Generation of full-thickness skin equivalents using hair follicle-derived primary human keratinocytes and fibroblasts

Skin equivalents are increasingly used as human-based test systems for basic and preclinical research. Most of the established skin equivalents are composed of primary keratinocytes and fibroblasts, isolated either from excised human skin or juvenile foreskin following circumcisions. Although the potential of hair follicle-derived cells for the generation of skin equivalents has been shown, this approach normally requires microdissections from the scalp for which there is limited subject compliance or ethical approval. In the present study, we report a novel method to isolate and cultivate keratinocytes and fibroblasts from plucked hair follicles that were then used to generate skin equivalents. The procedure is non-invasive, inflicts little-pain, and may allow easy access to patient-derived cells without taking punch biopsies. Overall, minor differences in morphology, ultrastructure, expression of important structural proteins, or barrier function were observed between skin equivalents generated from hair follicle-derived or interfollicular keratinocytes and fibroblasts. Interestingly, improved basal lamina formation was seen in the hair follicle-derived skin equivalents. The presented method here allows easy and non-invasive access to keratinocytes and fibroblasts from plucked hair follicles that may be useful particularly for the generation of skin disease equivalents

Generation of full‐thickness skin equivalents using hair follicle‐derived primary human keratinocytes and fibroblasts

Skin equivalents are increasingly used as human-based test systems for basic and preclinical research. Most of the established skin equivalents are composed of primary keratinocytes and fibroblasts, isolated either from excised human skin or juvenile foreskin following circumcisions. Although the potential of hair follicle-derived cells for the generation of skin equivalents has been shown, this approach normally requires microdissections from the scalp for which there is limited subject compliance or ethical approval. In the present study, we report a novel method to isolate and cultivate keratinocytes and fibroblasts from plucked hair follicles that were then used to generate skin equivalents. The procedure is non-invasive, inflicts little-pain, and may allow easy access to patient-derived cells without taking punch biopsies. Overall, minor differences in morphology, ultrastructure, expression of important structural proteins, or barrier function were observed between skin equivalents generated from hair follicle-derived or interfollicular keratinocytes and fibroblasts. Interestingly, improved basal lamina formation was seen in the hair follicle-derived skin equivalents. The presented method here allows easy and non-invasive access to keratinocytes and fibroblasts from plucked hair follicles that may be useful particularly for the generation of skin disease equivalents

Corpus luteal angiogenesis in a high milk production dairy breed differs from that of cattle with lower milk production levels

Globally, high producing, young dairy cows often have health and fertility problems such as ovarian, uterine and placental dysfunction, mastitis and impaired wound healing. These health issues can result in a shortened average lifespan of fewer than three lactations. We hypothesise that many of these health issues may be an effect of altered angiogenesis. Hence, the aim of this pilot study was to investigate the status of vascularisation in the corpus luteum of a high milk-producing cattle breed (Holstein Friesian) compared with that of low milk producing beef cattle (Limousin) and of dual purpose cattle (Fleckvieh, Rotbunt). The corpus luteum was chosen because as a transient endocrine gland it is one of the few tissues with physiological angiogenesis in the adult. Blood vessels were labelled in paraffin sections of corpora lutea with the lectin Bandeiraea simplicifolia agglutinin I and the following angiogenesis parameters were analysed morphometrically using image analysis: (a) number of blood capillaries per mm(2), (b) intercapillary distance, (c) percentage area occupied by blood vessels and (d) area of blood vessel lumina (mu m(2)). This analysis revealed that the extent of corpus luteal vascularisation is greater in high milk-producing cattle than in beef and dual purpose cattle as shown by a significantly higher number of blood capillaries per mm(2), significantly shorter intercapillary distances and a higher percentage of the corpus luteum area covered by blood vessels (non-significant). Only the average value of the luminal area of the luteal blood capillaries in the high milk-producing cattle is smaller than that measured in the low producing cattle

The effect of endothelialization on the epidermal differentiation in human three-dimensional skin constructs – A morphological study

INTRODUCTION: Inducing vascularization in three-dimensional skin constructs continues to be difficult. In this study, two variations of human full-thickness skin constructs were examined. Type KCFB consists of keratinocytes (epidermal equivalent) and fibroblasts that were embedded in a collagen matrix (dermal equivalent). Type KCFB-EC consists of keratinocytes as well as fibroblasts and vascular endothelial cells. The epidermal equivalent of KCFB-EC constructs underwent cellular alterations in their differentiation possibly induced by the presence of endothelial cells. The objective of the study was to assess the effect of endothelial cells, i.e., endothelialization of the dermal equivalent on the differentiation of keratinocytes by comparing the morphology and ultrastructure of the two types of skin constructs, as well as to excised normal human skin. Hypothesis: The differentiation of keratinocytes is influenced by the presence of endothelial cells. METHODS, PATIENTS, MATERIAL: KCFB constructs (keratinocytes, fibroblasts) and KCFB-EC skin constructs (kera-tinocytes, fibroblasts, endothelial cells) were prepared according to Kuchler et al. [25]. After two weeks, the skin constructs were processed for analysis by light microscopy (LM) and electron microscopy (TEM), followed by quantitative, semi-quantitative as well as qualitative assessment. For comparison, analysis by LM and TEM of excised normal human skin was also performed. RESULTS: Both KCFB and KCFB-EC skin constructs and the human skin had all strata of stratified soft-cornified epidermis present. The comparison of the respective layers of the skin constructs brought the following characteristics to light: The KCFBEC constructs had significantly more mitotic cells in the stratum spinosum, more cell layers in the stratum granulosum and more keratohyalin granules compared to KCFB skin constructs. Additionally, the epidermal architecture was unorganized in the endothelialized constructs and features of excessive epidermal differentiation appeared in KCFB-EC skin constructs. CONCLUSION: The endothelialization of the dermal equivalent caused changes in the differentiation of the epidermis of KCFB-EC skin constructs that may be interpreted as an unbalanced, i.e., uncontrolled or enhanced maturation proces

Human and equine endothelial cells in a live cell imaging scratch assay in vitro

BACKGROUND: Human and equine patients are known to frequently develop vascular complications, particularly thrombosis both in veins and arteries as well as in the microvasculature. OBJECTIVE: The aim of the present study was to investigate and compare the angiogenic response of human and equine endothelial cells to lesions in an in vitro scratch assay. METHODS: Endothelial cells from human umbilical vein (HUVEC), abdominal aorta (HAAEC) and dermal microvasculature (HDMEC) as well as equine carotid artery (EACEC) and jugular vein (EVJEC) were cultured and an elongated defect was created (scratch or "wound"). Cultures were monitored over a period of 90 hours in a life cell imaging microscope. RESULTS: In the human endothelial cell cultures, there was a uniform and continuous migration of the cells from the scratch fringe into the denuded area, which was closed after 17 (HUVEC), 15 (HAAEC) and 26 (HDMEC) hours. In the equine endothelial cell cultures, a complete closure of the induced defect occurred after 17 (EVJEC) and 35 (EACEC) hours. CONCLUSIONS: In the equine arterial cells, the delay in closure of the denuded area seems to be the results of a disoriented and uncoordinated migration of endothelial tip cells resulting in slow re-endothelialization

Impact of intercellular crosstalk between epidermal keratinocytes and dermal fibroblasts on skin homeostasis

Dermal fibroblasts seem critical for epidermal maturation and differentiation and recent work demonstrated that diseased fibroblasts may drive pathophysiological processes. Nevertheless, still very little is known about the actual crosstalk between epidermal keratinocytes and dermal fibroblasts and the impact of dermal fibroblasts on epidermal maturation and differentiation. Aiming for a more fundamental understanding of the impact of the cellular crosstalk between keratinocytes and fibroblasts on the skin homeostasis, we generated full-thickness skin equivalents with and without fibroblasts and subsequently analysed them for the expression of skin differentiation markers, their barrier function, skin lipid content and epidermal cell signalling. Skin equivalents without fibroblasts consistently showed an impaired differentiation and dysregulated expression of skin barrier and tight junction proteins, increased skin permeability, and a decreased skin lipid/protein ratio. Most interestingly, impaired Ras/Raf/ERK/MEK signalling was evident in skin equivalents without fibroblasts. Our data clearly indicate that the epidermal-dermal crosstalk between keratinocytes and fibroblasts is critical for adequate skin differentiation and that fibroblasts orchestrate epidermal differentiation processes

Human and equine endothelial cells in a live cell imaging scratch assay in vitro

BACKGROUND:Human and equine patients are known to frequently develop vascular complications, particularly thrombosis both in veins and arteries as well as in the microvasculature. OBJECTIVE:The aim of the present study was to investigate and compare the angiogenic response of human and equine endothelial cells to lesions in an in vitro scratch assay. METHODS:Endothelial cells from human umbilical vein (HUVEC), abdominal aorta (HAAEC) and dermal microvasculature (HDMEC) as well as equine carotid artery (EACEC) and jugular vein (EVJEC) were cultured and an elongated defect was created (scratch or “wound”). Cultures were monitored over a period of 90 hours in a live cell imaging microscope. RESULTS:In the human endothelial cell cultures, there was a uniform and continuous migration of the cells from the scratch fringe into the denuded area, which was closed after 17 (HUVEC), 15 (HAAEC) and 26 (HDMEC) hours. In the equine endothelial cell cultures, a complete closure of the induced defect occurred after 17 (EVJEC) and 35 (EACEC) hours. CONCLUSIONS:In the equine arterial cells, the delay in closure of the denuded area seems to be the results of a disoriented and uncoordinated migration of endothelial tip cells resulting in slow re-endothelialization