The advantages of hair follicle pluripotent stem cells over embryonic stem cells and induced pluripotent stem cells for regenerative medicine

Multipotent adult stem cells have many potential therapeutic applications. Our recent findings suggest that hair follicles are a promising source of easily accessible multipotent stem cells. Stem cells in the hair follicle area express the neural stem cell marker nestin, suggesting that hair-follicle stem cells and neural stem cells have common features. Nestin-expressing hair follicle stem cells can form neurons and other cell types, and thus adult hair follicle stem cells could have important therapeutic applications, particularly for neurologic diseases. Transplanted hair follicle stem cells promote the functional recovery of injured peripheral nerve and spinal cord. Recent findings suggest that direct transplantation of hair-follicle stem cells without culture can promote nerve repair, which makes them potentially clinically practical. Human hair follicle stem cells as well as mouse hair follicle stem cells promote nerve repair and can be applied to test the hypothesis that human hair follicle stem cells can provide a readily available source of neurologically therapeutic stem cells. The use of hair follicle stem cells for nerve regeneration overcomes critical problems of embryonic stem cells or induced pluripotent stem cells in that the hair follicle stem cells are multipotent, readily accessible, non-oncogenic, and are not associated with ethical issues.     

诱导多能干细胞在皮肤病研究的应用进展

诱导多能干细胞是将一系列转录因子转入动物或人多种分化成熟的体细胞,经过重组的程序诱导获得多能干细胞,其在形态、增殖能力、表面抗原、基因和蛋白表达、分化能力等方面与胚胎干细胞相似,因此,科研工作者利用诱导多能干细胞代替胚胎干细胞来研究皮肤病.因诱导多能干细胞除具有多能性还具有无限增殖的能力,这些特性能使研究者获得无限的特定细胞,进而建立遗传性皮肤病模型.将致病的突变基因在诱导多能干细胞水平矫正,矫正后的诱导多能干细胞能产生基因正常的健康皮肤组织细胞,从而可望治疗遗传性皮肤病.     

诱导多潜能干细胞在皮肤病治疗中的应用

诱导多潜能干细胞拥有胚胎干细胞所有特征,包括多能性和生成各种体细胞.用皮肤细胞产生诱导多潜能干细胞,不仅起始细胞易获取,而且这些诱导多潜能干细胞更容易定向分化为角质形成细胞、黑素细胞和成纤维细胞等多种功能性皮肤细胞.患者自体来源的诱导多潜能干细胞是细胞疗法理想的细胞库,用诱导多潜能干细胞分化后的细胞治疗皮肤病,不仅细胞量充足,且可避免伦理问题和免疫排斥反应.利用回复突变体嵌合体,结合诱导多潜能干细胞技术,能获得充分的患者特异性功能性回复体细胞而用于治疗遗传性皮肤病.该技术可避免常规基因治疗中出现的免疫排斥和插入诱变.     

Differentiation of mouse induced pluripotent stem cells into a multipotent keratinocyte lineage

Recent breakthroughs in the generation of induced pluripotent stem cells (iPSCs) have provided a novel renewable source of cells with embryonic stem cell-like properties, which may potentially be used for gene therapy and tissue engineering. Although iPSCs have been differentiated into various cell types, iPSC-derived keratinocytes have not yet been obtained. In this study, we report the in vitro differentiation of mouse iPSCs into a keratinocyte lineage through sequential applications of retinoic acid and bone-morphogenetic protein-4 and growth on collagen IV-coated plates. We show that iPSCs can be differentiated into functional keratinocytes capable of regenerating a fully differentiated epidermis, hair follicles, and sebaceous glands in an in vivo environment. Keratinocytes derived from iPSCs displayed characteristics similar to those of primary keratinocytes with respect to gene and protein expression, as well as their ability to differentiate in vitro and to reconstitute normal skin and its appendages in an in vivo assay. At present, no effective therapeutic treatments are available for many genetic skin diseases. The development of methods for the efficient differentiation of iPSCs into a keratinocyte lineage will enable us to determine whether genetically corrected autologous iPSCs can be used to generate a permanent corrective therapy for these diseases.     

Induced pluripotent stem cells from individuals with recessive dystrophic epidermolysis bullosa

Recessive dystrophic epidermolysis bullosa (RDEB) is an inherited blistering skin disorder caused by mutations in the COL7A1 gene-encoding type VII collagen (Col7), the major component of anchoring fibrils at the dermal-epidermal junction. Individuals with RDEB develop painful blisters and mucosal erosions, and currently, there are no effective forms of therapy. Nevertheless, some advances in patient therapy are being made, and cell-based therapies with mesenchymal and hematopoietic cells have shown promise in early clinical trials. To establish a foundation for personalized, gene-corrected, patient-specific cell transfer, we generated induced pluripotent stem (iPS) cells from three subjects with RDEB (RDEB iPS cells). We found that Col7 was not required for stem cell renewal and that RDEB iPS cells could be differentiated into both hematopoietic and nonhematopoietic lineages. The specific epigenetic profile associated with de-differentiation of RDEB fibroblasts and keratinocytes into RDEB iPS cells was similar to that observed in wild-type (WT) iPS cells. Importantly, human WT and RDEB iPS cells differentiated in vivo into structures resembling the skin. Gene-corrected RDEB iPS cells expressed Col7. These data identify the potential of RDEB iPS cells to generate autologous hematopoietic grafts and skin cells with the inherent capacity to treat skin and mucosal erosions that typify this genodermatosis.     

Induced pluripotent stem cells for modeling neurological disorders

Several diseases have been successfully modeled since the development of induced pluripotent stem cell(i PSC) technology in 2006. Since then, methods for increased reprogramming efficiency and cell culture maintenance have been optimized and many protocols for differentiating stem cell lines have been successfully developed, allowing the generation of several cellular subtypes in vitro. Gene editing technologies have also greatly advanced lately, enhancing disease-specific phenotypes by creating isogenic cell lines, allowing mutations to be corrected in affected samples or inserted in control lines. Neurological disorders have benefited the most from i PSC-disease modeling for its capability for generating disease-relevant cell types in vitro from the central nervous system, such as neurons and glial cells, otherwise only available from post-mortem samples. Patient-specific i PSC-derived neural cells can recapitulate the phenotypes of these diseases and therefore, considerably enrich our understanding of pathogenesis, disease mechanism and facilitate the development of drug screening platforms for novel therapeutic targets. Here, we review the accomplishments and the current progress in human neurological disorders by using i PSC modeling for Alzheimer's disease, Parkinson's disease, Huntington's disease, spinal muscular atrophy, amyotrophic lateral sclerosis, duchenne muscular dystrophy, schizophrenia and autism spectrum disorders, which include Timothy syndrome, Fragile X syndrome, Angelman syndrome, Prader-Willi syndrome, PhelanMc Dermid, Rett syndrome as well as Nonsyndromic Autism.     

诱导性多功能干细胞在皮肤科研究与应用的进展

【摘要】 诱导性多功能干细胞（iPSC）是通过向体细胞中导入一些特定的多能性转录因子使其重编程为干细胞。相比于传统干细胞,iPSC具有多方面优势,如来源广泛并且可以全能分化,以及更少涉及胚胎干细胞长期争议的取材和伦理问题。在皮肤科,iPSC可以用于制造再生皮肤,构建皮肤疾病模型,为研究皮肤疾病的发病机制提供新的平台,还可以针对特定皮肤疾病进行细胞治疗或基因修正治疗。目前其效果已在动物模型中得到验证,临床试验也正在逐步进行。     

Co-culture of melanocytes with adipose-derived stem cells as a potential substitute for co-culture with keratinocytes

Cell-to-cell interactions between melanocytes and keratinocytes increase the proliferation and migration of melanocytes. In fact, mixed keratinocyte and melanocyte cultures have been used for autologous cell transplantation for treatment of vitiligo. However, this may require taking an amount of skin tissue large enough to leave scars. In this study, the in vitro effect of adipose-derived stem cells (ADSCs) on proliferation, differentiation and migration of melanocytes was compared with that of keratinocytes using immunohistochemistry and a Boyden chamber migration assay. The proliferation and migration of melanocytes was significantly stimulated by co-culture with ADSCs compared with melanocyte monocultures, al-though the effect of ADSCs was less powerful than that of keratinocytes. This may be related to increases in stem cell factor and basic fibroblast growth factor, growth factors for melanocytes, produced by the ADSCs. The ratios of melanocytes stained with antibodies against Trp-2, E-cadherin and N-cadherin were significantly increased by co-culturing with ADSCs compared with co-culturing with keratinocytes as well as melanocyte monocultures. The proportion of less-pigmented melanocytes was also increased and sustained for a longer duration in the presence of ADSCs. Our data show that co-culturing with ADSCs results in increased melanocyte proliferation and migration while reducing differentiation, and could provide a means to treat disorders such as vitiligo.     

Nestin-positive hair follicle pluripotent stem cells can promote regeneration of impinged peripheral nerve injury

Nestin-positive, keratin 15 (K15)-negative multipotent hair follicle stem cells are located above the hair follicle bulge. We have termed this location the hair follicle pluripotent stem cell area. We have previously shown that transplantation of nestin-expressing hair follicle stem cells can regenerate peripheral nerve and spinal cord injuries. In the present study, we regenerated the impinged sciatic nerve by transplanting hair follicle pluripotent stem cells. Human hair follicle stem cells were transplanted around the impinged sciatic nerve of ICR nude (nu/nu) mice. The hair follicle stem cells were transplanted between impinged sciatic nerve fragments of the mouse where they differentiated into glial fibrillary acidic protein-positive Schwann cells and promoted the recovery of pre-existing axons. The regenerated sciatic nerve functionally recovered. These multipotent hair follicle stem cells thereby provide a potential accessible, autologous source of stem cells for regeneration therapy of nerves degenerated by compression between bony or other hard surfaces.     

体外诱导小鼠骨髓间充质干细胞分化为黑素细胞的观察

【摘要】 目的 探讨骨髓间充质干细胞体外诱导成为黑素细胞的可能性。方法　6周龄雄性C57BL/6小鼠股骨基质细胞行原代培养,6次传代后以氢化可的松、重组人胰岛素、转铁蛋白和成纤维细胞生长因子诱导黑素细胞。倒置光学显微镜观察细胞分化;透射电镜观察黑素小体成熟;免疫荧光染色观察黑素细胞相关表位表达;流式细胞仪检测黑素细胞的细胞周期及获得率。结果　6次传代间充质干细胞数量近109,免疫荧光检测CD44阳性率94.3%和CD105阳性率82.3%。培养180 d,细胞形态接近于黑素细胞,树突增多,胞质内出现黑素小体样结构,生长周期加快为3 ~ 4 d,肉眼可见棕黑色细胞沉淀。电镜观察显示Ⅳ期为主的黑素小体。免疫荧光显示酪氨酸酶相关蛋白-1,酪氨酸酶相关蛋白-2和小眼畸形相关转录因子阳性。流式细胞仪分析显示细胞基本处于G1和S期。酪氨酸酶相关蛋白-1阳性的黑素细胞获得率约为80%。结论　骨髓间充质干细胞可以被大量诱导分化为黑素细胞;诱导黑素细胞的形态学、超微结构、特异性表位等皆接近于正常黑素细胞,具有一定的增殖活性,获得率较高。 【关键词】 间质干细胞; 骨髓; 黑素细胞; 体外研究     

人脂肪间充质干细胞体外向黑素细胞分化的研究

【摘要】 目的 建立体外诱导人脂肪间充质干细胞（hADSC）向黑素细胞分化的方法。 方法 由人皮下脂肪分离培养获得hADSC,流式细胞仪检测细胞表型,成骨成脂分化证明分化潜能。将干细胞生长因子（SCF）、内皮素3 （EDN-3）、碱性成纤维细胞生长因子（bFGF）等加到条件培养基中,促使第5代hADSC向黑素细胞方向诱导,诱导至第10周。未诱导组为正常对照组。使用实时荧光定量PCR检测黑素相关基因小眼畸形转录因子（MITF）,酪氨酸激酶受体c-Kit（KIT）,多巴色素异构酶（DCT）,酪氨酸酶（TYR）,酪氨酸酶相关蛋白1（TYRP1）,性别决定区域相关转录因子10（SOX10） mRNA的表达量,统计学分析采用单因素方差分析及LSD-t检验。诱导结束后,通过免疫细胞化学法、细胞免疫荧光法进行鉴定。 结果 流式细胞仪显示分离培养获得的hADSC高表达CD29、CD44、CD73、CD90、CD105、CD166,阳性率均在95%以上;低表达或不表达CD31、CD34、CD45,人白细胞DR抗原（HLA-DR）。成骨成脂分化结果显示hADSC具有分化潜能。实时荧光定量PCR结果显示,诱导10周后,MITF、KIT、DCT、TYR、TYRP1、SOX10 mRNA的表达水平分别为0.325 ± 0.012,0.042 ± 0.006,0.046 ± 0.013,0.036 ± 0.005,0.041 ± 0.003,0.225 ± 0.014,与诱导0周组相比,均有上调（P < 0.05）。诱导结束后,免疫细胞化学MITF、HMB45染色阳性,细胞免疫荧光TYRP1、S100阳性表达。 结论 由人脂肪间充质干细胞诱导所得细胞具有一定的黑素细胞生物学特性。     

人表皮黑素细胞、毛囊无色素黑素细胞和鼠黑素瘤细胞的原子力显微镜观察

目的：观察培养的人表皮黑素细胞、毛囊无色素黑素细胞和S91鼠黑素瘤细胞的形态结构。方法：培养并纯化来自正常人包皮的黑素细胞以及来自毛囊的无色素黑素细胞，同时复苏S91细胞株，传代后接种到内置云母片的培养板中，细胞贴附到云母片上后固定，用原子力显微镜扫描观察。结果：正常人表皮黑素细胞有3级分支，在主干及分支的顶端和侧缘可见膨出的球形结构。鼠黑素瘤细胞仅有很短的2级分支，在2级树突近端可见黑素小体。毛囊无色素黑素细胞只有1级树突，并只在树突近端有少数黑素小体。结论：表皮黑素细胞在形态上比黑素瘤细胞、毛囊无色素黑素细胞更成熟，有更多的黑素颗粒从树突的顶端和侧缘以胞吐的形式被输出。     

Human melanocytes cultured from nevi and melanomas

The requirements for growth factors of human melanocytes in culture may be dependent on the stage of malignant transformation. Several factors synergistically promote the viability and proliferation of human neonatal melanocytes in culture. They are TPA (12-O-tetradecanoyl-phorbol-13-acetate), isobutylmethyl xanthine, cholera toxin, and as yet unidentified factors from extracts derived from several cell lines and human placenta. Neonatal melanocytes can maintain at least 50 population doublings during a period of 6 months, whereas melanocytes from adult skin proliferate only for 1 month and at less than 1% of the proliferative rate of melanocytes derived from newborn foreskins. In contrast, melanocytes from dysplastic and congenital nevi proliferate well in the presence of mitogens during the initial 4-6 weeks of culture, but then become quiescent. Melanocytes from primary melanomas are the most difficult to grow in culture. They need the mitogens, but their rate of proliferation is slow. Most of the metastatic melanocyte strains that do not need the mitogens in order to proliferate, are strongly inhibited by TPA, and to a lesser extent by WI-38 cell extract. We conclude that the acquisition of independence from mitogens in culture is a late event in the transformation of melanocytes to melanomas.     

Morphometric and ultrastructural analyses of melanocytes,nevus cells,and melanoma cells

Summary Cytological atypia, revealed in the course of routine light microscopy, is considered a valuable indicator of malignancy in melanocytic lesions. A clear definition of the term cytological atypia, however, is lacking. Therefore, by morphometric analysis of ultrathin sections of 11 malignant melanomas (7 invasive, 3 in situ, and 1 lentigo maligna melanoma) and 10 compound nevi, we evaluated the discriminating power of the various facets of cytological atypia, i. e., nuclear area, area of the nucleolus, area of the total cell, and nuclear irregularity. In each case, at least 50 intraepidermal melanocytic cells were examined.The two-sided U-test showed significant differences between intraepidermal nevus and melanoma cells, with regard to the mean values (x) and standard deviations (s) of the nuclear area (x and s, p=0.00011), area of the nucleolus (x, p=0.00043; s, p=0.00011), and area of the total cell (x, p=0.00011; s, p=0.00093). However, only the mean values and standard deviations of the nuclear area allowed a clear distinction in each individual case.The area of the nucleus can be estimated in the course of routine histology. We therefore think that the size and variation of the nuclear area should be considered in the histological differential diagnosis between malignant melanomas and benign nevi.Presented in part at the 12th Colloquium of the Society for Ultrastructural Research and the 6th International Dermatopathology Colloquium, 17–20 April 1985, Florence, Italy     

内皮素-1诱导黑素细胞树突生成研究

目的:探讨311 nm UVB对人角质形成细胞(KC)分泌内皮素1(ET-1)的影响及ET-1通过Rho家族小GTP酶对人黑素细胞树突生成的调节作用.方法:应用ELISA检测311 nm UVB照射人KC后收集的培养上清液中IL-1、ET-1和bFGF的浓度;应用相差显微镜观察人ET-1对人黑素细胞树突生成的影响;并应用Pull-down方法检测ET-1对人黑素细胞GTP-RhoA和GTP-Rac1蛋白的表达情况.结果:在25和50 mJ/cm2剂量时,人KC培养上清液中IL-1和ET-1的浓度与对照组比较明显升高.黑素细胞的树突数量在ET-1处理24 h后明显增多,其中大于3个树突以上的细胞占到(63.67±5.51)%.Pull-down实验显示GTP-Rac1的表达在ET-1处理后逐渐升高.结论:ET-1可通过活化Rac1和抑制RhoA双重途径促进人黑素细胞树突生成的作用.     

维A酸促毛囊外毛根鞘无色素黑素细胞分化的实验研究

目的：研究维A酸(all-transretinoic acid，ATA)对毛囊无色素黑素细胞（famelanotic melangeytes，AMMC)的激活作用。方法：以高、中、低3种不同浓度的ATA作用于培养的人毛囊外毛根鞘AMMC，倒置显微镜观察细胞形态的变化，细胞计数法测定ATA对AMMC增殖率的影响，通过间接免疫荧光法结合激光共聚焦显微镜半定量分析药物作用前后AMMC酪氨酸酶(tyrosinase，TYR)、酪氨酸酶相关蛋白-1(tyrosinase related protein-1，TRP-1)和酪氨酸酶相关蛋白-2(TRP-2)表达的变化。结果：ATA能抑制AMMC的增殖，并能促进AMMC表达TYR和TRP-1，但对TRP-2的表达没有影响。结论：ATA能够促进AMMC的分化，同时抑制增殖，其抑制机制可能与凋亡有关。