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 first 48 h of culture11; however, it has been reported that this method has the potential to isolate a heterogeneous population, phenotypically and functionally, at least for bone marrow12. The proposed culture method also selects cells by plastic adhesion capacity and isolates mesenchymal cells from a tissue that has many cell types, without the necessity of sorting the desirable cells. Due to the fact that mesenchymal stem cells express numerous receptors that play an important role in cell adhesion8, it was hypothesized that if a population of cells with rapid adhesion were selected, it would result in a more homogeneous population at the first passage, with a reliable MSC profile. The representative results provide evidence that a shorter duration plastic adhesion assay can be used to isolate a morphologically homogeneous population of MSCs at the first passage, without the necessity of waiting for more advanced passages or cell sorting.
A critical step in this protocol is to change the media 3 h after the beginning of the culture. As shown in the representative results section, the stem cells obtained from canine ovaries met the three criteria established for the characterization of MSCs: they exhibited a fibroblast-like morphology, were positive for the presence of MSC markers, and were able to differentiate into such mesenchymal lineages as osteogenic, adipogenic and chondrogenic lineages. Cells isolated in this experiment were also successfully differentiated into endodermal precursors, ectodermal lineages, and primordial germ cell lineages. The cells should reach confluence approximately 5 - 7 days after the initial plating. The cells from animals with an advanced age may grow more slowly than those of young animals, and of those, fewer cells may adhere to the plastic. In these cases, the concentration of FBS in the media can be increased to 15%. 
Therefore, the selection for MSCs based on the cells with more rapid adherence capacity represents an inexpensive, streamlined, and effective procedure. From a therapeutic perspective, it is most important to highlight that these cells represent a promising tool for regenerative medicine, especially because of their wide range of differentiation.

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

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 rapidly adhered to the plastic surface of the culture dish and grew into a morphologically homogeneous monolayer culture with typical MSC-like morphology (Figure 3).
Characterization of Ovarian MSC In Vitro
The goal of MSC characterization is to ensure that isolated cells conform to standard MSC criteria. These include adherence to plastic, a positive detection of mesenchymal surface markers, and an absence of hematopoietic surface markers, as well as the capacity to undergo mesodermal differentiation.
As shown in Figure 3, ovarian-derived cells were adherent to the plastic and exhibited a fibroblast-like morphology, fulfilling the first criterion that defines MSCs. Moreover, the isolated cells showed the expression of transcripts for the canine MSC markers CD44, CD90, and CD105. In addition, MSC-specific cell surface antigens CD90 and CD44 were detected by early passage FACS analysis and immunocytochemistry. The absence of the markers CD45 and CD34, which are hematopoietic-specific cell surface antigens, was also confirmed by the same techniques (Figure 4). This further confirmed that the ovary contains cells expressing an MSC-specific phenotype9. The antibodies that were used in this experiment for canine MSC characterization are listed in the Table of Materials.
The next step in the characterization of MSCs is to attain proof of their ability to differentiate into mesodermal lineages. After 30 days of exposure and culture in lineage-specific differentiation protocols, staining was performed to identify a commitment to different lineages (Figure 5A). A commitment to the osteogenic lineage was confirmed through Von Kossa staining, which identified calcium deposits. Safranin O proteoglycan staining confirmed chondrogenic commitment, and Oil Red O lipid staining confirmed adipogenic commitment. Continuing the investigation of the differentiability of these cells, they were able to undergo differentiation into the ectodermic lineage, as shown by immunostaining for two neuroectodermal stem cell markers: &#946;-tubulin and Nestin, following exposure of ovary-derived MSCs to the neuronal lineage differentiation protocol for 10 days (Figure 5C). Transcripts for SOX17 and CD184, as well as cell morphologies specific to the endodermal lineage, were observed after exposure to specific differentiation protocols for 5 days (Figure 5B). Finally, after 14 days of exposure of ovarian-derived MSCs to germ cell induction media, OCT 4 and DDX4 were identified (Figure 5D). Together, these results support a robust and diverse capacity of differentiation for MSCs derived from ovarian tissue, when exposed to specific differentiation protocols9.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58163/58163fig1.jpg" /> 
Figure 1: Main steps of the isolation procedure. (1) After surgery, transfer the ovaries to sterile tubes with PBS, on ice. (2) In order to wash the ovaries, they should be transferred to a 100 mm plate with PBS. (3) Mince the tissue into small pieces, approximately 1 mm in size. (4) In order to liberate the cells from the tissue, transfer the pieces of tissue to 35 mm plates and add collagenase for a 3 h digestion in the incubator. (5) Transfer the contents of the plate to a conical tube for centrifugation. (6) Plate the pellet in a T25 bottle and incubate for 3 h. (7) After the first 3 h of culture, change the culture media. <a href="https://www.jove.com/files/ftp_upload/58163/58163fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58163/58163fig2v2.jpg" /> 
Figure 2: Hemocytometer. The five small squares to be counted are marked in red.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58163/58163fig3.jpg" /> 
Figure 3: Cell morphology. When the media was changed 3 h after the beginning of the culture it resulted in a homogeneous population of mesenchymal cells, which exhibited a fibroblast-like morphology. On the contrary, when the first media change is done after 48 h other cell types besides the fibroblast-like cells can be observed. The scale bar = 1,000 &#181;m. This figure has been modified from Hill et al.9. <a href="https://www.jove.com/files/ftp_upload/58163/58163fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58163/58163fig4.jpg" /> 
Figure 4: Molecular profile of ovarian MSC. (A) The isolated cells exhibited a classic MSC marker profile, being positive for three MSC markers by real-time PCR (CD90, CD105, and CD44). (B) By protein analysis, CD44 was positive in those cells, and they lacked the expression of two hematopoietic markers (CD34 and CD45). The scale bar = 70 &#181;m. This figure has been modified from Hill et al.9. <a href="https://www.jove.com/files/ftp_upload/58163/58163fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58163/58163fig5.jpg" /> 
Figure 5: Differentiation of ovarian MSC. The isolated cells were able to differentiate into (A) mesodermal lineages (chondrogenic, osteogenic, and adipogenic; the scale bar = 70 &#181;m). Moreover, the cells were able to differentiate into both (B) endodermal precursors (the scale bar = 70 &#181;m) and (C) neurogenic lineages (the scale bar = 50 &#181;m). Interestingly, the cells underwent (D) primordial germ cell differentiation, demonstrating a wide capacity of differentiation for ovarian mesenchymal stem cells. This figure has been modified from Hill et al.9.&#160;<a href="https://www.jove.com/files/ftp_upload/58163/58163fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Derivation and Differentiation of Canine Ovarian Mesenchymal Stem Cells at JoVE.com

Derivation and Differentiation of Canine Ovarian Mesenchymal Stem Cells

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 ...

The overall goal of this procedure is to isolate, expand and differentiate mesenchymal stems sells derived from canine ovarian tissue. 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 Sao Paulo State University, FVAC.Representative results.Discussion.Conclusion.

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5.7K Views.  São Paulo State University (UNESP). The overall goal of this procedure is to isolate, expand and differentiate mesenchymal stems sells derived from canine ovarian tissue. 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 Sao Paulo State University, FVAC.Representative results.Discussion.Conclusion.

Video: Derivation and Differentiation of Canine Ovarian Mesenchymal Stem Cells

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs, differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result, numerous strategies have been developed to derive CPCs from ESCs. In this protocol, differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs, ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus, CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs.

In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines.

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great ...

An Enzymatic Method to Rescue Mesenchymal Stem Cells from Clotted Bone Marrow Samples

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade clots in the bone marrow and release MSCs for further use. This protocol provides a rapid and inexpensive alternative to bone marrow resampling. Bone marrow is a major source of MSCs, which are interesting for tissue engineering and autologous stem cell therapies. Upon withdrawal bone marrow may clot, as it comprises all of the hematopoietic system. The resulting clots contain also MSCs that are lost for expansion culture or direct stem cell therapy. We experienced that 74% of canine bone marrow samples contained clots and yielded less than half of the stem cell number expected from unclotted samples. Thus, we developed a protocol for enzymatic digestion of those clots to avoid labor-intense and costly bone marrow resampling. Urokinase - a clinically approved and readily available thrombolytic drug &#8211; clears away the bone marrow clots almost completely. As a consequence, treated bone marrow aspirates yield similar numbers of MSCs as unclotted samples. Also, after urokinase treatment the cells kept their metabolic activity and the ability to differentiate into chondrogenic, osteogenic and adipogenic lineages. Our protocol salvages clotted blood and bone marrow samples without affecting the quality of the cells. This obsoletes resampling, considerably reduces sampling costs and enables the use of clotted samples for research or therapy.

Mesenchymal stem cells are usually obtained from bone marrow and require expansion culture. When samples clot before processing, a protocol using the (enzymatic) thrombolytic drug urokinase can be applied to degrade the clot. Thus, cells are released and available for expansion culture. This protocol provides a rapid and inexpensive alternative to resampling.

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade ...

An Orthotopic Mouse Model of Ovarian Cancer using Human Stroma to Promote Metastasis

Ovarian cancer is characterized by early, diffuse metastasis with 70% of women having metastatic disease at the time of diagnosis. While elegant transgenic mouse models of ovarian cancer exist, these mice are expensive and take a long time to develop tumors. Intraperitoneal injection xenograft models lack human stroma and do not accurately model ovarian cancer metastasis. Even patient derived xenografts (PDX) do not fully recapitulate the human stromal microenvironment as serial PDX passages demonstrate significant loss of human stroma. The ability to easily model human ovarian cancer within a physiologically relevant stromal microenvironment is an unmet need. Here, the protocol presents an orthotopic ovarian cancer mouse model using human ovarian cancer cells combined with patient-derived carcinoma-associated mesenchymal stem cells (CA-MSCs). CA-MSCs are stromal progenitor cells, which drive the formation of the stromal microenvironment and support ovarian cancer growth and metastasis. This model develops early and diffuses metastasis mimicking clinical presentation. In this model, luciferase expressing ovarian cancer cells are mixed in a 1:1 ratio with CA-MSCs and injected into the ovarian bursa of NSG mice. Tumor growth and metastasis are followed serially over time using bioluminescence imaging. The resulting tumors grow aggressively and form abdominal metastases by 14 days post injection. Mice experienced significant decreases in body weight as a marker of systemic illness and increased disease burden. By day 30 post injection, mice met endpoint criteria of &gt;10% body weight loss and necropsy confirmed intra-abdominal metastasis in 100% of mice and 60%-80% lung and parenchymal liver metastasis. Collectively, orthotopic engraftment of ovarian cancer cells and stroma cells generates tumors that closely mimic the early and diffuse metastatic behavior of human ovarian cancer. Furthermore, this model provides a tool to study the role of ovarian cancer cell: stroma cell interactions in metastatic progression.

Orthotopic engraftment of ovarian cancer cells mixed with human stromal cells provides a mouse model that exhibits rapid, diffuse metastatic behavior that is characteristic of human ovarian cancer. This model also allows for the study of tumor cell and stromal cell interactions, as well as their role in tumor progression and metastasis.

Ovarian cancer is characterized by early, diffuse metastasis with 70% of women having metastatic disease at the time of diagnosis. While elegant ...

Cancer Research

Evaluation of Stem Cell Properties in Human Ovarian Carcinoma Cells Using Multi and Single Cell-based Spheres Assays

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem cells&#8221; (CSCs) responsible for tumor initiation, maintenance and relapse following conventional chemotherapies. Identification of ovarian CSCs is therefore an important goal. A commonly used method to assess CSC potential in vitro is the spheres assay in which cells are plated under non-adherent culture conditions in serum-free medium supplemented with growth factors and sphere formation is scored after a few days. Here, we review currently available protocols for human ovarian cancer spheres assays and perform a side-by-side analysis between commonly used multi cell-based assays and a more accurate system based on single cell plating. Our results indicate that both multi cell-based as well as single cell-based spheres assays can be used to investigate sphere formation in vitro. The more laborious and expensive single cell-based assays are more suitable for functional assessment of individual cells and lead to overall more accurate results while multi cell-based assays can be strongly influenced by the density of plated cells and require titration experiments upfront. Methylcellulose supplementation to multi cell-based assays can be effectively used to reduce mechanical artifacts.

In vitro spheres assays are commonly used to identify cancer stem cells. Here we compare single with multi cell-based spheres assays. The more laborious single cell-based assays or methylcellulose supplementation give more accurate results while multi cell-based assays performed in liquid medium can be highly influenced by cell density.

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem ...

Medicine

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Biology

Derivation of Mouse Trophoblast Stem Cells from Blastocysts

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the trophectoderm mediates implantation and generates the fetal part of the placenta. As a result, the development of this lineage is essential for embryo survival. Derivation of trophoblast stem (TS) cells from mouse blastocysts was first described by Tanaka et al. 1998. The ability of TS cells to preserve the trophoblast specific property and their expression of stage- and cell type-specific markers after proper stimulation provides a valuable model system to investigate trophoblast lineage development whereby recapitulating early placentation events. Furthermore, trophoblast cells are one of the few somatic cell types undergoing natural genome amplification. Although the molecular pathways underlying trophoblast polyploidization have begun to unravel, the physiological role and advantage of trophoblast genome amplification remains largely elusive. The development of diploid stem cells into polyploid trophoblast cells in culture makes this ex vivo system an excellent tool for elucidating the regulatory mechanism of genome replication and instability in health and disease. Here we describe a protocol based on previous reports with modification published in Chiu et al. 2008.

In this video, we demonstrate the isolation of mouse blastocysts and the derivation of trophoblast stem cells from blastocysts. We also describe conditions for maintenance of the stem cell property as well as induction of differentiation in culture.

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the ...

Differentiation and Characterization of Osteoclasts from Human Induced Pluripotent Stem Cells

This protocol details the propagation and passaging of human iPSCs and their differentiation into osteoclasts. First, iPSCs are dissociated into a single-cell suspension for further use in embryoid body induction. Following mesodermal induction, embryoid bodies undergo hematopoietic differentiation, producing a floating hematopoietic cell population. Subsequently, the harvested hematopoietic cells undergo a macrophage colony-stimulating factor maturation step and, finally, osteoclast differentiation. After osteoclast differentiation, osteoclasts are characterized by staining for TRAP in conjunction with a methyl green nuclear stain. Osteoclasts are observed as multinucleated, TRAP+ polykaryons. Their identification can be further supported by Cathepsin K staining. Bone and mineral resorption assays allow for functional characterization, confirming the identity of bona fide osteoclasts. This protocol demonstrates a robust and versatile method to differentiate human osteoclasts from iPSCs and allows for easy adoption in applications requiring large quantities of functional human osteoclasts. Applications in the areas of bone research, cancer research, tissue engineering, and endoprosthesis research could be envisioned.

This protocol presents the differentiation of human osteoclasts from induced pluripotent stem cells (iPSCs) and describes methods for the characterization of osteoclasts and osteoclast precursors.

This protocol details the propagation and passaging of human iPSCs and their differentiation into osteoclasts. First, iPSCs are dissociated into a ...

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

Studying germ cell formation and differentiation has traditionally been very difficult due to low cell numbers and their location deep within developing embryos. The availability of a "closed" in vitro based system could prove invaluable for our understanding of gametogenesis. The formation of oocyte-like cells (OLCs) from somatic stem cells, isolated from newborn mouse skin, has been demonstrated and can be visualized in this video protocol. The resulting OLCs express various markers consistent with oocytes such as Oct4 , Vasa , Bmp15, and Scp3. However, they remain unable to undergo maturation or fertilization due to a failure to complete meiosis. This protocol will provide a system that is useful for studying the early stage formation and differentiation of germ cells into more mature gametes. During early differentiation the number of cells expressing Oct4 (potential germ-like cells) reaches ~5%, however currently the formation of OLCs remains relatively inefficient. The protocol is relatively straight forward though special care should be taken to ensure the starting cell population is healthy and at an early passage.

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

In recent years the formation of germ cells using in vitro culture methods has been demonstrated using several different types of somatic stem cells. This article will visually present the differentiation of newborn mouse derived stem cells to early oocyte-like cells.

Studying  germ cell formation and differentiation has traditionally been very difficult  due to low cell numbers and their location deep within developing ...

Adipose Tissue-Derived Stem Cell Isolation and Characterization

Isolation and Identification of Mesenchymal Stem Cells Derived from Adipose Tissue of Sprague Dawley Rats

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in addition to their great capacity for self-renewal, migration, and proliferation. Adipose tissue is one of the least invasive and most accessible sources of mesenchymal cells. It has also been reported to have higher yields compared to other sources, as well as superior immunomodulatory properties. Recently, different procedures for obtaining adult mesenchymal cells from different tissue sources and animal species have been published. After evaluating the criteria of some authors, we standardized a methodology that is applicable to different purposes and easily reproducible. A pool of stromal vascular fraction (SVF) from perirenal and epididymal adipose tissue allowed us to develop primary cultures with optimal morphology and functionality. The cells were observed adhered to the plastic surface for 24 h, and exhibited a fibroblast-like morphology, with prolongations and a tendency to form colonies. Flow cytometry (FC) and immunofluorescence (IF) techniques were used to assess the expression of the membrane markers CD105, CD9, CD63, CD31, and CD34. The ability of adipose-derived stem cells (ASCs) to differentiate into the adipogenic lineage was also assessed using a cocktail of factors (4 &#181;M insulin, 0.5 mM 3-methyl-iso-butyl-xanthine, and 1 &#181;M dexamethasone). After 48 h, a gradual loss of fibroblastoid morphology was observed, and at 12 days, the presence of lipid droplets positive to oil red staining was confirmed. In summary, a procedure is proposed to obtain optimal and functional ASC cultures for application in regenerative medicine.

Author Spotlight: Isolation and Identification of Mesenchymal Stem Cells Derived from Adipose Tissue of Sprague Dawley Rats  

This protocol describes a methodology for isolating and identifying adipose tissue-derived mesenchymal stem cells (MSCs) from Sprague Dawley rats.

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in ...

Characterizing Surface Antigens and Proliferation Ability in hiPSC-Driving MSCs

Comparison of Two Representative Methods for Differentiation of Human Induced Pluripotent Stem Cells into Mesenchymal Stromal Cells

Mesenchymal stromal cells (MSCs) are adult pluripotent stem cells which have been widely used in regenerative medicine. As somatic tissue-derived MSCs are restricted by limited donation, quality variations, and biosafety, the past 10 years have seen a great rise in efforts to generate MSCs from human induced pluripotent stem cells (hiPSCs). Past and recent efforts in the differentiation of hiPSCs into MSCs have been centered around two culture methodologies: (1) the formation of embryoid bodies (EBs) and (2) the use of monolayer culture. This protocol describes these two representative methods in deriving MSC from hiPSCs. Each method presents its advantages and disadvantages, including time, cost, cell proliferation ability, the expression of MSC markers, and their capability of differentiation in vitro. This protocol demonstrates that both methods can derive mature and functional MSCs from hiPSCs. The monolayer method is characterized by lower cost, simpler operation, and easier osteogenic differentiation, while the EB method is characterized by lower time consumption.

Author Spotlight: Advancements in iPSCs and Genetic Disease Research 

This protocol describes and compares two representative methods for differentiating hiPSCs into mesenchymal stromal cells (MSCs). The monolayer method is characterized by lower cost, simpler operation, and easier osteogenic differentiation. The embryoid bodies (EBs) method is characterized by lower time consumption.

Mesenchymal stromal cells (MSCs) are adult pluripotent stem cells which have been widely used in regenerative medicine. As somatic tissue-derived MSCs are ...

Derivation and Differentiation of Canine Ovarian Mesenchymal Stem Cells

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Summary

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Evaluation of Stem Cell Properties in Human Ovarian Carcinoma Cells Using Multi and Single Cell-based Spheres Assays

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Derivation of Mouse Trophoblast Stem Cells from Blastocysts

Differentiation and Characterization of Osteoclasts from Human Induced Pluripotent Stem Cells

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

Isolation and Identification of Mesenchymal Stem Cells Derived from Adipose Tissue of Sprague Dawley Rats

Comparison of Two Representative Methods for Differentiation of Human Induced Pluripotent Stem Cells into Mesenchymal Stromal Cells