Meraj Tabatabaei, Amir Hossein Zarnani, Shohreh Nikoo, Bahareh Keshavarzi, Fahimeh Ramzani Tehrani, Soheila Arefi, Ebrahim Mirzadegan, Nariman Mosaffa,
Volume 37, Issue 1 (4-2013)
Abstract
Background: Human amniotic epithelial cells (hAECs) have been introduced as a valuable source of stem cells with therapeutic applications. Despite the extensive study and emerging evidence, cell surface marker signature of hAECs remained controversial. The aim of the present study was to establish an efficient and optimized isolation procedure of hAECs and their characterization in terms of proliferation and stem cell markers.
Materials and methods: Four human placentas were collected from healthy women undergoing elective caesarian delivery at term labor under sterile condition. Amniotic membranes were carefully isolated and subjected to trypsin digestion. Single cell suspensions were obtained and the epithelial origin was confirmed by assessment of cytokeratin expression. Expression of CD9, CD10, CD29, CD34, CD38, CD44, CD45, CD73, CD105, CD133, MHC-I, HLA-DR, HLA-G, SSEA-4, STRO-1 and OCT-4 was then evaluated by flow cytometry.
Results: The overall yield of hAEC ranged from 80-130×106 cells per placenta with more than 98% viability. Microscopic examination revealed that hAECs are large and refractive cells with great capacity to adhere to plastic surfaces. The cells substantially expressed cytokeratin implying their epithelial origin. Flow cytometry data revealed that hAECs are a heterogenic population consisting of immune phenotypically mixed cells. Nearly all purified cells expressed mesenchymal markers, CD9, CD10, CD29, and CD73. A large proportion of cells also expressed HLA-I, HLA-G, STRO-1, OCT-4 and SSEA-4. Isolated cells, however, failed to express CD34, CD38, CD44, CD45, CD105, CD133 and HLA-DR.
Conclusion: The procedure presented here is a simple and cost-effective protocol for isolation of a large numbers of viable hAECs. Surface markers and such features as proliferation capacity of hAECs are among the characteristics that are strongly dependent upon isolation and cell culture conditions. Thus such variables should be taken in to consideration when stem cell properties of hAECs are to be interpreted.
Dr Farnaz Khadivi, Miss Mahya Ramesh, Tayebeh Rastegar, Mrs Maryam Fakhraee Farooji,
Volume 47, Issue 2 (7-2023)
Abstract
Background and Aim: Testicular torsion leads the a disorder of contralateral testicular function in addition to destructive effects on the ipsilateral testicle through the impairment of the blood- testis barrier, release of sperm into the blood, activation of immune responses, and production of anti-sperm antibody. Detorsion surgery also resulted in inflammatory reactions and the production of reactive oxygen species (ROS), which was finally accompanied by the destruction of the contralateral testicular tissue and infertility. The application of mesenchymal stem cells conditioned medium recently has been considered in the treatment of male infertility due to the presence of various growth factors and anti-inflammatory properties. The aim of this study was to evaluate the effect of conditioned medium derived from human amniotic membrane mesenchymal stem cells on the promotion of spermatogenesis and sperm chromatin structure in the contralateral testis following the torsion of the unilateral spermatic cord.
Methods: In this experimental study 40 adult male NMRI mice were randomly divided into 4 groups. Control group without surgical intervention. Left spermatic cord torsion / detorsion group. Negative control group: torsion / detorsion surgery and injection of culture medium into the left testis. Treatment group: torsion / detorsion surgery and injection of conditioned medium derived from human amniotic membrane mesenchymal stem cells into the left testis. The Histomorphometric evaluation of the right testicle was done by preparation of tissue sections and hematoxylin and eosin (H&E) staining. This evaluation included counting different cells of testicular tissue, the number of seminiferous tubules per field, measurement of outer diameter and epithelium thickness of tubules, Johnson's score, and tubules spermatogenic criteria by Image J software. Also, semen samples were prepared from the cauda epididymis and toluidine blue staining was performed for evaluation of sperm chromatin integrity. One- way analysis of variance (ANOVA) and Tukey's test were performed for comparisons between groups, p-value less than 0.05 (P ≤ 0.05) is statistically significant.
Results: The number of spermatogonial cells, primary spermatocyte, spermatid, Sertoli, Leydig, Johnson's score, outer diameter and thickness of seminiferous tubules germinal epithelium, TDI, SPI, MI, and the diameter of the spermatogonial cell nucleus in right testis significantly decreased in the torsion / detorsion group (P < 0.01). A remarkable improvement in the above parameters was observed after treatment with the conditioned medium in comparison with the torsion / detorsion group, although there was a significant difference as compared to the control group (P < 0.01). The percentage of sperms with abnormal chromatin in the spermatic cord torsion / detorsion group increased significantly compared to the control group (19.6 ± 8.54) (P < 0.001). A significant improvement in the sperm chromatin structure was observed compared to the torsion/ detorsion group after treatment (14.4± 3.6) (P< 0.05).
Conclusion: It seems that Conditioned medium derived from human amniotic membrane mesenchymal stem cells leadsto a relative improvement in sperm chromatin and spermatogenic indices of the contralateral testicular tissue after unilateral ischemia / reperfusion injury of the spermatic cord.
