Bioinformatic Analysis of Genes Associated with Autophagy in Vitiligo.

Background: As vitiligo progresses, autophagy becomes more and more important. Objectives: To validate potential genes associated with autophagy in vitiligo through bioinformatics analysis and experimental testing. Materials and Methods: Dataset GSE75819 of mRNA expression profiles was obtained from GEO. After data normalisation, gene set enrichment analyse enrichment analysis and abundance analysis of infiltrating immune cells were performed. A list of autophagy-related differentially expressed genes (ARDEGs) associated with vitiligo was generated using R software. Protein-protein interaction (PPI) analysis, correlation analysis, and enrichment analysis on gene ontology (GO) and Kyoto encyclopaedia of genes and genome (KEGG) pathways were conducted on the ARDEG data. The microRNAs associated with hub genes were predicted using the TargetScan database. Finally, RNA expression of 10 hub genes and Western blotting (WB) of autophagy pathway factors were further verified. Results: From the lesions of 15 vitiligo patients, 44 ARDEGs were identified. PPI analysis demonstrated that these ARDEGs interacted with each other. GO and KEGG analyses of ARDEGs revealed that several enriched terms were associated with macroautophagy (biological process), vacuolar membranes (cellular components), cysteine-type peptidase activity (molecular function), and autophagy in animals, neurodegeneration-multiple disease pathways, and apoptosis. In vitiligo lesions, qRT-PCR and sequencing validation analyses showed expression levels of CCL2, RB1CC1, TP53, and ATG9A that were consistent with bioinformatic analysis of the microarray. WB results also showed that autophagy-related proteins were differentially expressed. Conclusions: Forty-four potential ARDEGs were identified in vitiligo by bioinformatic analysis. Vitiligo may be affected by autophagy regulation through CCL2, RB1CC1, TP53, and ATG9A.

Clinical Observation on the Therapeutic Effect of Port-Wine Stains with Intravenous Injection of Hematoporphyrin Monomethyl Ether (HMME).

Background: Hematoporphyrin monomethyl ether (HMME) is a promising photosensitizer for photodynamic therapy (PDT) and has found wide application in the treatment of port-wine stains (PWS). Objective: This study aims to observe and analyze the clinical efficacy and safety of HMME-PDT in the treatment of PWS patients. It also aims to evaluate the usefulness of color Doppler flow imaging (CDFI), an ultrasound technique for detecting blood flow in skin lesions, in assessing clinical efficacy. Methods: Thirty-three patients with PWS underwent HMME-PDT at our dermatology outpatient clinic between January 2019 and March 2020. Data on patient demographics, lesion location, lesion type (pink, purple, nodular thickening), treatment frequency, and pre- and post-treatment images were collected and retrospectively analyzed. CDFI was performed on three patients. Results: All patients received intravenous HMME and underwent irradiation with 532 nm green LED light. Of these, 5 patients received 1 session of HMME-PDT, 14 received 2 sessions, 9 received 3 sessions and the remaining 5 patients received more than 3 sessions. Of the 33 patients, 9 were cured (27.27%), 10 showed improvement (30.30%), 11 experienced a reduction in symptoms (33.33%), and 3 showed no significant improvement (9.09%). Most patients reported local pain and oedema, and no systemic adverse effects were observed. Clinical efficacy correlated with lesion type and total number of treatment sessions. CDFI appears to be an excellent technique for assessing clinical efficacy. Conclusion: HMME-PDT is a safe and effective method for the treatment of PWS. CDFI examination appears to be a promising assessment tool. However, further validation with larger sample sizes is warranted.

Characterization of CM-Dil-labeled Muse cells in culture and in skin wounds in rats.

To investigate the characteristics of multilineage-differentiating stress-enduring (Muse) cells labeled with chloromethyl dialkylcarbocyanine (CM-Dil) in culture and in skin wounds of rats. Normal human dermal fibroblasts (NHDFs) were obtained from foreskins and were confirmed by immunocytochemistry with vimentin. Muse cells were derived from NHDFs using long-term trypsinization (LTT), were confirmed using immunocytochemistry with antibodies against stage specific embryonic antigen-3 (SSEA-3) and CD105 and were expanded in suspension cultures. The Muse cells were labeled with CM-Dil and were further evaluated with respect to their biological properties using CCK-8 assays and scratch tests. One hundred µl CM-Dil-labeled Muse cells at a concentration of 5 × 103/µl were injected subcutaneously at the edges of skin wounds in adult male SD rats. At weeks 1, 3 and 5 after the injection, the distribution of CM-Dil-labeled Muse cells in skin tissues was observed using immunofluorescence microscopy. Muse cells were double-positive for CD105 and SSEA-3. ALP staining of the M-clusters were positive and they displayed orange-red fluorescence after labelling with CM-Dil, which had no adverse effects on their viability, migration or differentiation capacity. One week after the subcutaneous injection of CM-Dil-labeled Muse cells, many cells with orange-red fluorescence were observed at the edges of the skin injuries; those fluorescent spots gradually decreased over time, and only a few Muse cells with fluorescence could be detected by week 5. CM-Dil can be used to label Muse cells without affecting their proliferation, migration or differentiation, and can be used for short-term tracking of Muse cells for the treatment of skin wounds in a rat model.

Characteristics of multilineage-differentiating stress-enduring cell clusters in different culture conditions.

OBJECTIVE: To observe the morphological characteristics of clusters of Muse cells from normal human dermal fibroblasts (NHDFs) under different culture conditions. METHODS: Muse cells were sorted by magnetic activated cell sorting (MACS) from NHDFs, and were evaluated by flow cytometry. Muse cells were cultured in suspension and in adherent conditions to obtain Muse cell clusters (M-clusters), which were further characterized by alkaline phosphatase (AP) staining, immunofluorescence (IF) staining and transmission electron microscopy (TEM). The M-clusters were further cultured on Lando artificial dermal regeneration matrix (LADRM) for analysis by scanning electron microscopy (SEM) and IF staining of frozen sections. RESULTS: The proportion of SSEA3 and CD105 double-positive cells obtained by MACS was 87.4%. The sorted cells rapidly formed M-clusters after suspension culture, and showed internal characteristics of stem cells under TEM. After adherent culture, M-clusters stained positively for AP, SSEA-3 and OCT-4. Each M-cluster on the surface of the LADRM displayed an outer membrane of amorphous materials under SEM. Frozen sections and fluorescence staining of LADRM loaded with M-clusters showed an uneven fluorescence intensity of SSEA-3 within the clusters. CONCLUSIONS: Muse cells sorted by MACS from NHDFs could generate M-clusters, which included cells of different stemness and are wrapped in membrane-like structures.

RNA-seq and ATAC-seq analyses of multilineage differentiating stress enduring cells: Comparison with dermal fibroblasts.

The aim of this study is to characterize the molecular properties of multilineage differentiating stress-enduring (Muse) cells compared with dermal fibroblasts (FBs) and to characterize differences in their transcriptomes and open chromatin regions that are involved in cellular plasticity. Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) and RNA sequencing (RNA-seq) analyses was then performed on FBs and Muse cells. Subsequently, cell type-selective gene regulatory regions were identified by coalition analysis. Expression patterns of transcription factors (TFs) and signaling pathways intermediates were verified using quantitative real-time polymerase chain reaction and Western blot analyses. RNA-seq identified 2355 significantly differentially expressed genes (DEGs) that regulate the transcriptome, including 1222 upregulated and 1133 downregulated DEGs. The general panorama of RNA-seq and ATAC-seq analyses confirmed the differences in TFs and open chromatin regions between FBs and Muse cells. ATAC-seq analysis showed that Muse cells had more reproducible and meaningful peaks than FBs, and the peak signals were concentrated near promoter-transcription start site areas. In genomic regions that can be preferentially accessed in FBs and Muse cells, more than 200 TFs had binding motif sequences. Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and coalition analyses identified differences in factors involved in the cell cycle and the protein kinase B (AKT) signaling pathway of FBs and Muse cells. The results of RNA-seq and ATAC-seq analyses clarified the genetic basis of the different biological properties of Muse cells and FBs. These results suggest that the cell cycle transition and the AKT signaling pathway may affect the morphology and biological characteristics of Muse cells.

Effects of keratinocyte-derived and fibroblast-derived exosomes on human epidermal melanocytes.

BACKGROUND: Exosomes have been demonstrated to carry proteins, membrane lipids, mRNAs and microRNAs which can be transferred to surrounding cells and regulate the functions of those recipient cells. OBJECTIVES: The objective of the study was to investigate the effects of exosomes released by keratinocytes and fibroblasts on the proliferation, tyrosinase activity and melanogenesis of melanocytes. METHODS: Melanocytes, keratinocytes and fibroblasts obtained from human foreskin were cultured and exosomes secreted by keratinocytes and fibroblasts were harvested from the culture supernatants by ultracentrifugation. Each exosome fraction was divided into two parts; one part was subjected to high-throughput sequencing using an Illumina HiSeq sequencer to characterize the microRNA expression profiles, while the other part was labeled with the fluorescent dye PKH67 and was then co-cultivated with epidermal melanocytes. RESULTS: High-throughput sequencing analysis showed 168 differentially expressed microRNA within exosomes derived from keratinocytes and from fibroblasts, 97 of those being up-regulated with the other 71 down-regulated. Gene ontology analysis showed that the target genes responsible for these differentially expressed microRNAs were mainly enriched in the protein-binding region of molecular functions. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that target genes regulated by differentially expressed microRNA were mainly involved in mitogen-activated protein kinase (MAPK) signaling pathway, Ras signaling pathway, cAMP signaling pathway and Wnt signaling pathway. Keratinocyte-derived exosomes were taken up by melanocytes co-cultured with them and promoted the proliferation, tyrosinase activity and melanin synthesis of those melanocytes. However, fibroblast-derived exosomes had no similar effects on melanocytes. CONCLUSION: Keratinocyte-derived exosomes but not fibroblast-derived exosomes were taken up by melanocytes in co-culture and significantly stimulated their proliferation, tyrosinase activity and melanin synthesis. Those different effects may be mainly due to the differential expression of microRNAs in exosomes derived from the different types of cells. LIMITATIONS: Electron microscopy of the obtained exosomes and in-depth study of apparently differentially expressed microRNAs were not performed.

A Case of Cutaneous Rosai-Dorfman Disease Treated by Intralesional Injections of Glucocorticoid.

A 45-year-old male presented with painless reddish-brown plaques and nodules that had infiltrated his shoulder and back for 3 months. From the clinical manifestations and histopathological findings, the patient was diagnosed with cutaneous Rosai-Dorfman disease. Intralesional injections of betamethasone (trade name: diprospan) were recommended and the lesions improved significantly after three treatments.

Dedifferentiation of human epidermal melanocytes in vitro by long-term trypsinization.

Human epidermal melanocytes can be induced to form melanocyte spheroids and revert to immature characteristics by long-term trypsinization (LTT). To further explore the biological characteristics of melanocytes after LTT and to study the underlying mechanism. Melanocytes were subjected to long-term (2 h) trypsinization in this study, after which their viability, proliferation and autophagy were characterized. The expression of melanocyte markers [human melanoma black45 (HMB45), tyrosinase (TYR) and Nestin] was detected and relative expression levels of mRNAs encoding TYR, Nestin, c-Kit and microphthalmia-associated transcription factor (MITF) were determined. After LTT, more short spindle-shaped melanocytes appeared and viability assays demonstrated that most melanocytes survived that treatment but had decreased proliferation rates compared to the untreated controls. There was a significant increase in autophagy of melanocytes after LTT and the expression of TYR was decreased compared with untreated control melanocytes. There were no significant differences in the expression of HMB45 or Nestin between the two groups. Compared with untreated melanocytes, levels of message ribonucleic acid (mRNAs) encoding TYR, c-Kit and MITF were decreased after LTT, while Nestin mRNA levels were increased. These results clarified that Long-term treatment with trypsin causes the dedifferentiation of mature epidermal melanocytes in vitro.

An unusual case of secondary syphilis misdiagnosed as allergic dermatitis for 2 years.

Dermatologists should be aware that the clinical manifestations of syphilis are very complex and changeable. Unilaterally distributed skin lesions and painless lip ulcers may also be the clinical manifestations of secondary syphilis.

Majocchi's granuloma on the forearm caused by Trichophyton tonsurans in an immunocompetent patient.

Majocchi's granuloma is an uncommon fungal infection of the dermis and subcutaneous tissue. The most frequently identified cause of Majocchi's granuloma is anthropophilic Trichophyton rubrum, and it is most commonly located on the anterior aspect of the lower limbs in women. Here, we report a case of Majocchi's granuloma on the forearm, a site that is rarely involved, in a 62-year-old woman who had been bitten by a dog. Histological examination revealed a dense dermal infiltrate composed of lymphoplasmacytic cells and neutrophils, with hyphae in the dermis. The presence of the fungus, Trichophyton tonsurans, was confirmed by mycological examination and molecular methods. Therefore, histological and mycological examination confirmed the diagnosis of Majocchi's granuloma. The patient was treated with local moxibustion and itraconazole, 200 mg/day, for 60 days, which facilitated a complete resolution of the lesions.

The tolerance of human epidermal cells to trypsinization in vitro.

To characterize the tolerance of different types of human epidermal cells to trypsinization in vitro and develop a new method to separate and purify melanocytes according to their tolerance to trypsinization. Epidermal cells were obtained by separating the epidermis from human foreskins. Some of those cells were used for routine culture, and then were subjected to differential trypsin digestion. The remaining epidermal cells were resuspended in a 0.25% trypsin solution and then were neutralized by the addition of bovine serum at different time points. Immunofluorescence staining of HMB45, K15 and vimentin was used to identify melanocytes, keratinocytes and fibroblasts, respectively. We found that Keratinocytes, melanocytes and fibroblasts are primary cells obtained from conventional cultures of human skin. Purified keratinocytes and melanocytes can be obtained by conventional differential trypsin digestion, but fibroblasts in the melanocyte population quickly gain a survival advantage after passage. With longer trypsin digestion times, the number of adherent cells decreased, the time required for cell attachment increased, and the proportion of melanocytes increased. There were no obvious keratinocytes in cell populations obtained after 12 h of trypsinization of epidermal cells, and more short spindle-shaped melanocytes appeared, all of which were HMB45-positive. In conclusion, the tolerance of human epidermal melanocytes to trypsinization in vitro was better than epidermal keratinocytes, and that property can be used to purify melanocytes and was better than traditional differential trypsin digestion. The morphology of cells that survived the long-term trypsin digestion changed and they had good proliferative activity, but seemed to be more immature.

Characterization of Induced Pluripotent Stem Cells from Human Epidermal Melanocytes by Transduction with Two Combinations of Transcription Factors.

OBJECTIVE: In order to generate induced Pluripotent Stem Cells (iPSCs) more efficiently, it is crucial to identify somatic cells that are easily accessible and possibly require fewer factors for conversion into iPSCs. METHODS: Human epidermal melanocytes were transduced with lentiviral vectors carrying 3 transcription factors (OCT-4, KLF-4 and c-MYC, 3F) or 4 transcription factors (OCT-4, KLF-4, c-MYC and SOX-2, 4F). Once the clones had formed, assays related to stem cell pluripotency, including alkaline phosphatase staining, DNA methylation levels, expression of stem cell markers and ultrastructure analysis were carried out. The iPSCs obtained were then induced to differentiate into the cells representing the three embryonic layers in vitro. RESULTS: Seven days after the transduction of epidermal melanocytes with 3F or 4F, clones were formed that were positive for alkaline phosphatase staining. Fluorescent staining with antibodies against OCT-4 and SOX-2 was strongly positive, and the cells showed a high nucleus-cytoplasm ratio and active karyokinesis. No melanosomes were found in the cytoplasm by ultrastructural analysis. There were obvious differences in DNA methylation levels between the cloned cells and their parental cells. However, there was not a significant difference between 3F or 4F transfected clonal cells. Meanwhile, the iPSCs successfully differentiated into the three germ layer cells in vitro. CONCLUSION: Human epidermal melanocytes do not require ectopic SOX-2 expression for conversion into iPSCs, and may serve as an alternative source for deriving patient-specific iPSCs with fewer genetic elements.

Experimental study and clinical observations of autologous hair follicle cell transplants to treat stable vitiligo.

BACKGROUND: Vitiligo is characterized by the loss and/or dysfunction of melanocytes in the skin and has a profound impact on the social interactions of patients. Although there are many treatment options for vitiligo, the outcome is frequently unsatisfactory, especially for patients with stable vitiligo. OBJECTIVES: To study the biological properties of melanocytes derived from human hair follicles and to observe the efficacy of using transplants of autologous hair follicle cells to treat patients with stable vitiligo. METHODS: From February 2014 to March 2017, 26 patients with stable vitiligo, who were refractory to all current routine therapy, underwent treatment with transplanted autologous hair follicle cells. The skin graft from each patient's occipital region was trimmed to remove excess adipose tissue and some of the upper part of the dermis. The remaining tissue, including hair follicles and dermal papillae, was cut into pieces and incubated in collagenase type IV and then in trypsin-ethylenediaminetetraacetic acid solutions. The cells were recovered, resuspended in the patient's own serum and then applied to the recipient area. Clinical observations continued for 6 months to 1 year. Laboratory experiments were also performed during this time on scalp specimens obtained from normal human volunteers. Cells migrating from the outer root sheath and the dermal papillae at various times of culture were observed using a microscope. RESULTS: Most of the repigmentation in the vitiligo areas appeared within 8 weeks of transplantation of autologous hair follicle cells. Early skin repigmentation was not uniform and appeared more repigmented than the surrounding normal skin. As time went by, the repigmentation became more obvious and matched the color of the skin around the lesion. Most of the pigmentation presented as a diffuse pattern and was not localized around the hair follicles. Among the 26 patients, 9 (34.6%) achieved excellent repigmentation, while 13 (50.0%) had good, 3 (11.5%) fair and 1 (3.9%) poor repigmentation. During the follow-up visit at 1 year, no excess hair growth was observed in the recipient areas and there was no scarring or ulcer formation in the donor or recipient areas. In the experimental part of the study, many keratinocytes, melanocytes and fibroblasts migrated from the adherent outer root sheath. In later subcultures using a specialized medium, pure melanocytes were obtained that had a strong proliferative capacity and had bipolar or poly-dendritic shapes. On the other hand, cells from the dermal papillae grew radially in primary culture and were almost fibroblast-like. However, a few bipolar melanocytes appeared in the later stage of culture. CONCLUSIONS: The results of our study show that transplantation of autologous hair follicle cells is a simple and effective method to treat patients with stable vitiligo. Hair follicles (especially the outer root sheath) harbor many melanocytes with potential proliferative ability. LIMITATIONS: There are a few limitations of the present study: a small sample size, a short follow-up period, no cell counting or viability testing.

Misdiagnosis of uncommon presentation of Angiolymphoid hyperplasia with Eosinophilia.

In 50-year-old female patients, the purple-red papules in the right upper arm were misdiagnosed as molluscum contagiosum or scars. The diagnosis of angiolymphoid hyperplasia with eosinophilia was determined on the basis of the clinicopathological features.

The distribution of melanocytes and the degradation of melanosomes in fetal hair follicles.

There have been many studies about the formation, storage, transport and degradation of melanosomes in epidermal melanocytes but studies of melanocytes and melanosomes in fetal hair follicles (HFs) have been limited and ambiguous. The goal of this study was to investigate the distribution of melanocytes and the degradation of melanosomes in fetal HFs. After obtaining approval and informed consent for the study, a scalp specimen from a 5 month gestational age fetus was obtained and was divided into two parts. One part was subjected to immunohistochemical staining with the melanocyte-specific marker HMB-45 and was then observed by light microscopy to detect the distribution of melanocytes in HFs. The other part underwent conventional processing for transmission electron microcopy (TEM). Subsequently, the morphology of melanosomes in HF melanocytes and their degradation in cortical keratinocytes were observed. Immunohistochemically, scattered round melanocytes lacking dendrites were mainly observed along the outer root sheath of the lower part of the HF. A few fusiform or tri-dendritic melanocytes were located at the bottom of the hair bulbs. Significantly melanized melanocytes with multiple dendrites were concentrated in the pigmented area in the center of the hair bulbs, only above the dermal papilla. Analysis by TEM revealed melanocytes containing melanosomes at all stages of development. Autophagosomes containing stage mature IV melanosomes were observed in some melanocytes. Many phagolysosomes containing numerous melanosomes were observed in the cortical keratinocytes. Some phagolysosomes were concentrically surrounded by 3-5 layers of endoplasmic reticulum. Melanosomes that had been degraded or were being degraded in phagolysosomes in keratinocytes had lost their integrity and had become an ill-defined melanosomal dust that were arranged irregularly. Partial melanin particles were released into the cytosol. Melanocytes in different regions of fetal HFs had different morphologies and were at various stages of differentiation. Fetal HF melanocytes contained not only melanosomes at different developmental stages, but autophagosomes were seen occasionally. Melanosomes were degraded into irregular pigment particles in the phagolysosomes of cortical keratinocytes. These results provide important clues to elucidate the mechanism of melanosome biodegradation.

Melanocyte spheroids are formed by repetitive long-term trypsinization.

BACKGROUND: Autologous melanocyte transplantation plays an important role in the treatment of vitiligo. OBJECTIVE: Previous studies have indicated that, compared with melanocytes growing in monolayers, melanocyte spheroids have a better survival in growth factor- and serum-deprived conditions. METHODS: Melanocyte spheroids were obtained from human epidermis by repetitive long-term trypsinization and maintained an aggregated morphology for a short period in certain conditions. RESULTS: Melanocyte spheroids were capable of growing into normal dendritic melanocytes in monolayer when they were harvested and reinoculated in 24-well plates. Immunohistochemical analysis of the melanocyte spheroids revealed that they were positive for HMB45, a melanosome-specific marker. No melanomas occurred when melanocyte spheroids were transplanted into mice. CONCLUSION: Our study provides a promising approach for melanocyte transplantation to treat vitiligo.

Generation of Induced Pluripotent Stem Cells from Human Epidermal Keratinocytes.

Induced pluripotent stem cells (iPSCs) play an important role in cell replacement therapy. Several studies have shown that keratinocytes are promising reprogrammed cells. We easily and efficiently enriched epidermal stem cells by attaching them for a limited time in culture dishes. Individual epidermal cells enriched in stem cells, which showed strong immunostaining for K15, were obtained and generated iPSCs within 10 days after transfection with lentiviruses encoding 4 transcription factors (OCT4, SOX2, KLF4, and NANOG). Immunofluorescent staining showed that those iPSCs expressed SOX2, OCT4, NANOG, and SSEA3 (a specific marker of embryonic stem cells). The embryoid bodies generated from those iPSCs stained positively for OCT4 and NANOG and also with the CDy1 dye that is specific for stem cells. When the iPSCs were subcutaneously injected into 4-week-old BALB/c nude mice, teratoma developed at the inoculation site. The iPSCs also demonstrated reduced DNA methylation compared with the original cells and could be induced to differentiate into adipocytes (mesodermal), hepatocytes (endodermal), and neural cells (ectodermal) in vitro. Our research provides an easy and efficient method for producing iPSCs from keratinocytes, which has important applications in cell replacement therapy.

Effects of acrylate/acrylamide polymers on the adhesion, growth and differentiation of Muse cells.

BACKGROUND: Acrylate/acrylamide copolymers have excellent optical properties and biocompatibility and are ideal biomaterials that have been widely used in tissue engineering. Multilineage-differentiating stress-enduring cells (Muse cells) are a specific subset of mesenchymal stem cells that have an excellent potential for the regenerative medicine. OBJECTIVE: This study was designed to investigate the effects of acrylate/acrylamide copolymers on the adhesion, proliferation and pluripotent-like properties of Muse cells, which were derived from normal human dermal fibroblasts by long-term trypsin incubation. METHODS: In an initial experiment, Muse cells were seeded on primary microarrays containing micro-spots of 275 different mixtures of acrylate/acrylamide. Each mixture was composed of two of 11 different monomers in various proportions, and was replicated in four micro-spots each. According to the adhesion and growth characteristics of Muse cells on those substrates, specific polymer candidates for Muse cells were selected and secondary microarrays were prepared. We then observed the effects of those specific polymer candidates on the adherence, proliferation and differentiation of Muse cells and suitable candidates for their optimal culture were identified. RESULTS: According to the adhesion and growth patterns of Muse cells on the primary microarrays, ten suitable mixtures of acrylate/acrylamide copolymers were identified. Muse cells grew well on six of those combinations and around the four other combinations of those polymer mixtures. Muse cells cultured on three of those combinations proliferated and differentiated into long spindle-shaped cells that looked like fibroblasts, while Muse cells cultured on one combination formed clusters that were ring-shaped. Muse cells cultured on some of those combinations of acrylate/acrylamide proliferated and formed clusters that appeared to be very healthy, whereas Muse cells cultured on other combinations formed clusters that expanded outwards. CONCLUSIONS: These results identified a polymer combination that was optimum for the adhesion, proliferation and maintenance of Muse cells in an undifferentiated state.

Construction of a three-dimensional in-vitro skin model on polycaprolactone fibers.

BACKGROUND: The aim of this study was to observe the morphological characteristics and the biological properties of human epidermal cells when cultured at an air-liquid interface in polycaprolactone (PCL) fibers as a three-dimensional scaffold for tissue engineering. METHODS: In this study, the melanocytes and keratinocytes were obtained from human scalp skin, seeded onto a PCL film, and cocultured for 2 weeks to construct a three-dimensional (3D) skin model. The cells were then characterized by hematoxylin and eosin staining, by immunohistochemical staining with antibodies to cytokeratin 15 (CK15), Ki-67, CD34, CD200 and HMB45 and by transmission electron microscopy. RESULTS: Keratinocytes and melanocytes grew well in the co-culture system. Hematoxylin and eosin staining revealed that the cells adhered to the PCL fiber scaffold well, the keratinocyte layer became a multilayered concentric structure and the surface became distinctly keratinized at the air-liquid interface. Immunohistochemical analyses exhibited a scattered distribution of cells expressing CK15, CD34, CD200, Ki-67 and/or HMB45. Transmission electron microscopy revealed that the keratinocytes contained a number of keratin fibrils and membrane-coated granules. CONCLUSIONS: The PCL scaffold has excellent adhesiveness and biocompatibility with human epidermal cells, and is suitable for constructing 3D skin models for tissue engineering in the future.