Study of lead-induced neurotoxicity in cholinergic cells differentiated from bone marrow-derived mesenchymal stem cells.

The developing brain is susceptible to the neurotoxic effects of lead. Exposure to lead has main effects on the cholinergic system and causes reduction of cholinergic neuron function during brain development. Disruption of the cholinergic system by chemicals, which play important roles during brain development, causes of neurodevelopmental toxicity. Differentiation of stem cells to neural cells is recently considered a promising tool for neurodevelopmental toxicity studies. This study evaluated the toxicity of lead acetate exposure during the differentiation of bone marrow-derived mesenchyme stem cells (bone marrow stem cells, BMSCs) to CCholinergic neurons. Following institutional animal care review board approval, BMSCs were obtained from adult rats. The differentiating protocol included two stages that were pre-induction with ß-mercaptoethanol (BME) for 24 h and differentiation to cholinergic neurons with nerve growth factor (NGF) over 5 days. The cells were exposed to different lead acetate concentrations (0.1-100 µm) during three stages, including undifferentiated, pre-induction, and neuronal differentiation stages; cell viability was measured by MTT assay. Lead exposure (0.01-100 µg/ml) had no cytotoxic effect on BMSCs but could significantly reduce cell viability at 50 and 100 µm concentrations during pre-induction and neuronal differentiation stages. MAP2 and choline acetyltransferase (ChAT) protein expression were investigated by immunocytochemistry. Although cells treated with 100 µm lead concentration expressed MAP2 protein in the differentiation stages, they had no neuronal cell morphology. The ChAT expression was negative in cells treated with lead. The present study showed that differentiated neuronal BMSCs are sensitive to lead toxicity during differentiation, and it is suggested that these cells be used to study neurodevelopmental toxicity.

Implications of immunotherapy with high-dose glatiramer acetate in acute phase of spinal cord injury in rats.

Objective: Recently, many researches with different viewpoints have focused on application of immunotherapy agents in treatment of spinal cord injury (SCI) according to neuroprotective results in some neurodegenerative disease. Glatiramer acetate (GA) is the most commonly used drug for Multiple sclerosis (MS) patients that exerts an immunomodulatory effect against Myelin basic protein (MBP) antigen. Materials and methods: High-dose (2mg/kg) treatment of GA for 28 consecutive days after SCI was compared with its low-dose (0.5 mg/kg) treatment, SCI control and Sham control rat groups. Results: High-dose GA group had significantly worsened outcome in standard functional recovery evaluation test (BBB) 12 weeks after SCI compared to SCI control and low-dose GA groups, which was confirmed by augmented spinal cavity volume and reduced ventral horn motor neurons in high-dose GA group; however, there was no significant difference between low-dose GA and control SCI group. In addition, proliferation test performed on lymphocytes from spleen and lymph nodes one week after SCI showed that high-dose GA injection has more significant effect on Division Index (DI) in response to MBP stimulation compared to low-dose GA and control SCI groups, which was associated with significant increase in IFN-γ, IL-4, and IL-17A secretion. Conclusion: Along with confirmation of deleterious aspects of autoimmunity resulting from autoreactive lymphocytes against myelin antigens in SCI, this study has shown that high-dose immunotherapy using GA, especially in acute phase after SCI, overwhelms any neuroprotective effect of adoptive immune system.

Motor Neuron Transdifferentiation of Neural Stem Cell from Adipose-Derived Stem Cell Characterized by Differential Gene Expression.

Adipose-derived stem cells (ADSC) are adult stem cells which can be induced into motor neuron-like cells (MNLC) with a preinduction-induction protocol. The purpose of this study is to generate MNLC from neural stem cells (NSC) derived from ADSC. The latter were isolated from the perinephric regions of Sprague-Dawley rats, transdifferentiated into neurospheres (NS) using B27, EGF, and bFGF. After generating NSC from the NS, they induced into MNLC by treating them with Shh and RA, then with GDNF, CNTF, BDNF, and NT-3. The ADSC lineage was evaluated by its mesodermal differentiation and was characterized by immunostaining with CD90, CD105, CD49d, CD106, CD31, CD45, and stemness genes (Oct4, Nanog, and Sox2). The NS and the NSC were evaluated by immunostaining with nestin, NF68, and Neurod1, while the MNLC were evaluated by ISLET1, Olig2, and HB9 genes. The efficiency of MNLC generation was more than 95 ± 1.4 % (mean ± SEM). The in vitro generated myotubes were innervated by the MNLC. The induced ADSC adopted multipolar motor neuron morphology, and they expressed ISLET1, Olig2, and HB9. We conclude that ADSC can be induced into motor neuron phenotype with high efficiency, associated with differential expression of the motor neuron gene. The release of MNLC synaptic vesicles was demonstrated by FM1-43, and they were immunostained with synaptophysin. This activity was correlated with the intracellular calcium ion shift and membrane depolarization upon stimulation as was demonstrated by the calcium indicator and the voltage-sensitive dye, respectively.

Human ciliary neurotrophic factor-overexpressing stable bone marrow stromal cells in the treatment of a rat model of traumatic spinal cord injury.

BACKGROUND AIMS: Traumatic injury to the central nervous system (CNS) often causes motor dysfunctions. However, because of the CNS complexity and variability in the clinical presentations, efforts to repair damaged CNS tissue and restoring its functions are particularly demanding. On the other hand, recent progress in the regenerative therapy field have led to novel approaches for the treatment of traumatic CNS injury and renewed hopes to overcome the obstacles. It appears that the balance between neurite re-growth-inhibiting and neurite re-growth-inducing molecules determines the axonal re-growth fate. Neurotrophic factors can tilt this balance and indeed promote cell survival and axonal re-growth over neurodegeneration. One of the promising neurotrophic factors in this field is ciliary neurotrophic factor (CNTF). METHODS: We transfected rat bone marrow stromal cells with a mammalian expression vector-inserted human CNTF gene through the use of a non-viral method to prepare human CNTF-overexpressing stem cells under ex vivo conditions. We transplanted these modified cells to the rat model of spinal cord traumatic injury to explore functional recovery after contusion induction. RESULTS: Our data from immunocytochemistry and behavioral tests showed that such cells can act as a powerful potential approach to treat traumatic CNS injuries because these modified cells improved the behavioral test scores in the rat model of spinal cord injury. CONCLUSIONS: CNTF-overexpressing bone marrow stromal cells can ameliorate spinal cord traumatic injury and can be used in the treatment of traumatic CNS injuries in the near future.

Decreased GFAP expression and improved functional recovery in contused spinal cord of rats following valproic acid therapy.

Many studies have illustrated that much of the post-traumatic degeneration of the spinal cord cells is caused by the secondary mechanism. The aim of this study is to evaluate the effect of the anti-inflammatory property of valproic acid (VPA) on injured spinal cords (SC). The rats with the contused SC received intraperitoneal single injection of VPA (150, 200, 300, 400 or 500 mg/kg) at 2, 6, 12 and 24 h post-injury. Basso-Beattie-Bresnahan (BBB) test and H-reflex evaluated the functional outcome for 12 weeks. The SC were investigated 3 months post-injury using morphometry and glial fibrillary acid protein (GFAP) expression. Reduction in cavitation, H/M ratio, BBB scores and GFAP expression in the treatment groups were significantly more than that of the untreated one (P < 0.05). The optimal improvement in the condition of the contused rats was in the ones treated at the acute phase of injury with 300 mg/kg of VPA at 12 h post-injury, they had the highest increase in BBB score and decrease in astrogliosis and axonal loss. We conclude that treating the contused rats with 300 mg/kg of VPA at 12 h post-injury improves the functional outcome and reduces the traumatized SC gliosis.

The Effect of Antinociceptive Dose of Morphine on Cell Therapy in Rats with Spinal Cord Injury.

Spinal cord injury (SCI) is a sensory-motor injury. Today, combined treatments such as cell therapy along with drug therapy and their interactions are of interest. Morphine is an opioid drug used to relieve intolerable pain. This study aims to evaluate the impact of an antinociceptive dose of morphine (with minimal tolerance/dependence but effective pain relief) on cell therapy in SCI. The antinociceptive dose of morphine was determined in rats with SCI through the Hargreaves and naloxone-induced morphine withdrawal tests. The rats were then allocated to 5 groups: laminectomy, SCI, SCI + Morphine, SCI + cell therapy, SCI + Morphine + cell therapy. The antinociceptive dose (5 mg/kg) was administered on days 1, 4, 10, and 13 (i.p.) post-SCI. On day 7, Neural-like stem cells derived from adipose tissue were transplanted intraspinally into the injured animals, and they were monitored for 12 weeks. The outcomes were assessed using the BBB test, somatosensory evoked potential (SSEP), and histology. The BBB test indicated that morphine significantly hindered functional recovery post-cell transplantation compared to animals receiving only cell therapy (p < 0.05). In the SSEP test, the analysis of amplitude and latency of waves did not reveal a significant difference (p > 0.05). The histological results showed that cell therapy reduced the cavity size post-SCI, while morphine had no significant impact on it. Morphine at the antinociceptive dose significantly impairs motor recovery despite cell therapy. Nonetheless, there was no significant difference between groups in terms of sensory pathway outcomes.

Improvement of contusive spinal cord injury in rats by co-transplantation of gamma-aminobutyric acid-ergic cells and bone marrow stromal cells.

BACKGROUND AIMS: Cell therapy is considered a promising option for treatment of spinal cord injury (SCI). The purpose of this study is to use combined therapy of bone marrow stromal cells (BMSCs) and BMSC-derived gamma-aminobutyric acid (GABA)ergic inhibitory neurotransmitter cells (BDGCs) for the contusion model of SCI in rats. METHODS: BDGCs were prepared from BMSCs by pre-inducing them with ß-mercaptoethanol followed by retinoic acid and then inducing them by creatine. They were immunostained with BMSC, proneuronal, neural and GABA markers. The BDGCs were intraspinally transplanted into the contused rats, whereas the BMSCs were delivered intravenously. The animals were sacrificed after 12 weeks. RESULTS: The Basso, Beattie and Bresnahan test showed improvement in the animals with the combined therapy compared with the untreated animals, the animals treated with GABAergic cells only and the animals that received BMSCs. The immunohistochemistry analysis of the tissue sections prepared from the animals receiving the combined therapy showed that the transplanted cells were engrafted and integrated into the injured spinal cord; in addition, a significant reduction was seen in the cavitation. CONCLUSIONS: The study shows that the combination of GABAergic cells with BMSCs can improve SCI.

Enhanced growth and differentiation of neural stem cells on alginate/collagen/reduced graphene oxide composite hydrogel incorporated with lithium chloride.

Introduction: Cell transplantation with hydrogel-based carriers is one of the advanced therapeutics for challenging diseases, such as spinal cord injury. Electrically conductive hydrogel has received much attention for its effect on nerve outgrowth and differentiation. Besides, a load of neuroprotective substances, such as lithium chloride can promote the differentiation properties of the hydrogel. Methods: In this study, alginate/collagen/reduced graphene oxide hydrogel loaded with lithium chloride (AL/CO/rGO Li+) was prepared as an injectable cell delivery system for neural tissue regeneration. After determining the lithium-ion release profile, an MTT assay was performed to check neural viability. In the next step, real-time PCR was performed to evaluate the expression of cell adhesion and neurogenic markers. Results: Our results showed that the combination of collagen fibers and rGO with alginates increased cell viability and the gene expression of collagen-binding receptor subunits such as integrin α1, and ß1. Further, rGO contributed to the controlled release of lithium-ion hydrogel in terms of its plenty of negatively charged functional groups. The continuous culture of NSCs on AL/CO/rGO Li+ hydrogel increased neurogenic genes' expressions of nestin (5.9 fold), NF200 (36.8 fold), and synaptophysin (13.2 fold), as well as protein expression of NF200 and synaptophysin after about 14 days. Conclusion: The simultaneous ability of electrical conduction and lithium-ion release of AL/CO/rGO Li+ hydrogel could provide a favorable microenvironment for NSCs by improving their survival, maintaining cell morphology, and expressing the neural marker. It may be potentially used as a therapeutic approach for stem cell transplantation in a spinal cord injury.

In vitro differentiation of bone marrow stromal cells into oligodendrocyte-like cells using triiodothyronine as inducer.

An in vitro technique was devised to induced autologous adult stem cells into oligodendrocyte-like cells. In this study, a protocol was developed for the induction of bone marrow stromal cells (BMSCs) into oligodendrocyte-like cells. BMSCs were incubated in one of these three pre-inducers: dimethyl sulfoxide (DMSO), ß-mercaptoethanol (ßME) or biotylated hydroxyanisol (BHA), each followed by retinoic acid (RA) treatment. The percentage of viable cells in BHA-RA preinduced cells was significantly lower than the others. The results showed that the preinduced cells were immunoreactive for nestin and NF-68; among the mentioned protocols, the immunoreactivity yielded by following the DMSO-RA protocol was significantly higher than the others. Moreover, no significant immunoreactivity was observed for preinduced cells to O4, O1, MBP (myelin basic protein), S100, and GFAP (glial fibrillary acidic protein). The cells were immunoreactive to oligo-2. Two phases of induction were done: the first was a combination of basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF) and heregulin (HRG), followed by either triiodothyronine (T3) or Forskolin (FSK) as the second phase. The conclusion is that the trans-differentiation of BMSCs by DMSO followed by RA (preinduction stage) then bFGF-PDGF-HRG followed by T3 (10 ng/ml) (induction stage) can be a potential source for oligodendrocyte-like cells preparation.

Differentiation of PC12 cell line into neuron by Valproic acid encapsulated in the stabilized core-shell liposome-chitosan Nano carriers.

Valproic acid (VPA) usage in high dose is teratogen with low bioavailability. Hence to improve its efficacy and reduce its side effect it was encapsulated by the Nano liposomes and stabilized by the chitosan at different concentrations. The cellular uptake, biocompatibility, loading and encapsulation efficiency of the six-different formulations (1:1, 2:1, and 4:1 of chitosan-phospholipids: VPA), PC12 differentiation to neuron cells assays (gene-expression level by qRT-PCR) were conducted for the efficacy assessment of the Nano carriers. The encapsulation efficiency (EE) results revealed that the encapsulation of the VPA corresponds to the phospholipids dose, where 2:1 formulations showed higher encapsulating rate (64.5% for non-coated and 80% for coated by chitosan). The time monitored released of VPA also showed that the chitosan could enhance its controlled release too. The cellular uptake exhibited similar uptake behavior for both the coated and the non-coated Nano carriers and cytoplasmic distribution. We witnessed no toxicity effects, at different concentrations, for both formulations. Moreover, the results indicated that the gene expression level of SOX2, NeuroD1, and Neurofilament 200 increased from 1 to 5 folds for different genes. The qRT-PCR data were confirmed by the immunofluorescence antibodies staining, where Neurofilament 68 and SOX2 cell markers were modulated during differentiation of PC12 cells. Finally, our findings suggest promising potential for the Lip-VPA-Chit Nano carrier in inducing the differentiation of PC12 into neuron for treating neurodegenerative disorders.

Self-organization of developing embryo using scale-invariant approach.

BACKGROUND: Self-organization is a fundamental feature of living organisms at all hierarchical levels from molecule to organ. It has also been documented in developing embryos. METHODS: In this study, a scale-invariant power law (SIPL) method has been used to study self-organization in developing embryos. The SIPL coefficient was calculated using a centro-axial skew symmetrical matrix (CSSM) generated by entering the components of the Cartesian coordinates; for each component, one CSSM was generated. A basic square matrix (BSM) was constructed and the determinant was calculated in order to estimate the SIPL coefficient. This was applied to developing C. elegans during early stages of embryogenesis. The power law property of the method was evaluated using the straight line and Koch curve and the results were consistent with fractal dimensions (fd). Diffusion-limited aggregation (DLA) was used to validate the SIPL method. RESULTS AND CONCLUSION: The fractal dimensions of both the straight line and Koch curve showed consistency with the SIPL coefficients, which indicated the power law behavior of the SIPL method. The results showed that the ABp sublineage had a higher SIPL coefficient than EMS, indicating that ABp is more organized than EMS. The fd determined using DLA was higher in ABp than in EMS and its value was consistent with type 1 cluster formation, while that in EMS was consistent with type 2.

Vitamins E and D3 attenuate demyelination and potentiate remyelination processes of hippocampal formation of rats following local injection of ethidium bromide.

Cognitive deficits have been observed in patients with multiple sclerosis (MS) due to hippocampal insults. Antioxidant vitamins D and E are suggested for patients suffering from neurodegenerative diseases like MS, while their mechanisms of action are not well understood. Here, we have tried to study the effects of these vitamins on demyelination, cell death, and remyelination of rat hippocampus following local ethidium bromide (EB) injection. Animals received 100 mg/kg vitamin E or 5 microg/kg of vitamin D3 for 2, 7, or 28 days. The extent of demyelination, myelin staining intensity, and expression of myelin basic protein and caspase-3 were investigated using histological and immunoblotting verification. Administration of EB alone caused demyelination, cell death, and afterward an endogenous repair. Vitamins E and D3 reduced the EB-induced damage and increased the endogenous remyelination of hippocampus. Although the anti-apoptotic effect of these vitamins and protection against demyelination were predictable based on their antioxidant effect, our results indicated the positive effect of vitamins E and D3 on process of remyelination by endogenous progenitor cells and supported their possible therapeutic effects in the context of demyelinating diseases like MS.

The effect of two different polyethylene glycol (PEG) derivatives on the immunological response of PEG grafted pancreatic islets.

Islet transplantation is one of the promising ways to treat diabetes. To reduce the immune system response, several methods have been developed, a novel one being the grafting of methoxy polyethylene glycol (mPEG) derivatives onto collagen capsules of islets. In this study, the effects of the first and second generations of activated mPEG on the immunological response of polyethylene glycol (PEG) grafted pancreatic islets were studied. mPEG-Succinimidyl carbonate (mPEG-SC) and mPEG-succinimidyl propionic acid (mPEG-SPA) (with nominal molecular weight 5 kDa), typical of the first and second generations of activated mPEG, were selected, respectively. Both activated mPEGs did not affect the morphology, viability, or functionality of PEGylated islets compared to free islets (naked islets). The amount of IL-2 secreted from lymphocytes co-cultured with mPEG-SPA grafted islets (131.83 ± 15.28 pg/ml) was not significantly different from that with mPEG-SC grafted islets (156.09 ± 27.94 pg/ml). These results indicated that both mPEG-SC and mPEG-SPA had the same effect for camouflaging Langerhans islets, but the former is more suitable due to its easier synthesis process.

Differential gene expression by lithium chloride induction of adipose-derived stem cells into neural phenotype cells.

OBJECTIVES: Adipose-derived stem cells (ADSCs), with suitable and easy access, are multipotential cells that have the ability for differentiation into other mesodermal and transdifferentiate into neural phenotype cells. In this study, Lithium chloride (LiCl) was used for in vitro transdifferentiation of rat ADSCs into neuron-like cells (NLCs). MATERIALS AND METHODS: ADSCs were isolated from the rats' perinephric region using Dulbecco΄s Modified Eagle΄s Medium (DMEM) with Fetal Bovine Serum (FBS), cultured for 3 passages, characterized by flowcytometry and differentiation into adipogenic and osteogenic phenotypes. The ADSCs were exposed to 0.1, 0.5, 1, 1.5, 2, 5, and 10 millimolar (mM) LiCl without serum for 24 hr. The optimum dose of LiCl was selected according the maximum viability of cells. The expression of neurofilament light chain (NfL), neurofilament high chain (NfH), and nestin was evaluated by immunocytochemistry. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to evaluate the amount of synaptophysin, neurogenin-1, neuroD1, NfL, NfH, and nestin genes' expression in ADSCs and NLCs. RESULTS: The optimum dose of LiCl was 1 mM in 24 hr. The transdifferentiated ADSCs showed cytoplasmic extension with synapse-like formation. Synaptophysin, neurogenin-1, neuroD1, NfL, NfH, and nestin genes were significantly expressed more in NLCs than in ADSCs. CONCLUSION: LiCl can induce ADSCs into neural phenotype cells with higher expression of neural and neuronal genes.

Conversion of Neural Stem Cells into Functional Neuron-Like Cells by MicroRNA-218: Differential Expression of Functionality Genes.

Conversion of mesenchymal stem cells (MSC) into neuron-like cells (NLC) is a feasible cell therapy strategy for replacing lost neurons in neuronal disorders. In this study, adipose-derived MSC (ADMSC) were converted into neural stem cells (NSC) via neurosphere. The resulting NSC were then differentiated into NLC by transduction with microRNA-218, using a lentiviral vector. ADMSC, NSC, and NLC were first characterized by flow cytometry, RT-PCR, and immunocytochemistry. The functionality of the NLC was evaluated by qRT-PCR and patch clamp recording. Immunophenotyping of ADMSC showed their immunoreactivity to MSC markers CD90, CD73, CD105, and CD49d, but not to CD31 and CD45. RT-PCR results demonstrated the expression of nestin, neurogenin, neurod1, neurofilament light, and GAP43 genes in NSC while NLC expressed synaptophysin, neurofilament heavy, and GAP43. In addition, NSC morphology changed into multipolar with long processes after transduction with miR-218. Moreover, using qRT-PCR, the expression levels of miR-218 and functionality genes CACNA1C, SNAP25, KCNH1, KCNMA1, and SCN9A were significantly increased in NLC, compared with NSC, and ADMSC at 3 weeks and 5 months post-transduction. Furthermore, the generated NLC expressed significantly higher protein levels of neurofilament heavy polypeptide (NFh) and enolase 2 (Eno2) neuronal markers, compared with ADMSC and NSC. Finally, action potentials were successfully recorded by the generated NLC, using patch clamp. In summary, ADMSC-derived NSC differentiated into functional NLC by transduction with miR-218. The generated NLC expressed functional SNAP25, CACNA1C, KCNH1, KCNMA1, and SCN9A and produced an action potential, which provides useful insights into the generation of functional neuronal cells.

PuraMatrix hydrogel enhances the expression of motor neuron progenitor marker and improves adhesion and proliferation of motor neuron-like cells.

OBJECTIVES: Cell therapy has provided clinical applications to the treatment of motor neuron diseases. The current obstacle in stem cell therapy is to direct differentiation of stem cells into neurons in the neurodegenerative disorders. Biomaterial scaffolds can improve cell differentiation and are widely used in translational medicine and tissue engineering. The aim of this study was to compare the efficiency of two-dimensional with a three-dimensional culture system in their ability to generate functional motor neuron-like cells from adipose-derived stem cells. MATERIALS AND METHODS: We compared motor neuron-like cells derived from rat adipose tissue in differentiation, adhesion, proliferation, and functional properties on two-dimensional with three-dimensional culture systems. Neural differentiation was analyzed by immunocytochemistry for immature (Islet1) and mature (HB9, ChAT, and synaptophysin) motor neuron markers. RESULTS: Our results indicated that the three-dimensional environment exhibited an increase in the number of Islet1. In contrast, two-dimensional culture system resulted in more homeobox gene (HB9), Choline Acetyltransferase (ChAT), and synaptophysin positive cells. The results of this investigation showed that proliferation and adhesion of motor neuron-like cells significantly increased in three-dimensional compared with two-dimensional environments. CONCLUSION: The findings of this study suggested that three-dimension may create a proliferative niche for motor neuron-like cells. Overall, this study strengthens the idea that three-dimensional culture may mimic neural stem cell environment for neural tissue regeneration.

Transdifferentiation of bone marrow stromal cells into cholinergic neuronal phenotype: a potential source for cell therapy in spinal cord injury.

BACKGROUND AIMS: Cholinergic neurons are very important cells in spinal cord injuries because of the deficits in motor, autonomic and sensory neurons. In this study, bone marrow stromal cells (BMSC) were evaluated as a source of cholinergic neurons in a rat model of contusive spinal cord injury. METHODS: BMSC were isolated from adult rats and transdifferentiated into cholinergic neuronal cells. The BMSC were pre-induced with beta-mercaptoethanol (BME), while the induction was done with nerve growth factor (NGF). Neurofilament (NF)-68, -160 and -200 immunostaining was used for evaluating the transdifferentiation of BMSC into a neuronal phenotype. NeuroD expression, a marker for neuroblast differentiation, and Oct-4 expression, a marker for stemness, were evaluated by reverse transcriptase (RT)-polymerase chain reaction (PCR). Choline acetyl transferase (ChAT) immunoreactivity was used for assessing the cholinergic neuronal phenotype. Anti-microtubule-associated protein-2 (MAP-2) and anti-synapsin I antibodies were used as markers for the tendency for synptogenesis. Finally, the induced cells were transplanted into the contused spinal cord and locomotion was evaluated with the Basso-Beattie-Bresnahan (BBB) test. RESULTS: At the induction stage, there was a decline in the expression of NF-68 associated with a sustained increase in the expression of NF-200, NF-160, ChAT and synapsin I, whereas MAP-2 expression was variable. Transplanted cells were detected 6 weeks after their injection intraspinally and were associated with functional recovery. CONCLUSIONS: The transdifferentiation of BMSC into a cholinergic phenotype is feasible for replacement therapy in spinal cord injury.

A comparative study of therapeutic benefits of intraspinal and intravenous bone marrow stromal cell administration to spinal cord injuries.

BACKGROUND: Recent reports demonstrated that intravenous route, as a minimally invasive method and similar to direct injection, is suitable for bone marrow stromal cell (BMSC) transplantation. In this study, we made a comparison of intraspinal and intravenous route of BMSC administration to repair injured spinal cord tissue. METHODS: Six groups of adult female rats were used in this study. Laminectomy and spinal cord injury were carried out at first lumbar vertebra level (L1). Labeled stromal cells were administered intraspinally and intravenously in experimental groups one week after spinal cord injury. In control groups, serum was administered in the same way. Another groups consisted of laminectomy alone and spinal cord injury. Behavioral testing was performed weekly to 5 weeks post injury. Tissue processing and immune-histochemical studies were performed four weeks after cell transplantation. RESULTS: Mean of Basso-Beattie-Bresnehan (BBB) scale scores in intraspinal and intravenous groups were 15.8 +/- 0.44 and 15.6 +/- 0.54, while in their controls were 10.6 +/- 0.33 and 10.6 +/- 0.56, respectively. BBB scale in laminectomy and spinal cord injury groups were 21 and 10.5 +/- 0.36, respectively. Immuno-histochemical staining visualized BMSC in the site of injury. Differentiation of a few implanted cells to neuron and glial cell was detected in intravenous group, while only differentiation to glial cells was detected in intraspinal group. CONCLUSION: The results of this study suggest that intravenous administration of BMSC, such as intraspinal method, provides therapeutic benefits for SCI.

In vitro transdifferentiation of bone marrow stromal cells into GABAergic-like neurons.

BACKGROUND: Cell therapy of many neurodegenerative diseases using bone marrow stromal cells (BMSC) requires the differentiation of BMSC into neuronal subtype. However, the transdifferentiation of BMSC into GABAergic phenotype requires more investigation. METHODS: In this study, BMSC of adult female rats were pre-induced into neuroblast-like cells using 1 mM beta-mercaptoethanol (betaME) and 10 microM retinoic acid (RA), followed by 40 mM potassium chloride as inducer. The BMSC were evaluated by fibronectin as well as Oct-4. The percentage of nestin, neurofilaments (NF 68, NF 160, and NF 200) and GABA immuno-reactive cells was used to evaluate the GABAergic differentiation at the pre-induction and induction stages. The statistical analysis was carried out using unpaired student's t-test and ANOVA with Tukey's multiple comparison. RESULTS: The BMSC in the fourth passage expressed fibronectin up to 91.24 +/- 0.82%. The pre-induced cells after 2 days of RA exposure showed the expression of neuroblastic markers of nestin and NF68 (81.56 +/- 2.64% and 82.12 +/- 2.65%, respectively). The yield of GABAergic neurons with beta-ME for 1 h and RA as pre-inducer for 2 days followed by potassium chloride as inducer (40 mM for 3 days) was 60.64% +/- 1.97%. In addition, NF160 and NF200 were detected in the transdifferentiated cells. RT-PCR showed no expression of Oct-4 after the induction and pre-induction stages. CONCLUSION: GABAergic-like neurons obtained from BMSC can be potentially used in cell transplanting for some neurodegenerative disorders.

Induction of bone marrow stromal cells into cholinergic-like cells by nerve growth factor.

BACKGROUND: Bone marrow stromal cells (BMSC) are used as a source for cell therapy in different model for neurological disorder such as stroke and spinal cord injury. However, the transdifferentiation of BMSC into cholinergic phenotype requires more investigation. METHODS: BMSC were isolated from adult rats, pre-induced with beta-mercaptoethanol (BME) and followed by nerve growth factor (NGF) induction. Neurofilaments of 68 kDa, 160 kDa and 200 kDa (NF-200, NF-160 and NF-68, respectively) immuno-staining were used for evaluating the transdifferentiation of BMSC into neuronal phenotype. The percentage of neurofilaments immuno-reactive cells was applied in order to evaluate the results at the pre-induction and the induction stages. Also, NeuroD and Oct-4 expressions, using RT-PCR, were used in assessing the progression of BMSC into neuronal lineage. Choline acetyltransferase immuno-reactive cells were used for estimating the percentage of cholinergic neuronal phenotype. Immuno-staining with anti-microtubule-associated protein-2 (MAP-2) and anti-synapsin-I antibodies was done in order to evaluate cell tendency for synaptogenesis. RESULTS: The yield of cholinergic neurons with BME as pre-inducer and NGF as inducer was 80%. Also, NF-200, NF-160, NF-68, MAP-2 and synapsin-I were detected in the transdifferentiated cells. RT-PCR showed the expression of NeuroD, while Oct-4 was not detected. CONCLUSION: BME as pre-inducer and NGF as inducer for BMSC transdifferentiation into cholinergic phenotype are potential sources in traumatic injury therapy in the central nervous system.