分段毛囊上皮细胞增生能力比较

目的 阐明分段毛囊上皮细胞之间的生物学特性和毛囊干细胞的定位。方法 将毛囊上皮细胞分成上段,下段的球部,分别培养于人成纤维细胞滋养层上,然后传代,或在纤维细胞胶原凝胶上行器官型培养。结果 上段细胞传代次数最多,形成的克隆数最多,细胞生长活力最强;而球部细胞的生长能力阳差,下段细胞有少数能形成进行性生长克隆。     

人毛囊干细胞的定位及增殖特性

目的 探讨人毛囊干细胞定位和增殖特性.方法 取人枕部头皮毛囊进行角蛋白19(K19)免疫组化染色,并测量阳性段距毛囊下端的距离;将包含毛球部上方阳性段的毛囊上皮组织分别置于含有和不含有间质细胞的凝胶表面气液界面培养,当出现增殖灶时,测量其距毛囊下端的距离,分析K19阳性部位与培养出现的增殖灶之间的关系,并通过透射电镜观察增殖灶的超微结构.结果 人枕部头皮毛囊有两个K19阳性区域,即隆突部和毛球部上方一段外根鞘,毛囊下段上皮组织只在含有间质细胞的凝胶表面培养时毛球部上方出现一增殖灶,统计学分析支持毛球部上方的K19阳性部位和毛囊下段上皮组织培养出现的增殖灶为同一部位.透射电镜显示增殖灶含有幼稚细胞、成熟角质形成细胞和凋亡细胞.结论 人头皮毛囊可能有两个干细胞库,即隆突部和球部上方外根鞘的一个局限区域,干细胞的增殖需要间质细胞(如毛乳头细胞)的存在,增殖的干细胞可能向形成新的干细胞、成熟角质形成细胞和凋亡细胞三个方向演变.     

A model system to analyse the ability of human keratinocytes to form hair follicles

Earlier studies showed that dermal cells lose trichogenic capacity with passage, but studies on the effect of keratinocyte passage on human hair follicle neogenesis and graft quality have been hampered by the lack of a suitable model system. We recently documented human hair follicle neogenesis in grafted dermal‐epidermal composites, and in the present study, we determined the effects of keratinocyte passage on hair follicle neogenesis. Dermal equivalents were made with cultured human dermal papilla cells and were overlaid with either primary or passaged human keratinocytes to form dermal‐epidermal composites; these were then grafted onto immunodeficient mice. Superior hair follicle neogenesis was observed using early keratinocyte cultures. Characteristics such as formation of hair shafts and sebaceous glands, presence of hair follicles with features of anagen or telogen follicles, and reproducible hair and skin function parameters make this model a tool to study human hair follicle neogenesis and development.     

Independent inheritance of genes regulating two subpopulations of mouse clonogenic keratinocyte stem cells

Mouse keratinocyte stem cells originate from the bulge of hair follicle, and, according to definition, possess a clonogenic activity in vitro. We have investigated seven inbred (C57BL/6, C3H, DBA/2, BALB/c, FVB) and outbred (SENCAR, CD-1) mouse strains and found that three genetically distinct subsets of mouse strains differ significantly in the frequency of clonogenic activity in vitro. The analysis of keratinocyte colonies in two reciprocal backcross [C57BL/6 x (BALB/c x C57BL/6); BALB/c x (BALB/c x C57BL/6)] and intercross [(BALB/c x C57BL/ 6)F2] of BALB/c and C57BL/6 mice allowed us to identify two subpopulations of clonogenic keratinocytes able to produce small (less than 2 mm2) and large (more than 2 mm2) colonies. We conducted linkage analysis and found that small colonies associated with mouse chromosomes 1, 6, 7, 8, and 9; but large colonies--with the chromosome 4. We defined locus on the chromosome 9 that associated with small colonies as keratinocyte stem cell locus 1 (Ksc1), and locus on the mouse chromosome 4 associated with large colonies-keratinocyte stem cell locus 2 (Ksc2). Ksc1 and loci on chromosomes 6 and 7 are close if not equal to loci associated with sensitivity to skin carcinogenesis. We conclude that two subpopulations of stem cells able to produce small and large colonies regulated by different genes and genes regulating small colonies might be responsible for sensitivity to skin carcinogenesis.     

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.     

毛乳头细胞体外诱导毛囊上皮细胞分化的实验研究

目的:探讨间质细胞对毛囊上皮细胞分化的调节作用,研究毛囊上皮细胞的分化特性。方法:分别用团块状的毛乳头细胞、皮肤成纤维细胞制成间质细胞胶原凝胶,表面接种毛囊上皮细胞,进行气-液界面培养。结果:毛囊上皮细胞有向毛乳头细胞移动集结的趋势;团块状的毛乳头细胞诱导毛囊上皮细胞形成球形结构;皮肤成纤维细胞诱导毛囊上皮细胞形成表皮样层化结构。结论:(1)毛囊上皮细胞具有双向分化特性,它既能分化形成毛囊,也能分化形成表皮结构;(2)毛乳头细胞对毛囊上皮细胞有趋化作用;(3)间质细胞的种类及分布在毛囊上皮细胞分化的调节中起着重要的作用。     

Differential expression of the 4F2 activation antigen on human follicular epithelium in hair cycle

The expression of the 4F2 activation molecule has been studied on keratinocytes of human skin and hair follicle using immunoperoxidase staining with three different anti-4F2 monoclonal antibodies. Membranes of basal layer keratinocytes of the skin uniformly expressed this antigen, whereas a differential expression of this antigen was located in specific areas of the hair follicle. Follicles in the complete anagen phase displayed a strong 4F2 positive staining at the matrix and the outer root sheath cells. This positive staining gradually decreased along the proliferation zone, and became negative at the migration zone. Positivity was recovered in follicular cells at the duct of the sebaceous gland and was maintained in the upper outer root sheath where those cells fuse with the keratinocyte basal monolayer. Changes were also detected on different phases of the hair cycle. Follicles in the catagen-telogen phase expressed a very low number of positive cells in the matrix. Positive labeling progressively increased when the follicle was at the initial anagen stage, reaching a complete staining pattern in hair at the anagen phase. These results suggest that the expression of this activation antigen on hair keratinocytes may be related to the proliferation, active metabolism, and/or activation states of these cell types.     

Three-dimensional culture of plucked human hair follicles inside the collagen gel matrix

Plucked human hair follicles were cultured in collagen gel matrix. Epithelial cells, possibly outer root sheath keratinocytes, appeared from the outer root sheath 4 to 5 days after culturing and continually grew into the gel to form spike-like structures for next 3 weeks. The number and size of the spikes differed in each follicle. Autoradiographically, many DNA-synthesizing cells were seen at the outer cell layer in the enlarged outer root sheath and at the edges in the newly formed spike-like structures. The culture method described here appears to be suitable to study the three-dimensional growth, morphogenesis and differentiation of the outer root sheath cells in vitro.     

Differential epithelial outgrowth of plucked and microdissected human hair follicles in explant culture

In the present study we prepared explant cultures of plucked total hair follicles and of fragments microdissected from the following regions: B1 (bulb region), B2 (intermediate region), B3-1 (lower central outer root sheath, ORS), B3-2 (upper central ORS) and B4 (area of fracture). The growth capacities, the start of epithelial outgrowth, the stages of differentiation and apoptosis were studied immunohistochemically in early and late explant cultures using a battery of antibodies against cytokeratins, growth factor receptors and cell adhesion molecules and proliferation markers. Whole plucked hair follicles showed epithelial outgrowths exclusively in the upper central ORS (B3-2) starting early, mostly by day 3. In microdissected fragments, in contrast, outgrowths were more widespread, mostly in B3-2 and B3-1, and started early, but were also of late onset in some cases of B2 and B4. Epithelial outgrowths exhibited a basal layer of small cuboidal cells in a low stage of differentiation and one to two suprabasal layers of large prickle-like cells expressing late differentiation markers. The former expressed the receptor of nerve growth factor (NGF) heterogeneously whereas epidermal growth factor (EGF) receptor was not detectable. This is similar to ORS cells of this area in vivo. The proliferative activity of the outgrowths was always restricted to peripheral cells. Thus no essential differences in differentiation of outgrowing cells were detected. These results suggest that keratinocytes with the highest growth capacities in plucked human hair follicles are localized in the lower central ORS (corresponding to B3-2) and some with a lower capacity in the upper central ORS (corresponding to B3-1) as established after microdissection. This is in agreement with the bulge activation theory. NGF may also play a role in hair growth.     

培养毛乳头细胞生物学特性和毛囊重建的研究

目的 观察毛乳头细胞在体内外诱导毛囊再生和支持毛囊生长情况。方法 采用免疫组化、原位杂交、毛囊器官型培养和裸鼠移植技术，观察不同传代培养的毛乳头细胞碱性成纤维细胞生长因子，内皮素和干细胞因子的表达变化情况。结果 低传代培养的毛乳头细胞的内皮素和干细胞因子表达较强，传代6代后减弱。用低传代培养的毛乳头细胞与毛囊上皮细胞在毛囊器官型培养模型中可见毛囊样结构形成，移植到裸鼠后可见较为完整的毛囊形成。用低传代培养的毛乳头细胞与毛囊上皮细胞按一定比例混合后直接注射到裸鼠皮下，也可见毛囊样结构形成。发现毛乳头细胞诱导毛囊再生的能力与其表达内皮素和干细胞因子的强弱相关。结论 低传代培养的毛乳头细胞在体内外均具有诱导毛囊再生的能力，并且与其表达内皮素和干细胞因子的强弱相关。     

Label-retaining cells (presumptive stem cells) of mice vibrissae do not express gap junction protein connexin 43

We investigated whether connexin 43, a gap junction protein present in human epidermis and mouse hair follicle, can serve as a negative marker for keratinocyte stem cells. Experiments carried out in mouse pelage and vibrissae hair follicles demonstrated that most of the slowly cycling cells, detected as label-retaining cells, do not express connexin 43. In humans, cells with immunohistochemically undetectable levels of connexin 43 are found in the epidermal basal layer of neonatal foreskin, and in the follicular bulge region. About 10% of the basal keratinocytes are connexin 43 negative, as determined by flow cytometry. These cells are uniformly small and low in granularity suggesting that presumptive keratinocyte stem cells can be identified and separated based on connexin 43 expression.     

永生化人毛乳头细胞系体内诱导毛囊的形成

目的研究永生化人毛乳头细胞(DPC)系DPC-hTERT在体内诱导毛囊形成的能力。方法将第30代DPC-hTERT与刚分离的毛囊上皮细胞混合后直接注射到裸鼠背部皮下,观察毛囊形成情况。结果 DPC-hTERT与刚分离的毛囊上皮细胞混合后注射到裸鼠背部皮下后,可见毛囊样结构形成,且此结构表达毛囊特有的角蛋白。结论 DPC-hTERT具有正常DPC的功能,具备在体内诱导毛囊样结构的能力。     

Human hair follicle bulge cells are biochemically distinct and possess an epithelial stem cell phenotype

Stem cells are vital for the homeostasis of self-renewing tissues and their manipulation may have wide ranging applications, including gene therapy, wound repair, and tissue transplantation. Although rodent hair follicle stem cells have been localized to the follicle bulge, the location of human hair follicle stem cells is less clear, and their characterization has been hampered by a lack of cellular markers for the bulge area. We demonstrate that the C8/144B monoclonal antibody, originally raised against a CD8 peptide sequence, immunostains the human hair follicle bulge. We show that this antibody recognizes cytokeratin 15 (K15) in keratinocytes, and that K15-positive bulge cells possess a stem cell phenotype characterized by their slowly cycling nature, proliferation at the onset of new hair follicle growth, and high level of beta1 integrin expression. These results localize human hair follicle stem cells to the bulge and suggest that K15 is preferentially expressed in epithelial stem cells.     

Nail stem cells

Our knowledge on stem cells of the hair follicle has increased exponentially after the bulge was characterized as the stem cell niche two decades ago. In contrast, little is known about stem cells in the nail unit. Whereas hair follicles are plentiful and easy to access, the human body has only twenty nails and they are rarely biopsied. Therefore, examining fetal material offers unique advantages. In the following mini‐review, our current knowledge on nail stem cells is summarized and analogies to the hair follicle stem cells are drawn.     

Syndecan-1 is strongly expressed in the anagen hair follicle outer root sheath and in the dermal papilla but expression diminishes with involution of the hair follicle

Syndecan-1 is the prototypic member of a family of heparan sulfate-bearing cell surface proteoglycans that function in adhesion, cell-extracellular matrix interactions, migration, and proliferation. During embryogenesis, syndecan-1 expression in the epithelium is downregulated when the epithelium gives rise to motile mesenchymal cells, whereas mesenchymal syndecan-1 expression is upregulated during organ formation. In aggressive basal cell carcinomas, syndecan-1 expression is evident in the stroma. Some neoplastic cells induce stroma to meet needs for growth, and it may be the mesenchymal cells that produce and shed syndecan-1 into the stroma. The physiologic mechanism by which the hair follicle undergoes its cyclic process of involution and formation of a new active hair follicle is not well understood. Sixty scalp biopsies and a large scalp resection were evaluated for syndecan-1 expression within hair follicles in the growing (anagen), involuting (catagen), and resting (telogen) phases. Strong syndecan-1 immunoreactivity was evident in the outer root sheath (ORS) of the anagen hair follicle, but this expression diminished in intensity with the involution and resting stages in the hair follicle cycle. The diminution of syndecan-1 immunoreactivity in the ORS of involuting and resting hair follicles may be a result of terminal keratinocyte differentiation. Syndecan-1 was also present in the dermal papilla of the anagen hair follicle, where it may promote growth factor-mediated cell signaling that induces and maintains growth of the hair shaft and the inner root sheath.     

Changing patterns of localization of putative stem cells in developing human hair follicles

In rodents, the hair follicle stem cells lie in a well-defined bulge in the outer root sheath; however, the bulge as a stem cell site of human hair follicle epithelium is still controversial. Epidermal stem cells are thought to express high levels of beta1 integrin and low levels of E-cadherin and beta- and gamma-catenin. In order to clarify the ontogenic distribution of possible stem cells during hair follicle development, the expression patterns of beta1 integrin subunits, E-cadherin, and beta- and gamma-catenins in the skin samples from human fetuses of a series of estimated gestational ages (EGA) were examined. beta1 integrin-rich, E-cadherin-, and beta- and gamma-catenin-poor cells, possible stem cells, were localized to the entire hair germ (65-84 d EGA) and later to the outermost cells of hair peg (85-104 d EGA). In the bulbous hair peg (105-135 d EGA) and in the differentiated lanugo hair follicle (>135 d EGA), they were settled in the bulge and the outermost layer of the outer root sheath. This sequential localization was similar to that of cells rich in epidermal growth factor receptor expression and positive with keratin 19, a putative marker of epidermal stem cells. In addition, these beta1 integrin-rich, E-cadherin-, and beta- and gamma-catenin-poor cells showed similar, undifferentiated morphologic features by electron microscopy. This information of ontogenic localization of possible hair follicle stem cells contributes to the further understanding of mechanisms of human hair follicle morphogenesis and supports the idea that the human fetal hair follicle bulge is a site of stem cells for follicular epithelium.     

Expression of the hair stem cell-specific marker nestin in epidermal and follicular tumors

Nestin, a marker of neural stem cells, is expressed in the stem cells of the mouse hair follicle. The nestin-expressing hair follicle stem cells give rise to the outer-root sheath. Nestin-expressing hair follicle stem cells that are negative for the keratinocyte marker keratin 15 (K15) can differentiate into neurons, glia, keratinocytes, smooth muscle cells, and melanocytes in vitro. Recent studies suggest that the epithelial stem cells are important in tumorigenesis. In this study, we immunohistochemically examined the expression of three hair follicle stem cell and progenitor cell markers, nestin, K15, and CD34, in normal human epidermis and hair follicles and in epidermal and follicular tumors, trichilemmoma, basal cell carcinoma (BCC), and squamous cell carcinoma (SCC). In normal human skin, the cells in the epidermal basal layer were positive for K15 and negative for nestin and CD34. The hair follicle cells below the sebaceous glands were also positive for nestin and K15 and negative for CD34. The outer-root sheath cells under this area could be divided into three parts: an upper part of the outer-root sheath cells that was partially positive for nestin and positive for K15 and negative for CD34; a middle part that was CD34-positive and K15-negative; and a lower part that was positive for K15 and negative for CD34. In the tumor tissues, nestin immunoreactivity was observed in trichilemmoma but not in BCC. Also, immunoreactivity for K15 was strong in BCC and weak in trichilemmoma, and SCC was negative for nestin and partially positive for K15. No CD34 immunoreactivity was observed in any of the cases. These results suggested that trichilemmoma originates in the nestin-positive/K15-positive/CD34-negative outer-root sheath cells below sebaceous glands, BCC tumor cells from the more mature nestin-negative/K15-positive/CD34-negative outer-root sheath cells, and SCC from the nestin-negative/K15-positive/CD34-negative keratinocytes of the basal cell layer in the epidermis.     

Epithelial cells in the hair follicle bulge do not contribute to epidermal regeneration after glucocorticoid-induced cutaneous atrophy

One of the major adverse effects of glucocorticoid therapy is cutaneous atrophy, often followed by the development of resistance to steroids. It is accepted that epithelial stem cells (SCs) located in the hair follicle bulge divide during times of epidermal proliferative need. We determined whether follicular epithelial SCs and their transit amplifying progeny were stimulated to proliferate in response to the chronic application of glucocorticoid fluocinolone acetonide (FA). After first two applications of FA, keratinocyte proliferation in the interfollicular epidermis (IFE) and hair follicles was minimal and resulted in significant epidermal hypoplasia. We observed that a 50% depletion of the interfollicular keratinocyte population triggered a proliferative response. Unexpectedly, less than 2% of the proliferating keratinocytes were located in the bulge region of the hair follicle, whereas 82% were in IFE. It is known that cell desensitization to glucocorticoids is mediated via temporary decrease of glucocorticoid receptor (GR) expression. We found that GR expression was significantly decreased in IFE keratinocytes after each FA treatment. In contrast, many bulge keratinocytes retained GR in the nucleus. Our results indicate that bulge keratinocytes, including follicular SCs, are more sensitive to the antiproliferative effect of glucocorticoids than basal keratinocytes, possibly due to the incomplete process of desensitization.     

Epidermale Stammzellen

Current understanding of the biology of epidermal stem cells opens a totally new perspective in the function of the epidermis and adjacent epithelial structures. A number of pathogenetic as well as clinical‐therapeutic approaches against a variety of dermatoses may become possible with knowledge about keratinocyte proliferation, differentiation and regeneration. The reservoir of epidermal stem cells is located in the interfollicular epidermis, the hair follicle area and the germinal hair follicle matrix. Endogenous stem cell clones exist here, giving rise to transient amplifying cells and postmitotic cells. The stem cell clones are organized in clusters and display high expression of adhesion proteins, which guarantee their stability in a specific environment consisting of different cell types and extracellular substrates in the stratum basale. Differentiation is determined by a specific cascade of chemical signals from the stem cell environment and from the genetic program of the cell. The clinical relevance of stem cells lies primarily in their therapeutic potential with reconstruction of epithelia by reimplantation of autologous stem cells or gene therapeutic applications such as targeted transfection. However, the benefit‐to‐risk ratio cannot yet be accurately estimated.     

Epidermal and hair follicle progenitor cells express melanoma-associated chondroitin sulfate proteoglycan core protein

Basal keratinocytes in the epidermis and hair follicle are biologically heterogeneous but must include a stable subpopulation of epidermal stem cells. In animal models these can be identified by their retention of radioactive label due to their slow cycle (label-retaining cells) but human studies largely depend on in vitro characterization of colony forming efficiency and clonogenicity. Differential integrin expression has been used to detect cells of increased proliferative potential but further stem cell markers are urgently required for in vivo and in vitro characterization. Using LHM2, a monoclonal antibody reacting with a high molecular weight melanoma-associated proteoglycan core protein, a subset of basal keratinocytes in both the interfollicular epidermis and the hair follicle has been identified. Coexpression of melanoma-associated chondroitin sulfate proteoglycan with keratins 15 and 19 as well as beta 1 and alpha 6 integrins has been examined in adult and fetal human skin from hair bearing, nonhair bearing, and palmoplantar regions. Although melanoma-associated chondroitin sulfate proteoglycan coexpression with a subset of beta 1 integrin bright basal keratinocytes within the epidermis suggests that melanoma-associated chondroitin sulfate proteoglycan colocalizes with epidermal stem cells, melanoma-associated chondroitin sulfate proteoglycan expression within the hair follicle was more complex and multiple subpopulations of basal outer root sheath keratinocytes are described. These data suggest that epithelial compartmentalization of the outer root sheath is more complex than interfollicular epidermis and further supports the hypothesis that more than one hair follicle stem cell compartment may exist.