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Summary

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

Abstract

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.

Introduction

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.

Protocol

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 Prepare or purchase 500 mL of sterile Dulbecco's phosphate-buffered saline (DPBS) without calcium or magnesium. Prepare a collagenase I stock solution by mixing 40 µg of the enzyme in 1 mL of DPBS. Filter using a 0.2 µm syringe filter, and store 2 mL aliquots at -20 °C. Prepare the culture media from Dulbecco's modified Eagle's medium (DMEM) low glucose with 10% fetal serum and 1% of antibiotics. Collect sterile materials: scissors and forceps, a tissue culture flask, dishes, tubes, and 10 mL and 5 mL pipettes. Use standard cell culture laboratory equipment: a biological safety cabinet (BSC), a cell culture incubator set at 5% CO2 and 38 °C, and a centrifuge. Clean the BSC: with gloved hands, sterilize it with 70% EtOH, using UV lights. 2. Isolation of Mesenchymal Stem Cells from the Ovary After surgery, keep the ovaries in PBS on ice in a conical tube, until their arrival at the lab. Fill two 100 mm Petri dishes with 10 mL of PBS. Remove the ovaries from the tube and transfer them to the first Petri dish with PBS. Wash them with mixing movements. 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. Transfer the ovarian tissue to a 35 mm Petri dish and add 2 mL of collagenase. Mince the tissue a little more. Place the Petri dish in the incubator at 38 °C for 3 h and gently shake the Petri dish with circular movements every 20 min. After 3 h, remove the Petri dish from the incubator. Transfer the contents of the dish to a 15 mL tube. Add 5 mL of expansion media. 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. Transfer the pellet to a T25 bottle. Place the bottle in the incubator with 5% CO2 at 38 °C for 3 h. NOTE: The most important part of the procedure is to change the media after 3 h of incubation. Remove the bottle from the incubator and remove the media with the remaining tissue. Add 3 mL of fresh expansion media to the bottle. 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. 3. Expansion of Mesenchymal Stem Cells from the Ovary Cell passage When the cells reach confluence, remove the media from the bottle. Wash the bottle with 3 mL of PBS. Remove the PBS. Add 1.5 mL of trypsin and put the bottle in the incubator at 38 °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. Add 3 mL of expansion media and transfer the contents to a conical tube. Centrifuge the tube at 2100 x g for 7 min. Remove the supernatant and add 1 mL of expansion media. Gently homogenize the contents of the tube. Take an aliquot of 10 µL of the sample to perform cell counting and put the tube with the cells back in the incubator. Place 10 µL of the mix into the hemocytometer. Counting the cells Use the microscope's 10X objective and focus on the grid lines of the hemocytometer. Count the cells in five small squares (Figure 2). Multiply the counted number by 50,000 to estimate the number of cells per milliliter. 4. Differentiation of Mesenchymal Stem Cells from the Ovary NOTE: Differentiation assays were performed according to the guidelines established in Hill et al.9. Seed 1.0 x 104 cells per well, in triplicate, using a 4-well culture dish. Add 1 mL of expansion media. Gently agitate the dish with circular movements. After 24 h, replace the culture media with the desirable differentiation media. 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. 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 µM hydrocortisone, 10 µM forskolin, 5 mM potassium chloride, 5 µg/mL insulin, and 200 µM butylated hydroxyanisole. For endoderm precursors, incubate for 5 d in endoderm media as per the reagent manufacturer's instructions. 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 µg/mL insulin, 0.1 mM β-mercaptoethanol, 60 µM putrescine, 20 µg/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.

Representative Results

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

Discussion

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…

Materials

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