Transplanting P75-Suppressed Bone Marrow Stromal Cells Promotes Functional Behavior in a Rat Model of Spinal Cord Injury

Background: Bone marrow stromal cells (BMSC) have been successfully employed for movement deficit recovery in spinal cord injury (SCI) rat models. One of the unsettled problems in cell transplantation is the relative high proportion of cell death, specifically after neural differentiation. According to our previous studies, p75 receptor, known as the death receptor, is only expressed in BMSC in a time window of 6-12 hours following neural induction. Moreover, we have recently reported a decreased level of apoptosis in p75-suppressed BMSC in vitro. Therefore, our objective in this research was to explore the functional effects of transplanting p75:siRNA expressing BMSC in SCI rats. Methods: Laminectomy was performed at L1 vertebra level to expose spinal cord for contusion using weight-drop method. PBS-treated SCI rats (group one) were used as negative controls, in which cavitations were observed 10 weeks after SCI. pRNA-U6.1/Hygro- (group two, as a mock) and pRNA-U6.1/Hygro-p75 shRNA- (group three) transfected BMSC were labeled with a fluorescent dye, CM-DiI, and grafted into the lesion site 7 days after surgery. The Basso-Beattie-Bresnehan locomotor rating scale was performed weekly for 10 weeks. Results: There was a significant difference (P≤0.05) between all groups of treated rats regarding functional recovery. Specifically, the discrepancy among p75 siRNA and mock-transfected BMSC was statistically significant. P75 siRNA BMSC also revealed a higher level of in vivo survival compared to the mock BMSC. Conclusion: Our data suggest that genetically modified BMSC that express p75:siRNA could be a more suitable source of cells for treatment of SCI. Key Words: Spinal cord injury (SCI), Apoptosis, Bone marrow stromal cells (BMSC)     

Microglial Activation in Rat Experimental Spinal Cord Injury Model

Background: The present study was designed to evaluate the secondary microglial activation processes after spinal cord injury (SCI). Methods: A quantitative histological study was performed to determine ED-1 positive cells, glial cell density, and cavitation size in untreated SCI rats at days 1, 2, and 4, and weeks 1, 2, 3, and 4. Results: The results of glial cell quantification along the 4900-µm long injured spinal cord showed a significant increase in glial cell density percentage at day 2 as compared to other days. Whereas the highest increase in ED-1 immunoreactive cells (monocyte/phagocyte marker in rats) was observed at day 2 (23.15%) post-injury. Evaluation of cavity percentage showed a significant difference between weeks 3 and 4 post-injury groups. Conclusions: This study provides a new insight into the multiphase immune response to SCI, including cellular inflammation, macrophages/microglia activation, glial cell density, and cavitation. Better understanding of the inflammatory processes associated with acute SCI would permit the development of better therapeutic strategies. Key Words: Spinal cord injuries, Inflammation, Microglia, Macrophages     

Effect of oleuropein on tissue myeloperoxidase activity in experimental spinal cord trauma

Background: Neutrophil infiltration plays an important role in inflammatory reactions following spinal cord injury (SCI) and these cells cause substantial secondary tissue damage. The purpose of this study was to determine the effect of oleuropein (OE) on myeloperoxidase (MPO) activity as an index of neutrophil infiltration. Methods: Rats were randomly divided into four groups of 7 rats each as follows: sham-operated group, trauma group, and OE treatment groups (20 mg/kg, i.p., immediately and 1 hour after SCI). Spinal cord samples were taken 24 hours after injury and studied for determination of MPO activity. Results: The results showed that MPO activity was significantly decreased in OE-treated rats. Conclusion: On the basis of our findings, we propose that OE may be effective in protecting rat spinal cord from secondary damage by modulating of neutrophil infiltration.Key Words: Oleuropein (OE), Neutrophil infiltration, Myeloperoxidase     

Differentiation of Umbilical Cord Lining Membrane-Derived Mesenchymal Stem Cells into Endothelial-Like Cells

Background: Stem cell therapy for the treatment of vascular-related diseases through functional revascularization is one of the most important research areas in tissue engineering. The aim of this study was to investigate the in vitro differentiation of umbilical CL-MSC into endothelial lineage cells. Methods: In this study, isolated cells were characterized for expression of MSC-specific markers and osteogenic and adipogenic differentiation. They were induced to differentiate into endothelial-like cells and then examined for expression of the endothelial-specific markers, karyotype, and functional behavior of cells. Results: Isolated cells expressed MSC-specific markers and differentiated into adipocytes and osteoblasts. After endothelial differentiation, they expressed CD31, vWF, VE-cadherin, VEGFR1, and VEGFR2 at both mRNA and protein level, but their morphological changes were not apparent when compared with those of undifferentiated cells. There were no significant changes in karyotype of differentiated cells. Furthermore, angiogenesis assay and LDL uptake assay showed that differentiated cells were able to form the capillary-like structures and uptake LDL, respectively. Conclusion: The results indicated that umbilical CL-MSC could differentiate into functional endothelial-like cells. Also, they are suitable for basic and clinical studies to cure several vascular-related diseases. Key Words: Endothelial differentiation, Endothelial-like cells, Mesenchymal stem cells, Umbilical cord lining membrane     

Healing potential of mesenchymal stem cells cultured on a collagen-based scaffold for skin regeneration

Background: Wound healing of burned skin remains a major goal in public health. Previous reports showed that the bone marrow stem cells were potent in keratinization and vascularization of full thickness skin wounds. Methods: In this study, mesenchymal stem cells were derived from rat adipose tissues and characterized by flowcytometry. Staining methods were used to evaluate their differentiation ability. A collagen-chitosan scaffold was prepared by freeze-drying method and crosslinked by carbodiimide-based crosslinker. Results: The results of immunecytochemistry and PCR experiments confirmed the adipose-derived stem cells (ASC) in differentiation to the keratinocytes under the treatment of keratinocyte growth factor. The isolated ASC were seeded on the scaffolds and implanted at the prepared wounds. The scaffolds without cells were considered as a control and implanted on the other side of the rat. Histopathological analyses confirmed the formation of new tissue on the scaffold-cell side after 14 days with the formation of dermis and epidermis. Conclusion: These results indicated the capacity of ASC in differentiation to keratinocytes and also wound healing in vivo. Key Words: Tissue engineering, Keratinocytes, Mesenchymal stem cells     

Chondrogenic differentiation of rat mesenchymal stem cells on silk fibroin/chondroitin sulfate/hyaluronic acid ternary scaffolds

Cartilage repair is a challenge in bone tissue reconstruction. In this study, silk fibroin (SF), chondroitin sulfate (CS) and hyaluronic acid (HA) were employed to fabricate scaffolds for tissue engineered cartilage by freeze drying technique. The secondary pores were formed in the main pores of SF/CS/HA scaffold which improved the pore connectivity and equilibrium swelling of the scaffold. Furthermore, rat bone marrow mesenchymal stem cells were seeded on the scaffolds to evaluate the cell adhesion and proliferation. Results of hematoxylin/eosin staining and cell counting kit-8 assay showed that the cells migration and differentiation of SF/CS/HA (80/15/5) scaffold were better than that of SF/CS/HA scaffolds with different ratios after 7 days culture. Moreover, immunohistochemistry and scanning electron microscope demonstrated that large amounts of collagen II and proteoglycans of the cells were expressed in the SF/CS/HA 3D scaffold, while the expression of collagen I was barely visible by immunohistochemistry. Abound of extracellular matrix was formed to morphologically round and distributed uniformly throughout the scaffolds. The 3D ternary scaffold could promote the cells chondrogenic differentiation without using any inductive agent and offer potential for cartilage tissue regeneration.     

Effects of valproic acid, a histone deacetylase inhibitor, on improvement of locomotor function in rat spinal cord injury based on epigenetic science

Background: The primary phase of traumatic spinal cord injury (SCI) starts by a complex local inflammatory reaction such as secretion of pro-inflammatory cytokines from microglia and injured cells that substantially contribute to exacerbating pathogenic events in secondary phase. Valproic acid (VPA) is a histone deacetylase inhibitor. Acetylation of histones is critical to cellular inflammatory and repair processes. Methods: In this study, rats were randomly assigned to five experimental groups (laminectomy, untreated, and three VPA-treated groups). For SCI, severe contusion was used. In treated groups, VPA was administered intraperitoneally at doses of 100, 200 and 400 mg/kg daily three hours after injury for 7 days. To compare locomotor improvement among experimental groups, behavioral assessments were performed by the Basso, Beattie and Bresnahan (BBB) rating scale. The expression of neurotrophins was evaluated by RT-PCR and real-time PCR. Results: VPA administration increased regional brain-derived neurotrophic factor and glial cell-derived neurotrophic factor mRNA levels. Local inflammation and the expression of the lysosomal marker ED1 by activated macrophages/microglial cells were reduced by VPA and immunoreactivity of acetylated histone and microtubule-associated protein were increased. Conclusion: The results showed a reduction in the development of secondary damage in rat spinal cord trauma with an improvement in the open field test (BBB scale) with rapid recovery.Key Words: Inflammation, Epigenetics, Valproic acid (VPA)     

Glycoconjugates Distribution during Developing Mouse Spinal Cord Motor Organizers

Background: The aim of this research was to study the distribution and changes of glycoconjugates particularly their terminal sugars by using lectin histochemistry during mouse spinal cord development. Methods: Formalin-fixed sections of mouse embryo (10-16 fetal days) were processed for lectin histochemical method. In this study, two groups of horseradish peroxidase-labeled specific lectins were used: N-acetylgalactosamine, including Dolichos biflorus, Wisteria floribunda agglutinin (WFA), Vicia villosa, Glycine max as well as focuse-binding lectins, including tetragonolobus, Ulex europaeus, and Orange peel fungus (OFA). All sections were counterstained with alcian blue (pH 2.5). Results: Our results showed that only WFA and OFA reacted strongly with the floor plate cells from early to late embryonic period of developing spinal cord. The strongest reactions were related to the 14, 15, and 16 days of tissue sections incubated with OFA and WFA lectins. Conclusion: The present study demonstrated that cellular and molecular differentiation of the spinal cord organizers is a wholly regulated process, and α-L-fucose, α-D-GalNAc, and α/β-D-GalNAc terminal sugars play a significant role during the prenatal spinal cord development.Key Words: Development, Glycoconjugates, Spinal cord     

The Role of Biodegradable Engineered Nanofiber Scaffolds Seeded with Hair Follicle Stem Cells for Tissue Engineering

Background

The aim of this study was to fabricate the poly caprolactone (PCL) aligned nanofiber scaffold and to evaluate the survival, adhesion, proliferation, and differentiation of rat hair follicle stem cells (HFSC) in the graft material using electrospun PCL nanofiber scaffold for tissue engineering applications.

Methods

The bulge region of rat whisker was isolated and cultured in DMEM: nutrient mixture F-12 supplemented with epidermal growth factor. The morphological and biological features of cultured bulge cells were observed by light microscopy using immunocytochemistry methods. Electrospinning was used for production of PCL nanofiber scaffolds. Scanning electron microscopy (SEM), 3-(4, 5-di-methylthiazol- 2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, and histology analysis were used to investigate the cell morphology, viability, attachment and infiltration of the HFSC on the PCL nanofiber scaffolds.

Results

The results of the MTT assay showed cell viability and cell proliferation of the HFSC on PCL nanofiber scaffolds. SEM microscopy images indicated that HFSC are attached, proliferated and spread on PCL nanofiber scaffolds. Also, immunocytochemical analysis showed cell infiltration and cell differentiation on the scaffolds.

Conclusion

The results of this study reveal that PCL nanofiber scaffolds are suitable for cell culture, proliferation, differentiation and attachment. Furthermore, HFSC are attached and proliferated on PCL nanofiber scaffolds.Key Words: Nanofiber, Electrospinning, Stem cells, Tissue engineering     

Chitosan-Alginate Biocomposite Containing Fucoidan for Bone Tissue Engineering

Over the last few years, significant research has been conducted in the construction of artificial bone scaffolds. In the present study, different types of polymer scaffolds, such as chitosan-alginate (Chi-Alg) and chitosan-alginate with fucoidan (Chi-Alg-fucoidan), were developed by a freeze-drying method, and each was characterized as a bone graft substitute. The porosity, water uptake and retention ability of the prepared scaffolds showed similar efficacy. The pore size of the Chi-Alg and Chi-Alg-fucoidan scaffolds were measured from scanning electron microscopy and found to be 62–490 and 56–437 µm, respectively. In vitro studies using the MG-63 cell line revealed profound cytocompatibility, increased cell proliferation and enhanced alkaline phosphatase secretion in the Chi-Alg-fucoidan scaffold compared to the Chi-Alg scaffold. Further, protein adsorption and mineralization were about two times greater in the Chi-Alg-fucoidan scaffold than the Chi-Alg scaffold. Hence, we suggest that Chi-Alg-fucoidan will be a promising biomaterial for bone tissue regeneration.     

Neuronal Cell Reconstruction with Umbilical Cord Blood Cells in the Brain Hypoxia-Ischemia

Background: Brain hypoxia-ischemia is a human neonatal injury that is considered a candidate for stem cell therapy. Methods: The possible therapeutic potential of human umbilical cord blood (HUCB) stem cells was evaluated in 14-day-old rats subjected to the right common carotid occlusion, a model of neonatal brain hypoxia-ischemia. Seven days after hypoxia-ischemia, rats received either saline solution or 4 × 10⁵ HUCB cells i.v. Rats in control group did not receive any injection. After two weeks, rats were assessed using two motor tests. Subsequently, rats were scarified for histological and immunohistochemical analyses. Results: Our immunohistochemical findings demonstrated selective migration of the injected HUCB cells to the ischemic area as well as reduction in infarct volume. Seven days after surgery, we found significant recovery in the behavioral performance in the test group (12.7 +/- 0.3) compared to the sham group (10.0 +/-0.05), a trend which continued to day 14 (15.3 ± 0.3 vs. 11.9 ± 0.5, P<0.05). Postural and motor asymmetries at days 7 and 14 in the test group showed a significant decrease in the percentage of right turns in comparison to the sham group (75% and 59% vs. 97% and 96%, P<0.05). Conclusion: The results show the potential of HUCB stem cells in reduction of neurologic deficits associated with neonatal hypoxia-ischemia. Key Words: Hypoxia-Ischemia, Nerve cell, Umbilical Cord Blood     

Conductive 3D structure nanofibrous scaffolds for spinal cord regeneration

The complex nature of spinal cord injuries has provided much inspiration for the design of novel biomaterials and scaffolds which are capable of stimulating neural tissue repair strategies. Recently, conductive polymers have gained much attention for improving the nerve regeneration. In our previous study, a three-dimensional (3D) structure with reliable performance was achieved for electrospun scaffolds. The main purpose in the current study is formation of electrical excitable 3D scaffolds by appending polyaniline (PANI) to biocompatible polymers. In this paper, an attempt was made to develop conductive nanofibrous scaffolds, which can simultaneously present both electrical and topographical cues to cells. By using a proper 3D structure, two kinds of conductive scaffolds are compared with a non-conductive scaffold. The 3D nanofibrous core-sheath scaffolds, which are conductive, were prepared with nanorough sheath and aligned core. Two different sheath polymers, including poly(lactic-co-glycolic acid) PLGA and PLGA/PANI, with identical PCL/PANI cores were fabricated. Nanofibers of PCL and PLGA blends with PANI have fiber diameters of 234±60.8 nm and 770±166.6 nm, and conductivity of 3.17×10^-5 S/cm and 4.29×10^-5 S/cm, respectively. The cell proliferation evaluation of nerve cells on these two conductive scaffolds and previous non-conductive scaffolds (PLGA) indicate that the first conductive scaffold (PCL/ PANI-PLGA) could be more effective for nerve tissue regeneration. Locomotor scores of grafted animals by developed scaffolds showed significant performance of non-conductive 3D scaffolds. Moreover, the animal studies indicated the ability of two new types of conductive scaffolds as spinal cord regeneration candidates.     

Mesenchymal Stem/Stromal-Like Cells from Diploid and Triploid Human Embryonic Stem Cells Display Different Gene Expression Profiles

Background:hESCs-MSCs open a new insight into future cell therapy applications, due to their unique characteristics, including immunomodulatory features, proliferation, and differentiation. Methods:Herein, hESCs-MSCs were characterized by IF technique with CD105 and FIBRONECTIN as markers and FIBRONECTIN, VIMENTIN, CD10, CD105, and CD14 genes using RT-PCR technique. FACS was performed for CD44, CD73, CD90, and CD105 markers. Moreover, these fibroblast-like cells, due to multipotent characteristics, differentiated to the osteoblast. Results:MSCs were derived from diploid and triploid hESC lines using sequential 3D and 2D cultures and characterized with the specific markers. IF showed the expression of FIBRONECTIN and CD105 in hESCs-MSCs. Flow cytometry data indicated no significant difference in the expression of MSC markers after 6 and 13 passages. Interestingly, gene expression profiles revealed slight differences between MSCs from diploid and triploid hESCs. The hESCs-MSCs displayed osteogenic differentiation capacity, which was confirmed by Alizarin red staining. Conclusion:Our findings reveal that both diploid and triploid hESC lines are capable of forming MSCs; however, there are some differences in their gene expression profiles. Generation of MSCs from hESCs, as a non-invasive procedure in large scale, will lend itself for the future cell-based therapeutic applications. Key Words: Human embryonic stem cells, Mesenchymal stem/stromal cells, Regenerative medicine     

Characteristics of Human Endometrial Stem Cells in Tissue and Isolated Cultured Cells: An Immunohistochemical Aspect

Background:

The aim of this study was to investigate the percentage of the stem cells population in human endometrial tissue sections and cultured cells at fourth passage.

Methods:

Human endometrial specimens were divided into two parts, one part for morphological studies and the other part for in vitro culture. Full thickness of human normal endometrial sections and cultured endometrial cells at fourth passage were analyzed via immunohistochemistry for CD146 and some stemness markers such as Oct4, Nanog, Sox2, and Klf4 and the expression of typical mesenchymal stem cell markers CD90, CD105.

Results:

11.88±1.29% of human endometrial cells within tissue sections expressed CD146 marker vs. 28±2.3% of cultured cells, CD90 and CD105 were expressed by functionalis stroma (85±2.4 and 89±3.2%) than basalis stroma (16±1.4 and 17±1.9%), respectively (P<0.05). Oct4 and Nanog-expressing cells comprise 1.43±0.08 and 0.54±0.01% of endometrial stromal cells in endometrial sections vs. 12±3.1% and 8±2.9% of cultured cells, respectively. They reside near the glands in the basal layer of endometrium. Sox2 and Klf4 were not commonly expressed in tissue samples and cultured cells. CD9 and EpCAM were expressed by epithelial cells of the endometrium, rather than by stroma or perivascular cells.

Conclusion:

The human endometrial stem cells and pluripotency markers may be localized more in basalis layer of endometrium. The immunostaining observations of endometrial cells at fourth passage were correlated with the immunohistochemistry data.Key Words: Endometrium, Immunohistochemistry, Mesenchymal stem cells     

The Effect of Melanocyte Stimulating Hormone and Hydroxyapatite on Osteogenesis in Pulp Stem Cells of Human Teeth Transferred into Polyester Scaffolds

Presently, tissue engineering is employed in the restoration and repair of tissue defects. Degradable scaffolds, stem cells and stimulating factors are employed in this method. In this study, the effect of melanocyte-stimulating hormone (MSH) and/or hydroxyapatite (HA) on proliferation, osteoblast differentiation, and mineralization of human dental pulp stem cells (hDPSCs) seeded on PLLA-PCL nanofibrous scaffolds was evaluated. For this aim, PLLA-PCL-HA nanofibrous scaffolds were fabricated using electrospinning method. FE-SEM images exhibited that all nanofibers had bead-free morphologies with average diameters ranging from 150–205 nm. Human DPSCs seeded into PLLA-PCL nanofibers were treated with MSH. Cell viability, proliferation, morphology, osteogenic potential, and the expression of tissue-specific genes were assessed by means of MTT assay, FE-SEM, alizarin red S staining, and RT-PCR analysis. hDPSCs exhibited improved adhesion and proliferation capacity on the PLLA-PCL-HA nanofibers treated with MSH compared to other groups (p<0.05). Additionally, PLLA-PCL-HA nanofibers treated with MSH exhibited significantly higher mineralization and alkaline phosphatase activity than other groups. RT-PCR results confirmed that PLLA-PCL-HA nanofibers enriched with MSH could significantly unregulated the gene expression of BMP2, osteocalcin, RUNX2 and DSPP that correlated to osteogenic differentiation (p<0.05). Based on results, incorporation of HA nanoparticles in PLLA-PCL nanofibers and addition of MSH in media exhibited synergistic effects on the adhesion, proliferation, and osteogenesis differentiation of hDPSCs, and therefore assumed to be a favorable scaffold for bone tissue engineering applications.     

Paroxetine Attenuates the Development and Existing Pain in a Rat Model of Neurophatic Pain

Background: P2X4 receptor (P2X4R), a purinoceptor expressed in activated spinal microglia, plays a key role in the pathogenesis of neuropathic pain. Spinal nerve injury induces up-regulation of P2X4R on activated microglia in the spinal cord, and blockade of this receptor can reduce neuropathic pain. The present study was undertaken to determine whether paroxetine, an inhibitor of P2X4R, could attenuate allodynia and hyperalgesia in chronic constriction injury (CCI) model of neuropathic pain when used preemptively or after the sciatic nerve injury. Methods: Male Wistar rats (150-200 g, n = 6) were divided into 3 different groups: 1- CCI vehicle-treated group, 2- Sham group, and 3- CCI paroxetine-treated group. Paroxetine (10 mg/kg, i.p.) was administered 1 h before surgery and continued daily until day 14. In other part of the study, paroxetine (10 mg/kg, i.p.) was administered at day 7 post injury and continued daily until day 14. von Frey filaments for mechanical allodynia and analgesia meter for thermal hyperalgesia were used to assay pain behavior. Results: In a preventive paradigm, paroxetine significantly attenuated both mechanical allodynia and thermal hyperalgesia (P<0.001). A significant decrease in pain behavior was seen with paroxetine on existing allodynia (P<0.001) and hyperalgesia (P<0.01) when initiated at day 7 post injury. Conclusion: It seems that paroxetine can attenuate pain behavior when administered before and also after sciatic nerve injury in CCI model of neuropathic pain. Key Words: Paroxetine, P2X4 receptor (P2X4R), Allodynia, Hyperalgesia     

Xyloketal B Suppresses Glioblastoma Cell Proliferation and Migration in Vitro through Inhibiting TRPM7-Regulated PI3K/Akt and MEK/ERK Signaling Pathways

Glioblastoma, the most common and aggressive type of brain tumors, has devastatingly proliferative and invasive characteristics. The need for finding a novel and specific drug target is urgent as the current approaches have limited therapeutic effects in treating glioblastoma. Xyloketal B is a marine compound obtained from mangrove fungus Xylaria sp. (No. 2508) from the South China Sea, and has displayed antioxidant activity and protective effects on endothelial and neuronal oxidative injuries. In this study, we used a glioblastoma U251 cell line to (1) explore the effects of xyloketal B on cell viability, proliferation, and migration; and (2) investigate the underlying molecular mechanisms and signaling pathways. MTT assay, colony formation, wound healing, western blot, and patch clamp techniques were employed. We found that xyloketal B reduced cell viability, proliferation, and migration of U251 cells. In addition, xyloketal B decreased p-Akt and p-ERK1/2 protein expressions. Furthermore, xyloketal B blocked TRPM7 currents in HEK-293 cells overexpressing TRPM7. These effects were confirmed by using a TRPM7 inhibitor, carvacrol, in a parallel experiment. Our findings indicate that TRPM7-regulated PI3K/Akt and MEK/ERK signaling is involved in anti-proliferation and migration effects of xyloketal B on U251 cells, providing in vitro evidence for the marine compound xyloketal B to be a potential drug for treating glioblastoma.