This work was supportedby the American Cancer Society Research Scholars Grant(RSG-12-148-01-DDC) and the CIRM Basic Biology Award (RB4-05779).

The human skin protocol was performed in accordance with the guidelines of the University of California, San Diego&#8217;s Research Ethics Committee. Human skin can be obtained from discarded surgical samples or bought from skin banks (skin bank is listed in the Material/Equipment Table). The location where the skin is derived from or age of the donor is not critical to the experiment as long as the basement membrane zone proteins (collagen/laminin) in the dermis are not degraded.
1. Preparation of Devitalized Human Dermis
<ol>
	<li>Upon receiving human skin, place the tissue in the tissue culture hood to thaw (~3-5 min). Depending on the size of the skin, place thawed tissue in a sterile disposable 50 ml conical tube or 125 ml bottle. The typical size of skin from the skin bank is 0.75 square feet.</li>
	<li>Fill the container 75% full with 4x penicillin and streptomycin (pen/strep) in 1x PBS. Shake vigorously for 5 min and dispose of the liquid. Repeat this step 3 more times.</li>
	<li>After the last wash, transfer the tissue to a new tube/bottle and add fresh 4x pen/strep/PBS to fill 75% of the container. Incubate this mixture in a tissue culture incubator at 37 &#176;C for 2 weeks to allow the separation of the dermis from the epidermis.</li>
	<li>Under sterile conditions, use forceps to peel away the epidermis from the dermis. The dermis is completely white while the epidermis will have coloring depending on the race of the donor (Figure 1A). Discard the epidermis according to the institution&#8217;s protocol for dealing with human tissue.</li>
	<li>Place the dermis in a tube or bottle and wash it in 4x pen/strep diluted in 1x PBS with vigorous shaking (5 min washes for 4 times). After the last wash, transfer the dermis to a new tube/bottle in 4x pen/strep diluted in 1x PBS and store at 4 &#176;C until ready to use. Dermis can be stored at 4 &#176;C for up to a year.</li>
</ol>
2. Genetically Modifying Primary Human Keratinocytes
NOTE: Use amphotropic phoenix cells grown in complete DMEM media [DMEM +10% fetal bovine serum (FBS)+pen/strep] to produce viruses to infect primary human keratinocytes. Viral packaging genes that encode proteins such as gag-pol and envelope are stably integrated into the phoenix cell genome which allows for virus production when transfected with a retroviral vector. Phoenix cells can be transfected with high efficiency allowing for high viral titer production. Virus produced from this packaging line can also infect a large variety of mammalian cells including human.
<ol>
	<li>Day 1: The day before transfection, seed phoenix cells in a 6-well plate at a density of 800,000 cells per well in complete DMEM media.</li>
	<li>Day 2: The day of transfection, use 3 &#181;g of retroviral vectors per well. Aliquot 3 &#181;g of retroviral vector into a 1.5 ml tube. Use a mix of 100 &#181;l DMEM plus 6 &#181;l of transfection reagent for every 3 &#181;g of vector. Mix 6 &#181;l of transfection reagent with 100 &#181;l DMEM in a 1.5 ml tube. (The retroviral vectors that are used are listed in the equipment/materials table).
	<ol>
		<li>Incubate for 5 min and add the 106 &#181;l mixture into the pre-aliquoted tube containing the 3 &#181;g of retroviral vector. Mix and incubate at RT for 30 min. Add this entire mixture dropwise to each well of the phoenix cells.</li>
	</ol>
	</li>
	<li>Day 3: The day after transfection, remove the media from the phoenix cells and add 2 ml of fresh complete DMEM media. On the same day, seed primary human keratinocytes into 6-well plates at a density of 75,000 cells per well. Maintain the keratinocytes in a keratinocyte serum free medium (KCSFM) with antibiotics (pen/strep). 
	NOTE: Primary human keratinocytes were purchased. Cells can be purchased from a variety of vendors (listed in the Material/Equipment table).</li>
	<li>Day 4: Harvest the media from the transfected phoenix cells, which now contains viral particles. Pass the media through a 0.45 &#181;m filter using a syringe (to remove any contaminating phoenix cells) and place 2 ml onto the keratinocytes (plated at 75,000 cells/well the previous day: see step 2.3). Add hexadimethrine bromide (5 &#181;g/ml) to the media to help mediate the infection process. Viral titers are ~1 x 107 TU/ml.
	<ol>
		<li>Spin the 6-well plates in a centrifuge at 200 x g for 1 hr at RT. After the spin, remove the media containing virus and wash the cells once with 1x PBS before adding KCSFM.</li>
	</ol>
	</li>
	<li>Day 5: Infect the same batch of keratinocytes again as in step 2.4. 
	NOTE: Select the keratinocytes using a drug if there is a selectable marker on the retroviral vector and expand for use in downstream analysis or applications such as organotypic cultures.</li>
</ol>
3. Setting Up Organotypic Skin Cultures
<ol>
	<li>Build the organotypic cassette from common materials found in the laboratory or the cassettes can be custom made through a metal fabrication shop (Figure 2A-F). To make these cassettes from a 3.5 cm tissue culture plate, use a cautery to remove a 1.0 cm x 1.0 cm square from the center of the lid of a 3.5 cm dish (Figure 2A-B). Do this under sterile conditions.
	<ol>
		<li>Attach square pegs to the bottom of the cassette (lid of 3.5 cm dish) using clear nail polish (Figure 2C). Allow 5 min for the nail polish to dry and flip the cassette over so that it is resting on the square pegs (Figure 2D). Place the cassette into a 6 cm dish. 
		NOTE: Square pegs can be purchased from a variety of arts and crafts stores.</li>
	</ol>
	</li>
	<li>Prepare keratinocyte growth medium (KGM) comprised of 66% DMEM, 22% Ham&#8217;s F12, 100 units/ml penicillin, 100 &#181;g/ml streptomycin, 10% FBS, 2.67 &#181;g/ml adenine, 40 ng/ml hydrocortisone, 10 ng/ml cholera toxin, 4.94 &#181;g/ml insulin, 5 &#181;g/ml transferrin, 1.36 ng/ml Triiodo-L-thyronine, 10 ng/ml EGF, and 10 &#181;g/ml ciprofloxacin hydrochloride. Filter the media through a 0.22 &#181;m filter and store at 4 &#176;C until ready to use.</li>
	<li>Take the dermis out of the 4x pen/strep + PBS solution and wash 2 times in KGM and then incubate in KGM at 37&#160;&#176;C for two days prior to use.</li>
	<li>Cut the dermis using a scalpel to 1.5 cm x 1.5 cm sized pieces and then place onto the organotypic cassette. Place the dermis with the top of the dermis facing up. The top of the dermis will be the side that comes into contact with the keratinocytes (Figure 1B).</li>
	<li>Place the pre-cut dermis onto the square hole of the organotypic cassette with the top side of the dermis facing up (Figure 3A).</li>
	<li>Flip the entire organotypic cassette containing the dermis over using forceps (Figure 3B). Thaw the extracellular matrix solution on ice. Use a needle and syringe to draw out 100 &#181;l of extracellular matrix solution per cassette. Add 5 drops to the bottom of the dermis (glossy side) and shake slightly to ensure even distribution throughout the dermis (Figure 3B).</li>
	<li>Allow 5 min for the commercial extra cellular matrix solution to completely solidify (Figure 3C). After solidification, use forceps to flip the cassette back over. Add 4 ml of KGM to the 6 cm dish.</li>
	<li>Place 0.5-1 million genetically modified keratinocytes onto the organotypic cassettes containing the dermis (Figure 3D).
	<ol>
		<li>Trypsinize keratinocytes by placing 4 ml of 0.05% Trypsin onto 10 cm plates for 5 min and quench with 4 ml of complete DMEM media.</li>
		<li>Count the keratinocytes using a hemocytometer and then spin down at 200 x g for 5 min. Resuspend 0.5-1 million cells (depending on the number of cells available) in 90 &#181;l of KGM and place all the cells onto the dermis. 
		NOTE: It is important to place the same number of cells on dermis within the same experiment to ensure that any differences seen in thickness of the regenerated epidermis is not due to differences in starting cell number. Placing less than 0.5 million cells on dermis can lead to poor stratification and differentiation.</li>
	</ol>
	</li>
	<li>Change KGM media on the organotypic cultures every other day. Harvest tissue 1-14 days after placement of keratinocytes on the dermis. Full stratification and differentiation of the epidermis usually occurs after day 5.</li>
</ol>

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We have nothing to disclose.

Genetic manipulation in human skin organotypic cultures offer many advantages to commonly studied 2D cultured cells as well as mouse models. 2D cultures lack the three dimensional cell-cell and cell-extracellular matrix interactions found in intact tissues and organs. Recent studies have also found tremendous differences between 2D and 3D cultured skin cancer cells with the 3D cultured cells showing much more gene expression similarities to primary human skin tumors16. Human organotypic skin cultures also have several advantages over mouse models. Mouse models are time consuming and expensive to generate as well as several notable differences between human and mouse skin. These include different tissue turnover rates as well as the thickness of the epidermis between murine and human epidermis8. With new technologies emerging such as CRISPR-CAS, specific genes can be modified/mutated to model human skin diseases using organotypic skin cultures23. Furthermore, genes can be delivered or knocked down in these cells through a wide variety of methods besides the retroviral gene transfer described here. As long as there is robust delivery through high transduction efficiency or drug selection, the method can be employed. These delivery methods include nucleofection of siRNAs, lentiviral, adenoassociated virus, and adeno virus11,12,24-26.
The organotypic skin cultures can be modified by adding different cell types to the system. For example, normal or genetically altered human fibroblasts can be added to the devitalized dermis to study mesenchymal and epithelial crosstalk. Immune cells can also be added to the dermis to study the inflammatory response to perturbations in the skin. One limitation of the system is that the desquamation process that occurs in vivo does not occur in organotypic cultures. This prevents the use of these cultures for long term and most analysis is done between 1-14 days after seeding of the epidermal cells. For longer term assays, the organotypic cultures can be grafted onto the backs of immune compromised mice17,27.
The skin organotypic culture presented here is unique in that it utilizes intact devitalized human dermis which is the natural substrate of the epidermis in vivo whereas most other systems use collagen or Matrigel as the substrate28. Setup of organotypic skin cultures using substrates such as Matrigel results in thinner and less robust stratification of the epidermis in our experience. The dermis contains basement membrane zone proteins including laminin and collagen which allows the epidermal cells to attach, proliferate, differentiate, and stratify29. Use of dermis allows for generation of epidermis that mimics their in vivo counterpart. Thus, organotypic skin cultures can be extremely valuable in toxicological and pharmacological studies in cases where animal studies are not permitted (cosmetics industry)30.
Using devitalized dermis also comes with its own drawbacks since the thickness of the dermis between different donors or even within the same donor can vary. Epidermal cells grown on thick dermis tend to not proliferate and generate as thick of epidermis as cells grown on thinner dermis. Thus, it is critical to choose similar thickness dermis when setting up the experiments comparing multiple samples. Another critical parameter is to seed the same number of epidermal cells when comparing different groups so that any differences seen is not due to the differences in initial number of cells seeded.
In conclusion, the organotypic skin culture can serve as an advanced and efficient in vitro model for validating molecular pathways in a more physiological context, which ultimately will accelerate the understanding of gene regulatory mechanisms that are important in epidermal growth and differentiation.

The human epidermis is a stratified epithelium that connects to the underlying dermis through an extracellular matrix known as the basement membrane zone.The epidermis not only serves as an impermeable barrier to prevent the loss of moisture but also as a first line of defense to protect the body from foreign and toxic substances1. The basal layer, which is the deepest layer of the epidermis, contains the epidermal stem and progenitor cells that give rise to the differentiated progeny that form the rest of the epidermis2. As epidermal progenitor cells differentiate they migrate upwards to form the first layer of differentiated cells known as the spinous layer3. In the spinous layer, cells turn on the expression of keratins 1 and 10, which then provide the strength to withstand physical stress for the differentiated layers of the epidermis. As the spinous layer cells further differentiate, they move upwards in the epidermis to form the granular layer which is characterized by the formation of keratohyalin and lamellar granules as well as structural proteins that are assembled below the plasma membrane. As the cells proceed in the differentiation process, the proteins beneath the plasma membrane are cross linked to each other while the lamellar granules are extruded from the cells to form a lipid rich barrier called the stratum corneum4.
Diseases that involve alterations in epidermal growth and differentiation impact ~20% of the population5. Thus, understanding the mechanisms of this process is of great importance. Since manifestation of many of these diseases is contingent upon cell-cell or cell-matrix contact, organotypic cultures where the human epidermis is reconstituted in a 3D environment have been created6-10. These methods typically involve the use of primary or transformed keratinocytes seeded on extracellular matrix such as devitalized human dermis, Matrigel, or collagen.
To understand gene regulatory mechanisms that are important in epidermal growth and differentiation, keratinocytes can be genetically manipulated through retroviral vectors to knockdown or overexpress genes in 2D culture and then reconstituted in 3D. These methods have been used extensively to characterize genes involved in epidermal stem and progenitor cell self-renewal and differentiation as well as progression to neoplasia11-21. Here, an in-depth protocol on how to alter gene expression in epidermal organotypic cultures through the use of retroviruses is provided.

<table>
	<tbody>
		<tr>
			<td>Human skin</td>
			<td>New York Firefighters Skin Bank</td>
			<td><a href="http://www.cornellsurgery.org/pro/services/burn-surgery/skin-bank.html">http://www.cornellsurgery.org/pro/services/burn-surgery/skin-bank.html</a></td>
		</tr>
		<tr>
			<td>PEN/STREP</td>
			<td>GIBCO</td>
			<td>15140-122</td>
		</tr>
		<tr>
			<td>amphotropic phoenix cell lines</td>
			<td>ATCC</td>
			<td>CRL-3213</td>
		</tr>
		<tr>
			<td>FUGENE 6 transfection reagent</td>
			<td>Promega</td>
			<td>E2691</td>
		</tr>
		<tr>
			<td>Keratinocyte Media (KCSFM)</td>
			<td>Life Technologies</td>
			<td>17005042</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>GIBCO</td>
			<td>11995</td>
		</tr>
		<tr>
			<td>Ham&#39;s F12</td>
			<td>Cambrex</td>
			<td>12-615F</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>GIBCO</td>
			<td>10437-028</td>
		</tr>
		<tr>
			<td>Adenine</td>
			<td>Sigma</td>
			<td>A-9795</td>
		</tr>
		<tr>
			<td>Cholera Toxin</td>
			<td>Sigma</td>
			<td>&#160;C-8052</td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>Calbiochem</td>
			<td>3896</td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma</td>
			<td>I-1882</td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Invitrogen</td>
			<td>13247-051</td>
		</tr>
		<tr>
			<td>Transferrin&#160;</td>
			<td>Sigma</td>
			<td>T-0665</td>
		</tr>
		<tr>
			<td>Ciprofloxacin Hydrochloride</td>
			<td>Serologicals</td>
			<td>89-001-1</td>
		</tr>
		<tr>
			<td>cautery</td>
			<td>Bovie Medical Corporation</td>
			<td>AA01</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>354234</td>
		</tr>
		<tr>
			<td>Keratin 1 antibody</td>
			<td>Biolegend</td>
			<td>PRB-149P</td>
		</tr>
		<tr>
			<td>square pegs</td>
			<td>Arts and crafts stores</td>
			<td></td>
		</tr>
		<tr>
			<td>human neonatal keratinocytes</td>
			<td>ATCC</td>
			<td>PCS-200-010</td>
		</tr>
		<tr>
			<td>human neonatal keratinocytes</td>
			<td>Cell Applications</td>
			<td>102K-05n</td>
		</tr>
		<tr>
			<td>MSCV retroviral vector</td>
			<td>Clontech</td>
			<td>634401</td>
		</tr>
		<tr>
			<td>LZRS retroviral vector</td>
			<td>Addgene</td>
			<td></td>
		</tr>
		<tr>
			<td>pSuper.Retro.Puro Retroviral vector</td>
			<td>Oligoengine</td>
			<td>VEC-PRT-0002&#160;</td>
		</tr>
		<tr>
			<td>hexadimethrine bromide&#160;</td>
			<td>Sigma</td>
			<td>H9268-5G</td>
		</tr>
	</tbody>
</table>

generation of genetically modified organotypic skin cultures using devitalized human dermis

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function and physiology of their in vivo tissue counterparts. This is exemplified by organotypic skin cultures which faithfully recapitulates the epidermal differentiation and stratification program. Primary human epidermal keratinocytes are genetically manipulable through retroviruses where genes can be easily overexpressed or knocked down. These genetically modified keratinocytes can then be used to regenerate human epidermis in organotypic skin cultures providing a powerful model to study genetic pathways impacting epidermal growth, differentiation, and disease progression. The protocols presented here describe methods to prepare devitalized human dermis as well as to genetically manipulate primary human keratinocytes in order to generate organotypic skin cultures. Regenerated human skin can be used in downstream applications such as gene expression profiling, immunostaining, and chromatin immunoprecipitations followed by high throughput sequencing. Thus, generation of these genetically modified organotypic skin cultures will allow the determination of genes that are critical for maintaining skin homeostasis.

The first step in generating organotypic human skin is to remove the epidermis from the dermis. The two week incubation of the skin at 37&#160;&#176;C in 4x pen/strep/PBS should allow the separation of the dermis from the epidermis (Figure 1A). If separating the epidermis and dermis is difficult then place the tissue at 37&#160;&#176;C in 4x pen/strep/PBS for another week and then try peeling again using forceps.
One of the keys to regenerating human epidermis on devitalized dermis is to ensure that the keratinocytes are placed on the correct side of the dermis. The dermis has a top and a bottom. The top of the dermis is the side that comes into contact with the keratinocytes in vivo as well as in organotypic cultures. The top of the dermis can be differentiated from the bottom of the dermis due to the glossiness of the bottom (Figure 1B). If keratinocytes are placed directly onto the bottom side (glossy) of the dermis, the epidermis will not differentiate or stratify properly. Thus, it is crucial to have the top of the dermis (non-glossy) facing up in the organotypic cassette. The dermis can be placed directly onto an organotypic cassette (Figure 2A-F). Cassettes made from tissue culture plates should be UV sterilized while metal fabricated cassettes can be autoclaved prior to the dermis being placed on. The genetically modified keratinocytes can then be placed onto the dermis. The keratinocytes are initially submerged in liquid since they were placed onto the dermis resuspended in KGM. After 24 hr the KGM will have diffused through the dermis which allows the keratinocytes to be exposed to the air-liquid interface which causes stratification and differentiation into epidermis7 (Figure 3).
RNA, protein, DNA/chromatin, as well as tissue sections can be harvested from the organotypic skin cultures which can all be used for a variety of downstream applications. RNA can be used to determine gene expression differences between control and knockdown/overexpression tissue using RT-QPCR, microarrays, or RNA-Seq. DNA/chromatin can be used for DNA or ChIP-Seq applications. The regenerated skin can be mounted in OCT and frozen sections can be used for immuno-staining or hematoxylin and eosin staining to assess tissue morphology, protein expression/localization in control and experimental groups.
An example of this is shown in Figure 4 with the hematoxylin and eosin staining of a typical organotypic skin culture. Note that all four distinct layers of the epidermis are formed with layer specific expression of differentiation related proteins (Figure 4)11,12,14,15. This system can be used to determine the impacts of overexpression or knockdown of different genes on the epidermis. Figure 5 shows regenerated epidermal tissue that expresses control, LACZ as well as tissue overexpressed for SNAI2. Overexpression of SNAI2 led to an inhibition of epidermal differentiation as noted by the lack of keratin 1 (K1) staining as well as loss of expression of differentiation induced structural genes such as TGM1 and SPRR1A (Figure 5A-C)13,22. The epidermal thickness shown in Figure 4 and Figure 5 may vary within the same sample due to more cells accumulating in the middle of the dermis versus the periphery when the cells were initially placed on the dermis. Thus, the middle sections tend to have thicker epidermis whereas the periphery tends to have thinner. For direct comparison between different samples the same regions of a section should be compared. However, the staining for the markers should stay consistent.
<img alt="Figure 1" src="/files/ftp_upload/53280/53280fig1.jpg" /> 
Figure 1.&#160;Preparation of the dermis from human skin. (A) After incubation of the human skin at 37&#160;&#176;C for 2 weeks, the dermis can be separated from the epidermis. Forceps are used to peel the epidermis (brown colored) away from the dermis (white). (B) The top of the dermis can be distinguished from the bottom by the glossiness of the tissue. The top of the dermis (the side that will naturally face the epidermis in vivo or where the keratinocytes will be seeded) is non-glossy. The bottom of the dermis is glossy. The tissue is pink due to incubation in KGM. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53280/53280fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53280/53280fig2.jpg" /> 
Figure 2. Generation of organotypic cassettes. (A) Use a cautery to remove a 1.0 cm x 1.0 cm square from the center of the lid of a 3.5 cm dish. (B) Remove the square from the center of the 3.5 cm dish. (C) Use clear nail polish to adhere square pegs. Allow 5 min&#160;for the nail polish to dry. (D) Flip the lid over so that it is resting on the square pegs. The cassette is now ready to be used. (E) Image of a metal fabricated organotypic cassette with its dimensions. (F) Image of a metal fabricated cassette flipped upside down to show the support pegs. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53280/53280fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53280/53280fig3.jpg" /> 
Figure 3. Generation of organotypic skin cultures. (A) Place the dermis with the top of the dermis (non-glossy side) facing up onto the organotypic cassette. (B) Flip the cassette over and add Matrigel to the glossy side of the dermis. (C) Allow 5 min for the Matrigel to solidify and then flip the cassette back over. (D) Add genetically modified keratinocytes to the dermis (0.5-1 million cells are resuspended in 90 &#181;l of KGM). Harvest the tissue 1-14 days after seeding of the keratinocytes. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53280/53280fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53280/53280fig4.jpg" /> 
Figure 4. Hematoxylin and eosin staining of organotypic skin cultures. Regenerated human skin contains both the epidermis and dermis. The epidermis is fully stratified and differentiated with 4 distinct layers. These layers include the undifferentiated basal layer and the 3 differentiated layers including stratum spinosum, granulosum and corneum. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53280/53280fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/53280/53280fig5.jpg" /> 
Figure 5. Immunofluorescence staining and gene expression analysis of genetically modified human epidermis. (A) Overexpression of SNAI2 inhibits epidermal differentiation. Staining for differentiation protein keratin 1 (K1) is shown in green and nuclei is marked in blue (Hoechst). (B) RT-QPCR analysis on the mRNA levels of SNAI2 in control LACZ and SNAI2 overexpressing tissue. Error bars = SD, n = 3. (C) RT-QPCR analysis on the mRNA levels of differentiation transcripts KRT1, TGM1, and SPRR1A in control LACZ and SNAI2 overexpressing tissue. Error bars = SD, n = 3. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53280/53280fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis at JoVE.com

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

The goal of this paper is to provide a comprehensive and detailed protocol on how to generate genetically modified human organotypic skin from epidermal keratinocytes and devitalized human dermis.

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

generation-genetically-modified-organotypic-skin-cultures-using

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

11.2K Views.  UCSD Stem Cell Program, University of California, San Diego. The goal of this paper is to provide a comprehensive and detailed protocol on how to generate genetically modified human organotypic skin from epidermal keratinocytes and devitalized human dermis. 

Video: Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This method maintains the characteristic topographical morphology of the hippocampus while allowing direct application of pharmacological agents to the developing hippocampal dentate gyrus. Additionally, slice cultures can be maintained for up to 4 weeks and thus, allow one to study the maturation process of newborn granule neurons. Slice cultures allow for efficient pharmacological manipulation of hippocampal slices while excluding complex variables such as uncertainties related to the deep anatomic location of the hippocampus as well as the blood brain barrier. For these reasons, we sought to optimize organotypic slice cultures specifically for postnatal neurogenesis research.

Here we describe a technique for studying hippocampal postnatal neurogenesis using the organotypic slice culture technique. This method allows for in vitro manipulation of adult neurogenesis and allows for the direct application of pharmacological agents to the cultured hippocampus.

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This ...

Generation of Human Adipose Stem Cells through Dedifferentiation of Mature Adipocytes in Ceiling Cultures

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature adipocytes can also be isolated from fresh adipose tissue for depot-specific characterization of their function and metabolic properties. Here, we describe a well-established protocol to isolate mature adipocytes from adipose tissues using collagenase digestion, and subsequent steps to perform ceiling cultures. Briefly, adipose tissues are incubated in a Krebs-Ringer-Henseleit buffer containing collagenase to disrupt tissue matrix. Floating mature adipocytes are collected on the top surface of the buffer. Mature cells are plated in a T25-flask completely filled with media and incubated upside down for a week. An alternative 6-well plate culture approach allows the characterization of adipocytes undergoing dedifferentiation. Adipocyte morphology drastically changes over time of culture. Immunofluorescence can be easily performed on slides cultivated in 6-well plates as demonstrated by FABP4 immunofluorescence staining. FABP4 protein is present in mature adipocytes but down-regulated through dedifferentiation of fat cells. Mature adipocyte dedifferentiation may represent a new avenue for cell therapy and tissue engineering.

Mature adipocytes may represent an abundant source of stem cells through dedifferentiation, which leads to a homogenous population of fibroblast-like cells. Collagenase digestion is used to isolate mature adipocytes from human fat. The goal of our protocol is to obtain multipotent, dedifferentiated fat cells from human mature adipocytes.

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature ...

Transfection, Selection, and Colony-picking of Human Induced Pluripotent Stem Cells TALEN-targeted with a GFP Gene into the AAVS1 Safe Harbor

Targeted transgene addition can provide persistent gene expression while circumventing the gene silencing and insertional mutagenesis caused by viral vector mediated random integration. This protocol describes a universal and efficient transgene targeted addition platform in human iPSCs based on utilization of validated open-source TALENs and a gene-trap-like donor to deliver transgenes into a safe harbor locus. Importantly, effective gene editing is rate-limited by the delivery efficiency of gene editing vectors. Therefore, this protocol first focuses on preparation of iPSCs for transfection to achieve high nuclear delivery efficiency. When iPSCs are dissociated into single cells using a gentle-cell dissociation reagent and transfected using an optimized program, >50% cells can be induced to take up the large gene editing vectors. Because the AAVS1 locus is located in the intron of an active gene (PPP1R12C), a splicing acceptor (SA)-linked puromycin resistant gene (PAC) was used to select targeted iPSCs while excluding random integration-only and untransfected cells. This strategy greatly increases the chance of obtaining targeted clones, and can be used in other active gene targeting experiments as well. Two weeks after puromycin selection at the dose adjusted for the specific iPSC line, clones are ready to be picked by manual dissection of large, isolated colonies into smaller pieces that are transferred to fresh medium in a smaller well for further expansion and genetic and functional screening. One can follow this protocol to readily obtain multiple GFP reporter iPSC lines that are useful for in vivo and in vitro imaging and cell isolation.

TALEN-mediated gene editing at the safe harbor AAVS1 locus enables high-efficiency transgene addition in human iPSCs. This protocol describes the procedures for preparing iPSCs for TALEN and donor vector delivery, transfecting iPSCs, and selecting and isolating iPSC clones to achieve targeted integration of a GFP gene to generate reporter lines.

Targeted transgene addition can provide persistent gene expression while circumventing the gene silencing and insertional mutagenesis caused by viral ...

3D Organotypic Co-culture Model Supporting Medullary Thymic Epithelial Cell Proliferation, Differentiation and Promiscuous Gene Expression

Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse this scaffold in a highly coordinated temporal and spatial order while sequentially passing obligatory check points, i.e., T cell lineage commitment, followed by T cell receptor repertoire generation and selection prior to their export into the periphery. The two major resident cell types forming this scaffold are cortical (cTECs) and medullary thymic epithelial cells (mTECs). A key feature of mTECs is the so-called promiscuous expression of numerous tissue-restricted antigens. These tissue-restricted antigens are presented to immature thymocytes directly or indirectly by mTECs or thymic dendritic cells, respectively resulting in self-tolerance.
Suitable in vitro models emulating the developmental pathways and functions of cTECs and mTECs are currently lacking. This lack of adequate experimental models has for instance hampered the analysis of promiscuous gene expression, which is still poorly understood at the cellular and molecular level. We adapted a 3D organotypic co-culture model to culture ex vivo isolated mTECs. This model was originally devised to cultivate keratinocytes in such a way as to generate a skin equivalent in vitro. The 3D model preserved key functional features of mTEC biology: (i) proliferation and terminal differentiation of CD80lo, Aire-negative into CD80hi, Aire-positive mTECs, (ii) responsiveness to RANKL, and (iii) sustained expression of FoxN1, Aire and tissue-restricted genes in CD80hi mTECs.

Studying medullary thymic epithelial cells in vitro has been largely unsuccessful, as current 2D culture systems do not mimic the in vivo scenario. The 3D culture system described herein - a modified skin organotypic culture model - has proven superior in recapitulating mTEC proliferation, differentiation and maintenance of promiscuous gene expression.

Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse ...

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 ...

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which&#160;allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies&#160;are: 1) the use of a 2A &#34;self-cleavage&#34; peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

Using Primary Neurosphere Cultures to Study Primary Cilia

The primary cilium is fundamentally important for the proliferation of neural stem/progenitor cells and for neuronal differentiation during embryonic, postnatal, and adult life. In addition, most differentiated neurons possess primary cilia that house signaling receptors, such as G-protein-coupled receptors, and signaling molecules, such as adenylyl cyclases. The primary cilium determines the activity of multiple developmental pathways, including the sonic hedgehog pathway during embryonic neuronal development, and also functions in promoting compartmentalized subcellular signaling during adult neuronal function. Unsurprisingly, defects in primary cilium biogenesis and function have been linked to developmental anomalies of the brain, central obesity, and learning and memory deficits. Thus, it is imperative to study primary cilium biogenesis and ciliary trafficking in the context of neural stem/progenitor cells and differentiated neurons. However, culturing methods for primary neurons require considerable expertise and are not amenable to freeze-thaw cycles. In this protocol, we discuss culturing methods for mixed populations of neural stem/progenitor cells using primary neurospheres. The neurosphere-based culturing methods provide the combined benefits of studying primary neural stem/progenitor cells: amenability to multiple passages and freeze-thaw cycles, differentiation potential into neurons/glia, and transfectability. Importantly, we determined that neurosphere-derived neural stem/progenitor cells and differentiated neurons are ciliated in culture and localize signaling molecules relevant to ciliary function in these compartments. Utilizing these cultures, we further describe methods to study ciliogenesis and ciliary trafficking in neural stem/progenitor cells and differentiated neurons. These neurosphere-based methods allow us to study cilia-regulated cellular pathways, including G-protein-coupled receptor and sonic hedgehog signaling, in the context of neural stem/progenitor cells and differentiated neurons.

The primary cilium is fundamentally important in neural progenitor cell proliferation, neuronal differentiation, and adult neuronal function. Here, we describe a method to study ciliogenesis and the trafficking of signaling proteins to cilia in neural stem/progenitor cells and differentiated neurons using primary neurosphere cultures.

The primary cilium is fundamentally important for the proliferation of neural stem/progenitor cells and for neuronal differentiation during embryonic, ...

A Modified Trier Social Stress Test for Vulnerable Mexican American Adolescents

The Trier Social Stress Test (TSST) is a well validated and widely used social stressor that has been shown to induce a 2-4 fold increase in cortisol, the biological output produced by the Hypothalamic-Pituitary-Adrenal (HPA) axis in humans. While studies have explored how modifications to the TSST influence stress responsivity, few studies have created a modified TSST appropriate for vulnerable youth that elicits a significant cortisol stress response. Thus, the current study sought to modify or adjust the TSST in a culturally sensitive way for a vulnerable sample of 14 year-old adolescents. The present study took place within the context of a longitudinal birth cohort study of Mexican American families in California called the Center for the Health Assessment of Mothers and Children of Salinas (CHAMACOS). The CHAMACOS sample was optimal to test the effectiveness of a modified culturally appropriate TSST, as it is comprised of Mexican American youth, who are often excluded from research. These youths also have experienced significant early life adversity. Example modifications included timed prompts, alternative math tasks, use of same-ethnicity peers as confederates, debriefing immediately after the conclusion of the TSST, and using an unknown youth examiner to administer the debrief. Saliva samples were collected at baseline (after a resting phase), and then again at 15, 30, and 45 min post-TSST onset to assess cortisol concentration. A pilot study of 50 participants (50% female) have been analyzed for cortisol reaction to the TSST. Results confirmed that this modified version of the TSST was successful at eliciting a significant cortisol reaction, with a wide range of variability likely due to individual differences. Goals for modifications and ethnicity considerations are discussed. This study provides the foundation for future research to utilize a modified TSST with vulnerable youth.

Here, we present a protocol that provoked cortisol reactivity in a vulnerable adolescent Mexican American sample utilizing a modified version of the Trier Social Stress Test (TSST). Saliva samples were collected at baseline, 15, 30, and 45 min post-TSST onset. Future research could utilize this modified TSST with vulnerable youth.

The Trier Social Stress Test (TSST) is a well validated and widely used social stressor that has been shown to induce a 2-4 fold increase in cortisol, the ...

Generation of Genetically Modified Mice through the Microinjection of Oocytes

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear injection of DNA expression constructs into fertilized oocytes remains the most commonly used technique to generate transgenic mice for overexpression. With the introduction of CRISPR technology for gene targeting, pronuclear injection into fertilized oocytes has been extended to the generation of both knockout and knockin mice. This work describes the preparation of DNA for injection and the generation of CRISPR guides for gene targeting, with a particular emphasis on quality control. The genotyping procedures required for the identification of potential founders are critical. Innovative genotyping strategies that take advantage of the "multiplexing" capabilities of CRISPR are presented herein. Surgical procedures are also outlined. Together, the steps of the protocol will allow for the generation of genetically modified mice and for the subsequent establishment of mouse colonies for a plethora of research fields, including immunology, neuroscience, cancer, physiology, development, and others.

The microinjection of mouse oocytes is commonly used for both classic transgenesis (i.e., the random integration of transgenes) and CRISPR-mediated gene targeting. This protocol reviews the latest developments in microinjection, with a particular emphasis on quality control and genotyping strategies.

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear ...

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. ...