中,包括胚胎干细胞、胚胎和新生动物的心肌细胞、骨骼肌成肌细胞、 骨髓干细胞、脂肪来源的干细胞、可诱导的多能干细胞等.但是,这些用于移植的细胞 存在成活率低、在心脏局部存留少、与宿主心肌细胞不能整合和免疫排斥等问题,这 些问题限制了它们的应用.心脏自身存在的干细胞因为没有其他来源细胞存在的种种 问题,因而成为备受关注的治疗心肌梗死的种子细胞.但是,心脏干/祖细胞也有自身 弊端,包括干细胞群的细胞生物学或遗传学标志没有统一,在心肌中数量极少,体外 扩增能力有限等,因而限制了心脏干/祖细胞的有效应用.如何能有效动员和促进心脏 干/祖细胞增殖,依赖于人们对心脏干/祖细胞增殖、分化、归巢的调控机制,包括心 脏干/祖细胞修复损伤心肌的分子机制的深入了解.本文将就近年来在心脏再生领域中 ,心脏干/祖细胞的研究新进展进行综述.     

Hair Follicle Development in Mouse Pluripotent Stem Cell-Derived Skin Organoids

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Dynamic multiphoton imaging of acellular dermal matrix scaffolds seeded with mesenchymal stem cells in diabetic wound healing

Significantly effective therapies need to be developed for chronic nonhealing diabetic wounds. In this work, the topical transplantation of mesenchymal stem cell (MSC) seeded on an acellular dermal matrix (ADM) scaffold is proposed as a novel therapeutic strategy for diabetic cutaneous wound healing. GFP‐labeled MSCs were cocultured with an ADM scaffold that was decellularized from normal mouse skin. These cultures were subsequently transplanted as a whole into the full‐thickness cutaneous wound site in streptozotocin‐induced diabetic mice. Wounds treated with MSC‐ADM demonstrated an increased percentage of wound closure. The treatment of MSC‐ADM also greatly increased angiogenesis and rapidly completed the reepithelialization of newly formed skin on diabetic mice. More importantly, multiphoton microscopy was used for the intravital and dynamic monitoring of collagen type I (Col‐I) fibers synthesis via second harmonic generation imaging. The synthesis of Col‐I fibers during diabetic wound healing is of great significance for revealing wound repair mechanisms. In addition, the activity of GFP‐labeled MSCs during wound healing was simultaneously traced via two‐photon excitation fluorescence imaging. Our research offers a novel advanced nonlinear optical imaging method for monitoring the diabetic wound healing process while the ADM and MSCs interact in situ. Schematic of dynamic imaging of ADM scaffolds seeded with mesenchymal stem cells in diabetic wound healing using multiphoton microscopy. PMT, photo‐multiplier tube.      

间充质干细胞对造血干/祖细胞分化为巨核细胞的影响

目的:探讨间充质干细胞(MSC)共培养对体外诱导脐带血单个核细胞来源的造血干/祖细胞生成巨核细胞的影响。方法:分离得到骨髓和脐带2种来源的MSC,并对它们进行表面标志和多向分化能力的鉴定,同时通过实时定量PCR及对RT-PCR产物的电泳分析,对比相同培养代数下2种MSC表达造血因子的情况;用梯度离心法分离得到单个核细胞,通过直接接触或Trans-well分隔的方式分别与MSC共培养,观察细胞增殖情况,并检测巨核系特异性的表面标志和相关基因的表达。结果:骨髓和脐带来源的MSC均分泌对巨核细胞增殖分化有促进作用的造血因子,与造血干/祖细胞直接共培养,对于巨核细胞的增殖有明显的促进作用,分化效果不明显;在非接触共培养的条件下,对巨核细胞的增殖及分化都产生促进作用,且骨髓来源的MSC较脐带来源的MSC效果更加明显。结论:MSC与脐带血造血干/祖细胞非接触培养,对其向巨核分化和增殖的促进作用明显,本实验所用的骨髓来源MSC促分化效果更好。本研究为今后进一步优化巨核系诱导分化体系奠定了基础,并对未来体外大规模制备巨核系祖细胞应用于临床治疗有一定的指导作用。