This research was supported by Netherlands Organisation for Scientific Research (NWO/ALW/MEERVOUD/836.12.010, H.Z.) (NWO/ALW/Open Competition/ALWOP 376, H.Z., J.G.A.S.); Radboud University fellowship (H.Z.); and Chinese Scholarship Council grant 201406330059 (J.Q.).

<ol>
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The authors have nothing to disclose.

This work describes a method for inducing human keratinocyte differentiation and subsequent characterization using RNA-seq analyses. In the current literature, many studies on human keratinocyte differentiation use two other methods, with a high calcium concentration or with serum&#160;as methods to induce differentiation2,3,23. A previous report carefully compared these three different methods3 and showe...

Human primary keratinocytes derived from the human skin are often used as a cellular model to study the biology of the epidermis1,2,3,4. The stratification of the epidermis can be modeled by keratinocyte differentiation, either in a 2D submerged monolayer fashion or 3D air-lift organotypic model2,3,5,6,7. Although 3D models have become increasingly important to assess the epidermal structure and function, 2D differentiation models are still widely used, due to their convenience and the possibility to generate large numbers of cells for analyses.
Various conditions have been applied for inducing keratinocyte differentiation in 2D, including addition of serum, high concentration of calcium, lower temperature and inhibition of epidermal growth factor receptors2,3. Each of these methods has been validated by a number of keratinocyte differentiation marker genes and shown to be effective in assessing keratinocyte differentiation, including under pathological conditions. However, these induction conditions also show differences in their differentiation efficiency and kinetics when specific panels of marker genes are examined2,3.
One of these methods involves keratinocyte contact inhibition and depletion of growth factors in the culture medium8. It has been shown that keratinocytes can differentiate spontaneously when cells reach full density. &#160;Excluding growth factors in the culture medium can further enhance differentiation. The method combining contact inhibition and depleting growth factors has been shown to generate differentiated keratinocytes with gene expression patterns similar to the normal stratified epidermis when using several epidermal markers3, suggesting that this model is suitable for studying normal keratinocyte differentiation. Recently, two comprehensive gene expression analyses of keratinocyte differentiation using this model have been reported9,10. Researchers validated this model at the molecular level and showed that it can be used to study normal and diseased keratinocyte differentiation.
This protocol describes the procedure for the in vitro differentiation method and molecular analysis of differentiated cells using RNA-seq. It also illustrates characterization of the transcriptome of cells on differentiation day 0 (proliferation stage), day 2, day 4, and day 7 (early, middle, and late differentiation, respectively). It is shown that differentiated keratinocytes display gene expression patterns that largely resemble cells during epidermal stratification. To examine whether this method can be used for studying skin pathology, we applied the same experimental and analysis pipeline to investigate keratinocytes carrying mutations of the transcription factor p63 that are derived from patients with ectrodactyly, ectodermal displasia and cleft lip/palate (EEC) syndrome11,12. This protocol focuses on the in vitro differentiation of keratinocytes as well as subsequent bioinformatic analysis of RNA-seq. Other steps in the complete procedure such as RNA extraction, RNA-seq sample preparation and library construction, are well documented and can be easily followed, especially when using many commonly used commercial kits. Therefore, these steps are only briefly described in the protocol. The data show that this pipeline is suitable for studying molecular events during epidermal differentiation in healthy and diseased keratinocytes.

<table><tbody><tr><td>Bioanalyzer 2100</td><td>Agilent</td><td>G2929BA</td><td></td></tr><tr><td>Bovine pituitary extract (BPE)</td><td>Lonza</td><td></td><td>Part of the bulletKit</td></tr><tr><td>CFX96 Real-Time system</td><td>Bio-Rad</td><td></td><td>qPCR machine</td></tr><tr><td>Dulbecco&#039;s Phosphate-Buffered Saline (DPBS)</td><td>Sigma-Aldrich</td><td>D8537</td><td></td></tr><tr><td>Epidermal Growth Factor (EGF)</td><td>Lonza</td><td></td><td>Part of the bulletKit</td></tr><tr><td>Ethanolamine &gt;= 98%</td><td>Sigma-Aldrich</td><td>E9508</td><td></td></tr><tr><td>High Sensitivity DNA chips</td><td>Agilent</td><td>5067-4626</td><td></td></tr><tr><td>Hydrocortison</td><td>Lonza</td><td></td><td>Part of the bulletKit</td></tr><tr><td>Insulin</td><td>Lonza</td><td></td><td>Part of the bulletKit</td></tr><tr><td>iQ SYBR Green Kit</td><td>BioRad</td><td>170-8886</td><td></td></tr><tr><td>iScript cDNA synthesis</td><td>Bio rad</td><td>1708890</td><td></td></tr><tr><td>KAPA Library Quant Kit</td><td>Roche</td><td>07960255001</td><td>Low concentration measure kit</td></tr><tr><td>KAPA RNA HyperPrep Kit with RiboErase</td><td>Roche</td><td>KK8540</td><td>RNAseq kit</td></tr><tr><td>KGM Gold Keratinocyte Growth Medium BulletKit</td><td>Lonza</td><td>192060</td><td></td></tr><tr><td>Nanodrop</td><td>deNovix</td><td>DS-11 FX (model)</td><td>Nanodrop and Qbit for DNA and RNA measurements</td></tr><tr><td>NEXTflex DNA barcodes -24</td><td>Illumnia</td><td>NOVA-514103</td><td>6 bp long primers</td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td>15140122</td><td></td></tr><tr><td>RNA Pico Chip</td><td>Agilent</td><td>5067-1513</td><td></td></tr></tbody></table>

characterization of in vitro differentiation of human primary keratinocytes by rna-seq analysis

Human primary keratinocytes are often used as in vitro models for studies on epidermal differentiation and related diseases. Methods have been reported for in vitro differentiation of keratinocytes cultured in two-dimensional (2D) submerged manners using various induction conditions. Described here is a procedure for 2D in vitro keratinocyte differentiation method by contact inhibition and subsequent molecular characterization by RNA-seq. In brief, keratinocytes are grown in defined keratinocyte medium supplemented with growth factors until they are fully confluent. Differentiation is induced by close contacts between the keratinocytes and further stimulated by excluding growth factors in the medium. Using RNA-seq analyses, it is shown that both 1) differentiated keratinocytes exhibit distinct molecular signatures during differentiation and 2) the dynamic gene expression pattern largely resembles cells during epidermal stratification. As for comparison to normal keratinocyte differentiation, keratinocytes carrying mutations of the transcription factor p63 exhibit altered morphology and molecular signatures, consistent with their differentiation defects. In conclusion, this protocol details the steps for 2D in vitro keratinocyte differentiation and its molecular characterization, with an emphasis on bioinformatic analysis of RNA-seq data. Because RNA extraction and RNA-seq procedures have been well-documented, it is not the focus of this protocol. The experimental procedure of in vitro keratinocyte differentiation and bioinformatic analysis pipeline can be used to study molecular events during epidermal differentiation in healthy and diseased keratinocytes.

Normal keratinocyte differentiation and RNA-seq analysis 
In this experiment, keratinocyte lines derived from five individuals were used for differentiation and RNA-seq analyses. Figure 1 summarizes the experimental procedure of differentiation and RNA-seq analysis results. An overview of in vitro differentiation procedures of normal keratinocytes and cell morphology changes during differentiation are illustrated in Figure 1A. Principle com...

Watch this Scientific Journal Video about Characterization of In Vitro Differentiation of Human Primary Keratinocytes by RNA-Seq Analysis at JoVE.com

Characterization of In Vitro Differentiation of Human Primary Keratinocytes by RNA-Seq Analysis

Presented here is a stepwise procedure for in vitro differentiation of human primary keratinocytes by contact inhibition followed by characterization at the molecular level by RNA-seq analysis.

Human primary keratinocytes are often used as in vitro models for studies on epidermal differentiation and related diseases. Methods have been reported ...

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JoVE Journal

Genetics

6.0K Views.  Radboud University, Nijmegen, The Netherlands. In dit protocol beschrijven we een procedure met twee delen. Ten eerste, een monolayer 2D keratinocyten differentiatie methode in vitro door contactremming. Ten tweede, de moleculaire karakterisering door RNA-seq.De 2D in vitro differentiatie methode is eenvoudig en eenvoudig uit te voeren. Het kan worden gebruikt om opperhuiddifferentiatie te bestuderen, vooral wanneer een groot aantal cellen nodig zijn. Bijvoorbeeld in epigenomische analyse.De gedetailleerde RNA-seq analyse pi...

Video: Characterization of In Vitro Differentiation of Human Primary Keratinocytes by RNA-Seq Analysis

Differentiation of a Human Neural Stem Cell Line on Three Dimensional Cultures, Analysis of MicroRNA and Putative Target Genes

Neural stem cells (NSCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes under specific local microenvironments. In here, we present a set of methods used for three dimensional (3D) differentiation and miRNA analysis of a clonal human neural stem cell (hNSC) line, currently in clinical trials for stroke disability (NCT01151124 and NCT02117635, Clinicaltrials.gov). HNSCs were derived from an ethical approved first trimester human fetal cortex and conditionally immortalized using retroviral integration of a single copy of the c-mycERTAMconstruct. We describe how to measure axon process outgrowth of hNSCs differentiated on 3D scaffolds and how to quantify associated changes in miRNA expression using PCR array. Furthermore we exemplify computational analysis with the aim of selecting miRNA putative targets. SOX5 and NR4A3 were identified as suitable miRNA putative target of selected significantly down-regulated miRNAs in differentiated hNSC. MiRNA target validation was performed on SOX5 and NR4A3 3&#8217;UTRs by dual reporter plasmid transfection and dual luciferase assay.

With the intent of characterizing changes in miRNAs on differentiated human neural stem cells (hNSCs) we describe hNSC differentiation on a three dimensional system,the evaluation of changes in microRNA expression by miRNA PCR array, and computational analysis for miRNA target prediction and its validation by dual luciferase assay.

Neural stem cells (NSCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes under specific local ...

Developmental Biology

Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using patient-specific hiPSCs allows the study of the underlying mechanism for pathogenesis, also providing a platform for the development of in vitro drug screening and gene therapy to improve treatment options. The promising potential of hiPSCs for regenerative medicine is also evident from the increasing number of publications (>7000) on iPSCs in recent years. Various cell types from distinct lineages have been successfully used for hiPSC generation, including skin fibroblasts, hematopoietic cells and epidermal keratinocytes. While skin biopsies and blood collection are routinely performed in many labs as a source of somatic cells for the generation of hiPSCs, the collection and subsequent derivation of hair keratinocytes are less commonly used. Hair-derived keratinocytes represent a non-invasive approach to obtain cell samples from patients. Here we outline a simple non-invasive method for the derivation of keratinocytes from plucked hair. We also provide instructions for maintenance of keratinocytes and subsequent reprogramming to generate integration-free hiPSC using episomal vectors.

This manuscript provides a step-by-step procedure for the derivation and maintenance of human keratinocytes from plucked hair and subsequent generation of integration-free human induced pluripotent stem cells (hiPSCs) by episomal vectors.

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and effect sizes of regulatory elements to a target gene. Some progress has been made with the bioinformatic prediction of the existence and function of proximal epigenetic modifications associated with activated gene expression using conserved transcription factor binding sites. Chromatin conformation capture studies have revolutionized our ability to discover physical chromatin contacts between sequences and even within an entire genome. Circular chromatin conformation capture coupled with next-generation sequencing (4C-seq), in particular, is designed to discover all possible physical chromatin interactions for a given sequence of interest (viewpoint), such as a target gene or a regulatory enhancer. Current 4C-seq strategies directly sequence from within the viewpoint but require numerous and diverse viewpoints to be simultaneously sequenced to avoid the technical challenges of uniform base calling (imaging) with next generation sequencing platforms. This volume of experiments may not be practical for many laboratories. Here, we report a modified approach to the 4C-seq protocol that incorporates both an additional restriction enzyme digest and qPCR-based amplification steps that are designed to facilitate a greater capture of diverse sequence reads and mitigate the potential for PCR bias, respectively. Our modified 4C method is amenable to the standard molecular biology lab for assessing chromatin architecture.

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

The identification of physical interactions between genes and regulatory elements is challenging but has been facilitated by chromosome conformation capture methods. This modification to the 4C-seq protocol mitigates PCR bias by minimizing over-amplification of PCR templates and maximizes the mappability of reads by incorporating an addition restriction enzyme digest step.

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

Isolating, Sequencing and Analyzing Extracellular MicroRNAs from Human Mesenchymal Stem Cells

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, milk and urine. One subset of these RNAs are the posttranscriptional regulators &#8211; microRNAs (miRNAs). To delineate the miRNAs produced by specific cell types, in vitro culture systems can be used to harvest and profile exRNAs derived from one subset of cells. The secreted factors of mesenchymal stem cells are implicated in alleviating numerous diseases and is used as the in vitro model system here. This paper describes the process of collection, purification of small RNA and library generation to sequence extracellular miRNAs. ExRNAs from culture media differ from cellular RNA by being low RNA input samples, which calls for optimized procedures. This protocol provides a comprehensive guide to small exRNA sequencing from culture media, showing quality control checkpoints at each step during exRNA purification and sequencing.

This protocol demonstrates how to purify extracellular microRNAs from cell culture media for small RNA library construction and next generation sequencing. Various quality control checkpoints are described to allow readers to understand what to expect when working with low input samples like exRNAs.

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, ...

Generation and Culturing of Primary Human Keratinocytes from Adult Skin

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. In addition, keratinocytes play an essential role in the initiation, maintenance, and regulation of epidermal immune responses by being part of the innate immune system responding to antigenic stimuli in a fast, nonspecific manner. Here, we describe a protocol for isolation of primary human keratinocytes from adult skin, and demonstrate that these cells respond to calcium-induced terminal differentiation, as measured by an increased expression of the differentiation marker involucrin. In addition, we show that the isolated keratinocytes are responsive to IL-1&#946;-induced activation of intracellular signaling pathways as measured by the activation of the p38 MAPK pathway. Taken together, we describe a method for isolation and culturing of primary human keratinocytes from adult skin. Because the keratinocytes are the predominant cell type in the epidermis, this method is useful to study molecular mechanisms in cutaneous biology in vitro.

The human skin acts as a first line of defense against the external environment. We present a method for isolating primary human keratinocytes from adult skin. These isolated keratinocytes are useful in numerous experimental setups, and are a highly suitable model for studying molecular mechanisms in cutaneous biology in vitro.

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. ...

A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical applications. The conventional isolation method of human keratinocytes involves a two-step sequential enzymatic digestion procedure, which has been proven to be inefficient in generating primary cells from adult tissues due to the low cell recovery rate and reduced cell viability. We recently reported an advanced method to isolate human primary epidermal progenitor cells from skin tissues that utilizes the Rho kinase inhibitor Y-27632 in the medium. Compared with the traditional protocol, this new method is simpler, easier, and less time-consuming, and increases epithelial stem cell yield and enhances their stem cell characteristics. Moreover, the new methodology does not require the separation of the epidermis from the dermis, and, therefore, is suitable for isolating cells from different types of adult tissues. This new isolation method overcomes the major shortcomings of conventional methods and is more suitable for producing large numbers of epidermal cells with high potency both for laboratory and for clinical applications. Here, we describe the new method in detail.

Here we present a protocol to efficiently isolate primary human keratinocytes from adult skin tissues. This method simplifies the conventional procedure by using the ROCK Inhibitor Y-27632 in the inoculation medium to spontaneously separate epidermal cells from dermal cells.

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical ...

RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome wide analysis of differentially expressed transcripts after such an experimental manipulation has the potential to uncover an almost complete picture of the downstream effects caused by the transfected construct. This work describes a simple method for comparing transcriptomes from samples of transfected embryonic spinal cords comprising all steps between electroporation and identification of differentially expressed transcripts. The first stage consists of guidelines for electroporation and instructions for dissection of transfected spinal cord halves from HH23 embryos in ribonuclease-free environment and extraction of high-quality RNA samples suitable for transcriptome sequencing. The next stage is that of bioinformatic analysis with general guidelines for filtering and comparison of RNA-Seq datasets in the Galaxy public server, which eliminates the need of a local computational structure for small to medium scale experiments. The representative results show that the dissection methods generate high quality RNA samples and that the transcriptomes obtained from two control samples are essentially the same, an important requirement for detection of differential expression genes in experimental samples. Furthermore, one example is provided where experimental overexpression of a DNA construct can be visually verified after comparison with control samples. The application of this method may be a powerful tool to facilitate new discoveries on the function of neural factors involved in spinal cord early development.

This video shows the basic steps for performing whole transcriptome analysis on dissected chick embryonic spinal cord samples after transfection with in ovo electroporation.

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome ...

Neuroscience

Establishing a High Throughput Epidermal Spheroid Culture System to Model Keratinocyte Stem Cell Plasticity

Epithelial dysregulation is a node for a variety of human conditions and ailments, including chronic wounding, inflammation, and over 80% of all human cancers. As a lining tissue, the skin epithelium is often subject to injury and has evolutionarily adapted by acquiring the cellular plasticity necessary to repair damaged tissue. Over the years, several efforts have been made to study epithelial plasticity using in&#160;vitro and ex vivo cell-based models. However, these efforts have been limited in their capacity to recapitulate the various phases of epithelial cell plasticity. We describe here a protocol for generating 3D epidermal spheroids and epidermal spheroid-derived cells from primary neonatal human keratinocytes. This protocol outlines the capacity of epidermal spheroid cultures to functionally model distinct stages of keratinocyte generative plasticity and demonstrates that epidermal spheroid re-plating can enrich heterogenous normal human keratinocytes (NHKc) cultures for integrin&#945;6hi/EGFRlo keratinocyte subpopulations with enhanced stem-like characteristics. Our report describes the development and maintenance of a high throughput system for the study of skin keratinocyte plasticity and epidermal regeneration.

Here we describe a protocol for the systematic cultivation of epidermal spheroids in 3D suspension culture. This protocol has wide-ranging applications for use in a variety of epithelial tissue types and for the modeling of several human diseases and conditions.

Epithelial dysregulation is a node for a variety of human conditions and ailments, including chronic wounding, inflammation, and over 80% of all human ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

Biology

MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types.

The microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human induced-pluripotent stem (iPS) cells (iPS-RPE), and fetal RPE, were compared.

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, ...

Characterization of In Vitro Differentiation of Human Primary Keratinocytes by RNA-Seq Analysis

Samenvatting

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