The endometrial layer of the uterus contains a population of cells with similar characteristics of mesenchymal stem cells (MSCs). In the present study, caprine endometrial mesenchymal stromal stem cells (En-MSCs) characters and differentiation potential to chondrogenic, osteogenic, and adipogenic cell lines as well as their growth kinetics in breeding and anestrous stages were evaluated. En-MSCs were enzymatically isolated from endometrial layer of the uterus of adult goats and were cultured and subcultured until passage 4. The growth kinetics and population doubling time (PDT) of caprine En-MSCs in breeding and anestrous stages were determined. En-MSCs in passage 4 were used for the karyotyping and differentiation into chondrocytes, osteocytes, and adipocytes. The PDT in anestrus phase was 40.6 h and in cyclic goats was 53 h. En-MSCs were fibroblast-like in all passages. The number of chromosomes was normal (
Friedenstein et al. [
As a large animal model and similarity of human knee joint, goat is widely investigated for bone tissue engineering [
Information on the goat MSCs characterization is relatively more recent and it is little compared to investigations in commonly used species such as the human and mouse. The bone marrow and adipose tissue are the reported sources of access to MSCs in goats [
Another source of MSCs is endometrial layer of the uterus [
All procedures are in accordance with animal guideline care of Ethical Committee of Shiraz University. All applicable institutional and national guidelines for the care and use of animals were also followed.
Uteri were collected from the healthy mature nonpregnant Fars native nanny goats with 2 to 3 years of age in breeding (February) and anestrous (June) stages from Shiraz Slaughter House in Shiraz, southern Iran. Breeding season of Fars native goats starts from late summer by decrease of daylight to darkness ratio and anestrus stage lasts from April to August when daylight to darkness ratio increases [
After removing of excess fat tissue surrounding the uterus, the uterine surfaces were rinsed well with warmed sterile normal saline. Under laminar flow (Class II, Jal Tajhiz, Iran) in sterile condition, six full-thickness samples of endometrial tissue (
Uteri of goats (a) in breeding stage with corpus luteum (black arrow) and (b) in anestrus stage. The locations of sampling of endometrial tissues are shown by white arrows on body and horn of the uterus.
The isolated cells were transferred to DMEM/F12 medium containing 10% FBS and incubated at 37°C and 5% CO2 and non-MSCs in primary culture were suspended on the medium and, by changing medium, they were removed. En-MSCs were attached to the bottom of the T25 culture flasks. Changing medium was done every three days and the cells after reaching 70–80% confluency were isolated by trypsin/EDTA (0.5%, Sigma, USA) and passaged. The cells were cultured for 4 passages.
In order to have a cell count, the cells were mixed with the equal volume of cell suspension and trypan blue and were stained, while the cells were later counted by hemocytometer. By seeding the cells, growth curve and population doubling time (PDT) were provided and calculated as previously described [
After counting the cells in each passage and recording them, the cells were divided into two parts, one part for transferring into the next passage and the other for freezing to evaluate their survival after thawing. In order to freeze the cells, the freezing medium containing 50% FBS, 40% DMEM, and 10% dimethyl sulphoxide (DMSO, MP Bio, USA) was used. The cells suspended in freezing medium were transferred to a freezer (−20°C) immediately for 1 h and then to another freezer (−70°C) for 24 h and finally into liquid nitrogen tank (Atocel, Austria) for long-term storage. To evaluate their survival after thawing, 10 days later, the cryotubes were removed from the liquid nitrogen and warmed in a 37°C water bath rapidly and were immediately transferred into a DMEM culture medium. They were centrifuged at 1200 rpm for 7 min and the cell pellet was suspended into DMEM culture medium. After thawing, the viability was evaluated by the trypan blue exclusion test (0.4% trypan blue in phosphate buffered saline, PBS). Cell viability was calculated as the number of viable cells divided by the total number of cells and expressed as a percentage.
Karyotype of adult goat cells from passage 4 was prepared. For this purpose, the cells were incubated for 2 h and 30 min in the vicinity colcemid. KCL (0.075 M) was used as a hypotonic solution. The cells were stabilized by methanol and acetic acid at a ratio of 3 : 1 and 2 : 1 and Giemsa staining was used to stained chromosomes. The internationally accepted cytogenetic system of nomenclature (ISCN: International System of Cytogenetic Nomenclature) is used for classification of the chromosomes.
A sample of cells from passage 4 was used. The 9-well plate was used where 6 wells were considered to differentiate into chondrocytes and 3 wells as control. The wells of the control group received just DMEM/F12 medium and were cultured. But, in the wells of differentiation group, chondrocytes differentiation medium was added. Briefly, 5
To evaluate the osteogenic differentiation, En-MSCs were seeded into a 9-well plate. At 80% confluency, the cells were cultured for 21 days with low glucose DMEM containing 100 nM dexamethasone (Sigma, USA), 0.05l M ascorbate-2-phosphate (Wako Chemicals, USA), 10 mM b-glycerophosphate (Sigma, USA), 1% penicillin/streptomycin, and 10% FBS. The medium was replaced every other day. After 21 days, osteogenic differentiation was determined using Alizarin red staining (Sigma, USA).
For adipogenic differentiation, En-MSCs were seeded in a 9-well plate. When they reached 80% confluency, they were induced to adipogenic differentiation with adipogenic induction medium containing DMEM low glucose, 10% FBS, 0.5 mM isobutyl-methylxanthine (Sigma-Aldrich), 10% FBS, 0.5 mM isobutyl-methylxanthine (Sigma-Aldrich), 1
The mean and SE of counted cells in growth curve analysis were compared using independent sample
The caprine endometrium differed from human in that it had glandular intercaruncles and aglandular caruncles. To evaluate anestrus and breeding uteri, cells were isolated from the whole caprine endometrium including both caruncle and intercaruncle areas.
During the primary cell culture, cells were observed with inverted microscope and after 24 h, the number of cells attached to the bottom of the flasks, and their shape was fibroblast-like and several colonies were found at the bottom of culture flasks. Two groups of colonized cells with different morphology were visible in primary culture. Some colonies were compact and round which were similar to endometrial epithelial cells colony and the others were stromal cells. After four days of culturing cells, it was found that the cells grew and the culture flask was full of the single layer of cells. After the first passage, most of epithelial cells were not detached from the primary culture flasks and the adhesion cells in subcultured flasks had formed the population of uniform fibroblast-like cells (Figure
Fibroblast-like morphology of caprine endometrial mesenchymal stromal stem cells in (a) anestrous and (b) breeding stages (magnification:
In anestrous goats, when 2.2
Comparison of growth curves of goat endometrial mesenchymal stromal stem cells after seeding of 2.2
Fifty metaphase cells from passage 4 from the cyclic adult goat sample were analyzed. The number of metaphase chromosomes in all cells was 60 normal acrocentric chromosomes (Figure
Karyotype of endometrial mesenchymal stromal stem cells in passage 4 of cyclic goats.
After 24 hours, before Alcian blue staining, undifferentiated cells in observed wells were round and dense concentric circular colonies were observed. After staining, extracellular matrix was taken to dark blue stain where the so-called Alcian blue stain had metachromatic appearance to extracellular matrix (Figure
Differentiation of endometrial mesenchymal stromal stem cells (En-MSCs) into (a) chondrocytes stained with Alcian blue shows methachromatic appearance (magnification:
En-MSCs also successfully differentiated into adipocytes and osteoblasts lineages (Figures
Due to the diverse and inclusive use of MSCs in cell therapy, investigations focused on the isolation of these cells from different sources and on the characteristics of them [
The importance of determination of self-renewal and growth behaviors of the caprine En-MSCs is on application of these cells in cell therapy which needs appropriate number of these cells after three or four passages. Our findings showed, in anestrus stage, a higher number of cells at the log phase and with a shorter PDT grown in comparison with breeding stage. It is shown that En-MSCs as stromal cells require several growth factors for colony-forming cells/units activity including epidermal growth factor (EGF), platelet-derived growth factor BB (PDGF-BB), transforming growth factor
After cell expansion, En-MSCs fibroblastic morphology is the most important qualitative parameter evaluated by light microscopy in a regular pattern. In this study, 24 h after the primary culture, some cells were attached to the flasks and morphology of cells was changed to fibroblast-like shape. After four passages, they also kept this fibroblast-like morphology. En-MSCs had high proliferative properties and colony forming unit (CFU) activity. MSCs are defined as plastic adherent cells [
Isolated En-MSCs of the goat endometrium were differentiated to chondrocytes, osteocytes, and adipocytes, and this differentiation was confirmed by staining with the Alcian blue, Alizarin red, and Oil Red O, respectively. This research result was similar to the research conducted by Wolff et al. [
In this study, result of En-MSCs karyotype showed that number of chromosomes was
In conclusion, the present study showed that caprine En-MSCs were morphologically similar to MSCs while their differentiation potential was also confirmed. The PDT of these cells revealed that anestrus goats could yield more cells in a shorter period of time. Therefore, according to normal karyotype of these cells and their multilineage differentiation potential properties, En-MSCs can open a new window in stem cell therapies and as a readily available source in regenerative medicine investigations on animal models.
The authors declare that no conflict of interests exists.
The authors would like to appreciate the kind financial support of Shiraz University and the Stem Cell and Transgenic Technology Research Center, Shiraz University of Medical Sciences, for laboratory cooperation. This study was funded by a DVM thesis grant from the Shiraz University. This study was funded by Shiraz University, Shiraz, Iran (Grant no. tez1440; date: December 15, 2014).