Improvement of neuroglial differentiation from human dental pulp stem cells using CSF

Background and purpose: Cerebrospinal fluid (CSF) has a broad set of molecules which is essential for neurogenesis. Human dental pulp stem cells (hDPSCs) are putatively neural crest cell-derived that can differentiate into neurons and glial cells under appropriate neurotrophic factors. The aim of this study was to induce differentiation of hDPSCs into neuroglial phenotypes using Retinoic acid (RA) and CSF. Materials and methods: The hDPSCs were isolated by mechanical enzymatic digestion from an impacted third molar and cultured. 2 � 105cells were treated by 10-7µM Retinoic acid (RA group) for 8 days, CSF (CSF group) for 8 days and pre-induced with RA for 4 days followed by inducing with CSF for 4 days (RC group). Nestin, βIII-tubulin and GFAP immunostaining were used for evaluating the differentiated cells. Axonal outgrowth was detected using Bielschowsky's silver impregnation method and Nissl bodies were stained in differentiated cells by Cresyl violet. Data analysis was performed in SPSS V.16 applying One-way ANOVA and Chi-square test. Results: The morphology of differentiated cells in treated groups significantly changed after 3-5 days. The immunocytochemistry results showed that nestin, the neuroprogenitor marker, was observed in all groups. Whereas, a high percentage of nestin positive cells and �III-tubulin, as mature neural markers, were seen at the pre-induction and induction stage, respectively. Nissl bodies were detected as dark-blue particles in the cytoplasm of treated cells. Conclusion: The findings suggest that the RA as pre-inducer and CSF as inducer could be used for in vitro differentiation of neuroglial cells from hDPSCs. © 2016, Mazandaran University of Medical Sciences. All rights reserved

In vitro anti-bacterial effect of nano-polyamidoamine-G5 dendrimer

Introduction: Progress in nanotechnology in the past decayed has created various opportunities for evaluation of biological effects such as anti-bacterial effects of nanoparticles. This study was aimed to examine synthesis and the antibacterial effect of Nano-Polyamidoamine-G5 (NPAMAM-G5) dendrimer on Klebsiella Pneumoniae, Pseudomonas Aeruginosa, Shigella Dysenteriae and Bacillus Subtilis. Materials and Methods: NPAMAM-G5 dendrimers was synthesized by Tomalia�s divergent growth approach. The antibacterial effects of NPAMAM-G5 dendrimer were studied by disc diffusion and micro-dilution method. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against gram-positive and gram-negative bacteria were determined according to Clinical and Laboratory Standards Institute (CLSI) guideline. Transmission electron microscopy (TEM) was used to analyze morphology and size of NPAMAM-G5. Results: Zone of inhibition in concentration 25μg/ml of NPAMAM-G5 dendrimers for Klebsiella Pneumoniae, Pseudomonas Aeruginosa, Shigella Dysenteriae and Bacillus Subtilis were 27, 13, 30 and 18 mm, respectively. There was a significant difference regarding the zone of inhibition between gram-negative and gram-positive bacteria (p<0.05). Remarkably, the MIC for Klebsiella Pneumoniae, Pseudomonas Aeruginosa and Shigella Dysenteriae was 2.5μg/ml and for Bacillus Subtilis was 25μg/ml. The MBC for Shigella Dysenteriae and Pseudomonas aeruginosa were 50 and 200 μg/ml, respectively and for Klebsiella Oxytoca and Bacillus Subtilis was100 μg/ml. It was found that NPAMAM-G5 particles had a spherical shape with a mean diameter size of 10 nm. Conclusion: According to the results, the NPAMAM-G5 dendrimer with end amine groups displayed a positive effect on the removal of standard strains of gram-positive and gram-negative bacteria. © 2019, Semnan University of Medical Sciences. All rights reserved

Co-transplantation of Human Embryonic Stem Cell-derived Neural Progenitors and Schwann Cells in a Rat Spinal Cord Contusion Injury Model Elicits a Distinct Neurogenesis and Functional Recovery

Co-transplantation of neural progenitors (NPs) with Schwann cells (SCs) might be a way to overcome low rate of neuronal differentiation of NPs following transplantation in spinal cord injury (SCI) and the improvement of locomotor recovery. In this study, we initially generated NPs from human embryonic stem cells (hESCs) and investigated their potential for neuronal differentiation and functional recovery when co-cultured with SCs in vitro and co-transplanted in a rat acute model of contused SCI. Co-cultivation results revealed that the presence of SCs provided a consistent status for hESC-NPs and recharged their neural differentiation toward a predominantly neuronal fate. Following transplantation, a significant functional recovery was observed in all engrafted groups (NPs, SCs, NPs+SCs) relative to the vehicle and control groups. We also observed that animals receiving co-transplants established a better state as assessed with the BBB functional test. Immunohistofluorescence evaluation five weeks after transplantation showed invigorated neuronal differentiation and limited proliferation in the co-transplanted group when compared to the individual hESC-NPs grafted group. These findings have demonstrated that the co-transplantation of SCs with hESC-NPs could offer a synergistic effect, promoting neuronal differentiation and functional recovery

Elevated calpain activity in acute myelogenous leukemia correlates with decreased calpastatin expression

Calpains are intracellular cysteine proteases that have crucial roles in many physiological and pathological processes. Elevated calpain activity has been associated with many pathological states. Calpain inhibition can be protective or lethal depending on the context. Previous work has shown that c-myc transformation regulates calpain activity by suppressing calpastatin, the endogenous negative regulator of calpain. Here, we have investigated calpain activity in primary acute myelogenous leukemia (AML) blast cells. Calpain activity was heterogeneous and greatly elevated over a wide range in AML blast cells, with no correlation to FAB classification. Activity was particularly elevated in the CD34+CD38− enriched fraction compared with the CD34+CD38+ fraction. Treatment of the cells with the specific calpain inhibitor, PD150606, induced significant apoptosis in AML blast cells but not in normal equivalent cells. Sensitivity to calpain inhibition correlated with calpain activity and preferentially targeted CD34+CD38− cells. There was no correlation between calpain activity and p-ERK levels, suggesting the ras pathway may not be a major contributor to calpain activity in AML. A significant negative correlation existed between calpain activity and calpastatin, suggesting calpastatin is the major regulator of activity in these cells. Analysis of previously published microarray data from a variety of AML patients demonstrated a significant negative correlation between calpastatin and c-myc expression. Patients who achieved a complete remission had significantly lower calpain activity than those who had no response to treatment. Taken together, these results demonstrate elevated calpain activity in AML, anti-leukemic activity of calpain inhibition and prognostic potential of calpain activity measurement

Differentiation of human dental pulp stem cells into functional motor neuron: In vitro and ex vivo study

There are several therapeutic options for spinal cord injury (SCI), among these strategies stem cell therapy is a potential treatment. The stem cells based therapies have been investigating in acute phase of clinical trials for promoting spinal repair in humans through replacement of functional neuronal and glial cells. The aim of this study was to evaluate the differentiation of Human Dental Pulp Stem Cells (hDPSCs) into functional motor neuron like cells (MNLCs) and promote neuroregeneration by stimulating local neurogenesis in the adult spinal cord slice culture. The immunocytochemistry analysis demonstrated that hDPSCs were positive for mesenchymal stem cell markers (CD73, CD90 and CD105) and negative for the hematopoietic markers (CD34 and CD45). hDPSCs were induced to neurospheres (via implementing B27, EGF, and bFGF) and then neural stem cells (NSC). The NSC differentiated into MNLCs in two steps: first by Shh and RA and; then with GDNF and BDNF administration. The NS and the NSC were assessed for Oct4, nestin, Nanog, Sox2 expression while the MNLCs were evaluated by ISLET1, Olig2, and HB9 genes. Our results showed that hDPSC can be differentiated into motor neuron phenotype with expression of the motor neuron genes. The functionality of MNLCs was demonstrated by FM1-43, intracellular calcium ion shift and co- culture with C2C12. We co-cultivated hDPSCs with adult rat spinal slices in vitro. Immunostaining and hoechst assay showed that hDPSCs were able to migrate, proliferate and integrate in both the anterolateral zone and the edges of the spinal slice

Transdifferentiation of Human Umbilical Cord-Derived Mesenchymal Stem Cells in Dopaminergic Neurons in a Three-Dimensional Culture

Introduction: The induction of human umbilical cord-derived mesenchymal stem cells (HUC-MSCs) toward dopaminergic neurons is a major challenge in tissue engineering and experimental and clinical treatments of various neurodegenerative diseases, including Parkinson disease. This study aims to differentiate HUC-MSCs into dopaminergic neuron-like cells. Methods: Following the isolation and characterization of HUC-MSCs, they were transferred to Matrigel-coated plates and incubated with a cocktail of dopaminergic neuronal differentiation factors. The capacity of differentiation into dopaminergic neuronlike cells in 2-dimensional culture and on Matrigel was assessed by real-time polymerase chain reaction, immunocytochemistry, and high-performance liquid chromatography. Results: Our results showed that dopaminergic neuronal markers' transcript and protein levels were significantly increased on the Matrigel differentiated cells compared to 2D culture plates. Conclusion: Overall, the results of this study suggest that HUC-MSCs can successfully differentiate toward dopaminergic neuron-like cells on Matrigel, having great potential for the treatment of dopaminergic neuron-related diseases. © 2022 Iran University of Medical Sciences. All rights reserved

Promoting motor functions in a spinal cord injury model of rats using transplantation of differentiated human olfactory stem cells: A step towards future therapy

Human olfactory ecto-mesenchymal stem cells (hOE-MSCs) derived from the human olfactory mucosa (OM) can be easily isolated and expanded in cultures while their immense plasticity is maintained. To mitigate ethical concerns, the hOE-MSCs can be also transplanted across allogeneic barriers, making them desirable cells for clinical applications. The main purpose of this study was to evaluate the effects of administering the hOE-MSCs on a spinal cord injury (SCI) model of rats. These cells were accordingly isolated and cultured, and then treated in the neurobasal medium containing serum-free Dulbecco's Modified Essential Medium (DMEM) and Ham's F-12 Medium (DMEM/F12) with 2 B27 for two days. Afterwards, the pre-induced cells were incubated in N2B27 with basic fibroblast growth factor (bFGF), fibroblast growth factor 8b (FGF8b), sonic hedgehog (SHH), and ascorbic acid (vitamin C) for six days. The efficacy of the induced cells was additionally evaluated using immunocytochemistry (ICC) and real-time polymerase chain reaction (RT-PCR). The differentiated cells were similarly transplanted into the SC contusions. Functional recovery was further conducted on a weekly basis for eight consecutive weeks. Moreover, cell integration was assessed via conventional histology and ICC, whose results revealed the expression of choline acetyltransferase (ChAT) marker at the induction stage. According to the RT-PCR findings, the highest expression level of insulin gene-enhancer protein (islet-1), oligodendrocyte transcription factor (Olig2), and homeobox protein HB9 was observed at the induction stage. The number of engraftment cells also rose (approximately by 2.5 ± 0.1) in the motor neuron-like cells derived from the hOE-MSCs-grafted group compared with the OE-MSCs-grafted one. The functional analysis correspondingly revealed that locomotor and sensory scores considerably improved in the rats in the treatment group. These findings suggested that motor neuron-like cells derived from the hOE-MSCs could be utilized as an alternative cell-based therapeutic strategy for SCI. © 2021 Elsevier B.V