Effects of amniotic fluid on human keratinocyte gene expression: Implications for wound healing.

Cutaneous wounds can lead to huge suffering for patients. Early fetal wounds have the capacity to regenerate without scar formation. Amniotic fluid (AF), containing hyaluronic acid (HA), may contribute to this regenerative environment. We aimed to analyse changes in gene expression when human keratinocytes are exposed to AF or HA. Human keratinocytes were cultured to subconfluence, starved for 12 h and then randomised to be maintained in (1) Dulbecco's modified Eagle's medium (DMEM), (2) DMEM with 50% AF, or (3) DMEM with 50% fetal calf serum (FCS). Transcriptional changes were analysed using microarray and enriched with WebGestalt and Enrichr. Additionally, eight diagnostic genes were analysed using semiquantitative real-time PCR to investigate epidermal differentiation and cellular stress after HA exposure as an alternative for AF exposure. The AF and FCS treatments resulted in enrichment of genes relating to varied aspects of epidermal and keratinocyte biology. In particular, p63-, AP1- and NFE2L2- (Nrf2) associated genes were found significantly regulated in both treatments. More genes regulated by FCS treatment were associated with inflammatory signalling, whilst AF treatment was dominantly associated with molecular establishment of epidermis and lipid metabolic activity. HA exposure mostly resulted in gene regulation that was congruent with the AF microarray group, with increased expression of ITGA6 and LOR. We conclude that AF exposure enhances keratinocyte differentiation in vitro, which suggests that AF constituents can be beneficial for wound-healing applications.

Non-occlusive topical exposure of human skin in vitro as model for cytotoxicity testing of irritant compounds.

Testing of irritant compounds has traditionally been performed on animals and human volunteers. Animal testing should always be restricted and for skin irritancy mice and rabbits hold poor predictive value for irritant potential in humans. Irritant testing on human volunteers is restricted by the duration subjects can be exposed, and by the subjectivity of interpreting the visual signs of skin irritation. We propose an irritant testing system using viable human full thickness skin with the loss of cell viability in the exposed skin area as end point measurement. Skin was exposed to sodium dodecyl sulfate (SDS) at 20% concentration by non-occluded topical exposure to establish a positive control response and subsequent test compounds were statistically compared with the 20% SDS response. Cell viability and metabolism were measured with 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The model presents correlation between increased concentration of SDS and decreased viability of cells in the exposed skin area (R(2) = 0.76). We propose the model to be used for cytotoxicity testing of irritant compounds. With fully intact barrier function, the model comprises all cells present in the skin with quantifiable end point measurement.

Salinomycin induces activation of autophagy, mitophagy and affects mitochondrial polarity: differences between primary and cancer cells.

The molecular mechanism of Salinomycin's toxicity is not fully understood. Various studies reported that Ca(2+), cytochrome c, and caspase activation play a role in Salinomycin-induced cytotoxicity. Furthermore, Salinomycin may target Wnt/ß-catenin signaling pathway to promote differentiation and thus elimination of cancer stem cells. In this study, we show a massive autophagic response to Salinomycin (substantially stronger than to commonly used autophagic inducer Rapamycin) in prostrate-, breast cancer cells, and to lesser degree in human normal dermal fibroblasts. Interestingly, autophagy induced by Salinomycin is a cell protective mechanism in all tested cancer cell lines. Furthermore, Salinomycin induces mitophagy, mitoptosis and increased mitochondrial membrane potential (∆Ψ) in a subpopulation of cells. Salinomycin strongly, and in time-dependent manner decreases cellular ATP level. Contrastingly, human normal dermal fibroblasts treated with Salinomycin show some initial decrease in mitochondrial mass, however they are largely resistant to Salinomycin-triggered ATP-depletion. Our data provide new insight into the molecular mechanism of preferential toxicity of Salinomycin towards cancer cells, and suggest possible clinical application of Salinomycin in combination with autophagy inhibitors (i.e. clinically-used Chloroquine). Furthermore, we discuss preferential Salinomycins toxicity in the context of Warburg effect.

Differentiation of human dermal fibroblasts towards endothelial cells.

The ultimate goal of vascular tissue engineering is the production of functional grafts for clinical use. Difficulties acquiring autologous endothelial cells have motivated the search for alternative cell sources. Differentiation of dermal fibroblasts towards several mesenchymal lineages as well as endothelial cells has been proposed. The aim of the present study was to investigate the endothelial differentiation capacity of human dermal fibroblasts on a gene expression, protein expression and functional physiological level. Endothelial differentiation of fibroblasts was induced by culturing cells in 30% human serum, but not in fetal calf serum. Expression of proteins and genes relevant for endothelial function and differentiation was increased after induction. Furthermore, fibroblasts exposed to 30% human serum displayed increased uptake of low-density lipoprotein and formation of capillary-like networks. The results of this study may have an impact on cell sourcing for vascular tissue engineering, and the development of methods for vascularization of autologous tissue engineered constructs.

A Study on Symptoms of Pelvic Floor Dysfunction in Assigned Female at Birth Patients Diagnosed with Gender Dysphoria Undergoing Vaginectomy.

Background: A person diagnosed with gender dysphoria who was assigned female at birth (AFAB) may request a vaginectomy as part of gender-affirming treatment. The aim of this study was to investigate the impact of vaginectomy on symptoms of pelvic floor dysfunction (PFD). Methods: This is a cohort study on patient-reported symptoms of PFD in patients who were AFAB, diagnosed with gender dysphoria, and undergoing vaginectomy in a single surgical center. Patients responded to a questionnaire preoperatively and 1 year postoperatively. The questionnaire consisted of 33 questions, including a modified short-form version of the Pelvic Floor Distress Inventory (PFDI-20). Results: Twenty-three consecutive patients were included in the study and 20 patients (87%) completed the 1-year follow-up. The preoperative median PFDI-20 score was 24 (0-114) compared with 32 (0-168) at the 1-year follow-up (P = 0.07). Patients who had previously undergone neophallus construction with a metoidioplasty (n = 15) had no significant change between the preoperative and the 1-year postoperative PFDI-20 score [median 17.5 (0-114) and 27.5 (0-145) (P = 0.65), respectively]; whereas those with a groin flap phalloplasty (n = 5) had a significant increase in reported symptoms [median 37 (10-95) and 124 (45-168), respectively (P = 0.04)]. Conclusions: Overall, vaginectomy could be performed without any major impact on symptoms of PFD. However, this seemed to be true mainly for patients with previous metoidioplasty, whereas patients with previous groin flap phalloplasty reported worsening of symptoms.

Interpreted gene expression of human dermal fibroblasts after adipo-, chondro- and osteogenic phenotype shifts.

Autologous cell-based therapies promise important developments for reconstructive surgery. In vitro expansion as well as differentiation strategies could provide a substantial benefit to cellular therapies. Human dermal fibroblasts, considered ubiquitous connective tissue cells, can be coaxed towards different cellular fates, are readily available and may altogether be a suitable cell source for tissue engineering strategies. Global gene expression analysis was performed to investigate the changes of the fibroblast phenotype after four-week inductions toward adipocytic, osteoblastic and chondrocytic lineages. Differential gene regulation, interpreted through Gene Set Enrichment Analysis, highlight important similarities and differences of induced fibroblasts compared to control cultures of human fibroblasts, adipocytes, osteoblasts and articular chondrocytes. Fibroblasts show an inherent degree of phenotype plasticity that can be controlled to obtain cells supportive of multiple tissue types.

Transplantation of autologous cells and porous gelatin microcarriers to promote wound healing.

Definitive treatment to achieve wound healing in major burns frequently include skin transplantation, where split-thickness skin grafts is considered gold standard. This method is associated with several drawbacks. To overcome these hurdles, efforts have been made to develop tissue engineered skin substitutes, often comprised of a combination of cells and biomaterials. In the present study, we aimed to investigate transplantation of autologous keratinocytes and fibroblasts seeded on porous gelatin microcarriers using a porcine wound model. Pre-seeded microcarriers were transplanted to a total of 168 surgical full-thickness wounds (2cm diameter) on eight adult female pigs and covered with occlusive dressings. The experimental groups included wounds transplanted with microcarriers seeded with the combination of keratinocytes and fibroblasts, microcarriers seeded with each cell type individually, microcarriers without cells, each cell type in suspension, and NaCl control. Wounds were allowed to heal for one, two, four or eight weeks before being excised and fixated for subsequent histological and immunohistochemical analysis. In vitro, we confirmed that viable cells populate the surface and the pores of the microcarriers. In vivo, the microcarriers were to a large extent degraded after two weeks. After one week, all treatment groups, with the exception of microcarriers alone, displayed significantly thicker neo-epidermis compared to controls. After two weeks, wounds transplanted with microcarriers seeded with cells displayed significantly thicker neo-epidermis compared to controls. After four weeks there was no difference in the thickness of neo-epidermis. In conclusion, the experiments performed illustrate that autologous cells seeded on porous gelatin microcarriers stimulates the re-epithelialization of wounds. This method could be a promising candidate for skin transplantation. Future studies will focus on additional outcome parameters to evaluate long-term quality of healing following transplantation.

Adipogenic, chondrogenic and osteogenic differentiation of clonally derived human dermal fibroblasts.

The apparent need of an autologous cell source for tissue engineering applications has led researchers to explore the presence of cells with stem cell plasticity in several human tissues. Dermal fibroblasts (FBs) are easy to harvest, expand in vitro and store, rendering them plausible candidates for cell-based therapies. The aim of the present study was to observe the effects of adipogenic, chondrogenic and osteogenic induction media on the phenotype of human FBs. Human preadipocytes obtained from fat tissue have been proposed as an adult stem cell source with suitable characteristics, and were used as control cells in regard to their differentiation potential. Routine staining, immunohistochemical analysis and alkaline phosphatase assay were employed, in order to study the phenotypic shift. FBs were shown to possess multilineage potential, giving rise to fat-, cartilage- and bone-like cells. To exclude contaminant progenitor cells or cell fusion giving rise to tissue with adipocyte-, chondrocyte- and osteoblast-like cells, single-cell cloning was performed. Single-cell-cloned FBs (sccFBs) displayed a similar differentiation potential as primary-culture FBs. The presence of 'stem-cell-specific' surface antigens was analyzed using flow cytometry. The results reveal that sccFBs have several of the markers associated with cells exhibiting stem cell plasticity. The findings presented here are corroborated by the findings of other groups, and suggest the use of human dermal FBs in cell-based therapies for the reconstruction of fat, cartilage and bone.

[Sex reassignment surgery in transsexuals]. / Kjønnskorrigerende kirurgi ved transseksualisme.

BACKGROUND: Rikshospitalet (The National Hospital) treats all transsexual patients in Norway. 50-70 patients are referred annually for assessment and about 20 of these are diagnosed as transsexual. Approximately 15 confirmed transsexuals, who have received hormone treatment, are referred to sex reassignment surgery per year. This article describes diagnosis and treatment of transsexualism with an emphasis on surgery. MATERIAL AND METHODS: The article is based on a non-systematic review of the literature and our own clinical experience. RESULTS: Patients are evaluated for sex reassignment surgery after at least one year of psychiatric evaluation and an additional year of endocrine treatment. Male-to-female patients are evaluated for breast augmentation if hormone therapy has given unsatisfactory results. Thereafter, genital surgery can be performed, i.e. the testis and corpus cavernosum are removed and a neovagina and neoclitoris are constructed. Female-to-male patients have the possibility to undergo breast reduction, hysterectomy, salpingoophorectomy and construction of a neopenis. If the patient responds well to male hormones, the clitoris can be straightened and reconstructed as a small neopenis with an erectile function (metaidoioplasty). Alternatively, phalloplasty is performed using the local groin flap or a microvascular free flap. INTERPRETATION: Sex reassignment surgery mainly consists of operations on breasts and genitalia.

Human dermal fibroblasts: a potential cell source for endothelialization of vascular grafts.

BACKGROUND: Recently, there has been an intense ongoing search for suitable cell sources for vascular tissue engineering. Previous studies report that cells with multilineage potential have been found within the connective stroma of the skin. In line with this, preliminary data from our group suggest that human dermal fibroblasts have the capacity to alter their phenotype into an endothelial cell-like phenotype in vitro. As a first step in using these cells in vascular tissue engineering, we investigated their ability to form an endothelial cell-like layer on a scaffold in vitro. Furthermore, we studied the possibility of seeding dermal fibroblasts on a scaffold and later commencing with induction toward an endothelial cell-like phenotype. METHODS: Cells cultured in either normal fibroblast medium or endothelial induction medium were seeded on a gelatin-based scaffold. To study the organization of cells, routine staining was performed. Differentiation was confirmed by Western blotting and immunohistochemistry with antibodies directed toward molecules commonly used to identify endothelial cells. RESULTS AND CONCLUSION: Our data support that human dermal fibroblasts differentiated toward endothelial cell-like cells prior to seeding showed histological resemblance to mature endothelial cells, while fibroblasts seeded and later induced into endothelial differentiation grew in multilayer. However, expression of various surface molecules indicative of an endothelial phenotype was seen using both techniques. In conclusion, the results presented in this study indicate that human dermal fibroblasts differentiated toward an endothelial cell-like phenotype may be a novel cell source for endothelialization of vascular grafts.

Accumulation of Silver and Delayed Re-epithelialization in Normal Human Skin: An ex-vivo Study of Different Silver Dressings.

Silver is commonly used in wound dressings and topical formulations to assist in the management of wounds that are infected or at risk of becoming infected. They provide potent broad-spectrum antimicrobial activity, but should not cause sustained staining of the skin, dermal or systemic accumulation of silver, or discomfort to the patient. However, clinicians and healthcare personnel have been concerned about topical staining of the skin and complaints of additional pain from patients treated with certain silver dressings. Some delay in re-epithelialization has also been noticed and reported. The reasons for this are not clear, and the authors believed further study regarding the possible effects of silver accumulation and silver dressings' effect on re-epithelialization was required. The authors studied possible silver accumulation and re-epithelialization in normal human dermal skin. The results showed that most of the dressings or treatments discolored the wound surface and that there was a dermal accumulation of what were assumed to be silver particles. Varying grades of accumulation were found in deep dermal tissue, particularly around blood vessels, depending on the dressing used. The results also indicated that all of the tested products delayed re-epithelialization in this model. .

Hyaluronic Acid Accelerates Re-epithelialization and Alters Protein Expression in a Human Wound Model.

BACKGROUND: Hyaluronic acid (HA), a large glycosaminoglycan involved in proliferation, migration, and tissue repair, is suggested to be an important factor for keratinocyte activation and re-epithelialization. The experimental hypothesis of this study was that HA accelerates re-epithelialization, and we aimed to investigate the effect of exogenous intradermal HA during deep dermal, incisional wound healing in vivo in humans, the primary endpoint being re-epithelialization. METHODS: A total of 8 standardized deep dermal incisional wounds (depth 1.6 mm, width 1.8 mm) per subject were induced in 10 healthy volunteers. Two of the wound sites per subject were pretreated with injections of HA and 2 with saline solution. At 2 time points (24 hours and 14 days), 2 biopsies for each treatment group (one for histology and one for proteomics) were taken. Skin erythema was measured at 24-hour intervals for 14 days as a surrogate measurement of inflammation. RESULTS: At 24 hours, 8 of 9 wounds pretreated with HA showed complete re-epithelization, whereas none of the wounds pretreated with saline had re-epithelized. Wounds pretreated with HA also showed a 10-fold regulation of 8 identified proteins involved in wound healing compared to wounds treated with saline solution. No difference in inflammation, as measured as erythema, could be seen between any of the groups. CONCLUSIONS: We conclude that HA accelerates re-epithelialization and stimulates an altered protein expression in vivo in human deep dermal incisional skin wounds, but has no effect on the inflammation process as measured by erythema.

Tracking keratinocytes and melanocytes using carboxyfluorescein hydroxysuccinimidyl ester staining.

INTRODUCTION: The treatment of burn wounds and hypopigmentation conditions often require autologous transplantation of keratinocytes and melanocytes. Tracking transplanted cells to ascertain their contribution to tissue recapitulation presents a challenge. This study demonstrates a methodology based on passive staining with carboxyfluorescein hydroxysuccinimidyl ester (CFSE) that enables localization of cells in tissue sections to investigate the fate of transplanted cells in wound re-epithelialisation. METHODS: Viability and migration of CFSE-stained keratinocytes and melanocytes were investigated using viability staining and scratch assays, while proliferation of cells was measured using flow cytometry. In addition, CFSE-stained keratinocytes and melanocytes were transplanted to a human ex vivo wound model, either in suspension, or with the aid of macroporous gelatine microcarriers. Wounds were analysed seven, 14 and 21 days post transplantation using cryosectioning and fluorescence microscopy. Sections from wounds with transplanted co-cultured keratinocytes and melanocytes were stained for pancytokeratin to distinguish keratinocytes. RESULTS: CFSE-staining of keratinocytes and melanocytes did not affect the viability, migration or proliferation of the cells. Transplanted cells were tracked in ex vivo wounds for 21 days, illustrating that the staining had no effect on wound re-epithelialisation. In conclusion, this study presents a novel application of CFSE-staining for tacking transplanted primary human keratinocytes and melanocytes.

Transplantation of cultured human keratinocytes in single cell suspension: a comparative in vitro study of different application techniques.

Transplantation of autologous cultured keratinocytes in single cell suspension is useful in the treatment of burns. The reduced time needed for culture, and the fact that keratinocytes in suspension can be transported from the laboratory to the patient in small vials, thus reducing the costs involved and be stored (frozen) in the clinic for transplantation when the wound surfaces are ready, makes it appealing. We found few published data in the literature about actual cell survival after transplantation of keratinocytes in single cell suspension and so did a comparative in vitro study, considering commonly used application techniques. Human primary keratinocytes were transplanted in vitro in a standard manner using different techniques. Keratinocytes were counted before and after transplantation, were subsequently allowed to proliferate, and counted again on days 4, 8, and 14 by vital staining. Cell survival varied, ranging from 47 to >90%, depending on the technique. However, the proliferation assays showed that the differences in numbers diminished after 8 days of culture. Our findings indicate that a great number of cells die during transplantation but that this effect is diminished if cells are allowed to proliferate in an optimal milieu. A burned patient's wounds cannot be regarded as the optimal milieu, and using less harsh methods of transplantation may increase the take rate and wound closing properties of autologous keratinocytes transplanted in a single cell suspension.

Employing human keratinocytes cultured on macroporous gelatin spheres to treat full thickness-wounds: an in vivo study on athymic rats.

Providing cutaneous wounds with sufficient epidermis to prevent infections and fluid loss is one of the most challenging tasks associated with surgical treatment of burns. Recently, application of cultured keratinocytes in this context has allowed this challenge to be met without several of the limitations connected with the use of split-thickness skin grafts. The continuous development of this novel approach has now revealed that transplantation of cultured autologous keratinocytes as single-cell suspensions exhibits several advantages over the use of cultured epidermal grafts. However, a number of methodological problems remain to be solved, primarily with regards to the complexity of culturing these cells; loss of viability and other negative effects during their preparation and transportation; the relatively long period of time required following transplantation to obtain a sufficiently protective epidermis. In the present investigation we attempted to eliminate these limitations by culturing the keratinocytes on macroporous gelatin spheres. Accordingly, the efficacies of normal human keratinocytes in single-cell suspension or growing on macroporous gelatin spheres, as well as of split-thickness skin grafts in healing wounds on athymic rats were compared. Human keratinocytes were found to adhere and proliferate efficiently both on the surface and within the pores of such spheres. Transplantation of such cells adherent to the spheres resulted in significantly more rapid formation of a stratified epidermis than did transplantation of single-cell suspensions or spheres alone. Twenty-three days after transplantation, the epidermis formed from the cells bound to the spheres was not as thick as the epidermis on wounds covered with split-thickness skin grafts, but significantly thicker than on wounds to which single-cell suspensions, spheres alone or no transplant at all was applied. Furthermore, fluorescence in situ hybridisation revealed that the transplanted keratinocytes, both those adherent to gelatin spheres and those in single-cell suspension, were components of the newly formed epidermis. These findings indicate that application of biodegradable macroporous spheres may prove to be of considerable value in designing cell-based therapies for the treatment of acute and persistent wounds.

Expression and growth dependency of the insulin-like growth factor I receptor in craniopharyngioma cells: a novel therapeutic approach.

Craniopharyngioma is a rare benign intracranial epithelial tumor that, however, often recurs and sometimes kills the affected patients, one-third of which are children. In many cases, the patients acquire growth hormone deficiency and postoperatively need substitution. Generally, growth hormone promotes local release of insulin-like growth factor I (IGF-I), which in turn activates the IGF-I receptor (IGF-IR) if present. Together, these circumstances raise the question whether IGF-IR may be involved in craniopharyngioma growth. To address this issue, we analyzed phenotypically well-characterized primary low-passage craniopharyngioma cell lines from nine different patients for IGF-IR expression and IGF-I dependency. Two of the cell lines showed no/very low expression of the receptor and was independent on IGF-I, whereas five cell lines exhibited a strong expression and was clearly contingent on IGF-I. The two remaining cell lines had low receptor expression and IGF-I dependency. Upon treatment with an IGF-IR inhibitor, cells with high IGF-IR expression responded promptly with decreased Akt phosphorylation followed by growth arrest. These responses were not seen in cells with no/very low receptor expression. Growth of cell lines with low IGF-IR expression was only slightly affected by IGF-IR inhibition. Taken together, our data suggest that IGF-IR may be involved in the growth of a subset of craniopharyngiomas and points to the possibility of the involvement of IGF-IR inhibitors as a treatment modality to obtain complete tumor-free conditions before growth hormone substitution.

Influence of acidic pH on keratinocyte function and re-epithelialisation of human in vitro wounds.

BACKGROUND: Chronic wounds are one of the greatest challenges for the healthcare system. Today, a plethora of dressings are used in the treatment of these wounds, each with specific influence on the wound environment. Due to differences in the permeability of the dressings the use will result in differences in the pH balance in the wound bed. However, little is known about how changes in the pH in the wound environment affect the different phases of the healing process. AIM: The aim of the present study was to investigate the effects of acidic pH on the regeneration phase by studying keratinocyte function in vitro and re-epithelialisation in an in vitro model of human skin. RESULTS: In vitro assays showed reduced viability and migration rates in human keratinocytes when pH was lowered. Real time PCR revealed differential expression of genes related to wound healing and environmental impairment. Tissue culture showed no re-epithelialisation of wounds subjected to pH 5.0 and moderate re-epithelialisation at pH 6.0, compared to controls at pH 7.4. CONCLUSION: The results indicate that lowering pH down to pH 5.0 in wounds is counterproductive in aspect of keratinocyte function which is crucial for successful wound healing.

Human melanocytes mitigate keratinocyte-dependent contraction in an in vitro collagen contraction assay.

Scarring is an extensive problem in burn care, and treatment can be especially complicated in cases of hypertrophic scarring. Contraction is an important factor in scarring but the contribution of different cell types remains unclear. We have investigated the contractile behavior of keratinocytes, melanocytes and fibroblasts by using an in vitro collagen gel assay aimed at identifying a modulating role of melanocytes in keratinocyte-mediated contraction. Cells were seeded on a collagen type I gel substrate and the change in gel dimensions were measured over time. Hematoxylin & Eosin-staining and immunohistochemistry against pan-cytokeratin and microphthalmia-associated transcription factor showed that melanocytes integrated between keratinocytes and remained there throughout the experiments. Keratinocyte- and fibroblast-seeded gels contracted significantly over time, whereas melanocyte-seeded gels did not. Co-culture assays showed that melanocytes mitigate the keratinocyte-dependent contraction (significantly slower and 18-32% less). Fibroblasts augmented the contraction in most assays (approximately 6% more). Non-contact co-cultures showed some influence on the keratinocyte-dependent contraction. Results show that mechanisms attributable to melanocytes, but not fibroblasts, can mitigate keratinocyte contractile behavior. Contact-dependent mechanisms are stronger modulators than non-contact dependent mechanisms, but both modes carry significance to the contraction modulation of keratinocytes. Further investigations are required to determine the mechanisms involved and to determine the utility of melanocytes beyond hypopigmentation in improved clinical regimes of burn wounds and wound healing.

Biodegradable Gelatin Microcarriers Facilitate Re-Epithelialization of Human Cutaneous Wounds - An In Vitro Study in Human Skin.

The possibility to use a suspended tridimensional matrix as scaffolding for re-epithelialization of in vitro cutaneous wounds was investigated with the aid of a human in vitro wound healing model based on viable full thickness skin. Macroporous gelatin microcarriers, CultiSpher-S, were applied to in vitro wounds and cultured for 21 days. Tissue sections showed incorporation of wound edge keratinocytes into the microcarriers and thicker neoepidermis in wounds treated with microcarriers. Thickness of the neoepidermis was measured digitally, using immunohistochemical staining of keratins as epithelial demarcation. Air-lifting of wounds enhanced stratification in control wounds as well as wounds with CultiSpher-S. Immunohistochemical staining revealed expression of keratin 5, keratin 10, and laminin 5 in the neoepidermal component. We conclude that the CultiSpher-S microcarriers can function as tissue guiding scaffold for re-epithelialization of cutaneous wounds.

The expression pattern of PFKFB3 enzyme distinguishes between induced-pluripotent stem cells and cancer stem cells.

Induced pluripotent stem cells (iPS) have become crucial in medicine and biology. Several studies indicate their phenotypic similarities with cancer stem cells (CSCs) and a propensity to form tumors. Thus it is desirable to identify a trait which differentiates iPS populations and CSCs. Searching for such a feature, in this work we compare the restriction (R) point-governed regulation of cell cycle progression in different cell types (iPS, cancer, CSC and normal cells) based on the expression profile of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase3 (PFKFB3) and phosphofructokinase (PFK1). Our study reveals that PFKFB3 and PFK1 expression allows discrimination between iPS and CSCs. Moreover, cancer and iPS cells, when cultured under hypoxic conditions, alter their expression level of PFKFB3 and PFK1 to resemble those in CSCs. We also observed cell type-related differences in response to inhibition of PFKFB3. This possibility to distinguish CSC from iPS cells or non-stem cancer cells by PFKB3 and PFK1 expression improves the outlook for clinical application of stem cell-based therapies and for more precise detection of CSCs.