This protocol delineates a methodological framework for the quantitative analysis of collagen architecture during the processes of skin regeneration and fibrosis. The protocol integrates skin regeneration modeling with a...This protocol delineates a methodological framework for the quantitative analysis of collagen architecture during the processes of skin regeneration and fibrosis. The protocol integrates skin regeneration modeling with advanced computational tools. The splinted wound model facilitates the observation of wound regeneration in mice and the evaluation of therapeutic interventions. The xenografting of human skin allows in vivo investigations of human skin regeneration and the monitoring of dynamic changes following therapy application. To objectively quantify these changes, we employ curvelet transform-based algorithms (CurveAlign and CT-FIRE), which allow clear fiber visualization at brightfield and fluorescent images and extraction of individual fiber parameters. We suggest an algorithm for standardizing data collection and analysis using open-access software for collagen fiber structure examination.
Wound healing is a complex, multifactorial process that is divided in sequential and overlapping phases in order to restore the skin barrier. For the study of wound healing, different in vivo, in vitro, and ex vivo model...Wound healing is a complex, multifactorial process that is divided in sequential and overlapping phases in order to restore the skin barrier. For the study of wound healing, different in vivo, in vitro, and ex vivo models have been used in the past. Here, we describe in detail the methodology of the human skin punch-in-a-punch ex vivo wound healing model.
There is a wide variety of models to study different aspects of infections such as cell lines, organoid cultures, ex vivo models, or even experiments with animals. Each of which mirrors a rising complexity level, and ref...There is a wide variety of models to study different aspects of infections such as cell lines, organoid cultures, ex vivo models, or even experiments with animals. Each of which mirrors a rising complexity level, and reflect the in vivo condition in humans to a varying degree. When infection models are used to test the therapeutic efficacy and tolerability of antimicrobials or antiseptics, the right choice of model is crucial to truly extract meaningful information and apply these findings to the in vivo circumstances in humans. In this chapter, we report on protocols for using infection models based on ex vivo human skin for the testing of antimicrobial formulations and antiseptics. We describe general protocols to create bacterial and viral infections and to extract and quantify the microorganisms. In addition, we describe the methods for the microscopical visualization of the ex vivo skin and wound infections.
Wound healing is a major medical challenge as acute injuries affect millions annually, while chronic wounds such as diabetic foot and venous leg ulcers persist in 1-2% of patients, driving substantial morbidity and ~4% o...Wound healing is a major medical challenge as acute injuries affect millions annually, while chronic wounds such as diabetic foot and venous leg ulcers persist in 1-2% of patients, driving substantial morbidity and ~4% of global health-care expenditure. In response to an injury, restoration of skin barrier is critical process and regulated at multiple levels involving coordinated communications and interaction among keratinocytes, immune cells, and fibroblasts. In vivo mouse models are essential for dissecting the cellular and molecular regulators of normal and pathological repair, especially given the availability of transgenic tools and cell-sorting methods. However, the most common back-punch excisional model heals predominantly by panniculus carnosus-mediated contraction (up to 90%), which limits granulation tissue formation and affects reepithelialization. The mouse tail excisional model provides an alternative to back-punch model as wound closure yielding robust granulation and epithelial coverage due to the "splinting" effect of the tail structure. Here, we describe a detailed, reproducible protocol for tail excisional wounds optimized for histology, cryosectioning, and downstream cell isolation for single-cell multi-omic analyses.
The isolation of sweat glands from human skin has long been a challenging task. The human scalp contains thousands of eccrine glands, while the axilla harbors both eccrine and apocrine glands. Recently, the close anatomi...The isolation of sweat glands from human skin has long been a challenging task. The human scalp contains thousands of eccrine glands, while the axilla harbors both eccrine and apocrine glands. Recently, the close anatomic relationship of the eccrine gland with the scalp hair follicle has been described. Taking advantage of this anatomic relationship, we describe here an efficient method to isolate sweat glands from the human scalp and the axilla using the micro-punch harvesting technique known as follicular unit excision (FUE), commonly employed in hair transplant surgery.Once the follicular unit (FU) has been extracted, it needs to be stained with Methylene Blue or Neutral Red in order to make the sweat gland visible for its dissection under the stereomicroscope. The efficiency of this isolation method should encourage further research into human sweat glands and opens possibilities for new translational applications.
Primary cicatricial alopecia is characterized by a permanent "scarring" alopecia. This condition is characterized by the irreversible loss of hair follicles (HFs) as a result of apoptosis and epithelial-mesenchymal trans...Primary cicatricial alopecia is characterized by a permanent "scarring" alopecia. This condition is characterized by the irreversible loss of hair follicles (HFs) as a result of apoptosis and epithelial-mesenchymal transition (EMT) of epithelial stem cells localized in the HF bulge.We here report the procedure for experimentally induced EMT in healthy human epidermal stem cells (eSCs) using full-length HF organ culture ex vivo. The present model can be used to recapitulate the complex processes observed in scarring alopecia patient tissues, to further investigate the mechanisms involved in EMT transformation of HFeSCs, and to test substances that could prevent and/or rescue HFeSCs from EMT for the management of scarring alopecias.
Alopecia areata (AA) is a hair follicle (HF) autoimmune disease leading to hair loss. Interferon gamma (IFNγ) is regarded as the key cytokine involved in AA development, which has been shown to play a role in the collaps...Alopecia areata (AA) is a hair follicle (HF) autoimmune disease leading to hair loss. Interferon gamma (IFNγ) is regarded as the key cytokine involved in AA development, which has been shown to play a role in the collapse of HF immune privilege (IP), CD8 T-cell-driven inflammation toward the HF bulb, and premature catagen development. We present here the procedure to induce IP collapse in human healthy HFs ex vivo. This assay can be suitable not only to investigate mechanisms involved in HF IP collapse but also to test substances for either preventing and/or reversing HF IP collapse for the management of AA or other autoimmune diseases sharing similar pathogenesis.
Surgical induction of alopecia areata (AA) via full-thickness grafting of spontaneous AA-affected C3H/HeJ mouse skin to naïve recipients has been a primary method of transferring the AA disease model phenotype. However,...Surgical induction of alopecia areata (AA) via full-thickness grafting of spontaneous AA-affected C3H/HeJ mouse skin to naïve recipients has been a primary method of transferring the AA disease model phenotype. However, this method is associated with the need to perform an invasive procedure that could negatively impact animal well-being. Therefore, a rodent model that rapidly develops AA at a predictable rate and without the need to perform invasive surgical procedures on the mice is essential for studying the pathogenesis of AA. Here, we describe a cell injection technique using cultured skin-draining lymph node cells (LNCs) injected intradermally into naïve recipients to induce rapid AA development. The cultured LNCs can reach ~ten fold expansion after 6 days with specific cytokine stimulation. The LNCs derived from a single AA-affected mouse donor can induce AA development in more than 80 naïve mice within 2-18 weeks. For comparative control studies, mice receiving cultured LNCs from normal donors remain normally haired. The method enables the production of large numbers of AA mice for use in research and treatment development studies while avoiding the use of surgical procedures. We anticipate that the protocol can also be adapted for use in other mouse autoimmune disease models.
Various hair follicle (HF)-associated disorders, such as acne vulgaris, hidradenitis suppurativa, and alopecia areata, are linked to dysbiosis, an imbalance between resident and pathogenic microbes. Characterization of t...Various hair follicle (HF)-associated disorders, such as acne vulgaris, hidradenitis suppurativa, and alopecia areata, are linked to dysbiosis, an imbalance between resident and pathogenic microbes. Characterization of the HF and skin microbiome employs techniques such as 16S rRNA gene sequencing and metagenomic shotgun sequencing, with the latter providing comprehensive taxonomic and functional insights. However, relic DNA from dead microbes and free environmental DNA can persist in samples, meaning that metagenomic data does not exclusively reflect living microbiota. For functional studies on HF dysbiosis or to assess potential therapeutic interventions, we describe here how propidium monoazide (PMA) treatment can be performed before (metagenomics) sequencing to distinguish viable microbial communities. Furthermore, we exemplify qPCR and (fluorescent) in situ hybridization (ISH) of two alternative viability screening methods for the HF and scalp microbiome.
Laser-capture microdissection (LCM) enables the study of the hair follicle (HF) microbiome in relation to hair health and disease with high spatial resolution. It allows the precise excision of specific HF regions, each...Laser-capture microdissection (LCM) enables the study of the hair follicle (HF) microbiome in relation to hair health and disease with high spatial resolution. It allows the precise excision of specific HF regions, each containing a unique and conserved microbiome, from full-length HFs encompassing all relevant HF compartments. With LCM, cross-contamination with microbiota from neighboring regions is minimized. Coupled with 16S rRNA gene or metagenomic shotgun sequencing, LCM offers great potential to assess region-specific microbiome changes, particularly in HF-associated disorders.
Modeling organoids with hair follicle germ-like properties provides an opportunity for developing strategies for alopecia drug discovery and replacement therapy, as well as investigating the molecular mechanisms underlyi...Modeling organoids with hair follicle germ-like properties provides an opportunity for developing strategies for alopecia drug discovery and replacement therapy, as well as investigating the molecular mechanisms underlying human hair follicle regeneration in vitro. Hair follicle germ reconstruction in vitro is based on dermal papilla hair-inducing abilities and the plasticity of skin epidermal keratinocytes. The current protocol describes a highly efficient approach suitable for adult human skin cell applications. This method allows to obtain hair follicle germs using tissues from one donor. Isolated and cultured for 2 weeks, adult hair follicle dermal papilla cells and skin epidermal keratinocytes self-organize in hanging drop cultures generating organoids that exhibit the features of folliculogenesis onset.
The culture of microdissected hair follicles (HFs) and scalp skin rich in terminal HFs are the best currently available preclinical assays for studying hair and skin biology/pathology in the human system. While microdiss...The culture of microdissected hair follicles (HFs) and scalp skin rich in terminal HFs are the best currently available preclinical assays for studying hair and skin biology/pathology in the human system. While microdissected HFs organ culture only allows the testing of test compounds into the culture medium, mimicking a systemic application, the scalp skin organ culture also is suitable to test topical and intradermal applications. Here, we describe different methods for isolation of human scalp HFs, the procedures for culturing the scalp skin and microdissected HFs, and we also outline different delivery techniques (e.g., topical, systemic) to test active and control substances.
The isolation and molecular characterization of distinct cell populations from heterogeneous tissues are essential for investigating cell-type-specific functions and regulatory mechanisms, particularly within complex org...The isolation and molecular characterization of distinct cell populations from heterogeneous tissues are essential for investigating cell-type-specific functions and regulatory mechanisms, particularly within complex organs such as the skin. Here, we present a comprehensive workflow for isolating hair matrix cells from postnatal mouse skin using fluorescence-activated cell sorting (FACS) for gene expression and high-resolution chromatin profiling. The protocol begins with enzymatic dissociation of the skin to obtain a single-cell suspension, followed by immunolabeling with cell surface markers to enable precise FACS-based enrichment of the desired cell populations. A low-input CUT&Tag assay is then used to map histone posttranslational modifications, transcription factor binding sites, and DNA modifications. Importantly, this cell isolation strategy is compatible with a range of integrative multi-omics approaches including RNA sequencing (RNA-seq), assay for transposase-accessible chromatin using sequencing (ATAC-seq), whole-genome bisulfite sequencing (WGBS), and high-throughput chromosome conformation capture (Hi-C) using input material as low as ~100,000 cells per assay. Together, this methodology allows comprehensive, cell-type resolved analysis of the transcriptional and epigenetic landscapes in hair matrix keratinocytes in postnatal skin.
Tissue whole mounts have long been used to assess morphological changes following genetic perturbation in mice. Traditional methods for preparing dorsal skin whole mounts, however, often lead to breakage of hair follicle...Tissue whole mounts have long been used to assess morphological changes following genetic perturbation in mice. Traditional methods for preparing dorsal skin whole mounts, however, often lead to breakage of hair follicle structures, such as sebaceous glands, making it difficult to perform unbiased quantitation. Here, we describe a modified technique for preparing whole mounts harvested from mouse dorsal skin. This protocol incorporates adhesives to stiffen the tissue and facilitate the separation of intact epidermis from the dermis. Critically, this method is compatible with downstream applications such as lipid staining of sebaceous glands and immunofluorescence, and we provide protocols for both approaches. Overall, the use of this modified "glue technique" for preparing skin whole mounts will help preserve tissue integrity for morphological analysis.
The skin deploys multiple barriers to protect internal tissues and organs, as well as the skin itself, from the external environment. Each of the skin constituents-epidermis, dermis, sebaceous glands, sweat glands, hair,...The skin deploys multiple barriers to protect internal tissues and organs, as well as the skin itself, from the external environment. Each of the skin constituents-epidermis, dermis, sebaceous glands, sweat glands, hair, and adipose tissue-contributes to making these barriers competent to maintain proper biological functions. Among these constituents, the epidermis (the outermost layer of skin) is the first line of defense, so it is most responsible for skin barrier functions. A competent skin barrier is needed to protect against excess loss of water and other endogenous compounds from body, oxidative stress, sun and other sources of light irradiation, thermal stress, microbial invasion, substance permeation, and mechanical stress. In this chapter, we describe methods to assess both the epidermal permeability barrier and the light irradiation barrier using in vitro and ex vivo skin organ-cultured systems.
Spatial genome organization in the cell nucleus plays a crucial role in the control of genome functions. Our knowledge about spatial genome organization relies on the advances in the genome imaging technologies and the b...Spatial genome organization in the cell nucleus plays a crucial role in the control of genome functions. Our knowledge about spatial genome organization relies on the advances in the genome imaging technologies and the biochemical approaches based on the spatially dependent ligation of the genomic regions. Fluorescent in situ hybridization using specific fluorescent DNA and RNA probes in cells and tissues with the spatially preserved nuclear and genome architecture (3D-FISH) provides a powerful tool for advancing our knowledge about genome structure and functions. Here, we describe the 3D-FISH protocols allowing such analysis in mammalian tissue in situ, including in the skin. These protocols include DNA probe amplification and labeling; tissue fixation; preservation and preparation for hybridization; hybridization of the DNA probes with genomic DNA in the tissue; and the post-hybridization tissue sample processing.
MicroRNAs (miRNAs) are a family of small noncoding RNAs (~19-24 nt), expressed in a wide range of animals and plants. It is estimated that more than one-third of protein-encoding mRNAs are regulated by miRNAs. MiRNAs lar...MicroRNAs (miRNAs) are a family of small noncoding RNAs (~19-24 nt), expressed in a wide range of animals and plants. It is estimated that more than one-third of protein-encoding mRNAs are regulated by miRNAs. MiRNAs largely contribute to the regulation of gene expression by fine-tuning and buffering the activity of signaling pathways. Therefore, miRNAs represent remarkably diverse regulatory networks, playing a key role in the execution of gene expression programs in various cells and tissues. Many technical challenges have been encountered when investigating miRNAs, in particular, determining the spatiotemporal expression pattern of miRNAs in cells and tissues. Therefore, we describe here a well-established in situ hybridization (ISH) protocols for use of the detection and analysis of spatiotemporal expression patterns of miRNAs in skin and its appendages such as the hair follicle in both frozen and paraffin-embedded tissue sections. We describe in detail the different steps that are associated with utilizing ISH procedure on either frozen or paraffin-embedded tissues for miRNAs localization. Post-fixation of tissues, tissues are hybridized with LNA double-labeled probes with digoxygenin. Detection of hybridized probes is performed by using an alkaline phosphatase-coupled antibody against digoxygenin. The final step involves the use of substrates to develop the color of alkaline phosphatase-LNA-probe structure, leading to the identification of the spatiotemporal of target miRNAs in target tissue and cells. We also discuss two options for substrate color development in these procedures: (i) NBT/BCIP and (ii) BM-Purple. This method is a simple and convenient way of determining the spatiotemporal expression pattern of miRNAs, which has been a challenge since their discovery due to their relatively small size. Knowledge gained from ISH will be crucial for better understanding of individual miRNA(s) during distinct stages of development in various cells and tissues, and these protocols will be beneficial to the wider scientific community.
In situ hybridization (ISH), fluorescent in situ hybridization (FISH), and the most advanced RNA in situ hybridization methodology termed RNAscope® enable us to detect the expression and localization of a specific RNA in...In situ hybridization (ISH), fluorescent in situ hybridization (FISH), and the most advanced RNA in situ hybridization methodology termed RNAscope® enable us to detect the expression and localization of a specific RNA in fixed cells or tissue sections. Here, we describe in detail three procedures adjusted to reveal specifically noncoding RNAs (ncRNAs) in normal human keratinocytes and in skin tissue samples. Examples of the results obtained by the three different approaches are also shown.
Immunofluorescence-based imaging can offer high signal-to-noise and overall superior image quality of fixed cells over that of live-cell imaging. This is especially true of tracking and quantifying DNA damage sites as th...Immunofluorescence-based imaging can offer high signal-to-noise and overall superior image quality of fixed cells over that of live-cell imaging. This is especially true of tracking and quantifying DNA damage sites as there is a dearth of reliable or easy-to-use methods in living cells. Here, I describe a useful workflow for DNA damage induction in cell culture, fixation, staining, and method for analyzing DNA double-strand breaks (DSBs). Further, I provide troubleshooting tips for common issues while performing image segmentation and analysis.
CRISPR/Cas9 is a straightforward genome-editing technique that is implemented across disciplines and research areas. However, in keratinocytes, CRISPR/Cas9 can be particularly difficult due to variable genome-editing eff...CRISPR/Cas9 is a straightforward genome-editing technique that is implemented across disciplines and research areas. However, in keratinocytes, CRISPR/Cas9 can be particularly difficult due to variable genome-editing efficiency, reduced cell viability, and difficulties during (sub)cloning of gene-edited keratinocyte populations. Here, we provide a step-by-step detailed protocol for the genetic manipulation of human (primary) keratinocytes, including widely accepted procedures for the analysis of CRISPR/Cas9 efficiency, (sub)cloning procedures to select heterozygous or homozygous keratinocytes, and off-target genome-editing analysis.