فهرست مطالب مهرداد نوروزی نیا

Hypertrophic cardiomyopathy is a common cardiac disease diagnosed in young adults and rarely detectable in childhood. Hypertrophic cardiomyopathy exhibits considerable diversity in its clinical and genetic characteristics. To date, mutations in multiple genes associated with HCM have been discovered, with the most common ones being MYBPC3 and MYH7 genes. The present study aimed to utilize whole exome sequencing for conducting a genetic analysis of Hypertrophic cardiomyopathy in four children belonging to Iranian families.

Patients underwent medical evaluation, including clinical assessment, electrocardiography, and echocardiography. Genetic testing was performed after DNA extraction using whole exome sequencing to identify genetic alterations that may be responsible for this disease. In addition, bioinformatic analysis of the genetic changes was carried out using software tools for alignment, variant calling, and interpretation. Finally, the Sanger sequencing method was employed to confirm the genetic variations in the affected individual's family members.

The patients were children presenting with initial symptoms, such as syncope and palpitations. They were diagnosed with Hypertrophic cardiomyopathy type 3 and 4 based on the results of electrocardiography and echocardiography. The genetic testing results revealed a pathogenic de novo mutation (c.1208G>A, p.Arg403Gln) in the MYH7 gene. In addition, another disease-causing homozygous nonsense genetic variation (c.3811C>T, p.Arg1271Ter) was identified in the MYBPC3 gene, resulting in the production of a premature protein.

This study not only expanded the spectrum of genetic variations associated with Hypertrophic cardiomyopathy disease and aided in genetic counseling for families affected by it but also presented the first variations of the sarcomere gene in Iranian children.

Control processes in osteoblastic differentiation of mesenchymal stem cells are not yet fully understood. Epigenetic mechanisms, especially the methylation of CpG Islands in the promoter of genes, are considered as one of the most important control mechanisms in stem cell differentiation. In the process of differentiation, it is debated whether only the methylation of specific genes changes or the methylation of global DNA. Therefore, in the present study, the state of global DNA methylation was evaluated during osteoblastic differentiation of mesenchymal stem cells by differentiation medium and also in treatment by zoledronic acid.

In this laboratory study, after isolation and proliferation of mesenchymal stem cells, induction of osteoblastic differentiation was done using differentiating medium and zoledronic acid. DNA extraction was performed at differentiation weeks 1, 2, and 3 as well as from undifferentiated mesenchymal stem cells. Global DNA methylation status was assessed by antibodies against 5-methyl cytosine. Repeated measurement design test was used to analyze the data.

Global DNA methylation status of the genome did not change during osteoblastic differentiation of mesenchymal stem cells (p=0.093). Also, treatment with zoledronic acid had no effect on global DNA methylation (p=0.057).

Osteoblastic differentiation of mesenchymal stem cells by differentiation medium and also in treatment with zoledronic acid is not associated with altered global methylation of the genome. Therefore, the differentiation process of mesenchymal stem cells to osteoblasts is a unique pathway, and possibly other genetic and epigenetic mechanisms play a role in controlling it.

The severity of the spinal muscular atrophy phenotype is inversely associated with the expression levels of the SMN2 gene; this correlation is not absolute, for this reason there is currently no effective treatment. The interference of other severity modifying factors, apart from SMN2 gene expression has been suggested. Here we investigate the effects of valproic acid as a STAT5 inducer not only on SMN2 expression but on STAT5A gene expression as well as on the rate of apoptosis.
Expression levels of the SMN2 and STAT5A genes were determined using Real-time PCR while apoptotic rates were determined using flow cytometry in lymphocyte cell lines derived from three patients and two normal controls in response to valproic acid.
STAT5A gene induction, enhanced FL-SMN2 expression and reduced apoptotic rate were seen in lymphocyte cell lines after valproic acid treatment (p Since STAT5A gene induction was accompanied with SMN2 transcriptional upregulation, these findings propose the involvement of STAT5A gene both as a component of the STAT5 pathway and as a target gene in drug responsiveness.

The response surface methodology was employed to optimize the effects of pH, temperature (˚C), time (min) and the ratio of enzyme to substrate (% of substrate) on the hydrolysis process of cuttlefish muscle by alcalase. Central composite rotatable design with 5 levels and 4 factors and α=2 was used for the optimization of the process to gain the highest degree of hydrolysis. pH, temperature, time, enzyme concentration, interaction of temperature-enzyme concentration, square of pH, temperature, time and enzyme concentration had significant effects on the process. The R2 = 0.95, lack of fit

Different regulation processes have an effect on osteoblastic differentiation of mesenchymal stem cells (MSCs), and among them Wnt signaling pathway is particularly desirable. In Wnt signaling pathway, Adenomatous Polyposis Coli (APC) bind to β-catenin and induce its degradation, thereby acting as a negative regulator of canonical Wnt pathway. In this study, gene expression and DNA methylation of APC gene during osteoblastic differentiation were determined.

In this experimental study, after the isolation of MSCs, the induction of osteoblastic differentiation was done. To confirm osteoblastic differentiation, alizarin red staining together with the expression of Alkaline Phosphatase (ALP) and osteocalcin as specific osteoblastic markers was performed. APC gene methylation status by MSP (Methylation Specific PCR) and gene expression status of APC gene using Real-Time PCR technique during different times were evaluated.

The results of alizarin red staining and the expression of ALP and osteocalcin confirmed osteoblastic differentiation. In addition, the results showed a significant decrease in the expression of APC gene on the 7th day of osteoblastic differentiation (P

It seems that the decreased expression APC gene will play an important role in Wnt signaling pathway regulation in different stages during osteoblastic differentiation of bone marrow-derived MSC. Also, according to the results, APC gene promoter methylation will play an important role in controlling gene expression.

Obesity is now considered as one of the main risk factors of certain known diseases such as cardio-vascular diseases، non- insulin-dependent diabetes، and common cancers. Moreover، the increase of white fat tissue is known as a main factor in the obesity process، in terms of physiology and pathology. Therefore، the understanding of adipocytes differentiation processes is crucial.

In this study، mesenchymal stem cells (MSCs) were isolated from human bone marrow by ficol-gradient، and then، their surface markers were confirmed by flow cytometry. Osteoblastic and adipocytes differentiation were done by dexamethasone protocol and confirmed by staining. Then qualitative and quantitative expressions of PPARgamma (PPAR-γ) gene as an important transcription factor in the differentiation of fat were studied by RT-PCR and REAL TIME PCR before and after differentiation into adipocytes. For statistical analysis، paired t-test was performed، using pfaffl and graph pad software.

PPAR-gamma gene expression showed a significant increase after differentiation of human bone marrow mesenchymal stem cells into adipocytes (p<0/05).

According to the results، the PPAR-γ gene acts as one of the important factors in the differentiation of MSCs into adipocytes. In brief، the inhibition of this gene''s expression to prevent obesity is suggested as an idea for treatment in the future.

Understanding the molecular mechanisms involved in the increased levels of HbF inducing drugs should be advised for effective induction. The aim of this study was to investigate the molecular effects of the drugs thalidomide and sodium butyrate considered as HbF inducer agents. In this experimental study, CD133+ cord blood stem cells carrying mutations of heterozygous β-thalassemia were isolated and differentiated into erythroid lineage. In order to evaluate the expression of the erythroid markers, CD71 and CD235a, was analysed. For this purpose, the RNA extracted from erythroid precursors at days 6 and 12 of erythroid differentiation and cDNA synthesized, and then the expression of these genes was performed by quantitative Real-time PCR technique. The results of this study showed the significant effect of thalidomide on erythroid proliferation as compared to sodium butyrate and control group (P<0.05). Also, thalidomide significantly increased CD71expression and decreased CD235a expression as compared to sodium butyrate and control groups (P<0.05). Thalidomide may play its role on HbF induction by increasing the proliferation of early erythroid precursors.

Bipolar I disorder is a common disorder with a complex etiology. A genetic approach is gaining increasing importance in this disorder. The dysbindin gene، located at 6p22. 3 is considered a susceptibility gene for schizophrenia. Certain genotypes of dysbindin are thought to be associated with other psychoses such as bipolar disorders. This study intends to assess the association in previously implicated dysbindin genotypes and haplotypes with bipolar I disorder in an Iranian population. We genotyped four previously reported SNPs: rs2619522، rs1018381، 2743852 and rs2619538. Their haplotypes were analyzed in a population of 124 patients that consisted of 44 confirmed bipolar I disorder patients and 80 control subjects. We used multiple allele-specific PCR method for genotyping، which was verified by direct sequencing. In concordance with previous reports in other populations our findings showed no association between the single SNPs and bipolar I disorder. Furthermore، none of the alleles showed a significant association with the disorder. In contrast to previous reports، haplotype analysis did not reveal any statistically significant associations with bipolar I disorder. Considering reports of previous studies regarding the implication of these dysbindin genotypes in bipolar I disorder، it is probable that allelic heterogeneity along with lack of an established causal variant in the dysbindin gene can be main factors for this discordance. With regards to ethnicities in other studies، population variation can also be considered an important factor in the observed variation.

Epigenetic reprogramming of somatic cells into embryonic stem cells has attracted much attention, because of the potential for stem cell transplantation and compatibility with recipient. However, the therapeutic application of either nuclear transfer or nuclear fusion of somatic cell has been hindered by technical complications as well as ethical objections. Recently, a new method is reported whereby ectopic expression of embryonic specific transcription factors was shown to induce fibroblasts to become embryonic like SCs (induced pluripotent stem cells). A major limitation of this method is the use of potentially harmful genome integrating viruses such as reto- or lentivirus. The main aim of this investigation was generation of human hematopoietic stem cells from induced fibroblasts by safe adenovectors carrying embryonically active genes. Isolated fibroblasts from foreskin were expanded and recombinant adenoviruses carrying human Sox2, Oct4, Klf4, cMyc genes were added to culture. After formation of embryonic like colonies and cell expansion, they were transferred to embryonic media without bFGF, and embryoid bodies were cultured on stromal and non-stromal differentiation media for 14 days. Expression of CD34 gene and antigenic markers, CD34, CD38 & CD133 in stromal culture showed significant difference with non-differentiation and non-stromal media. These findings show high hematopoietic differentiation rate of Adeno-iPS cells in stromal culture and no need to use growth factors. While, there was no difference between non-differentiation and non-stromal media.

Human spermatogonial stem cells (SSCs)، are the foundation of spermatogenesis. Because of low number and lack of significant marker in human SSCs، studying their characteristics، could provide better understanding about the biology of male fertility. This study was designed to examine the effects of in vitro co-culture with sertoli cells on SSC colonization and germ cells specific gene expression of human spermatogonial stem cells. Testicular cells were isolated from testis biopsies by using two step enzymatic digestion and differential plating. two culture system were designed: co-culture with patient Sertoli cells and culture of SSC without co-culture (as control group). The number and diameter of colonies were evaluated during 3 weeks of culture. The expression of alpha 6 integrin، beta1 integrin and PLZF، as germ stem cell specific markers، was assessed using quantitative RT-PCR. Statistical analysis was performed using one way ANOVA in SPSS vesion 16 software with 95% Confidence interval. Our results were showed that the number and diameter of colonies increased significantly in co-culture with sertoli cells (P<0. 05). The expression profile of genes in 2nd and 3rd weeks of culture revealed that there is significant higher expression of germ stem cell markers in our co-culture group versus control group. Based on the optimal effects of sertoli cells on spermatogonial stem cells، co culture of the human SSCs with the feeder layer sertoli may be used as a suitable method for the enrichment of human spermatogonial stem cells.

A recent field of research in epigenetics is DNA methylation which involves the CpG island in the genome that subsequently controls transcription and translation of targeted genes. In the hepatitis B virus (HBV) genome، there are three CpG islands which tend to be methylated. The aim of the current study is to determine the methylation pattern of the HBV X gene in chronically infected HBV patients. Study participants comprised 45 chronically infected HBV patients. According to the presence of the HBeAg، patients were divided into two groups، HBeAg positive (n=24) and HBeAg negative (n=21). Initially، viral DNA was treated with natrium bisulfate. Then، analysis was performed with two sets of methylated and non-methylated primers by the MSP method. The overall methylation rate in serum samples of hepatitis B infected patients was 35. 5%; the rate in the HBeAg positive patients was 20. 8%، whereas it was 52. 3% in HBeAg negative patients. There was a significantly higher rate of methylation in serum samples of HBeAg negative patients compared to HBeAg positive patients (student''s t-test; P=0. 02). Methylation of HBV can be used as a new mechanism to control the progression of viral infection. This methodology can be useful for determining the characteristics of clinical stages of this infection.

cancer/testis genes (CT-genes) are a family gene which only express in normal testis tissue; some of them are randomly expressed in some types of cancers. The aim of this study was to evaluate the frequency of expression of CT-genes in the patients with breast cancer.

Thirty two breast cancer tissue samples were prepared. Expression of NY-ESO-1 1a، NY-ESO-1 1b، SCP1، SSX-2 and MAGE-3 genes، as well as GAPDH (internal control)، were studied by multiplex RT-PCR method.

Three (9%) of 32 tumor samples expressed mRNA of NY-ESO-1 1a، while six (19%) of 32 tumor samples expressed mRNA of NY-ESO-1 1b. Seven (22%) and two (6%) of 32 tumor samples expressed mRNA of SCP1 and mRNA of MAGE3، rtepectively. Overall، Thirteen (41%) samples expressed one of the studied genes.

NY-ESO-1 and SCP1 genes had the highest frequency of expression of mRNA. It is suggested that more number of breast cancer tumor samples should be examined to evaluate expression of CT-genes. SCP1 and NY-ESO-1 proteins may promote future breast cancer immunotherapy.

syndrome is a very rare hereditary lethal skeletal dysplasia. There are few reports of milder phenotypes that patients were survived till late childhood. Radiologic investigations showed increased bone density and osteosclerosis. The increased density of bones in head and face causes a characteristic dysmorphic features that includes: prominent and narrow forehead، proptosis، small hypoplstic nose with depressed nasal bridge، mid-face hypoplasia، triangular moth، coanal atresia، and intracranial cerebral calsification. Osteosclerosis is severe enough that would be mistaken with osteopetrosis. Raine syndrome is a hereditary autosomal recessive disease. The cause of it is a homozygous or compound heterozygous mutation in the FAM20C gene. The gene encodes a phosphorylase-kinase which is responsible for biomineralization of skeleton. Here we report a typical patient of this syndrome from Iran، and to our knowledge this is the first confirmed one in our population. Molecular analysis showed a homozygous novel mutation in the FAM20C gene. This patient is the 17th reported case in the relevant literature.

Specific differentiation processes to various cell lineages are closely associated with factors such as transcription factors، tumor suppressor elements and internal signaling pathways including vHL، Ecad، and Runx3. Epigenetics is an effective control mechanism of these factors، including several mechanisms such as methylation and acetylation. The main objective of this study was discovering the expression status of vHL، Runx3 and Ecad genes and determining the methylation patterns in the respective genes in hematopoietic cord blood stem cells. After isolating the stem cells from cord blood، expansion of the desired cells and DNA purification from the cells، in the first stage cDNA was constructed from the RNA، and PCR was performed for the genes under study. In later stages، the processed DNA from the previous stages was used for MSP reaction. After PCR using designed primers to be used in Real-Time PCR and RNA derived cDNA of stem cells، it was found that all the genes under study were expressed in CD34+ stem cells. MSP in another stage of the study showed that Ecad and Runx3 genes are partially methylated and partially expressed، and vHL gene is not methylated and is completely expressed in this stage. There is substantial agreement between gene expression and epigenetic alterations، and study of all the three genes indicates their effective role in biology and function of these stem cells in Hematopoietic stem cells.

Cell signaling elements such as P15 and P16 play an important role in differentiation of primitive cells into various cell types. These factors are involved in controlling gene expression by different mechanisms, of which epigenetics especially methylation is noteworthy. The main objectives of this study were discovering the expression status of target genes in CD34+ cord blood stem cells and determining the methylation changes in the respective genes is this same stage. After collection of cord blood bags and purification and proliferation of stem cells, cellular DNA was isolated. In later stages, cDNA and Bisulfite treated DNAs were synthesized from RNA and DNA of primary cells, respectively. Polymerase Chain Reaction and Methylation Specific PCR were later performed for both genes. After MSP, it was found that the P15 has partial methylation and expression in CD34+ stem cells, and P16 lacks methylation in this stage and is completely expressed. The results of PCR indicated expression of both genes in CD34+ stem cells. Gene expression profile of each tissue or cell is in accordance with its functions; the specific expression of P15 and P16 can indicate a possible role of these genes in biology of CD34+ stem cells in umbilical cord blood. There is always substantial agreement between expression of a gene and its epigenetic changes.

RUNX2 is the most specific transcription factor in osteoblastic differentiation of MSCs. In this research، RUNX2 expression was quantified in MSCs differentiated by osteogenic differentiation medium (ODM) and zoledronic acid (ZA). In this experimental study، hMSCs were treated by osteogenic differentiation medium and ZA. RNA extraction was carried out from both osteoblastic differentiated cells in the first، second and third weeks of differentiation، and also from undifferentiated MSCs. RUNX2 expression was quantified by quantitative Real Time-PCR. Gene expression of RUNX2 in the first، second and third weeks of osteogenic differentiation by ODM compared to undifferentiated MSCs showed 1. 76±0. 09-fold، 3. 54±0. 25-fold and 3. 40±0. 17-fold increase in expression، respectively. Zoledronic acid increased the expression of RUNX2 2. 91±0. 13-fold، 3. 25±0. 3-fold and 3. 36±0. 23-fold at the same time points، respectively. Comparison of RUNX2 expression by ODM (1. 76±0. 09-fold) and ZA (2. 91±0. 13-fold) in the first week of differentiation showed statistical differences (P<0. 05). Whereas، RUNX2 expression by both ODM and ZA in the second and third weeks of differentiation were approximately equal. RUNX2 expression was increased in osteoblastic differentiation by both ODM and ZA. However، it seems that ZA can cause more expression of RUNX2 in the first week of osteoblastic differentiation.

Utilization of fetal hemoglobin inducing drugs is of crucial importance in novel therapeutic approach of β-thalassemia and sickle cell disease. These drugs induce fetal hemoglobin production by several mechanisms such as increasing the proliferation and differentiation of erythroid progenitors. Thalidomide and sodium butyrate are two fetal hemoglobin inducers used as single and combination treatments.This research performed on erythroid progenitors derived from CD133+ cord blood stem cells. Flow cytometry was used for evaluation of the homogeneity of isolated CD133+ cells. Then, the colony formation potential of defined treated groups was evaluated using colony formation assay. Flow cytometry analysis showed the homogeneity of isolated CD133+ cord blood stem cells more than 95%. Also, the results of colony formation assay showed the potential of thalidomide in increasing the hematopoietic and erythroid colony count (1.3- and 1.5- fold increase, respectively) as compared to the control (p<0.05). The results of this study showed that thalidomide has a high potential in colony formation without cytotoxic effects on erythroid progenitors as compared to sodium butyrate and combination treatment (p<0.05).

Background and ObjectivesFLT3 mutations are associated with poor outcome in acute myeloblastic leukemia(AML) patients. Only limited information is available about effects of FLT3 mutation on Acute Promyelocytic Leukemia (APL). We investigated the prevalence and impact of FLT3 mutations on the clinical characteristics and the response to treatment in APL patients treated with arsenic trioxide (As2O3).Materials and MethodsBlood samples were collected from 115 untreated APL patients and genomic DNA was extracted by the salting-out method. FLT3-ITD and FLT3-D835 mutations were investigated by PCR-RFLP. Mann-Whitney U test and Chi-square were used for data analysis. ResultsFLT3-ITD and FLT3-D835 mutations were detected in 16 (14%) and 13(11%) of the patients, respectively. Both mutations were identified in two patients, so overall frequency of FLT3 mutations was estimated to be 23.5%. Patients positive for FLT3–ITD mutation had a higher rate of white cell counts (p= 0.005) and more frequent bcr3 type of PML/RARA fusion (p=0.04). We have not found any significant association between FLT3-D835 mutation and the clinical characteristics of patients. Between the group with FLT3 Mutations and the group without, there was no significant difference in response to therapy. ConclusionsComplete remission induction with As2O3 may be independent of FLT3 mutation status, so As2O3 may be the first choice of APL especially in patients with FLT3 mutations. However, further studies on a large group of patients are necessary to confirm our findings.

Estrogen receptor alpha protein status is determined by routine immunohistochemistry analysis in all malignant breast tumors. This assay has its limitations. RNA based techniques are potential complements for immunohistochemistry but it must be noticed that gene silencing may occur at different levels from RNA to protein. The aim of this study was the comparison of the results from these two assays and characterizing the tumors subgroup in which gene expression occurs at RNA level but the target protein is absent. 92 primary breast tumors including their clinical and IHC results were collected before treatment. Estrogen receptor gene expression of tumors was studied by Reverse Transcription Polymerase Chain Reaction (RT PCR). In this assay, GAPDH was used as a reference gene. 36.6 % of tumors with negative estrogen receptor protein showed gene expression at mRNA level. In this subgroup most of the patient were older than 50 years and in stages 3 or 4 of breast cancer and had poor prognosis according to Nottingham prognostic index. Most cases of the perineural invasion have been seen in this subgroup. It seems that RT-PCR assay would enable us to recognize a subgroup of breast tumors with poor prognosis which expresses RNA but not protein.

Low density Lipo-protein Receptor- related Protein (LRP) is the most important cholesterol receptor in neurons. It serves as a receptor for APOE protein which is the most important risk factor for Alzheimer’s Disease. LRP also contributes to the ligation of lipoproteins with APOE in neurons. Association between LRP C766T and Alzheimer’s disease in Iranian patients with late onset Alzheimer’s disease (LOAD) was investigated in this research. 100 patients with LOAD were selected based on DSM-IV-TR and NINCDS-ADRDA diagnostic criteria and 100 normal controls without any personal and family history of Alzheimer’s disease or dementia were included in this case- control study. AD patients and control subjects were matched for age and sex. PCR-RFLP was set up to detect LRP C766T polymorphism. LRP C/C genotype and C allele distribution were more frequent in AD patients than in control subjects. However, this difference was not statistically significant. When association between LRP C/C genotype and AD was categorized by the gender, in both genders, there was not any significant correlation. Our findings indicate that 766C allele of LRP gene could not significantly alter the risk of developing late-onset Alzheimer's disease in Iranian patients. Analysis of other genetic factors and environmental factors are promoted in Iranian population.

Induction of fetal hemoglobin (HbF) is one of the most promising therapies of β-thalassemia and sickle cell disease, as HbF permanent induction can substantially ameliorate the clinical symptoms of these genetic disorders. Several classes of therapeutics have been implemented to induce γ-globin expression in beta-thalassemia patients and subsequently to raise total hemoglobin levels. Sodium butyrate and Talidomide are two of the known agents. This study was conducted on erythroid cells differentiated from cord blood CD133 + stem cells. Four groups were designed and gamma globin was induced by thalidomide and sodium butyrate treatment (100 μM concentrations). Gamma gene expression was evaluated using Quantitative Real Time PCR techniques. Among the treated groups with sodium butyrate and thalidomide, it was shown that although these two agents both affected the induction of gamma and beta genes, thalidomide had a greater potency on beta cluster genes than sodium butyrate. Our results showed that the pharmaceutical regime of thalidomide and sodium butyrate could be useful for the treatment of β-Thalassemia and sickle cell patients because of the induction of the beta gene cluster.

Cancer development is not restricted to the genetic changes, but also to epigenetic changes. Epigenetic processes are very important in the development of hematological malignancies. The main epigenetic alterations are aberrations in DNA methylation, post-translational modifications of histones, chromatin remodeling and microRNAs patterns, and these are associated with tumor genesis. All the various cellular pathways contributing to the neoplastic phenotype are affected by epigenetic genes in cancer. These pathways can be explored as biomarkers in clinical use for early detection of disease, malignancy classification and response to treatment with classical chemotherapy agents and epigenetic drugs. A literature review was performed using PUBMED from 1985 to 2008. Cross referencing of discovered articles was also reviewed. In chronic lymphocytic leukemia, regional hypermethylation of gene promoters leads to gene silencing. Many of these genes have tumor suppressor phenotypes. In myelodysplastic syndrome (MDS), CDKN2B (alias, P15), a cyclin-dependent kinase inhibitor that negatively regulates the cell cycle, has been shown to be hypermethylated in marrow stem (CD34+) cells in patients with MDS. At present both Vidaza and Decitabine (DNA methyltransferase inhibitors) are approved for the treatment of MDS. Unlike mutations or deletions, DNA hypermethylation and histone deacetylation are potentially reversible by pharmacological inhibition, therefore those epigenetic changes have been recognized as promising novel therapeutic targets in hematopoietic malignances. In this review, we discussed molecular mechanisms of epigenetics, epigenetic changes in hematological malignancies and epigenetic based treatments.

Bone sialoprotein (BSP) is a specific marker of osteoblastic differentiation. In this research, the effect of Zoledronic Acid on BSP expression and methylation status during osteoblastic differentiation of mesenchymal stem cells (MSCs) was evaluated. In this experimental study, MSCs were isolated from human bone marrow. For osteogenic differentiation, hMSCs were pulse treated with zoledronic acid, and were incubated in osteogenic differentiation medium for 3 weeks. The DNA and RNA were extracted after the first, second and third weeks of culture and also from undifferentiated MSCs. After Sodium bisulfate (SBS) treatment, gene specific methylation analysis for BSP was carried out using Methylation Specific PCR technique. BSP expression was observed in osteoblastic differentiated cells whereas it was not seen in MSCs. MSP showed that BSP was unmethylated during osteoblastic differentiation. BSP was expressed from the first week of differentiation. This confirms that zoledronic acid accelerates osteoblastic differentiation. Unmethylation status of BSP indicates that zoledronic acid does not have any effect on BSP methylation status. Other genetic or epigenetic mechanisms may control BSP expression during osteoblastic differentiation induction by zoledronic acid.

The study of erythropoiesis needs to develop the methods for erythroid progenitor cells (EPCs) culture using stem cell potency to differentiate into variety of hematopoietic cells lineages. In this study, we induced differentiation of cord blood stem cells into erythroid progenitor cells in a semisolid culture media. Cord blood mononuclear cells were isolated and cultured in liquid culture media consist of erythroid differentiation factors (phase I). Non-adherent cells were cultured in semisolid media containing SCF, IL-3, IL-6 and EPO (phase II). After one week, the appeared erythroid colonies were picked up. Characterization of differentiated cells was performed by assessment of CD235a and CD36 using flowcytometry, giemsa cytological staining and gene expression analysis of GATA-1, EKLF, α-globin genes by RT-PCR. Flowcytometry analysis to detect CD235a and CD36 positive cells showed that 94.3% and 95.5% of differentiated cells have erythroid specific cell markers, respectively. Morphology of the cells in giemsa stained slides demonstrated erythroid progenitor cells, ranged from proerythroblast to orthochromatic erythroblast. The RT-PCR results, confirmed the precursor state of erythroid differentiated cells by expression of GATA-1, EKLF, α-globin genes. Cord blood stem cells have high potency to differentiate into erythroid progenitor cells using described method that can be utilized in the experimental studies.