Elevated expression of DNMT1 is associated with increased expansion and proliferation of hematopoietic stem cells co-cultured with human MSCs

BACKGROUND: Mesenchymal stem cells (MSCs) play an important role in hematopoietic stem cell (HSC) maintenance, proliferation, and apoptosis. DNA methyltransferase 1 (DNMT1) is considered an essential factor in the maintenance of HSCs in mammalian cells. Therefore, this study was conducted to evaluate the mRNA expression level of DNMT1 during cord blood (CB)-HSC ex vivo expansion with MSCs. METHODS: Ex vivo cultures of CB-HSCs were performed in three culture conditions for 7 days: cytokines, cytokines with MSCs, and only MSCs. Total and viable cell numbers were counted after 5 and 7 days using trypan blue stain, and the stem cell percentage was then evaluated by flow cytometry. Moreover, in vitro colony-forming unit assay was carried out to detect clonogenic potential of HSCs at days 0 and 7 using MethoCult H4434. Finally, DNMT1 mRNA expression level was evaluated by real-time polymerase chain reaction. RESULTS: Maximum CB-CD34⁺ cell expansion was observed on day 7 in all the three cultures. After 7 days, ex vivo expansion of CB-CD34⁺ cells indicated a significant decrease in DNMT1 expression in the cytokine cultures, whereas in the two co-culture conditions DNMT1 expression was increased. A significant difference between the number of CD34⁺ and CD34⁻ cells in the cytokine co-culture system was observed. CONCLUSION: These data indicated that an elevated expression of DNMT1 is associated with increased expansion and proliferation of HSCs co-cultured with human MSCs. Hence, DNMT1 may be a potential factor in the maintenance of expanded HSCs co-cultured with human MSCs.

immunosuppressive activity of amniotic membrane mesenchymal stem cells on T lymphocytes

Mesenchymal Stem Cells [MSCs] are isolated from different sources like placenta. The placenta and its membranes like Amniotic Membrane [AM] are readily available and easy to work with. There is only limited knowledge on the immunomodulatory properties of human Amniotic Membrane-derived Mesenchymal Stem Cells [hAM-MSCs]. The aim of this study was to survey the suppressive activity of hAM-MSCs on T lymphocytes in vitro. Human AMs were obtained after caesarean section births from healthy women. After enzymatic digestion, cells were cultured and hAM-MSCs were obtained. In addition, human T lymphocytes were isolated and co-cultured with hAM-MSCs for 72 hr in the presence or absence of phytohemagglutinin [PHA]. Subsequently, proliferation of T cells was analyzed using BrdU and subsequently flow cytometry technique. Besides, the production of IL-4 and IFN-gamma was examined by ELISA method. Additionally, the expression of activation markers [CD38, HLA-DR] was studied on T lymphocytes by flow cytometry technique. It was revealed that hAM-MSCs could significantly suppress the proliferation of T lymphocytes [p

[Comparison of three cord blood processing methods: hydroxyethyl starch, simple centrifugation and Sepax automated system]

Background: Different processing methods are being used to improve the quality of hematopoietic stem cell transplantation. Using hydroxyethyl starch, simple centrifugation and Sepax automation, this study was aimed to compare these three conventional methods.

Material and Methods: 90 cord blood samples were taken and processed by hydroxyethyl starch, simple centrifugation and Sepax automation methods. Then they were subjected to total nucleated cell [TNC] counting and CD34 positive counting as well as colony assay. Finally, all data were analyzed using one-way analysis of variance [ANOVA] and ps less than 0.05 were considered statistically significant.

Results: The TNC recoveries in hydroxyethyl starch, simple centrifugation and Sepax automation methods were 76%, 71% and 80%, respectively [p> 0.05]. The CD34+ cell recoveries in the Sepax automation and in the other two methods were 91% and 85%, respectively [p> 0.05]. Also, the colony assay recoveries were not significantly different among the three methods [p> 0.05].

Conclusion: No significant difference was seen in TNC number, CD34 positive counting and colony formation among the three different methods.

[TGF-b downregulation by RNAi technique in ex vivo-expanded HSCs on 3D DBM scaffold]

Bone Marrow Transplantations [BMT] are limited by low CD34+ cell counts in umbilical cord blood [UCB] and these cells need to be expanded for success in such procedures. To achieve this goal, ex vivo expansion of hematopoietic stem cells [HSCs] by enhancing their self-renewal activity on demineralized bone matrix [DBM] scaffold coated with mesenchymal progenitor cells [MPCs] and unrestricted somatic stem cells [USSCs] was recommended. TGF-b pathway is a key inhibitory factor for HSCs self-renewal. In this study ex vivo expansion and downregulation of TGF-b pathway were simultaneously performed. USSC cells were isolated from UCB and then coated on DBM scaffold as a feeder layer. UCB CD34+ cells were isolated from UCB by magnetic activated cell sorting [MACS] method and were transfected by siRNA against TGFbR2 in two-dimensional [2D] and three-dimensional [3D] cultures by co-cultivation with USSC. TGFbR2 expression levels were evaluated by quantitative real-time PCR. Cell count and flow cytometry were performed and clonogenic activity was evaluated. Ex vivo expansion of CD34+ cells was significantly enhanced [41 +/- 0.7 folds] by TGFbR2 downregulation, especially in 2D than 3D cultures. Finally, 2D culture showed less TGFbR2 expression levels and higher increase in the percentage of CD34 markers by flow cytometry assay. The 3D siRNA delivery system would be of lower efficiency in contrast to 2D settings where the cells have less freedom and are in more contact with the feeder layer

[TGF-bReceptor2 knocked down by SiRNA increases cord blood CD34+ HSCs self-renewal]

Nowadays, cord blood Hematopoietic stem cells [HSCs] are known as a valuable source for bone marrow transplantation but unfortunately their insufficient number is a limiting factor for using them in adult bone marrow transplantation. Cord blood HCSs expansion is an approach to overcome this problem, by inducing their self-renewal. TGF-b signaling pathway is a key inhibitory agent for HSCs self-renewal. In this study, we tried to enhance self-renewal of long term culture initiating cell by inhibiting TGFbR2 expression. CD34+ HSCs were isolated from cord blood units with MACS column. SiRNA against TGFbR2 was transfected by Lipofectamine [TM] RNAiMAX as transfection reagent. HSCs were cultured in IMDM medium containing 10% FBS and early acting cytokines [Flt3L, SCF, Tpo] for 8 days. Then we evaluated TGFbR2 expression by QRT-PCR. The CD34+ subpopulation of cultured cells were examined by flow cytometry on the 8th day. Finally the expanded cells were evaluated for the presence of early hematopoietic stem cells by LT-CIC and clonogenic assays. According to our results, TGFbR2 down regulation increases CD34+ subpopulation of HSCs. In addition, LT-CIC assay showed an enhancement in primitive hematopoietic stem cell capable of self-renewal. All in all, it seems that positive regulators have attracted more attention in the field of HSCs expansion while negative regulators have same importance in self-renewal process of HSCs and their inhibition can be a beneficial tool for enhancement of HSCs self-renewa

[ effects of p-benzoquinone and hydroquinone on the RUNX2 gene expression and osteoblastic differentiation of human marrow derived mesenchymal stem cells]

Evaluating the effects of p-benzoquinone and hydroquinone on the RUNX2 expression and osteoblastic differentiation of human marrow derived mesenchymal stem cells [MSCs]. Bone marrow MSCs obtained by cultivating marrow mononuclear cells, were exposed to 10?M of either p-benzoquinone or hydroquinone. Following chemical treatment, RUNX2 gene expression was assessed by Real-time RT PCR 1, 6, 24 and 48 hours later and osteogenic differentiation was analyzed using alizarin red and alkaline phosphatase staining methods on days 7 and 14 after ostegenic induction. RUNX2 expression was significantly elevated [up to approximately 8 times] due to chemical exposure but the applied chemicals exert no considerable effect on MSCs osteogenic differentiation. According to the literature, despite the necessity of RUNX2 overexpression on the induction of osteogenic differentiation, but it is not sufficient for osteogenesis to occure so increase in RUNX2 expression observed in our study is not the indicator of the induced osteogenic differentiation. Instead, this elevated expression could be the sign of increased activity of the canonical Wnt signaling pathway thereby its involvement in the development of AML due to exposure to benzene and its metabolites. Moreover, this augmented expression of RUNX2 in MSCs can indicate the RUNX2 overexpression in myeloid progenitors as an expected similar effect of exposure to benzene and its metabolites to contribute in myeloid malignancies developed due to benzene exposure

miR-155 down Regulation by LNA inhibitor can reduce cell growth and proliferation in PC12 cell line

MicroRNAs [miRNAs] are a class of small non coding regulatory RNAs that have key functions in multiple cell processes. Deregulation of these tiny miRNAs are involved in various human diseases. MiR-155 is one of the multifunctional miRNA that its over-expression has been found to be associated with different kinds of cancer such as leukemia, breast and colon cancers. It is thought that deregulation and over-expression of this microRNA may be associated with PC12 cell proliferation. So, the aim of this study was to investigate the role of miR-155 expression on PC12 cell growth. For this reason, PC12 cells were cultured and transfected by 3 different concentration [25, 50 and 75 nmol] of either LNA anti-miR-155 or scramble antisense in 24-well plate. Then, total RNA was extracted from transfected cells. miRNA cDNAs were synthesized from isolated total RNA. In the second step, miR-155 expression level was analyzed using the quantitative real-time polymerase chain reaction [QRT-PCR]. MTT test was performed to evaluate cell viability. In the next step, apoptosis assay was assessed to investigate anti miR-155 effect on PC12 cells death. Obtained results were analyzed with t-test. MTT test revealed that cell viability of transfected cells with 75 nM of anti-miR- 155 to be reduced by half of the control and scramble groups [0.5 vs. 0.97 and 0.94]. Our data suggest that miR-155 over-expression is associated with PC12 cell growth. So, miR-155 down regulation by anti-miR-155 could open up new ways to restrain brain tumor growth, as anti-miR-155 causes PC12 cells to repress

Molecular spectrum of beta-globin mutations in transfusion-dependent patients with thalassemia in Qazvin province, Iran

Beta thalassemia is a common inherited disease, resulting from one or more of 200 different mutations in the beta-globin gene. Qazvin province has attracted migrations of several different populations due to industrialization during the past five decades. The aim of this study was to define the molecular spectrum of beta-thalassemia mutations in Qazvin province Ethylen diamin acetic acid-containing venous blood samples were collected from 100 patients with transfusion-dependent beta-thalassemia from the department of Pediatrics in Qods hospital. Age, sex, history, and consanguinity between the parents were recorded by reviewing the patients' files. DNA was isolated from leukocytes using the standard procedure. Amplification refractory mutation system [ARMS] technique was used for molecular detection of mutations. Direct sequencing analysis was applied for DNA samples when no mutation was detected with ARMS. Of the 200 chromosomes investigated, 11 types of mutations were identified by ARMS technique while direct sequencing revealed the remaining alleles [9 types of mutations]. Total 20 different mutations discovered by this two-step approach. Abundant alleles [IVS II-1, IVS I-10, FSC 8/9] accounted for 59.3% of the mutations. IVS II-1 with a frequency of 31.3% was the most common while HbS, Cd 74/75 and Cd 15, each with a frequency of 0.55%, had the least frequencies. Beta thalassemia mutations are very heterogeneous in Qazvin province. Extensive ethnic and genetic admixture has resulted in unexpectedly high number of different mutations, most of them similar to that of north and north-western provinces of Iran. Different mutations in this region suggest migration of chromosomes from distant places and genetic admixture

Development of a novel three-dimensional biocompatible nanofibrous scaffold for the expansion and hepatogenic differentiation of human bone marrow mesenchymal stem cells

In this present study, we examined the differentiation potential of human bone marrow derived mesenchymal stem cells [hBMSCs] into hepatocytes on a three-dimentional [3D] nanofibrous scaffold formed by Poly [e-caprolactone] [PCL], collagen and polyethersulfone [PES]. The nanofiber was prepared by the electrospining technique. HBMSCs were isolated using combining gradient density centrifugation with plastic adherence. Flow cytometric analysis was used to identify the isolated MSCs. The performance of the cells on the scaffold was evaluated by scanning electron microscopy [SEM] and MTT assay. The hBMSCs were then cultured in a hepatic differentiation medium containing hepatocyte growth factor [HGF], oncostatin M [OSM] and dexamethasone [DEX] for up to 21 days. The results showed that the isolated hBMSCs expressed specific markers such as CD44, CD166, CD105 and CD13. The integrity of the MSCs was further confirmed by their differentiation potential to osteogenic and adipogenic lineages. Scanning electron micrographs and MTT analysis revealed that the cells adhered and proliferated well on the nanofibrous hybrid scaffolds. Immunocytochemical analysis of albumin and a-fetoprotein [AFP] showed the accumulation of these markers in the differentiated cells on the scaffold. Hepatocyte differentiation was further confirmed by showing expression of albumin, AFP and cytokeratin-19 [CK-19] at mRNA levels in differentiated cells. In conclusion, the evidences presented in this study show that the engineered scaffold is promising for maintenance of hepatocyte-like cells suitable for transplantation