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., Atala, A., Lanza, R. <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.">Mesenchymal stem cells.</a> Handbook of Stem Cells (2nd Edition). , 513-527 (2013).</li><li>Zipori, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nature+of+stem+cells:+state+rather+than+entity.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+surface+epithelium:+biology,+endocrinology,+and+pathology.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial-mesenchymal+interconversions+in+normal+ovarian+surface+epithelium+and+ovarian+carcinomas:+an+exception+to+the+norm.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oogenesis+in+cultures+derived+from+adult+human+ovaries.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell-like+cells+established+by+culture+of+adult+ovarian+cells+in+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germline+stem+cells+and+follicular+renewal+in+the+postnatal+mammalian+ovary.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells:+biology+and+potential+clinical+uses.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal-like+stem+cells+in+canine+ovary+show+high+differentiation+potential.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimal+criteria+for+defining+multipotent+mesenchymal+stromal+cells.+The+International+Society+for+Cellular+Therapy+position+statement.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteogenesis+in+transplants+of+bone+marrow+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-colony+derived+strains+of+human+marrow+stromal+fibroblasts+form+bone+after+transplantation+in+vivo.">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 border="0" cellpadding="0" cellspacing="0" style="width:665px;" width="666">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">DPBS&#160;</td>
			<td style="width:151px;">Thermo Fisher</td>
			<td style="width:123px;">14190144</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Collagenase I</td>
			<td style="width:151px;">Thermo Fisher</td>
			<td style="width:123px;">17100017</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Tissue flask</td>
			<td style="width:151px;">Corning</td>
			<td style="width:123px;">CLS3056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">DMEM low glucose</td>
			<td style="width:151px;">Thermo Fisher</td>
			<td style="width:123px;">11054020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">FBS</td>
			<td style="width:151px;">Thermo Fisher</td>
			<td style="width:123px;">12484-010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">TrypLE express</td>
			<td style="width:151px;">Thermo Fisher</td>
			<td style="width:123px;">12604021</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:221px;">StemPro Adipogenesis Differentiation Kit</td>
			<td style="width:151px;">Thermo Fisher</td>
			<td style="width:123px;">A1007001</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:221px;">StemPro Chondrogenesis Differentiation Kit</td>
			<td style="width:151px;">Thermo Fisher</td>
			<td style="width:123px;">A1007101</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:221px;">StemPro Osteogenesis Differentiation Kit</td>
			<td style="width:151px;">Thermo Fisher</td>
			<td style="width:123px;">A1007201</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:221px;">STEMdiff Definitive Endoderm Kit</td>
			<td style="width:151px;">StemCell</td>
			<td style="width:123px;">5110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Penicillin-Streptomycin</td>
			<td style="width:151px;">Thermo Fisher</td>
			<td style="width:123px;">15070063</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">CD45</td>
			<td style="width:151px;">AbD Serotec</td>
			<td style="width:123px;">MCA 2035S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">CD34</td>
			<td style="width:151px;">AbD Serotec</td>
			<td style="width:123px;">MCA 2411GA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">CD90</td>
			<td style="width:151px;">AbD Serotec</td>
			<td style="width:123px;">MCA 1036G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">CD44</td>
			<td style="width:151px;">AbD Serotec</td>
			<td style="width:123px;">MCA 1041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Nestin</td>
			<td style="width:151px;">Milipore</td>
			<td style="width:123px;">MAB353</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">&#946;-Tubulin&#160;</td>
			<td style="width:151px;">Milipore</td>
			<td style="width:123px;">MAB1637</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">DDX4</td>
			<td style="width:151px;">Invitrogen</td>
			<td style="width:123px;">PA5 -23378</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">IgG- FITC</td>
			<td style="width:151px;">AbD Serotec</td>
			<td style="width:123px;">STAR80F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">IgG- FITC</td>
			<td style="width:151px;">AbD Serotec</td>
			<td style="width:123px;">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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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 ...

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5.6K Views.  São Paulo State University (UNESP). Herein, we describe a method for the isolation, expansion, and differentiation of mesenchymal stem cells from canine ovarian tissue.

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 ...

Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

No cure has been discovered for age-related macular degeneration (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex multifactorial disease with an unknown etiology, although it is largely thought to occur due to death or dysfunction of the retinal pigment epithelium (RPE), a monolayer of cells that underlies the retina and provides critical support for photoreceptors. RPE cell replacement strategies may hold great promise for providing therapeutic relief for a large subset of AMD patients, and RPE cells that strongly resemble primary human cells (hRPE) have been generated in multiple independent labs, including our own. In addition, the uses for iPS-RPE are not limited to cell-based therapies, but also have been used to model RPE diseases. These types of studies may not only elucidate the molecular bases of the diseases, but also serve as invaluable tools for developing and testing novel drugs. We present here an optimized protocol for directed differentiation of RPE from stem cells. Adding nicotinamide and either Activin A or IDE-1, a small molecule that mimics its effects, at specific time points, greatly enhances the yield of RPE cells. Using this technique we can derive large numbers of low passage RPE in as early as three months.

Stem cell-derived retinal pigment epithelium (RPE) cells may be used for multiple applications including cell-based therapies for retinal degeneration, disease modeling, and drug studies. Here we present a simple protocol for reproducibly deriving RPE from stem cells.

No cure has been discovered for age-related macular degeneration  (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex ...

Derivation of Highly Purified Cardiomyocytes from Human Induced Pluripotent Stem Cells Using Small Molecule-modulated Differentiation and Subsequent Glucose Starvation

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes available for basic research and translational applications. To differentiate hiPSCs into cardiomyocytes, various protocols including embryoid body (EB)-based differentiation and growth factor induction have been developed. However, these protocols are inefficient and highly variable in their ability to generate purified cardiomyocytes. Recently, a small molecule-based protocol utilizing modulation of Wnt/&#946;-Catenin signaling was shown to promote cardiac differentiation with high efficiency. With this protocol, greater than 50%-60% of differentiated cells were cardiac troponin-positive cardiomyocytes were consistently observed. To further increase cardiomyocyte purity, the differentiated cells were subjected to glucose starvation to specifically eliminate non-cardiomyocytes based on the metabolic differences between cardiomyocytes and non-cardiomyocytes. Using this selection strategy, we consistently obtained a greater than 30% increase in the ratio of cardiomyocytes to non-cardiomyocytes in a population of differentiated cells. These highly purified cardiomyocytes should enhance the reliability of results from human iPSC-based in vitro disease modeling studies and drug screening assays.

Here, we describe a robust protocol for human cardiomyocyte derivation that combines small molecule-modulated cardiac differentiation and glucose deprivation-mediated cardiomyocyte purification, enabling production of purified cardiomyocytes for the purposes of cardiovascular disease modeling and drug screening.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes ...

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can direct the differentiation of hPSCs into the cell type of interest by manipulating the culture conditions to recapitulate signals seen during development. One such cell type is the melanocyte, a pigment-producing cell of neural crest (NC) origin responsible for protecting the skin against UV irradiation. This protocol presents an extension of a currently available in vitro Neural Crest differentiation protocol from hPSCs to further differentiate NC into fully pigmented melanocytes. Melanocyte precursors can be enriched from the Neural Crest protocol via a timed exposure to activators of WNT, BMP, and EDN3 signaling under dual-SMAD-inhibition conditions. The resultant melanocyte precursors are then purified and matured into fully pigmented melanocytes by culture in a selective medium. The resultant melanocytes are fully pigmented and stain appropriately for proteins characteristic of mature melanocytes.

This work describes an in vitro differentiation protocol to produce pigmented, mature melanocytes from human pluripotent stem cells via a neural crest and melanoblast intermediate stage using a feeder-free, 25 day protocol.

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated by transcription factors which bind genomic regulatory regions including enhancers and promoters of cardiac constitutive genes. DNA is wrapped around histones that are subjected to chemical modifications. Modifications of histones further lead to repressed, activated or poised gene transcription, thus bringing another level of fine tuning regulation of gene transcription. Embryonic Stem cells (ES cells) recapitulate within embryoid bodies (i.e., cell aggregates) or in 2D culture the early steps of cardiac development. They provide in principle enough material for chromatin immunoprecipitation (ChIP), a technology broadly used to identify gene regulatory regions. Furthermore, human ES cells represent a human cell model of cardiogenesis. At later stages of development, mouse embryonic tissues allow for investigating specific epigenetic landscapes required for determination of cell identity. Herein, we describe protocols of ChIP, sequential ChIP followed by PCR or ChIP-sequencing using ES cells, embryoid bodies and cardiac specific embryonic regions. These protocols allow to investigating the epigenetic regulation of cardiac gene transcription.

A fine tuning regulation of gene transcription underlies embryonic cell fate decision. Herein, we describe chromatin immunoprecipitation assays used to investigate epigenetic regulation of both cardiac differentiation of stem cells and cardiac development of mouse embryos.

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated ...

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 ...

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading cause of mortality worldwide. Mesenchymal stem cells (MSCs) are a promising option for cell-based therapies to replace current, invasive techniques. MSCs can differentiate into mesenchymal lineages, including cardiac cell types, but complete differentiation into functional cells has not yet been achieved. Previous methods of differentiation were based on pharmacological agents or growth factors. However, more physiologically relevant strategies can also enable MSCs to undergo cardiomyogenic transformation. Here, we present a differentiation method using MSC aggregates on cardiomyocyte feeder layers to produce cardiomyocyte-like contracting cells.
Human umbilical cord perivascular cells (HUCPVCs) have been shown to have a greater differentiation potential than commonly investigated MSC types, such as bone marrow MSCs (BMSCs). As an ontogenetically younger source, we investigated the cardiomyogenic potential of first-trimester (FTM) HUCPVCs compared to older sources. FTM HUCPVCs are a novel, rich source of MSCs that retain their in utero immunoprivileged properties when cultured in vitro. Using this differentiation protocol, FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to BMSCs, as indicated by the increased expression of cardiomyocyte markers (i.e., myocyte enhancer factor 2C, cardiac troponin T, heavy chain cardiac myosin, signal regulatory protein &#945;, and connexin 43). They also maintained significantly lower immunogenicity, as demonstrated by their lower HLA-A expression and higher HLA-G expression. Applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells clusters within 1 week of co-culture on cardiac feeder layers, becoming the first MSC type to do so. 
Our results demonstrate that this differentiation strategy can effectively harness the cardiomyogenic potential of young MSCs, such as FTM HUCPVCs, and suggests that in vitro pre-differentiation could be a potential strategy to increase their regenerative efficacy in vivo.

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Here, we present a method to efficiently harness the cardiac differentiation potential of young sources of human mesenchymal stem cells in order to generate functional, contracting, cardiomyocyte-like cells in vitro.

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver failure&#8212;which can be caused by chronic alcohol intake, hepatitis, acute poisoning, or other insults&#8212;is a severe condition that can culminate in bleeding, jaundice, coma, and eventually death. However, approaches to treat liver failure, as well as studies of liver function and disease, have been stymied in part by the lack of a plentiful supply of human liver cells. To this end, this protocol details the efficient differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells, guided by a developmental roadmap that describes how liver fate is specified across six consecutive differentiation steps. By manipulating developmental signaling pathways to promote liver differentiation and to explicitly suppress the formation of unwanted cell fates, this method efficiently generates populations of human liver bud progenitors and hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. This is achieved through the temporally-precise control of developmental signaling pathways, exerted by small molecules and growth factors in a serum-free culture medium. Differentiation in this system occurs in monolayers and yields hepatocyte-like cells that express characteristic hepatocyte enzymes and have the ability to engraft a mouse model of chronic liver failure. The ability to efficiently generate large numbers of human liver cells in vitro has ramifications for treatment of liver failure, for drug screening, and for mechanistic studies of liver disease.

This protocol details a monolayer, serum-free method to efficiently generate hepatocyte-like cells from human pluripotent stem cells (hPSCs) in 18 days. This entails six steps as hPSCs sequentially differentiate into intermediate cell-types such as the primitive streak, definitive endoderm, posterior foregut and liver bud progenitors before forming hepatocyte-like cells.