Name: isolation of cd 90+ fibroblast/myofibroblasts from human frozen gastrointestinal specimens
Uploaded: 2016-01-31T14:00:00
Description: Watch this Scientific Journal Video about Isolation of CD 90+ Fibroblast/Myofibroblasts from Human Frozen Gastrointestinal Specimens at JoVE.com

This work was supported by National Institute of Health (1R01DK103150-01A1, T32 DK007639, R01CA175803, K08CA125209) and the Institute for Translational Sciences at the University of Texas Medical Branch, and a Clinical and Translational Science Award (8UL1TR000071).

The protocol for obtaining discarded human tissue from surgical patients and the establishment of primary cultures was approved by the University of Texas Medical Branch and University of New Mexico Institutional Review Boards. The general requirements for the procurement of human tissue specimens is described below. Note that since MFs may be isolated from frozen tissue, biobanks and commercial vendors are also viable options.
1. Obtain Human Tissue
<ol>
	<li>Submit a protocol to obtain hum...

<ol>
	<li>Yamaguchi, H., 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=Stromal+Fibroblasts+Mediate+Extracellular+Matrix+Remodeling+and+Invasion+of+Scirrhous+Gastric+Carcinoma+Cells.">Stromal Fibroblasts Mediate Extracellular Matrix Remodeling and Invasion of Scirrhous Gastric Carcinoma Cells.</a> PLoS ONE. 9, e85485 (2014).</li><li>Mullan, N., Hughes, K. R., Mahida, Y. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+Human+Colonic+Myofibroblasts+Are+Resistant+to+Clostridium+difficile+Toxin+A-Induced,+but+Not+Toxin+B-Induced,+Cell+Death.">Primary Human Colonic Myofibroblasts Are Resistant to Clostridium difficile Toxin A-Induced, but Not Toxin B-Induced, Cell Death.</a> Infect Immun. 79, 1623-1630 (2011).</li><li>Powell, D. W., Pinchuk, I. V., Saada, J. I., Chen, X., Mifflin, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+Cells+of+the+Intestinal+Lamina+Propria.+Annu+Rev+Physiol.">Mesenchymal Cells of the Intestinal Lamina Propria. Annu Rev Physiol.</a> Annu Rev Physiol . 73, 213-237 (2011).</li><li>Latella, G., Sferra, R., Speca, S., Vetuschi, A., Gaudio, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+we+prevent,+reduce+or+reverse+intestinal+fibrosis+in+IBD?.">Can we prevent, reduce or reverse intestinal fibrosis in IBD?.</a> Eur Rev Med Pharmacol Sci. 17, 1283-1304 (2013).</li><li>Saada, J. I., 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=Subepithelial+myofibroblasts+are+novel+nonprofessional+APCs+in+the+human+colonic+mucosa.">Subepithelial myofibroblasts are novel nonprofessional APCs in the human colonic mucosa.</a> J. Immunol. 177, 5968-5979 (2006).</li><li>Mahida, Y. R., 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=Adult+human+colonic+subepithelial+myofibroblasts+express+extracellular+matrix+proteins+and+cyclooxygenase-1+and+-2.">Adult human colonic subepithelial myofibroblasts express extracellular matrix proteins and cyclooxygenase-1 and -2.</a> Am J Physiol. 273, G1341-G1348 (1997).</li><li>Roncoroni, L., 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=Isolation+and+culture+of+fibroblasts+from+endoscopic+duodenal+biopsies+of+celiac+patients.">Isolation and culture of fibroblasts from endoscopic duodenal biopsies of celiac patients.</a> J Transl Med. 7 (40), (2009).</li><li>Pinchuk, I. V., 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=Stromal+Cells+Induce+Th17+during+Helicobacter+pylori+Infection+and+in+the+Gastric+Tumor+Microenvironment.">Stromal Cells Induce Th17 during Helicobacter pylori Infection and in the Gastric Tumor Microenvironment.</a> PLoS ONE. 8, e53798 (2013).</li><li>Pinchuk, I. V., 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=Monocyte+chemoattractant+protein-1+production+by+intestinal+myofibroblasts+in+response+to+staphylococcal+enterotoxin+a:+relevance+to+staphylococcal+enterotoxigenic+disease.">Monocyte chemoattractant protein-1 production by intestinal myofibroblasts in response to staphylococcal enterotoxin a: relevance to staphylococcal enterotoxigenic disease.</a> J. Immunol. 178, 8097-8106 (2007).</li><li>Paulsson, J., Micke, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prognostic+relevance+of+cancer-associated+fibroblasts+in+human+cancer.">Prognostic relevance of cancer-associated fibroblasts in human cancer.</a> Sem Cancer Biol. 25, 61-68 (2014).</li><li>Valcz, G., Sipos, F., Tulassay, Z., Molnar, B., Yagi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Importance+of+carcinoma-associated+fibroblast-derived+proteins+in+clinical+oncology.">Importance of carcinoma-associated fibroblast-derived proteins in clinical oncology.</a> J. Clin. Pathol. 67, 1026-1031 (2014).</li><li>Minonzio, G., 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=Frozen+adipose-derived+mesenchymal+stem+cells+maintain+high+capability+to+grow+and+differentiate.">Frozen adipose-derived mesenchymal stem cells maintain high capability to grow and differentiate.</a> Cryobiology. 69, 211-216 (2014).</li><li>Gargus, M., Niu, C., Shaker, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Myofibroblasts+from+Mouse+and+Human+Esophagus.">Isolation of Myofibroblasts from Mouse and Human Esophagus.</a> J. Vis. Exp. (95), e52215 (2015).</li><li>Khalil, H., Nie, W., Edwards, R. A., Yoo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+primary+myofibroblasts+from+mouse+and+human+colon+tissue.">Isolation of primary myofibroblasts from mouse and human colon tissue.</a> J. Vis. Exp. (80), e50611 (2013).</li><li>Schiller, J. H., Bittner, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+the+Tumorigenic+Phenotype+with+in+Vitro,+but+not+in+Vivo,+Passaging+of+a+Novel+Series+of+Human+Bronchial+Epithelial+Cell+Lines:+Possible+Role+of+an+&#945;5/&#946;1-Integrin-Fibronectin+Interaction.">Loss of the Tumorigenic Phenotype with in Vitro, but not in Vivo, Passaging of a Novel Series of Human Bronchial Epithelial Cell Lines: Possible Role of an &#945;5/&#946;1-Integrin-Fibronectin Interaction.</a> Cancer Res. 55, 6215-6221 (1995).</li><li>Augello, A., Kurth, T. B., De Bari, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells:+a+perspective+from+in+vitro+cultures+to+in+vivo+migration+and+niches.">Mesenchymal stem cells: a perspective from in vitro cultures to in vivo migration and niches.</a> Eur Cell Mater. 20, 121-133 (2010).</li><li>Meller, D., Pires, R. T. F., Tseng, S. C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+preservation+and+expansion+of+human+limbal+epithelial+stem+cells+on+amniotic+membrane+cultures.">Ex vivo preservation and expansion of human limbal epithelial stem cells on amniotic membrane cultures.</a> Br J Ophthalmol. 86, 463-471 (2002).</li><li>Kuhlmann, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+prophylactic+use+of+antibiotics+in+cell+culture.">The prophylactic use of antibiotics in cell culture.</a> Cytotechnology. 19, 95-105 (1995).</li><li>Morris, K. T., Nofchissey, R. A., Pinchuk, I. V., Beswick, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+Macrophage+Migration+Inhibitory+Factor+Exposure+Induces+Mesenchymal+Epithelial+Transition+and+Promotes+Gastric+and+Colon+Cancers.">Chronic Macrophage Migration Inhibitory Factor Exposure Induces Mesenchymal Epithelial Transition and Promotes Gastric and Colon Cancers.</a> PLoS ONE. 9, e98656 (2014).</li></ol>

While this protocol was developed for the research application only, in the light of growing critical importance of stromal cells as a potential cancer prognostic biomarkers and therapeutic target, the ability to freeze &#34;functional&#34; biospecimens for later use offers a significant advantage. This represents a unique advantage for creation of clinical biorepository, which may serve as additional tools serving to advance the development of personalized medicine10, 11. Similar to findings previously report...

Myofibroblasts/fibroblasts (MFs) represent an abundant population of cells in gastrointestinal (GI) mucosa. These stromal cells are located just beneath the epithelium and form an interconnected network within mucosal lamina propria in the gut. MFs are not only responsible for the deposition of the extracellular matrix, but through paracrine regulation may influence electrolyte transport, restitution, and barrier function of the adjacent epithelium1, 2. Furthermore, MFs have been shown to play a key role in inflammation and tissue remodeling3, 4. Myofibroblasts are a critical part of the tumor microenvironment, where they are also known as cancer-associated fibroblasts, and contribute to the tumor cell growth and serve as a niche for cancer stem cells3. Emerging data suggests that MFs may also serve as local antigen presenting cells. Additionally, MFs function as important regulators of innate and adaptive immune responses, producing a variety of cytokines and growth factors5.
In healthy individuals, MFs cells are believed to differentiate from mesenchymal stem cells and express on their cell surface, the mesenchymal marker, CD903, 5. These cells are also positive for vimentin, but negative for epithelial and hematopoietic cell marker, EpCAM and CD45, respectively. Myofibroblasts are suggested to be an activated form of fibroblasts and can be distinguished from non-activated fibroblasts by the expression of &#945;-SMA5.
Over the past decade, multiple approaches for the isolation of myofibroblasts from human colonic mucosa have been published-mostly based on the method originally described by Mahida et al.5-8. While individual studies report isolation procedures from various gut mucosa, no universal protocol for the isolation of MFs from multiple areas of the GI tract (i.e., gastric, small and large intestinal mucosa) has been published. The protocol presented herein has been tested and successfully used for all three type of tissue mentioned above. . Furthermore, procedures for isolating MFs from frozen GI mucosa have not been reported.
Here, we present an optimized method, which is based on enzymatic digestion, and concurrently allows for the isolation of human gut mucosal MFs for culture and flow cytometry analysis in freshly-digested, single cell, mucosal preparations. This technique reliably yields primary cultures with an MF phenotype. Furthermore, the same methods can be used to isolate MFs from frozen specimens of gastric and small intestinal tissues&#160;as well. Isolation of myofibroblasts from fresh GI tissue has been previously described; however, the utilization of frozen specimens presents many benefits. Namely, researchers would be able to collect and store tissues from any number of collaborators across the world who have the capability to ship frozen tissue samples. Moreover, researchers may find the collection of discarded tissue from the operating room and/or endoscopy suite and immediate processing the tissue for isolation to conflict with their current experimentation schedule. Also, due to the unpredictable nature of surgery, tissue procurement may occur very late in the day, which will limit the time left for processing tissue. Freezing tissue for later processing will ameliorate these challenges.
Lastly, these methods have been successfully utilized in the isolation of colonic, gastric and small intestinal myofibroblasts in disease states such as colorectal carcinoma and inflammatory bowel diseases.

<table border="0" cellpadding="0" cellspacing="0" width="719">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">MEM, 1X</td>
			<td style="width:111px;">Corning</td>
			<td style="width:119px;">MT-10-010-CV</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Hanks&#39; Balanced Salt Solution</td>
			<td style="width:111px;">Sigma-Aldrich</td>
			<td style="width:119px;">H6648</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Sodium pyruvate</td>
			<td style="width:111px;">Sigma-Aldrich</td>
			<td style="width:119px;">S8636</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Antibiotic-Antimycotic, 100X</td>
			<td style="width:111px;">Gibco</td>
			<td style="width:119px;">15240-062</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Ciprofloxacin HCl</td>
			<td style="width:111px;">Corning</td>
			<td style="width:119px;">61-277-RF</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">L-Glutamine, 100x</td>
			<td style="width:111px;">Corning</td>
			<td style="width:119px;">25-005-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">MEM Nonessential Amino Acids</td>
			<td style="width:111px;">Corning</td>
			<td style="width:119px;">25-025-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Dimethyl Sulfoxide (DMSO) Hybri-Max</td>
			<td style="width:111px;">Sigma-Aldrich</td>
			<td style="width:119px;">D2650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">DL-Dithiothreitol solution (DTT)</td>
			<td style="width:111px;">Sigma-Aldrich</td>
			<td style="width:119px;">646563</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">0.5M EDTA, pH 8.0</td>
			<td style="width:111px;">Cellgro</td>
			<td style="width:119px;">46-034-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">DNAse</td>
			<td style="width:111px;">Worthington</td>
			<td style="width:119px;">LS002139</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:323px;">Collagenase from&#160;Clostridium histolyticum, Type I</td>
			<td style="width:111px;">Sigma-Aldrich</td>
			<td style="width:119px;">C1639</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:323px;">Collagenase from&#160;Clostridium histolyticum, Type II</td>
			<td style="width:111px;">Sigma-Aldrich</td>
			<td style="width:119px;">C1764</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:323px;">Collagenase from Clostridium histolyticum, Type IV</td>
			<td style="width:111px;">Sigma-Aldrich</td>
			<td style="width:119px;">C5138</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Accumax cell dissociation solution</td>
			<td style="width:111px;">Sigma-Aldrich</td>
			<td style="width:119px;">A7089</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">gentleMACS C Tubes</td>
			<td style="width:111px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-093-237</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">gentleMACS Dissociator</td>
			<td style="width:111px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-093-235</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Countess Automated Cell Counter</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:119px;">C10227</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Cell Strainer (70&#181;m)</td>
			<td style="width:111px;">Corning</td>
			<td style="width:119px;">352350</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">PE Mouse Anti-Human Vimentin</td>
			<td style="width:111px;">BD Biosciences</td>
			<td style="width:119px;">562337</td>
			<td>Clone RV202</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">FITC Monoclonal Anti-Actin, &#945;-Smooth Muscle</td>
			<td style="width:111px;">Sigma-Aldrich</td>
			<td style="width:119px;">F3777</td>
			<td>Clone 1A4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">APC Mouse Anti-Human CD90</td>
			<td style="width:111px;">BD Biosciences</td>
			<td style="width:119px;">559869</td>
			<td>Clone 5E10</td>
		</tr>
	</tbody>
</table>

isolation of cd 90+ fibroblast/myofibroblasts from human frozen gastrointestinal specimens

Fibroblasts/myofibroblasts (MFs) have been gaining increasing attention for their role in pathogenesis and their contributions to both wound healing and promotion of the tumor microenvironment. While there are currently many techniques for the isolation of MFs from gastrointestinal (GI) tissues, this protocol introduces a novel element of isolation of these stromal cells from frozen tissue. Freezing GI tissue specimens not only allows the researcher to acquire samples from worldwide collaborators, biobanks, and commercial vendors, it also permits the delayed processing of fresh samples. The described protocol will consistently yield characteristic spindle-shaped cells with the MF phenotype that express the markers CD90, &#945;-SMA and vimentin. As these cells are derived from patient samples, the use of primary cells also confers the benefit of closely mimicking MFs from disease states-namely cancer and inflammatory bowel diseases. This technique has been validated in gastric, small bowel, and colonic MF primary culture generation. Primary MF cultures can be used in a vast array of experiments over a number of passage and their purity assessed by both immunocytochemistry and flow cytometry analysis.

Using this enzymatic digestion protocol, we have consistently been able to isolate and grow MF population gut CD90+ mucosal stromal cells from GI surgical specimens and biopsies (Figure 1). Visible MF colony proliferation could be observed on day 2 - 5 after seeding mononuclear cell suspensions into 6 well plates (Figure 1A-B). The MF primary cultures reach ~ 50 - 70 % confluence by day 7 - 11 (Figure 1C-D).
<p class="jove_...

Watch this Scientific Journal Video about Isolation of CD 90+ Fibroblast/Myofibroblasts from Human Frozen Gastrointestinal Specimens at JoVE.com

Isolation of CD 90+ Fibroblast/Myofibroblasts from Human Frozen Gastrointestinal Specimens

Here, a protocol to isolate and establish primary fibroblast/myofibroblast (MF) cultures from frozen gastric, small intestinal, and colonic tissue-yielding cells with a MF phenotype-is presented. These cells express CD90, &#945;-SMA and vimentin. MFs can be used for a variety of functional assays including enzymatic activity and cytokine production.


Fibroblasts/myofibroblasts (MFs) have been gaining increasing attention for their role in pathogenesis and their contributions to both wound healing and ...

The overall goal of this method is to isolate viable mucosal myofibroblasts and fibroblasts from frozen gastrointestinal tissue samples, originally obtained from biopsies or surgical specimens. The isolation of myofibroblasts and fibroblasts from properly frozen gastrointestinal tissue allows processing of fresh samples to be delayed. This facilitates the use of samples from different sources worldwide.If the tissue was preserved in freeze medium, place the cryovial in a 37 degree Celsius water bath until the tissue and medium are thawed. For four to five two square millimeter tissue fragments, add 10 milliliters of HBSS without calcium and magnesium, tightly close the lid, and wash the tissue by gently inverting the tube one time. Once the tissue has settled by gravity, carefully discard the supernatant, and wash the tissues again.Then, add 10 millilites of collagenase solution to the tube, and transfer the tissue suspension into a sterile DNase/RNase free tube with built-in rotors. Tightly close the rotor tube and attach it upside down onto the sleeve of a dissociator. Then, run the preset H-tumor zero one program.At the termination of the program, incubate the tissue slurry for 45 minutes at 37 degrees Celsius, under continuous 140 RPM rotation on a shaker. Then, return the tube to the dissociator, and run the H-tumor zero two program. After the second dissociation, add 50 microliters of DNA stock solution to the cell solution for 30 minutes at 37 degrees Celsius, under continuous 60 RPM rotation on the shaker.At the end of the second incubation, run the H-tumor zero three program. Now, filter the suspension through a sterile 70 micron cell strainer, into a 50 milliliter conical tube for centrifugation. Discard the supernatant, and wash the cell pellet in 25 milliliters of HBSS without calcium and magnesium two times, counting the cells after the first wash.For myofibroblast primary culture generation, resuspend the pellet in up to 4 x 10 to the 6th cells per three milliliters of medium, and seed them in 6 well cell culture-treated plates. Grow the cells at 37 degrees Celsius, and five percent carbon dioxide, changing the medium every two to three days until the formation of an 80 percent confluent myofibroblast monolayer. The cells are then ready to be passaged in T25 culture flasks.To analyze or sort the isolated myofibroblasts by flow cytometry, plate up to 2 x 10 to the 6th cells per two milliliters of fibroblast isolation medium, in a 24 well low binding plate for overnight incubation at 37 degrees Celsius and five percent carbon dioxide. Using this enzymatic digestion protocol, myofibroblasts and fibroblasts got CD-90 positive mucosal stromal cell populations, can be consistently isolated and grown from gastrointestinal surgical specimens and biopsies. While there is a slight decrease in the viability of the mucosal single cell preparation after freezing the surgical specimens, there is an increase in the efficiency of the myofibroblast and fibroblast culture generation due to a concomitant decrease in the bacterial and fungal concentration.After two passages, the myofibroblasts and fibroblasts are positive for CD90, the mesenchymal marker lineage marker, Vimentin, and smooth muscle actin, a marker of differentiated mesenchymal cells, as determined by both confocal microscopy and flow cytometric analysis. Once mastered, if performed properly, this technique can be completed in approximately two hours. While attempting this procedure, it is important to remember to allow the necessary time for the tissue to be fully enzymatically digested.Following this procedure, other methods, like flow cytometry, immunocytochemistry, and various functional assays may be performed. They may be used to determine the contribution of myofibroblasts and fibroblasts to gut mucosal homeostasis as well as to the pathology of acute and chronic inflammatory gastrointestinal disease. After watching this video, you should have a good understanding of how to isolate myofibroblasts and fibroblasts from frozen gastrointestinal tissue.Don't forget, unfixed human tissue can be contaminated by pathogenic microorganisms. Universal precautions while working with human biological samples should always be taken while performing this procedure.

isolation-cd-90-fibroblastmyofibroblasts-from-human-frozen

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 from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

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

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are distributed along the dendrites. Their morphology is largely dependent on neuronal activity, and they are dynamic. Dendritic spines express glutamatergic receptors (AMPA and NMDA receptors) on their surface and at the levels of postsynaptic densities. Each spine allows the neuron to control its state and local activity independently. Spine morphologies have been extensively studied in glutamatergic pyramidal cells of the brain cortex, using both in vivo approaches and neuronal cultures obtained from rodent tissues. Neuropathological conditions can be associated to altered spine induction and maturation, as shown in rodent cultured neurons and one-dimensional quantitative analysis 1. The present study describes a protocol for the 3D quantitative analysis of spine morphologies using human cortical neurons derived from neural stem cells (late cortical progenitors). These cells were initially obtained from induced&#160;pluripotent stem cells. This protocol allows the analysis of spine morphologies at different culture periods, and with possible comparison between induced&#160;pluripotent stem cells obtained from control individuals with those obtained from patients with psychiatric diseases.

Dendritic spines of pyramidal neurons are the sites of most excitatory synapses in mammalian brain cortex. This method describes a 3D quantitative analysis of spine morphologies in human cortical pyramidal glutamatergic neurons derived from induced&#160;pluripotent stem cells.

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation

Genome-editing of human pluripotent stem cells (hPSCs) provides a genetically controlled and clinically relevant platform from which to understand human development and investigate the pathophysiology of disease. By employing site-specific nucleases (SSNs) for genome editing, the rapid derivation of new hPSC lines harboring specific genetic alterations in an otherwise isogenic setting becomes possible. Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 are the most commonly used SSNs. All of these nucleases function by introducing a double stranded DNA break at a specified site, thereby promoting precise gene editing at a genomic locus. SSN-meditated genome editing exploits two of the cell&#39;s endogenous DNA repair mechanisms, non-homologous end joining (NHEJ) and homology directed repair (HDR), to either introduce insertion/deletion mutations or alter the genome using a homologous repair template at the site of the double stranded break. Electroporation of hPSCs is an efficient means of transfecting SSNs and repair templates that incorporate transgenes such as fluorescent reporters and antibiotic resistance cassettes. After electroporation, it is possible to isolate only those hPSCs that incorporated the repair construct by selecting for antibiotic resistance. Mechanically separating hPSC colonies and confirming proper integration at the target site through genotyping allows for the isolation of correctly targeted and genetically homogeneous cell lines. The validity of this protocol is demonstrated here by using all three SSN platforms to incorporate EGFP and a puromycin resistance construct into the AAVS1 safe harbor locus in human pluripotent&#160;stem cells.

Genome editing of human pluripotent stem cells (hPSCs) can be done quickly and efficiently. Presented here is a robust experimental procedure to genetically engineer hPSCs as exemplified by editing the AAVS1 safe harbor locus to express EGFP and introduce antibiotic resistance.

Genome-editing of human pluripotent stem cells (hPSCs) provides a genetically controlled and clinically relevant platform from which to understand human ...

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 dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.

Herein we describe methods for the dissection of fetal and maternal tissues from human term placenta, followed by isolation and expansion of mesenchymal stem/stromal cells (MSC) from these tissues.

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

Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their anatomical tissue location remained unclear. The recent discovery of defined perivascular and MSC cell marker expression by perivascular cells in multiple tissues by our group and other researchers has provided an opportunity to prospectively isolate and purify specific homogenous subpopulations of multipotent perivascular precursor cells. We have previously demonstrated the use of fluorescent activated cell sorting (FACS) to purify microvascular CD146+CD34- pericytes and vascular CD34+CD146- adventitial cells from human skeletal muscle. Herein we describe a method to simultaneously isolate these two perivascular cell subsets from human myocardium by FACS, based on the expression of a defined set of cell surface markers for positive and negative selections. This method thus makes available two specific subpopulations of multipotent cardiac MSC-like precursor cells for use in basic research and/or therapeutic investigations.

Human cardiac tissue harbours multipotent perivascular precursor cell populations that may be suitable for myocardial regeneration. The technique described here allows for the simultaneous isolation and purification of two multipotent stromal cell populations associated with native blood vessels, i.e. CD146+CD34- pericytes and CD34+CD146- adventitial cells, from the human myocardium.

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their ...

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly gained attention because they do not pose any ethical or moral concerns. Current methods to isolate MSCs from UC yield low amounts of cells with variable proliferation potentials. Since UC is an anatomically-complex organ, differences in MSC properties may be due to the differences in the anatomical regions of their isolation. In this study, we first dissected the cord/placenta samples into three discrete anatomical regions: UC, cord-placenta junction (CPJ), and fetal placenta (FP). Second, two distinct zones, cord lining (CL) and Wharton&#39;s jelly (WJ), were separated. The explant culture technique was then used to isolate cells from the four sources. The time required for the primary culture of cells from the explants varied depending on the source of the tissue. Outgrowth of the cells occurred within 3 - 4 days of the CPJ explants, whereas growth was observed after 7 - 10 days and 11 - 14 days from CL/WJ and FP explants, respectively. The isolated cells were adherent to plastic and displayed fibroblastoid morphology and surface markers, such as CD29, CD44, CD73, CD90, and CD105, similarly to bone marrow (BM)-derived MSCs. However, the colony-forming efficiency of the cells varied, with CPJ-MSCs and WJ-MSCs showing higher efficiency than BM-MSCs. MSCs from all four sources differentiated into adipogenic, chondrogenic, and osteogenic lineages, indicating that they were multipotent. CPJ-MSCs differentiated more efficiently in comparison to other MSC sources. These results suggest that the CPJ is the most potent anatomical region and yields a higher number of cells, with greater proliferation and self-renewal capacities in vitro. In conclusion, the comparative analysis of the MSCs from the four sources indicated that CPJ is a more promising source of MSCs for cell therapy, regenerative medicine, and tissue engineering.

Here, we present a protocol for the dissection of human umbilical cord (UC) and fetal placenta sample into cord lining (CL), Wharton&#39;s jelly (WJ), cord-placenta junction (CPJ), and fetal placenta (FP) for the isolation and characterization of mesenchymal stromal cells (MSCs) using the explant culture technique.

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and colleagues1. Later, others documented the existence of similar types of EPCs originating from bone marrow2,3. More recently, Yoder and Ingram showed that EPCs derived from umbilical cord blood had a higher proliferative potential compared to ones isolated from adult peripheral blood4,5,6. Apart from being involved in postnatal vasculogenesis, EPCs have also shown promise as a cell source for creating tissue-engineered vascular and heart valve constructs7,8. Various isolation protocols exist, some of which involve the cell sorting of mononuclear cells (MNCs) derived from the sources mentioned earlier with the help of endothelial and hematopoietic markers, or culturing these MNCs with specialized endothelial growth medium, or a combination of these techniques9. Here, we present a protocol for the isolation and culture of EPCs using specialized endothelial medium supplemented with growth factors, without the use of immunosorting, followed by the characterization of the isolated cells using Western blotting and immunostaining.

The goal of this protocol is to isolate endothelial progenitor cells from umbilical cord blood. Some of the applications include using these cells as a biomarker for identifying patients with cardiovascular risk, treating ischemic diseases, and creating tissue-engineered vascular and heart valve constructs.

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and ...

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 the heart after ischemic injury. When activated, epicardial cells undergo a process known as epithelial to mesenchymal transition (EMT) to provide cells to the regenerating myocardium. Furthermore, the epicardium contributes via secretion of essential paracrine factors. To fully appreciate the regenerative potential of the epicardium, a human cell model is required. Here we outline a novel cell culture model to derive primary epicardial derived cells (EPDCs) from human adult and fetal cardiac tissue. To isolate EPDCs, the epicardium is dissected from the outside of the heart specimen and processed into a single cell suspension. Next, EPDCs are plated and cultured in EPDC medium containing the ALK 5-kinase inhibitor SB431542 to maintain their epithelial phenotype. EMT is induced by stimulation with TGF&#946;. This method enables, for the first time, the study of the process of human epicardial EMT in a controlled setting, and facilitates gaining more insight in the secretome of EPDCs that may aid heart regeneration. Furthermore, this uniform approach allows for direct comparison of human adult and fetal epicardial behavior.

The epicardium plays a crucial role in the development and repair of the heart by providing cells and growth factors to the myocardial wall. Here, we describe a method to culture human primary epicardial cells that enables the study and comparison of their developmental and adult characteristics.

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

Isolation and Characterization of Human Umbilical Cord-derived Mesenchymal Stem Cells from Preterm and Term Infants

Mesenchymal stem cells (MSCs) have considerable therapeutic potential and attract increasing interest in the biomedical field. MSCs are originally isolated and characterized from bone marrow (BM), then acquired from tissues including adipose tissue, synovium, skin, dental pulp, and fetal appendages such as placenta, umbilical cord blood (UCB), and umbilical cord (UC). MSCs are a heterogeneous cell population with the capacity for (1) adherence to plastic in standard culture conditions, (2) surface marker expression of CD73+/CD90+/CD105+/CD45-/CD34-/CD14-/CD19-/HLA-DR- phenotypes, and (3) trilineage differentiation into adipocytes, osteocytes, and chondrocytes, as currently defined by the International Society for Cellular Therapy (ISCT). Although BM is the most widely used source of MSCs, the invasive nature of BM aspiration ethically limits its accessibility. Proliferation and differentiation capacity of MSCs obtained from BM generally decline with the age of the donor. In contrast, fetal MSCs obtained from UC have advantages such as vigorous proliferation and differentiation capacity. There is no ethical concern for UC sampling, as it is typically regarded as medical waste. Human UC starts to develop with continuing growth of the amniotic cavity at 4-8 weeks of gestation and keeps growing until reaching 50-60 cm in length, and it can be isolated during the whole newborn delivery period. To gain insight into the pathophysiology of intractable diseases, we have used UC-derived MSCs (UC-MSCs) from infants delivered at various gestational ages. In this protocol, we describe the isolation and characterization of UC-MSCs from fetuses/infants at 19-40 weeks of gestation.

Human umbilical cord (UC) can be obtained during the perinatal period as a result of preterm, term, and postterm delivery. In this protocol, we describe the isolation and characterization of UC-derived mesenchymal stem cells (UC-MSCs) from fetuses/infants at 19-40 weeks of gestation.

Mesenchymal stem cells (MSCs) have considerable therapeutic potential and attract increasing interest in the biomedical field. MSCs are originally ...

Isolation, Culture, Characterization, and Differentiation of Human Muscle Progenitor Cells from the Skeletal Muscle Biopsy Procedure

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle regenerative process. There are recognized challenges to working with human primary adult stem cells, particularly human muscle progenitor cells (hMPCs) derived from skeletal muscle biopsies, including low cell yield from collected tissue and a large degree of donor heterogeneity of growth and death parameters among cultures. While incorporating heterogeneity into experimental design requires a larger sample size to detect significant effects, it also allows us to identify mechanisms that underlie variability in hMPC&#160;expansion capacity, and thus allows us to better understand heterogeneity in skeletal muscle regeneration. Novel mechanisms that distinguish the expansion capacity of cultures have the potential to lead to the development of therapies to improve skeletal muscle regeneration.

We present techniques for isolating, culturing, characterizing, and differentiating human primary muscle progenitor cells (hMPCs) obtained from skeletal muscle biopsy tissue. hMPCs obtained and characterized through these methods can be used to subsequently address research questions related to human myogenesis and skeletal muscle regeneration.