The stem cell-specific long non-coding RNAs in leukemia

Leukemia is defined as a heterogeneous group of hematological cancers whose prevalence is on the rise worldwide. Despite the large body of studies, the etiology of leukemia has not been fully elucidated. Leukemia stem cells (LSCs) are a subpopulation of cancer cells that sustain the growth of the leukemic clone and are the main culprit for the maintenance of the neoplasm. In contrast to most leukemia cells, LSCs are resistant to chemo- and radiotherapy. Several recent studies demonstrated the altered expression profile of long non-coding RNAs (lncRNAs) in LSCs and shed light on the role of lncRNAs in the survival, proliferation, and differentiation of LSCs. LncRNAs are transcripts longer than 200 nucleotides that are implicated in several cellular and molecular processes such as gene expression, apoptosis, and carcinogenesis. Likewise, lncRNAs have shown a prognostic marker in leukemia patients and represent novel treatment options. Herein, we review the current knowledge concerning lncRNAs’ implication in the pathogenesis of LSCs and discuss their prognostic, diagnostic, and therapeutic potential (AU)

The stem cell-specific long non-coding RNAs in leukemia.

Leukemia is defined as a heterogeneous group of hematological cancers whose prevalence is on the rise worldwide. Despite the large body of studies, the etiology of leukemia has not been fully elucidated. Leukemia stem cells (LSCs) are a subpopulation of cancer cells that sustain the growth of the leukemic clone and are the main culprit for the maintenance of the neoplasm. In contrast to most leukemia cells, LSCs are resistant to chemo- and radiotherapy. Several recent studies demonstrated the altered expression profile of long non-coding RNAs (lncRNAs) in LSCs and shed light on the role of lncRNAs in the survival, proliferation, and differentiation of LSCs. LncRNAs are transcripts longer than 200 nucleotides that are implicated in several cellular and molecular processes such as gene expression, apoptosis, and carcinogenesis. Likewise, lncRNAs have shown a prognostic marker in leukemia patients and represent novel treatment options. Herein, we review the current knowledge concerning lncRNAs' implication in the pathogenesis of LSCs and discuss their prognostic, diagnostic, and therapeutic potential.

Mesenchymal stem cells derived from the kidney can ameliorate diabetic nephropathy through the TGF-ß/Smad signaling pathway.

Diabetic nephropathy (DN) has been introduced as one of the main microvascular complications in diabetic patients, the most common cause of end-stage renal disease (ESRD). Based on the therapeutic potential of mesenchymal stem cells in tissue repair, we aimed to test the hypothesis that kidney stem cells (KSCs) might be effective in the kidney regeneration process. Stem cells from rat kidney were separated, and the surface stem cell markers were determined by flow cytometry analysis. Thirty-two Sprague Dawley rats were divided into four groups (control, control that received kidney stem cells, diabetic, diabetic treated with stem cells). To establish diabetic, model STZ (streptozotocin) (60 mg/kg) was used. The KSCs were injected into experimental groups via tail vein (2 × 106 cells/rat). In order to determine the impact of stem cells on the function and structure of the kidney, biochemical and histological parameters were measured. Further, the expression of miRNA-29a, miR-192, IL-1ß, and TGF-ß was determined through the real-time PCR technique. Phosphorylation of Smad2/3 was evaluated by using the standard western blotting. The KSCs significantly reduced blood nitrogen (BUN), serum creatinine (Scr), and 24-h urinary proteins in DN (P < 0.05). IL-1ß and TGF-ß significantly increased in the kidney of diabetic rats. In addition, the expression of miR-29a is significantly increased, whereas miR-192 decreased after treatment with KSCs (P < 0.05). Diabetic rats showed an increased level of phosphorylation of both Smad2 and Smad3 (P < 0.05). Periodic acid-Schiff (PAS) staining showed improved histopathological changes in the presence of KSCs. Stem cells derived from adult rat kidney may be an option for treating the early DN to improve the functions and structure of kidneys in rats with DN.

Evaluation of the Protective Effects of Doxycycline on Acetaminophen-Induced Hepatotoxicity in Mice.

Acetaminophen (APAP) toxicity threatens human health due to increased mortality associated with its overdose. Doxycycline (DC) because of its properties such as antioxidant and anti-inflammatory can be a good therapeutic strategy to treat the acute toxicity induced by APAP. Male mice were divided into six groups in two periods of 3 h and 24 h as normal saline, APAP 400 mg/kg, DC 100 mg/kg and groups treated by 25, 50 and 100 mg/kg DC just before APAP, respectively. At the end of the 3 h and 24 h periods, the hepatic index, biochemical parameters including serum aspartate transaminase (AST) and alanine transaminase (ALT) activity and hepatic catalase activity, glutathione (GSH) and malondialdehyde (MDA) levels in liver and histopathological changes were evaluated. The results indicated that DC had no apparent effect on the hepatic index but significantly normalized the level of biochemical parameters and reduced APAP induced liver damage. Overall, it could be concluded that DC can inhibit or resolve harmful effects of APAP through antioxidant and anti-inflammatory properties. However, more studies are needed to understand exact mechanism of DC and its application for clinical use.

Protective effect of Zingerone against mouse testicular damage induced by zinc oxide nanoparticles.

The purpose of the present study was to evaluate the effect of Zingerone (Zing) on zinc oxide nanoparticle (ZNP)-induced spermatogenesis defects in mice. To this end, 50 mg/kg of ZNP was prescribed to the mice as an intoxicated group for 35 days. In protection groups, Zing (10, 20, and 40 mg/kg) was given prior to ZNP treatment for seven days and then co-administration of ZNP for 35 days. Epididymal sperm parameters, testicular histology, Johnsen's scoring, morphometric parameters, TUNEL staining, oxidative stress, and serum testosterone level were evaluated for determining ZNP and Zing effects on the mouse testicles. Effects of Zing and ZNP on the viability of mouse Leydig (TM3) and mouse Sertoli (TM4) cell lines were also done. Testicular weights, testosterone levels, sperm quality, morphometric parameters, Johnsen's score, and superoxide dismutase (SOD) and catalase (CAT) activities were significantly decreased in ZNP-intoxicated mice, while apoptotic index, Malondialdehyde (MDA) content, and histological features, including epithelial vacuolization, sloughing, and germ cell detachment, were improved significantly in ZNP-intoxicated mice. Pretreatment with 20 or 40 mg/kg Zing significantly reduced the histological criteria, increased morphometric parameters, enhanced testosterone levels, attenuated apoptotic index, improved sperm quality, and reversed oxidative stress by reducing the level of MDA and incrementing the activity level of SOD and CAT enzymes. Zing dose-dependently enhanced the viability of ZNP-treated TM3 and TM4 cells in comparison with only ZNP-exposed cells. According to the results of our study, Zing effectively prevented the defects in spermatogenesis among mice treated by ZNP.

Effects of Pravastatin in Adriamycin-Induced Nephropathy in Rats.

The aim of this study is to evaluate the effects of pravastatin on Adriamycin (ADR)-induced nephropathy and the mechanisms involved. Forty rats were divided into the following 4 groups: control, ADR (15 mg.kg-1, IP), ADR plus pravastatin (20 mg.kg-1 which was started 5 days prior to ADR injection), and ADR plus pravastatin (20 mg.kg-1 which was started 5 days after ADR injection). On day 20 after ADR injection, the animals were sacrificed. The results showed that administration of pravastatin decreased the levels of 24-h urinary protein (24-h UP), blood urea nitrogen (BUN), and creatinine (p < 0.05) which had increased after the injection of ADR; in addition, pravastatin reversed structural changes seen in ADR group. Furthermore, pravastatin elevated mRNA and protein expression of nephrin (p < 0.05) which had been reduced in ADR group. We conclude that pravastatin protects and treats renal injury induced by ADR.

Protective Effect of Gemfibrozil on Hepatotoxicity Induced by Acetaminophen in Mice: the Importance of Oxidative Stress Suppression.

Purpose: Gemfibrozil (GEM) apart from agonist activity at peroxisome proliferator-activated receptor-alpha (PPAR-α) has antioxidant and anti-inflammatory properties. Accordingly, the present study was designed to investigate the protective effect of GEM on acute liver toxicity induced by acetaminophen (APAP) in mice. Methods: In this study, mice divided in seven groups include, control group, APAP group, GEM group, three APAP groups pretreated with GEM at the doses of 25, 50 and 100 mg/kg respectively and APAP group pretreated with N-Acetyl cysteine. GEM, NAC or vehicle were administered for 10 days. In last day, GEM and NAC were gavaged 1 h before and 1 h after APAP injection. Twenty four hours after APAP, mice were sacrificed. Serum parameters include alanine aminotransferase (ALT), aspartate aminotransferase (AST) and liver tissue markers including catalase enzyme activity, reactive oxygen species (ROS), malondialdehyde and reduced glutathione (GSH) levels determined and histopathological parameters measured. Results: GEM led to significant decrease in serum ALT and AST activities and increase in catalase activity and hepatic GSH level and reduces malondialdehyde and ROS levels in the liver tissue. In confirmation, histopathological findings revealed that GEM decrease degeneration, vacuolation and necrosis of hepatocytes and infiltration of inflammatory cells. Conclusion: Present data demonstrated that GEM has antioxidant properties and can protect the liver from APAP toxicity, just in the same pathway that toxicity occurs by toxic ROS and that GEM may be an alternative therapeutic agent to NAC in APAP toxicity.

Radiotherapy Enhancement with Electroporation in Human Intestinal Colon Cancer HT-29 Cells

Background: The efficiency of radiotherapy for tumors can be enhanced with different radiosensitizers. Previous studies have shown that electroporation (EP) can sensitize some cancer cell lines to ionizing radiation (IR). HT-29 is a radiation resistant colorectal cancer cell line, representative of a cancer type which is the second cause of cancer mortalities in developed countries. The present study aimed to evaluate radiosensitizing effects of EP on HT-29 cells in vitro exposed to 6 MV X-ray photon beams. Methods: HT-29 cells were exposed to a 6 MV X-ray photon beam as the control or to a combination of electroporation and irradiation. The response of cells was evaluated by colony formation assay and survival curves. Results: The survival fraction of the HT-29 cells was significantly decreased by electroporation prior to radiotherapy. A single electric pulse increased colorectal HT-29 cancer cell sensitivity to megavoltage radiation by a factor of 1.36. Conclusion: Our findings showed that EP before radiotherapy can significantly enhance tumor cell sensitivity. This combined treatment modality should be assessed for its applicability in clinic settings for employment against radioresistant cancers. However, to facilitate achieving this goal, many different tumors with a broad range of radiosensitivities should be evaluated.

Gold nanoparticles and electroporation impose both separate and synergistic radiosensitizing effects in HT-29 tumor cells: an in vitro study.

BACKGROUND AND OBJECTIVE: Radiation therapy (RT) is the gold standard treatment for more than half of known tumors. Despite recent improvements in RT efficiency, the side effects of ionizing radiation (IR) in normal tissues are a dose-limiting factor that restricts higher doses in tumor treatment. One approach to enhance the efficiency of RT is the application of radiosensitizers to selectively increase the dose at the tumor site. Gold nanoparticles (GNPs) and electroporation (EP) have shown good potential as radiosensitizers for RT. This study aims to investigate the sensitizing effects of EP, GNPs, and combined GNPs-EP on the dose enhancement factor (DEF) for 6 MV photon energy. METHODS: Radiosensitizing effects of EP, GNPs, and combinations of GNPs-EP were comparatively investigated in vitro for intestinal colon cancer (HT-29) and Chinese hamster ovary (CHO) cell lines by MTT assay and colony formation assay at 6 MV photon energy in six groups: IR (control group), GNPs+IR, GNPs (24 h)+IR, EP+IR, GNPs+EP+IR, and GNPs (24 h)+EP+IR. RESULTS: Treatment of both cell lines with EP, GNPs, and combined GNPs-EP significantly enhanced the response of cells to irradiation. However, the HT-29 showed higher DEF values for all groups. In addition, the DEF value for HT-29 cells for GNPs+IR, GNPs (24 h)+IR, EP+IR, GNPs+EP+IR, and GNPs (24 h)+EP+IR was, respectively, 1.17, 1.47, 1.36, 2.61, and 2.89, indicating synergistic radiosensitizing effect for the GNPs (24 h)+EP+IR group. Furthermore, the synergistic effect was observed just for HT-29 tumor cell lines. CONCLUSION: Combined GNPs-EP protocols induced synergistic radiosensitizing effect in HT-29 cells, and the effect is also tumor specific. This combined therapy can be beneficially used for the treatment of intrinsically less radiosensitive tumors.

A dermal equivalent developed from adipose-derived stem cells and electrospun polycaprolactone matrix: an in vitro and in vivo study.

Polycaprolactone (PCL) is used as a material of choice for surgical sutures, wound dressings, contraceptives, fixation devices and dentistry in paramedical sciences. In addition, adipose-derived stem cells (ASCs) have been shown to be effective in the treatment of acute and chronic wounds. This study aimed to evaluate the effect of electrospun PCL fibers on keratinocyte differentiation of ASCs and wound healing. PCL solution was electrospun and characterized. Isolated and characterized ASCs were differentiated into keratinocyte-like cells on a tissue culture plate (TCP) and PCL matrices and compared. PCL nano-/microfibers cultured with ASCs (test group) or alone (control) were implanted as a dermal substitute for wound healing. There were significant increases in the proliferation rate and expression level of cytokeratin 14, filaggrin and involucrin in cells cultured on PCL matrices compared to TCP (p < 0.05). After histological and immunological evaluation of the reconstituted skin, a thick epidermal layer with several skin appendages was evidently observed in the ASC/PCL group, whereas no real and mature epidermis was formed, especially in the central area of the healing wound in the pure PCL group on day 14. Pure PCL, if possessing suitable properties including good adhesiveness, high proliferative capability, inductive elasticity and stiffness for migration and differentiation, could drive the keratinocyte differentiation of ASCs and act as an efficient dermal equivalent to promote wound healing.

Effects of Exendine-4 on The Differentiation of Insulin Producing Cells from Rat Adipose-Derived Mesenchymal Stem Cells.

OBJECTIVE: To evaluate the effect of Exendine-4 (EX-4), a Glucagon-like peptide 1 (GLP-1) receptor agonist, on the differentiation of insulin-secreting cells (IPCs) from rat adipose-derived mesenchymal stem cells(ADMSCs). MATERIALS AND METHODS: In this experimental study, ADMSCs were isolated from rat adi- pose tissue and exposed to induction media with or without EX-4. After induction, the existence of IPCs was confirmed by morphology analysis, expression pattern analysis of islet-specific genes (Pdx-1, Glut-2 and Insulin) and insulin synthesis and secretion. RESULTS: IPCs induced in presence of EX-4 were morphologically similar to pancre- atic islet-like cells. Expression of Pdx-1, Glut-2 and Insulin genes in EX-4 treated cells was significantly higher than the cells exposed to differentiation media without EX-4. Compared to EX-4 untreated ADMSCs, insulin release from EX-4 treated ADMSCs showed a nearly 2.5 fold (P<0.05) increase when exposed to a high glucose (25 mM) medium. The percentage of insulin positive cells in the EX-4 treated group was ap- proximately 4-fold higher than in the EX-4 untreated ADMSCs. CONCLUSION: The present study has demonstrated that EX-4 enhances the differen- tiation of ADMSCs into IPCs. Improvement of this method may help the formation of an unlimited source of cells for transplantation.

Enrichment of skin-derived neural precursor cells from dermal cell populations by altering culture conditions.

BACKGROUND: As stem cells play a critical role in tissue repair, their manipulation for being applied in regenerative medicine is of great importance. Skin-derived precursors (SKPs) may be good candidates for use in cell-based therapy as the only neural stem cells which can be isolated from an accessible tissue, skin. Herein, we presented a simple protocol to enrich neural SKPs by monolayer adherent cultivation to prove the efficacy of this method. METHODS: To enrich neural SKPs from dermal cell populations, we have found that a monolayer adherent cultivation helps to increase the numbers of neural precursor cells. Indeed, we have cultured dermal cells as monolayer under serum-supplemented (control) and serum-supplemented culture, followed by serum free cultivation (test) and compared. Finally, protein markers of SKPs were assessed and compared in both experimental groups and differentiation potential was evaluated in enriched culture. RESULTS: The cells of enriched culture concurrently expressed fibronectin, vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors as compared to control culture. In addition, they possessed a multipotential capacity to differentiate into neurogenic, glial, adipogenic, osteogenic and skeletal myogenic cell lineages. CONCLUSIONS: It was concluded that serum-free adherent culture reinforced by growth factors have been shown to be effective on proliferation of skin-derived neural precursor cells (skin-NPCs) and drive their selective and rapid expansion.

Three-dimensional differentiation of adipose-derived mesenchymal stem cells into insulin-producing cells.

The aim of this study is to evaluate the collagen/hyaluronic acid (Col/HA) scaffold effect on the differentiation of insulin-producing cells (IPCs) from adipose-derived mesenchymal stem cells (ASCs). In this experimental study, ASCs were cultured and seeded in a Col/HA scaffold (3D culture) and then treated with induction media. After induction, the presence of IPCs was evaluated using gene expression (PDX-1, GLUT-2 and insulin) analysis and immunocytochemistry, while functional maturity was determined by measuring insulin release in response to low- and high-glucose media. The induced IPCs were morphologically similar to pancreatic islet-like cells. Expression of the islet-associated genes PDX-1, GLUT-2 and insulin genes in 3D-cultured cells was markedly higher than the 2D-cultured cells exposure differentiation media. Compared to the 2D culture of ASCs-derived IPCs, the insulin release from 3D ASCs-derived IPCs showed a nearly 4-fold (p < 0.05) increase when exposed to a high glucose (25 mmol) medium. The percentage of insulin-positive cells in the 3D experimental group showed an approximately 4-fold increase compared to the 2D experimental culture cells. The results of this study demonstrated that the COL/HA scaffold can enhance the differentiation of IPCs from rat ASCs.

Three-dimensional differentiation of bone marrow-derived mesenchymal stem cells into insulin-producing cells.

Fibrin glue (FG) is used in a variety of clinical applications and in the laboratory for localized and sustained release of factors potentially important for tissue engineering. The aim of this study was to evaluate FG scaffold effect on differentiation of insulin-producing cells (IPCs) from bone marrow-derived mesenchymal stem cells (BM-MSCs). In this experimental study BM-MSCs were cultured and the cells characterized by analysis of cell surface markers using flow cytometry. BM-MSCs were seeded in FG scaffold (3D culture) and then treated with induction media. After induction, the presence of IPCs was demonstrated using gene expression profiles for pancreatic cell differentiation markers (PDX-1, GLUT-2 and insulin) and insulin detection in cytoplasm. Release of insulin by these cells was confirmed by radioimmunoassay. Expression of the islet-associated genes PDX-1, GLUT-2 and Insulin genes in 3D cultured cells was markedly higher than the 2D cultured cells exposure differentiation media. Compared to 2D culture of BM-MSCs-derived IPCs, the insulin release from 3D BM-MSCs-derived IPCs showed a nearly 3 fold (p<0.05) increase when exposed to a high glucose (25 mM) medium. Percentage of insulin positive cells in 3D experimental group showed an approximately 3.5-fold increase in compared to 2D experimental culture cells. The results of this study demonstrated that FG scaffold can enhance the differentiation of IPCs from rats BM-MSCs.

Dusty Air Pollution is Associated with an Increased Risk of Allergic Diseases in Southwestern Part of Iran.

Concerns have been raised about the adverse impact of dusty air pollution (DAP) on human health. The aim of this study was to find the association between dusty air pollution based on air quality index (AQI) and the risk of allergic diseases in southwestern provinces of Iran, with assessing cytokine profiles and lymphocyte immunophenotypes.In this case control study 148 individuals participated. The sampling was done in hazardous condition (AQI>300) as the case and clean air (AQI<50) as the control. We measured cytokine production by using ELISA method and phenotypes of T-lymphocytes (CD4+ and CD8+), CD19+ B-lymphocytes, CD25+, CD4+ CD25+ cells by FACSort flow cytometer.The mean serum level of IL-4 (33.4 ± 2.9 vs 0.85 ± 0.65 pg/dl) and IL-13 (15.1 ± 4.4 vs. 0.12 ± 0.7 pg/dl) in the subjects exposed to ambient DAP was increased significantly compared to the individuals in the clean air condition. Also, CD19+ B-lymphocytes (12.6 ± 4.9 vs 8.9 ± 3.2%) and CD4+ CD25+ cell count (13.6 ± 4.6 vs 7.7 ± 3.8%) in peripheral blood were increased significantly in subjects exposed to ambient DAP compared with the controls.The result of our study suggested that ambient DAP affected immune system in a way that might lead to allergic diseases in the population.

The effects of exendine-4 on insulin producing cell differentiation from rat bone marrow-derived mesenchymal stem cells.

OBJECTIVE: The aim of this study was to evaluate the effect of exendin-4 (EX-4) on differentiation of insulin-producing cells (IPCs) from rat bone marrow-derived mesenchymal stem cells (RAT-BM-MSCs). MATERIALS AND METHODS: In this experimental study, RAT-BM-MSCs were cultured and the cells characterized by flow cytometry analysis of cell surface markers. RAT-BM-MSCs were subsequently treated with induction media with or without EX-4. After induction, the presence of IPCs was demonstrated with dithizone (DTZ) staining and gene expression profiles for pancreatic cell differentiation markers (PDX-1, GLUT-2, insulin) were assessed using reverse transcription polymerase chain reaction (RT-PCR). Insulin excreted from differentiated cells was analyzed with radioimmunoassay (RIA). The two-tailed student's t-test was used for comparison of the obtained values. RESULTS: The percentage of DTZ-positive cells significantly increased in EX-4 treated cells (p<0.05). Expression of the islet-associated genes PDX-1, GLUT-2 and insulin genes in EX-4 treated cells was markedly higher than in the cells exposed to differentiation media without EX-4. RIA analysis demonstrated significant release of insulin with the glucose challenge test in EX-4 treated cells compared to EX-4 untreated cells. CONCLUSION: The results of this study have demonstrated that EX-4 can enhance differentiation of IPCs from RAT-BM-MSCs.

Exendin-4 effects on islet volume and number in mouse pancreas

The aim of this study was to evaluate Exendin-4 (EX-4) effects on islet volume and number in the mouse pancreas. Thirty-two healthy adult male NMRI mice were randomly divided into control and experimental groups. EX-4 was injected intraperitoneally (i. p.) at doses of 0.25 (E1 group), 0.5 (E2 group), and 1 µg/kg (E3 group), twice a day for 7 consecutive days. One day after the final injection, the mice were sacrificed, and the pancreas from each animal dissected out, weighed, and fixed in 10% formalin for measurement of pancreas and islet volume, and determination of islet number by stereological assessments. There was a significant increase in the weight of pancreases in the E3 group. Islet and pancreas volumes in E1 and E2 groups were unchanged compared to the control group. The E3 group showed a significant increase in islet and pancreas volume (P < 0.05). There were no significant changes in the total number of islets in all three experimental groups. The results revealed that EX-4 increased pancreas and islet volume in non-diabetic mice. The increased total islet mass is probably caused by islet hypertrophy without the formation of additional islets.

O objetivo deste estudo foi avaliar os efeitos do Exendin-4 (EX-4) sobre o volume e número de ilhotas no pâncreas. Trinta e dois camundongos NMRI machos saudáveis e adultos foram divididos ao acaso em grupos controle e grupos experimentais. EX-4 foi injetado intraperitonealmente (i. p.) nas doses de 0,25 (grupo E1), 0,5 (grupo E2) e 1 (grupo E3), duas vezes por dia durante 7 dias consecutivos. Um dia após a injeção final, os camundongos foram sacrificados e o pâncreas de cada animal foi dissecado, pesado e fixado em solução de formaldeído 10% para avaliação do volume do pâncreas e ilhotas e do número de ilhotas por métodos estereológicos. Observou-se aumento significativo no peso de pâncreas no grupo E3. O volume do pâncreas assim como das ilhotas não apresentou alterações nos grupos E1 e E2, quando comparados ao grupo controle No grupo E3 houve aumento significativo no volume do pâncreas e das ilhotas (P<0,05). Não se observaram alterações significativas no número de ilhotas nos três grupos experimentais. Os resultados revelaram que o EX-4 provoca aumento no volume do pâncreas, bem como no volume das ilhotas em camundongos não-diabéticos. O aumento no volume total de ilhotas deve-se, provavelmente, a hipertrofia das ilhotas sem a formação de ilhotas adicionais.

Kinetics of gene expression during exposure of mouse stem cells to activin A.

This study aimed to evaluate the pattern of gene expression induced by activin A in mouse Embryonic Stem Cells (ESCs). Mouse ES cells cultured in undifferentiated state by leukemia inhibitory factor and feeder layer cells. Following removing these two anti differentiation factors for 5 days and forming Embryoid Bodies (EBs), the cells divided to 8 equal cells per groups. Differentiation procedure was performed in a two staged protocol; Formed EBs for 4 days (Stage one); expanded differentiated ESCs on gelatin coated dishes for one week (stage two). In the stage one, the media of groups 2-7 contained 10, 30 and 100 ng mL(-1) Activin A. The media in stage two was the same for all groups and contained only Fetal Bovine Serum (FBS). The expression of undifferentiated, ectoderm, mesoderm and endoderm markers were compared with relative RT-PCR method and statistically analyzed. The expression of an undifferentiating marker; Nanog was increased in the Activin A treated groups of stage one. The expression of OCT4 reduced in Activin A treated groups in stage two. In the stage one, the expression of Nodal increased by Activin A. expression of sonic hedgehog (Shh) was suppressed in Activin A treated groups of both stages. In stage two, there were significant decrease for the expression of mesoderm (Brachyury) and Nodal and visceral endoderm (GATA4) markers (p < 0.01). The expression of definitive endoderm markers (PDX1, TAT) showed significantly increased in Activin A treated groups (p < 0.01). Activin A induced differentiation in high concentration by imbalance in undifferentiating markers. Nodal has a dual role, undifferentiating effect and regulation of visceral endoderm towards definitive endoderm. Overexpression of Nanog, alteration in the expression of Nodal and Shh inhibition are three mechanisms for explanation of differentiation induced by activin A in ES cells. These mechanisms induces cascade of gene expression that commits ESCs towards definitive endodermal cells.