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Differentiation of human bone marrow mesenchymal stem cells to neural- like cells in vitro
Nemati Sh,Zare Mehrjerdi N,Baharvand H
Tehran University Medical Journal , 2009,
Abstract: "n Normal 0 false false false EN-US X-NONE AR-SA MicrosoftInternetExplorer4 /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-qformat:yes; mso-style-parent:""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin:0in; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri","sans-serif"; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"Times New Roman"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:Arial; mso-bidi-theme-font:minor-bidi;} Background: Human bone marrow mesenchymal stem cells (hMSCs) can differentiate into several types of mesenchymal cells, including osteocytes, chondrocytes, and adipocytes, but can also differentiate into non-mesenchymal cells, such as neural cells, under appropriate experimental conditions. Until now, many protocols for inducing neuro-differentiation in MSCs in vitro have been reported. In this study, we induced differentiation into neural phenotype in the hMSCs population by new protocol. In this treatment, hMSCs could express neural markers more than other reports, associating with remarkable morphological modifications. "n"nMethods: The Bone marrow specimens were aspirated from the iliac crest of normal men. hMSCs were isolated and cultured in DMEM containing 15% FBS. Between 4-8 passages conversion of hMSCs into neurosphere-like structures and induction this cells to nerve precursors in the low-attachment plastic bacterial dishes with bFGF, EGF & RA were initiated. After seven days terminal neural differentiation was initiated by plating the cells on poly-L-ornithin and Laminin coated dishes. Cells were differentiated for 7-14 days. We used flowcytometry and immunocytochemistry analysis for assessment of specific neural stem cell markers in induced cells."n"nResults: Flowcytometery analysis showed that after induction, 90±2.52 percent of the cells will express neuronal marker Nestin and about 41±1 percent of the cells will express Tuj-1 and about 67±1.05 percent of the cells will express GFAP. Immunocytochemistry and morphologically modifications revealed the same results. "n"nConclusion: Results showed that hMSCs treatment with bFGF, EGF & RA the number of Tuj1 neurons. These data confirmed that hMSCs can exhibit neuronal differentiation potential in vitro, depending on the protocols of inducement.
Differentiation of human embryonic stem cells into insulin- secreting cells
H Baharvand,H Jafary,M Massumi,S Mollamohammadi
Journal of Shahid Sadoughi University of Medical Sciences , 2006,
Abstract: Introduction: Type I diabetes mellitus is caused by autoimmune destruction of the insulin-producing β-cells. A new potential method for curing the disease is transplantation of differentiated insulin- secreting cells from human embryonic stem cells. Methods: Human embryonic stem cell lines (Royan H1) were used to produce embryoid bodies. Differentiation carried out by growth factor-mediated selection of nestin positive cells. In final stage, these cells were expanded in the presence of bFGF, followed by addition of nicotinamide to promote differentiation of insulin- secreting cells. Cells were assayed by immunocytochemistry, RT-PCR, insulin secreting assay with Radio-immuno assay kit and Transmission Electron Microscopy. The cells were transplanted into immunosuppressed mice. Results: Analysis of differentiation cells immunocytochemistry showed that these cells were insulin, glucagon, somatostatin and pancreatic polypeptide positive. RT-PCR reaction demonstrated the expression of pancreatic endocrine genes. Differentiation cells secreted insulin in response to glucose, but no significance difference in insulin concentration was observed with an increase in concentration of glucose. The implanted cells were vascularized and remained immunoreactive with insulin and glucagon. Transmission Electron microscopy of differentiate cells showed Golgi complexes, rough endoplasmic reticulum and a few granules but no true β granules. Conclusion: The data showed that human embryonic stem cells can produce insulin secreting cells. However, more studies are needed to generate true beta cells.
In-Vitro Differentiation Of Human Embryonic Stem Cells Into Hemangioblasts
F Ganji,S Abruon,H Baharvand,M Ebrahimi
Tehran University Medical Journal , 2012,
Abstract: Background: Human embryonic stem cells (hESCs) are capable of self-renewal and large-scale expansion. They also have the capacity to differentiate into a variety of cell types including liver, cardiac and neuron cells. However, it is not yet clear whether hESCs can differentiate to hemangioblasts under in-vitro conditions. Hemangioblasts are bipotential progenitors that can generate hematopoietic lineages and endothelial cells. The aim of this study was to identify the potential of human Royan H5 embryonic stem cells in differentiating into hemangioblast cells. Methods: HESCs were cultured at suspension system in DMEM/F12 supplemented with bFGF. 7-day old cells differentiated into blast cells under defined condition consisting of hematopoietic cytokines including BMP4, VEGF, etc. Blast cell markers kinase insert domain receptor (KDR), CD31, and CD34 were evaluated by flow cytometry and blast gene expressions (TAL-1, Runx-1 and CD34) were detected by qRT-PCR. Clonogenic assays were performed in semisolid medium by colony forming unit-assays. Results: The hESCs (Royan H5) had the capacity of differentiating into hemangioblast cells. We could detect colonies that expressed 79%±12.5 KDR+, 5.6%±2.8 CD31+-CD34+ and 6%±2.12 KDR+-CD31+ on day 8 in the hESCs. Up-regulation of TAL-1, Runx-1 and CD34 occurred during hemangioblast commitment (P≤0.05 and P≤0.01, respectively). Moreover, hemangioblast cells generated mixed-type and endothelial-like colonies in semi-solid media. Conclusion: Our results showed that hESCs (Royan H5) were able to differentiate into hemangioblasts under in-vitro conditions. The hemangioblasts had the potential to generate two non-adherent (Mixed-type) and adherent (endothelial-like) cell populations.
The comparison of ultrastructural development of mouse embryonic stem cell-derived cardiomyocytes and normal invivo cardiomyocytes
H Baharvand,R Rohani,A Piryaei,A Taei
Journal of Shahid Sadoughi University of Medical Sciences , 2006,
Abstract: Introduction: Stem cell biology has been the subject of much recent discussion. Embryonic stem (ES) cells, derived from the inner cell mass of the blastocyst stage of early mammalian embryos are expected to become a powerful tool in future regenerative medicine and developmental biology due to their capacity of self-renewal and pluripotency. In the present study, the ultrastructural development of mouse ES cell derived cardiomyocytes was compared with invivo cardiomyocytes.. Methods: Cardiomyocytes were derived from mouse ES line (Royan B1) which developed spontaneously. The cultured cardiomyocytes were processed 3, 7, 14 and 21days after plating (day 7) for immuno histochemistry and transmission electron microscopy (TEM). The in vivo cardiomyocytes were derived from16 days old fetuses and 2 and 8 days old pups. Results: The beating cells expressed α-actinin. The maturation of the ultrastructure of cardiomyocytes depended on enhancement of development and expressed as myofibrillar bundle organization and exhibited intercalated discs, Z-disc, A, I, and H-bands, and M-line. While 7+21 days old cardiomyocytes showed all sarcomeric components such as A, I, and H-bands, Z-disc, and also M-line, T-tubule, sarcoplasmic reticulum and intercalated discs, early stage (7+3d, 7+7d and 7+14d) cardiomyocytes had few primary characteristics of subcellular structure. In fetal and 2-days old pups, the M-line was not visible. M-line was present in 8-days old pups frequently. Conclusion: Based on our data, mature cardiomyocytes can be produced from ES cells, and ES cell provide a good model for cardiomyocyte development. The cells can be used for cell therapy in future.
Matrix Metalloproteinase’s Role in Producing and Curing of Liver Fibrosis
Vahideh Rabani,Hossein Baharvand
Cell Journal , 2009,
Abstract: Matrix metalloproteinases are a zinc and calcium dependent endopeptidase family that areexpressed in injured tissue such as cardiovascular or hepatic disease. Complex efforts of thisenzymes on the extra cellular matrix structure is related to up and down regulation of themand their tissue inhibitors. Configuration of extra cellular matrix during pathogenesis, curingand development is affected by two key mechanisms: matrix metalloproteinase and hepaticstellate cell activity. The important role of these enzymes on liver injuries and regenerationare indicated when their effects on migration of bone marrow stem cells and hepatic stemcells was discoverd.
Management of Vascular Malformations in Dangerous Area of Oral Cavity: The Iranian Experience and Review of Treatment Modalities  [PDF]
Somayeh Alirezaei, Maryam Baharvand, Masood Rezaei, Arash Azizi, Bita Tavakoli
Open Journal of Stomatology (OJST) , 2014, DOI: 10.4236/ojst.2014.43018
Abstract: Aim: To evaluate the effect of a highly potent corticosteroid (dexamethasone) in the treatment of vascular malformation when the location is difficult to reach and complications such as uncontrolled bleeding is predictable in surgery. Background: Vascular malformation is not a common lesion in oral cavity especially in alveolar ridge with extension to the pillar uvula. These lesions arise from capillary or venous malformations with various surgical or non-surgical treatment modalities. Case Description: We performed weekly intralesional injection of dexamethasone in a patient with a vascular malformation in alveolar ridge extending to the lingual side of alveolar ridge and posterior extension to the uvula. Complete resolution of lesion was observed after 6th injection. Conclusion: Intralesional injection of dexamethasone is a potentially curative method to treat oral vascular malformation. Clinical Significance: Injection of dexamethasone is a simple and cost-effective therapy that can be used as a safe treatment for vascular malformations prior to or as a substitute for surgery.
Construction of expression vectors carrying mouse peroxisomal protein gene (PeP) with GST and Flag labels
MN Jahantigh, K Ghaedi, MHN Isfahani, S Tanhaei, F Rabiee, KH Karbalaei, MO Sharif, M Nematollahi, H Baharvand, SH Razavi, M Miroliaei
African Journal of Biotechnology , 2009,
Abstract: The aim of this study was to construct expression vectors carrying mouse peroxisomal protein gene (PEP-cDNA) in prokaryotic and mammalian expression vectors in chimeric cDNA types, encompassing GST and FLAG with PEP-cDNA. PEP-cDNA was sub-cloned in pGEX6p2 prokaryotic expression vector in order to label this gene with GST to purify PEP protein for further biochemical analysis and identifying related proteins thereafter. FLAG-PEP recombinant DNA was produced and sub-cloned in pUcD3 eukaryotic expression vector to express tagged-PEP protein for transient transfection analysis and identifying intracellular localization of PEP protein in future experiments. PEP-cDNA was amplified in different PCR reactions using pEGFP-PEP vector and 2 sets of primers introducing specific restriction sites at the ends of PEP. PCR products with BamHI/SalI restriction sites were treated by restriction enzymes and inserted into the pGEX6p2, downstream of GST tag. PEP-cDNA containing BamHI/ApaI restriction sites and FLAG gene (which amplified using pUcD3-FLAG-PEX3 vector) were used as templates in secondary PCR for amplifying FLAG-PEP recombinant DNA. FLAG-PEP fragment was treated by enzymatic digestion and inserted into the pUcD3 eukaryotic expression vector. pGEX6p2-PEP and pUcD3-FLAG-PEP constructed vectors were transformed into the one shot TOP10 and JM105 bacterial competent cells, respectively. Positive colonies were selected for plasmid preparation. Results confirmed correct amplification of the expected products. PEP-cDNA in both PCR reactions encompasses 630 bp. FLAG fragment containing designed sites was 77 bp and FLAG-PEP fragment was 700 bp. Sequencing of constructed vectors confirmed that PEP-cDNA was tagged appropriately and inserted free of mutation and in frame with GST and FLAG.
Induction of Human Embryonic Stem Cells into neuronal differentiation by increasing cyclic Adenosine Mono Phosphate
Hossein Baharvand,Bijan Hatami,Mohammad Massumi
Physiology and Pharmacology , 2006,
Abstract: Introduction: To evaluate the cAMP -mediated IBMX (3-IsoButyle -1-Methyl Xanthin) and db-cAMP (dibutyryl cAMP) effects on differentiation of human Embryonic Stem Cells (hESCs) into nerve cells were the objectives of this study. Methods: We have used Royan H1 hESC- derived embryoid bodies with four treatment groups: six days treatment with IBMX (5×10 -4M) and db-cAMP (10 -9M) (referred to as cAMP), retinoic acid (RA, 10 -6M), IBMX + db-cAMP + RA, and control (no treatment). Immunocytochemistry was carried out for neural specific antibodies including β-Tubulin III, Microtubule Associated Protein 2 (MAP-2), Neurofilament Protein-Heavy chain (NF-H), Glial Fibrilary Acidic Protein (GFAP) and Synaptophysin as well as morphological studies. Semiquantitative RT-PCR was also used to evaluate gene expression involved in neurogenesis. Results: In the 4+6+4 days the neuronal process were apparently observed. Immunocytochemical studies using nerve specific antibodies for proteins such as β- Tubulin III, MAP-2, NF-H, GFAP and Synaptophysin showed the presence of these neuronal and astrocyte markers in differentiated cells by cAMP. Evaluation of expression of genes involved in neurogenesis showed that Hash1, Synaptophysin, β-Adrenergic Receptor and Acetylcholine Receptor- which were silent in embryoid bodies - switched on after treatment with cAMP and/or RA. Relative expression of nerve specific genes showed a significant enhancement in expression of Synaptophysin, NFM and β-Adrenergic Receptor during differentiation, which, with the enhancement in cAMP treated groups were more than those treated with RA and control (p < 0.05). Conclusion: In conclusion, this study showed that cAMP could be a neurogenic agent for human embryonic stem cells differentiation.
The Effect of Low Level Laser Irradiation on Human Embryonic Stem Cells
Hossein Baharvand,Masoud Soleimani,Hamid Gourabi
Cell Journal , 2005,
Abstract: Introduction: Different effects of low level laser irradiation (LLLI) on various cell types have already been demonstrated. However, its effects on embryonic stem cells have not yet been shown. The present study evaluates the morphological and immunocytochemical effects of LLLI on human embryonic stem cell (hESC) colonies. Material and Methods: Equal-sized pieces of hESC line (Royan H1) were irradiated with a single dose of 830-nm Ga-Al-As diode laser (3, 5, and 8 jcm-2, 30mW) and cultured on mouse embryonic fibroblasts. The morphology of the colonies was evaluated qualitatively by observation under an inverted microscope (grades A, B, C, and D exhibited 0-30%, 30-50%, 50-80%, and 80-100% differentiation, respectively). The stemness area was assessed by expression of surface antigens using anti-Tra-1-60 and anti-Tra-1-81. Results: Our data demonstrated a dose-dependent stimulatory effect of LLLI on hESC differentiation. Two doses of 5 and 8jcm-2 induced statistically significant differentiation (grades C and D). Conclusions: These data showed that LLLI influenced hESC differentiation, which might be used for cell therapy after transplantation
Methods for Isolation of Bone Marrow Stem Cells: Comparative Analysis
Mehrnaz Namiri,Hossein Baharvand,Nasser Aghdami
Cell Journal , 2011,
Abstract: During the past decade, regenerative medicine has emerged as a key technology in thenext generation of medical care, and cell therapy and organ repair using stem cells havebecome very attractive options for regenerative medicine. The application of stem cells inregenerative medicine has required modified methods for isolation. Furthermore, the processof cell separation plays an important role in cell therapy and regenerative medicineusing stem cells. So, in this review, we compare different methods for the separation ofcells from bone marrow for transplantation to humans, with emphasis on the advantagesand disadvantages of each method.
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