Sanambar Sadighi , Ahad Khoshzban , Amir Hossein Tavakoli , Ramin Khatib Semnani, Zahra Sobhani , Nayer Dadashpur Majidabad,
Volume 72, Issue 1 (4-2014)
Abstract
Background: Currently, autologous and allogeneic adipose tissues represent a ubiqui-tous source of material for fat reconstructive therapies. However, these approaches are limited, and often accompanied by a 40-60% reduction in graft volume following transplantation, limited proliferative capacity of mature adipocytes for ex vivo expansion, and extensive adipocyte damage encountered when harvested with conventional liposuction techniques. Recently, cell-based approaches utilizing adipogenic progenitor cells for fat tissue engineering have been developed and were reported to promote both short-term in vivo adipogenesis and to repair defect sites. The aim of this study was to isolate stem cells from fat tissue than examine the growth of stem cells by invitro tests.
Methods: For human adipose stem cell isolation (hASC), subcutaneous adipose tissue sites were obtained from female subjects undergoing elective procedures. Tissues were washed 3-4 times in phosphate buffered saline (PBS) and suspended in an equal volume of PBS supplemented with 1% FCS and 0.1% collagenase type I. The tissue was placed in an agitated water bath at 37 1C. The supernatant containing mature adipocytes, was aspirated. Portions of the SVF were suspended in DMEM medium. hASCs were selected based on their ability to adhere to tissue culture plastic and subsequently expanded to 75-90% confluence. Adipose stem cells were isolated and cultured on DMEM. To assess mesenchymal origin of stem cells we used flow-cytomery technique as well as differentiation to osteocyte and chondrocyte lines.
Results: The nature of the mesenchymal cells was confirmed by flow -cytometry tech-niques, based on the expression of CD90, CD105, CD166, and lack of expression of hematopoietic markers of CD34, CD31, and CD45. The successful differentiation of our stem cells to osteocyte, chondrocyte had been showed by specific Alizarin-Red and Toluidine-blue staining of cells.
Conclusion: Although we have not the results of in vivo tests to support in vivo adipo-genesis either alone or in combination with natural or synthetic matrix, the results showed that stem cells isolation from adipose tissue was successful, and we provided an environment for differentiation of stem cells.
Mohammad Miryounesi , Zeinab Jamali , Masoumeh Razipour , Elahe Alavinejad , Mohammad Hossein Modarressi ,
Volume 72, Issue 11 (2-2015)
Abstract
Background: About 15% of couples have fertility problems and male factor in fertility accounts for half of the cases. In vitro generation of germ cells introduces a novel approach to male infertility and provides an effective system in gene tracking studies, however many aspects of this process have remained unclear. We aimed to promote mouse embryonic stem cells (mESCs) differentiation into germ cells and evaluate its effectiveness with tracking the expression of the Testis specific 10 (Tsga10) during this process.
Methods: This is an in vitro study that was performed in department of Medical Genetics in Tehran University of Medical Sciences from February 2012 to March 2013. Mouse embryonic stem cells were cultured on mouse embryonic fibroblast as feeder layer. Then mESCs were differentiated into germ cells in the presence of Retinoic Acid. Based on developmental schedule of the postnatal testis, samples were taken on the 7th, 12th and 25th days of the culture and were subjected to expression analysis of a panel of germ cell specific genes (Stra8 as pre-meiotic, Dazl and Sycp3 as meiotic and Protamin1 and Spata19 as Post-meiotic). Expression of Testis Specific Gene 10 (Tsga10) at RNA and protein levels was then analyzed.
Results: It was shown that transition of embryonic stem cells from mitosis to meiosis occurred between 7th and 12th days of mESC culture and post-meiotic gene expression did not occur until 25th day of the culture. Results showed low level of Tsga10 expression in undifferentiated stem cells. During transition from meiotic to post-meiotic phase, Tsga10 expression increased in 6.6 folds. This finding is in concordance with in vivo changes during transition from pre-pubertal to pubertal stage. Localization of processed and unprocessed form of the related protein was similar to those in vivo as well.
Conclusion: Expression pattern of Tsga10, as a gene with critical function in spermatogenesis, is similar during in vitro and in vivo germ cell generation. The results suggest that in vitro derived germ cells could be a trusted model to study genes behavior during spermatogenesis.
