fatemeh akyash

There are ethical and technical challenges in studying human germ cell development. Therefore, the aim ofthe study is in vitro differentiation of human embryonic stem cells (hESCs), as pluripotent cells, to the germ cells whichis a valuable tool for studying molecular and cellular aspects of gametogenesis and understanding causes of infertility. In this experimental study, two different complete media [Dulbecco’s Modified Eagle Medium(DMEM)+20% fetal bovine serum (FBS) and embryoid bodies (EBs) medium; KOSR/HES without basic fibroblastgrowth factor (bFGF)] were used in the both of test groups using testicular cells derived conditioned medium (TCCM)and control groups spontaneously differentiated (SD). Thereby, EBs from hESCs (Yazd2; 46XY) were cultured indifferent conditions EB medium; EB medium and conditioned EB medium; EB medium, DMEM, and FBS withoutconditioning; EB medium, conditioned DMEM, and FBS medium. EBs were collected after 4, 7, and 14 days and theirgene expression profiles were assessed and compared to hESCs, as day 0, using IF and relative reverse transcriptionquantitative polymerase chain reaction (RT-qPCR). An increase in the gametogenesis gene expression level in TCCM groups was showed in comparison withSD groups. Additionally, immunostaining of differentiated cells in all groups showed in vitro gametogenesis (IVG). Our findings showed that human TCCM could be used as a natural niche for in vitro male and femalegerm cell development. However, further studies are needed to define the factors and metabolites within the humanTCCM.

Xeno-free generation of human embryonic stem cells (hESCs) is important to prevent potential animal contaminations in culture for advanced cell-based therapeutic applications. Xeno-free production of hESCs is the first step for manufacturing clinical-grade hESC lines.

To produce new hESC lines in xeno-free condition.

This lab resources report was conducted at Stem Cell Biology Research Center, Yazd, Iran from 2019-2022. 4 new hESC lines from 11 (10 fresh and 1 frozen) donated surplus discarded human embryos were established. In this study, we report the xeno-free derivation of new Yazd hESC lines (Yazd4-7), without using immunosurgery, by culturing intact zona-free blastocysts obtained from discarded embryos onto the YhFF#8 cells as a feeder layer in a microdrop culture system. The pluripotency gene expression profile of the cell lines was assessed by reverse transcription polymerase chain reaction and the expression of specific surface markers was detected using immunofluorescent staining. In vitro differentiation was induced using embryoid body formation and gene expression profile of 3 germ layers and germ cells. Reverse transcriptase polymerase chain reaction was investigated to prove their pluripotent capacity.

In sum, we have been able to generate 4 new hESC lines (Yazd4-7) from 11 discarded embryos in xeno-free culture conditions using a micro drop culture system and YhFF#8 as a human source feeder layer.

The outcome of this work can be the foundation for the future allogeneic cell-based therapeutic application using clinical grade good manufacturing practice-derived hESC derivatives.

It has been proven that human mesenchymal stem cells (MSCs) conditioned medium (hMSCs-CM) can influence human embryonic stem cells (hESCs) chondrogenic differentiation. In this study, we hypothesized that conditioned medium (CM) from hESCs-derived MSCs in a sequential 3D-2D culture system could facilitate the induction of chondrogenesis in hESCs.

CM was collected from Yazd2 (hESCs; 46, XY) derived MSCs confluent cultures and stored at -20 °C. Yazd4 hESC line (46, XX) was induced for differentiation using EB formation as 3D culture into SD (spontaneously differentiation) and CM groups (differentiation using conditioned medium) for four days. Cell culture continued in a 2D (monolayer) culture system for both groups till day 14. Ultimately, chondrogenic differentiation was assessed by Alcian blue and masson′s trichrome staining at 4 and 14 days of differentiation, and quantitative real-time polymerase chain reaction (PCR) for NANOG, MEOX1, SOX5, SOX6, SOX9, ACAN, COL2A1 and RUNX2 genes for SD and CM groups on days 0 and 4.

  The gene expression profile for chondrogenic genes in the CM group was significantly more than the SD group (p< 0.05). Furthermore, chemical staining assessment illustrated a significant GAG and collagen II difference between the CM and SD groups at days 4 and 14 (p< 0.05).

Our findings would pave the way for creating an in vitro human chondrogenesis model for further studies in the developmental biology of articular cartilage tissue, which lend itself to cell-based therapy to cure joint diseases such as osteoarthritis.

Synchronization between the embryonic stage and the uterine endometrial lining is important in the outcomes of the vitrified-warmed embryo transfer (ET) cycles.

The aim was to investigate the effect of the exact synchronization between the cleavage stage of embryos and the duration of progesterone administration on the improvement of clinical outcomes in frozen embryo transfer (FET) cycles.

312 FET cycles were categorized into two groups: (A) day-3 ET after three days of progesterone administration (n = 177) and (B) day-2 or -4 ET after three days of progesterone administration (n = 135). Group B was further divided into two subgroups: B1: day-2 ET cycles, that the stage of embryos were less than the administrated progesterone and B2: day-4 ET cycles, that the stage of embryos were more than the administrated progesterone. The clinical outcome measures were compared between the groups.

The pregnancy outcomes between groups A and B showed a significant differences in the chemical (40.1% vs 27.4%; p = 0.010) and clinical pregnancies (32.8% vs 22.2%; p = 0.040), respectively. The rate of miscarriage tended to be higher and live birth rate tended to be lower in group B than in group A. Also, significantly higher rates were noted in chemical pregnancy, clinical pregnancy, and live birth in group A when compared with subgroup B2.

Higher rates of pregnancy and live birth were achieved in day-3 ET after three days of progesterone administration in FET cycles.

Human embryonic stem cell-mesenchymal stem/stromal cell (hESCs-MSCs) open a new insight into future cell therapy applications, due to their unique characteristics, including immunomodulatory features, proliferation, and differentiation.

Herein, hESCs-MSCs were characterized by immunofluorescence technique with CD105 and FIBRONECTIN as markers and FIBRONECTIN, VIMENTIN, CD10, CD105, and CD14 genes using reverse transcription-polymerase chain reaction technique. Fluorescence-activated cell sorting was performed for CD44, CD73, CD90, and CD105 markers. Moreover, these fibroblast-like cells, due to multipotent characteristics, differentiated to the osteoblast.

MSCs were derived from diploid and triploid hESC lines using sequential three dimensional and two dimensional cultures and characterized with the specific markers. Immunofluorescence showed the expression of FIBRONECTIN and CD105 in hESCs-MSCs. Flow cytometry data indicated no significant difference in the expression of MSC markers after 6 and 13 passages. Interestingly, gene expression profiles revealed slight differences between MSCs from diploid and triploid hESCs. hESCs-MSCs displayed osteogenic differentiation capacity, which was confirmed by Alizarin red staining.

Our findings reveal that both diploid and triploid hESC lines are capable of forming MSCs; however, there are some differences in their gene expression profiles. Generation of MSCs from hESCs, as a non-invasive procedure in large scale, will lend itself for the future cell-based therapeutic applications.

Human endometrium with consecutive regeneration capability undergoes monthly hormonal changes for probable implantation, which confirms the presence of the cells in the basalis layer known as stem cell.

Previously, we reported the isolation and culture of the mesenchymal-like cells from human endometrium. In this study, we evaluated the biological and stemness characteristics of these cells.

The characterization of Yazd human endometrial-derived mesenchymal stem/stromal cells (YhEnMSCs) was assessed using immunofluorescence (IF) staining for CD105, VIMENTIN, and FIBRONECTIN as markers and RT-PCR for CD166, CD10, CD105, VIMENTIN, FIBRONECTIN, MHCI, CD14, and MHCII genes. Flow cytometry (FACS) was performed for CD44, CD73, CD90, and CD105 markers. Moreover, the differentiation capacity of the YhEnMSCs to the osteoblast and adipocytes was confirmed by Alizarin Red and Oil Red staining.

YhEnMSCs expressed CD105, VIMENTIN, FIBRONECTIN, CD44, CD73, and CD90 markers and CD166, CD10, CD105, VIMENTIN, FIBRONECTIN, and MHCI, but, did not express CD14, MHCII.

Our data confirm previous reports by other groups indicating the application of endometrial cells as an available source of MSCs with self-renewal and differentiation capacity. Accordingly, YhEnMSCs can be used as a suitable source for cell-based therapies.

Testicular cell conditioned medium (TCCM) has been shown to induce female germ cell developmentin vitro from embryonic stem cells (ESCs). Testicular cells (TCs) secrete a variety of growth factors such as growthdifferentiation factor-9 (GDF-9), bone morphogenetic protein 4 (BMP-4), stem cell factor (SCF), leukemia inhibitoryfactor (LIF), and other, that could improve oocyte maturation. Here we have investigated the effect of human TCCM(hTCCM) on in vitro maturation (IVM) and morphology of mouse oocytes.

In this experimental study, 360 germinal vesicle (GV) oocytes were obtained from NMRImice, aged 4-6 weeks that had received 5 IU pregnant mare's serum gonadotropin (PMSG) 48 hours before. GVoocytes were subjected to IVM. 120 GV oocytes were cultured in each medium; hTCCM as the test group, DMEM+ 20%FBS as the control group and Ham’s F10 + HFF medium as the sham group. The rates of the IVM and perivitellinespace (PVS) changes were recorded at 8, 16 and 24 hours after culture. The metaphase II (MII) oocytes weresubjected for in vitro fertilization (IVF) and the fertilization rate was evaluated after 1, 2, and 3 days.

There was a significant difference between the maturation rates in hTCCM (31.67% MII) and the control [0% MII,p <0.05, (7.5% MI, 52.5% deg. and 40%GV)] groups but there was not a significant difference between the maturation ratesin hTCCM and the sham group (53.33% MII, P>0.05). IVF success rate for MII oocytes obtained from IVM in the hTCCMgroup was 28.94% (n=11). Our data showed that hTCCM is an effective medium for GV oocyte growth and maturationcompared to the control medium.

Our findings show that TCCM supports oocyte IVM in mice and affect oocyte morphology.

Previous studies have shown that adipose-derived mesenchymal stem/ stromal cells are one of the sources of mesenchymal stem cells (MSCs) with the capacity to differentiate into various mesodermal cell lineages. MSCs with cytokines secretion capability, which contributes to repair damaged tissues have gained wide credence for future cell-based therapeutic applications. In this study, the effect of the different dosages of vitamin E and Selenium was assessed on the stemness of the human adipose tissue-derived MSCs (AD-MSCs).

Following 24 hours of cell treatments with different dosages of vitamin E and Selenium, MTT assay was used to assess the effect of them on cell proliferation. Moreover, the stemness of the AD-MSCs was assessed using osteogenic and adipogenic induction medium supplemented with the different dosages of the vitamin E and Selenium. Finally, Alizarin red and Oil-red O staining were performed to detect matrix mineralization and lipid droplet accumulation, respectively.

MTT data revealed that the optimal concentration for vitamin E and Selenium were 125 µM and 121 µM for the viability of the AD-MSCs. Moreover, the effect of vitamin E and Selenium were assessed by osteogenic and adipogenic differentiation by optimal dosages obtained by MTT assay, respectively. Maximum mineralization and lipid droplet aggregation of the differentiated cells were detected at IC50 in comparison with the control group.

These results suggest that different dosages of vitamin E and Selenium could have various impacts on the proliferation and differentiation induction of human AD-MSCs.

Cell banking of initial outgrowths from newly derived human embryonic stem cells (hESCs) requires an efficient freezing method. Vitrification is used for the preservation of gametes and early embryos in assisted reproduction techniques (ART). Moreover, vitrification was applied for cryopreservation of hESCs using open pulled straws.

To derive and characterize new hESC lines and then use Cryotech and Cryowin tools for their vitrification.

Human ESC lines were generated in a microdrop culture system using mouse embryonic fibroblasts (MEFs) as the feeder layer; this was later scaled up using both MEFs and Yazd human foreskin fibroblasts batch 8 (YhFF#8). To bank the cell lines, master cell banks of 100 Cryotech and Cryowin tools were produced for each individual cell line using the vitrification method; flasks of hESC lines were also cryopreserved using a conventional slow-freezing method.

The pluripotency of cell lines was assessed by their expression of pluripotency-associated genes (OCT4/POU5F1, NANOG, and SOX2) and markers such as SSEA4, TRA-1-60, and TRA-2-49. Their in vitro capacity to differentiate into germ layers and germ cells using embryoid body (EB) formation and monolayer culture was assessed by screening the expression of differentiation-associated genes. The chromosomal constitution of each hESC line was assessed by G-banding karyotyping.

Cryotech and Cryowin tools used to vitrify new hESCs at an early stage of derivation is an efficient method of preserving hESCs.

 Monthly regeneration of endometrium after cyclical mensturation confirmed the ability of specific population of the cells that presence in the basalis layer and undergone consecutive hormonal changes that could prepared the endometrial layer for probable implantation. These cells, known as, stem cell. The aim of this study was the isolation and culture of human endometrial derived mesenchymal stem/stromal cells (EnMSCs).

.In this study, human endometrial tissues were collected after fully-informed patient consent, which attended the Research and Clinical Center for Infertility. After washing and enzymatic treatment, EnMSCs were isolated and cultured in vitro.

Cells from endometrium were successfully isolated using enzymatically treatment and mechanically dissociation, then cultured and expanded for several passages for further characterization and evaluations. Endometrium derived cells were morphologically similar to mesenchymal stem/stromal cells (MSCs).

The results of the present study confirm previous reports in vitro studies for the isolation and culture of human EnMSCs. Endometrial tissue is a part of uterus with available source of MSCs with self-renewal and differentiation capacity that undergoes a cyclical regeneration every month in normal women’s life span. In this regard, human EnMSCs could be used for future novel therapeutic methods in regenerative medicine for treatment of uterine-factor infertile patients, which can lead to recurrent pregnancy loss (RPL) and finally resolve of surrogacy problems.

Sperm is particularly susceptible to reactive oxygen species (ROS) during critical phases of spermiogenesis. However, the level of seminal ROS is restricted by seminal antioxidants which have beneficial effects on sperm parameters and developmental potentials. Mitochondria and sperm plasma membrane are two major sites of ROS generation in sperm cells. Besides, leukocytes including polymer phonuclear (PMN) leukocytes and macrophages produce broad category of molecules including oxygen free radicals, non-radical species and reactive nitrogen species. Physiological role of ROS increase the intracellular cAMP which then activate protein kinase in male reproductive system. This indicates that spermatozoa need small amounts of ROS to acquire the ability of nuclear maturation regulation and condensation to fertilize the oocyte. There is a long list of intrinsic and extrinsic factors which can induce oxidative stress to interact with lipids, proteins and DNA molecules. As a result, we have lipid peroxidation, DNA fragmentation, axonemal damage, denaturation of the enzymes, over generation of superoxide in the mitochondria, lower antioxidant activity and finally abnormal spermatogenesis. If oxidative stress is considered as one of the main cause of DNA damage in the germ cells, then there should be good reason for antioxidant therapy in these conditions.