Name: a simplified and efficient method to isolate primary human keratinocytes from adult skin tissue
Uploaded: 2018-08-25T13:00:00
Description: Watch this Scientific Journal Video about A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue at JoVE.com

This work was supported by the National Key Research and Development Program of China (2017YFA0104604), the General Program of National Natural Science Foundation of China (NSFC, 81772093), the Science and Technology Development Program of Suzhou (ZXL2015128), the Natural Science Foundation of Jiangsu Province (BK20161241), and a Shandong Taishan Scholar Award (tshw201502065).

Human tissues used in this protocol have been handled according to the guidelines of the Institution&#39;s human research ethics committee (NO.2015120401, date: May 12, 2015).
1. Preparations
<ol>
	<li>Collect fresh adult abdominal skin tissues discarded from plastic surgery at the hospital in a 50-mL tube with 10 mL of ice-cold Dulbecco&#39;s modified Eagle medium (DMEM). The specimen can be kept at 4 &#176;C for up to 72 h without significantly affecting the cell viability.</li>
	<li>Prepa...

<ol>
	<li>Ojeh, N., Pastar, I., Tomic-Canic, M., Stojadinovic, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+Cells+in+Skin+Regeneration,+Wound+Healing,+and+Their+Clinical+Applications.">Stem Cells in Skin Regeneration, Wound Healing, and Their Clinical Applications.</a> International Journal of Molecular Sciences. 16 (10), 25476-25501 (2015).</li><li>Sotiropoulou, P. A., Blanpain, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+homeostasis+of+the+skin+epidermis.">Development and homeostasis of the skin epidermis.</a> Cold Spring Harbor Perspectives in BIology. 4 (7), a008383 (2012).</li><li>Kamstrup, M., Faurschou, A., Gniadecki, R., Wulf, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+stem+cells+-+role+in+normal,+wounded+and+pathological+psoriatic+and+cancer+skin.">Epidermal stem cells - role in normal, wounded and pathological psoriatic and cancer skin.</a> Current Stem Cell Research &amp; Therapy. 3 (2), 146-150 (2008).</li><li>Blanpain, C., Fuchs, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+stem+cells+of+the+skin.">Epidermal stem cells of the skin.</a> Annual Review of Cell and Developmental Biology. 22 (22), 339-373 (2006).</li><li>Guo, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Building+a+microphysiological+skin+model+from+induced+pluripotent+stem+cells.">Building a microphysiological skin model from induced pluripotent stem cells.</a> Stem Cell Research &amp; Therapy. 4 (S1), S2 (2013).</li><li>Aasen, T., Izpisua Belmonte, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+cultivation+of+human+keratinocytes+from+skin+or+plucked+hair+for+the+generation+of+induced+pluripotent+stem+cells.">Isolation and cultivation of human keratinocytes from skin or plucked hair for the generation of induced pluripotent stem cells.</a> Nature Protocols. 5 (2), 371-382 (2010).</li><li>Bayati, V., Abbaspour, M. R., Neisi, N., Hashemitabar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin-derived+precursors+possess+the+ability+of+differentiation+into+the+epidermal+progeny+and+accelerate+burn+wound+healing.">Skin-derived precursors possess the ability of differentiation into the epidermal progeny and accelerate burn wound healing.</a> Cell Biology International. 41 (2), 187-196 (2017).</li><li>Hirsch, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+the+entire+human+epidermis+using+transgenic+stem+cells.">Regeneration of the entire human epidermis using transgenic stem cells.</a> Nature. 551 (7680), 327-332 (2017).</li><li>Hentzer, B., Kobayasi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+human+epidermal+cells+from+fibroblasts+in+primary+skin+culture.">Separation of human epidermal cells from fibroblasts in primary skin culture.</a> Archiv f&#252;r dermatologische Forschung. 252 (1), 39-46 (1975).</li><li>Terunuma, A., Limgala, R. P., Park, C. J., Choudhary, I., Vogel, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+procurement+of+epithelial+stem+cells+from+human+tissue+specimens+using+a+Rho-associated+protein+kinase+inhibitor+Y-27632.">Efficient procurement of epithelial stem cells from human tissue specimens using a Rho-associated protein kinase inhibitor Y-27632.</a> Tissue Engineering Part A. 16 (4), 1363-1368 (2010).</li><li>Zhou, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ROCK+inhibitor+Y-27632+increases+the+cloning+efficiency+of+limbal+stem/progenitor+cells+by+improving+their+adherence+and+ROS-scavenging+capacity.">ROCK inhibitor Y-27632 increases the cloning efficiency of limbal stem/progenitor cells by improving their adherence and ROS-scavenging capacity.</a> Tissue Engineering Part C: Methods. 19 (7), 531-537 (2013).</li><li>Kurosawa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+Rho-associated+protein+kinase+(ROCK)+inhibitor+to+human+pluripotent+stem+cells.">Application of Rho-associated protein kinase (ROCK) inhibitor to human pluripotent stem cells.</a> Journal of Bioscience and Bioengineering. 114 (6), 577-581 (2012).</li><li>Wen, J., Zu, T., Zhou, Q., Leng, X., Wu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Y-27632+simplifies+the+isolation+procedure+of+human+primary+epidermal+cells+by+selectively+blocking+focal+adhesion+of+dermal+cells.">Y-27632 simplifies the isolation procedure of human primary epidermal cells by selectively blocking focal adhesion of dermal cells.</a> Journal of Tissue Engineering and Regenerative Medicine. 12 (2), e1251-e1255 (2018).</li><li>Zou, D., Pan, J., Zhang, P., Wu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+to+isolate+human+epidermal+keratinocytes.">A new method to isolate human epidermal keratinocytes.</a> Journal of Clinical Dermatology (in Chinese). 6, 424-429 (2016).</li><li>Cerqueira, M. T., Frias, A. M., Reis, R. L., Marques, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interfollicular+epidermal+stem+cells:+boosting+and+rescuing+from+adult+skin).">Interfollicular epidermal stem cells: boosting and rescuing from adult skin).</a> Methods in Molecular Biology. 989, 1-9 (2013).</li><li>Candi, E., Schmidt, R., Melino, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cornified+envelope:+a+model+of+cell+death+in+the+skin.">The cornified envelope: a model of cell death in the skin.</a> Nature Reviews Molecular Cell Biology. 6 (4), 328-340 (2005).</li><li>Breyer, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+Rho+kinases+increases+directional+motility+of+microvascular+endothelial+cells.">Inhibition of Rho kinases increases directional motility of microvascular endothelial cells.</a> Biochemical Pharmacology. 83 (5), 616-626 (2012).</li><li>Wozniak, M. A., Modzelewska, K., Kwong, L., Keely, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focal+adhesion+regulation+of+cell+behavior.">Focal adhesion regulation of cell behavior.</a> Biochimica et Biophysica Acta. 1692 (2-3), 103-119 (2004).</li></ol>

Cultured primary HKCs have been widely utilized to treat wounds in clinics for more than three decades and, since that time, it has been always important to efficiently obtain sufficient numbers of cells for clinical applications in a timely manner. Therefore, in practice, the conventional isolation method, which requires the separation of the epidermis from the dermis, makes it difficult to meet these demands, due to the low yield of cells and the low ability to passage adult cells. Here, we describe a new simple method...

The goal was to develop a simple and efficient protocol to isolate primary human keratinocytes (HKCs) from adult tissues, especially for clinical applications. Skin epidermal stem cells, localized in the basal layer of the skin, possess a high potential to proliferate and differentiate and provide keratinocytes to maintain the functions of the skin1,2,3,4. HKCs isolated from skin tissues are widely used for skin tissue engineering and regeneration purposes, especially in the repair of damaged skin and in gene therapy for clinical applications5,6. The key issue for HKC-based applications is to efficiently isolate and expand large numbers of HKCs with high potential in vitro7,8. Although various research groups have developed methods to produce cultures of stem-like HKCs, these methods are sometimes time-consuming and complicated to perform and have other limitations, such as low cell yields and being limited by the type of skin specimen used9. For instance, the traditional method to isolate HKCs from skin tissues involves a two-step enzymatic digestion with a separation of the epidermis from the dermis6. That method usually works well for neonatal tissues, but it becomes very difficult when used to isolate cells from adult tissues.
Y-27632, an inhibitor of Rho-associated protein kinase (ROCK), has been reported to significantly enhance the efficiency of epidermal stem cell isolation and colony growth10,11,12. In a previous study, we discovered that Y-27632 facilitates the clonal growth of epidermal cells but reduces the yield of dermal cells by differentially controlling the expression of adhesion molecules13. We also established a new conditioned inoculation medium, called G-medium, which supports the growth and yield of primary epidermal cells. By combining G-medium with Y-27632, this novel method can spontaneously separate epidermal and dermal cells after enzyme digestion, thus removing the step of epidermis-dermis separation13,14. Based on previous reports, we now describe the detailed procedure of this new method to isolate HKCs from adult skin tissue.

<table border="0" cellpadding="0" cellspacing="0" style="width:1344px;" width="1342">
	<tbody>
		<tr height="23">
			<td height="23" style="height:23px;width:263px;">Countess automated cell counter</td>
			<td style="width:291px;">Invitrogen Inc.&#160;</td>
			<td style="width:264px;">C10227</td>
			<td style="width:527px;">Automatic cell counting&#160;</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">CO2 Incubator</td>
			<td>Thermo Scientific</td>
			<td>51026333</td>
			<td>For cell incubating</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Sorvall ST 16R Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>75004380</td>
			<td>Cell centrifuge</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Constant Temperature Shaker</td>
			<td>Shanghai Boxun</td>
			<td>150036</td>
			<td>For water bath</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Electronic Scale</td>
			<td>Harbin Zhonghui</td>
			<td>1171193</td>
			<td>For tissue weighing</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Cell Culture Dish</td>
			<td>Eppendorf</td>
			<td>30702115</td>
			<td>For cell culture</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">50ml Centrifuge Tube</td>
			<td>KIRGEN</td>
			<td>171003</td>
			<td>For cell centrifuge</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Cell Strainer</td>
			<td>Corning incorporated</td>
			<td>431792</td>
			<td>Cell filtration</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Phosphate buffered solution</td>
			<td>Solarbio Life Science&#160;</td>
			<td>P1020-500</td>
			<td>Washing solution</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">DMEM</td>
			<td>Thermo Scientific</td>
			<td>C11995500</td>
			<td>Component of neutralization medium</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Defined K-SFM</td>
			<td>Life Technologies</td>
			<td>10785-012</td>
			<td>Epidermal cells culture medium</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Penicillin Streptomycin</td>
			<td>Thermo Scientific</td>
			<td>15140-122</td>
			<td>Antibiotics</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Fetal Bovine Serum</td>
			<td>Biological Industries</td>
			<td>04-001-1AC5</td>
			<td>Component of neutralization medium</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">0.05% Trypsin</td>
			<td>Life Technologies</td>
			<td>25300-062</td>
			<td>For HKC dissociation</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">0.25% Trypsin&#160;</td>
			<td>Beijing Solarbio Science &#38; Technology</td>
			<td>T1350-100</td>
			<td>For HKC dissociation</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Coating Matrix Kit</td>
			<td>Life Technologies</td>
			<td>R-011-K</td>
			<td>For coating matrix</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Dispase</td>
			<td>Gibco</td>
			<td>17105-041</td>
			<td>For HKC isolation</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Collagenase Type I</td>
			<td>Life Technologies</td>
			<td>17100-017</td>
			<td>For HKC isolation</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Deoxyribonuclease I</td>
			<td>Sigma</td>
			<td>9003-98-9</td>
			<td>For HKC isolation</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">F12 Nutrient Mix, Hams</td>
			<td>Life Technologies</td>
			<td>31765035</td>
			<td>Component of G-medium</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">B27 Supplement</td>
			<td>Life Technologies</td>
			<td>17504044</td>
			<td>Growth factor in G-medium</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">FGF-2 Millipore</td>
			<td>Merck Biosciences</td>
			<td>341595</td>
			<td>Growth factor in G-medium</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Y-27632</td>
			<td>Sigma-Aldrich</td>
			<td>Y0503</td>
			<td>ROCK inhibitor</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Fungizone</td>
			<td>Gibco</td>
			<td>15290026</td>
			<td>Preparation for G-medium</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">EGF Recombinant Human Protein</td>
			<td>Gibco</td>
			<td>PHG0311</td>
			<td>Growth factor in G-medium</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Cell Counting Kit-8</td>
			<td>Thermo Scientific</td>
			<td>NC9864731</td>
			<td>cell proliferation and cytotoxicity assays</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Mouse Anti-Human Cytokeratin5</td>
			<td>Hewlett-Packard Development Company</td>
			<td>MA-20142</td>
			<td>For immunofluorescence staining to check differentiation marker of HKCs</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Rabbit Anti-Human Loricrin</td>
			<td>Covance</td>
			<td>PRB-145p</td>
			<td>For immunofluorescence staining to check differentiation marker of HKCs</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Mouse anti-human Vimentin</td>
			<td>Cell Signaling Technology</td>
			<td>3390</td>
			<td>For immunofluorescence staining of dermal fibroblasts</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Integrin &#945;6(GOH3)</td>
			<td>Santa Cruz&#160;</td>
			<td>SC-19622</td>
			<td>flow cytometry analysis of HKCs</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:263px;">Rat IgG2a FITC</td>
			<td>Santa Cruz&#160;</td>
			<td>SC-2831</td>
			<td>negative control antibody of &#945;6-integrin&#160; in flow cytometry analysis&#160;</td>
		</tr>
	</tbody>
</table>

a simplified and efficient method to isolate primary human keratinocytes from adult skin tissue

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

Schematic diagrams of the new method (Figure 1A) and the conventional method (Figure 1B) are presented in Figure 1. The conventional method is a two-step digestion, which requires a 2-day procedure. By contrast, the new method is a one-step digestion, which takes around 3 hours to perform. Importantly, the one-step new method can obtain two populations (epidermal and dermal cells) at the same time, w...

Watch this Scientific Journal Video about A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue at JoVE.com

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

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

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

The general goal of this video is to introduce a new simple method to isolate and culture primary human epidermal cells from an adult skin tissue. This advanced method is more suitable for producing a large number of high-potential epidermal cells for both laboratory and clinical applications. Wash the tissue with PBS before weighing.Weigh the skin tissue by an electronic scale. Rinse the skin tissue with 70%ethanol for 30 seconds. Incubate the tissue in PBS with antibiotics twice for five minutes each time.Transfer the tissue to another sterile dish and homogenize thoroughly by using scalpel blades. Add 200 microliters of PBS every five minutes to keep the tissue wet. Transfer the homogenized tissue into a 50 milliliter tube.Add 10 milliliters of enzyme mixture for every one gram of skin tissue. Mix the enzymes thoroughly with the homogenized tissues. Incubate the mixture in a 37 degree Celsius water bath with shaking.After that, add 1/5 the volume of 0.25%trypsin. Continue the incubation for 30 minutes in a 37 degree Celsius water bath. Add Dnase I solution to the mixture at a one to 100 ratio.Then incubate for another five minutes. Stop the digestion process by adding the same volume of neutralization medium. Pipette the solution up and down for about 20 times.Filter the dissociated cells through a 100 micrometer cell strainer to remove tissue debris. Centrifuge at 200 g for five minutes. Observe the pellet at the bottom of the tube.Remove the supernatants and wash the cell pellet with 10 milliliters of neutralization medium. Then centrifuge at 200 g for five minutes. Resuspend the cell pellet in the inoculation medium with 10 micromolar ROCK inhibitor.Plate two times 10 to the sixth cells in a 100 millimeter culture dish. After three days, replace the inoculation medium with serum-free keratinocyte medium. Change the medium every two days.When the cells have reached about 80%of the density, passage the cells. Wash the cells with PBS. If necessary, trypsinize for two minutes and wash the cells with PBS to remove contaminated dermal cells.Add the same volume of neutralization medium. Centrifuge at 200 g for five minutes. Finally, remove the supernatants and resuspend the cell pellet.The isolated keratinocytes from human skin tissues were incubated separately by two methods. The conventional method is a two-step digestion which involves a two-day procedure. By contrast, the new method is a one-step digestion which takes around three hours to perform.On day three, we can easily find many cell clusters cultured by the new method while this phenomenon does not occur when using the conventional method. On the fifth day, it was observed that the new method produced a larger amount of primary cells. To test the differentiation status of cultured keratinocytes, we analyzed the basal cell marker K5 and terminal differentiation marker Loricrin.We found that 90%of the total cells were K5 positive, but less than 10%of the cells expressed Loricrin at passage three, indicating that they are undifferentiated keratinocytes. We checked the expression of vimentin which is expressed in the dermal fibroblast to examine the contamination of the dermal cells during isolation. We found that around 3%of the dermal cells appeared in the initial passage, but only around 0.02%of the dermal cells were detected at passage three, indicating a high purity of the epidermal cells after passage.

Research

JoVE Journal

Developmental Biology

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

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 ...

A Quick and Efficient Method for the Purification of Endoderm Cells Generated from Human Embryonic Stem Cells

The differentiation capabilities of pluripotent stem cells such as embryonic stem cells (ESCs) allow a potential therapeutic application for cell ...

Epigenetic Conversion as a Safe and Simple Method to Obtain Insulin-secreting Cells from Adult Skin Fibroblasts

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such ...

Isolating Hair Follicle Stem Cells and Epidermal Keratinocytes from Dorsal Mouse Skin

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by ...

An Efficient Method to Obtain Dedifferentiated Fat Cells

Tissue engineering and cell therapy hold great promise clinically. In this regard, multipotent cells, such as mesenchymal stem cells (MSCs), may be used ...

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

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. ...

The Isolation and Culture of Primary Epicardial Cells Derived from Human Adult and Fetal Heart Specimens

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of ...

A Direct and Simple Method to Assess Drosophila melanogaster's Viability from Embryo to Adult

A Direct and Simple Method to Assess Drosophila melanogaster's Viability from Embryo to Adult

In Drosophila melanogaster, viability assays are used to determine the fitness of certain genetic backgrounds. Allelic variations can result in partial or ...