Direct lineage conversion of astrocytes to induced neural stem cells or neurons

Since the generation of induced pluripotent stem cells in 2006, cellular reprogramming has attracted increasing attention as a revolutionary strategy for cell replacement therapy. Recent advances have revealed that somatic cells can be directly converted into other mature cell types, which eliminates the risk of neoplasia and the generation of undesired cell types. Astrocytes become reactive and undergo proliferation, which hampers axon regeneration following injury, stroke, and neurodegenerative diseases. An emerging technique to directly reprogram astrocytes into induced neural stem cells(i NSCs) and induced neurons(i Ns) by neural fate determinants brings potential hope to cell replacement therapy for the above neurological problems. Here, we discuss the development of direct reprogramming of various cell types into i Ns and i NSCs, then detail astrocyte-derived i NSCs and i Ns in vivo and in vitro. Finally, we highlight the unsolved challenges and opportunities for improvement.     

Efficient production of neural stem cells and neurons from embryonic stem cells

We have developed a simple method to efficiently produce a large number of neural stem cells and neurons from mouse embryonic stem (ES) cells. When cultured in astrocyte-conditioned medium (ACM) with mitogens (FGF-2 and EGF) under free-floating conditions, colonies of undifferentiated ES cells give rise to neural stem spheres (NSSs), composed of plentiful neural stem cells. Subsequent culture of the NSSs on an adhesive substrate with mitogens results in the migration of neural stem cells onto the substrate. These cells can be expanded, preserved by freezing, and differentiated into functional neurons. Neural stem cells and neurons provided by this NSS method may be valuable as potential donor cells for neuronal transplantation and also as convenient alternatives to tissue-derived neural cells.     

海马干细胞分化的神经元电生理特征的初步探讨

目的探讨大鼠海马干细胞分化的神经元的电生理特征。方法无血清方法分离、培养新生大鼠海马干细胞,诱导其分化为神经元,采用全细胞膜片钳技术记录分化的神经元的静息膜电位、动作电位及离子单通道等电生理指标。结果海马干细胞分化的神经元具有一定的电生理特征,记录到静息膜电位及动作电位,其50%和90%复极化动作电位时程分别为69.75±4.57ms及79.75±6.45ms,并同时记录到三种外向钾电流。结论在现有培养条件下海马干细胞可分化为具有一定电生理特征的神经元。     

Fibroblast growth factor 2 negatively regulates the induction of neuronal progenitors from neural stem cells

Fibroblast growth factor 2 (FGF2) exhibits pleiotropic functions during embryogenesis. In neural development, both pro- and antineurogenic activities of FGF2 have been described in the differentiation of neuronal progenitors into postmitotic neurons. We used cultured neural stem cells (NSCs) derived from rat embryonic day 14.5 cortex to determine the FGF2 effect on the induction of early neuronal progenitors. Our data showed that the presence of FGF2 during serum-induced differentiation of NSCs reduced the number of Tuj1(+) neurons. A bromodeoxyuridine (BrdU)/Tuj1 double-labeling assay and expression analyses of the pro- and antineurogenic basic helix-loop-helix (bHLH) factors showed that FGF2 blocked the generation of early neuronal progenitors, but not the cell-cycle exit of dividing neurons. This negative regulation of neuronal induction by FGF2 was associated with the persistent expression of an antineurogenic bHLH, hairy and enhancer of split (HES)-1. A gene-profiling study demonstrated that the developmental programs underlying neuronal differentiation were altered as a whole and identified several developmentally regulated, neural-enriched genes. This work shows that FGF2 exerts an antineurogenic effect during the developmental window when neuronal progenitors are first induced from NSCs. It also provides a novel experimental system that can be used to prospectively identify genes expressed at different stages of neuronal differentiation.     

Immortalized neural stem cells differ from nonimmortalized cortical neurospheres and cerebellar granule cell progenitors

Pluripotent neural stem cells (NSCs) have been used as replacement cells in a variety of neurological disease models. Among the many different NSCs that have been used to date, most robust results have been obtained with the immortalized neural stem cell line (C17.2) isolated from postnatal cerebellum. However, it is unclear if other NSCs isolated from different brain regions are similar in their potency as replacement therapies. To assess the properties of NSC-like C17.2 cells, we compared the properties of these cells with those reported for other NSC populations identified by a variety of different investigators using biological assays, microarray analysis, RT-PCR, and immunocytochemistry. We show that C17.2 cells differ significantly from other NSCs and cerebellar granule cell precursors, from which they were derived. In particular, they secrete additional growth factors and cytokines, express markers that distinguish them from other progenitor populations, and do not maintain karyotypic stability. Our results provide a caution on extrapolating results from C17.2 to other nonimmortalized stem cell populations and provide an explanation for some of the dramatic effects that are seen with C17.2 transplants but not with other cells. We suggest that, while C17.2 cells can illustrate many fundamental aspects of neural biology and are useful in their own right, their unique properties cannot be generalized.     

海马神经干细胞不同传代方法的比较

背景：体外培养神经干细胞,在悬浮培养时由于自身增殖特性会形成球,传代时将会面临如何将细胞球分离成单细胞的问题。 目的：寻求理想的大鼠海马神经干细胞传代方法,以获得大量可增生的神经干细胞以供研究。 方法：分离新生1 d SD大鼠海马神经干细胞,原代培养至五六天时,分别用机械吹打法、胰蛋白酶、TrypLE和Accutase消化法分离神经干细胞球。之后每7 d传代1次,连续传代3次。分别于每次传代后第1天和传代后第4天计数活细胞比例和细胞球数目,实验重复3次。 结果与结论：神经干细胞球经3种酶消化后获得的均是单细胞;经机械吹打后既有单个细胞,也有小细胞球分布于培养液中。在酶消化法中,Accutase消化法传代后神经干细胞的活细胞比例明显高于胰蛋白酶消化(P < 0.01)和TrypLE消化法 (P < 0.05)。同时,Accutase消化法传代后新形成的细胞球数目也较其余各组多(P < 0.01)。提示在实验条件下,Accutase消化法能够较好地将神经干细胞球分离成存活率较高、能快速形成新的克隆球的单个细胞,是较为理想的神经干细胞分离传代方法。     

Ontogeny and plasticity of adult hippocampal neural stem cells

We have investigated the ontogenetic origin and the degree of plasticity of adult hippocampal neural stem cells. Wnt1-expressing cells are located at the dorsal aspect of the embryonic neural tube and some of them are predestined to give rise to neural crest stem cells. Whereas the majority of adult hippocampal neural stem cells do not originate from cells that express Wnt1, a subset does express Wnt1 transiently during embryogenesis, as determined in the double transgenic mouse, Wnt1-cre/R26R. Hippocampal stem cells from adult ROSA 26 mice differentiate into chondrocytes, melanocytes (pigment cells) and smooth muscle cells when cocultured with neural crest cells from quail embryos. Neural crest cell-generated stimuli have a short-range of action and are recognized across species. These observations provide evidence for the heterogeneity in the hippocampal neural stem cell pool with regard to Wnt1 expression. Furthermore, they show plasticity and a remarkably wide range of developmental options of adult hippocampal stem cells.     

通心络对大鼠胚胎神经干细胞源性神经细胞谱系的影响*★

目的：前期体内动物模型实验说明，通心络可能具有影响神经干细胞增殖及向各种神经细胞分化的作用。实验拟观察通心络对大鼠胚胎神经干细胞源性神经细胞谱系的影响以及时效、量效的关系。 方法：实验于2007-06/10在南方医科大学机能学实验室完成。①实验材料：孕12~14 d SD大鼠由南方医科大学实验动物中心提供，实验过程中对动物处置符合动物伦理学标准。通心络，主要成分为人参、水蛭、全蝎、土鳖虫、蜈蚣、蝉蜕、赤芍、冰片等，由石家庄以岭药业股份有限公司生产，国药准字Z19980015。通心络含药血清的制备：按大剂量组1 g/（d?kg）、小剂量组0.5 g/（d?kg）分别予大鼠通心络混悬液灌胃，7 d后抽血离心，吸取血清，过滤消毒，分装，-70 ℃冻存备用。②实验方法：自孕12~14 d大鼠胚胎中分离培养神经干细胞，取第3代细胞分别给予大、小剂量组通心络含药血清干预。以添加普通血清干预的为对照。③实验评估：于培养1，3，7 d通过免疫荧光染色观察各种类型神经细胞所占比例。 结果：①神经干细胞经通心络含药血清干预后1 d，仅在大剂量组海马齿状回有极少数细胞呈BrdU(+)GFAP(+)，其余细胞都呈BrdU(+)Nestin(+)，小剂量组和对照组均为BrdU(+)Nestin(+)细胞。②经通心络含药血清干预后3d，大剂量组、小剂量组和对照组Nestin(+)、βtubulin(+)、GFAP(+)细胞比例差异有显著性意义（P < 0.01），GalC(+)细胞比例差异无统计学意义（P > 0.05）。③大鼠胚胎神经干细胞经通心络含药血清干预后：各组Nestin(+)细胞比例先降后升；βtubulin(+)细胞比例大剂量组持续上升，小剂量组和对照组先升后降；GFAP(+)细胞比例大剂量组先升后降，小剂量组和对照组持续上升，GalC(+)细胞比例大、小剂量组均较平稳，对照组7 d时明显上升。 结论：通心络有促进大鼠胚胎神经干细胞增殖及向神经元分化的效应，并存在时效、量效关系，剂量越大效应越明显及持久。     

Ablation of proliferating neural stem cells during early life is sufficient to reduce adult hippocampal neurogenesis

Environmental exposures during early life, but not during adolescence or adulthood, lead to persistent reductions in neurogenesis in the adult hippocampal dentate gyrus (DG). The mechanisms by which early life exposures lead to long‐term deficits in neurogenesis remain unclear. Here, we investigated whether targeted ablation of dividing neural stem cells during early life is sufficient to produce long‐term decreases in DG neurogenesis. Having previously found that the stem cell lineage is resistant to long‐term effects of transient ablation of dividing stem cells during adolescence or adulthood (Kirshenbaum, Lieberman, Briner, Leonardo, & Dranovsky, 2014 ), we used a similar pharmacogenetic approach to target dividing neural stem cells for elimination during early life periods sensitive to environmental insults. We then assessed the Nestin stem cell lineage in adulthood. We found that the adult neural stem cell reservoir was depleted following ablation during the first postnatal week, when stem cells were highly proliferative, but not during the third postnatal week, when stem cells were more quiescent. Remarkably, ablating proliferating stem cells during either the first or third postnatal week led to reduced adult neurogenesis out of proportion to the changes in the stem cell pool, indicating a disruption of the stem cell function or niche following stem cell ablation in early life. These results highlight the first three postnatal weeks as a series of sensitive periods during which elimination of dividing stem cells leads to lasting alterations in adult DG neurogenesis and stem cell function. These findings contribute to our understanding of the relationship between DG development and adult neurogenesis, as well as suggest a possible mechanism by which early life experiences may lead to lasting deficits in adult hippocampal neurogenesis.     

Plasticity‐related gene 1 is important for survival of neurons derived from rat neural stem cells

Plasticity‐related gene 1 (Prg1) is a membrane‐associated lipid phosphate phosphatase. In this study, we first investigated the role of Prg1 in the survival of neurons derived from rat neural stem cells (NSCs) using small interfering RNA (siRNA). Prg1 knock‐down decreased the cell number. Interestingly, Prg1 knock‐down increased genomic DNA fragmentation, suggesting the possible induction of apoptosis. Exogenously expressed Prg1 rescued the cells from death and restored the loss of 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) activity induced with Prg1 siRNA. However, exogenously expressed mutated‐Prg1 (the 253rd amino acid, histidine253, had been changed to alanine) did not rescue the cell death or restore the MTT activity. Histidine253 of Prg1 has been reported to be important for lipid phosphate phosphatase activity. These results suggest that Prg1 is important for survival of neurons through its dephosphorylation activity. © 2013 Wiley Periodicals, Inc.     

Rhotekin modulates differentiation of cultured neural stem cells to neurons

Rhotekin is a downstream signal of Rho and is expressed in the central nervous system. However, the physiological role of rhotekin in the development of neural stem cells (NSCs) into neurons is unknown. In this study, we knocked down the expression of rhotekin protein with small interfering RNA (siRNA) in the NSCs and in neural differentiated cells and measured cell proliferation, differentiation, neurite length, and survival. By using immunocytochemistry and Western blot, the production of rhotekin was observed in NSCs and neuronal cells. Furthermore, rhotekin production was increased in accordance with neural differentiation. Rhotekin knock-down reduced 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) activity and increased the cell death 72 hr after transfection in neurons. On the other hand, in NSCs, rhotekin knock-down increased MTT activity and the number of 5-bromo-2'-deoxyuridine (BrdU)-positive cells. In the present study, we demonstrated that rhotekin is required for maintenance and survival of neurons and positively regulates differentiation and neurite outgrowth. Moreover, we found that rhotekin is produced in NSCs and that the role of rhotekin is to regulate cell proliferation negatively. In conclusion, these results suggest that rhotekin is one of the key molecules in the differentiation of NSCs into neurons.     

Changes in the proliferative activity of hippocampal neural stem cells from manganismus mice

BACKGROUND: Manganese neurotoxicity presents in the form of not only extracorticospinal tract injury of central nervous system (CNS), but also learning and memory ability damage. So, the mechanism of manganese neurotoxicity will be further studied from the angle of hippocampus. OBJECTIVE: To observe the effects of manganism on learning and memory ability and the proliferation of neural stem cells (NSCs) in hippocampus of mouse brains, and analyze whether this effect has dose-dependence. DESIGN: Randomized controlled experiment. SETTING: Department of Human Anatomy, and Department of Industrial Hygiene and Occupational Diseases, Guangxi Medical University. MATERIALS: Twenty-eight male Kunming mice, aged 2 weeks, were involved in this experiment. The involved mice were randomized into 4 groups, with 7 in each: control group, low-dose manganism group, middle-dose manganism group and high-dose manganism group. Manganese chloride was purchased from Shantou Chemicals Factory. METHODS: This experiment was carried out in the Experimental Center for Preclinical Medicine, Guangxi Medical University from November 2005 to August 2006. Mice in the low-, middle- and high-dose manganism groups were intraperitoneally injected with 5, 20 and 50 mg/kg per day manganese chloride, once a day, for 2 weeks successively. Mice in the control group were injected with the same amount of stroke-physiological saline solution. Neurobehavioral detection of all the animals was performed in Morris water maze constantly from the 7th day after the first injection of manganese chloride solution. Learning ability was detected in the place navigation test. Mice were trained for 5 consecutive days with four trials per day. The time to find the platform was latency. Memory ability was detected in spatial probe test. Platform was withdrawn on the following day of place navigation. The mice were placed in the water from a random start in the edge of the pool. The number of times they traversed the plateform's region was recorded as the performance of spatial memory. At the final two days of the water maze tests, all the animals were daily intraperitoneally injected with 50 mg/kg BrdU three times successively, once every 4 hours. At 24 hours after the final BrdU injection, all the animals were sacrificed and perfused, and their brains were harvested, fixed and successively sliced at coronary plane on a freezing microtome. Distribution and number of BrdU-positive cells in the subgranular zone of hippocampus of brains of experimental animas were detected respectively by immunohistochemistry for reflecting the proliferation of NSCs. Single-factor analysis of variance was used for comparing the difference of measurement data. Linear correlation analysis was used among the performance record in Morris water maze test, the number of BrdU-immunopositive cells and the dose of manganism. MAIN OUTCOME MEASURES: Learning and memory ability and the number of hippocampal NSCs of mice in each group. RESULTS: ①Performance of mice in Morris water maze: In the place navigation test, there was a significant retarded learning in mice of high-dose manganism group from the 3rd day as compared with control group (P < 0.01). Till the 5th day, escape latency of mice in each manganism group was prolonged, and learning performance was significantly decreased (P < 0.05), while swimming speed did not affect above results. In the spatial probe test, the average frequency of middle- and high-dose manganism groups was 1.17±1.60 and 0.80±1.10, respectively, and decreased remarkably than that of control group which was 4.86±1.35 (P < 0.01), indicating memory ability was decreased; while the average frequency of low-dose manganism group did not differ obviously from that of control group (P =0.066) although it was 2.67±3.27. The difference of swimming speed in each group was still of no statistic significance (P > 0.05). ②Effect of manganism on the number of NSCs: After counting, the average number of BrdU- immunopositive cells of one side in the control group, low-, middle- and high-dose manganism groups was 69.20±4.48, 36.63±4.50, 31.00±6.87, 26.76±4.83, individually (P < 0.01). ③Results of linear correlation analysis: The ability of spatial memory was significantly in positive correlation with the number of BrdU-labeled cells ( r =0.734, P < 0.01), and in negative correlation with the intensity of manganese poisoning (r =–0.598,P < 0.01).Meanwhile, there was a significant negative correlation between the number of BrdU-labeled cells and the intensity of manganese poisoning(r =–0.666, P < 0.01). CONCLUSION: Manganese exposure in mice can affect the ability of learning and memory, which is probably caused by the inhibition of manganese to the neurogenesis of NSCs in hippocampus in dose-dependent manner.     

嗅鞘细胞与神经干细胞共培养后增殖及向神经元的分化

背景：影响神经干细胞增殖分化的外在因素包括细胞因子和微环境,嗅鞘细胞能分泌多种细胞因子,与神经干细胞共培养时改变其微环境。 目的：观察不同浓度嗅鞘细胞对神经干细胞增殖及分化的影响。 方法：体外分别培养Wistar大鼠神经干细胞和嗅鞘细胞,5×107 L-1神经干细胞分别和1×107 L-1,1×109 L-1,1×1011 L-1嗅鞘细胞共培养,同时设立正常对照组,不加嗅鞘细胞。倒置荧光显微镜下观察神经干细胞增殖情况,诱导7 d后行NSE免疫细胞化学染色,计算阳性细胞/细胞总数得出阳性细胞百分比。 结果与结论：①3种浓度嗅鞘细胞与神经干细胞共培养3 d后,均促进了神经干细胞增殖,以1×109 L-1嗅鞘细胞共培养组作用最显著,明显优于1×107 L-1嗅鞘细胞组、1×1011 L-1嗅鞘细胞组。②神经干细胞与1×109 L-1嗅鞘细胞共培养7 d后,神经干细胞分化为神经元样细胞的百分比最高,与正常对照组相比差异有显著性意义(P < 0.01)。     

Isolation and transplantation of dopaminergic neurons and neural stem cells

Although transplantation of mesencephalic tissue is considered a promising therapy for Parkinson's disease (PD), its clinical use is still restricted to a very few cases. A major limiting factor of this therapy is the difficulty of obtaining sufficient quantities of viable embryonic mesencephalic tissue. To overcome this limitation, techniques to produce dopaminergic (DA) neurons in vitro have been developed. However, these cultures are likely to contain a variety of unidentified cells, which must be removed before implantation. Specific cell-surface markers to sort DA neurons or their precursors are not available. We have developed an alternative strategy, by which these cells can be labeled with green fluorescent protein and isolated with fluorescent activated cell sorter. Transplantation of the sorted cells resulted in recovery of a rat model of the PD. This strategy should be useful for developing new therapies for PD.     

Amphiregulin is a mitogen for adult neural stem cells

Neurons are continuously generated from stem cells in the hippocampus and along the lateral ventricles in the adult brain. Neural stem cells can be propagated in vitro in the presence of epidermal growth factor (EGF) or fibroblast growth factor-2. We report here that amphiregulin, a growth factor related to EGF, is a mitogen for adult mouse neural stem cells in vitro and displays potency similar to that of EGF. Neural stem cell cultures can be initiated and the cells propagated as efficiently in the presence of amphiregulin only as with EGF. Furthermore, we show that amphiregulin is expressed in the choroid plexus of the ventricular system and in the hippocampus in the adult brain, suggesting that amphiregulin may participate in the regulation of neural stem cell proliferation and neurogenesis in the adult brain.     

Estrogen promotes differentiation and survival of dopaminergic neurons derived from human neural stem cells

To investigate the effect of estrogen on neuronal differentiation, especially on dopaminergic (DA) neurons, human neural stem cells (NSCs) were differentiated in the presence of 17beta-estradiol. NSCs gave rise to tyrosine hydroxylase (TH)-positive neurons in vitro, the proportion of which was increased by 17beta-estradiol. Increase in TH-positive neurons was abrogated by an estrogen receptor (ER) antagonist, ICI182780, suggesting ERs play a role in differentiation of DA neurons. The observation that ERs were expressed in both proliferating NSCs and postmitotic DA neurons suggested that increase in TH-positive neurons was due to induction and support of DA neurons. 17beta-Estradiol also increased the number of DA neurons derived from human NSCs in vivo when the cells were grafted into mouse brains. These results support a possible role for estrogen in the transplantation of NSCs for Parkinson's disease.     

Increase in dopaminergic neurons from mouse embryonic stem cell-derived neural progenitor/stem cells is mediated by hypoxia inducible factor-1alpha

A reliable method to induce neural progenitor/stem cells (NPCs) into dopaminergic (DAergic) neurons has not yet been established. As well, the mechanism involved remains to be elucidated. To induce DAergic differentiation from NPCs, a cytokine mixture (C-Mix) of interleukin (IL)-1beta, IL-11, leukemia-inhibitory factor (LIF), and glial-derived neurotrophic factor or low oxygen (3.5% O(2): L-Oxy) was used to treat embryonic stem (ES) cell-derived NPCs. Treatment with C-Mix increased the number of tyrosine hydroxylase (TH)-positive cells compared with controls (2.20-fold of control). The C-Mix effect was induced by mainly LIF or IL-1beta treatment. Although L-Oxy caused an increase in TH-positive cells (1.34-fold), the combination of L-Oxy with C-Mix did not show an additive effect. Increases in DA in the medium were shown in the presence of C-Mix, LIF, and L-Oxy by high-performance liquid chromatography. Gene expression patterns of neural markers [tryptophan hydroxylase (TPH), GAD67, GluT1, beta-tubulin III, glial fibrillary acidc protein, and TH] were different in C-Mix and L-Oxy treatments. Because increases in hypoxia-inducible factor (HIF)-1alpha protein were found in both treatments, we investigated the effect of HIF-1alpha on differentiation of NPCs to DAergic neurons. Inhibition of HIF-1alpha by the application of antisense oligodeoxynucleotides (ODNs) to NPCs caused a decrease in TH-positive cells induced by LIF treatment. Gene expressions of TH, GAD67, and GluT1 were decreased, and those of TPH, beta-tubulin III, and S-100beta were increased by treatment with just ODNs, indicating the importance of the endogenous effect of HIF-1alpha on neuronal differentiation. These data suggest that enhanced differentiation into DAergic neurons from ES cell-derived NPCs was induced by C-Mix or L-Oxy mediated by HIF-1alpha.