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1.
The aim of this study was to compare the neural differentiation potential and the expression of neurotrophic factors (NTFs) in differentiated adipose-derived stem cells (ADSCs) using three established induction protocols, serum free (Protocol 1), chemical reagents (Protocol 2), and spontaneous (Protocol 3) protocols. Protocol 1 produced the highest percentage of mature neural-like cells (MAP2ab+). Protocol 2 showed the highest percentage of immature neural-like cells (β-tubulin III+), but the neural-like state was transient and reversible. Protocol 3 caused ADSCs to differentiate spontaneously into immature neural-like cells, but not into mature neural cell types. The neural-like cells produced by Protocol 1 lived the longest in culture with little cell death, but Protocol 2 and 3 led to the significant cell death. Therefore, Protocol 1 is the most efficient among these protocols. Additionally, soon after differentiation, the mRNA levels of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) in dADSCs were sharply decreased by Protocol 1 and 2 (acute induction protocol), but not by Protocol 3 (chronic induction protocol). The results indicate that NTFs played an important role in neural differentiation via acute responses to NGF and BDNF, but not chronically during the transdifferentiation process.  相似文献   

2.
Calcitonin gene-related peptide (CGRP) promotes neuron recruitment and neurogenic activity. However, no evidence suggests that CGRP affects the ability of stem cells to differentiate toward neurogenesis. In this study, we genetically modified rat adipose-derived stem cells (ADSCs) with the CGRP gene (CGRP-ADSCs) and subsequently cultured in complete neural-induced medium. The formation of neurospheres, cellular morphology, and proliferative capacity of ADSCs were observed. In addition, the expression of the anti-apoptotic protein Bcl-2 and special markers of neural cells, such as Nestin, MAP2, RIP and GFAP, were evaluated using Western blot and immunocytochemistry analysis. The CGRP-ADSCs displayed a greater proliferation than un-transduced (ADSCs) and Vector-transduced (Vector-ADSCs) ADSCs (p<0.05), and lower rates of apoptosis, associated with the incremental expression of Bcl-2, were also observed for CGRP-ADSCs. Moreover, upon neural induction, CGRP-ADSCs formed markedly more and larger neurospheres and showed round cell bodies with more branching extensions contacted with neighboring cells widely. Furthermore, the expression levels of Nestin, MAP2, and RIP in CGRP-ADSCs were markedly increased, resulting in higher levels than the other groups (p<0.05); however, GFAP was distinctly undetectable until day 7, when slight GFAP expression was detected among all groups. Wnt signals, primarily Wnt 3a, Wnt 5a and β-catenin, regulate the neural differentiation of ADSCs, and CGRP gene expression apparently depends on canonical Wnt signals to promote the neurogenesis of ADSCs. Consequently, ADSCs genetically modified with CGRP exhibit stronger potential for differentiation and neurogenesis in vitro, potentially reflecting the usefulness of ADSCs as seed cells in therapeutic strategies for spinal cord injury.  相似文献   

3.
It is reported that adipose-derived stem cells (ADSCs) had multilineage differentiation potential, and could differentiate into neuron-like cells induced by special induction media, which may provide a new idea for restoration of erectile dysfunction (ED) after cavernous nerve injury. The aim of this research was to explore the neuronal differentiation potential of ADSCs in vitro. ADSCs isolated from inguinal adipose tissue of rat were characterized by flow cytometry, and results showed that ADSCs were positive for mesenchymal stem cell markers CD90 and CD44, but negative for hematopoietic stem cell markers. ADSCs maintained self-renewing capacity and could differentiate into adipocytes and neurocytes under special culture condition. In this research, two methods were used to induce ADSCs. In method 1, ADSCs were treated with the preinduction medium including epithelium growth factor, basic fibroblast growth factor, and brain derived neurotrophic factor (BDNF) for 3?days, then with the neurogenic induction medium containing isobutylmethylxanthine, indomethacin, and insulin. While in method 2, BDNF was not used to treat ADSCs. After induction, neuronal differentiation of ADSCs was evaluated. Neuronal markers, glial fibrillary acidic protein (GFAP), and ??-tubulin III (Tuj-1) were detected by immunofluorescence and Western Blot analyses. The expressions of GFAP and Tuj-1 in method 1 were obviously higher then those in method 2. In addition, the positive rate of the neuron-like cells was higher in method 1. It suggested that ADSCs are able to differentiate into neural-like cells in vitro, and the administration of BDNF in the preinduction medium may provide a new way to modify the culture method for getting more neuron-like cells in vitro.  相似文献   

4.
The differentiation of human mesenchymal stem cells (hMSCs) into neural cells in vitro provides a potential tool to be utilized for cell therapy of neurodegenerative disorders. Although previous studies repeated different protocols for the induction of neural cells from hMSCs in vitro, the results were not in complete agreement. In this study, we have attempted to compare three of these neural induction methods; retinoic acid (RA) treatment, RA treatment in serum reduced conditions, and treatment using other chemical compounds (dimethyl sulfoxide and potassium chloride) along with RA by real-time cell analysis and immunofluorescent staining of neural markers. RA treatment led to a slow progression of cells into neural-like morphology with the expression of neural protein neurofilament whereas reducing serum during RA treatment caused a much more extended differentiation process. Additionally, neural-like morphology was persistent in the later periods of differentiation in RA treatment. On the other hand, chemical induction caused cell shrinkages mimicking neural-like morphology in a short time and loss of this morphology along with increased cell death in later periods. Among the three methods compared, RA treatment was the most reliable one in terms of stability of differentiation and neural protein expressions.  相似文献   

5.
Pluripotent stem cell lines have been generated in several domestic animal species; however, these lines traditionally show poor self-renewal and differentiation. Using canine embryonic stem cell (cESC) lines previously shown to have sufficient self-renewal capacity and potency, we generated and compared canine neural stem cell (cNSC) lines derived by lineage selection with epidermal growth factor (EGF) or Noggin along the neural default differentiation pathway, or by directed differentiation with retinoic acid (RA)-induced floating sphere assay. Lineage selection produced large populations of SOX2+ neural stem/progenitor cell populations and neuronal derivatives while directed differentiation produced few and improper neuronal derivatives. Primary canine neural lines were generated from fetal tissue and used as a positive control for differentiation and electrophysiology. Differentiation of EGF- and Noggin-directed cNSC lines in N2B27 with low-dose growth factors (BDNF/NT-3 or PDGFαα) produced phenotypes equivalent to primary canine neural cells including 3CB2+ radial progenitors, MOSP+ glia restricted precursors, VIM+/GFAP+ astrocytes, and TUBB3+/MAP2+/NFH+/SYN+ neurons. Conversely, induction with RA and neuronal differentiation produced inadequate putative neurons for further study, even though appropriate neuronal gene expression profiles were observed by RT-PCR (including Nestin, TUBB3, PSD95, STX1A, SYNPR, MAP2). Co-culture of cESC-derived neurons with primary canine fetal cells on canine astrocytes was used to test functional maturity of putative neurons. Canine ESC-derived neurons received functional GABA(A)- and AMPA-receptor mediated synaptic input, but only when co-cultured with primary neurons. This study presents established neural stem/progenitor cell populations and functional neural derivatives in the dog, providing the proof-of-concept required to translate stem cell transplantation strategies into a clinically relevant animal model.  相似文献   

6.
Electrospun nanofibrous scaffolds show huge potential to improve the neurological outcome in central nervous system disorders. In this study, we cultured mouse embryonic stem cells (mESCs) on an electrospun nanofibrous polylactic acid/Chitosan/Wax (PLA/CS/Wax) scaffold and surveyed the attachment, behavior, and differentiation of mESCs into neural cells. Differentiation in neural-like cells (NLCs) was investigated with a medium containing SB431542 as a small molecule and conjugated linolenic acid after 20 days. We used Immunocytochemistry and quantitative real-time polymerase chain reaction (RT-PCR) techniques to assess neural marker expression in differentiated cells. SEM imaging demonstrated that mESCs could strongly attach, stretch, and differentiate on PLA/CS/Wax scaffolds. MESCs that were cultured on PLA/CS/Wax scaffolds showed enhanced numbers of neural structures and neural markers including Nestin, NF-H, Tuj-1, and Map2 in neural induction medium compared to the control sample. These results revealed that electrospun PLA/CS/Wax scaffolds associated with the induction medium can assemble proper conditions for stem cell differentiation into NLCs. We hope that the development of new technologies in neural tissue engineering may pave a new avenue for neural tissue regeneration.  相似文献   

7.
This study comparatively investigated the effectiveness of calcium and other well‐known inducers such as isobutylmethylxanthine (IBMX) and insulin in differentiating human adipose‐derived stem cells (ADSCs) into neuronal‐like cells. ADSCs were immunophenotyped and differentiated into neuron‐like cells with different combinations of calcium, IBMX, and insulin. Calcium mobilization across the membrane was determined. Differentiated cells were characterized by cell cycle profiling, staining of Nissl bodies, detecting the gene expression level of markers such as neuronal nuclear antigen (NeuN), microtubule associated protein 2 (MAP2), neuron‐specific enolase (NSE), doublecortin, synapsin I, glial fibrillary acidic protein (GFAP), and myelin basic protein (MBP) by quantitative real‐time polymerase chain reaction (quantitative real‐time polymerase chain reaction (qRT‐PCR) and protein level by the immunofluorescence technique. Treatment with Ca + IBMX + Ins induced neuronal appearance and projection of neurite‐like processes in the cells, accompanied with inhibition of proliferation and halt in the cell cycle. A significantly higher expression of MBP, GFAP, NeuN, NSE, synapsin 1, doublecortin, and MAP2 was detected in differentiated cells, confirming the advantages of Ca + IBMX + Ins to the other combinations of inducers. Here, we showed an efficient protocol for neuronal differentiation of ADSCs, and calcium fostered differentiation by augmenting the number of neuron‐like cells and instantaneous increase in the expression of neuronal markers.  相似文献   

8.
Although the avian primordial germ cells (PGCs) have been used to produce transgenic birds, their characteristics largely remain unknown. The isolation, culture, biological characterization, and directed neural differentiation of duck EG cells were assayed in this study. The Results showed that the EG cells were got by isolating embryonic gonad and surrounding tissue from 7-day-old duck embryo. The PGCs co-cultured with their gonadal somatic cells were well grown. After passaging, the EG cells were incubated in medium with cytokines and Mitomycin C on inactivated duck embryonic fibroblasts (DEFs) feeder layers. After several passages, alkaline phosphatase (ALP) and periodic acid-Schiff (PAS) resulted positive, cellular markers detection positive for SSEA-1, SSEA-4, TRA-1-60, and TRA-1-81. Karyotype analysis showed the EG cells kept diploid condition and the hereditary feature was stable in accordance with varietal characteristics of duck. These cells grew continuously for 11 passages on DEFs. Under induction of medium with BME, RA, and IBMX, the EG cells lost undifferentiated state, large amount of neural cells appeared with the formation of neural cells networks. Special Nissl body was found by toluidine blue stain after induced for 7 days. Immunofluorescence staining results indicated that differentiated EG cells expressed Nestin, NSE, and GFAP positive. The expression of Nestin, NSE, and GFAP mRNA were positive by RT-PCR. The results revealed that RA can obviously promote the directed differentiation of duck EG cells into neural lineage. The duck EG cells will be useful for the production of transgenic birds, for cell replacement therapy and for studies of germ cell differentiation.  相似文献   

9.
In order to characterize the potency of menstrual blood stem cells (MenSCs) for future cell therapy of neurological disorders instead of bone marrow stem cells (BMSCs) as a well-known and conventional source of adult stem cells, we examined the in vitro differentiation potential of these stem cells into neural-like cells. The differentiation potential of MenSCs to neural cells in comparison with BMSCs was assessed under two step neural differentiation including conversion to neurosphere-like cells and final differentiation. The expression levels of Nestin, Microtubule-associated protein 2, gamma-aminobutyric acid type B receptor subunit 1 and 2, and Tubulin, beta 3 class III mRNA and/or protein were up-regulated during development of MenSCs into neurosphere-like cells (NSCs) and neural-like cells. The up-regulation level of these markers in differentiated neural-like cells from MenSCs was comparable with differentiated cells from BMSCs. Moreover, both differentiated MenSCs and BMSCs expressed high levels of potassium, calcium and sodium channel genes developing functional channels with electrophysiological recording. For the first time, we demonstrated that MenSCs are a unique cell population with differentiation ability into neural-like cells comparable to BMSCs. In addition, we have introduced an approach to generate NSCs from MenSCs and BMSCs and their further differentiation into neural-like cells in vitro. Our results hold a promise to future stem cell therapy of neurological disorders using NSCs derived from menstrual blood, an accessible source in every woman.  相似文献   

10.
Bone marrow mesenchymal stem cells (MSC) are multipotent cells. To explain their plasticity, we postulated that undifferentiated MSC may express proteins from other tissues such as neuronal tissues. MSC are obtained by two different approaches: plastic adhesion or negative depletion (RosetteSep and magnetic beads CD45/glycophorin A). MSC are evaluated through FACS analysis using a panel of antibodies (SH2, SH3, CD14, CD33, CD34, CD45, etc.). To confirm the multipotentiality in vitro, we have differentiated MSC into adipocytes, chondrocytes, osteocytes, and neuronal/glial cells using specific induction media. We have evaluated neuronal and glial proteins (Nestin, Tuj-I, betaIII Tubulin, tyrosine hydroxylase [TH], MAP-2, and GFAP) by using flow cytometry, Western blots, and RT-PCR. We found that MSC constituently express native immature neuronal proteins such as Nestin and Tuj-1. After only five passages, MSC can already express more mature neuronal or glial proteins, such as TH, MAP-2, and GFAP, without any specific induction. We noticed an increase in the expression of more mature neuronal/glial proteins (TH, MAP-2, and GFAP) after exposure to neural induction medium, thus confirming the differentiation of MSC into neurons and astrocytes. The constitutive expression of Nestin or Tuj-1 by MSC suggests that these cells are "multidifferentiated" cells and thus can retain the ability for neuronal differentiation, enhancing their potentiality to be employed in the treatment of neurological diseases.  相似文献   

11.
Mesenchymal stem cells (MSCs) can differentiate into neurons in an appropriate cellular environment. Retinoid signaling pathway is required in neural development. However, the effect and mechanism through retinoid signaling regulates neuronal differentiation of MSCs are still poorly understood. Here, we report that all‐trans‐retinoic acid (ATRA) pre‐induction improved neuronal differentiation of rat MSCs. We found that, when MSCs were exposed to different concentrations of ATRA (0.01–100 μmol/L) for 24 h and then cultured with modified neuronal induction medium (MNM), 1 μmol/L ATRA pre‐induction significantly improved neuronal differentiation efficiency and neural‐cell survival. Compared with MNM alone induced neural‐like cells, ATRA/MNM induced cells expressed higher levels of Nestin, neuron specific enolase (NSE), microtubule‐associated protein‐2 (MAP‐2), but lower levels of CD68, glial fibrillary acidic protein (GFAP), and glial cell line‐derived neurotrophic factor(GDNF), also exhibited higher resting membrane potential and intracellular calcium concentration, supporting that ATRA pre‐induction promotes maturation and function of derived neurons but not neuroglia cells from MSCs. Endogenous retinoid X receptors (RXR) RXRα and RXRγ (and to a lesser extent, RXRβ) were weakly expressed in MSCs. But the expression of RARα and RARγ was readily detectable, whereas RARβ was undetectable. However, at 24 h after ATRA treatment, the expression of RARβ, not RARα or RARγ, increased significantly. We further found the subnuclear redistribution of RARβ in differentiated neurons, suggesting that RARβ may function as a major mediator of retinoid signaling during neuronal differentiation from MSCs. ATRA treatment upregulated the expression of Vimentin and Stra13, while it downregulated the expression of Brachyury in MSCs. Thus, our results demonstrate that pre‐activation of retinoid signaling by ATRA facilitates neuronal differentiation of MSCs.  相似文献   

12.
目的研究人胚不同脑区神经前体细胞(neural progenitor cells,NPCs)培养及增殖分化特性。方法取14-17周人胚脑区组织,分为新皮质、纹状体、间脑、中脑、后脑和延髓组,悬浮培养。鉴定细胞球巢蛋白抗原的表达,分化及自我更新能力。观察各脑区培养细胞的生长、增殖状况。新皮质、纹状体及间脑来源的神经球分化后,运用免疫荧光细胞化学法比较神经元及星形胶质细胞的比例。结果各脑区培养出的悬浮细胞球巢蛋白抗原阳性,可分化为MAP2或GFAP阳性细胞,且BrdU掺入实验阳性。体外培养第3d,纹状体及间脑组均可见大量神经球,且纹状体组明显多于间脑组;新皮质组传代后可见较多神经球;其它组仅见个别神经球。新皮质、纹状体、间脑来源的NPCs诱导分化后,MAP2或GFAP阳性细胞率各组间比较差异无显著性。结论人胚不同脑区均可培养出NPCs,从易到难依次为纹状体、间脑、新皮质及其它脑区。新皮质、纹状体、间脑来源的NPCs体外分化比例一致。  相似文献   

13.
Retinoic acid (RA) plays multiple roles in the nervous system, including induction of neural differentiation, axon outgrowth and neural patterning. Previously, RA for neural differentiation of embryonic stem (ES) cells always relies on embryoid bodies (EBs) formation. Here we report an in vitro adherent monoculture system to induce mouse ES cells into neural cells accompanied with RA. RA (1 μM) treatment, during initial 2 days of differentiation, can enhance the expression of neural markers, such as Nestin, Tuj1 and MAP2, and result in an earlier neural differentiation of ES cells. Furthermore, RA promotes a significant increase in neurite elongation of ES-derived neurons. Our study also implies that RA induced to express Wnt antagonist Dickkopf-1 (Dkk-1) for neural differentiation. However, the mechanisms of RA triggering neural induction remain to be determined. Our simple and efficient strategy is proposed to provide a basis for studying RA signaling pathways in neural differentiation in vitro.  相似文献   

14.
目的:研究孤儿核受体相关基因1(Nurr-1)对脂肪干细胞(adipose tissue-derived stem cells,ADSC)向神经元方向分化的潜在作用。方法:流式细胞术与成骨、成脂诱导技术鉴定脂肪干细胞;Nurrr-1基因转染脂肪干细胞后,应用神经特异性标志物MAP-2,β-tubulin的免疫荧光染色评估其向神经方向分化的能力。结果:流式细胞术结果表明培养的细胞CD29,CD44表达90%以上,CD45,CD90表达均低于1.5%,经过诱导后,油红O、茜素红S染色均呈阳性,表明所培养的细胞为脂肪干细胞;慢病毒转染Nurr-1基因后,免疫荧光染色检测MAP-2,β-tubulin的免疫荧光强度显著增加;RT-PCR结果显示Nurr-1转染的脂肪干细胞的MAP-2、β-tubulin、NF200的表达量显著提高。结论:Nurr-1基因转染能促进脂肪干细胞向神经方向分化,为神经损伤和神经退行性病变的治疗提供了新途径。  相似文献   

15.
Stem cells from human exfoliated deciduous teeth (SHED) are highly proliferative, clonogenic and multipotent stem cells with a neural crest cell origin. Additionally, they can be collected with minimal invasiveness in comparison with other sources of mesenchymal stem cells (MSCs). Therefore, SHED could be a desirable option for potential therapeutic applications. In this study, SHEDs were established from enzyme-disaggregated deciduous dental pulp obtained from 6 to 9 year-old children. The cells had typical fibroblastoid morphology and expressed antigens characteristic of MSCs, STRO1, CD146, CD45, CD90, CD106 and CD166, but not the hematopoietic and endothelial markers, CD34 and CD31, as assessed by FACS analysis. Differentiation assessment revealed a strong osteogenic and adipogenic potential of SHEDs. In order to further evaluate the in vitro differentiation potential of SHED into neural cells, a simple short time growth factor-mediated induction was used. Immunofluorescence staining and flow cytometric analysis revealed that SHED rapidly expressed nestin and b-III tubulin, and later expressed intermediate neural markers. In addition, the intensity and percentages of nestin and b-III tubulin and mature neural markers (PSA-NCAM, NeuN, Tau, TH, or GFAP) increased significantly following treatment. Moreover, RT-PCR and Western blot analyses showed that the neural markers were strongly up-regulated after induction. In conclusion, these results provide evidence that SHED can differentiate into neural cells by the expression of a comprehensive set of genes and proteins that define neural-like cells in vitro. SHED cells might be considered as new candidates for the autologous transplantation of a wide variety of neurological diseases and neurotraumatic injuries.  相似文献   

16.
The purpose of this study was to investigate miR-7 overexpression effects on neural differentiation of mesenchymal stem cells (MSCs) on both two-dimensional (2D) and three-dimensional (3D) culture systems. We upregulated miR-7 through lentiviral vector in trabecular meshwork MSCs (TMMSCs) and polymers of poly l -lactic acid/polycaprolactone fibrous scaffold were fabricated by electrospinning and characterized using scanning electron microscopy (SEM) and Fourier transform infrared (FTIR). Neural markers expression was evaluated through quantitative-polymerase chain reaction (q-PCR) and immunostaining. The results showed that the high percentage of cell transduction (84.9%) and miR-7 expression (folds: 677.93 and 556.4) was detected in TMMSCs-miR-7(+). SEM and FTIR established the fabrication of the hybrid scaffold. q-PCR analysis showed that on days 14 and 21 of transduction, the expression level of Nestin and glial fibrillary acidic protein (GFAP) genes were significantly higher in the scaffold (3D) compared with tissue culture polystyrene (2D) culture. The expression of microtubule-associated protein-2 (MAP-2) and GFAP genes in TMMSCs-miR-7(+) cells were significantly higher than those miR-7(−) cells after 21 days of cell culture. Also, MAP-2 and Nestin proteins were detected in TMMSCs-miR-7(+) cells. Our results demonstrate that miR-7 is involved in neural differentiation of TMMSCs and scaffold can improve differentiate into glial and neural progenitor cells. These findings provided some information for future use of microRNAs and scaffold in tissue engineering and cell therapy for neurological diseases.  相似文献   

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The beneficial impact of lowering oxygen tension to physiological levels has been demonstrated in a number of stem cell differentiation protocols. The majority of these studies compare normal laboratory oxygen tension with one physiological condition (typically 2-5% O(2) ). In this article, we investigated whether the full spectrum of physiological oxygen tensions (0-20% O(2) ) and step-changes in oxygen tension could enhance the production of neural populations from of embryonic stem cells (ESCs). We used a model system for the conversion of mouse ESCs into cells expressing one neuroectoderm stem cell marker (nestin) and two neural markers (βIII tubulin and microtubule-associated protein (MAP2)). 4-10% O(2) was associated with large increases in the total production of viable cells and the highest number of cells expressing Nestin, βIII tubulin, and MAP2. However, 4-10% O(2) also caused a reduction in the percentage of cells expressing all three markers. Step changes in oxygen tension at the mid-point of the differentiation process affected the total production of viable cells and the percentage of cells expressing all three markers. We found that the initial oxygen tension and the magnitude of the step-change were critical variables. A step increase from 0 to 2% O(2) mid-way through the protocol resulted in the highest percentage of cells expressing βIII tubulin (86.5%). In conclusion, we have demonstrated that the full spectrum of physiological oxygen tensions and step changes in oxygen tension represent a powerful tool for the optimisation of neural differentiation processes.  相似文献   

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