Qom University of Medical Sciences Journal

Introduction
The ovary is an endocrine organ that need granulosa and theca cells for secretions. In the ovary tissue, in addition to follicles, there are stem cells. These stem cells can be located in the cortex, epithelium, and the area between the cortex and medulla of the ovary. For many years, researchers believed that the number of primordial follicles in the ovary is constant after birth. However, new studies have shown that ovarian stem cells (OSCs) have the ability to differentiate into oocyte-like cells. In addition, OSCs are able to differentiate into theca cells. There are stem cells located in different tissues of the body after birth. These cells are multipotent type and are called mesenchymal stem cells. These cells can be extracted from a variety of tissues such as bone marrow and adipose tissue. Mesenchymal stem cells also express some markers of stem cells, such as Sox2, Pou5f, and Nanog. These fibroblast-like cells are non-hematopoietic and undifferentiated with adhesive property. They can proliferate extensively and can differentiate into several types of mesodermal and non-mesodermal cells, such as bone cells and adipocytes. In this study, we aim to investigate the characteristics of OSCs in rats in terms of the expression of some stem cell markers and their ability to differentiate into osteocyte-like and adipocyte-like in vitro.
Methods
First, five newborn Syrian rats (DBA2) were euthanized by cervical dislocation method. Then, their ovaries (n=10) were removed and transferred to the culture dish under the loop. After slicing the ovarian tissue, their remnants were placed in collagenase and incubated for 15 minutes. Then, it was neutralized with serum, and the contents were passed through a 70-mesh filter. Afterwards, the cells were washed twice with phosphate buffered saline (PBS). The sediment of the cells was transferred into a 25-cm flask under the cell culture hood and cultured in DMEM/F12 culture medium containing 10% FBS. Then, they were kept in the incubator. After 24h, the medium was changed. After 3-4 days, the cells filled the bottom of the flasks due to multiplication. At this time, the first passaging was done for cell cultivation. In this method, the cells were separated from the bottom of the flask using the trypsin/EDTA solution. The cells were separated from each other by pipetting, and then were centrifuged. After centrifugation, the media of the Falcon tube were drained. Afterward, some culture medium was added to the tube, and pipetting was performed again. The contents of the falcon tube were again poured into three culture flasks and placed inside the incubator at 37°C. After 3 passages, completely uniform cells with a completely similar appearance remained in the bottom of the flask, which was ready for examination. A part of the cells was used for molecular study, and the other part was used for the differentiation process. For the molecular study, after RNA extraction and cDNA synthesis, the expression level of genes related to OSCs, including Sox2, Oct4, and Nanog, was measured by the real-time PCR method. Beta-tubulin gene was used as the reference gene. Amplified fragments from each sample were analyzed by 1% agarose gel electrophoresis. After the third passage, some of the stem cells were centrifuged and then cultured in the prepared stem cell culture differentiation media specifically for bone and fat cells for 21 days. The differentiation potential of these cells into osteocyte-like and adipocyte-like cells was investigated by Alizarin red and Oil red staining, respectively after 21 days.
Results
When the stem cells were placed on the plate, they were round and did not adhere to the bottom of the plate. After 8 hours, these cells were gradually adhered to the bottom of the plate, and 24 hours later, they started to form colonies in the initial stage of culture. These colonies consisted of flattened, spindle-shape, round, and long cells. After the third passage, these cells became uniform; almost all of them were adhered to the bottom of the plate and were spindle-shaped and elongated. These cells had an appearance similar to fibroblastic cells. Genes related to transcription factors Sox2, Oct4, and Nanog were expressed in these stem cells extracted from the ovary using the PCR method. Following the differentiation of theca stem cells into adipocyte-like cells, fat droplets were seen in the differentiated cells. These droplets were stained with oil red and shown in red particles. After the differentiation of theca stem cells into osteocyte-like cells, a calcareous matrix was formed, which turned into red after staining by alizarin red.
Discussion
The OSCs include multipotent stem cells that can express the markers of stem cells and differentiate into osteocyte-like and adipocyte-like cells. According to the characteristics of OSCs, these cells can be used in cell therapy and regenerative medicine.


Ethical Considerations
Compliance with ethical guidelines
The Ethics Committee of Islamic Azad University of Tehran Medical Sciences Branch, approved the study (IR.IAU.TMU.REC.1401.172).
Funding
This study was conducted through self-funding the author.
Conflicts of interest
The authors declared no conflict of interest.
Acknowledgements
The author would like to thank Dr. Mehdi Alikhani.