This work is supported by NIH NIAMS grant R37 AR 060306, R01 AR 047364, and RO1 AR078050. The work is also supported by a collaborative research contract between USC and China Medical University in Taiwan. We thank the USC BISC 480 Developmental Biology 2023 class for successfully testing this avian skin culture protocol during several lab modules.

1. Chicken skin explant culture (Figure 1)
<ol>
	<li>Incubate fertilized chicken eggs in a humidified incubator at 38 &#176;C and stage them according to Hamburger and Hamilton14.
	<ol>
		<li>At stage 28 (~E5.5), the limb&#39;s second digit and third toe are longer than the others; three digits and four toes are distinct.</li>
		<li>At stage 29 (~E6), the wing is bent at the elbow; the second digit is distinctly longer than the others.</li>
		<li>At stage 30 (~E6.5), two dorsal feather bud rows to either side of the spinal cord are visible at the brachial level and three rows are seen at the level of the legs.</li>
		<li>At stage 31 (~E7), there are ~7 rows of feathers at the jumbo-sacral levels.</li>
		<li>At stage 32, eleven or more rows of feather buds are present at the level of the legs.</li>
	</ol>
	</li>
	<li>Place a stage 28-32 chicken embryo in a 60 mm Petri dish containing Hanks&#39;s buffered saline solution (HBSS) or phosphate-buffered saline (PBS).
	<ol>
		<li>Dissect out the embryo dorsal skin between the lower neck to the tail region. Use scissors to remove the head from the neck. Gently pull the limb buds to position the ventral side of the body downwards with the appendages spreading outwards.</li>
		<li>Make an anterior to posterior incision in the skin along the two flanking sides of the embryo body using watchmaker&#39;s forceps or sharp spring scissors. Be sure to stabilize the body with a pair of forceps by pinching the limb buds longitudinally. Gently peel the skin from the neck to the tail region or from the tail to the neck using watchmaker&#39;s forceps.</li>
	</ol>
	</li>
	<li>Gently remove the subcutaneous tissues that remain attached to the peeled skin and smooth the edges of the skin with the sharp spring scissors.</li>
	<li>Add 2 mL/well of Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and Penicillin/streptomycin solution (diluted 1:100) to a well in a 6-well dish. Once the medium and antibiotics have been added, place a sterile tissue culture insert inside the well, such that the media is on the exterior of the culture insert.</li>
	<li>Transfer the skin with a spatula to the culture insert with the epidermis face up and the mesenchyme face down on the insert membrane. To ensure the skin lays flat without any creases, pull the skin onto the spatula in HBSS. Slide the skin off the spatula with the liquid to facilitate the transfer of the skin without folding.</li>
	<li>Remove excess HBSS from the culture insert with a 200 &#181;L pipette. Leave a thin layer of HBSS inside the culture insert to keep the explant semi-wet to provide an air-liquid interface.</li>
	<li>Incubate the skin explant cultures at 37 &#176;C using 5% CO2 and 95% air. Change the medium every 2 days. 
	&#8203;NOTE: This procedure has been published4. This culture system can also be used with the skin of other birds such as quail or finch skin explant cultures.</li>
</ol>
2. Chicken skin epithelial-mesenchymal recombination (Figure 2)
<ol>
	<li>Incubate fertilized chicken eggs in a humidified incubator at 38 &#176;C and stage the embryos according to Hamburger and Hamilton14 (section 1.1).</li>
	<li>Prepare 2x calcium-magnesium-free (CMF) saline with 0.25% ethylenediaminetetraacetic acid (EDTA) buffer.
	<ol>
		<li>Make 10x CMF buffer (NaCl (1.37 M) 80 g, KCl (0.04 M) 3 g, NaH2PO4 (0.004 M) 0.5 g, KH2PO4 (2 M) 0.25 g, NaHCO3 (0.12 M) 10 g, glucose (0.1 M) 20 g in 1,000 mL of distilled water. Adjust pH to 7.3. To make 100 mL of 2x CMF with 0.25% EDTA buffer, the EDTA should be dissolved in 80 mL of distilled water first, then add 20 mL of 10x CMF buffer to make 2x CMF with 0.25% EDTA working solution.</li>
	</ol>
	</li>
	<li>Dissect stage 30-32 chicken embryo dorsal skins in HBSS as described above (section 1.2) and incubate them in 2x CMF saline with 0.25% EDTA on ice for 15-20 min.</li>
	<li>Carefully separate the epithelium and mesenchyme using watchmaker&#39;s forceps. Transfer the separated epithelium and mesenchyme to a clean dish containing HBSS.</li>
	<li>Recombine the epithelium with the mesenchyme by first placing the mesenchyme on a Petri dish containing HBSS, then place the epithelium on top of the mesenchyme. Place the epithelium along the same anterior-posterior axis as the mesenchyme or turn the epithelium 90o from the anterior-posterior axis of the mesenchyme to determine which component regulates different aspects of skin morphogenesis.</li>
	<li>Transfer the recombined skins to culture inserts in 6-well culture dishes for growth and remove excess HBSS around the recombined skin.</li>
	<li>Place 2 mL of DMEM culture medium with 10% FBS in the well outside the insert. Place a thin layer of DMEM inside the insert chamber to keep the explant semi-wet to provide an air-liquid interface.</li>
	<li>Incubate the skin recombinants at 37 &#176;C in a mixture of 5% CO2 and 95% air. Change the medium every 2 days. Document phenotypic changes in initial skin phases of skin development using time-lapse photography with a camera mounted on a dissecting microscope. 
	NOTE: This procedure has been published11.</li>
</ol>
3. Chicken skin reconstitution (Figure 3)
<ol>
	<li>Incubate fertilized chicken eggs in a humidified incubator at 38 &#176;C and stage the embryos according to Hamburger and Hamilton14 (section 1.1).</li>
	<li>Dissect stage 30-33 chicken embryo dorsal skins in HBSS. Incubate the skins in 2x CMF saline with 0.25% EDTA on ice for 15-20 min as described above (section 2.3).</li>
	<li>Carefully separate the epithelium and mesenchyme using watchmaker&#39;s forceps. Transfer the separated epithelium and mesenchyme to HBSS on ice. 
	NOTE: Be careful not to damage the basal layer of the epithelium when separating the epithelium from the mesenchyme.</li>
	<li>Collect the separated mesenchyme in a 15 mL centrifuge tube and incubate with 0.1% collagenase/trypsin solution made in PBS in a 37 &#176;C water bath for 10-15 min.</li>
	<li>Dissociate the skin mesenchyme with a Pasteur pipet to make a single-cell suspension. Add DMEM with 10% FBS to stop the digestion.</li>
	<li>Pellet and centrifuge the cells at 233 &#215; g for 5 min. Resuspended the cells with culture medium at a concentration of 2 &#215; 107 cells/mL. 
	NOTE: At this step, the mesenchymal cells can also be resuspended in virus-containing medium for 2-3 h on ice for gene transduction.</li>
	<li>Place a cell culture insert in one well of a 6-well dish. Drop 10 &#181;L of 2 &#215; 107 cells/mL mesenchymal cells on the tissue culture insert. Place 2 ml of DMEM culture medium with 10% FBS in the well outside of the insert. Incubate the plated mesenchymal cells at 37 &#176;C using a 5% CO2 and 95% air incubator for 1 h to allow the cells to settle on the insert membrane.</li>
	<li>Transfer the intact epithelium on top of the plated mesenchymal droplet with the epithelium basal cell layer facing the mesenchymal cells using a 1 mL pipet. 
	NOTE: When transferring epithelium to the mesenchymal droplet, the mesenchymal cells should not be disturbed.</li>
	<li>Flatten the intact epithelium over the mesenchyme to make the reconstituted skin explant. Leave a thin layer of the medium on the insert to keep the reconstituted skin explant semi-wet to provide an air-liquid interface. Incubate the reconstituted skin explant at 37 &#176;C in a 5% CO2 and 95% air incubator. Change the medium every 2 days. 
	NOTE: This procedure has been published13.</li>
</ol>

A Comprehensive Review of Avian Embryonic Skin Explant Culture Protocols

<ol><li>Lucas, A. M., Stettenheim, P. R. Avian anatomy: Integument part I and part II. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aLucas%2C+AM+%22Agriculture+Handbook%22">Agriculture Handbook</a>. 362, Agricultural Research Service, U.S. Department of Agriculture, Washington D.C.. (1972).</li>
<li>Sengel, P. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aSengel%2C+P+%22In+Morphogenesis+of+Skin%2C+l-277%22">In Morphogenesis of Skin, l-277</a>. , Cambridge Univ. Press. New York. (1976).</li>
<li>Dhouailly, D., Wilehm Roux, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Formation+of+cutaneous+appendages+in+dermo-epidermal+recombinations+between+reptiles%2C+birds+and+mammals%5BTitle%5D)+AND+%22Archives+of+Developmental+Biology%22%5BJournal%5D)">Formation of cutaneous appendages in dermo-epidermal recombinations between reptiles, birds and mammals.</a> Archives of Developmental Biology. 177 (4), 323-340 (1975).</li>
<li>Jiang, T. -X., Chuong, C. -M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanism+of+feather+morphogenesis%3A+I.+Analyses+with+antibodies+to+Adhesion+Molecules+Tenascin%2C+N-CAM+and+Integrin%5BTitle%5D)+AND+%22Developmental+Biology%22%5BJournal%5D)">Mechanism of feather morphogenesis: I. Analyses with antibodies to Adhesion Molecules Tenascin, N-CAM and Integrin.</a> Developmental Biology. 150 (1), 82-98 (1992).</li>
<li>Li, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Shaping+organs+by+a+Wnt+%2F+Notch+%2F+non-muscle+myosin+module+which+orients+feather+bud+elongation%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences%2C+USA%22%5BJournal%5D)">Shaping organs by a Wnt / Notch / non-muscle myosin module which orients feather bud elongation.</a> Proceedings of the National Academy of Sciences, USA. 110 (16), E1452-E1461 (2013).</li>
<li>Li, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Calcium+oscillations+coordinate+feather+mesenchymal+cell+movement+by+SHH+dependent+modulation+of+gap+junction+networks%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">Calcium oscillations coordinate feather mesenchymal cell movement by SHH dependent modulation of gap junction networks.</a> Nature Communications. 9 (1), 5377(2018).</li>
<li>Ting-Berreth, S. A., Chuong, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Local+delivery+of+TGF+beta2+can+restore+epithelium+dependent+organization+of+mesenchymal+condensation+during+skin+appendage+morphogenesis%5BTitle%5D)+AND+%22Developmental+Biology%22%5BJournal%5D)">Local delivery of TGF beta2 can restore epithelium dependent organization of mesenchymal condensation during skin appendage morphogenesis.</a> Developmental Biology. 179 (2), 347-359 (1996).</li>
<li>Widelitz, R. B., Jiang, T. -X., Noveen, A., Chen, C. -W. J., Chuong, C. -M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((FGF+induces+new+feather+buds+from+developing+avian+skin%5BTitle%5D)+AND+%22Journal+of+Investigation+Dermatology%22%5BJournal%5D)">FGF induces new feather buds from developing avian skin.</a> Journal of Investigation Dermatology. 107 (6), 797-803 (1996).</li>
<li>Rawles, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tissue+interactions+in+scale+and+feather+development+as+studies+in+dermal-epidermal+recombinations%5BTitle%5D)+AND+%22Journal+of+Embryology+and+Experimental+Morphology%22%5BJournal%5D)">Tissue interactions in scale and feather development as studies in dermal-epidermal recombinations.</a> Journal of Embryology and Experimental Morphology. 11, 765-789 (1963).</li>
<li>McAleese, S. R., Sawyer, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Correcting+the+phenotype+of+the+epidermis+from+chick+embryos+homozygous+for+the+gene+scaleless+%28sc%2Fsc%29%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Correcting the phenotype of the epidermis from chick embryos homozygous for the gene scaleless (sc/sc).</a> Science. 214 (4524), 1033-1034 (1981).</li>
<li>Chuong, C. M., Widelitz, R. B., Ting-Berreth, S., Jiang, T. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Early+events+during+avian+skin+appendage+regeneration%3A+dependence+on+epithelial-mesenchymal+interaction+and+order+of+molecular+reappearance%5BTitle%5D)+AND+%22J+Invest+Dermatol%22%5BJournal%5D)">Early events during avian skin appendage regeneration: dependence on epithelial-mesenchymal interaction and order of molecular reappearance.</a> J Invest Dermatol. 107 (4), 639-646 (1996).</li>
<li>Jiang, T. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Global+feather+orientations+changed+by+electric+current%5BTitle%5D)+AND+%22iScience%22%5BJournal%5D)">Global feather orientations changed by electric current.</a> iScience. 24 (6), 102671(2021).</li>
<li>Jiang, T. X., Jung, H. S., Widelitz, R. B., Chuong, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Self-organization+of+periodic+patterns+by+dissociated+feather+mesenchymal+cells+and+the+regulation+of+size%2C+number+and+spacing+of+primordia%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Self-organization of periodic patterns by dissociated feather mesenchymal cells and the regulation of size, number and spacing of primordia.</a> Development. 126 (22), 4997-5009 (1999).</li>
<li>Hamburger, V., Hamilton, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+series+of+normal+stages+in+the+development+of+the+chick+embryo%5BTitle%5D)+AND+%22Journal+of+Morphology%22%5BJournal%5D)">A series of normal stages in the development of the chick embryo.</a> Journal of Morphology. 188, 49-92 (1951).</li>
<li>Chen, Y. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Conservation+of+early+odontogenic+signaling+pathway+in+Aves%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences%2C+USA%22%5BJournal%5D)">Conservation of early odontogenic signaling pathway in Aves.</a> Proceedings of the National Academy of Sciences, USA. 97 (18), 10044-10049 (2000).</li>
<li>Chuong, C. -M., Ting, S. A., Widelitz, R. B., Lee, Y. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanism+of+Skin+Morphogenesis%3A+II.+Retinoic+acid+gradient+modulates+axis+orientation+and+phenotypes+of+skin+appendages%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Mechanism of Skin Morphogenesis: II. Retinoic acid gradient modulates axis orientation and phenotypes of skin appendages.</a> Development. 115 (3), 839-852 (1992).</li>
<li>Noveen, A., Jiang, T. -X., Chuong, C. -M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protein+kinase+A+and+protein+kinase+C+modulators+have+reciprocal+effects+on+mesenchymal+condensation+during+skin+appendage+morphogenesis%5BTitle%5D)+AND+%22Developmental+Biology%22%5BJournal%5D)">Protein kinase A and protein kinase C modulators have reciprocal effects on mesenchymal condensation during skin appendage morphogenesis.</a> Developmental Biology. 171 (2), 677-693 (1995).</li>
<li>Wu, X. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Self-assembly+of+biological+networks+via+adaptive+patterning+revealed+by+avian+intradermal+muscle+network+formation%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences%2C+USA%22%5BJournal%5D)">Self-assembly of biological networks via adaptive patterning revealed by avian intradermal muscle network formation.</a> Proceedings of the National Academy of Sciences, USA. 116 (22), 10858-10867 (2019).</li>
</ol>

The authors have no conflicts of interest to declare.

Tissue recombination provides an assay to explore the unique contributions of the epithelium and mesenchyme. In chickens, feathers begin to develop at embryonic day 7 (E7) while scales begin at E9. When E9 scale mesenchyme is recombined with E7 feather epithelium, the recombined tissue forms scales, and when E7 feather mesenchyme is recombined with E9 scale epithelium feathers are formed11. These studies have demonstrated that the mesenchyme controls the pattern formation spacing and organ identit...

Avian embryo skin development is an excellent model for studying the mechanisms of morphogenesis because of the distinct patterns and the accessibility to microsurgery and manipulation1,2. However, evaluating cellular and molecular events in intact tissues can be difficult because the presence of extraneous tissues can complicate microscopic observations. Furthermore, the ability to manipulate gene expression to test their role in skin morphogenesis is not always a simple task. We find we can test gene functions using retroviral transduction with a higher success rate using skin explant models. Here we discuss the advantages of three skin explant models that have been developed.
Avian embryonic skin culture is a powerful system to assess cell behavior, gene regulation, and function during skin feather bud development3,4,5,6. It allows for the evaluation of the molecular mechanisms of feather bud development through the global addition of growth factors placed in the culture media or their local release from growth factor-coated beads. Developmental regulatory genes can also be manipulated using viral gene transduction of intact or dominant negative forms for functional studies evaluating their roles in specific morphogenetic events 7,8.
Avian epithelial-mesenchymal recombination culture enables investigators to determine the contributions of each skin component during the early stages of skin morphogenesis. Rawles&#39; use of this approach revealed that interactions between the mesenchyme and epithelium are essential to forming skin appendages9. The mesenchyme can form condensations and the epithelium is needed to induce and maintain mesenchymal condensation formations2. Later, this approach was used to assess why Scaleless chickens fail to form feathers. The defect was discovered to be in mesenchyme10. Dhouailly performed tissue epithelial-mesenchymal recombination studies in embryos from different species. These studies provided developmental and evolutionary insights into epithelial-mesenchymal communications that promote skin morphogenesis3.
This study was used to better understand factors that control feather growth. The method also improves the visualization of cellular and molecular events involved in skin patterning that take place during feather initiation, development, and elongation along the anterior-posterior axis. When the epithelium is separated from the mesenchyme and the two components are then recombined, new interactions re-establish skin patterning. This approach allows us to evaluate mesenchymal inducing signals and epithelial competence molecules that enable the epidermis to respond to the mesenchymal signals11. The subsequent downstream molecular expression that is required for feather bud development and pattern formation can also be examined. These studies have established that the location of buds is controlled by the mesenchyme. Rotation of the epithelium 90o before recombination with the mesenchyme demonstrates that the direction of feather bud elongation is controlled by the epithelium. This method was essential for us to study the molecular mechanism regulating feather bud orientation12.
Avian skin reconstitution culture, in which the skin mesenchyme is dissociated to single cells before plating at high cell density and overlaid with intact epithelium, resets dermal cells to a primordial state. The explant then self-organizes to form a new periodic pattern independent of the previous cues13. This skin reconstitution model can be used to study the initial processes of feather periodic patterning. We used this approach to explore how modulating the ratio of mesenchymal cells to a single piece of epithelium can influence the size or number of feather buds. The number of buds was found to increase but not the size of buds as the ratio of mesenchymal cells increased. Another advantage to this approach is that mesenchymal cell viral transduction shows higher efficiency than in the other two culture conditions and can produce more obvious phenotypes.

<table><tbody><tr><td>6-well culture dish&amp;nbsp;</td><td>Falcon</td><td>REF 353502</td><td>Air-Liquid Interface (ALI) Cultures &amp;nbsp;</td></tr><tr><td>Cell culture inset&amp;nbsp;</td><td>Falcon</td><td>REF 353090.</td><td>0.4 &amp;micro;m Transparent PET Membrane</td></tr><tr><td>Collagenase Type 1</td><td>Worthington Biochemical</td><td>LS004196</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s modified Eagle&amp;rsquo;s medium&amp;nbsp;</td><td>Corning</td><td>10-013-CV</td><td>4.5 g/L glucose</td></tr><tr><td>Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA)</td><td>Sigma-Aldrich</td><td>E5134</td><td></td></tr><tr><td>Fetal bovine serum</td><td>ThermoFisher</td><td>16140-071</td><td></td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G8270</td><td></td></tr><tr><td>Hanks&amp;rsquo;s buffered saline solution</td><td>Gibco</td><td>14170-112</td><td>No calcium, no magnesium</td></tr><tr><td>Penicillin/streptomycin&amp;nbsp;</td><td>Gibco</td><td>15-140-122</td><td></td></tr><tr><td>Pogassium phosphate monobasic (KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>P5379</td><td></td></tr><tr><td>Potassium chloride (KCl)</td><td>Sigma-Aldrich</td><td>P9333</td><td></td></tr><tr><td>Sodium bicarbonate (NaHCO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>S6014</td><td></td></tr><tr><td>Sodium chloride (Nacl)</td><td>EMD&amp;nbsp;</td><td>CAS 7647-14-5</td><td></td></tr><tr><td>Sodium phosphate monobasic (NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>S0751</td><td></td></tr><tr><td>Trypsin</td><td>Gibco</td><td>27250-042</td><td></td></tr></tbody></table>

using avian skin explants to study tissue patterning and organogenesis

The developing avian skin during embryogenesis is a unique model that can provide valuable insights into tissue patterning. Here three variations on skin explant cultures to examine different aspects of skin development are described. First, ex vivo organ cultures and manipulations offer researchers opportunities to observe and study the development of feather buds directly. Skin explant culture can grow for 7 days enabling direct analysis of cellular behavior and 4D imaging at intervals during this growth period. This also allows for physical and molecular manipulations of culture conditions to visualize tissue response. For example, growth factor-coated beads can be applied locally to induce changes in feather patterning in a limited area. Alternatively, viral transduction can be delivered globally in the culture media to up or downregulate gene expression. Second, the skin recombination protocol allows researchers to investigate tissue interactions between the epidermis and mesenchyme that are derived from different skin regions, different life stages, or different species. This affords an opportunity to test the time window in which the epithelium is competent to respond to signals and its ability to form different skin appendages in response to signals from different mesenchymal sources. Third, skin reconstitution using dissociated dermal cells overlaid with intact epithelium resets skin development and enables the study of the initial processes of periodic patterning. This approach also enhances our ability to manipulate gene expression among the dissociated cells before creating the reconstituted skin explant. This paper provides the three culture protocols and exemplary experiments to demonstrate their utility.

Skin explant cultures 
Feather bud development from ex vivo skin organ cultures can directly be observed under the microscope. Using the skin explant culture model of chicken stage 30&#160;dorsal skin, the placodes are visible along the midline. The morphogenetic front then gradually propagates laterally toward the skin periphery with the formation of new feather primordia. These feather primordia will develop into short feather buds after 2 days in culture and long feather buds after 4 day...

Watch this Scientific Journal Video about Using Avian Skin Explants to Study Tissue Patterning and Organogenesis at JoVE.com

Author Spotlight: Insight into the Current Experimental Avian Skin Explant Methodologies

Here we describe protocols for three types of avian embryonic skin explant cultures that can be used to examine tissue interactions, 4D imaging timelapse movie (3D plus time), global or local perturbation of molecular function, and systems biology characterization.

Using Avian Skin Explants to Study Tissue Patterning and Organogenesis

The developing avian skin during embryogenesis is a unique model that can provide valuable insights into tissue patterning. Here three variations on skin ...

using-avian-skin-explants-to-study-tissue-patterning-and-organogenesis

Research

JoVE Journal

Developmental Biology

1.1K Views.  Keck School of Medicine of University of Southern California. Here we describe protocols for three types of avian embryonic skin explant cultures that can be used to examine tissue interactions, 4D imaging timelapse movie (3D plus time), global or local perturbation of molecular function, and systems biology characterization. 

Video: Author Spotlight: Insight into the Current Experimental Avian Skin Explant Methodologies

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 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 ...

A 3D Organotypic Melanoma Spheroid Skin Model

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased metastatic potential. Recently, mono-chemotherapies continue to improve by melanoma specific combination therapies with targeted kinase inhibitors. Still, metastatic melanoma remains a life-threatening disease because tumors exhibit primary resistance or develop resistance to novel therapies, thereby regaining tumorigenic capacity. In order to improve the therapeutic success of malignant melanoma, the determination of molecular mechanisms conferring resistance against conventional treatment approaches is necessary; however, it requires innovative cellular in vitro models. Here, we introduce an in vitro three-dimensional (3D) organotypic melanoma spheroid model that can portray the in vivo architecture of malignant melanoma and may warrant new insights into intra-tumoral as well as tumor-host interactions. The model incorporates defined numbers of mature and differentiated melanoma spheroids in a 3D human full skin reconstruction model consisting of primary skin cells. The cellular composition and differentiation status of the embedded melanoma spheroids is similar to the one of cutaneous melanoma metastasis in vivo. Using this organotypic melanoma spheroid model as a drug screening platform may support the identification of responders to selected combination therapies, while sparing the unnecessary treatment burden for non-responders, thereby increasing the benefit of therapeutic interventions.

Here, we present a protocol to generate a 3D organotypic melanoma spheroid skin model that recapitulates both the architecture and multicellular complexity of an organ/tumor in vivo but at the same time accommodates systematic experimental intervention.

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased ...

Cancer Research

Cell Population Analyses During Skin Carcinogenesis

Cancer development is a multiple-step process involving many cell types including cancer precursor cells, immune cells, fibroblasts and endothelial cells. Each type of cells undergoes signaling and functional changes during carcinogenesis. The current challenge for many cancer researchers is to dissect these changes in each cell type during the multiple-step process in vivo. In the last few years, the authors have developed a set of procedures to isolate different cell populations during skin cancer development using K14creER/R26-SmoM2YFP mice. The procedure is divided into 6 parts: 1) generating appropriate mice for the study (K14creER+ and R26-SmoM2YFP+ mice in this protocol); 2) inducing SmoM2YFP expression in mouse skin; 3) preparing mouse skin biopsies; 4) isolating epidermis from skin; 5) preparing single cells from epidermis; 6) labeling single cell populations for flow cytometry analysis. Generation of sufficient number of mice with the right genotype is the limiting step in this protocol, which may take up to two months. The rest of steps take a few hours to a few days. Within this protocol, we also include a section for troubleshooting. Although we focus on skin cancer, this protocol may be modified to apply for other animal models of human diseases.

Carcinogenesis is a process involving interactions between cancer cells and the cancer microenvironment. To dissect the molecular events, one needs to isolate different cell populations at different stages during cancer development. Using a mouse model for basal cell carcinoma, we describe a protocol for cell analyses during carcinogenesis.

Cancer development is  a multiple-step process involving many cell types including cancer precursor  cells, immune cells, fibroblasts and endothelial ...

Medicine

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily functions. Biomimetics is the imitation of the models, systems, and elements of nature for the purpose of solving complex human problems1. Skin biomimetics is a useful tool for in vitro disease research and in vivo regenerative medicine. Human induced pluripotent stem cells (iPSCs) have the characteristic of unlimited proliferation and the ability of differentiation to three germ layers. Human iPSCs are generated from various primary cells, such as blood cells, keratinocytes, fibroblasts, and more. Among them, cord blood mononuclear cells (CBMCs) have emerged as an alternative cell source from the perspective of allogeneic regenerative medicine. CBMCs are useful in regenerative medicine because human leukocyte antigen (HLA) typing is essential to the cell banking system. We provide a method for the differentiation of CBMC-iPSCs into keratinocytes and fibroblasts and for generation of a 3D skin organoid. CBMC-iPSC-derived keratinocytes and fibroblasts have characteristics similar to a primary cell line. The 3D skin organoids are generated by overlaying an epidermal layer onto a dermal layer. By transplanting this 3D skin organoid, a humanized mice model is generated. This study shows that a 3D human iPSC-derived skin organoid may be a novel, alternative tool for dermatologic research in vitro and in vivo.

We propose a protocol that shows how to differentiate induced pluripotent stem cell-derived keratinocytes and fibroblasts and generate a 3D skin organoid, using these keratinocytes and fibroblasts. This protocol contains an additional step of generating a humanized mice model. The technique presented here will improve dermatologic research.

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern tissue engineering. A technical bottleneck is the deposition of collagen in vitro, as it is notoriously slow, resulting in sub-optimal formation of connective tissue and subsequent tissue cohesion, particularly in skin models. Here, we describe a method which involves the addition of differentially-sized sucrose co-polymers to skin cultures to generate macromolecular crowding (MMC), which results in a dramatic enhancement of collagen deposition. Particularly, dermal fibroblasts deposited a significant amount of collagen I/IV/VII and fibronectin under MMC in comparison to controls.
The protocol also describes a method to decellularize crowded cell layers, exposing significant amounts of extracellular matrix (ECM) which were retained on the culture surface as evidenced by immunocytochemistry. Total matrix mass and distribution pattern was studied using interference reflection microscopy. Interestingly, fibroblasts, keratinocytes and co-cultures produced cell-derived matrices (CDM) of varying composition and morphology. CDM could be used as &#34;bio-scaffolds&#34; for secondary cell seeding, where the current use of coatings or scaffolds, typically from xenogenic animal sources, can be avoided, thus moving towards more clinically relevant applications.
In addition, this protocol describes the application of MMC during the submerged phase of a 3D-organotypic skin co-culture model which was sufficient to enhance ECM deposition in the dermo-epidermal junction (DEJ), in particular, collagen VII, the major component of anchoring fibrils. Electron microscopy confirmed the presence of anchoring fibrils in cultures developed with MMC, as compared to controls. This is significant as anchoring fibrils tether the dermis to the epidermis, hence, having a pre-formed mature DEJ may benefit skin graft recipients in terms of graft stability and overall wound healing. Furthermore, culture time was condensed from 5 weeks to 3 weeks to obtain a mature construct, when using MMC, reducing costs.

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

We present a protocol to obtain cell-derived matrices rich in extracellular matrix proteins, using macromolecular crowders (MMC). In addition, we present a protocol which incorporates MMC in 3D organotypic skin co-culture generation, which reduces culture time while maintaining maturity of construct.

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern ...

Bioengineering

Generation of Self-assembled Vascularized Human Skin Equivalents

Human skin equivalents (HSEs) are tissue engineered constructs that model epidermal and dermal components of human skin. These models have been used to study skin development, wound healing, and grafting techniques. Many HSEs continue to lack vasculature and are additionally analyzed through post-culture histological sectioning which limits volumetric assessment of the structure. Presented here is a straightforward protocol utilizing accessible materials to generate vascularized human skin equivalents (VHSE); further described are volumetric imaging and quantification techniques of these constructs. Briefly, VHSEs are constructed in 12 well culture inserts in which dermal and epidermal cells are seeded into rat tail collagen type I gel. The dermal compartment is made up of fibroblast and endothelial cells dispersed throughout collagen gel. The epidermal compartment is made up of keratinocytes (skin epithelial cells) that differentiate at the air-liquid interface. Importantly, these methods are customizable based on needs of the researcher, with results demonstrating VHSE generation with two different fibroblast cell types: human dermal fibroblasts (hDF) and human lung fibroblasts (IMR90s). VHSEs were developed, imaged through confocal microscopy, and volumetrically analyzed using computational software at 4- and 8-week timepoints. An optimized process to fix, stain, image, and clear VHSEs for volumetric examination is described. This comprehensive model, imaging, and analysis techniques are readily customizable to the specific research needs of individual labs with or without prior HSE experience.

The goal of this protocol is to describe the generation and volumetric analysis of vascularized human skin equivalents using accessible and simple techniques for long term culture. To the extent possible, the rationale for steps is described to allow researchers the ability to customize based on their research needs.

Human skin equivalents (HSEs) are tissue engineered constructs that model epidermal and dermal components of human skin. These models have been used to ...

Xenograft Skin Model to Manipulate Human Immune Responses In Vivo

The human skin xenograft model, in which human donor skin is transplanted onto an immunodeficient mouse host, is an important option for translational research in skin immunology. Murine and human skin differ substantially in anatomy and immune cell composition. Therefore, traditional mouse models have limitations for dermatological research and drug discovery. However, successful xenotransplants are technically challenging and require optimal specimen and mouse graft site preparation for graft and host survival. The present protocol provides an optimized technique for transplanting human skin onto mice and discusses necessary considerations for downstream experimental aims. This report describes the appropriate preparation of a human donor skin sample, assembly of a surgical setup, mouse and surgical site preparation, skin transplantation, and post-surgical monitoring. Adherence to these methods allows for maintenance of xenografts for over 6 weeks post-surgery. The techniques outlined below allow maximum grafting efficiency due to the development of engineering controls, sterile technique, and pre- and post-surgical conditioning. Appropriate performance of the xenograft model results in long-lived human skin graft samples for experimental characterization of human skin and preclinical testing of compounds in vivo.

Xenograft Skin Model to Manipulate Human Immune Responses In Vivo

The present protocol describes how to graft human skin onto non-obese diabetic (NOD)-scid interleukin-2 gamma chain receptor (NSG) mice. A detailed description of the preparation of human skin for transplant, preparation of mice for transplant, transplantation of split-thickness human skin, and post-transplantation recovery procedure are included in the report.

The human skin xenograft model, in which human donor skin is transplanted onto an immunodeficient mouse host, is an important option for translational ...

Immunology and Infection

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts migrate through the epidermis of the embryo where they subsequently colonize the developing hair follicles1,2. Neural crest cell migration is extensively studied in vitro but in vivo methods are still not well developed, especially in mammalian systems. One alternative is to use ex vivo organotypic culture3-6. Culture of mouse embryonic skin requires the maintenance of an air-liquid interface (ALI) across the surface of the tissue3,6. High resolution live-imaging of mouse embryonic skin has been hampered by the lack of a good method that not only maintains this ALI but also allows the culture to be inverted and therefore compatible with short working distance objective lenses and most confocal microscopes. This article describes recent improvements to a method that uses a gas permeable membrane to overcome these problems and allow high-resolution confocal imaging of embryonic skin in ex vivo culture6. By using a melanoblast specific Cre-recombinase expressing mouse line combined with the R26YFPR reporter line we are able to fluorescently label the melanoblast population within these skin cultures. The technique allows live-imaging of melanoblasts and observation of their behavior and interactions with the tissue in which they develop. Representative results are included to demonstrate the capability to live-image 6&#160;cultures in parallel.

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

We describe the dissection and ex vivo culture of mouse embryonic skin. The culture system maintains an air-liquid interface across the tissue surface and allows imaging on an inverted microscope. Melanoblasts, a component of the developing skin, are fluorescently labeled allowing their behavior to be observed using confocal microscopy.

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts ...

Biology

An Introduction to Organogenesis

Organogenesis is the process by which organs arise from one of three germ layers during the later stages of embryonic development. Researchers studying organogenesis want to better understand the genetic programs, cell-cell interactions, and mechanical forces involved in this process. Ultimately, scientists hope to use this knowledge to create therapies and artificial organs that will help treat human diseases.
This video offers a comprehensive overview of organogenesis, including historical highlights starting with breakthrough studies done in the 1800s. Next, key questions asked by developmental biologists are introduced, followed by a discussion of how tissue transplantations, imaging, and in vitro culture techniques can be used to answer these queries. Finally, we describe how these methods are currently being employed in developmental biology laboratories.

Organogenesis, Fate Mapping and Tissue Engineering

Organogenesis is the process by which organs arise from one of three germ layers during the later stages of embryonic development. Researchers studying ...

Education

JoVE Science Education

Advanced Biology

Organotypic 3D Skin Culture: A Cassette-based In Vitro Culture Technique to Regenerate Human Epidermis on Acellular Dermis

Source: Jingting Li et al. <a href="https://www.jove.com/v/53280"> Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis</a>, J. Vis. Exp. (2015).
This video protocol describes a cassette-based in vitro culture technique to generate&#160;human organotypic skin using epidermal keratinocytes and devitalized human dermis. They mimic the function and physiology of their in vivo tissue counterparts.

Source: Jingting Li et al.  Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis, J. Vis. Exp. (2015).
This video ...

Using Avian Skin Explants to Study Tissue Patterning and Organogenesis

Summary

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Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

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Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

Generation of Self-assembled Vascularized Human Skin Equivalents

Xenograft Skin Model to Manipulate Human Immune Responses In Vivo

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

An Introduction to Organogenesis

Organotypic 3D Skin Culture: A Cassette-based In Vitro Culture Technique to Regenerate Human Epidermis on Acellular Dermis