Name: Derivation and Differentiation of Canine Ovarian Mesenchymal Stem Cells (Video) | JoVE Journal
Uploaded: 2018-12-16T13:00:00.000+00:00
Duration: 11 min 41 s
Description: Learn to isolate, expand and differentiate mesenchymal stem cells from canine ovarian tissue. This method enables researchers to study stem cell biology and explore potential regenerative medicine applications.

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5 minutes

The authors acknowledge the canine sterilization program at UNESP-FCAV for kindly providing the ovaries. This work was supported by grants from FAPESP (process no. 2013/14293-0) and CAPES.

This experiment was performed with the ovaries of four mongrel female dogs donated after elective surgery at a canine sterilization program. This experiment was approved by the Ethics Committee on the use of animals of UNESP-FCAV (protocol no. 026991/13).
1. Experimental Preparation
<ol>
	<li>Prepare or purchase 500 mL of sterile Dulbecco&#39;s phosphate-buffered saline (DPBS) without calcium or magnesium.</li>
	<li>Prepare a collagenase I stock solution by mixing 40 &#181;g of the enzyme in 1 mL of DPBS. Filter using a 0.2 &#181;m syringe filter, and store 2 mL aliquots at -20 &#176;C.</li>
	<li>Prepare the culture media from Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) low glucose with 10% fetal serum and 1% of antibiotics.</li>
	<li>Collect sterile materials: scissors and forceps, a tissue culture flask, dishes, tubes, and 10 mL and 5 mL pipettes.</li>
	<li>Use standard cell culture laboratory equipment: a biological safety cabinet (BSC), a cell culture incubator set at 5% CO2 and 38 &#176;C, and a centrifuge.</li>
	<li>Clean the BSC: with gloved hands, sterilize it with 70% EtOH, using UV lights.</li>
</ol>
2. Isolation of Mesenchymal Stem Cells from the Ovary
<ol>
	<li>After surgery, keep the ovaries in PBS on ice in a conical tube, until their arrival at the lab.</li>
	<li>Fill two 100 mm Petri dishes with 10 mL of PBS.</li>
	<li>Remove the ovaries from the tube and transfer them to the first Petri dish with PBS. Wash them with mixing movements.</li>
	<li>Gently retrieve the ovary and place it into another dish. With sterile tweezers and scissors, mince the tissue into very small pieces, approximately 1 mm in size.</li>
	<li>Transfer the ovarian tissue to a 35 mm Petri dish and add 2 mL of collagenase. Mince the tissue a little more.</li>
	<li>Place the Petri dish in the incubator at 38 &#176;C for 3 h and gently shake the Petri dish with circular movements every 20 min.</li>
	<li>After 3 h, remove the Petri dish from the incubator.</li>
	<li>Transfer the contents of the dish to a 15 mL tube. Add 5 mL of expansion media.</li>
	<li>Gently invert the tube before centrifugation. Centrifuge the tube at 2100 x g for 7 min. Remove the supernatant and resuspend the pellet with 3 mL of expansion media.</li>
	<li>Transfer the pellet to a T25 bottle. Place the bottle in the incubator with 5% CO2 at 38 &#176;C for 3 h. 
	NOTE: The most important part of the procedure is to change the media after 3 h of incubation.</li>
	<li>Remove the bottle from the incubator and remove the media with the remaining tissue. Add 3 mL of fresh expansion media to the bottle.</li>
	<li>Change the media every 48 h and observe the bottle for cellular confluence. 
	NOTE: The main steps of the procedure can be observed in Figure 1.</li>
</ol>
3. Expansion of Mesenchymal Stem Cells from the Ovary
<ol>
	<li>Cell passage
	<ol>
		<li>When the cells reach confluence, remove the media from the bottle.</li>
		<li>Wash the bottle with 3 mL of PBS. Remove the PBS.</li>
		<li>Add 1.5 mL of trypsin and put the bottle in the incubator at 38 &#176;C for 3 min. Gently tap the bottle to help the cells to detach. 
		NOTE: Cells should reach confluence approximately 5 - 7 d after the initial plating.</li>
		<li>Add 3 mL of expansion media and transfer the contents to a conical tube.</li>
		<li>Centrifuge the tube at 2100 x g for 7 min. Remove the supernatant and add 1 mL of expansion media.</li>
		<li>Gently homogenize the contents of the tube.</li>
		<li>Take an aliquot of 10 &#181;L of the sample to perform cell counting and put the tube with the cells back in the incubator.</li>
		<li>Place 10 &#181;L of the mix into the hemocytometer.</li>
	</ol>
	</li>
	<li>Counting the cells
	<ol>
		<li>Use the microscope&#39;s 10X objective and focus on the grid lines of the hemocytometer.</li>
		<li>Count the cells in five small squares (Figure 2).</li>
		<li>Multiply the counted number by 50,000 to estimate the number of cells per milliliter.</li>
	</ol>
	</li>
</ol>
4. Differentiation of Mesenchymal Stem Cells from the Ovary
NOTE: Differentiation assays were performed according to the guidelines established in Hill et al.9.
<ol>
	<li>Seed 1.0 x 104 cells per well, in triplicate, using a 4-well culture dish.</li>
	<li>Add 1 mL of expansion media.</li>
	<li>Gently agitate the dish with circular movements.</li>
	<li>After 24 h, replace the culture media with the desirable differentiation media.</li>
	<li>For osteogenic, adipogenic, and chondrogenic differentiation, incubate the cells with the induction media for 30 d, with media replacement every 3 d. Specific differentiation kits were used for each of these assays.</li>
	<li>For neurogenic lineage differentiation, incubate the cells for 10 d in the induction media, with media replacement every 3 d. The media used here contained DMEM low glucose, 2 mM valproic acid, 1 &#181;M hydrocortisone, 10 &#181;M forskolin, 5 mM potassium chloride, 5 &#181;g/mL insulin, and 200 &#181;M butylated hydroxyanisole.</li>
	<li>For endoderm precursors, incubate for 5 d in endoderm media as per the reagent manufacturer&#39;s instructions.</li>
	<li>For primordial germ cell lineage differentiation, incubate the cells in the induction media for 14 d, with media replacement every 3 d. The media used here contained DMEM, 10% FBS, 1 mM sodium pyruvate, 10 ng/mL LIF, 1 mM nonessential amino acids, 2 mM L-glutamine, 5 &#181;g/mL insulin, 0.1 mM &#946;-mercaptoethanol, 60 &#181;M putrescine, 20 &#181;g/mL transferrin, 10 ng/mL mouse epidermal growth factor, 1 ng/mL human basic fibroblast growth factor, 40 ng/mL human glial cell line-derived neurotrophic factor, and 15 mg/L penicillin.</li>
</ol>

<ol><li>Gazit, Z., Pelled, G., Sheyn, D., Kimelman, N., Gazit, D. Mesenchymal stem cells. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aGazit%2C+Z+%22Handbook+of+Stem+Cells+%282nd+Edition%29%22">Handbook of Stem Cells (2nd Edition)</a>. Atala, A., Lanza, R. , Academic Press. 513-527 (2013).</li>
<li>Zipori, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+nature+of+stem+cells%3A+state+rather+than+entity%5BTitle%5D)+AND+%22Nature+Reviews+Genetics%22%5BJournal%5D)">The nature of stem cells: state rather than entity.</a> Nature Reviews Genetics. 5 (11), 873-878 (2004).</li>
<li>Auersperg, N., Wong, A. S., Choi, K. C., Kang, S. K., Leung, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ovarian+surface+epithelium%3A+biology%2C+endocrinology%2C+and+pathology%5BTitle%5D)+AND+%22Endocrine+Reviews%22%5BJournal%5D)">Ovarian surface epithelium: biology, endocrinology, and pathology.</a> Endocrine Reviews. 22 (2), 255-288 (2001).</li>
<li>Ahmed, N., Thompson, E. W., Quinn, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epithelial-mesenchymal+interconversions+in+normal+ovarian+surface+epithelium+and+ovarian+carcinomas%3A+an+exception+to+the+norm%5BTitle%5D)+AND+%22Journal+of+Cellular+Physiology%22%5BJournal%5D)">Epithelial-mesenchymal interconversions in normal ovarian surface epithelium and ovarian carcinomas: an exception to the norm.</a> Journal of Cellular Physiology. 213 (3), 581-588 (2007).</li>
<li>Bukovsky, A., Svetlikova, M., Caudle, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oogenesis+in+cultures+derived+from+adult+human+ovaries%5BTitle%5D)+AND+%22Reproductive+Biology+and+Endocrinology%22%5BJournal%5D)">Oogenesis in cultures derived from adult human ovaries.</a> Reproductive Biology and Endocrinology. 3 (1), 17(2005).</li>
<li>Gong, S. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Embryonic+stem+cell-like+cells+established+by+culture+of+adult+ovarian+cells+in+mice%5BTitle%5D)+AND+%22Fertility+and+Sterility%22%5BJournal%5D)">Embryonic stem cell-like cells established by culture of adult ovarian cells in mice.</a> Fertility and Sterility. 93 (8), 2594-2601 (2010).</li>
<li>Johnson, J., Canning, J., Kaneko, T., Pru, J. K., Tilly, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Germline+stem+cells+and+follicular+renewal+in+the+postnatal+mammalian+ovary%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Germline stem cells and follicular renewal in the postnatal mammalian ovary.</a> Nature. 428 (6979), 145(2004).</li>
<li>Deans, R. J., Moseley, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mesenchymal+stem+cells%3A+biology+and+potential+clinical+uses%5BTitle%5D)+AND+%22Experimental+Hematology%22%5BJournal%5D)">Mesenchymal stem cells: biology and potential clinical uses.</a> Experimental Hematology. 28 (8), 875-884 (2000).</li>
<li>Trinda Hill, A. B. T., Therrien, J., Garcia, J. M., Smith, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mesenchymal-like+stem+cells+in+canine+ovary+show+high+differentiation+potential%5BTitle%5D)+AND+%22Cell+Proliferation%22%5BJournal%5D)">Mesenchymal-like stem cells in canine ovary show high differentiation potential.</a> Cell Proliferation. 50 (6), 12391(2017).</li>
<li>Dominici, M. L. B. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Minimal+criteria+for+defining+multipotent+mesenchymal+stromal+cells.+The+International+Society+for+Cellular+Therapy+position+statement%5BTitle%5D)+AND+%22Cytotherapy%22%5BJournal%5D)">Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement.</a> Cytotherapy. 8, 4315-4317 (2006).</li>
<li>Friedenstein, A. J., Piatetzky-Shapiro, I. I., Petrakova, K. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Osteogenesis+in+transplants+of+bone+marrow+cells%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Osteogenesis in transplants of bone marrow cells.</a> Development. 16 (3), 381-390 (1966).</li>
<li>Kuznetsov, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-colony+derived+strains+of+human+marrow+stromal+fibroblasts+form+bone+after+transplantation+in+vivo%5BTitle%5D)+AND+%22Journal+of+Bone+and+Mineral+Research%22%5BJournal%5D)">Single-colony derived strains of human marrow stromal fibroblasts form bone after transplantation in vivo.</a> Journal of Bone and Mineral Research. 12 (9), 1335-1347 (1997).</li>
</ol>

The authors have nothing to disclose.

Herein we provide evidence that MSCs can be isolated from canine ovarian tissue, which is considered biological waste after ovariectomy. Due to the fact that many cell types can be found in the ovary, we proposed a protocol to select MSCs based on their rapid adherence to plastic, which successfully selected cells that grew in a monolayer with a fibroblast-like morphology. 
The first report of the derivation of MSCs from bone marrow was based on the plastic adhesion capacity of the MSCs during...

The number of published studies that focus on stem cells has increased substantially over the past decade, a research effort that has been fueled by the collective goal of discovering powerful regenerative medicine therapies. Stem cells have two primary defining markers: self- renovation and differentiation. Mesenchymal stem cells are responsible for regular tissue turnover and have a more restricted capacity of differentiation when compared to embryonic stem cells1. Recently, many studies have shown a wide range of differentiation of MSCs, and a topic under discussion is whether differences between embryonic and adult stem cells exist at all2.
The ovarium surface epithelium is an uncommitted layer of cells, relatively less differentiated, which expresses both epithelial and mesenchymal markers3, retaining the capacity to differentiate into different types of cells in response to environmental signals4. The exact location of stem cells in the ovary is not well known; however, it has been proposed that bipotential progenitors in the tunica albuginea give rise to germ cells5. Immunological studies have hypothesized that these cells have a stromal origin6 or are located in or proximal to the ovary surface7. Since mesenchymal stem cells express numerous receptors that play an important role in cell adhesion8, an experiment was designed to test the hypothesis that selecting a population of cells with rapid adhesion would isolate a population of cells clearly characterizable as mesenchymal in nature. Recently, our group reported the derivation of MSCs from ovarian tissue based on their capacity of adhesion to the plastic surface of the culture dish in the first 3 h of culture, in order to obtain a purified population of cells exhibiting rapid adhesion9. Here, we describe the developed method for mesenchymal stem cell isolation from the ovarian tissue.

<table><tbody><tr><td>DPBS&amp;nbsp;</td><td>Thermo Fisher</td><td>14190144</td><td></td></tr><tr><td>Collagenase I</td><td>Thermo Fisher</td><td>17100017</td><td></td></tr><tr><td>Tissue flask</td><td>Corning</td><td>CLS3056</td><td></td></tr><tr><td>DMEM low glucose</td><td>Thermo Fisher</td><td>11054020</td><td></td></tr><tr><td>FBS</td><td>Thermo Fisher</td><td>12484-010</td><td></td></tr><tr><td>TrypLE express</td><td>Thermo Fisher</td><td>12604021</td><td></td></tr><tr><td>StemPro Adipogenesis Differentiation Kit</td><td>Thermo Fisher</td><td>A1007001</td><td></td></tr><tr><td>StemPro Chondrogenesis Differentiation Kit</td><td>Thermo Fisher</td><td>A1007101</td><td></td></tr><tr><td>StemPro Osteogenesis Differentiation Kit</td><td>Thermo Fisher</td><td>A1007201</td><td></td></tr><tr><td>STEMdiff Definitive Endoderm Kit</td><td>StemCell</td><td>5110</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Thermo Fisher</td><td>15070063</td><td></td></tr><tr><td>CD45</td><td>AbD Serotec</td><td>MCA 2035S</td><td></td></tr><tr><td>CD34</td><td>AbD Serotec</td><td>MCA 2411GA</td><td></td></tr><tr><td>CD90</td><td>AbD Serotec</td><td>MCA 1036G</td><td></td></tr><tr><td>CD44</td><td>AbD Serotec</td><td>MCA 1041</td><td></td></tr><tr><td>Nestin</td><td>Milipore</td><td>MAB353</td><td></td></tr><tr><td>&amp;beta;-Tubulin&amp;nbsp;</td><td>Milipore</td><td>MAB1637</td><td></td></tr><tr><td>DDX4</td><td>Invitrogen</td><td>PA5 -23378</td><td></td></tr><tr><td>IgG- FITC</td><td>AbD Serotec</td><td>STAR80F</td><td></td></tr><tr><td>IgG- FITC</td><td>AbD Serotec</td><td>STAR120F</td><td></td></tr></tbody></table>

derivation and differentiation of canine ovarian mesenchymal stem cells

Derivation and Differentiation of Canine Ovarian Mesenchymal Stem Cells

Interest in mesenchymal stem cells (MSCs) has increased over the past decade due to their ease of isolation, expansion, and culture. Recently, studies have demonstrated the wide differentiation capacity that these cells possess. The ovary represents a promising candidate for cell-based therapies due to the fact that it is rich in MSCs and that it is frequently discarded after ovariectomy surgeries as biological waste. This article describes procedures for the isolation, expansion, and differentiation of MSCs derived from the canine ovary, without the necessity of cell-sorting techniques. This protocol represents an important tool for regenerative medicine because of the broad applicability of these highly differentiable cells in clinical trials and therapeutic uses.

Mesenchymal Stem Cell Isolation from Canine Ovary:
The ovarian MSC isolation procedure is summarized in Figure 1. After surgery, tissue mincing, collagenase digestion, and a media change 3 h after the beginning of the culture, a putative MSC population with rapid plastic-adhesive properties was successfully isolated from canine ovarian tissue. The harvested cells ra...

Herein, we describe a method for the isolation, expansion, and differentiation of mesenchymal stem cells from canine ovarian tissue.

Interest in mesenchymal stem cells (MSCs) has increased over the past decade due to their ease of isolation, expansion, and culture. Recently, studies ...

derivation-differentiation-canine-ovarian-mesenchymal-stem

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Learn to isolate, expand and differentiate mesenchymal stem cells from canine ovarian tissue. This method enables researchers to study stem cell biology and explore potential regenerative medicine applications.

Derivation and Differentiation of Canine Ovarian Mesenchymal Stem Cells

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