Bone Marrow Stromal Cell Transdifferentiation into Oligodendrocyte-Like Cells Using Triiodothyronine as a Inducer with Expression of Platelet-Derived Growth Factor α 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 µM all-trans-retinoic acid during the pre-induction stage and then induced by heregulin (HRG), platelet-derived growth factor AA (PDGFR-α), 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-α 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-α 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. Key Words: Bone marrow stromal cell, Triiodothyronine, Platelet-derived growth factor α     

Evaluation of Neurogenic Potential of Human Umbilical Cord Mesenchymal Cells; a Time- and Concentration-Dependent Manner

Background:

Retinoic acid as one of the most important regulators for cell differentiation was examined in this study for differentiation of human umbilical mesenchymal cells (hUCM).

Methods:

After isolation, hUCM were evaluated for mesenchymal stem cell properties by flow cytometry and alkaline phosphatase assay. Also, doubling time of the cells and their differentiation potential into adipogenic and osteogenic cells were tested. hUCM were then cultured with different concentrations of retinoic acid, and on days 1, 7, and 12, the percentage of differentiated cells was determined by immunostaining for nestin, anti-microtubule associated protein 2 (MAP₂), glutamic acid decarboxylase (GAD), and gamma-aminobutyric acid (GABA) markers.

Results:

The isolated cells were negative for the hematopoietic markers and positive for the mesenchymal markers. They showed the population doubling time 60 ± 3 hours and differentiated into osteogenic and adipogenic cells. A descending trend in nestin and an ascending trend in MAP₂, GAD, and GABA expression were observed from the first day until the last day between different concentrations of retinoic acid.

Conclusion:

hUCM cells may have the potential to differentiate into neural cells in the presence of different incubation period and concentration of retinoic acid.Key Words: Cell differentiation, Neural stem cells, Retinoic acid     

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)     

利用米糠外添GAD制备γ-氨基丁酸的工艺条件优化

以稻米加工副产品米糠为原料,采用单因素和正交实验确定了外添谷氨酸脱羧酶(GAD)制备γ-氨基丁酸(GABA)的最佳工艺条件:GAD酶用量175u/g,酶解时间2h,pH5.5,酶解温度45℃。经验证实验,在最佳工艺条件下GABA生成量为1530.7mg/100g米糠。     

大豆萌发过程中谷氨酸脱羧酶活性变化研究

研究了大豆在浸泡、萌发两个过程中谷氨酸脱羧酶(GAD)的活力变化。通过测定大豆中γ-氨基丁酸(GABA)的含量变化,间接测定大豆中GAD的活性。结果表明,大豆在25℃,浸泡10h,大豆中的GAD活性达到浸泡中最高水平,酶活性为2.45U。浸泡后的大豆在30℃,萌发40h时,酶活性达到萌发中最高水平,为1.28U。大豆在浸泡和萌发两个状态下,GAD活性较储藏期都有很大提高,且浸泡时酶活总体水平高于萌发期酶活总体水平,其中大豆GAD在25℃,浸泡10h时活性最高,为2.45U。     

Fucoidan from Laminaria japonica Inhibits Expression of GLUT9 and URAT1 via PI3K/Akt,JNK and NF-κB Pathways in Uric Acid-Exposed HK-2 Cells

This work aimed to investigate the effect of fucoidan (FPS) on urate transporters induced by uric acid (UA). The results showed that UA stimulated the expression of glucose transporter 9 (GLUT9) and urate transporter 1 (URAT1) in HK-2 cells, and FPS could reverse the effect. Moreover, UA could activate NF-κB, JNK and PI3K/Akt pathways, but both pathway inhibitors and FPS inhibited the UA-induced activation of these three pathways. These data suggested that FPS effectively inhibited the expression induction of reabsorption transporters URAT1 and GLUT9 by UA, through repressing the activation of NF-κB, JNK and PI3K/Akt signal pathways in HK-2 cells. The in vitro research findings support the in vivo results that FPS reduces serum uric acid content in hyperuricemia mice and rats through inhibiting the expression of URAT1 and GLUT9 in renal tubular epithelial cells. This study provides a theoretical basis for the application of FPS in the treatment of hyperuricemia.     

The histopathalogical effects of retinoic acid on the tissues

The aim of this study is to sum up the important information that has emerged from the last 10 years of experimental investigations over the effects of retinoic acid (RA) on embryonic structure and adult tissues. Administration of exogenous RA can affect the connective tissues including enhancement of myeloid compartment and suppression of erythroid cells and conversion of hematopoietic stem cells to erythroid progenitors. Also, it is able to induce osteogenic differentiation of stem cells derived from adipose tissues and etc. Examining the neural tissue highlighted that disruption of RA signaling in the adult leads to degeneration of motor neurons and development of some diseases. In vitro administration of All-Trans Retinoic Acid (ATRA) increased dendritic growth and synaptophysin puncta intensity and increased expressions of neuronal nuclei, neuron specific enolase, synaptophysin. RA also promotes expression of a marker of mature astrocytes. On muscular tissue, it can inhibit proliferation of smooth muscle cells (SMC) while promoting differentiation of SMC in vitro instead. The ATRA stimulates skeletal myogenesis while inhibiting cardiomyogenesis and hypertrophy and proliferation of cultured neonatal cardiomyocytes and cardiofibroblasts. In addition, differences in levels of embryonic RA may contribute to variability in great artery anomalies. In epithelial tissue, the squamous epithelium exposed to ATRA showed the columnar differentiation independent to proliferation. Also RA seems able to rescue the regeneration process of injured gut and revealing a better wound healing of the intestine undergone intra-operative radiotherapy. It can interrupt the process of progressive fibrosis, enhancements of the langerhans islets, exocrine pancreas, modulate the health of the mammary glands and repairs the lung cell. Thus, differences in levels of endogenous RA in embryonic and adult tissues may contribute to anomalies and pathogenesis of disease, furthermore RA has paradoxical effects on the parts forming the connective and muscles tissue in equal conditions.     

Mesenchymal Stem Cells as an Alternative for Schwann Cells in Rat Spinal Cord Injury

Background: Spinal cord has a limited capacity to repair; therefore, medical interventions are necessary for treatment of injuries. Transplantation of Schwann cells has shown a great promising result for spinal cord injury (SCI). However, harvesting Schwann cell has been limited due to donor morbidity and limited expansion capacity. Furthermore, accessible sources such as bone marrow stem cells have drawn attentions to themselves. Therefore, this study was designed to evaluate the effect of bone marrow-derived Schwann cell on functional recovery in adult rats after injury. Methods: Mesenchymal stem cells were cultured from adult rats’ bone marrow and induced into Schwann cells in vitro. Differentiation was confirmed by immunocytochemistry and RT-PCR. Next, Schwann cells were seeded into collagen scaffolds and engrafted in 3 mm lateral hemisection defects. For 8 weeks, motor and sensory improvements were assessed by open field locomotor scale, narrow beam, and tail flick tests. Afterwards, lesioned spinal cord was evaluated by conventional histology and immunohistochemistry. Results: In vitro observations showed that differentiated cells had Schwann cell morphology and markers. In this study, we had four groups (n = 10 each): laminectomy, control, scaffold and scaffold + Schwann cells. Locomotor and sensory scores of cell grafted group were significantly better than control and scaffold groups. In histology, axonal regeneration and remyelination were better than control and scaffold groups. Conclusion: This study demonstrates that bone marrow-derived Schwann cells can be considered as a cell source for Schwann cells in SCI treatment. Key Words: Rats, Spinal cord injuries (SCI), Bone marrow, Schwann cells, Cell transdifferentiation     

Neuronal Differentiation of Rat Hair Follicle Stem Cells: the Involvement of the Neuroprotective Factor Seladin-1 (DHCR24)

Background: The seladin-1 (selective Alzheimer disease indicator-1), also known as DHCR24, is a gene found to be down-regulated in brain region affected by Alzheimer disease (AD). Whereas, hair follicle stem cells (HFSC), which are affected in with neurogenic potential, it might to hypothesize that this multipotent cell compartment is the predominant source of seladin-1. Our aim was to evaluate seladin-1 gene expression in hair follicle stem cells. Methods: In this study, bulge area of male Wistar rat HFSC were cultured and then characterized with Seladin-1 immunocytochemistry and flow cytometry on days 8 to 14. Next, 9-11-day cells were evaluated for seladin-1 gene expression by real-time PCR. Results: Our results indicated that expression of the seladin-1 gene (DHCR24) on days 9, 10, and 11 may contribute to the development of HFSC. However, the expression of this gene on day 11 was more than day 10 and on 10^th day was more than day 9. Also, we assessed HFSC on day 14 and demonstrated these cells were positive for β-ш tubulin, and seladin-1 was not expressed in this day. Conclusion: HFSC express seladin-1 and this result demonstrates that these cells might be used to cell therapy for AD in future. Key Words: Seladin-1 (selective Alzheimer disease indicator-1), Alzheimer disease (AD), Hair follicle stem cells     

The Anticancer Effect of Fucoidan in PC-3 Prostate Cancer Cells

Fucoidan, a sulfated polysaccharide, has a variety of biological activities, such as anti-cancer, anti-angiogenic and anti-inflammatory. However, the mechanisms of action of fucoidan as an anti-cancer agent have not been fully elucidated. The present study examined the anti-cancer effect of fucoidan obtained from Undaria pinnatifida in PC-3 cells, human prostate cancer cells. Fucoidan induced the apoptosis of PC-3 cells by activating both intrinsic and extrinsic pathways. The induction of apoptosis was accompanied by the activation of extracellular signal-regulated kinase mitogen-activated protein kinase (ERK1/2 MAPK) and the inactivation of p38 MAPK and phosphatidylinositol 3-kinase (PI3K)/Akt. In addition, fucoidan also induced the up-regulation of p21^Cip1/Waf and down-regulation of E2F-1 cell-cycle-related proteins. Furthermore, in the Wnt/β-catenin pathway, fucoidan activated GSK-3β that resulted in the decrease of β-catenin level, followed by the decrease of c-myc and cyclin D1 expressions, target genes of β-catenin in PC-3 cells. These results suggested that fucoidan treatment could induce intrinsic and extrinsic apoptosis pathways via the activation of ERK1/2 MAPK, the inactivation of p38 MAPK and PI3K/Akt signaling pathway, and the down-regulation of Wnt/β-catenin signaling pathway in PC-3 prostate cancer cells. These data support that fucoidan might have potential for the treatment of prostate cancer.     

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     

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     

Delivery of epidermal neural crest stem cells (EPI-NCSC) to hippocamp in Alzheimer's disease rat model

Background: Alzheimer’s disease (AD) is characterized by progressive neuronal loss in hippocamp. Epidermal neural crest stem cells (EPI-NCSC) can differentiate into neurons, astrocytes and oligodendrocytes. The purpose of this study was to evaluate the effects of transplanting EPI-NCSC into AD rat model. Methods: Two weeks after induction of AD by injection of Amyloid-β 1-40 into CA1 area of rat hippocamp, Y-maze and single-trial passive avoidance tests were used to show deficit of learning and memory abilities. EPI-NCSC were obtained from the vibrissa hair follicle of rat, cultured and labeled with bromodeoxyuridine. When Alzheimer was proved by behavioral tests, EPI-NCSC was transplanted into CA3 area of hippocamp in AD rat model. The staining of EPI-NCSC markers (nestin and SOX10) was done in vitro. Double-labeling immunofluorescence was performed to study survival and differentiation of the grafted cells. Results: We showed that transplanted EPI-NCSC survive and produce many neurons and a few glial cells, presenting glial fibrillary acidic protein. Total number of granule cells in hippocamp was estimated to be more in the AD rat model with transplanted cells as compared to AD control group. We observed that rats with hippocampal damage made more errors than control rats on the Y-maze, when reward locations were reversed. Conclusion: Transplanted cells were migrated to all areas of hippocamp and the total number of granule cell in treatment group was equal compared to control group. Transplantation of EPI-NCSC into hippocamp might differentiate into cholinergic neurons and could cure impairment of memory in AD rat model.Key Words: Alzheimer’s disease, Cholinergic neuron, Hair follicle     

α-Conotoxins and α-Cobratoxin Promote,while Lipoxygenase and Cyclooxygenase Inhibitors Suppress the Proliferation of Glioma C6 Cells

Among the brain tumors, glioma is the most common. In general, different biochemical mechanisms, involving nicotinic acetylcholine receptors (nAChRs) and the arachidonic acid cascade are involved in oncogenesis. Although the engagement of the latter in survival and proliferation of rat C6 glioma has been shown, there are practically no data about the presence and the role of nAChRs in C6 cells. In this work we studied the effects of nAChR antagonists, marine snail α-conotoxins and snake α-cobratoxin, on the survival and proliferation of C6 glioma cells. The effects of the lipoxygenase and cyclooxygenase inhibitors either alone or together with α-conotoxins and α-cobratoxin were studied in parallel. It was found that α-conotoxins and α-cobratoxin promoted the proliferation of C6 glioma cells, while nicotine had practically no effect at concentrations below 1 µL/mL. Nordihydroguaiaretic acid, a nonspecific lipoxygenase inhibitor, and baicalein, a 12-lipoxygenase inhibitor, exerted antiproliferative and cytotoxic effects on C6 cells. nAChR inhibitors weaken this effect after 24 h cultivation but produced no effects at longer times. Quantitative real-time polymerase chain reaction showed that mRNA for α4, α7, β2 and β4 subunits of nAChR were expressed in C6 glioma cells. This is the first indication for involvement of nAChRs in mechanisms of glioma cell proliferation.     

Linagliptin Protects Human SH-SY5Y Neuroblastoma Cells against Amyloid-β Cytotoxicity via the Activation of Wnt1 and Suppression of IL-6 Release

Background:Alzheimer’s disease is one of the neurodegenerative disorders typified by the aggregate of Aβ and phosphorylated tau protein. Oxidative stress and neuroinflammation, because of Aβ peptides, are strongly involved in the pathophysiology of AD. Linagliptin shows neuroprotective properties against AD pathological processes through alleviation of neural inflammation and AMPK activation. Methods:We assessed the benefits of linagliptin pretreatment (at 10, 20, and 50 nM concentrations), against Aβ1-42 toxicity (20 μM) in SH-SY5Y cells. The concentrations of secreted cytokines, such as TNF-α, IL-6, and IL-1β, and signaling proteins, including pCREB, Wnt1, and PKCε, were quantified by ELISA. Results:We observed that Aβ led to cellular inflammation, which was assessed by measuring inflammatory cytokines (TNF-α, IL-1β, and IL-6). Moreover, Aβ1-42 treatment impaired pCREB, PKCε, and Wnt1 signaling in human SH-SY5Y neuroblastoma cells. Addition of Linagliptin significantly reduced IL-6 levels in the lysates of cells, treated with Aβ1-42. Furthermore, linagliptin prevented the downregulation of Wnt1 in Aβ1-42-treated cells exposed. Conclusion: The current findings reveal that linagliptin alleviates Aβ1-42-induced inflammation in SH-SY5Y cells, probably through the suppression of IL-6 release, and some of its benefits are mediated through the activation of the Wnt1 signaling pathway. Key Words: Alzheimer disease, Interleukin-6, Linagliptin, Wnt1 protein