OBJECTIVETo identify the localization of hair follicles stem cell (HFSC) in different stages of hair and explore the differentiating capacity of HFSC into epidermis in vitro.

METHODSHFSC were detected by K19 immunostaining in normal human skin. Then, the isolated HFSC through enzyme digestion were seeded on dermal equivalent (DE) and cultured between the air-liquid interfaces for 14 days. Skin-equivalents was harvested and used for evaluation.

RESULTSHFSC mainly located in outer root sheet in hair follicle and human anagen hair follicles containing two distinct reservoirs for K19-positive cells located in the bulge and bulb of the follicle. These two reservoirs fused in line of outer root sheets during the catagen-telogen transition phase and individualized again in the newly forming anagen hair follicle. Based on DE, growing HFSC built a multilayered and confined epidermis.

CONCLUSIONHFSC located in outer root sheets can promote hair cycle and differentiate into epidermis in vitro.

Cell Differentiation ; physiology ; Cells, Cultured ; Epidermis ; cytology ; Hair Follicle ; cytology ; Humans ; Stem Cells ; cytology

OBJECTIVETo observe the differentiation and distribution of epidermal stem cell (ESC) after skin soft tissue expansion, and to initially probe into the growth mechanism of expanded skin tissue.

METHODSSamples of normal skin and expanded skin (mean effusion period 45 days) were harvested from head and cervical region in 15 patients who underwent II stage surgery after skin expansion. Samples were divided into scalp adjacent to the center of expander group (expanded scalp, 3 cm from the vertical axis of the expander), scalp from lateral part of the expander group (expanded scalp, 5 - 7 cm lateral to the vertical axis of the expander), cervical skin expansion group, un-expanded scalp control group, and un-expanded cervical skin control group, according to the position of skin harvested. The tissue structure of skin in each group was observed with HE staining, and the differentiation and distribution characteristics of cytokeratin 19 (CK19) positive cells were observed with immunohistochemical staining.

RESULTSCompared with those in the un-expanded control groups, uneven, relatively thickened and obviously folded epidermis with more cell layers and cells with obvious aggregation close to the basal layer were observed in the expanded groups, but those cells were not well-arranged and the transition of polarity was not obvious. The continuity of CK19 positive cells in the basal layer of skin was observed in each of the expanded group with immunohistochemical staining, and positive cells increased obviously and arranged in multilayer in certain parts of basal layer. Clustered or dispersed CK19 positive cells were also observed outside the basal layer. No above-mentioned phenomenon was observed in the un-expanded control group.

CONCLUSIONSThe proliferation and differentiation of ESC with ectopic distribution may enhance the repair process after skin soft tissue expansion.

Cell Proliferation ; Dermis ; cytology ; Epidermis ; cytology ; Humans ; Stem Cells ; cytology ; Tissue Expansion ; Wound Healing

An experimental study concerning the effect of chronic irritation of corneocytes was made in relation to their number, size and shape. The desquamating portion of the stratum corneum was sampled with the detergent scrub technique using Triton X-100. The experimental subjects were scrub nurses who had worked in the operating room for more than 3 years and ward nurses were used as a control group. The corneocytes of skin irritated by daily scrubbing differed, from those of the non-irritated skin of the ward nurses. About twice as many cells were collected per cm2 skin surface from the scrub nurses on the first experimental day. Two and four days later the number was markedly decreased and became similar to that of the control group. The surface of the corneocytes was 15% smaller in the experimental group than that of the control group, through out the experiment. There was no significant difference between the two groups as regards corneocyte morphology.
Adult ; Epidermis/cytology* ; Female ; Handwashing* ; Human ; Soaps* ; Surface-Active Agents*

This study was designed to investigate the effects on count, size, and morphology of human corneocytes when the skin is irritated with a rough towel. The desquamating portion of the stratum corneum was sampled with the detergent scrub technique every other day for 12 days. The following parameters were measured; numerical count, size (surface (mu)2), and shape (regular, irregular). Corneocytes from skin irritated with a rough towel differed from those of normal skin in that they were diminished by about 60% in count and were 14% smaller in size on the first experimental day. These parameters became normalized in count after 10 days and in size after 5 days respectively. On the morphological classification of the cell outlines, there were no significant differences from the control groupts except that there were slightly increased numbers of irregular cells in the experimental groups.
Adult ; Baths* ; Bedding and Linens* ; Epidermis/cytology* ; Human ; Male

OBJECTIVETo investigate the feasibility of construction of tissue-engineered skin in vitro.

METHODSFibroblasts were seeded on the dermal surface of acellular dermal matrix (ADM). 7 days later, epidermal cells (5 x 10(5)/cm2) were mixed with fibroblasts (0.2 x 10(5)/cm2) and then seeded on epidermal surface of ADM. The culture medium was the mixture liquor containing K-SFM in half and the culture supernatants of fibroblasts in half. In the control group, only epidermal cells (5 x 10(5)/cm2) were seeded and cultured with K-SFM. After composite skin was cultured for 1 week and 3 week, samples were harvested respectively for morphological study and to receive identification by immunohistochemistry.

RESULTSAfter 3-week culture, there were 3-4 continuous layers of cells in the epidermis in the experiment group. The epidermis was attached tightly to the dermis with trochanterellus. But in the control group, there were just 1-2 layers of cells in the epidermis which was not connected to the dermis. Strong positive dye of Laminin indicated that basement membrane was thoroughly formed in the experiment group. The same result was demonstrated by transmission electron microscope.

CONCLUSIONSThe mix-seeding of epidermal cells and few fibroblasts would promote the adhesion and proliferation of epidermal cells on ADM, and the formation of basement membrane.

Animals ; Cells, Cultured ; Coculture Techniques ; Epidermis ; cytology ; Fibroblasts ; cytology ; Skin, Artificial ; Swine ; Tissue Engineering ; Tissue Scaffolds

UNLABELLED[Abstract]

OBJECTIVEThe study was (1) to investigate the biological character changes of human epidermal cells during proliferation culture in vitro and (2) to provide data for construction of engineered skin.

METHODSThe foreskin was collected from 20 healthy children. The epidermal cells were isolated with digestion of the foreskin and cultured in vitro. Growth curve was obtained from the data of cell counting. Cell growth kinetics was observed. Meanwhile, clonal analysis and cell size measurement was performed. The rate of keratin 19 (K19) and involucrin expression-positive cells was counted by flow cytometer. Expression of K19 and involucrin mRNA was detected by RT-PCR.

RESULTSWhen passaged to P6, the keratinocytes from child foreskin could be expanded by (700 +/- 37) times. Flow cytometer results showed that the percentage of K19 expression-positive cells decreased from (66.97 +/- 3.14)% to (4.65 +/- 1.38)% while the percentage of involucrin expression-positive cells increased from (11.65 +/- 1.62)% to (97.03 +/- 2.66)% at P0 and P6 respectively. RT-PCR results showed that expression of K19 mRNA decreased from P0 to P6 while involucrin mRNA kept stable with passage in vitro.

CONCLUSIONSHuman epidermal cells of passage 5 maintain proliferation phenotype, which are suitable for skin tissue engineering. Decrease of proliferation phenotype content is partially responsible for the proliferation capacity loss of in vitro cultured epidermal cells.

Cell Proliferation ; Cells, Cultured ; Child ; Epidermis ; cytology ; Foreskin ; cytology ; Humans ; Male ; Tissue Engineering ; methods

OBJECTIVETo study the promotion effect of stromal cell-derived factor 1 (SDF-1) on the migration of epidermal stem cells (ESC) in the healing process of frostbite-wound model ex vivo.

METHODSA three-dimensional model of full-thickness frostbite of skin was constructed (with slot-like wound) out of skin equivalent. The expression of SDF-1 in wound stroma was observed with immunohistochemistry staining on post injury days (PID) 3 and 7. The model frostbite wounds were divided into control group (treated with PBS 50 microL per wound), SDF-1 group (treated with 100 ng/mL SDF-1, 50 microL per wound), and AMD3100 group [treated with 100 ng/mL AMD3100 (50 microL per wound) for 30 minutes, and then SDF-1 50 microL was added per wound]. The redistribution of ESC around wound was observed.

RESULTSThe expression of SDF-1 in wound stroma increased gradually on PID 3 and 7. Compared with those in control and AMD3100 groups, there were more ESC and epithelial cell layers, and more integrin beta(1)-positive cells appeared at the basal layer of wound in SDF-1 group, and some of the positive cells migrated upward to epidermis.

CONCLUSIONSSDF-1 contributes to wound repair through promoting ESC to migrate toward and gather around wound edge. This may be one of the mechanisms of ESC participating in wound repair.

Cell Movement ; Chemokine CXCL12 ; metabolism ; Epidermis ; cytology ; Frostbite ; metabolism ; therapy ; Humans ; Stem Cells ; cytology ; Wound Healing

OBJECTIVETo explore the mechanisms of hair follicle regeneration by injection of follicular cells isolated from murine skin.

METHODSEpidermis was peeled off from the dermis of 3-5 d C57BL/6J mouse by 0.2% Dispase digestion at 37 degrees C for 2 hours. Dermis was cut into small pieces and digested in 0.2% collagenase at 37 degrees C for 30 minute with low speed stirring to isolate hair follicles from dermis. Hair follicles were collected through filtration, low-speed centrifugation and density gradient centrifugation. Collagenase and trypsin were added to digest hair follicles into dissociated cells which were marked by Dio and injected into the nude mouse skin.

RESULTS2 d after intradermal injection of hair follicle cells, a cyst was formed containing lots of round and elliptical cells and homogeneous eosin stained cell-free tissues. The cyst wall was composed of many spindle shaped fibroblast cells and showed sparsely localized green fluorescence. The contents of the cyst showed bright green fluorescence. 4 d after injection, the skin became slightly thicken with grey appearance, a lots of hair follicles formed with black bulb. 1 weeks after injection, the injection site became black and evaluated with a lots of black hair follicles and hyperproliferation of capillary blood. Newly formed hair follicles showed bright green fluorescence. 3 weeks after injection, a cyst containing lots of black hairs formed in the injection site. Newly formed hair follicles showed positive for Dio. Sebaceous gland can be seen accompanied with hair follicles. 6 weeks after injection, the cyst contained lots of sheded club hair shafts and hair follicles on the stage of anagen. Cultured follicular cells and injection below 1 x 10(5) failed to regenerate hairs. While the regenerated hair follicle was few when the hair follicle cells were injected subcutaneously.

CONCLUSIONSFollicular cells can aggregate spontaneously and develop synergistically into hair follicles with normal growth cycle after implantation. The regeneration depends on the interactions between follicular cells, as well as on the recipient sites and cell numbers.

Alopecia ; surgery ; Animals ; Cell Transplantation ; methods ; Dermis ; cytology ; Epidermis ; cytology ; Hair Follicle ; cytology ; Injections, Intradermal ; Mice ; Mice, Inbred C57BL ; Mice, Nude ; Regeneration ; Skin ; cytology ; Tissue Engineering ; methods

OBJECTIVETo investigate the migrating effect of adipose derived stem cells (ADSCs) on the wound model of human epidermal keratinocyte (HEKa).

METHODSRat ADSCs (rADSCs) were isolated and cultured (n = 10), rADSCs were direct co-cultured with HEKa cells in experiment group (experimental group, n = 10). In the control groups, rADSCs were indirect co-cultured with HEKa cells in transwell chamber (indirect group, n = 8), or HEKa was cultured alone (single group, n = 8). Then confluent HEKa cells were scraped to establish a wound model under invert microscope. After scraped 24, 48, and 72 h, cell numbers of which migrated across the edge of the wound was measured, the rate of wound healing was calculated by using SigmaScan Pro 5 software, and the proliferating effect of rADSCs on HEKa were examined by incorporation of [(3)H] thymidine.

RESULTSThe cells migrated across the edge of wound after 24 hours in experimental group, indirect group, and single group were (9.2 + or - 0.2), (5.0 + or - 0.3), (4.2 + or - 0.3), and were (58.5 + or - 0.4), (26.5 + or - 0.3), (20.7 + or - 0.5) 48 hours after, and were (125.8 + or - 0.4), (43.0 + or - 0.5), (35.6 + or - 0.5) cells/HP 72 hours after, respectively; the numbers were all significantly higher in experimental group than those in control groups (P < 0.05). The rates of wound healing after scraped 72 hours were 61.0% + or - 3.0%, 35.0% + or - 2.5% and 32.0 + or - 2.1%, the outcome in experimental group was significantly better than in the control groups (P < 0.05). And the thymidine feeding displayed the proliferation of HEKa in the three groups were (1440 + or - 210), (1050 + or - 280) and (1130 + or - 390) cpm/10(5) cell, and there was significant difference between the experimental and the control groups (P < 0.05).

CONCLUSIONSThe rADSCs can promote the migration of HEKa by direct contact with it.

Adipose Tissue ; cytology ; Animals ; Cell Count ; Cell Proliferation ; Cells, Cultured ; Coculture Techniques ; Epidermis ; cytology ; Humans ; Keratinocytes ; cytology ; Rats ; Rats, Sprague-Dawley ; Stem Cells ; cytology ; Wound Healing

OBJECTIVETTo explore the method for inducing the dedifferentiation of epidermal cells into their progenitor stem cells in vitro without external gene intervention.

METHODSHEK cells obtained from Casacade were induced to reverse their differentiated process and produce immature stem-like cells, namely the dedifferentiation derived epidermal stem cells (dESCs), by induction with basic fibroblasts growth factors (bFGF) in vitro. Immunochemical staining, flow FACS analysis, RT-PCR and immunofluorescent staining were used to detect the phenotypic and functional changes of the differentiated epidermal cells, using human epidermal stem cells (ESCs) as the positive control.

RESULTSImmunohistochemical staining revealed that the expressions of β₁-integrin, CK19 and CK14 were up-regulated, while CK10 expression was down-regulated significantly after bFGF treatment. Two-color flow cytometric analysis of α₆-integrin and CD71 showed that the percentages of α₆(+)CD71(-), α₆(+)CD71(+) and CD71(+) expressing populations reached 13.24%, 58.26% and 23.12% of the total isolated cells, as compared with those of the control (0.12%, 3.06%, 51.50%) and positive control cells (37.49%, 45.13%, 5.86%). RT-PCR analysis indicated that the relative gene expressions of β₁-integrin, CK19 and CK14 increased in bFGF treatment group, whereas the expression of CK10 was significantly suppressed. Although there was no significant difference in the expression levels of β₁ integrin, CK19 and CK10 between the bFGF-treated and the positive controls, the expression of CK14 in bFGF-treated cells showed a 1.4-fold increase as compared with that in ESCs (P < 0.05). Immunofluorescent staining showed that a regional difference in the subcellular localization of telomerase between dESCs and ESCs.

CONCLUSIONbFGF can induce the epidermal cells to convert into epidermal precursor cells. Although they are more likely to be transient amplifying cells, the method for reprogramming somatic epidermal cells into their progenitors by bFGF induction other than genetic manipulation offers a new approach to generate residual healthy stem cells for wound repair and regeneration.

Cell Dedifferentiation ; Cell Proliferation ; Cells, Cultured ; Epidermis ; cytology ; Fibroblast Growth Factor 2 ; pharmacology ; Humans ; Induced Pluripotent Stem Cells ; cytology