Let-7 family regulates HaCaT cell proliferation and apoptosis via the ΔNp63/PI3K/AKT pathway.

We evaluated the expression profiles of differentially expressed miRNAs (DEmiRNAs) involved in human fetal skin development via high-throughput sequencing to explore the expression difference and the regulatory role of miRNA in different stages of fetal skin development. Analysis of expression profiles of miRNAs involved collecting embryo samples via high-throughput sequencing, then bioinformatics analyses were performed to validate DEmiRNAs. A total of 363 miRNAs were differentially expressed during the early and mid-pregnancy of development, and upregulated DEmiRNAs were mainly concentrated in the let-7 family. The transfection of let-7b-5p slowed down HaCaT cell proliferation and promoted apoptosis, as evidenced by the cell counting kit-8 assay, quantitative real-time polymerase chain reaction, and flow cytometry. The double luciferin reporter assay also confirmed let-7b-5p and ΔNp63 downregulation through the combination with the 3'-untranslated region of ΔNp63. Moreover, treatment with a let-7b-5p inhibitor upregulated ΔNp63 and activated the phosphoinositide 3-kinase (PI3K)-protein kinase B (AKT) signaling pathway. The let-7b-5p caused a converse effect on HaCaT cells because of Np63 upregulation. Let-7b-5p regulates skin development by targeting ΔNp63 via PI3K-AKT signaling, contributing to future studies on skin development and clinical scar-free healing.

Nature-Inspired Scarless Healing: Guiding Biomaterials Design for Advanced Therapies.

The use of biomaterials in the treatment of skin wounds has been steadily increasing over the last two decades. The key to the successful application of biomaterials in scar reduction is the up-to-date knowledge of the actors involved in accelerated healing and the cellular factors that can be implemented in bioinspired materials. Natural models of scarless healing such as oral mucosa, fetal skin and the skin of amphibians, fish, and reptiles are a great source of information. By investigating their microenvironments, cellular factors, and inflammatory self-regulatory systems, a general model of scarless healing can be defined. This review introduces the basic and current concepts of skin wound healing and focuses on providing a detailed overview of the main processes of accelerated healing without scarring. The article outlines the common features and key points that develop and promote scar-free healing. The tissues and healing processes of the selected natural models are described individually (tissue organization, structural components, ratios of cellular factors such as Collagen and transforming growth factor and their mechanisms of regulation of inflammation and scar overgrowth). A comparative work of each natural model concerning healing in human skin is included in the discussion. Finally, the patterns identified through the analysis of each model and their differences from normal healing are presented to facilitate the knowledge for the implementation of new treatments.

Risk factors associated with accidental fetal skin lacerations during cesarean delivery.

OBJECTIVE: To identify risk factors associated with accidental fetal skin lacerations (AFL) during cesarean section (CS). METHODS: This retrospective cohort study was obtained from the registry of two large medical centers between 2014 and 2019. The study group comprised all newborns identified with AFL. The rates of various potential risk factors were compared between the study group and a group of CS at which no AFL had occurred (the control group). RESULTS: Of the 14 666 CS deliveries, 48 cases of AFL (0.33%) were documented, 52% of these following urgent CS. Compared with the control group (n = 14 618), the only risk factors associated with AFL were premature rupture of membranes (PROM) (odds ratio [OR] 5.38, 95% convidence interval [CI] 2.97-9.74) and meconium-stained amniotic fluid (OR 6.50, 95% CI 2.55-16.54). In subgroup analysis by CS urgency, no significance for these factors was noted in elective CS group; but higher rates of both PROM and meconium-stained amniotic fluid were noted in the AFL during urgent CS (OR 14.23, 95% CI 6.30-32.16 and OR 15.36, (95% CI 5.65-41.75, respectively). CONCLUSIONS: During urgent CS, the surgeon should bear in mind that the presence of PROM or meconium-stained amniotic fluid should prompt extra care and application of preventive measures to decrease the rates of AFL.

Human fetal skin derived merkel cells display distinctive characteristics in vitro and in bio-engineered skin substitutes in vivo.

Human skin contains specialized neuroendocrine Merkel cells responsible for fine touch sensation. In the present study, we performed in-depth analysis of Merkel cells in human fetal back skin. We revealed that these Merkel cells expressed cytokeratin 20 (CK20), were positive for the neuroendocrine markers synaptophysin and chromogranin A, and the mechanosensitive ion channel Piezo2. Further, we demonstrated that Merkel cells were present in freshly isolated human fetal epidermal cells in vitro, and in tissue-engineered human dermo-epidermal skin substitutes 4 weeks after transplantation on immune-compromised rats. Merkel cells retained the expression of CK20, synaptophysin, chromogranin A, and Piezo2 after isolation and in culture, and in the skin substitutes after transplantation. Interestingly, we observed that in fetal skin and in skin substitutes, only Merkel cells were positive for CK8, while in culture, also non-Merkel cells showed positivity for CK8. In summary, human fetal Merkel cells showed phenotypical features confirming their cell identity. This findings are of pivotal importance for the future application of fetal tissue-engineered skin in clinics.

Off-the-shelf acellular fetal skin scaffold as a novel alternative to buccal mucosa graft: the development and characterization of human tissue-engineered fetal matrix in rabbit model of hypospadiasis.

AIM: In this study, we aimed to develop a novel alternative to buccal mucosal graft from the acellular human fetal skin to manage hypospadias in a rabbit model. We optimized the decellularization protocol to develop and characterize the human tissue-engineered fetal dermal matrix as an "off-the-shelf" natural biomaterial. MATERIAL AND METHODS: Human fetal skin was obtained at 16-19 weeks gestational age with respect to a signed informed consent from parents under the university ethical committee approval. The dissected full-thickness fetal skin tissues were placed into SDS and Triton X-100 in different dosages to achieve the optimum decellularization protocol. Histopathology of the acellular fetal matrix was assessed by Hematoxylin & Eosin (H&E) and DAPI staining to confirm the removal of all cell materials, Masson's trichrome staining for collagen evaluation, DNA quantification for confirmation of DNA content, and scanning electron microscopy (SEM) for evaluation of scaffold microstructure. Immunohistochemistry (IHC) staining was used to detect specific dermal markers, namely vimentin, type I collagen, cytokeratin (CK)19. The prepared dermal scaffolds were then grafted on the 8 rabbit models of hypospadias. The rabbits underwent evaluations at 1, 2, 3, and 6 months postoperatively. RESULTS: H&E, Masson's trichrome, DAPI staining, and SEM confirmed the significant removal of cells; meanwhile, the ECM was completely preserved. At the time of biopsy, after 2, 4, and 6 months, no evidence of inflammation, fibrosis, necrosis, or rejection was observed. The grafted dermal scaffolds appeared histologically and anatomically normal. It was observed that the scaffolds were recellularized by circulating CD 34 + bone marrow stem cells (BMSCs) inside the body, implicating the body as a natural bioreactor. CONCLUSION: The application of acellular fetal skin (AFS) is a safe and feasible method that can decrease surgical time in a complex hypospadias reconstruction. Moreover, AFS demonstrated excellent angiogenesis characteristics and migration of the stem cells to the scaffold observed during the course of treatment. Novel natural AFS scaffold without cell seeding is an excellent alternative to buccal mucosal graft; hence, it can overcome the limitations concerning the graft size and prevent the creation of wounds in oral mucosal tissue.

Single-cell transcriptome analysis reveals the dynamics of human immune cells during early fetal skin development.

The immune system of skin develops in stages in mice. However, the developmental dynamics of immune cells in human skin remains elusive. Here, we perform transcriptome profiling of CD45+ hematopoietic cells in human fetal skin at an estimated gestational age of 10-17 weeks by single-cell RNA sequencing. A total of 13 immune cell types are identified. Skin macrophages show dynamic heterogeneity over the course of skin development. A major shift in lymphoid cell developmental states occurs from the first to the second trimester that implies an in situ differentiation process. Gene expression analysis reveals a typical developmental program in immune cells in accordance with their functional maturation, possibly involving metabolic reprogramming. Finally, we identify transcription factors (TFs) that potentially regulate cellular transitions by comparing TFs and TF target gene networks. These findings provide detailed insight into how the immune system of the human skin is established during development.

Use of ovine acellular peritoneal matrix combined with honey and ovine fetal skin extract in the healing of full-thickness infected burn wounds in a rat model.

Treatment of infected burn wounds remains a challenge in burn units. Silver-sulfadiazine (SSD) is the most commonly used topical antimicrobial agent in managing these wounds. We aimed to accelerate the healing of burn wounds by combined application of ovine acellular peritoneal matrix (OAPM), honey (H), and ovine fetal skin extract (OFSE). Sixty-four standardized burn wounds were created on the dorsum of 16 rats and were subsequently inoculated with Staphyloccocus aureus and Pseudomonas aeruginosa. After 48 hr, the wounds were surgically debrided and received either physiologic saline (control group) or SSD, OAPM+SSD, OAPM+H+SSD, and OAPM+H+OFSE+SSD. The healing wounds were evaluated for size, bacterial counts, histopathology, and biomechanical properties on days 3, 7, 14, 21, and 28 after surgery. All treatments had effectively reduced the level of S. aureus and P. aeruginosa on wounds compared to the control group by day 3 and 7. The wounds treated with combined application of OAPM+H+OFSE+SSD demonstrated considerable inflammation reduction, fibroplasia, complete re-epithelialization, and wound contraction together with significantly lesser scar tissue formation. Treatment with OAPM+H+OFSE+SSD showed superior biomechanical properties of the healing wounds. The findings suggested that the synergistic effect of dressing the wounds with OAPM, H, and OFSE was a very effective approach in accelerating the healing process of the experimentally induced infected full-thickness burn wounds in rats.

Bioengineering and in utero transplantation of fetal skin in the sheep model: A crucial step towards clinical application in human fetal spina bifida repair.

An intricate problem during open human fetal surgery for spina bifida regards back skin closure, particularly in those cases where the skin defect is much too large for primary closure. We hypothesize that tissue engineering of fetal skin might provide an adequate autologous skin substitute for in utero application in such situations. Eight sheep fetuses of four time-mated ewes underwent fetoscopic skin biopsy at 65 days of gestation. Fibroblasts and keratinocytes isolated from the biopsy were used to create fetal dermo-epidermal skin substitutes. These were transplanted on the fetuses by open fetal surgery at 90 days of gestation on skin defects (excisional wounds) created during the same procedure. Pregnancy was allowed to continue until euthanasia at 120 days of gestation. The graft area was analyzed macroscopically and microscopically. The transplanted fetal dermo-epidermal skin substitutes was well discernable in situ in three of the four fetuses available for analysis. Histology confirmed healed grafts with a close to natural histological skin architecture four weeks after in utero transplantation. This experimental study generates evidence that laboratory grown autologous fetal skin analogues can successfully be transplanted in utero. These results have clinical implications as an analogous procedure might be applied in human fetuses undergoing prenatal repair to facilitate primary skin closure. Finally, this study may also fertilize the field of fetal tissue engineering in general, particularly when more interventional, minimally invasive, and open fetal surgical procedures become available.

Unbiased and quantitative proteomics reveals highly increased angiogenesis induction by the secretome of mesenchymal stromal cells isolated from fetal rather than adult skin.

Scarless wound healing and functional regeneration are typical processes of the fetus, gradually lost during postnatal life, and maximally attributed to fetal skin tissue and induced by fetal skin fibroblasts. The latter have been successfully applied to postnatal wounds, with clear advantages compared with autologous dermis grafts or adult fibroblast applications. Our goal was to functionally identify and uncover key factors and mechanisms through the analysis of secretomes, the principal players in all cell therapies based on mesenchymal stromal cells (MSCs). Cell secretomes also putatively mediate skin regenerative effects achieved in clinical applications of fetal skin fibroblasts. An innovative and unbiased approach of comparative and quantitative proteomics of cell conditioned media enabled us to gain knowledge of key molecules and processes from a translational perspective. Using banks of fetal and adult skin fibroblasts that we previously characterized as being MSCs, we discovered secretome changes by identification and comparative quantification, distinguishing secretome signatures of fetal skin MSCs putatively relevant for therapeutic microenvironment modulation. The uncovered proteins can trigger, directly and by modulation of extracellular matrix, angiogenesis, thus highlighting its key role towards scarless wound healing. The angiogenic trigger was functionally validated and corroborated in vitro, with fetal skin MSC secretomes stabilizing and inducing the formation of capillary-like networks by endothelial cells and fetal liver MSCs, respectively. Our approach and our results may aid in the development of cell-based and cell-free products for skin regeneration in acute or chronic injury, and also for wound healing in the regeneration of other tissues. Copyright © 2017 John Wiley & Sons, Ltd.

Global gene expression changes of amniotic fluid cell free RNA according to fetal development.

OBJECTIVE: This study aimed to evaluate the effect of in utero fetal development on the cell-free transcriptome of amniotic fluid by analyzing global gene expression in the amniotic fluid supernatant obtained at different gestational ages from euploid fetuses STUDY DESIGN: Thirteen amniotic fluid samples were obtained from five individuals at 28 gestational weeks and eight individuals at full term pregnancy. Transcriptome data previously analyzed by our group from 14 euploid mid-trimester amniotic fluid samples were used for comparative analysis. RNA was extracted from amniotic fluid supernatants, hybridized to Affymetrix GeneChip Human arrays, and the transcriptome was analyzed using the DAVID toolkit. RESULTS: We evaluated 27 samples, which were divided into three groups as follows: 14 subjects between 16 and 18 gestational weeks from our previous study (group 1), five subjects in late second trimester (group 2), and eight subjects at full term pregnancy (group 3). No genes were significantly differentially regulated between group 3 and group 2. We identified 545 probe sets that were significantly differentially expressed between group 1 and group 2 and 3 samples (FDR P-value <0.05). Based on tissue expression analysis, 396 genes that were upregulated in group 1 were enriched in the nervous system including brain and endocrine organs such as pancreas and adrenal gland. In addition, 136 genes that were upregulated in group 2 and 3 were specific to bronchioepithelial cells. Functional pathway analysis revealed that there was no significantly enriched pathway in terms of genes that were upregulated in either group 2 or group 3. Comparing the amniotic fluid cell-free transcriptome of group 1 and 2 with that of group 3, 18 genes were significantly differently modulated. CONCLUSIONS: Fetal development affects the amniotic fluid cell-free transcriptome. Fetal skin keratinization, which begins at 19 gestational weeks, might play an important role in changes in global gene expression in the amniotic fluid.

The methylome and transcriptome of fetal skin: implications for scarless healing.

AIM: Fetal skin is known to heal without scarring. In mice, the phenomenon is observed until the 16-17 day of gestation - the day of transition from scarless to normal healing. The study aims to identify key methylome and transcriptome changes following the transition. MATERIALS & METHODS: Methylome and transcriptome profiles were analyzed in murine dorsal skin using microarray approach. RESULTS & CONCLUSION: The genes associated with inflammatory response and hyaluronate degradation showed increased DNA methylation before the transition, while those involved in embryonic morphogenesis, neuron differentiation and synapse functions did so after. A number of the methylome alterations were retained until adulthood and correlated with gene expression, while the functional associations imply that scarless healing depends on epigenetic regulation.

Immunohistochemical Expression of Placental Form of Glutathione S-transferase (GST-pi) in Fetal Skin / 대한피부과학회지

BACKGROUND: Glutathione S-transferases(GST) are a family of multi-functional enzymes involved in cellular detoxification and excretion of a variety of exogenous and endogenous toxic or carcinogenic compounds. The GST family has been divided into three classes, alpha, mu, and pi, based on substrate specificity and sequence homology. GST-pi is an acidic type and predominant in skin, small intestine, breast, lung and prostate. The overexpression of GST-pi associated with skin tumor and tumor-like lesion suggests that GST-pi is a major detoxifying enzyme in skin tumors. OBJECTIVE: The purpose of this study was to observe the expression and the distribution pattern of GST-pi in the human fetal skin. METHODS: Skin was obtained from the scalp, chest, and sole of 49 human fetuses, ranging from 8th to 40th weeks of gestational age. Immunohistochemical staining was performed using avidin biotin peroxidase complex method on paraffin embedded tissue using antirabbit polyclonal antibody against the human GST-pi. RESULTS: GST-pi was expressed in intermediated layer of epidermis at 8th week, and gradually increased in strength of expression stronger in suprabasal layer. In hair unit, GST-pi was expressed in sebaceous gland, bulge, hair matrix cell and outer root sheath cell from 15th week. In eccrine gland, also GST-pi was expressed in central differentiated cells of intradermal eccrine duct from 18th week, and in terminal duct and acini from 26th week of fetal age. CONCLUSION: GST-pi was expressed from the 8th week of gestation suggesting that GST-pi plays an important role in detoxification for the protection of the skin in fetal stage from the various toxic agent.

Gene expression of mitogen-activated protein kinases in fetal skin at different developmental stages and its potentially biological siginificance / 解放军医学杂志

Objective To explore the change in gene expression of extracellular signal regulated protein kinase1 (erk1),erk2,p38MAPK and 3 c-Jun N- terminal kinases (jnk1,jnk2,jnk3) in fetal skin at different developmental stages and children skin. Methods After morphological characteristics of fetal skin at different developmental stages were examined histologically,gene expressions of erk1,erk2,p38MAPK,jnk1,jnk2 and jnk3 were determined with reverse transcription-polymerase chain reaction analysis (RT-PCR). Results Compared with early gestational fetal skin,the levels of gene expression of erk1 and erk2 showed no substantial change in late gestational fetal skin,while the contents of transcripts of p38MAPK and jnk1 were significantly decreased,the expressions of mRNA of jnk2 and jnk3 were obviously elevated. In children skin,gene expressions of erk2,p38MAPK and jnk1 were even more remarkably lowered. In contrary,gene expressions of jnk2 and jnk3 were marked enhanced. Conclusion The relative elevation of gene transcription of erk2 and p38MAPK and the inhibition of gene expression of jnk2 and jnk3 in fetal skin of earlier developmental stage might be related to fetal scarless healing.

Expression patterns and significance of different genes in developing stage of human fetal skin / 解放军医学杂志

Objective To comprehensively analyse the transcriptional changes of genes and their biologic significance that occurred in the process of development of human fetal skin by using high-density oligonucleotide DNA array. Methods Samples of human fetal skin were obtained from aborted fetuese of 10W, 15W, 24W, 32W EGA (estimated gestational age) respectively. Total RNAs were isolated from of skin specimens of fetuses of different EGA, and mRNAs were purified and labeled with incorporation of fluorescent dUTP to prepare the hybridization probes by using reverse transcription polymerase chain reaction (RT-PCR). Approximately 21 329 human genes were spotted on a chemical-material-coated-glass plate in array. Results According to the hybridization results from oligonucleotide DNA microarray, gene expresion patterns and functions were analysed. Gene-chip disclosed a large scale of information in developmental human fetal skin, rendering a convenient way to investigate the temporal and spatial expressions of gene profile among skin cells. Many specific genes transcription expressed differently at different stages of development of fetal skin, suggesting their key roles in development, differentiation and regulation. Conclusions Microarray or DNA chip technology has revolutioned biological research by empowering to broaden the scope of collecting genomic information. Therefore, microarray-based study is able to reveal a substantial number of genes which might participate in embryogenesis and development of human skin. The present study demonstrated a previously unrecongnized role of gene expression in the control of human fetal skin growth and structure during its developmental stage. A complicated network of skin development process was fairly well characterized.

CHARACTERISTICS OF INSULIN-LIKE GROWTH FACTOR-Ⅰ RECEPTOR EXPRESSION IN FETAL AND ADULT SKINS / 解放军医学杂志

Localization and expression of two subunits (IGF ⅠR? and IGF ⅠR?) of insulin like growth factor Ⅰ receptor and phosphorylated tyrosine proteins (P Tyr) in skins at different developmental stages were studied in order to explore their potential biological significance. Immunohistochemistry and pathological methods were used to detect the expression intensity and distribution of IGF ⅠR?, IGF ⅠR? and P Tyr in skins of 12 fetuses with different gestational ages and 8 adults. The results showed that positive immunohistochemical signals of IGF ⅠR?, IGF ⅠR? and P Tyr could be found in fetal and adult skins. Along with growth and development of the fetus, the positive cell rates of IGF ⅠR?, IGF ⅠR? and P Tyr in skins elevated progressively. In adult skins, IGF ⅠR was mainly located in the cell membrane of epidermal cells, while P Tyr was chiefly distributed in epidermal cells and some fibroblasts. These results suggested that IGF ⅠR and its mediating signaling pathway might be involved in cutaneous development at embryonic stage, in cutaneous structure and function maintenance, and in wound healing at postnatal stage.

CHANGE OF EXTRACELLULAR-SIGNAL REGULATED-PROTEIN KINASE1/2 SIGNALING PATHWAY IN SKIN AT DIFFERENT DEVELOPMENT STAGES / 解放军医学杂志

The purpose of this study was to investigate the localization and expression characteristics of phosphorylated form of extracellular-signal regulated-protein kinasel/2 (p-ERKl/2), Ras and C-fos in skin at different development stages and to explore their potential biological significance. Immunohistochemistry and pathological methods were used to detect the expression intensity and distribution of p-ERKl/2, Ras and C-fos in skin of 8 fetuses with different gestational ages (13 to 31 weeks) and 8 adults. Positive immunohistochemical signals of p-ERK1/2, Ras and C-fos. could be found in fetal and postnatal skins. Along with the increment in gestational age, the positive cell rates of p-ERK1/2, Ras and C-fos in the skin elevated progressively. In skins derived from the fetus of late-trimester pregnancy and adult, the positive rates of these three proteins were significantly increased in comparison with skin from the early-trimester fetus (P

Localization and expression of TGF-? _(1,2) in fetal and adult skins / 中国病理生理杂志

AIM: To explore the localization and expression of transfo rming grow th factor-? 1,2 (TGF-? 1,2 ) and alpha-smooth muscle actin (?- ASMA) in fetal a nd adult skins. METHODS: Skins of 15 cases of fetuses with different gestational ages and 5 cases of adults were taken, embedded with paraffin wax, and sectione d. Immunohistochemistry method and pathological method were used to detect the e xpression intensity and distribution of TGF-? 1,2 and ?-ASMA. RESULTS: Positive immunohistochemical signals of TGF-? 1,2 and ?-A SMA were found in fetal and adult skins. In skins derived from young fet us, the positive signals of these three proteins were very weak. Along with the incr ement in gestational age, the positive cellular rates of TGF-? 1,2 and ?- ASMA were elevated pro gressively. In elder fetal and adult skins, TGF-? 1,2 were mostly distributed i n epidermal cells, endothelial cells and some fibroblasts, while ?-ASMA was mainly located in myofibroblasts and sweat gland epithelial cells. CONCLUSION: The endo genous TGF-? 1,2 might be involved in the cutaneous development at embryoni c stage, in the cutaneous structure maintenance at adult stage, and in the wound healing af ter injury.

The Changes of Cytokeratin 19 and bcl-2 Expression during Hair Follicle Morphogenesis in Human Fetal Skin / 대한피부과학회지

BACKGROUND: During human skin development, hair follicles dynamically develop to adult hair structures through the hair germ, hair peg and bulbous hair peg. In this perspective, it is thought that the distributions of stem cells changes following the stage of the hair follicle morphogenesis. OBJECTIVE: The purpose of this study was to observe the distribution of stem cells following the stage of hair follicle morphogenesis. METHODS: Skin was obtained from the abdomen skin of 15 human fetuses, ranging from 10 to 23 weeks of gestational age. Immunoperoxidase staining was performed on frozen sections using monoclonal antibodies to cytokeratin 19, known as a stem cell marker, and bcl-2, a factor to prevent cells from entering the apoptotic pathway. RESULTS: 1. Cytokeratin 19 was expressed in the periderm, basal cell layer and condenced cells constituting the hair germ and peg. In the early bulbous hair peg stage, it was expressed in the entire outer root sheath cells, the bulge area and basal cells of the hair bulb. In the late bulbous hair peg stage, it was expressed in the bulge area, outer root sheath cells only of inferior portion and some of the germinative cells of the hair bulb. 2. Bcl-2 was expressed in the basal cells intermittently, condenced cells constituting the hair germ and aggregates of dermal mesenchymal cells concentrating along the hair germ. In the hair peg stage, it was expressed in the entire basal cell layer, condenced cells constituting the hair peg and aggregates of dermal mesenchymal cells along the hair peg. In t.he bulbous hair peg stage it was expressed in the basal cells of the infundibulum, sebaceous gland, bulge area, germinative cells of the hair bulb and the dermal papilla cells. CONCLUSION: From the results obtained, we thought that the distribution of stem cells is dynamically changing following the stages of the hair follicle morphogenesis. It was also speculated that bcl-2 might have an important role in dermoepidermal interactions during hair follicle morphogenesis.

Isolation and pure culture of microvascular endothelial cells from the fetal skin

Microvascular endothelial cells were purely isolated from human fetal skin using magnetic particles. The principle of this technique is based on the selective binding of the lectin Ulex europaeus I (UEA I) to the endothelial cell surface via fucose residues. Initially UEA I was covalently bound to tosyl-activated magnetic polydisperse polymer particles (Dynabeads) and then the UEA I-coated beads were collected using a magnetic particle concentrator (MPC). Endothelial cells were isolated by extracting microvascular segments from trypsin-treated fetal skin tissue and were purified by sieving with nylon mesh and by 35% Percoll gradient centrifugation. For further purification, the obtained cells were incubated with UEA I-coated Dynabeads. The endothelial cells bound to the Dynabeads were collected using MPC. This is a simple and reproducible technique for isolating a pure population of microvascular endothelium from the fetal skin.

Morphological Observations on the Epidermal Development of Human Fetal Skin

To observe developing process of human fetal skin during intrauterine life, morphological studies in light microscopic level were made based on 27 human embryos and 76 fetuses ranging from 4 to 40 gestation weeks. The fetuses were the products of induced abortion and were found to have no associated diseases of congenital anomalies at the autopsy. Ten different portions of the body were sampled and examined. They were scalp, forehead, face, chest, abdomen, back, palm, sole, finger and toe. In embryos two different portions; cephalic and caudal portions were examined: The following results were obtained: 1) A single layer of undifferentiated cell was the primitive epidermis at the 4th week and it was followed by two layered epidermis consisting of periderm and primitive basal cell layer. Epidermal ridges started to develop along with primitive eccrine and hair germs as clustering of basal cells at the llth week. Stratum inter-medium was formed at the 12th week, and primitive granular cell layers and keratin formation in association with hair follicles at the 19th week forming earliest adult type epidermis, followed by progressive maturation. 2) The thickness of the fetal epidermis and keratin layer increased as the fetal age approached to the term with its slightly different developmental pattern by the site of body. Cephalic protions developed slightly earlier than the other parts. 3) The developmental pattern of various portions of epidermis could be categorized into three groups; (1) scalp, forehead and face; (2) chest, abdomen and back; (3) palm, sole, finger and toe.