Reserach Paper: Trans-differentiation of human dental pulp stem cells into cholinergic-like neurons via nerve growth factor

Introduction: Cell therapy has been widely considered as a therapeutic approach for neurodegenerative diseases and nervous system damage. Cholinergic neurons as one of the most important neurons that play a significant role in controlling emotions, mobility, and autonomic systems. In this study, Human Dental Pulp Stem Cells (hDPSCs) were differentiated into the cholinergic neurons by β-mercaptoethanol in the preinduction phase and also by the nerve growth factor (NGF) in the induction phase. Methods: The hDPSCs were evaluated for CD73, CD31, CD34, and Oct-4. Concentration-time relationships for NGF were assessed by evaluating the viability rate of cells and the immune response to nestin, neurofilament 160, microtubule-associated protein-2, and choline acetyltransferase. Results: The hDPSCs had a negative response to CD34 and CD31. The optimal dose for the NGF was 50 ng/mL seven days after the induction when the highest percentage of expressing markers for the Cholinergic neurons (ChAT) was detected. Conclusion: The results of this study provided a method for producing cholinergic neurons by hDPSCs, which can be used in cytotherapy for degenerative diseases of the nervous system and also spinal cord injury

Self-organization of developing embryo using scale-invariant approach

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Methods</p> <p>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 <it>C. elegans </it>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.</p> <p>Results and conclusion</p> <p>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.</p

Astrogliosis in different zones of the spinal cord gray matter after sciatic nerve axotomy in the newborn rat: A morphometric and immunohistochemical study

Astrocytic response following unilateral sciatic nerve axotomy was examined in the spinal gray matter of newborn rats. Using an antiserum to glial fibrillary acidic protein (GFAP), immunoreactive astrocytes were studied in the ventral, dorsal and transitional region between the dorsal and ventral gray matters (TDVG) at intervals of one day, one week, two weeks and one month postaxotomy. The axotomized side showed an obvious increase in the number of immunoreactive astrocytes at one week, two weeks and one month after surgery. The numerical density per area of the glial cells (N(a)) was determined in all groups on both the intact and axotomized sides, and it increased in all groups at the axotomized sides. The percentage of glial cell increase (Pgi) was also determined. At the ventral horn Pgi increased at day one and continued to increase in all groups, while the increase in TDVG and the dorsal horn occurred at later time points. The total motoneuron count in the ventral horn at the axotomized and intact sides was done at all time points, and the percentage of motoneuron reduction (Pmr) was calculated, the highest Pmr being noticed at one month (41%). A nonlinear regression for Pmr and Pgi showed that the rate of Pgi was approximately double that of Pmr. The rate of glial cell increase at each time point (one day, one week, two weeks and one month groups) was calculated, and the highest rate of glial cell increase in the ventral horn occurred one week after axotomy, while the highest rate in the dorsal horn and TDVG occurred at the second week. The conclusion of the study is that there may be an initial post-axotomic proliferative phase of the glial cells, which was followed by a differentiation phase. Also a gradient of an increase in the rate glial cell proliferation was noticed from the ventral horn toward the dorsal horn, maybe due to stimulation by a paracrine factor

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 ethidiumbromide 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

Genetic Changes during Differentiation of Spermatogonial Stem Cells into Oligoprogenitor Cells

BACKGROUND AND OBJECTIVE: The culture of spermatogonial cells and the production of embryonic stem cells (ES-like cells), suggest these cells as a sufficient new source for cell therapy and for the treatment of diseases, including neurodegenerative diseases. The aim of this study is to evaluate the pattern of genetic changes during differentiation of spermatogonial cells into oligodendrocyte precursor cells. METHODS: In this experimental study, spermatogonial cells were isolated from the testicles of 2 – 6 days old newborn mice (6 – 10 mice each time) through two stages of enzymatic digestion. The cells were divided into three groups of quasi-embryonic stem cells, neuro-progenitive and oligodendrocyte precursor. Specific markers stra8, mvh, piwil2, C-myc, Nanog, NF68, Nestin, Olig2, and NG2 were evaluated using Real Time-PCR and immunocytochemistry method at each differentiation step. FINDINGS: Molecular evaluations showed that increase in Nestin gene expression in neuronal precursor cells was 1.3 times more than quasi-embryonic stem cells. In the molecular evaluations at the end of the second stage of differentiation, it was determined that culture at the end of the induction steps resulted in a significant increase in the expression of the genes of Olig2 and NG2 and decrease in the expression of Nestin gene (p<0.05). Molecular evaluations showed that this increase in oligodendrocyte-like cells was respectively 1.4 and 1.6 times more than neuronal precursor cells. CONCLUSION: In this study, it was demonstrated that quasi-embryonic stem cells have the potential to express the NF68 and Nestin neuron markers. The quasi-embryonic stem cells of NG2 and Olig2 genes were expressed in the cells after the induction stage

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 post-injury 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

Creatine and retinoic acid effects on the induction of autophagy and differentiation of adipose tissue-derived stem cells into GABAergic-like neurons

BACKGROUND AND OBJECTIVE: Deficit of inhibitory GABAergic neurons as a part of central nervous system (CNS) pathogenesis was reported in neurodegenerative disorders; and adipose-derived stem cells (ADSCs) were shown to be a feasible option for cell transdifferentiation in neuronal disorders therapy. In this study, the role of autophagy in differentiation was considered by evaluating the expression of the autologous genes of LC3, P62 and GABARAP in fatty stem cells and after the pre-induction stage. METHODS: In this experimental study, under sterile conditions ADSCs were obtained from pararenal fat of two male adult rats. The cells were divided into three groups of fatty stem cells, pre-induction and induction. Following third passages of cell culture, ADSCs were preinduced to neural-like cells (NLCs) using 1mM β-mercaptoethanol (βME) and 10μM retinoic acid (RA), and then NLCs were induced by creatine(Cr) in 1, 5, 10, 20 millimolar for 5 days. In induction stage, the effects of creatine on differentiation were studied by anti nestin and GABA antibody immunostainig. The roles of GABARAP, LC3 and p62 autophagy genes in transdifferentiation were assessed by RT-PCR. FINDINGS: Immunocytochemical studies on ADSCs using CD49d indicated that cultured cells were ADSCs. In the immunochemical studies of the induction stage, at a dose of 10 mM creatinine for 5 days, the expression of the GABA neurons and the nestin-like neuronal cell marker were 58±2 and 56±5, respectively which had a significant difference with other doses and control group (p<0.05). RT-PCR results indicated that in pre-induced cells autophagy genes of GABARAP, LC3 and p62 were expressed but only P62 gene was expressed in fatty stem cells. CONCLUSION: This study demonstrated that fatty stem cells after induction are able to express nestin and GABA neuronal markers. GABARAP, LC3 and p62 autophagy genes were expressed in pre-induced cells, which indicates the potential role of autophagy in the differentiation of fatty stem cells into nerve-like cells. © 2017, Babol University of Medical Sciences. All rights reserved

Combined effects of 3D bone marrow stem cell-seeded wet-electrospun poly lactic acid scaffolds on full-thickness skin wound healing

© 2017 Taylor & Francis. Tissue engineering has emerged as an alternative treatment to traditional grafts for skin wound healing. Three-dimensional nanofibers have been used extensively for this purpose due to their excellent biomedical-related properties. In this study, high porous 3D poly lactic acid nanofibrous scaffolds (PLA-S) were prepared by wet-electrospinning technique and seeded with rat bone-marrow stem cells (BMSCs) to characterize the biocompatibility and therapeutic efficacy of these fibers on the treating full-thickness dermal wounds. The results of in vitro andin vivo studies indicate that the 3D fibrous PLA-S can be a potential wound dressing for wound repair, particularly when seeded with BMSCs. GRAPHICAL ABSTRACT

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

Objective(s): 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