Caveolin-1 downregulation promotes the dopaminergic neuron-like differentiation of human adipose-derived mesenchymal stem cells

Previous studies have shown that caveolin-1 is involved in regulating the differentiation of mesenchymal stem cells. However, its role in the differentiation of human adipose mesenchymal stem cells into dopaminergic neurons remains unclear. The aim of this study was to investigate whether caveolin-1 regulates the differentiation of human adipose mesenchymal stem cells into dopaminergic-like neurons. We also examined whether the expression of caveolin-1 could be modulated by RNA interference technology to promote the differentiation of human adipose mesenchymal stem cells into dopaminergic-like neurons. The differentiation of human adipose mesenchymal stem cells into dopaminergic neurons was evaluated morphologically and by examining expression of the markers tyrosine hydroxylase, Lmx1 a and Nurr1. The analyses revealed that during the differentiation of human adipose mesenchymal stem cells into dopaminergic neurons, the expression of caveolin-1 is decreased. Notably, the downregulation of caveolin-1 promoted the differentiation of human adipose mesenchymal stem cells into dopaminergic-like neurons, and it increased the expression of tyrosine hydroxylase, Lmx1 a and Nurr1. Together, our findings suggest that caveolin-1 plays a negative regulatory role in the differentiation of dopaminergic-like neurons from stem cells, and it may therefore be a potential molecular target for strategies for regulating the differentiation of these cells. This study was approved by the Medical Ethics Committee of the First Affiliated Hospital of Dalian Medical University of China(approval No. PJ-KS-KY-2020-54) on March 7, 2017.     

Differentiation of embryonic versus adult rat neural stem cells into dopaminergic neurons in vitro

BACKGROUND:It has been reported that the conversion of neural stem cells into dopaminergic neurons in vitro can be increased through specific cytokine combinations. Such neural stem cell-derived dopaminergic neurons could be used for the treatment of Parkinson’s disease. However, little is known about the differences in dopaminergic differentiation between neural stem cells derived from adult and embryonic rats. OBJECTIVE: To study the ability of rat adult and embryonic-derived neural stem cells to differen...     

Growth and differentiation of neural stem cells in a three-dimensional collagen gel scaffold

Collagen protein is an ideal scaffold material for the transplantation of neural stem cells. In this study, rat neural stem cells were seeded into a three-dimensional collagen gel scaffold, with suspension cultured neural stem cells being used as a control group. Neural stem cells, which were cultured in medium containing epidermal growth factor and basic fibroblast growth factor, actively expanded and formed neurospheres in both culture groups. In serum-free medium conditions, the processes extended from neurospheres in the collagen gel group were much longer than those in the suspension culture group. Immunofluorescence staining showed that neurospheres cultured in collagen gels were stained positive for nestin and differentiated cells were stained positive for the neuronal marker βIII-tubulin, the astrocytic marker glial fibrillary acidic protein and the oligodendrocytic marker 2’,3’-cyclic nucleotide 3’-phosphodiesterase. Compared with neurospheres cultured in suspension, the differentiation potential of neural stem cells cultured in collagen gels increased, with the formation of neurons at an early stage. Our results show that the three-dimensional collagen gel culture system is superior to suspension culture in the proliferation, differentiation and process outgrowth of neural stem cells.     

EGFP基因修饰大鼠胚胎中脑神经干细胞的建立

目的建立增强型绿色荧光蛋白（EGFP）基因修饰大鼠胚胎中脑神经干细胞。方法以质粒pEGFPNl转染培养第三代的大鼠胚胎中脑神经干细胞（mNSCs）,经G418筛选后,分别进行nestin免疫细胞化学鉴定和诱导分化后ρ-Ⅲ-／ubulin、GFAP、CNPase免疫细胞化学鉴定。结果EGFP在基因转染12h后开始表达,24h后明显增加,48h达到高峰,经G418筛选1月后有阳性克隆形成,EGFP基因修饰不影响大鼠胚胎mNSCs的增殖与分化。结论成功建立EGFP基因修饰大鼠胚胎mNSCs,为进一步开展帕金森病的细胞移植治疗研究奠定基础。     

星形胶质细胞源性因子对神经干细胞分化的实验研究

目的探讨星形胶质细胞源性因子对神经干细胞分化的影响。方法分离和培养新生大鼠脑组织的神经干细胞;采用差速贴壁法和振荡法分离纯化星形胶质细胞,用免疫细胞化学染色法,胶质纤维酸性蛋白(GFAP)标记星形胶质细胞,进行细胞的纯度鉴定;将星形胶质细胞和神经干细胞在互不接触的情况下进行共培养,免疫荧光法观察神经干细胞分化后神经元特异性烯醇化酶(NSE)、GFAP和酪氨酸羟化酶(TH)的表达。结果纯化的星形胶质细胞GFAP抗体标记阳性,细胞纯度达98%;星形胶质细胞与神经干细胞共培养时,神经干细胞贴壁分化加快,NSE阳性细胞及TH阳性细胞明显多于对照组(P<0·05)。结论星形胶质细胞源性因子可快速诱导神经干细胞向神经元细胞、包括多巴胺神经元细胞分化,提示星形胶质细胞支持神经元发生。     

Differentiation of Wharton's jelly mesenchymal stem cells into neurons in alginate scaffold

Alginate scaffold has been considered as an appropriate biomaterial for promoting the differentiation of embryonic stem cells toward neuronal cell lineage. We hypothesized that alginate scaffold is suitable for culturing Wharton's jelly mesenchymal stem cells(WJMSCs) and can promote the differentiation of WJMSCs into neuron-like cells. In this study, we cultured WJMSCs in a three-dimensional scaffold fabricated by 0.25% alginate and 50 m M Ca Cl2 in the presence of neurogenic medium containing 10 μM retinoic acid and 20 ng/m L basic fibroblast growth factor. These cells were also cultured in conventional two-dimensional culture condition in the presence of neurogenic medium as controls. After 10 days, immunofluorescence staining was performed for detecting β-tubulin(marker for WJMSCs-differentiated neuron) and CD271(motor neuron marker). β-Tubulin and CD271 expression levels were significantly greater in the WJMSCs cultured in the three-dimensional alginate scaffold than in the conventional two-dimensional culture condition. These findings suggest that three-dimensional alginate scaffold cell culture system can induce neuronal differentiation of WJMSCs effectively.     

Estradiol promotes proliferation of dopaminergic precursors resulting in a higher proportion of dopamine neurons derived from mouse embryonic stem cells

Estradiol protects dopamine neurons of the substantia nigra from toxic insults. Such neurons succumb in Parkinson's disease; one strategy for restoring dopamine deficiency is cell therapy with neurons differentiated from embryonic stem cells. We investigated the effects of 17β-estradiol on dopaminergic induction of embryonic stem cells using the 5-stage protocol. Cells were incubated with different steroid concentrations during the proliferation (stage 4) or differentiation (stage 5) phases. Estradiol added at nM concentrations only during stage 4 increases the proliferation of dopaminergic precursors expressing Lmx1a, inducing a higher proportion of dopamine neurons at stage 5. These actions were mediated by activation of estrogen receptors, because co-incubation of cells with estradiol and ICI 182,780 completely abolished the positive effect on both proliferation of committed precursors, and subsequent differentiation to dopaminergic neurons. Our results suggest that estradiol should be useful in producing higher proportions of dopamine neurons from embryonic stem cells aimed for treating Parkinson's disease.     

Panax notoginseng saponins influence on transplantation of neural stem cell-derived dopaminergic neurons in a rat model of Parkinson’s disease *☆

BACKGROUND： Dopaminergic neurons differentiated from neural stem cells have been successfully used in the treatment of rat models of Parkinson＇s disease; however, the survival rate of transplanted cells has been low. Most cells die by apoptosis as a result of overloaded intracellular calcium and the formation of oxygen free radicals.
OBJECTIVE： To observe whether survival of transplanted cells, transplantation efficacy, and dopaminergic differentiation from neural stem cells is altered by Panax notoginseng saponins （PNS） in a rat model of Parkinson＇s disease.
DESIGN, TIME AND SETTING： Cellular and molecular biology experiments with randomized group design. The experiment was performed at the Animal Experimental Center, First Hospital of Sun Yat-sen University from April to October 2007.
MATERIALS： Thirty-two adult, healthy, male Sprague Dawley rats, and four healthy Sprague Dawley rat embryos at gestational days 14-15 were selected. The right ventral mesencephalon was injected with 6-hydroxydopamine to establish a model of Parkinson＇s disease. 6-hydroxydopamine and apomorphine were purchased from Sigma, USA.
METHODS： Neural stem cells derived from the mesencephalon of embryonic rats were cultivated and passaged in serum-free culture medium. Lesioned animals were randomly divided into four groups （n = 8）： dopaminergic neuron, dopaminergic neuron ＋ PNS, PNS, and control. The dopaminergic neuron group was injected with 3 μL cell suspension containing dopaminergic neurons differentiated from neural stem cells. The dopaminergic neurons ＋ PNS group received 3 μ L dopaminergic cell suspension combined with PNS （250 mg/L）. The PNS group received 3 μL PNS （250 mg/L）, and the control group received 3 μL DMEM/F12 culture medium.
MAIN OUTCOME MEASURES： The rats were transcardially perfused with 4% paraformaldehyde at 60 days post-grafting for immunohistochemistry. The rats were intraperitoneally injected with apomorphine （0.5 mg/kg） to induce rotational behavior. RESU     

A simple method for large-scale generation of dopamine neurons from human embryonic stem cells

Dopamine (DA) neurons derived from human embryonic stem cells (hESCs) are potentially valuable in drug screening and as a possible source of donor tissue for transplantation in Parkinson's disease. However, existing culture protocols that promote the differentiation of DA neurons from hESCs are complex, involving multiple steps and having unreliable results between cultures. Here we report a simple and highly reproducible culture protocol that induces expandable DA neuron progenitors from hESCs in attached cultures. We found that the hESC-derived neuronal progenitors retain their full capacity to generate DA neurons after repeated passaging in the presence of basic fibroblast growth factor (bFGF) and medium conditioned with PA6 stromal cells. Using immunocytochemistry and RT-PCR, we found that the differentiated DA neurons exhibit a midbrain phenotype and express, e.g., Aldh1a, Ptx3, Nurr1, and Lmx1a. Using HPLC, we monitored their production of DA. We then demonstrated that the expanded progenitors are possible to cryopreserve without loosing the dopaminergic phenotype. With our protocol, we obtained large and homogeneous populations of dopaminergic progenitors and neurons. We conclude that our protocol can be used to generate human DA neurons suitable for the study of disease mechanisms, toxicology, drug screening, and intracerebral transplantation.     

NGF-β、GFP基因修饰大鼠胚胎中脑神经干细胞的建立

目的建立神经生长因子β（NGFβ）基因修饰大鼠胚胎中脑神经干细胞。方法以质粒pcDNA3-hNGFb、pEGFPN1共转染大鼠胚胎中脑神经干细胞，荧光显微镜观察绿色荧光蛋白（GFP）在细胞内的表达，免疫细胞化学、Westernblot鉴定NGF-β在细胞内的表达。体外诱导分化，免疫细胞化学鉴定其分化能力。结果GFP在基因转染12h后开始表达，免疫细胞化学、Westernblot结果表明NGF-β能在细胞中正确表达且NGF-β、GFP基因修饰不影响其增殖与分化。结论成功建立NGF-β、GFP基因修饰大鼠胚胎中脑神经干细胞。     

Are newborn rat-derived neural stem cells more sensitive to lead neurotoxicity?

Lead ion (Pb(2+) ) has been proven to be a neurotoxin due to its neurotoxicity on mammalian nervous system, especially for the developing brains of juveniles. However, many reported studies involved the negative effects of Pb(2+) on adult neural cells of humans or other mammals, only few of which have examined the effects of Pb(2+) on neural stem cells. The purpose of this study was to reveal the biological effects of Pb(2+) from lead acetate [Pb (CH 3 COO) 2 ] on viability, proliferation and differentiation of neural stem cells derived from the hippocampus of newborn rats aged 7 days and adult rats aged 90 days, respectively. This study was carried out in three parts. In the first part, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT viability assay) was used to detect the effects of Pb(2+) on the cell viability of passage 2 hippocampal neural stem cells after 48-hour exposure to 0-200 μM Pb(2+) . In the second part, 10 μM bromodeoxyuridine was added into the culture medium of passage 2 hippocampal neural stem cells after 48-hour exposure to 0- 200 μM Pb(2+) , followed by immunocytochemical staining with anti-bromodeoxyuridine to demonstrate the effects of Pb(2+) on cell proliferation. In the last part, passage 2 hippocampal neural stem cells were allowed to grow in the differentiation medium with 0-200 μM Pb(2+) . Immunocytochemical staining with anti-microtubule-associated protein 2 (a neuron marker), anti-glial fibrillary acidic protein (an astrocyte marker), and anti-RIP (an oligodendrocyte marker) was performed to detect the differentiation commitment of affected neural stem cells after 6 days. The data showed that Pb(2+) inhibited not only the viability and proliferation of rat hippocampal neural stem cells, but also their neuronal and oligodendrocyte differentiation in vitro. Moreover, increased activity of astrocyte differentiation of hippocampal neural stem cells from both newborn and adult rats was observed after exposure to high concentration of lead ion in vitro. These findings suggest that hippocampal neural stem cells of newborn rats were more sensitive than those from adult rats to Pb(2+) cytotoxicity.     

Are human dental papilla-derived stem cell and human brain-derived neural stem cell transplantations suitable for treatment of Parkinson's disease?

Transplantation of neural stem cells has been reported as a possible approach for replacing impaired dopaminergic neurons. In this study, we tested the efficacy of early-stage human dental papilla-derived stem cells and human brain-derived neural stem cells in rat models of 6-hydroxydopamine-induced Parkinson’s disease. Rats received a unilateral injection of 6-hydroxydopamine into right medial forebrain bundle, followed 3 weeks later by injections of PBS, early-stage human dental papilla-derived stem cells, or human brain-derived neural stem cells into the ipsilateral striatum. All of the rats in the human dental papilla-derived stem cell group died from tumor formation at around 2 weeks following cell transplantation. Postmortem examinations revealed homogeneous malignant tumors in the striatum of the human dental papilla-derived stem cell group. Stepping tests revealed that human brain-derived neural stem cell transplantation did not improve motor dysfunction. In apomorphine-induced rotation tests, neither the human brain-derived neural stem cell group nor the control groups (PBS injection) demonstrated significant changes. Glucose metabolism in the lesioned side of striatum was reduced by human brain-derived neural stem cell transplantation. [18 F]-FP-CIT PET scans in the striatum did not demonstrate a significant increase in the human brain-derived neural stem cell group. Tyrosine hydroxylase (dopaminergic neuronal marker) staining and G protein-activated inward rectifier potassium channel 2 (A9 dopaminergic neuronal marker) were positive in the lesioned side of striatum in the human brain-derived neural stem cell group. The use of early-stage human dental papilla-derived stem cells confirmed its tendency to form tumors. Human brain-derived neural stem cells could be partially differentiated into dopaminergic neurons, but they did not secrete dopamine.     

Glial cell line‐derived neurotrophic factor enhances in vitro differentiation of mid‐/hindbrain neural progenitor cells to dopaminergic‐like neurons

Parkinson's disease (PD) affects the motor system through the degeneration of the dopaminergic neurons of the substantia nigra. The use of human embryonic stem cell (hESC)‐derived human neural progenitor (hNP) cells provides a potential cell source for cell therapies and drug screens for future treatments. Glial cell line‐derived neurotrophic factor (GDNF) is a known dopaminergic neuroprotectant agent; however, its potential role in neural differentiation remains largely unknown. Addition of 25 ng/ml GDNF to hNP cell differentiation media, over a 21‐day period, induced a significantly (P < 0.05) greater portion of hNP cells to differentiate into dopaminergic neurons than non‐GDNF cultures, 50% compared with 2.9% of cells expressing tyrosine hydroxylase (TH), respectively. The hNP cells exposed to GDNF selectively expressed dopamine receptors 1, 4, and 5 and were evoked to release dopamine with KCl. This is the first report of GDNF and leukemia inhibitory factor enriching hESC‐derived hNP cells toward dopaminergic‐like neurons. © 2010 Wiley‐Liss, Inc.     

BDNF、EGFP基因修饰大鼠胚胎腹侧中脑神经干细胞的建立

目的建立脑源性神经营养因子（BDNF）、增强型绿色荧光蛋白（EGFP）基因修饰大鼠胚胎腹侧中脑神经干细胞（mNSCs）。方法以FuGENEHD转染试剂介导质粒pcDNA3-BDNF、pEGFPN1共转染第三代E14大鼠胚胎mNSCs。荧光显微镜观察EGFP表达情况；免疫细胞化学方法和Western blot鉴定BDNF的表达；体外诱导分化后免疫细胞化学鉴定其分化能力。结果基因转染12h后EGFP开始表达：免疫细胞化学、Western blot结果表明BDNF能在细胞中正确表达；体外诱导分化研究表明BDNF、EGFP基因修饰不影响大鼠胚胎mNSCs的增殖与分化。结论成功建立了BDNF、EGFP基因修饰大鼠胚胎mNSCs，可为进一步开展帕金森病的细胞移植治疗研究奠定基础。     

A Disintegrin and Metalloprotease 10 in neuronal maturation and gliogenesis during cortex development

The multiple-layer structure of the cerebral cortex is important for its functions. Such a structure is generated based on the proliferation and differentiation of neural stem/progenitor cells. Notch functions as a molecular switch for neural stem/progenitor cell fate during cortex development but the mechanism remains unclear. Biochemical and cellular studies showed that Notch receptor activation induces several proteases to release the Notch intracellular domain (NICD). A Disintegrin and Metalloprotease 10 (ADAM10) might be a physiological rate-limiting S2 enzyme for Notch activation. Nestin-driven conditional ADAM10 knockout in mouse cortex showed that ADAM10 is critical for maintenance of the neural stem cell population during early embryonic cortex development. However, the expression pattern and function of ADAM10 during later cerebral cortex development remains poorly understood. We performed in situ hybridization for ADAM10 mRNA and immunofluorescent analysis to determine the expression of ADAM10 and NICD in mouse cortex from embryonic day 9 (E14.5) to postnatal day 1 (P1). ADAM10 and NICD were highly co-localized in the cortex of E16.5 to P1 mice. Comparisons of expression patterns of ADAM10 with Nestin (neural stem cell marker), Tuj1 (mature neuron marker), and S100β (glia marker) showed that ADAM10 expression highly matched that of S100β and partially matched that of Tuj1 at later embryonic to early postnatal cortex developmental stages. Such expression patterns indicated that ADAM10-Notch signaling might have a critical function in neuronal maturation and gliogenesis during cortex development.     

X-box-binding protein 1-modified neural stem cells for treatment of Parkinson’s disease

X-box-binding protein 1-transfected neural stem cells were transplanted into the right lateral ventricles of rats with rotenone-induced Parkinson’s disease. The survival capacities and differentiation rates of cells expressing the dopaminergic marker tyrosine hydroxylase were higher in X-box-binding protein 1-transfected neural stem cells compared to non-transfected cells. Moreover, dopamine and 3,4-dihydroxyphenylacetic acid levels in the substantia nigra were significantly increased, α-synuclein expression was decreased, and neurological behaviors were significantly ameliorated in rats following transplantation of X-box-binding protein 1-transfected neural stem cells. These results indicate that transplantation of X-box-binding protein 1-transfected neural stem cells can promote stem cell survival and differentiation into dopaminergic neurons, increase dopamine and 3,4-dihydroxyphenylacetic acid levels, reduce α-synuclein aggregation in the substantia nigra, and improve the symptoms of Parkinson’s disease in rats.     

Early expressions of hypoxia-inducible factor 1alpha and vascular endothelial growth factor increase the neuronal plasticity of activated endogenous neural stem cells after focal cerebral ischemia

Endogenous neural stem cells become "activated" after neuronal injury, but the activation sequence and fate of endogenous neural stem cells in focal cerebral ischemia model are little known. We evaluated the relationships between neural stem cells and hypoxia-inducible factor-1α and vascular endothelial growth factor expression in a photothromobotic rat stroke model using immunohistochemistry and western blot analysis. We also evaluated the chronological changes of neural stem cells by 5-bromo-2′-deoxyuridine(BrdU) incorporation. Hypoxia-inducible factor-1α expression was initially increased from 1 hour after ischemic injury, followed by vascular endothelial growth factor expression. Hypoxia-inducible factor-1α immunoreactivity was detected in the ipsilateral cortical neurons of the infarct core and peri-infarct area. Vascular endothelial growth factor immunoreactivity was detected in bilateral cortex, but ipsilateral cortex staining intensity and numbers were greater than the contralateral cortex. Vascular endothelial growth factor immunoreactive cells were easily found along the peri-infarct area 12 hours after focal cerebral ischemia. The expression of nestin increased throughout the microvasculature in the ischemic core and the peri-infarct area in all experimental rats after 24 hours of ischemic injury. Nestin immunoreactivity increased in the subventricular zone during 12 hours to 3 days, and prominently increased in the ipsilateral cortex between 3–7 days. Nestin-labeled cells showed dual differentiation with microvessels near the infarct core and reactive astrocytes in the peri-infarct area. BrdU-labeled cells were increased gradually from day 1 in the ipsilateral subventricular zone and cortex, and numerous BrdU-labeled cells were observed in the peri-infarct area and non-lesioned cortex at 3 days. BrdU-labeled cells rather than neurons, were mainly co-labeled with nestin and GFAP. Early expressions of hypoxia-inducible factor-1α and vascular endothelial growth factor after ischemia made up the microenvironment to increase the neuronal plasticity of activated endogenous neural stem cells. Moreover, neural precursor cells after large-scale cortical injury could be recruited from the cortex nearby infarct core and subventricular zone.     

脂肪干细胞向神经元诱导分化的研究

目的研究脂肪干细胞向神经元分化的可行性,为神经系统疾病的替代治疗寻求行之有效的可种植细胞。方法用β-2-巯基乙醇和丁酸酯羟基茴香醚诱导脂肪干细胞向神经元分化,对干预后细胞进行形态学观察和细胞免疫组化鉴定。结果人类腹部脂肪来源的基质细胞进行原代和传代培养后细胞形态类似于成纤维细胞,经过诱导分化后细胞形态表现为典型的神经元形态。神经元特异性烯醇化酶鉴定绝大部分有阳性染色,但在不到一周的时间内死亡。结论脂肪组织中存在能分化为神经元的干细胞．有巨大的研究价值和临床应用前景。