Derivation and Differentiation of Canine Ovarian Mesenchymal Stem Cells

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 all

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

1. Prepare or purchase 500 mL of sterile Dulbecco's phosphate-buffered saline (DPBS) without calcium or magnesium.
2. 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.
3. Prepare the culture media from Dulbecco....

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

List of materials used in this article

Name	Company	Catalog Number	Comments
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