毛囊细胞——一种新的皮肤组织工程种子细胞

毛囊的上皮细胞和真皮细胞与皮肤的表皮角朊细胞和真皮成纤维细胞具有很大的相似性，但其具有更强的增殖分化能力和更多的生物学特性，并且毛囊真皮细胞具有干细胞的一些特性，作为皮肤组织工程的种子细胞具有更独特的优势，在构建带有皮肤附属器的组织工程皮肤上有潜在的前景。     

毛囊真皮鞘细胞的生物学特性研究进展

真皮鞘包绕于毛囊外周,是维持和再生毛乳头的重要物质基础,是毛囊再生的必要条件。近年研究表明,毛囊真皮鞘细胞不但参与皮肤创伤修复,而且具有成体干细胞特性,有望为皮肤创伤愈合研究和组织工程学研究提供新的思路。本文就毛囊真皮鞘细胞生物学特性研究进展作一综述。     

ES细胞源性表皮干细胞与类真皮构建组织工程皮肤的体内分化

目的以ES细胞源性表皮干细胞为种子细胞与类真皮构建组织工程皮肤,探讨其在体内的分化。方法胎鼠皮肤成纤维细胞和大鼠骨髓基质干细胞(BMSCs),分别与复合凝胶-明胶海绵构建类真皮(类真皮Ⅰ、Ⅱ),植入小鼠全层皮肤缺损创面,以生物膜为载体,把羊膜诱导后带有核标记的表皮干细胞覆盖在类真皮上,术后1～8周连续取材,苏木精-伊红染色,β1整合素、CK15、CK19、CK10和CEA免疫荧光双标和免疫组化观察。结果两组组织工程皮肤植入皮肤缺损3～4周后,创面完全长合,较厚新生皮完全覆盖创面,基底层细胞增生,形成短的细胞柱突向真皮层。新生表皮中可见核标记的细胞呈β1整合素、CK15、CK19阳性,真皮中的管腔样结构呈核荧光和CEA免疫组化双标阳性,4～8周新生表皮基底层细胞呈CK19、CK10阳性,新生表皮下可见毛囊样、皮脂腺样结构。结论ES细胞源性表皮干细胞为种子细胞与类真皮构建的两种组织工程皮肤在体内具有修复缺损皮肤及分化为表皮及毛囊样、皮脂腺样和汗腺样等皮肤附属结构的潜能。     

表皮干细胞与毛囊干细胞生物学特性的比较

背景：获取更合适的组织工程皮肤种子细胞可使皮肤功能得到更好的修复。 目的：分离、培养表皮干细胞和毛囊干细胞,并比较两种细胞的生物学特性。 方法：体外分离培养2月龄新西兰兔表皮干细胞和毛囊干细胞,取生长良好的第2,3,6代细胞观察其生物学特性。 结果与结论：毛囊干细胞较表皮干细胞贴壁快,具有更高的增殖活性。免疫组化及荧光定量PCR检测显示毛囊干细胞β1整合素、角蛋白19蛋白及mRNA表达均明显高于表皮干细胞。提示作为皮肤组织工程的种子细胞,毛囊干细胞较表皮干细胞更具优势。     

皮肤干细胞将成为细胞治疗神经系统疾病的新的细胞来源

皮肤干细胞主要有表皮干细胞、毛囊干细胞和皮肤前体细胞(SKP),具有多向分化潜能及强大的增殖能力,主要应用于皮肤组织工程。随着皮肤干细胞的不断研究深入,发现在体外可诱导皮肤干细胞成为神经干细胞,有些皮肤干细胞本身就具有神经干细胞性质,它们在一定的条件下可以分化为神经元和神经胶质细胞等。所以,皮肤干细胞的这一特性为神经系统病伤的细胞治疗提供了新的细胞来源。     

毛囊真皮鞘细胞中过表达Noggin对皮肤及毛囊生长的作用

背景:Noggin蛋白是一个抑制BMP信号通路的重要分子,可以与BMP2/4结合形成复合物,从而阻断BMP信号,影响生物有机体的正常发育过程。研究认为真皮鞘细胞是一类能够长期自我更新的真皮干细胞,参与毛囊再生过程中真皮毛乳头和真皮鞘的形成。在毛囊发育过程中,真皮毛乳头中Noggin参与毛囊的起始,Noggin的缺失会导致毛囊数量的减少和毛囊生长缓慢,但人们对于Noggin蛋白在毛囊真皮鞘中的作用所知甚少。目的:研究Noggin蛋白在毛囊真皮鞘中的生物学功能。方法:利用真皮鞘特异的αSMA-Cre ERT2工具鼠在真皮鞘中特异过表达Noggin蛋白,分别在出生后8,9 d对实验组αSMA-CreER;pMES-Noggin小鼠和对照组αSMA-CreER小鼠注射4-羟基他莫昔芬,在出生后21,23,28 d获取皮肤组织,苏木精-伊红染色观察毛囊生长情况和皮下脂肪层厚度,免疫组化染色分析表型。结果与结论:通过苏木精-伊红染色结果发现毛囊生长并没有受到影响,但毛囊皮下脂肪组织生长发育受到严重影响,小鼠皮下脂肪层变薄。免疫组化结果说明BMP信号降低可能是导致毛囊脂肪层变薄的原因。这一发现拓展了人们对于真皮鞘细胞和Noggin蛋白的认识,也为人们更加准确理解毛囊再生和组织生长机制提供了重要依据。     

载荷角质细胞生长因子聚乳酸-羟基乙酸共聚物纳米缓释微球在组织工程皮肤构建中的应用

背景：聚乳酸-羟基乙酸共聚物具有良好的生物相容性和可降解性性。 目的：制备载荷角质细胞生长因子聚乳酸-羟基乙酸共聚物控释载药系统用于组织工程皮肤。 方法：采用乳化-溶剂挥发法、冷冻干燥法制备载有角质细胞生长因子的聚乳酸-羟基乙酸共聚物纳米微球,并构建组织工程皮肤。扫描电镜、倒置显微镜、纳米粒度分析仪、紫外分光及ELISA法对微球评价其特性。 结果与结论：纳米微球载药量为(14.05±0.56)%,包封率为(59.86±2.38)%,角质细胞生长因子活性保留率(78.26±5.63)%,体外释放30 d的累积释药率达75%以上,微球形态规则圆润。微球形态规整,在脱细胞真皮表面分布均匀,与其联接良好。毛囊干细胞群在荷载聚乳酸-羟基乙酸共聚物纳米微囊脱细胞真皮上生长活跃,细胞形态良好,并呈克隆团生长。说明实验用组织工程材料制备工艺合理,材料相容性好,可用于构建新型组织工程皮肤。     

组织工程化皮肤的新种子细胞-毛囊干细胞

以干细胞为种子细胞构建组织工程化皮肤成为当前研究的热点之一。皮肤干细胞主要有表皮干细胞和毛囊干细胞。毛囊干细胞在体内表现为慢周期性,在体外培养时具有高克隆形成能力,毛囊干细胞作为组织工程化皮肤的新种子细胞受到关注。     

脱细胞真皮基质及其临床应用

脱细胞真皮基质（acellular dermal matrix,ADM）作为一种永久性真皮支架已成功用于皮肤创面修复、组织充填、重建及美容整形领域。由于ADM去除了皮肤组织中的细胞成分和部分可溶性蛋白,使其免疫原性显著降低,并且提高了组织相容性,被广泛应用于烧创伤所致的皮肤缺损以及口腔黏膜修复、腹壁缺损修补、硬脑膜修补、软硬组织充填及乳房重建等领域,此外,作为人工真皮的支架材料还被应用于组织工程皮肤的构建。     

异体脱细胞真皮基质作为组织工程皮肤真皮支架的可行性*

背景：组织工程皮肤是目前研究皮肤损伤修复重建的重要手段之一,异体脱细胞真皮基质不存在免疫原性,在异体移植时不会发生排斥反应,是比较理想的真皮替代物。 目的：观察异体脱细胞真皮基质的组织相容性。 方法：以正常人体真皮组织作为对照,通过体外、体内细胞毒性实验检测异体脱细胞真皮基质的组织相容性,以膨胀度、饱和含水量及生物力学分析检测异体脱细胞真皮基质的亲水性及机械性能。 结果与结论：真皮基质中未见任何细胞成分,其网孔直径介于100~180 μm 之间。脱细胞真皮基质组饱和含水量为(69.6±3.97)%,膨胀度2.30±0.42,最大断裂力为(3.082±0.046) N,与对照组相比,差异无显著性意义(P > 0.05)。体内外细胞毒性检测,未见明显细胞生长抑制及免疫排斥反应。提示异体脱细胞真皮基质机械性能接近正常皮肤,组织相容性好,免疫排斥反应小,是构建组织工程皮肤理想的真皮材料。     

Steady and temporary expressions of smooth muscle actin in hair, vibrissa, arrector pili muscle, and other hair appendages of developing rats

The hair erection muscle, arrector pili, is a kind of smooth muscle located in the mammalian dermis. The immunohistochemical study using an antibody against smooth muscle alpha actin (SMA) showed that the arrector pili muscle develops approximately 1-2 weeks after birth in dorsal and ventral skin, but thereafter they degenerate. The arrector pili muscle was not detected in the mystacial pad during any stage of development, even in the neighboring pelage-type hair follicle. A strong signal of SMA in the skin was located in the dermal sheath as well as in some outer root sheath cells in the hair and vibrissal follicles. Positive areas in the dermal and outer root sheaths were restricted to a lower moiety, particularly areas of similar height, where keratinization of the hair shaft occurs. This rule is valid for both pelage hair follicles and vibrissal follicles. At medium heights of the follicle, SMA staining in the dermal sheath was patchy and distant from the boundary between dermis and epidermis. In contrast to SMA, vimentin was expressed over the entire height of the dermal sheath. Unlike the arrector pili muscle, the expression of SMA in the dermal sheath was observed during fetal, neonatal, and adult stages. The presence of actin-myosin and vimentin fibers in supporting cells is thought to be beneficial for the hair follicle to cope with the movement of the hair shaft, which may be caused by physical contacts with outside materials or by the contraction of internal muscles.     

Wnt signaling maintains the hair-inducing activity of the dermal papilla

The formation of the hair follicle and its cyclical growth, quiescence, and regeneration depend on reciprocal signaling between its epidermal and dermal components. The dermal organizing center, the dermal papilla (DP), regulates development of the epidermal follicle and is dependent on signals from the epidermis for its development and maintenance. GFP specifically expressed in DP cells of a transgenic mouse was used to purify this population and study the signals required to maintain it. We demonstrate that specific Wnts, but not Sonic hedgehog (Shh), maintain anagen-phase gene expression in vitro and hair inductive activity in a skin reconstitution assay.     

Nitric oxide in the human hair follicle: constitutive and dihydrotestosterone-induced nitric oxide synthase expression and NO production in dermal papilla cells

The free radical nitric oxide, generated by different types of epidermal and dermal cells, has been identified as an important mediator in various physiological and pathophysiological processes of the skin, such as regulation of blood flow, melanogenesis, wound healing, and hyperproliferative skin diseases. However, little is known about the role of NO in the human hair follicle and in hair cycling processes. Here we demonstrate for the first time that dermal papilla cells derived from human hair follicles spontaneously produce NO by measuring nitrate and nitrite levels in culture supernatants. This biomolecule is apparently formed by the endothelial isoform of nitric oxide synthase, which was detected at the mRNA and protein levels. Remarkably, basal NO level was enhanced threefold by stimulating dermal papilla cells with 5alpha-dihydrotestosterone (DHT) but not with testosterone. Addition of N-[3-(aminomethyl)benzyl]acetamidine (1400W), a highly selective inhibitor of inducible nitric oxide synthase, restrained the elevation in NO level induced by DHT. Analyses of DHT-stimulated cells at the mRNA and protein levels confirmed the expression of inducible nitric oxide synthase. These findings suggest NO as a signaling molecule in human dermal papilla cells and implicate basal and androgen-mediated NO production to be involved in the regulation of hair follicle activity.     

Embryonic development of hair follicle pluripotent stem (hfPS) cells

Our laboratory discovered nestin-expressing hair follicle stem cells and demonstrated their pluripotency. We have shown that nestin-positive and K15-negative multipotent hair follicle stem cells are located above the hair follicle bulge, and we termed these cells hair follicle pluripotent stem (hfPS) cells. We have previously shown that hair follicle stem cells can regenerate peripheral nerve and spinal cord. In the present study, we describe the embryonic development of the hair follicle stem cell area (hfPSCA), which is located above the bulge and below the sebaceous glands in the adult mouse. At embryonic day 16.5 (E16.5) of nestindriven GFP (ND-GFP) transgenic mice, which express nestin in hfPS cells, the ND-GFP hair follicle stem cells are located in mesenchymal condensates. At postnatal day 0 (P0), the ND-GFP-expressing cells are migrating to the upper part of the hair follicle from the dermal papilla. At P3, keratin 15 (K15)-positive cells, derived from ND-GFP dermal papilla cells, are located in the outer-root sheath and basal layer of the epidermis. By P10, the ND-GFP have formed the K15-positive outer-root sheath as well as the ND-GFP hfPSA. These results suggest that ND-GFP hfPS cells in the dermal papilla form nestin-expressing hair follicle stem cells in the first hair cycle. These observations provide new insight into the origins of hfPS cells and the hfPSCA.     

Shh maintains dermal papilla identity and hair morphogenesis via a Noggin-Shh regulatory loop

During hair follicle morphogenesis, dermal papillae (DPs) function as mesenchymal signaling centers that cross-talk with overlying epithelium to regulate morphogenesis. While the DP regulates hair follicle formation, relatively little is known about the molecular basis of DP formation. The morphogen Sonic hedgehog (Shh) is known for regulating hair follicle epithelial growth, with excessive signaling resulting in basal cell carcinomas. Here, we investigate how dermal-specific Shh signaling contributes to DP formation and hair growth. Using a Cre-lox genetic model and RNAi in hair follicle reconstitution assays, we demonstrate that dermal Smoothened (Smo) loss of function results in the loss of the DP precursor, the dermal condensate, and a stage 2 hair follicle arrest phenotype reminiscent of Shh(-/-) skin. Surprisingly, dermal Smo does not regulate cell survival or epithelial proliferation. Rather, molecular screening and immunostaining studies reveal that dermal Shh signaling controls the expression of a subset of DP-specific signature genes. Using a hairpin/cDNA lentiviral system, we show that overexpression of the Shh-dependent gene Noggin, but not Sox2 or Sox18, can partially rescue the dermal Smo knockdown hair follicle phenotype by increasing the expression of epithelial Shh. Our findings suggest that dermal Shh signaling regulates specific DP signatures to maintain DP maturation while maintaining a reciprocal Shh-Noggin signaling loop to drive hair follicle morphogenesis.     

Comparative characterization of hair follicle dermal stem cells and bone marrow mesenchymal stem cells

We compared the growth and differentiation characteristics of hair follicle-derived dermal stem cells with bone marrow mesenchymal stem cells (MSCs). Follicular dermal cells were isolated from whisker hairs of Wistar rats and bone marrow MSCs were isolated from femora of the same animals. The adherent hair follicle dermal cells showed a fibroblastic morphology in serum-containing culture medium, were CD44(+), CD73(+), CD90(+), and CD34(), and had a population doubling time of 27 h. MSCs isolated from the bone marrow showed a similar morphology and population doubling time and expressed the same cell-surface markers. Following exposure to appropriate induction stimuli, both cell populations had the capacity to differentiate into various mesenchymal lineages, such as osteoblasts, adipocytes, chondrocytes, and myocytes and expressed neuroprogenitor cell markers. The rate and extent of differentiation were remarkably similar for both hair follicleand bone marrow-derived cells, whereas interfollicular dermal cells failed to differentiate. We identified telomerase activity in follicle dermal stem cells and marrow MSCs and demonstrated that they were capable of clonal expansion. In ex vivo analyses, we identified the presence of putative dermal stem cells in the dermal sheath and dermal papillae of the hair follicle. Consequently, the hair follicle may represent a suitable, accessible source for MSCs.     

Control of hair follicle cell fate by underlying mesenchyme through a CSL–Wnt5a–FoxN1 regulatory axis

Epithelial–mesenchymal interactions are key to skin morphogenesis and homeostasis. We report that maintenance of the hair follicle keratinocyte cell fate is defective in mice with mesenchymal deletion of the CSL/RBP-Jκ gene, the effector of “canonical” Notch signaling. Hair follicle reconstitution assays demonstrate that this can be attributed to an intrinsic defect of dermal papilla cells. Similar consequences on hair follicle differentiation result from deletion of Wnt5a, a specific dermal papilla signature gene that we found to be under direct Notch/CSL control in these cells. Functional rescue experiments establish Wnt5a as an essential downstream mediator of Notch–CSL signaling, impinging on expression in the keratinocyte compartment of FoxN1, a gene with a key hair follicle regulatory function. Thus, Notch/CSL signaling plays a unique function in control of hair follicle differentiation by the underlying mesenchyme, with Wnt5a signaling and FoxN1 as mediators.