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.     

Side population keratinocytes resembling bone marrow side population stem cells are distinct from label-retaining keratinocyte stem cells

Very primitive hematopoietic stem cells have been identified as side population cells based on their ability to efflux a fluorescent vital dye, Hoechst 33342. In this study we show that keratinocytes with the same side population phenotype are also present in the human epidermis. Although side population keratinocytes have the same dye-effluxing phenotype as bone marrow side population cells and can be blocked by verapamil, they do not express increased levels of the ABCG2 transporter that is believed to be responsible for the bone marrow side population phenotype. Because bone marrow side population cells have stem cell characteristics, we sought to determine if side population keratinocytes represent a keratinocyte stem cell population by comparing side population keratinocytes with a traditional keratinocyte stem cell candidate, label-retaining keratinocytes. Flow cytometric analyses demonstrated that side population keratinocytes have a different cell surface phenotype (low beta1 integrin and low alpha6 integrin expression) than label-retaining keratinocytes and represent a unique population of keratinocytes distinctly different from the traditional keratinocyte stem cell candidate. Future in vivo studies will be required to analyze the function of side population keratinocytes in epidermal homeostasis and to determine if side population keratinocytes have characteristics of keratinocyte stem cells.     

Cellular senescence induced loss of stem cell proportion in the skin in vitro

BACKGROUND: It is known that cellular senescence could affect culture results. A previous study on K19 found that the loss of stem cell proportion is the reason for difficulties experienced when culturing aged keratinocytes. But the situation is unclear, because K19 is not generally accepted as an epidermal stem cell marker. OBJECTIVE: The aim of this study was to investigate the effects of cellular senescence caused by chronological aging or by repeated subcultures. METHODS: The effects of cellular senescence were investigated using monolayer cultures of keratinocytes and reconstructed epidermis. We prepared keratinocytes from donors of different ages and by repeated subcultures. Flow cytometric analysis was performed using alpha6 integrin and CD71 antibodies, and candidate keratinocyte stem cell proportions were separated according to reactivities to these antibodies. Living skin equivalents (LSEs) were reconstructed using keratinocytes from child, adult and elderly donors. RESULTS: Flow cytometric analysis showed a decrease in the candidate stem cell proportion in an age- or culture passage-dependent manner. LSE experiments showed that a reconstructed epidermis using child's keratinocytes was well formed compared to epidermis reconstructed using an elderly donor's keratinocytes. Different expression of proliferation markers was also observed according to donor age. CONCLUSION: Our results showed that cellular senescence by chronological aging or repeated sub-culture induced the loss of candidate stem cell proportion in keratinocyte cultures. This seems to be the reason why it is difficult to culture keratinocytes from the elderly or by repeatedly culturing keratinocytes in vitro.     

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.     

Fibronectin expression determines skin cell motile behavior

Mouse keratinocytes migrate significantly slower than their human counterparts in vitro on uncoated surfaces. We tested the hypothesis that this is a consequence of differences in the extracellular matrix (ECM) that cells deposit. In support of this, human keratinocyte motility was markedly reduced when plated onto the ECM of mouse skin cells, whereas the latter cells migrated faster when plated onto human keratinocyte ECM. The ECM of mouse and human keratinocytes contained similar levels of the α3 laminin subunit of laminin-332. However, mouse skin cells expressed significantly more fibronectin (FN) than human cells. To assess whether FN is a motility regulator, we used small interfering RNA (siRNA) to reduce the expression of FN in mouse keratinocytes. The treated mouse keratinocytes moved significantly more rapidly than wild-type mouse skin cells. Moreover, the FN-depleted mouse cell ECM supported increased migration of both mouse and human keratinocytes. Furthermore, the motility of human keratinocytes was slowed when plated onto FN-coated substrates or human keratinocyte ECM supplemented with FN in a dose-dependent manner. Consistent with these findings, the ECM of α3 integrin-null keratinocytes, which also migrated faster than wild-type cells, was FN deficient. Our results provide evidence that FN is a brake to skin cell migration supported by laminin-332-rich matrices.     

miRNA miR‐17‐92 cluster is differentially regulated in the imiqumod‐treated skin but is not required for imiqumod‐induced psoriasis‐like dermatitis in mice

MicroRNAs (miRNAs) play very important roles in the control of immune cell and keratinocyte development and function and are implicated in skin inflammatory diseases, including psoriasis. miRNA miR‐17‐92 was reported to promote the differentiation of Th1 and Th1 cells and to regulate cell proliferation and apoptosis. Here we showed that imiquimod (IMQ) differentially regulates the expression of miR‐17‐92 cluster in the mouse skin, upregulating miR‐17 and miR‐19 families and downregulating miR‐92. To investigate whether miR‐17‐92 cluster is functionally involved in the psoriasis, we have generated three mutant mice with specific deletion or overexpression of miR‐17‐92 cluster in keratinocytes, or with deletion of miR‐17‐92 cluster in T cells. Interestingly, deletion or overexpression of miR‐17‐92 cluster in keratinocytes, or deletion of miR‐17‐92 in T cells did not significantly affect IMQ‐induced psoriasis‐like dermatitis development in the mutant mice compared with wild‐type littermates. Thus, miRNA miR‐17‐92 cluster may not be a key factor regulating imiqumod‐induced psoriasis‐like dermatitis.     

Complexity of VEGF responses in skin carcinogenesis revealed through ex vivo assays based on a VEGF-A null mouse keratinocyte cell line

Vascular endothelial growth factor (VEGF-A) is a critical player in cutaneous angiogenesis. However, the relative contribution of VEGF-A from different sources including epithelial and mesenchymal cells has not been fully characterized during skin repair and tumorigenesis. Moreover, the actual involvement of other vascular-specific acting molecules has remained elusive in part due to the masking and/or overlapping effects of VEGF-A. To shed light on these uncertainties we generated and characterized a clonal VEGF-null mouse keratinocyte cell line, through in vitro adenoviral gene transfer of Cre recombinase to VEGF-LoxP primary keratinocytes followed by repeated cell passaging under controlled conditions and cloning. In vitro and in vivo assays demonstrated that VEGF-null keratinocytes were nontumorigenic and expressed normal differentiation markers after calcium switch. Hras-induced tumorigenesis of immortalized VEGF-null keratinocytes upon subcutaneous injection was markedly reduced but not fully suppressed. However, the metastatic ability of Hras-transformed VEGF-null keratinocytes was abolished. These ex vivo approaches suggest the existence of VEGF-dependent and independent angiogenic stimuli in skin carcinogenesis. The VEGF-null mouse keratinocyte cell line arises as an important tool to assess the actual contribution of keratinocyte-derived VEGF with respect to other angiogenic factors in skin homeostasis and malignancy.     

Production of retroviral vectors in primary human keratinocytes after DNA-mediated gene transfer leads to prolonged gene expression

Prolonged stability and controlled expression of gene constructs transferred directly to human skin improve the possibility of using this tissue in somatic gene therapy. We aim to develop a simple transfection method resulting in retroviral mediated gene transfer to keratinocyte stem cells in situ. We here show that after DNA-mediated gene transfer into primary human keratinocytes it is possible to achieve production of retroviral vectors, leading to the transduction of co-cultured keratinocytes and prolonged reporter gene expression. The method is a first step in a strategy to generate retroviral producer cells in situ in the skin furthermore the method can be used for rapid analysis of the possible effects of transgenes in cultured human keratinocytes without preparatory retroviral vector production in packaging cell lines.     

Mutant loricrin is not crosslinked into the cornified cell envelope but is translocated into the nucleus in loricrin keratoderma

Loricrin is a major constituent of the epidermal cornified cell envelope. We have recently identified heterozygous loricrin gene mutations in two dominantly inherited skin diseases, the ichthyotic variant of Vohwinkel syndrome and progressive symmetric erythrokeratoderma, collectively termed loricrin keratoderma. In order to see whether the mutant loricrin molecules predicted by DNA sequencing are expressed in vivo and to define their pathologic effects, we raised antibodies against synthetic peptides corresponding to the mutated sequences of loricrin. Immunoblotting of horny cell extracts from loricrin keratoderma patients showed specific bands for mutant loricrin. Immunohistochemistry of loricrin keratoderma skin biopsies showed positive immunoreactivity to the mutant loricrin antibodies in the nuclei of differentiated epidermal keratinocytes. The immunostaining was localized to the nucleoli of the lower granular cell layer. As keratinocyte differentiation progressed the immunoreactivity moved gradually into the nucleoplasm leaving nucleoli mostly nonimmunoreactive. No substantial staining was observed along the cornified cell envelope. This study confirmed that mutant loricrin was expressed in the loricrin keratoderma skin. Mutant loricrin, as a dominant negative disrupter, is not likely to affect cornified cell envelope crosslinking directly, but seems to interfere with nuclear/nucleolar functions of differentiating keratinocytes. In addition, detection of the mutant loricrin in scraped horny layer could provide a simple noninvasive screening test for loricrin keratoderma. J Invest Dermatol 115:1088-1094 2000     

Enhancement of Keratinocyte Differentiation by Rose Absolute Oil

Background

Through differentiation processes, keratinocytes provide a physical barrier to our bodies and control skin features such as moisturization, wrinkles and pigmentation. Keratinocyte differentiation is disturbed in several skin diseases such as psoriasis and atopic dermatitis.

Objective

The aim of this study is to evaluate the keratinocyte differentiation-enhancing effect of rose absolute oil (RAO).

Methods

Primary cultured human normal keratinocytes were treated with RAO, and differentiation then checked by the expression of marker genes.

Results

RAO did not induce cytotoxicity on cultured keratinocytes at a dose of 10µM. The level of involucrin, an early marker for keratinocyte differentiation, was significantly increased by RAO. Concomitantly, RAO increased involucrin promoter activity, indicating that RAO increased involucrin gene expression at the mRNA level. Furthermore, RAO increased the level of filaggrin in cultured keratinocytes, and in the granular layer of mouse skin. In line with these results, RAO decreased the proliferation of keratinocytes cultured in vitro. When RAO was applied topically on the tape-stripped mouse skins, it accelerated the recovery of disturbed barrier function.

Conclusion

These results suggest that RAO may be applicable for the control of skin texture and keratinocyte differentiation-related skin diseases.     

Insulin-like growth factor binding protein-3 (IGFBP-3) localizes to and modulates proliferative epidermal keratinocytes in vivo

BACKGROUND: The colocalization of insulin-like growth factor binding protein-3 (IGFBP-3) and IGF-I receptor (IGF-IR) in the basal/germinative layer of the epidermis suggests a key role in modulating epidermal homeostasis. OBJECTIVES: We aimed to clarify both the specific cellular localization and the effect of excess epidermal IGFBP-3 on keratinocyte proliferation. METHODS: (i) Total RNA was isolated from fluorescence-activated cell sorted basal human keratinocyte subtypes [keratinocyte stem cells, transit amplifying keratinocytes (TA), postmitotic differentiating keratinocytes (PMD)], and real-time polymerase chain reaction analysis was used to determine the abundance of IGFBP-3 and IGF-IR mRNAs. (ii) An IGFBP-3 transgenic mouse model was then used to assess the effect of excess epidermal IGFBP-3 on keratinocyte proliferation. Excess epidermal IGFBP-3 mRNA and protein was determined by in situ hybridization and immunohistochemistry, respectively. RESULTS: (i) The highest levels of IGFBP-3 mRNA were detected in TA keratinocytes, in contrast to IGF-IR mRNA levels which were highest in PMD keratinocytes. (ii) Elevated human IGFBP-3 mRNA and protein was confirmed in the epidermis of skin derived from transgenic mice. Excess IGFBP-3 reduced the relative percentage of proliferative keratinocytes (Ki67 positive) irrespective of skin location (belly, back and tail). Thus, in the epidermis, IGFBP-3 mRNA is highly expressed by proliferative keratinocytes (TA) and overexpression of IGFBP-3 inhibits keratinocyte proliferation. CONCLUSIONS: We conclude that in vivo IGFBP-3 ensures epidermal homeostasis via downregulation of keratinocyte proliferation, and thus modulates the early stages of keratinocyte differentiation.     

Exploration of the functional hierarchy of the basal layer of human epidermis at the single‐cell level using parallel clonal microcultures of keratinocytes

Please cite this paper as: Exploration of the functional hierarchy of the basal layer of human epidermis at the single‐cell level using parallel clonal microcultures of keratinocytes. Experimental Dermatology 2010. Abstract: The basal layer of human epidermis contains both stem cells and keratinocyte progenitors. Because of this cellular heterogeneity, the development of methods suitable for investigations at a clonal level is dramatically needed. Here, we describe a new method that allows multi‐parallel clonal cultures of basal keratinocytes. Immediately after extraction from tissue samples, cells are sorted by flow cytometry based on their high integrin‐α6 expression and plated individually in microculture wells. This automated cell deposition process enables large‐scale characterization of primary clonogenic capacities. The resulting clonal growth profile provided a precise assessment of basal keratinocyte hierarchy, as the size distribution of 14‐day‐old clones ranged from abortive to highly proliferative clones containing 1.7 × 10⁵ keratinocytes (17.4 cell doublings). Importantly, these 14‐day‐old primary clones could be used to generate three‐dimensional reconstructed epidermis with the progeny of a single cell. In long‐term cultures, a fraction of highly proliferative clones could sustain extensive expansion of >100 population doublings over 14 weeks and exhibited long‐term epidermis reconstruction potency, thus fulfilling candidate stem cell functional criteria. In summary, parallel clonal microcultures provide a relevant model for single‐cell studies on interfollicular keratinocytes, which could be also used in other epithelial models, including hair follicle and cornea. The data obtained using this system support the hierarchical model of basal keratinocyte organization in human interfollicular epidermis.     

An extended epidermal response heals cutaneous wounds in the absence of a hair follicle stem cell contribution

Hair follicles have been observed to provide a major cellular contribution to epidermal healing, with emigration of stem-derived cells from the follicles aiding in wound reepithelialization. However, the functional requirements for this hair follicle input are unknown. Here we have characterized the keratinocyte stem cell status of mutant mice that lack all hair follicle development on their tail, and analyzed the consequent alterations in epidermal wound healing rate and mechanisms. In analyzing stem cell behavior in embryonic skin we found that clonogenic keratinocytes are relatively frequent in the ectoderm prior to hair follicle formation. However, their frequency in the interfollicular epidermis drops sharply by birth, at which time the majority of stem cells are present within the hair follicles. We find that in the absence of hair follicles cutaneous wounds heal with an acute delay in reepithelialization. This delay is followed by expansion of the region of activated epidermis, beyond that seen in normal haired skin, followed by appropriate wound closure. JID Journal Club article: for questions, answers, and open discussion about this article please go to http://network.nature.com/group/jidclub.     

Upregulation of N-acetylglucosaminyltransferase-V by heparin-binding EGF-like growth factor induces keratinocyte proliferation and epidermal hyperplasia

Oligosaccharide modification by N-acetylglucosaminyltransferase-V (GnT-V), a glycosyltransferase encoded by the Mgat5 gene that catalyses the formation of β1,6 GlcNAc (N-acetylglucosamine) branches on N-glycans, is thought to be associated with cancer growth and metastasis. Overexpression of GnT-V in cancer cells enhances the signalling of growth factors such as epidermal growth factor (EGF) and transforming growth factor-β by increasing galectin-3 binding to polylactosamine structures on receptor N-glycans. We previously demonstrated that transgenic mice overexpressing GnT-V fail to develop spontaneous tumors in any organs, but phenotypes reminiscent of epithelial-to-mesenchymal transition were observed in their skin. However, the biological function of GnT-V in normal skin remained unknown. In this study, we examined the role of GnT-V in keratinocyte proliferation using GnT-V-deficient mice. Proliferation of human keratinocytes was suppressed by treatment with GnT-V siRNA. Mgat5(-/-) mouse keratinocytes also showed impaired cell proliferation through the reduction in EGF receptors on the cell surface. Although the skin of Mgat5(-/-) mice appeared normal, epidermal hyperplasia and proliferation of keratinocytes induced by the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) were downregulated in these mutants. Moreover, a dramatic increase in GnT-V expression was observed by treatment with TPA or heparin-binding EGF-like growth factor (HB-EGF) in normal human epidermal keratinocytes. This increase was inhibited by an EGF receptor inhibitor. These results indicate that a high expression of GnT-V in keratinocytes contributes to HB-EGF-mediated epidermal hyperproliferation by inhibiting endocytosis of EGF receptors bearing β1,6 GlcNAc on their N-glycans. Our findings demonstrate a novel role for GnT-V in epidermal homoeostasis, particularly in hyperproliferative conditions.     

非节段型白癜风患者皮损单细胞转录图谱的初步研究

【摘要】 目的 采用单细胞RNA测序技术鉴定和区分白癜风皮损真表皮细胞亚群，并研究它们之间的关系。方法 2019年9月在杭州市第三人民医院皮肤科门诊收集2例健康人（无免疫及系统性疾病）正常皮肤和2例非节段型稳定期白癜风患者皮损样本，采用10 × Genomics单细胞RNA-Seq技术检测，对所有样本的11 000个细胞进行单细胞转录组测序。通过Seurat软件分析、筛选和统计细胞亚群。结果 对2例正常皮肤基因表达的聚类分析发现包括角质形成细胞、成纤维细胞、神经及黑素细胞、内皮细胞、组织干细胞和以树突细胞及T细胞为主的免疫细胞群。2例白癜风皮损中分化和数量异常的有成纤维细胞和4类角质形成细胞亚群，其中，成纤维细胞占比为0，低于正常皮肤（0.4%），角质形成细胞亚群5、6、10、12占比（8.03%、7.36%、3.52%、0.91%）均显著高于正常皮肤（4.47%、3.53%、2.69%、0.28%，均P＜0.01）。上述亚群的角质形成细胞处于细胞分化的末端，同时还具有极显著且特异的标记基因，分析显示，标记基因主要与细胞间相互作用和细胞稳态密切有关，GO和KEGG分析显示，角质形成细胞亚群5、6主要与细胞间连接和细胞黏附及细胞骨架功能相关，角质形成细胞亚群10与细胞稳态紧密相关。结论 国内首次报道通过单细胞测序手段研究白癜风皮损的转录表达谱，初步发现4群数量及功能差异的角质形成细胞，提示角质形成细胞亚群的异常分化与功能异常可能影响白癜风的发生发展。     

Dysfunctional γδ T cells contribute to impaired keratinocyte homeostasis in mouse models of obesity

Skin complications and chronic non-healing wounds are common in obesity, metabolic disease, and type 2 diabetes. Epidermal γδ T cells normally produce keratinocyte growth factors, participate in wound repair, and are necessary for keratinocyte homeostasis. We have determined that in γδ T cell-deficient mice, there are reduced numbers of keratinocytes and the epidermis exhibits a flattened, thinner structure with fewer basal keratinocytes. This is important in obesity, where skin-resident γδ T cells are reduced and rendered dysfunctional. Similar to γδ T cell-deficient mice, keratinocytes are reduced and the epidermal structure is altered in two obese mouse models. Even in regions where γδ T cells are present, there are fewer keratinocytes in obese mice, indicating that dysfunctional γδ T cells are unable to regulate keratinocyte homeostasis. The impact of absent or impaired γδ T cells on epidermal structure is exacerbated in obesity as E-cadherin localization and expression are additionally altered. These studies reveal that γδ T cells are unable to regulate keratinocyte homeostasis in obesity and that the obese environment further impairs skin structure by altering cell-cell adhesion. Together, impaired keratinocyte homeostasis and epidermal barrier function through direct and indirect mechanisms result in susceptibility to skin complications, chronic wounds, and infection.     

Optimization of a transplant model to assess skin reconstitution from stem cell-enriched primary human keratinocyte populations

Given that an important functional attribute of stem cells in vivo is their ability to sustain tissue regeneration, we set out to establish a simple and easy technique to assess this property from candidate populations of human keratinocyte stem cells in an in vivo setting. Keratinocytes were inoculated into devitalized rat tracheas and transplanted subcutaneously into SCID mice, and the epithelial lining regenerated characterized to establish the validity of this heterotypic model. Furthermore, the rate and quality of epidermal tissue reconstitution obtained from freshly isolated unfractionated vs. keratinocyte stem cell-enriched populations was tested as a function of (a) cell numbers inoculated; and (b) the inclusion of irradiated support keratinocytes and dermal cells. Rapid and sustained epidermal tissue regeneration from small numbers of freshly isolated human keratinocyte stem cells validates the utilization of this simple and reliable model system to assay for enrichment of epidermal tissue-reconstituting cells.     

A novel genodermatosis caused by mutations in plakophilin 1, a structural component of desmosomes

Desmosomes are adhesive intercellular junctions that link adjacent cells and provide anchoring points for the keratin filament cytoskeleton. The mechanical integrity of desmosomes depends on a complex network of transmembranous and cytoplasmic proteins and glycoproteins each encoded by distinct genes. Recently, naturally occurring human mutations in one of these desmosomal structural components, plakophilin 1, have been described. The clinical features of the affected individuals, who have total ablation of plakophilin 1, comprise a combination of skin fragility and ectodermal dysplasia with loss of hair, reduced sweating and nail dystrophy. Desmosomes in the skin are small and poorly formed and there is widening of intercellular spaces between keratinocytes as well as detachment of the keratin filament network from the cell membrane. These clinicopathological observations demonstrate the relevance of plakophilin 1 to keratinocyte adhesion and epidermal morphogenesis. This new form of genodermatosis represents the first example of human desmosome gene mutations and its clinical and ultrastructural characteristics are highlighted in this article.