Generation of Retinal Pigmented Epithelium-Like Cells from Pigmented Spheres Differentiated from Bone Marrow Stromal Cell-Derived Neurospheres

BACKGROUND: Retinal degeneration causes blindness, and cell replacement is a potential therapy. The purpose of this study is to formation of pigmented neurospheres in a simple medium, low-cost, high-performance manner over a short period of time while expressing markers of RPE cells and the activation of specific genes of the pigment cells. Also, these neurospheres have the ability to produce a monolayer of retinal pigment epithelium-like cells (RPELC) with the ability of photoreceptor outer segment phagocytosis. METHODS: BMSC were isolated from pigmented hooded male rats and were immunoreactive to BMSC markers, then converted into neurospheres, differentiated into pigmented spheres (PS), and characterized using Retinal pigment epithelium-specific 65 kDa protein (RPE65), Retinaldehyde-binding protein 1 (CRALBP) and orthodenticle homeobox 2 (OTX2) markers by immunocytochemistry, RT-PCR and RT-qPCR. The PS were harvested into RPELC. The functionality of RPELC was evaluated by phagocytosis of fluorescein-labeled photoreceptor outer segment. RESULTS: The BMSC immunophenotype was confirmed by immunostained for fibronectin, CD90, CD166 and CD44. These cells differentiated into osteogenic and lipogenic cells. The generated neurospheres were immunoreactive to nestin and stemness genes. The PS after 7–14 days were positive for RPE65 (92.76–100%), CRALBP (95.21–100%) and OTX2 (94.88–100%), and after 30 days RT-PCR, qPCR revealed increasing in gene expression. The PS formed a single layer of RPELC after cultivation and phagocyte photoreceptor outer segments. CONCLUSION: Bone marrow stromal stem cells can differentiate into functional retinal pigmented epithelium cells in a simple, low-cost, high-performancemanner over a short period of time. These cells due to expressing theRPELCgenes andmarkers can be used in cell replacement therapy for degenerative diseases including age-relatedmacular degeneration as well as retinitis pigmentosa.

Decrease in cavity size and oligodendrocyte cell death using Neurosphere-Derived oligodendrocyte-like cells in spinal cord contusion model

Background: Oligodendrocyte cell death is among the important features of spinal cord injury, which appears within 15 min and occurs intensely for 4 h after injury, in the rat spinal contusion model. Accordingly, the number of oligodendrocytes progressively reduced within 24 h after injury. Administration of oligodendrocyte-like cells [OLCs] into the lesion area is one of the approaches to counterbalance this condition

Methods: Bone marrow stromal cells were transdifferentiated into neurospheres and then into neural stem cells and later were differentiated into OLCs using triiodothyronine and transplanted into the spinal cord contusion rats. The postinjury functional recovery was explored and compared with the control group using Basso-Beattie-Bresnahan and narrow beam behavioral tests. At the end of 12th week, spinal cord segments T12-L1 were histomorphologically studied by immunohistochemistry

Results: Motor improvement was more obvious during 2nd to 4th weeks and got less prominent during 4th to 12th weeks. Histomorphometric findings indicated that cavity formation decreased in epicenter of transplantation area in experimental groups in comparison with the control groups

Conclusion: The findings obtained in the present study showed that OLC therapy is a potential approach in the treatment of spinal cord traumatic injuries

new multistep induction protocol for the transdifferentiation of bone marrow stromal stem cells into GABAergic neuron-like cells

Background: bone marrow stromal stem cells [BMSC] are appropriate source of multipotent stem cells that are ideally suited for use in various cell-based therapies. It can be differentiated into neuronal-like cells under appropriate conditions. This study examined the effectiveness of co-stimulation of creatine and retinoic acid in increasing the differentiation of BMSC into GABAergic neuron-like cells [GNLC]

Methods: BMSC isolated from the femurs and tibias of adult rats were cultured in DMEM/F12 medium supplemented with 10% FBS, pre induced using [beta]-mercaptoethanol [[beta]ME] and induced using retinoic acid [RA] and creatine. Immunostaining of neurofilament 200 kDa, neurofilament 160 kDa, nestin, fibronectin, Gamma-amino butyric acid [GABA] and glutamic acid decarboxylase [GAD] 65/67 were used to evaluate the transdifferentiation of BMSC into GNLC and to evaluate the effectiveness of pre-induction and induction assays. The expression of genes that encode fibronectin, octamer-binding transcription factor 4 [Oct-4], GAD 65/67 and the vesicular GABA transporter was examined in BMSC and GNLC by using RT-PCR assays during transdifferentiation of BMSC into GLNC

Results: co-stimulation with RA and creatine during the induction stage doubled the rates of GABAergic differentiation compared with induction using creatine alone, resulting in a 71.6% yield for GLNC. RT-PCR showed no expression of Oct-4 and fibronectin after the induction stage

Conclusion: the results of this study showed that the application of [beta]ME, RA, and creatine induced the transdifferentiation of BMSC into GLNC. Iran. Biomed. J. 17 [1]: 8-14, 2013

Bone marrow stromal cell transdifferentiation into oligodendrocyte-like cells using triiodothyronine as a inducer with expression of platelet-derived growth factor ? as a maturity marker

Background: the present study investigated the functional maturity of oligodendrocyte derived from rat bone marrow stromal cells [BMSC]

Methods: the BMSC were isolated from female Sprague-Dawley rats and evaluated for different markers, such as fibronectin, CD106, CD90, Oct-4 and CD45. Transdifferentiation of OLC from BMSC was obtained by exposing the BMSC to DMSO and 1 [micro]M all-trans-retinoic acid during the preinduction stage and then induced by heregulin [HRG], platelet-derived growth factor AA [PDGFR-alpha], fibroblast growth factor and T3. The neuroprogenitor cells [NPC] were evaluated for nestin, neurofilament 68, neurofilament 160 and glial fibrillary acidic protein gene expression using immunocytochemistry. The OLC were assessed by immunocytochemistry for O4, oligo2, O1 and MBP marker and gene expression of PDGFR-alpha was examined by RT-PCR

Results: our results showed that the fibronectin, CD106, CD90, CD45 and Oct-4 were expressed after the fourth passage. Also, the yield of OLC differentiation was about 71% when using the O1, O4 and oligo2 markers. Likewise, the expression of PDGFR-alpha in pre-oligodendrocytes was noticed, while MBP expression was detected in oligodendrocyte after 6 days of the induction

Conclusion: the conclusion of the study showed that BMSC can be induced to transdifferentiate into mature OLC. Iran. Biomed. J. 17 [2]: 62-70, 2013

In vitro labeling of neural stem cells with poly-L-lysine coated super paramagnetic nanoparticles for green fluorescent protein transfection

Background: the magnetic nanoparticle-based transfection method is a relatively new technique for delivery of functional genes to target tissues. We aimed to evaluate the transfection efficiency of rat neural stem cell [NSC] using poly-L-lysine hydrobromide [PLL]-coated super paramagnetic iron oxide nanoparticles [SPION]

Methods: the SPION was prepared and coated with PLL as transfection agent and the transfection efficiency was evaluated in rat NSC using enhanced green fluorescent protein-N1 plasmid containing GFP as a reporter gene. NSC was incubated for 24 h in cell culture media containing 25 [micro]g/ml SPION and in different concentrations of PLL [0.25, 0.50, 0.75, 1 and 2 [micro]g/ml]. Cell viability was determined before and after transfection for every concentration using Trypan blue assay. Characterization of prepared uncoated [SPION] and coated [SPION-PLL] complexes were evaluated by a transmission electron microscope and the zeta potential

Results: PLL at 0.75 [micro]g/ml showed optimal results with 25 [micro]g/ml SPION concentration compared with other PLL concentrations [0.25, 0.50, 1 and 2 [micro]g/ml]. The 18% efficiency of the transfected cells showed green fluorescence

Conclusion: transfection with SPION is an efficient, non-viral gene transfere method. Iran. Biomed. J. 17 [2]: 71-76, 2013

Microglial activation in rat experimental spinal cord injury model

The present study was designed to evaluate the secondary microglial activation processes after spinal cord injury [SCI]. 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. The results of glial cell quantification along the 4900- Micro m 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. 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

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

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. 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. 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. 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

Trans-differentiation of the adipose tissue-derived stem cells into neuron-like cells expressing neurotrophins by selegiline

Adult stem cells [ASC] are undifferentiated cells found throughout the body. These cells are promising tools for cell replacement therapy in neurodegenerative disease. Adipose tissue is the most abundant and accessible source of ASC. This study was conducted to evaluate effect of selegiline on differentiation of adiposederived stem cells [ADSC] into functional neuron-like cells [NLC], and also level of the neurotrophin expression in differentiated cells. ADSC were transdifferentiated into NLC using selegiline where CD90, CD49d, CD31, CD106 and CD45 were used as markers for ADSC identification. Lipogenic and osteogenic differentiation of ADSC were used to characterize the ADSC. ADSC were treated with selegiline at different concentrations [from 10[-6] to 10[-11] mM] and time points [3, 6, 12, 24 and 48 h]. Percentage of viable cells, nestin and neurofilament 68 [NF-68] immunoreactive cells were used as markers for differentiation. The optimal dose for neurotrophin expressions in differentiating cells was evaluated using reverse transcriptase-PCR. NLC function was evaluated by loading and unloading with FM1-43 dye. ADSC were immunoreactive to CD90 [95.67 +/- 2.26], CD49d [71.52 +/- 6.64] and CD31 [0.6 +/- 0.86], but no immunoreactivity was detected for CD106 and CD45. The results of neural differentiation showed the highest percentage of nestin and NF-68 positive cells at 10[-9] mM concentration of selegiline [exposed for 24 h]. The differentiated cells expressed synapsin and neurotrophin genes except brainderived neurotrophic factor. ADSC can be an alternative source in cell-based therapy for neurodegenerative diseases using selegiline to induce ADSC differentiation to neuronal lineage

Localization of herpes simplex virus type 1 DNA in latently infected BALB/c mice neurons using in situ polymerase chain reaction

Herpes simplex virus type-1 [HSV-1] establishes a lifelong latent infection in neurons following primary infection. The existence of latent HSV-1 DNA in the trigeminal ganglia of infected BALB/c mice was examined using a direct in situ PCR technique, based on Digoxigenin-11-dUTP detection system with anti-digoxigenin-peroxidase and 3,3f-diaminobenzidine [DAB] substrate. Eight-week-old male BALB/c mice were inoculated via the eye by 10[4] plaque forming unit of wild type Iranian isolates of HSV-1. After establishment of latency, trigeminal ganglia were removed and examined using in situ PCR to detect HSV-1 genome. Finally, the results of in situ PCR were verified by a two-round PCR method, using amplification cocktail of in situ reaction, as a template for a conventional gel base PCR. The results suggest that a direct in situ PCR method using a peroxidase and DAB detection system is a useful means for detection of latent HSV-1 DNA in the latently infected ganglia

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

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. 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. 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. 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

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

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. In this study, BMSC of adult female rats were pre-induced into neuroblast-like cells using 1 mM beta-mercaptoethanol [PME] and 10 micro M 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. 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. GABAergic-like neurons obtained from BMSC can be potentially used in cell transplanting for some neurodegenerative disorders

Graft efficiency of Co-cultured spermatogonial cells using sperm assay in epididymal lumen of recipient mice

Transplantation of germ cells restores the male fertility. Nevertheless, a lot of questions remain incompletely resolved. The aim of this study was to evaluate in vitro colonization efficiency of germ cells and sperm production capacity of spermatogonial cells before and after culture by sperm number assay in epididymis of recipient mice. Materials and We developed a Sertoli cell feeder in a co-culture system with spermatogonial cells and the cells were co-cultured for 2 months. The cells were isolated from mouse neonates. Colony assay was performed during culture using light microscopy. The transplanted cells were traced using BrdU incorporation. Sperm parameters were assessed 2 months after transplantation.Our findings showed that spermatogonial cells created colonies during culture. Transplantation of fresh spermatogonial cells at a concentration of 2'10[5] cells/ml did not show significant difference However, after transplantation of 2'10[5] cells/ml cultured for 2 weeks, the number of epididymal sperms in recipients increased significantly in groups with more fresh cells. Epididymal sperm number in recipient mice can be increased by enrichment of type A spermatogonial cells using an in vitro co-culture system. Other important factors include the source of donor cells and the number of transplanted cells

Induction of apoptosis and micronuclei by bleomycin sulfate in different cell cycle stages of human lymphocytes

Introduction: Bleomycin sulfate is a DNA damaging agent used in cancer chemotherapy. The effect of this drug on various cell cycle stages might be different, thus inducing different modes of death [apoptotic or mitotic death]. The aim of this investigation was to study the effects of bleomycin on human peripheral blood lymphocytes at various cell cycle stages by two different end points [induction of apoptosis or micronuclei]

Material and Methods: Human peripheral blood lymphocytes were treated with various doses of bleomycin at G0, G1, and G2 phases of the cell cycle and the percentages of apoptosis [AP] and micronuclei [MN] were determined. The peripheral lymphocytes were isolated by ficoll hypaque and suspended in RPMI-1640 containing 15 % fetal calf serum. The isolated lymphocytes were stimulated by phytohemagglutinin [PHA], cultured again inRPMI-1640, harvested after 64 hrs and 96 hrs, and stained with acridine orange and ethidium bromide to determine the percentage of apoptotic cells. MN assay was done according to the standard in vitro micronucleus assay

Results: The results showed that the percentages of apoptotic cells and MN at G2 stage were significantly higher than those of G0 and G1 stages. At higher doses, MN formation and apoptotic cells were increased; however with increasing time, the percentage of MN decreased while the percentage of apoptotic cells generally increased in all the cell cycle stages

Conclusion: The results indicate that bleomycin is a potent inducer of both micronuclei and apoptosis. The incidence of apoptotic cells following bleomycin treatment in G0 and G1 was much higher than the incidence of micro nucleated cells at the two sampling times. The percentage of AP cells following bleomycin treatment remained constant across cell cycle stages