Name: Characterization of In Vitro Differentiation of Human Primary Keratinocytes by RNA-Seq Analysis (Video) | JoVE Journal
Uploaded: 2020-05-16T13:00:00.000+00:00
Duration: 7 min 29 s
Description: Learn in vitro differentiation of human primary keratinocytes through contact inhibition and characterize molecular changes using RNA-seq analysis. This protocol enables large-scale studies in epidermal biology and epigenomics.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

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><li>Hardman, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Skin+equivalents+for+studying+the+secrets+of+skin%3A+from+development+to+disease%5BTitle%5D)+AND+%22British+Journal+of+Dermatology%22%5BJournal%5D)">Skin equivalents for studying the secrets of skin: from development to disease.</a> British Journal of Dermatology. 173 (2), 320-321 (2015).</li>
<li>Borowiec, A. S., Delcourt, P., Dewailly, E., Bidaux, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimal+differentiation+of+in+vitro+keratinocytes+requires+multifactorial+external+control%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Optimal differentiation of in vitro keratinocytes requires multifactorial external control.</a> PLoS One. 8 (10), 77507(2013).</li>
<li>Van Ruissen, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Induction+of+normal+and+psoriatic+phenotypes+in+submerged+keratinocyte+cultures%5BTitle%5D)+AND+%22Journal+of+Cellular+Physiology%22%5BJournal%5D)">Induction of normal and psoriatic phenotypes in submerged keratinocyte cultures.</a> Journal of Cellular Physiology. 168 (2), 442-452 (1996).</li>
<li>Ojeh, N., Pastar, I., Tomic-Canic, M., Stojadinovic, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stem+Cells+in+Skin+Regeneration%2C+Wound+Healing%2C+and+Their+Clinical+Applications%5BTitle%5D)+AND+%22International+Journal+of+Molecular+Sciences%22%5BJournal%5D)">Stem Cells in Skin Regeneration, Wound Healing, and Their Clinical Applications.</a> International Journal of Molecular Sciences. 16 (10), 25476-25501 (2015).</li>
<li>Ali, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Skin+equivalents%3A+skin+from+reconstructions+as+models+to+study+skin+development+and+diseases%5BTitle%5D)+AND+%22British+Journal+of+Dermatology%22%5BJournal%5D)">Skin equivalents: skin from reconstructions as models to study skin development and diseases.</a> British Journal of Dermatology. 173 (2), 391-403 (2015).</li>
<li>Luis, N. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulation+of+human+epidermal+stem+cell+proliferation+and+senescence+requires+polycomb-+dependent+and+-independent+functions+of+Cbx4%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">Regulation of human epidermal stem cell proliferation and senescence requires polycomb- dependent and -independent functions of Cbx4.</a> Cell Stem Cell. 9 (3), 233-246 (2011).</li>
<li>Mulder, K. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diverse+epigenetic+strategies+interact+to+control+epidermal+differentiation%5BTitle%5D)+AND+%22Nature+Cell+Biology%22%5BJournal%5D)">Diverse epigenetic strategies interact to control epidermal differentiation.</a> Nature Cell Biology. 14 (7), 753-763 (2012).</li>
<li>Poumay, Y., Pittelkow, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+density+and+culture+factors+regulate+keratinocyte+commitment+to+differentiation+and+expression+of+suprabasal+K1%2FK10+keratins%5BTitle%5D)+AND+%22Journal+of+Investigative+Dermatology%22%5BJournal%5D)">Cell density and culture factors regulate keratinocyte commitment to differentiation and expression of suprabasal K1/K10 keratins.</a> Journal of Investigative Dermatology. 104 (2), 271-276 (1995).</li>
<li>Kouwenhoven, E. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transcription+factor+p63+bookmarks+and+regulates+dynamic+enhancers+during+epidermal+differentiation%5BTitle%5D)+AND+%22EMBO+Rep%22%5BJournal%5D)">Transcription factor p63 bookmarks and regulates dynamic enhancers during epidermal differentiation.</a> EMBO Rep. 16 (7), 863-878 (2015).</li>
<li>Qu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mutant+p63+Affects+Epidermal+Cell+Identity+through+Rewiring+the+Enhancer+Landscape%5BTitle%5D)+AND+%22Cell+Reports%22%5BJournal%5D)">Mutant p63 Affects Epidermal Cell Identity through Rewiring the Enhancer Landscape.</a> Cell Reports. 25 (12), 3490-3503 (2018).</li>
<li>Brunner, H. G., Hamel, B. C., Van Bokhoven, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+p63+gene+in+EEC+and+other+syndromes%5BTitle%5D)+AND+%22Journal+of+Medical+Genetics%22%5BJournal%5D)">The p63 gene in EEC and other syndromes.</a> Journal of Medical Genetics. 39 (6), 377-381 (2002).</li>
<li>Browne, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differential+altered+stability+and+transcriptional+activity+of+DeltaNp63+mutants+in+distinct+ectodermal+dysplasias%5BTitle%5D)+AND+%22Journal+of+Cell+Science%22%5BJournal%5D)">Differential altered stability and transcriptional activity of DeltaNp63 mutants in distinct ectodermal dysplasias.</a> Journal of Cell Science. 124, Pt 13 2200-2207 (2011).</li>
<li>KGM Gold Keratinocyte Growth Medium BulletKit. , Available from: 
 <a target="_blank" href="https://bioscience.lonza.com/Lonza_bs/CH/en/Culture-Media-and-Reagents/p/000000000000192060/KGM-Gold-Keratinocyte-Growth-Medium-BulletKit">https://bioscience.lonza.com/Lonza_bs/CH/en/Culture-Media-and-Reagents/p/000000000000192060/KGM-Gold-Keratinocyte-Growth-Medium-BulletKit</a> (2019).</li>
<li>Doyle, K. aM. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aDoyle%2C+KaM+%22Protocols+and+applications+guide%22">Protocols and applications guide</a>. , Promega Corporation. Madison. (1996).</li>
<li>Roche. Hyperprep Kit. , Available from: 
 <a target="_blank" href="https://sequencing.roche.com/en-us/products-solutions/by-category/Library-preparation/rna-library-preparation/kapa-rna-hyperprep-kit-with-riboerasehmr.html">https://sequencing.roche.com/en-us/products-solutions/by-category/Library-preparation/rna-library-preparation/kapa-rna-hyperprep-kit-with-riboerasehmr.html</a> (2019).</li>
<li>Felix Krueger. TrimGalore. , Available from: 
 <a target="_blank" href="https://github.com/FelixKrueger/TrimGalore">https://github.com/FelixKrueger/TrimGalore</a> (2019).</li>
<li>Dobin, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((STAR%3A+ultrafast+universal+RNA-seq+aligner%5BTitle%5D)+AND+%22Bioinformatics%22%5BJournal%5D)">STAR: ultrafast universal RNA-seq aligner.</a> Bioinformatics. 29 (1), 15-21 (2013).</li>
<li>Li, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Sequence+Alignment%2FMap+format+and+SAMtools%5BTitle%5D)+AND+%22Bioinformatics%22%5BJournal%5D)">The Sequence Alignment/Map format and SAMtools.</a> Bioinformatics. 25 (16), 2078-2079 (2009).</li>
<li>Ryan, D. Convert chromosome names in a bigWig file. , Available from: 
 <a target="_blank" href="https://gist.github.com/dpryan79/39c70b4429dd4559d88fb079b8669721">https://gist.github.com/dpryan79/39c70b4429dd4559d88fb079b8669721</a> (2019).</li>
<li>Love, M. I., Huber, W., Anders, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Moderated+estimation+of+fold+change+and+dispersion+for+RNA-seq+data+with+DESeq2%5BTitle%5D)+AND+%22Genome+Biol%22%5BJournal%5D)">Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2.</a> Genome Biol. 15 (12), 550(2014).</li>
<li>Eden, E., Navon, R., Steinfeld, I., Lipson, D., Yakhini, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((GOrilla%3A+a+tool+for+discovery+and+visualization+of+enriched+GO+terms+in+ranked+gene+lists%5BTitle%5D)+AND+%22BMC+Bioinformatics%22%5BJournal%5D)">GOrilla: a tool for discovery and visualization of enriched GO terms in ranked gene lists.</a> BMC Bioinformatics. 10, 48(2009).</li>
<li>Yu, G., Wang, L. G., Han, Y., He, Q. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((clusterProfiler%3A+an+R+package+for+comparing+biological+themes+among+gene+clusters%5BTitle%5D)+AND+%22Omics%22%5BJournal%5D)">clusterProfiler: an R package for comparing biological themes among gene clusters.</a> Omics. 16 (5), 284-287 (2012).</li>
<li>Kretz, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Control+of+somatic+tissue+differentiation+by+the+long+non-coding+RNA+TINCR%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Control of somatic tissue differentiation by the long non-coding RNA TINCR.</a> Nature. 493 (7431), 231-235 (2013).</li>
<li>Mullegama, S. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nucleic+Acid+Extraction+from+Human+Biological+Samples%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">Nucleic Acid Extraction from Human Biological Samples.</a> Methods in Molecular Biology. 1897, 359-383 (2019).</li>
<li>Ma, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+comparison+between+whole+transcript+and+3%27+RNA+sequencing+methods+using+Kapa+and+Lexogen+library+preparation+methods%5BTitle%5D)+AND+%22BMC+Genomics%22%5BJournal%5D)">A comparison between whole transcript and 3' RNA sequencing methods using Kapa and Lexogen library preparation methods.</a> BMC Genomics. 20 (1), 9(2019).</li>
<li>Chao, H. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Systematic+evaluation+of+RNA-Seq+preparation+protocol+performance%5BTitle%5D)+AND+%22BMC+Genomics%22%5BJournal%5D)">Systematic evaluation of RNA-Seq preparation protocol performance.</a> BMC Genomics. 20 (1), 571(2019).</li>
<li>Podnar, J., Deiderick, H., Huerta, G., Hunicke-Smith, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Next-Generation+Sequencing+RNA-Seq+Library+Construction%5BTitle%5D)+AND+%22Current+Protocols+in+Molecular+Biology%22%5BJournal%5D)">Next-Generation Sequencing RNA-Seq Library Construction.</a> Current Protocols in Molecular Biology. 106, 21(2014).</li>
<li>Haque, A., Engel, J., Teichmann, S. A., Lonnberg, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+practical+guide+to+single-cell+RNA-sequencing+for+biomedical+research+and+clinical+applications%5BTitle%5D)+AND+%22Genome+Medicine%22%5BJournal%5D)">A practical guide to single-cell RNA-sequencing for biomedical research and clinical applications.</a> Genome Medicine. 9 (1), 75(2017).</li>
<li>Oyola, S. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimizing+Illumina+next-generation+sequencing+library+preparation+for+extremely+AT-biased+genomes%5BTitle%5D)+AND+%22BMC+Genomics%22%5BJournal%5D)">Optimizing Illumina next-generation sequencing library preparation for extremely AT-biased genomes.</a> BMC Genomics. 13, 1(2012).</li>
<li>Quail, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimal+enzymes+for+amplifying+sequencing+libraries%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">Optimal enzymes for amplifying sequencing libraries.</a> Nature Methods. 9 (1), 10-11 (2011).</li>
<li>Quail, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+tale+of+three+next+generation+sequencing+platforms%3A+comparison+of+Ion+Torrent%2C+Pacific+Biosciences+and+Illumina+MiSeq+sequencers%5BTitle%5D)+AND+%22BMC+Genomics%22%5BJournal%5D)">A tale of three next generation sequencing platforms: comparison of Ion Torrent, Pacific Biosciences and Illumina MiSeq sequencers.</a> BMC Genomics. 13, 341(2012).</li>
<li>Ross, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterizing+and+measuring+bias+in+sequence+data%5BTitle%5D)+AND+%22Genome+Biology%22%5BJournal%5D)">Characterizing and measuring bias in sequence data.</a> Genome Biology. 14 (5), 51(2013).</li>
<li>Soneson, C. ARMOR. , Available from: 
 <a target="_blank" href="https://github.com/csoneson/ARMOR">https://github.com/csoneson/ARMOR</a> (2019).</li>
<li>Sande, S. F. snakemake-workflows. , Available from: 
 <a target="_blank" href="https://github.com/vanheeringen-lab/snakemake-workflows">https://github.com/vanheeringen-lab/snakemake-workflows</a> (2019).</li>
</ol>

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

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 compo...

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 ...

characterization-vitro-differentiation-human-primary-keratinocytes

Research

JoVE Journal

Genetics

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

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

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.

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

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

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

RNA-sequencing and bioinformatics analyses were used to identify significantly and differentially expressed transcription factors in Lin-CD34+ and Lin-CD34- subpopulations of mouse EMLcells. These transcription factors might play important roles in determining the switch between self-renewing Lin-CD34+ and partially differentiated Lin-CD34- cells.

Identification of Key Factors Regulating Self-renewal and Differentiation in EML Hematopoietic Precursor Cells by RNA-sequencing Analysis

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.

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

Here, we present a protocol to efficiently generate human trophoblastic cells from human pluripotent stem cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal pathways. This method is suitable for the efficient differentiation of human pluripotent stem cells and can generate large quantities of cells for genetic manipulation.

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

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.

Generation and Culturing of Primary Human Keratinocytes from Adult Skin

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.

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

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.

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

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.

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

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.

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

Learn in vitro differentiation of human primary keratinocytes through contact inhibition and characterize molecular changes using RNA-seq analysis. This protocol enables large-scale studies in epidermal biology and epigenomics.

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

Explore More Videos