Cell Journal (Yakhteh)
Volume:12 Issue: 4, Winter 2011

During the past decade, regenerative medicine has emerged as a key technology in the next generation of medical care, and cell therapy and organ repair using stem cells have become very attractive options for regenerative medicine. The application of stem cells in regenerative medicine has required modified methods for isolation. Furthermore, the process of cell separation plays an important role in cell therapy and regenerative medicine using stem cells. So, in this review, we compare different methods for the separation of cells from bone marrow for transplantation to humans, with emphasis on the advantages and disadvantages of each method.

Basement membranes are specialized extracellular matrices which play important roles such as cell regulation, proliferation and migration. Collagen fibers, especially type IV, are the most important basement membrane constituents. As retina is one of the target organs in diabetes mellitus, and nephropathy is a major cause of end stage renal and retinal diseases resulting in increased morbidity and mortality, early diagnosis leads to better treatment. Hence, in this investigation, the appearance and distribution of collagen IV during gestational days and early postnatal periods were observed. 24 intact female Balb/c mice were kept under normal conditions. After mating, appearance of a vaginal plug was assumed as day zero of the pregnancy. From days 13-18 of gestation, the pregnant mice were euthanised and their embryos as well as pups from days 1 to 5 were collected. For histochemichal studies, heads of the specimens were fixed, serially sectioned and immunohistochemicall studies were performed by using monoclonal antibodies for tracing of collagen type IV. Our findings revealed that the amount of collagen IV in the internal limiting membrane (ILM) and extra cellular matrix (ECM) of the retina, as well as vessels of the vitreus body appear on embryonic day 16. Also, a patchy distribution was observed in the pigmented epithelium which continued to further develop until the end stage of embryonic life. Strong labeling was observed until postnatal day 3 but did not increase significantly thereafter. These findings establish the importance of collagen IV during the critical period of retinal development. In addition, this study indicates that high levels of collagen IV are present in the basal membrane (BM) of the inner limiting membranes and pigmented epithelium (3rd post natal on the 3rd postnatal day.

The production of heterologous proteins in Escherichia coli is strongly affected by codon bias. This phenomenon occurs when the codon usage of mRNA coding for the foreign protein differs from that of the bacterium. The ribosome pauses upon encountering a rare codon and may detach from mRNA, thereby the yield of recombinant protein production reduces. The aim of this study is to investigate the effect of these codon numbers reductions on the recombinant protein production. Since most amino acids are encoded by more than one codon, codons were changed in order to their usage in a special host such as E. coli without any transformation in amino acids sequence. Silent mutations in 5' codons of human basic fibroblast growth factor cDNA carried out by site-directed mutagenesis and the expression level of the recombinant protein is analyzed by means of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Expression level in mutant and wild-type genes indicated a considerable difference. In contrast with the remarkable bands of wild-type gene in all the strains particularly in codon plus strain, there were no significant bands related to mutant gene in SDS-PAGE analysis. Because of the same conditions of mutant and wild-type genes during the translation and transcription, this significant difference may relate to mRNA efficiency for translation. Our results indicate that increased stability of 5' mRNA secondary structures in E. coli prevents efficient translation initiation. Furthermore, wild-type gene significant bands in codon plus strain support the hypothesis that the possible elimination of translational pauses that increase translation rate leads to over expression.

Melatonin is a powerful endogenous antioxidant and it may play a role in prevention of radiation-induced damage. The aim of this study was to investigate the effect of melatonin on bone mineral density in rats receiving radiation. Sprague Dawley rats were divided into four groups. Group 1 (control group) received neither melatonin nor radiation (control group). Group 2 (Mel group) was administered intraperitoneal injections of 5mg/kg melatonin daily for ten days. Group 3 (RT group) and Group 4 were exposed to total cranium radiation of 5 Gy in a single dose by using a cobalt-60 teletherapy unit. In addition to irradiation, group 4 (RT + Mel group) was administered 5mg/kg of melatonin intraperitoneally. At the end of the 10th day, the rat's cranium and vertebrae bone mineral densities (BMDs) were measured. When cranial BMDs were evaluated, statistically more significant BMD increases were seen in the Mel group and the RT + Mel groups than in the control group. No significant difference was seen in the Mel group versus the RT + Mel group; however, there was a significant difference between RT and RT + Mel groups. When vertebral BMDs were evaluated, the only significant difference was found between the control and Mel groups. We think that melatonin is a radioprotective agent. However, we would like to emphasize that further studies are needed before clinical trials with melatonin are initiated.

To determine the effect of orthodontic tooth movement on the expression of interleukin-1β mRNA in rats using RT-PCR. Sample consisted of eighteen 8-week-old male Wistar rats. The right maxillary first molar of each animal was protracted using an orthodontic protraction appliance. The left maxillary first molar received no treatment and was assigned as the control group. On day 21, all rats were sacrifice and divided in two equal groups. The first group, group (A), was histologically evaluated for the presence and size of potential resorptive lacunae. The second group, group (B), was investigated using RT-PCR in order to determine IL-1β mRNA expression. Measurements revealed that the mean tooth movement was 0.23 mm in group A and 0.24 mm in group B. The mean depth of the resorptive lacunae was 0.17×10-11 mm2 in the control group and 4.9×10-11 mm2 in the intervention group (control group: left maxillary first molars; right maxillary first molars were divided to group A & B, histologic study of group A assures the existence of resorptive lacunae and its extent relative to control group). The difference between the two groups was statistically significant (p<0.05). The RT-PCR evaluation showed no significant differences in IL-1β mRNA expressions of resorptive lacunae between the treated and untreated groups. Although interleukin1-beta is the most potent stimulator of bone resorption and mediator of inflammatory response, the present study showed that the IL-1beta mRNA was not expressed more significantly in root resorption lacunae of the treated molars relative to the control group.

Nowadays, bone constructs elaborated according to tissue engineering principles are being regarded as an ideal choice for the reconstruction of segmental bone defects. In this study, proliferation and bone differentiation of marrow-derived mesenchymal stem cells (MSCs) were compared in different composite scaffolds containing varying morphologies of nano hydroxyapatite (nHAP). Needle nHAP/PLLA (poly (L-lactide acid)), spherical nHAP/PLLA and rod nHAP/PLLA scaffolds were prepared and 3D cultures of passaged-3 rat MSCs were established using the scaffolds. The loading of the cells onto the scaffold internal spaces was confirmed with microscopy and their proliferation was determined by MTT assay. To compare the osteogenic differentiation of the cells on the scaffold surfaces, osteogenic 3D cultures were established and kept for 21 days. At the end of this period culture mineralization and relative bone-related gene expression were quantified using the alizarin red quantification assay and semi quantitative RT-PCR analysis respectively. ANOVA was used to compare the data. According to the MTT assays, cells adhered to all the studied scaffold surfaces tended to proliferate. In this respect the microenvironment provided by the needle nHAP/PLLA appeared much better than that of either the spherical or rod nHAP/PLLA scaffolds (P<0.05). Similarly, mineralization was observed to be heavier for the needle nHAP/PLLA scaffold compared to the two other composite scaffolds. In addition, the relative expression of coll I, osteocalcin, runx2 and ALP genes all appeared to be significantly higher in the cells cultivated on needle nHAP/PLLA scaffolds versus their spherical and rod counterparts. Overall, needle nHAP/PLLA scaffolds appear to provide the most appropriate matrix for producing bone construct using MSCs.

The significant factor contributing to the distant invasion of cancer cells is the ability of tumors to produce large numbers of new blood vessels, known as angiogenesis. Many natural products inhibit angiogenesis. Herein, ethanol extract of Salvia officinalis (SO) has been analyzed for its anti-angiogenic, anti-proliferation and anti-migration activities. The anti-angiogenic effect of the SO extract was evaluated on chicken chorioallantoic membrane (CAM) neovascularisation model, microscopically. The inhibitory effect of the extract on human umbilical vein endothelial cells (HUVECs) migration was tested on the wound-healing model with an inverted microscope. In addition, SO extract was screened for its possible anti-proliferative effects by separately counting HUVECs, Wehi and K562 cells with cell counter against their control wells. So extract exhibited a significant inhibitory activity in CAM assay in a dose dependent manner. CAM angiogenesis was gradually prevented to from at 100 µg/ml of SO extract, but completely inhibited to form at 200 µg/ml. After human umbilical vein endothelial cells (HUVECs) were suppressed by dose-dependent SO extract, their migrations were detected by wound healing model, yet they were unable to show a dose response effect on proliferation of the different cells (50-200 µg/ml). As observing in this study, SOextract could inhibit proliferation of the different cells at the concentrations above 200 µg/ml without toxic effect on the cells in doses ranged from 0-500 µg/ml. These findings indicated that SO extract might be a promising candidate for anti-angiogenic treatment.

The human papillomavirus as an etiological agent of cervical cancer does not grow adequately in tissue culture systems. The tumor cell line TC-1 continuously expresses the E6 and E7 oncogenic proteins of HPV, and is considered a suitable tool in laboratory investigations and vaccine researches against cervical cancer. The TC-1 cell line was grown in RPMI 1650 supplemented with 10% FBS, glutamine and antibiotics, and was used for tumor development in mice. Six to seven week-old tumor bearing C57BL/6 mice were divided into 3 groups consisting of 7 mice per group. The first group received pcDNA-E7, the second group received pcDNA3, and the third group received phosphate buffered saline (PBS). The treated animals were monitored for their tumor size progression and survival. At last, the tumoric tissues from autopsied animals were fixed and examined with Mayer's hematoxylin and eosin (H&E). All experiments were done in accordance with guidelines of the Laboratory Animal Ethical Commission of Tarbiat Modares University. Data analysis was performed using the one-way ANOVA followed by Tukey's test in both experimental and control groups. A p-value <0.05 was considered significant. There were significant decreases in tumor growth; there were also improvements in survival among mice in the treated groups (p<0.041). H&E stained sections from untreated mice were studied independently in a blinded fashion by two observers and showed malignant neoplasms composed of severely pleomorphic tumor cells with nuclear enlargement, high nuclear-cytoplasmic (N/C) ratios, and prominent nucleoli in solid and fascicular patterns of growth. High mitotic activity with extensive necrosis was also noted in both test and control groups. The TC-1 lung metastatic model can be used to test the efficacy of various E7-based therapeutic cancer vaccine strategies for cervical cancer and the prevention of HPV-related neoplasia.

Previous studies have shown that morphine consumption during pregnancy may delay embryo development or cause abnormal nervous system function. The present study focused on the effects of maternal morphine consumption on brain cavities and central canal development in Wistar rats. In this study Wistar rats (average weight: 170-200 g) were used. The experimental group, after pregnancy, received 0.05 mg/ml of morphine by tap water while the control group received water. On the 17th day of pregnancy, the pregnant animals were anesthetized by chloroform and embryos were surgically removed. The samples were fixed in 10% formalin for four weeks. Then, tissues were processed and sectioned. Sections were stained with hematoxylin and eosin (H&E) and examined for ventricle, central canal and choroid plexus development by light microscopy and MOTIC software. Severe reductions of the third and lateral ventricles were observed in the experimental group. In addition, an increase in the choroid plexus (CP) area in the experimental group with regards to the control group was identified. The study showed that oral morphine consumption lead to reduction in the third and lateral brain cavities and an increase in the CP area. This defect may cause behavioral changes observed in the F1 generation from addicted pregnant animals.

Plant extracts are of considerable interest because of their antiepileptic activities. However, the mechanisms of action are not clearly defined. Here, the effects of Artemisia dracunculus L. (tarragon) leaves extract on excitability and electrophysiological characteristics of snail neurones were investigated, using an intracellular recording technique. Application of tarragon extract (0.05%) resulted in complete disappearance of paroxysmal depolarization shift (PDS) as elicited by pentylenetetrazol (PTZ), an epileptogenic drug. It also significantly decreased the firing frequency and shifted the firing pattern from bursting in the presence of PTZ to an irregular doublet activity. Changes in excitability properties were associated with a significant increase and decrease in the duration of action potential, and in the amplitude of after-hyperpolarization (AHP), respectively. When tarragon extract was applied alone, spontaneous activity became irregular and was interrupted by large inhibitory postsynaptic potentials (IPSPs), which disappeared following application of picrotoxin (100 µM). Tarragon also caused a significant decrease both in the amplitude of action potentials and AHP, and broadened the action potentials. However, pretreatment with extract did not prevent the induction of epileptiform activity by PTZ. The findings suggest that tarragon extract may affect membrane ion channels and/or GABAA receptors leading to a reduction in neuronal excitability.

Chronic hypoxia exists in many diseases, including cancer. The subject of our study is analysis of molecular pathways affected in the chronically hypoxic mouse brain. Using the emPAI protocol, we performed a quantitative proteomic approach to characterize the global proteome in the mouse brain exposed to 7% O2 for 48 hours. Utilizing the emPAI protocol to estimate protein abundance and assign molar concentrations to all proteins, we were able to identify 33 proteins with significant changes in their expression. Deregulated proteins were mainly involved in cell metabolism, apoptosis, Ca2+ signaling, pentose phosphate pathway, 14-3-3 protein mediated signaling cascades and protein degradation. The obtained data will provide some clues for understanding mechanisms with which cells respond and adapt to chronic hypoxia.

Members of the transforming growth factor-β superfamily, including bone morphogenetic protein 4 (BMP4), have been implicated as regulators of neural differentiation. The aim of this study was to establish whether BMP4 could influence neuronal differentiation of mesenchymal stem cells (MSCs). Therefore, neuronal differentiation of MSCs was induced by basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) and treatment. The expression of neuronal specific markers such as Nestin, MAP2, β-Tubulin III and NKX6.1 were detected by RT-PCR, flow cytometery and/or immunostaining. While the percentage of Nestin positive cells was increased significantly during treatment, the addition of BMP4 during the first 4 days of treatment with bFGF and EGF reduced Nestin expression as showed by flow cytometry. This observation was further confirmed by relative gene expression which showed the reduction in expression of neural markers such as Nestin, MAP2 and NKX6.1 following treatment with BMP4. The results of this study suggest that BMP4 downregulates the neural fate of induced mouse MSCs.

This study is an attempt to examine the transdifferentiation of bone marrow stromal cells (BMSCs) into tyrosine hydroxylase immunoreactive cells in parkinsonian rats associated with angiogenesis. In this study, Sprague-Dawley rats received unilateral stereotaxic injections of 6-hydroxydopamine(6-OHDA) into the left corpus striatum and then were divided into two groups. One group, the negative control, received only medium while the other group was treated with BMSCs. BMSCs were harvested from femur bones, labeled with bromodeoxyuridine (BrdU) and then transplanted into parkinsonian rats, where a behavioral study and immunohistochemistry were used to evaluate the treatment. The results showed statistically significant improvement in rotational behavior. Anti-BrdU antibody showed engraftment of the transplanted cells at the transplantation site. Additionally, double immunolabeling confirmed that these cells were positive for neurofilament-200 and tyrosine hydroxylase (TH). It may be concluded that BMSCs transplants could engraft and differentiate into TH immunoreactive cells which may cause recovery from motor deficits. Also, BMSCs may contribute to angiogenesis at the transplantation site.

Angiogenesis, the formation of new blood vessels, is a key process in cancer development and metastasis. In this study, the anti-angiogenic and anti-proliferative potentials of Ficus carica latex extract have been investigated using human umbilical vein endothelial cells (HUVECs).Different doses of latex extract were prepared and added to a three-dimensional culture of HUVEC in a collagen matrix. After 3-5 days of treatment, the anti-angiogenic effects of the extracts were monitored microscopically. For the anti-proliferation assay, different doses of the extracts were examined on HUVECs.The results clearly indicated that latex extract could inhibit proliferation and capillary tube formation of HUVECs in a dose-dependent manner at the range of 100-400 µg/ml. In addition, the extract was not cytotoxic up to 450 µg/ml as assessed by trypan blue and lactate dehydrogenase (LDH) cytotoxicity assays.It is concluded that latex extracts of F. carica contain strong anti-angiogenic and anti-proliferative activities. Our data indicates that latex extract could be a candidate as a potential agent for the prevention of angiogenesis in cancer and other chronic disorders.