Isolation and characterization of a novel single-chain variable fragment (scFv) against Lymphocyte function-associated antigen-1 (LFA-1) using phage display method.

Lymphocyte function-associated antigene-1 (LFA-1) is a well-described integrin found on lymphocytes and other leukocytes, which is known to be overexpressed in leukemias and lymphomas. This receptor plays a significant role in immune responses such as T-cell activation, leukocyte cell-cell interactions, and trafficking of leukocyte populations. Subsequently, binders of LFA-1 emerge as potential candidates for cancer and autoimmune therapy. This study used the phage display technique to construct and characterize a high-affinity single-chain fragment variable (scFv) antibody against LFA-1. After expression, purification, dialysis, and concentration of the recombinant LFA-1 protein, four female BALB/c mice were immunized, splenocyte's mRNA was extracted, and cDNA was synthesized. A scFv library was constructed by linking the amplified VH/Vκ fragments through a 72-bp linker using SOEing PCR. Next, the scFv gene fragments were cloned into the pComb-3XSS phagemid vector; thus, the phage library was developed. The selection process involved three rounds of phage-bio-panning, polyclonal, and monoclonal phage ELISA. AF17 was chosen and characterized among the positive clones through SDS-PAGE, Western blotting, indirect ELISA, and in-silico analyses. The results of the study showed the successful construction of a high-affinity scFv library against LFA-1. The accuracy of the AF17 production and its ability to bind to the LFA-1 were confirmed through SDS-PAGE, Western blot, and ELISA. This study highlights the potential application of the high-affinity AF17 against LFA-1 for targeting T lymphocytes for therapeutic purposes.

An enzymatic nucleic acid vertical flow assay.

Vertical flow assays have been developed in recent years addressing limitations of the lateral flow assays, including limited multiplexing capability, quantitation, and hook effect. In the present study, the first passive paper-based vertical flow assay is developed for the detection of the nucleic acid target. Horseradish peroxidase was used as an enzymatic tracer with a high potential for signal amplification. In order to achieve the best signal-to-noise ratio, different parameters of paper-based assays were optimized. The sample is heat denatured and hybridized with a specific probe to form a dual-labeled hybridization complex. A small volume of diluted sample, 12 µl, can be analyzed within 6 min on the assay in a sandwich format. Assay specificity was evaluated by testing different unrelated samples, and also, 1.7 nM was obtained as the limit of detection (LOD) using the 0 + 3SD method, which is equivalent to 8.5 fmol of double-stranded DNA in the 12 µl sample volume. The linear range of 3-194 nM with a 0.978 correlation coefficient was obtained according to the calibration curve. The developed assay was evaluated with 45 hepatitis B virus clinical plasma samples, and the result showed 100% consistency of the assay with the real-time PCR benchmark. In the present study, we sought to develop a mere detection system for nucleic acid targets, and to investigate the possibility of using enzyme reporter in a passive vertical flow assay.

Role of Endocytosis Pathways in Electropermeablization of MCF7 Cells Using Low Voltage and High Frequency Electrochemotherapy.

OBJECTIVE: The cell membrane is a major barrier for delivery of hydrophilic drugs and molecules into the cells. Although low voltage and high frequency electric fields (LVHF) are proposed to overcome the cell membrane barrier, the mechanism of membrane permeabilization is unclear. The aim of study is to investigate endocytosis pathways as a possible mechanism for enhancing uptake of bleomycin by LVHF. MATERIALS AND METHODS: In this experimental study, MCF-7 cells were exposed to bleomycin or to electric fields with various strengths (10-80 V/cm), frequency of 5 kHz, 4000 electric pulse and 100 µs duration in the presence and absence of three endocytosis inhibitors-chlorpromazine (Cpz), amiloride (Amilo) and genistein (Geni). We determined the efficiency of these chemotherapeutic agents in each group. RESULTS: LVHF, depending on the intensity, induced different endocytosis pathways. Electric field strengths of 10 and 20 V/cm stimulated the macropinocytosis route. Clathrin-mediated endocytosis was observed at electric field intensities of 10, 30, 60 and 70 V/cm, whereas induction of caveolae-mediated endocytosis was observed only at the lowest electric field intensity (10 V/cm). CONCLUSION: The results of this study imply that LVHF can induce different endocytosis pathways in MCF-7 cells, which leads to an increase in bleomycin uptake.

Endocytosis induction by high-pulsed magnetic fields to overcome cell membrane barrier and improve chemotherapy efficiency.

Cell membrane acts as a barrier to the entry of impermeable drugs into cells. Recent studies have suggested that using magnetic fields can enable molecules to overcome the cell membrane barrier. However, the mechanism of membrane permeabilization remains unclear. Therefore, we evaluated the increases in bleomycin (CT) uptake, a non-permanent chemotherapy agent, using high-pulsed magnetic fields and investigated whether endocytosis was involved in the process. This study exposed MCF-7 cells to magnetic fields (2.2 T strength, different number of 28 and 56 pulses, and frequency of 1 and 10 Hz) in order to investigate whether this approach could promote the cell-killing efficiency of bleomycin. The involvement of endocytosis as a possible mechanism was tested by exposing cells to three endocytosis inhibitors, namely chlorpromazine, genistein, and amiloride. Our results illustrated that magnetic fields, depending on their conditions, could induce different endocytosis pathways. In such conditions as 10 Hz-28 pulses, 10 Hz-56 pulses, and 1 Hz-56 pulse, clathrin-mediated endocytosis was observed. Moreover, macropinocytosis was induced by the 10 Hz magnetic field and caveolae-mediated endocytosis occurred in all the magnetic field conditions. The findings imply that high-pulsed magnetic fields generate different endocytosis pathways in the MCF-7 cells, thus increasing the efficiency of chemotherapy agents.

Designer Exosomes: A New Platform for Biotechnology Therapeutics.

Desirable features of exosomes have made them a suitable manipulative platform for biomedical applications, including targeted drug delivery, gene therapy, cancer diagnosis and therapy, development of vaccines, and tissue regeneration. Although natural exosomes have various potentials, their clinical application is associated with some inherent limitations. Recently, these limitations inspired various attempts to engineer exosomes and develop designer exosomes. Mostly, designer exosomes are being developed to overcome the natural limitations of exosomes for targeted delivery of drugs and functional molecules to wounds, neurons, and the cardiovascular system for healing of damage. In this review, we summarize the possible improvements of natural exosomes by means of two main approaches: parental cell-based or pre-isolation exosome engineering and direct or post-isolation exosome engineering. Parental cell-based engineering methods use genetic engineering for loading of therapeutic molecules into the lumen or displaying them on the surface of exosomes. On the other hand, the post-isolation exosome engineering approach uses several chemical and mechanical methods including click chemistry, cloaking, bio-conjugation, sonication, extrusion, and electroporation. This review focuses on the latest research, mostly aimed at the development of designer exosomes using parental cell-based engineering and their application in cancer treatment and regenerative medicine.

Metastasis inhibition by BRMS1 and miR-31 replacement therapy in claudin-low cell lines.

OBJECTIVES: The growing trend of research demonstrates that dynamic expression of two metastasis repressor classes (metastasis suppressor genes and anti-metastatic miRNA) has a close relationship with tumor invasion and metastasis. Using different strategies, it was revealed that cellular levels of miR-31 and Breast cancer Metastasis Suppressor1 (BRMS1) protein, which are among the most significant modulators of metastasis, have a correlation with the cell's capability for invading and metastasizing; cells containing higher levels of miR-31 or BRMS1 were less metastatic. This project was carried out to determine whether the combinations of miR-31 and BRMS1 genes are able to enhance the capability of repressing the claudin-low breast cancer cell (MDA-MB-231) invasion. MATERIALS AND METHODS: This study used a restoration-based approach by miR-31 mimic and optimized BRMS1 gene sequences, which were cloned into a chimeric construct and transfected to the MDA-M231cells. RESULTS: Our data revealed that the simultaneous expression of anti-metastasis miR and metastasis suppressor might inhibit migration and invasion in MDA-MB-231 cells efficiently. CONCLUSION: This combinatorial use of anti-metastatic miR and gene suggests a new therapeutic intervention for metastasis inhibition in MDA-MB-231.

Delivery of LNA-antimiR-142-3p by Mesenchymal Stem Cells-Derived Exosomes to Breast Cancer Stem Cells Reduces Tumorigenicity.

Exosomes, nano-sized cell-derived vesicles, have been employed as non-synthetic carriers of various pharmaceutics in numerous studies. As higher expression levels of miR-142-3p and miR-150 in breast cancer stem cells (BCSCs) are associated with their clonogenic and tumorigenic capabilities, the present study aims to exploit the mesenchymal stem cells-derived exosomes (MSCs-Exo) to deliver LNA-antimiR-142-3p into MCF7-derived cancer stem-like cells to suppress expression levels of miR-142-3p and miR-150 in order to reduce clonogenicity and tumorigenicity. Our results indicated that the MSCs-Exo can efficiently deliver the LNA-antimiR-142-3p to breast cancer stem-like cells to reduce the miR-142-3p and miR-150 expression levels. Furthermore, the inhibition of the oncomiRs with the delivery of LNA-antimiR-142-3p resulted in a significant reduction of clone-formation and tumor-initiating abilities of the MCF7-derived cancer stem-like cells. In conclusion, we showed that MSCs-derived exosomes could be used as a feasible nanovehicles to deliver RNA-based therapeutics into BCSCs to improve the cancer treatment. HIGHLIGHTS: Exosomes secreted by bone marrow-derived mesenchymal stem cells efficiently transfer the LNA-antimiR-142-3p to breast cancer stem cells. Exosomes-mediated delivery of LNA-antimiR-142-3p to the breast cancer stem cells leads to downregulation of miR-142-3p and miR-150 and the overexpression of target genes. Delivery of LNA-antimiR-142-3p by the exosomes reduces the colony formation capability of breast cancer stem cells in vitro. Inhibition of miR-142-3p and miR-150 by the LNA-antimiR-142-3p loaded exosomes reduces the tumorigenicity of breast cancer stem cells in vivo.

Targeted delivery of doxorubicin to HER2 positive tumor models.

BACKGROUND: Exosomes are natural nanovesicles with unique characteristics, such as long circulating half-life, the intrinsic ability to target tissues, biocompatibility, and minimal or no inherent systemic toxicity. Mesenchymal stem cells produce large amounts of exosomes with regenerative properties and more stability in human plasma. TUBO breast cancer cell lines overexpress rat HER2/neu protein. METHODS: Targeted exosomes were isolated from transduced bone marrow mesenchymal stem cells. Doxorubicin was encapsulated into exosomes by electroporation. Flow cytometry was used to assess the attachment of exosomes to the target cells. The in vitro cytotoxicity effect of targeted doxorubicin-loaded exosomes on TUBO cells was determined using MTT assay. Selective delivery of doxorubicin to tumor tissues was analyzed by measuring the auto-fluorescence of doxorubicin by in vivo imaging system. Moreover, tumor growth inhibition and body weight were monitored following injection of free doxorubicin, and targeted and untargeted doxorubicin-loaded exosomes in a TUBO breast cancer model. Finally, mouse tissues were examined for the presence of intrinsic fluorescence of doxorubicin. RESULTS: Flow cytometry results revealed significant differences in binding of targeted exosomes to HER2-positive (46.05%) and HER2-negative (13.9%) cells. The results of MTT assay showed that cytotoxicity of targeted doxorubicin-loaded exosomes was higher than free doxorubicin at 72 hours. Selective distribution of targeted doxorubicin-loaded exosomes in the target tissues of the murine breast cancer model suggested specific delivery of doxorubicin by targeted exosomes, rather than untargeted exosomes. Free doxorubicin and untargeted doxorubicin-loaded exosomes showed insignificant effects, whereas targeted doxorubicin-loaded exosomes reduced the tumor growth rate. CONCLUSION: Herein, we report efficient delivery of targeted doxorubicin-loaded exosomes in vitro, corroborated with a significant reduction of murine breast cancer model tumor growth rate.

A cell ELISA based method for exosome detection in diagnostic and therapeutic applications.

OBJECTIVE: Exosomes are nano-scaled carriers of miRNA, mRNA and proteins which are secreted from viable cells. Exosome detection within serum, saliva and semen offers diagnostic value for detection of various diseases including cancer. In the present study, we have lunched an in vitro study to develop a more efficient method for exosome detection. In this regard, the recombinant LAMP-DARPins (capable of Her2 targeting) gene was packed within the lentiviral particles and stably transferred into the HEK293T cells. The morphology and sizes of the obtained exosomes were characterized by TEM and zeta sizer. The expression of LAMP-DARPins antigen on the exosome surface was verified by western blotting. Ultimately, the efficiency of cell surface ELISA in suspension method was examined for exosome detection. RESULTS: The exosome particles were successfully harvested and purified from transfected cells. The sizes of exosome particles were determined to be 90 nm using zeta sizer instrument. The TEM scan showed that the exosomes are cap like shaped and their sizes range between 40 and 150 nm. An observed 120 kDa band on western blotting paper indicated the LAMP-DARPins antigen expression on exosome surfaces. The results of cell surface ELISA in suspension method were superior to the results of conventional cell ELISA. CONCLUSIONS: It could be concluded that the cell surface ELISA in suspension method could be an amenable method to detect exosome particles within the biological samples. Moreover, the method could be modified to evaluate the ability of exosomes to interact with target cells in both diagnostic and therapeutic applications.

Additive effect of metastamiR-193b and breast cancer metastasis suppressor 1 as an anti-metastatic strategy.

BACKGROUND: It has been reported that enhancing the cellular levels of miR-193b as well as breast cancer-metastasis-suppressor-1 (BRMS1) protein is associated with diminished metastatic characteristics in breast cancer. In view of these facts, as a new therapeutic intervention, we employed a restoration-based strategy using both miR-193b-3p mimic and optimized BRMS1 in the context of a chimeric construct. METHODS: miR-193b-3p and BRMS1 genes were cloned and the resulting plasmids were transfected into the MDA-MB231, MCF-7 and MCF-10A cell lines. microRNA expression levels were assessed by rea time PCR using LNA-primer and protein expression was confirmed by western blot method. Then, apoptosis, MTT, colony formation and invasion assays were carried out. RESULTS: The expression levels of miR-146a, miR-146b and miR-373 were up-regulated, while the miR-520c, miR-335 and miR-10b were down-regulated following the exogenous BRMS1 expression. The exogenous over-expression of BRMS1 was associated with higher amounts of endogenous miR-193b-3p expression and enabled more efficient targeting of the 3'UTR of uPA. Although, miR-193b-3p and BRMS1 are individually capable of suppressing breast cancer cell growth, migration and invasion abilities, their cistronic expression was capable of enhancing the ability to repress the breast cancer cells invasion. CONCLUSIONS: Our results collectively indicated the existence of an additive anti-metastatic effect between miR-193b-3p and BRMS1. Moreover, it has been hypothesized that the exogenous expression of a protein can effect endogenous expression of non-relevant microRNA. Our findings provide new grounds for miR-restoration therapy applications as an amenable anti-metastatic strategy.

Alteration of cellular and immune-related properties of bone marrow mesenchymal stem cells and macrophages by K562 chronic myeloid leukemia cell derived exosomes.

Leukemic cells can impact the bone marrow niche to create a tumor-favorable microenvironment using their secreted factors. Little knowledge is available about immunosuppressive and tumor-promoting properties of chronic myeloid leukemia derived exosomes in bone marrow stromal components. We report here that K562-derived exosomes can affect the gene expression, cytokine secretion, nitric oxide (NO) production, and redox potential of bone marrow mesenchymal stem cells (BM-MSCs) and macrophages. Human BM-MSCs and mouse macrophages were treated with K562-derived exosomes. Our results demonstrated that the expression of the genes involved in hematopoietic developmental pathways and immune responses, including C-X-C motif chemokine 12 (Cxcl12), Dickkopf-related protein 1 (DKK1), wnt5a, interleukin 6 (IL-6), transforming growth factor-beta, and tumor necrosis factor-alpha (TNF-alpha), changed with respect to time and exosome concentration in BM-MSCs. The TNF-alpha level was higher in exosome-treated BM-MSCs compared with the control. Exosome treatment of BM-MSCs led to an increased production of NO and a decreased production of reactive oxygen species (ROS) in a time- and concentration-dependent manner. We have shown that K562-derived exosomes induce overexpression of IL-10 and TNF-alpha and downregulation of iNOS transcript levels in macrophages. The enzyme-linked immunosorbent assay results showed that TNF-alpha and IL-10 secretions increased in macrophages. Treatment of macrophages with purified exosomes led to reduced NO and ROS levels. These results suggest that K562-derived exosomes may alter the local bone marrow niche toward a leukemia-reinforcing microenvironment. They can modulate the inflammatory molecules (TNF-alpha and NO) and the redox potential of BM-MSCs and macrophages and direct the polarization of macrophages toward tumor-associated macrophages.

Exosome-mediated delivery of functionally active miRNA-142-3p inhibitor reduces tumorigenicity of breast cancer in vitro and in vivo.

BACKGROUND: Exosomes, widely recognized natural nanovesicles, represent one of the recently discovered modes of intercellular communication due to their ability to transmit crucial cellular information that can be engineered to have robust delivery and targeting capacity. MiR-142-3p, one of the upregulated microRNAs (miRNAs) in many types of breast cancer, activates the canonical Wnt signaling pathway and transactivates the miR-150 expression, and results in the hyperproliferation of cancer cells in vitro and mammary glands in vivo. MATERIALS AND METHODS: In this study, we exploited the exosomes isolated from bone marrow-derived mesenchymal stem cells (MSCs-Exo) to deliver LNA (locked nucleic acid)-modified anti-miR-142-3p oligonucleotides to suppress the expression level of miR-142-3p and miR-150 in 4T1 and TUBO breast cancer cell lines. RESULTS: The in vitro results showed that the MSCs-Exo can efficiently deliver anti-miR-142-3p to reduce the miR-142-3p and miR-150 levels and increase the transcription of the regulatory target genes, APC and P2X7R. We also evaluated in vivo distribution of the MSCs-Exo in tumor-bearing mice. The in vivo result indicated that MSCs-Exo can penetrate the tumor site and are suitable nanovehicles to deliver the inhibitory oligonucleotides into the tumor tissues to downregulate the expression levels of miR-142-3p and miR-150. CONCLUSION: We showed that MSCs-derived exosomes could be used as a feasible nanovehicle to deliver drug molecules like LNA-anti-miR-142-3p in both in vitro and in vivo studies.

Development of a novel anti-HER2 scFv by ribosome display and in silico evaluation of its 3D structure and interaction with HER2, alone and after fusion to LAMP2B.

HER2 is a member of epidermal factor receptor (EGFR) family which is overexpressed in breast cancer, ovarian cancer and gastric cancer. Development of new binders for cancer cell surface receptors and expressing them at the surface of exosomes would be a great approach in targeted cancer therapy. We found a high affinity scFv against HER2 using ribosome display with the approach of applying it as a targeting moiety at the surface of exosomes by fusion to lysosomal associated membrane protein 2B (LAMP2B). We also provide some structural information about the ribosome display selected scFv (scFv HFS2) through modeling the 3D structure of scFv HFS2 using RosettaAntibody and docked it at the extracellular domain of HER2. We also evaluated the structure of scFv HFS2 and its binding to HER2 after fusion to LAMP2B. Our results showed no significant change in 3D structure of scFv HFS2 when fused to LAMP2B (RMSD 1.3) and interaction analysis represented that scFv HFS2 binds HER2 domain III before and after fusion to LAMP2B. Although binding domain of scFv HFS2 on HER2 was the same at both state, residues involved in their interactions showed significant differences as it was probably due to the spatial hindrance of scFv HFS2 when fused to LAMP2B through a short linker and it should be considered before proceeding to experiment.

Targeted cancer therapy using engineered exosome as a natural drug delivery vehicle.

PURPOSE: Exosomes are small 30-100 nm vesicles secreted by various cell types. They are released by most cell types, indicating their important role in physiological and pathological processes, including signaling pathways, cell-to-cell communication, tumor progression, and molecule transferring. As natural nanovesicles, exosomes can be a good candidate for drug delivery due to low immunogenicity and ability to enter tissues and even cross the blood-brain barrier. In an effort to improve the efficiency of exosomes for targeted drug delivery with minimal effect on normal cells, we expressed ligands against HER2+ cells. METHODS: To purify exosomes, transduced mesenchymal stromal cells were cultured to reach 80% confluency. Next, the cells were cultured in serum-free media for 48 hours and the supernatant was harvested to purify exosomes. These exosomes were then labeled with PKH67 and added to BT-474, SKBR3 (HER2+), and MDA-MB231 (HER2-), cell lines and their binding to HER2+ was evaluated by flow cytometry. Exosomes were loaded with doxorubicin and quantified using intrinsic fluorescence of doxorubicin at 594 nm. RESULTS: Targeted exosomes were preferably uptaken by HER2+ cells. Therefore, untargeted exosomes showed lower binding to HER2+ cells compared to their targeted counterparts. MTT assay was performed to analyze cytotoxic effect of exo-DOX (exosome encapsulated with doxorubicin). Efficiency of exo-DOX and free DOX (doxorubicin) delivery with different concentrations, to the BT-474 cell line, was compared, and no significant difference was observed. CONCLUSION: Our results imply that targeted exosomes are preferentially uptaken by HER2+ cells relative to HER2- cells and have the potential to be used as an efficient drug delivery system.

Embryonic stem cell derived germ cells induce spermatogenesis after transplantation into the testes of an adult mouse azoospermia model.

The present study aimed to: (i) identify the exogenous factors that allow in vitro differentiation of mouse spermatogonial stem cells (SSCs) from embryonic stem cells (ESCs); (ii) evaluate the effects of Sertoli cells in SSC enrichment; and (iii) assess the success of transplantation using in vitro differentiated SSCs in a mouse busulfan-treated azoospermia model. A 1-day-old embryoid body (EB) received 5 ng/ml of bone morphogenetic protein 4 (BMP4) for 4 days, 3 µM retinoic acid (RA) in a SIM mouse embryo-derived thioguanine and ouabain resistant (STO) co-culture system for 7 days, and was subsequently co-cultured for 2 days with Sertoli cells in the presence or absence of a leukaemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF) and RA composition, and in the presence of these factors in simple culture medium. Higher viability, proliferation and germ cell gene expression were seen in the presence of the LIF, bFGF and RA composition, on top of Sertoli cells. Immunocytochemistry results showed higher CDH1 expression in this group. Sertoli co-culture had no effects on SSC proliferation. Eight weeks after transplantation, injected cells were observed at the base of the seminiferous tubules and in the recipient testes. The number of spermatogonia and the mass of the testes were higher in transplanted testes relative to the control group. It seems that transplantation of these cells can be useful in infertility treatment.

Lung cancer and miRNAs: a possible remedy for anti-metastatic, therapeutic and diagnostic applications.

INTRODUCTION: Lung cancer still accounts for the leading cause of cancer-related deaths worldwide and despite the emerging advances in diagnostic and therapeutic techniques it remains to be a serious global public health concern. Micro-ribonucleic acids (microRNAs) are responsible for invasion and metastasis of various tumors including lung cancer which underscores the necessity of understanding their functions. Areas covered: Herein, we aim to summarize the recent advances made in our understanding of the miRNAs with special reference to lung cancer. Moreover, the role of miRNAs in crucial cellular processes will be elucidated. Various applications of the miRNAs would be explained and different kinds of them would be discussed to delineate their significance in lung cancer biology, therapy and diagnosis. Expert commentary: the miRNA study in the field of respiratory disease and specially lung cancer has emerged lately. Given the several miRNAs, which are in the clinical trials, this field is passing through its maturation phase which ultimately could rise to a robust tool for lung cancer therapy, diagnosis and prevention.

Proliferation in culture of primordial germ cells derived from embryonic stem cell: induction by retinoic acid.

An in vitro system that supports primordial germ cells (PGCs) survival and proliferation is useful for enhancement of these cells and efficient transplantation in infertility disorders. One approach is cultivation of PGCs under proper conditions that allow self-renewal and proliferation of PGCs. For this purpose, we compared the effects of different concentrations of retinoic acid (RA), and the effect of PGCs co-culture (Co-C) with SIM mouse embryo-derived thioguanine- and ouabain-resistant (STO) cells on the proliferation of embryonic stem cells (ESCs)-derived PGCs. One-day-old embryoid body (EB) was cultured for 4 days in simple culture system in the presence of 5 ng/ml bone morphogenetic protein-4 (BMP4) (SCB group) for PGC induction. For PGC enrichment, ESCs-derived germ cells were cultured for 7 days in the presence of different doses (0-5  µM) of RA, both in the simple and STO Co-C systems. At the end of the culture period, viability and proliferation rates were assessed and expression of mouse vasa homologue (Mvh),  α6 integrin,  ß1 integrin, stimulated by retinoic acid 8 (Stra8) and piwi (Drosophila)-like 2 (Piwil2) was evaluated using quantitative PCR. Also, the inductive effects were investigated immunocytochemically with Mvh and cadherin1 (CDH1) on the selected groups. Immunocytochemistry/PCR results showed higher expression of Mvh, the PGC-specific marker, in 3  µM RA concentrations on the top of the STO feeder layer. Meanwhile, assessment of the Stra8 mRNA and CDH1 protein, the specific makers for spermatogonia, showed no significant differences between groups. Based on the results, it seems that in the presence of 3 µM RA on top of the STO feeder layer cells, the majority of the cells transdifferentiated into germ cells were PGCs.

In vitro germ cell differentiation from embryonic stem cells of mice: induction control by BMP4 signalling.

The present study aims to confirm and analyse germ cell-related patterns and specific gene expressions at a very early stage of cell commitment. Following the XY cytogenetic confirmation of the CCE mouse embryonic stem cells (mESCs) line, cells were cultured to form embryoid bodies (EBs). Expression pattern assessment of the mouse vasa homologue (Mvh), Stra8, α6 and ß1 integrin genes in ESC and 1-3-day-old EB showed that all genes except α6 integrin were expressed in the ESC. The mean calibration of Mvh, Stra8 and α6 integrin expression significantly increased upon EB formation compared with the ESCs. During mouse embryogenesis, the signalling of bone morphogenetic protein (BMP) is essential for germ-line formation. To investigate its role in germ-line induction in vitro, mESCs were cultured as 1-day-old EB aggregates with BMP4 for 4 days in STO co-culture systems, in the presence and absence of 5 ng/ml BMP4. At the end of the culture period, colony assay (number and diameter) was performed and the viability percentage and proliferation rate was determined. There were no significant statistical differences in the abovementioned criteria between these two groups. Moreover, the expression of Mvh, α6 and ß1 integrins, Stra8 and Piwil2 genes was evaluated in co-culture groups. The molecular results of co-culture groups showed higher-but insignificant-Piwil2 and significant α6 integrin expressions in BMP4 treated co-culture systems. These results confirmed that the EB system and the presence of BMP4 in a STO co-culture system improve the differentiation of ESCs to germ cell.

Bone morphogenetic protein 4 is an efficient inducer for mouse embryonic stem cell differentiation into primordial germ cell.

Presence of specific growth factors and feeder layers are thought to be important for in vitro embryonic stem cell (ESCs) differentiation. In this study, the effect of bone morphogenetic protein 4 (BMP4) and mouse embryonic fibroblasts (MEFs) co-culture system on germ cell differentiation from mouse ESCs was evaluated. One-day-old embryoid body was cultured for 4 d in simple culture systems or on top of the MEFs, both in the presence or absence of BMP4. Data showed significant higher viability percent and proliferation rate in simple culture media compared to co-culture systems. Analysis of gene expression indicated that the germ cell-specific genes (VASA and Stra8) were expressed in a significant higher ratio in BMP4-treated cells in simple culture system. Also, the results of immunocytochemistry in simple culture systems showed that the mean percentage of immunostaining cells of VASA, the primordial germ cell (PGC) marker, was increased significantly in BMP4-treated cells compared with BMP4-free group. Meanwhile, CDH1, the late premiotic germ cell marker, showed no significant difference between these two groups. The results suggest that BMP4 is an efficient inducer in PGC derivation from mouse ESC. However, the employment of MEFs as feeder has no apparent effect on PGC derivation.

The correlation between the endometrial integrins and osteopontin expression with pinopodes development in ovariectomized mice in response to exogenous steroids hormones.

BACKGROUND: The ovariectomized animals are good models to evaluate the effect of different steroid hormone treatments on implantation events and the pattern of integrin expression. Therefore, this study was performed to compare the expression of integrins and osteopontin (OPN) in correlation with pinopode development in ovariectomized mice endometrium which was subjected to steroid hormones. METHODS: Ovariectomized mice were subjected to estrogen, progesterone and estrogen-progesterone hormones. Their uterine horns were evaluated for integrin expression by immunohistochemistry and real-time RT-PCR and for pinopode development by transmission and scanning electron microscopic studies. RESULTS: No immunostaining for integrin and OPN molecules were detected in the endometrium of non-ovariectomized mice except in metestrus phase. The α4 and ß1 integrin genes were expressed in all phases of estrous cycle. In ovariectomized mice, no reaction was detected in the endometrium of control, sham and estrogen-treated groups, but in both progesterone-treated groups, all examined genes were expressed. There was not any correlation between pinopodes and integrin expression in ovariectomized mice. CONCLUSION: The progesterone is more effective on endometrial integrin expression than estrogen and differences in the expression pattern of integrins reflect their important and different roles in embryo implantation. The pinopodes may have minor effect in mice implantation or have some delay in their expressions in ovariectomized mice which were subjected to exogenous hormones.