Efficient cultivation of neural stem cells with controlled delivery of FGF-2

Neural stem cells (NSCs) raised the hope for cell-based therapies in human neurodevelopmental and neurodegenerative diseases. Current research strategies aim to isolate, enrich, and propagate homogeneous populations of neural stem cells. Unfortunately, several concerns with NSC cultures currently may limit their therapeutic promise. Exhaustion of growth factors and/or their uncontrolled release with burst and fall in their concentration may greatly affect the in vitro behavior of NSCs. In this context, we investigate whether a device containing heparan sulfate (HS), which is a co-factor in growth factor-mediated cell proliferation and differentiation, could potentiate and prolong the delivery of fibroblast growth factor-2 (FGF-2) and thus improve in vitro NSC cultivation. We demonstrated that NSCs cultivated in media with a controlled release of FGF-2 from a polyelectrolyte polymer showed a higher proliferation rate, and reduced apoptosis and senescence. In these experimental conditions NSCs preserve their stemness properties for a longer period of time compared with controls. Also of interest is that cell fate properties are conserved as well. The controlled release of FGF-2 reduced the level of oxidative stress and this is associated with a lower level of damaged DNA. This result may explain the reduced level of senescence and apoptosis in NSCs cultivated in the presence of hydrogel-releasing FGF-2.     

Relief of hypoxia by angiogenesis promotes neural stem cell differentiation by targeting glycolysis

Blood vessels are part of the stem cell niche in the developing cerebral cortex, but their in vivo role in controlling the expansion and differentiation of neural stem cells (NSCs) in development has not been studied. Here, we report that relief of hypoxia in the developing cerebral cortex by ingrowth of blood vessels temporo‐spatially coincided with NSC differentiation. Selective perturbation of brain angiogenesis in vessel‐specific Gpr124 null embryos, which prevented the relief from hypoxia, increased NSC expansion at the expense of differentiation. Conversely, exposure to increased oxygen levels rescued NSC differentiation in Gpr124 null embryos and increased it further in WT embryos, suggesting that niche blood vessels regulate NSC differentiation at least in part by providing oxygen. Consistent herewith, hypoxia‐inducible factor (HIF)‐1α levels controlled the switch of NSC expansion to differentiation. Finally, we provide evidence that high glycolytic activity of NSCs is required to prevent their precocious differentiation in vivo. Thus, blood vessel function is required for efficient NSC differentiation in the developing cerebral cortex by providing oxygen and possibly regulating NSC metabolism.     

Kuwanon V Inhibits Proliferation,Promotes Cell Survival and Increases Neurogenesis of Neural Stem Cells

Neural stem cells (NSCs) have the ability to proliferate and differentiate into neurons and glia. Regulation of NSC fate by small molecules is important for the generation of a certain type of cell. The identification of small molecules that can induce new neurons from NSCs could facilitate regenerative medicine and drug development for neurodegenerative diseases. In this study, we screened natural compounds to identify molecules that are effective on NSC cell fate determination. We found that Kuwanon V (KWV), which was isolated from the mulberry tree (Morus bombycis) root, increased neurogenesis in rat NSCs. In addition, during NSC differentiation, KWV increased cell survival and inhibited cell proliferation as shown by 5-bromo-2-deoxyuridine pulse experiments, Ki67 immunostaining and neurosphere forming assays. Interestingly, KWV enhanced neuronal differentiation and decreased NSC proliferation even in the presence of mitogens such as epidermal growth factor and fibroblast growth factor 2. KWV treatment of NSCs reduced the phosphorylation of extracellular signal-regulated kinase 1/2, increased mRNA expression levels of the cyclin-dependent kinase inhibitor p21, down-regulated Notch/Hairy expression levels and up-regulated microRNA miR-9, miR-29a and miR-181a. Taken together, our data suggest that KWV modulates NSC fate to induce neurogenesis, and it may be considered as a new drug candidate that can regenerate or protect neurons in neurodegenerative diseases.     

Long-term self-renewal of naïve neural stem cells in a defined condition

During embryonic development, neural stem cells (NSCs) emerge as early as the neural plate stage and give rise to the nervous system. Early-stage NSCs express Sry-related-HMG box-1 (Sox1) and are biased towards neuronal differentiation. However, long-term maintenance of early-stage NSCs in vitro remains a challenge. Here, we report development of a defined culture condition for the long-term maintenance of Sox1-positive early-stage mouse NSCs. The proliferative ability of these Sox1-positive NSCs was confirmed by clonal propagation. Compared to the NSCs cultured using the traditional culture condition, the long-term self-renewing Sox1-positive NSCs efficiently differentiate into neurons and exhibit an identity representative of the anterior and midbrain regions. These early-stage Sox1-positive NSCs could also be switched to late-stage NSCs by being cultured with bFGF/EGF, which can then differentiate into astrocytes and oligodendrocytes. The long-term self-renewing Sox1-positive NSCs were defined as naïve NSCs, based on their high neuronal differentiation capacity and anterior regional identity. This culture condition provides a robust platform for further dissection of the NSC self-renewal mechanism and promotes potential applications of NSCs for cell-based therapy on nervous system disorders.     

Maintenance and Neuronal Cell Differentiation of Neural Stem Cells C17.2 Correlated to Medium Availability Sets Design Criteria in Microfluidic Systems

Background

Neural stem cells (NSCs) play an important role in developing potential cell-based therapeutics for neurodegenerative disease. Microfluidics has proven a powerful tool in mechanistic studies of NSC differentiation. However, NSCs are prone to differentiate when the nutrients are limited, which occurs unfavorable by fast medium consumption in miniaturized culture environment. For mechanistic studies of NSCs in microfluidics, it is vital that neuronal cell differentiation is triggered by controlled factors only. Thus, we studied the correlation between available cell medium and spontaneous neuronal cell differentiation of C17.2 NSCs in standard culture medium, and proposed the necessary microfluidic design criteria to prevent undesirable cell phenotype changes.

Methodology/Principal Findings

A series of microchannels with specific geometric parameters were designed to provide different amount of medium to the cells over time. A medium factor (MF, defined as the volume of stem cell culture medium divided by total number of cells at seeding and number of hours between medium replacement) successfully correlated the amount of medium available to each cell averaged over time to neuronal cell differentiation. MF smaller than 8.3×10⁴ µm³/cell⋅hour produced significant neuronal cell differentiation marked by cell morphological change and significantly more cells with positive β-tubulin-III and MAP2 staining than the control. When MF was equal or greater than 8.3×10⁴ µm³/cell⋅hour, minimal spontaneous neuronal cell differentiation happened relative to the control. MF had minimal relation with the average neurite length.

Significance

MFs can be controlled easily to maintain the stem cell status of C17.2 NSCs or to induce spontaneous neuronal cell differentiation in standard stem cell culture medium. This finding is useful in designing microfluidic culture platforms for controllable NSC maintenance and differentiation. This study also offers insight about consumption rate of serum molecules involved in maintaining the stemness of NSCs.     

Insulin concentration is critical in culturing human neural stem cells and neurons

Cell culture of human-derived neural stem cells (NSCs) is a useful tool that contributes to our understanding of human brain development and allows for the development of therapies for intractable human brain disorders. Human NSC (hNSC) cultures, however, are not commonly used, mainly because of difficulty with consistently maintaining the cells in a healthy state. In this study, we show that hNSC cultures, unlike NSCs of rodent origins, are extremely sensitive to insulin, an indispensable culture supplement, and that the previously reported difficulty in culturing hNSCs is likely because of a lack of understanding of this relationship. Like other neural cell cultures, insulin is required for hNSC growth, as withdrawal of insulin supplementation results in massive cell death and delayed cell growth. However, severe apoptotic cell death was also detected in insulin concentrations optimized to rodent NSC cultures. Thus, healthy hNSC cultures were only produced in a narrow range of relatively low insulin concentrations. Insulin-mediated cell death manifested not only in all human NSCs tested, regardless of origin, but also in differentiated human neurons. The underlying cell death mechanism at high insulin concentrations was similar to insulin resistance, where cells became less responsive to insulin, resulting in a reduction in the activation of the PI3K/Akt pathway critical to cell survival signaling.     

Short-Term Withdrawal of Mitogens Prior to Plating Increases Neuronal Differentiation of Human Neural Precursor Cells

Background

Human neural precursor cells (hNPC) are candidates for neural transplantation in a wide range of neurological disorders. Recently, much work has been done to determine how the environment for NPC culture in vitro may alter their plasticity. Epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2) are used to expand NPC; however, it is not clear if continuous exposure to mitogens may abrogate their subsequent differentiation. Here we evaluated if short-term removal of FGF-2 and EGF prior to plating may improve hNPC differentiation into neurons.

Principal Findings

We demonstrate that culture of neurospheres in suspension for 2 weeks without EGF-FGF-2 significantly increases neuronal differentiation and neurite extension when compared to cells cultured using standard protocols. In this condition, neurons were preferentially located in the core of the neurospheres instead of the shell. Moreover, after plating, neurons presented radial rather than randomly oriented and longer processes than controls, comprised mostly by neurons with short processes. These changes were followed by alterations in the expression of genes related to cell survival.

Conclusions

These results show that EGF and FGF-2 removal affects NPC fate and plasticity. Taking into account that a three dimensional structure is essential for NPC differentiation, here we evaluated, for the first time, the effects of growth factors removal in whole neurospheres rather than in plated cell culture.     

Melatonin antagonizes interleukin‐18‐mediated inhibition on neural stem cell proliferation and differentiation

Neural stem cells (NSCs) are self‐renewing, pluripotent and undifferentiated cells which have the potential to differentiate into neurons, oligodendrocytes and astrocytes. NSC therapy for tissue regeneration, thus, gains popularity. However, the low survivals rate of the transplanted cell impedes its utilities. In this study, we tested whether melatonin, a potent antioxidant, could promote the NSC proliferation and neuronal differentiation, especially, in the presence of the pro‐inflammatory cytokine interleukin‐18 (IL‐18). Our results showed that melatonin per se indeed exhibited beneficial effects on NSCs and IL‐18 inhibited NSC proliferation, neurosphere formation and their differentiation into neurons. All inhibitory effects of IL‐18 on NSCs were significantly reduced by melatonin treatment. Moreover, melatonin application increased the production of both brain‐derived and glial cell‐derived neurotrophic factors (BDNF, GDNF) in IL‐18‐stimulated NSCs. It was observed that inhibition of BDNF or GDNF hindered the protective effects of melatonin on NSCs. A potentially protective mechanism of melatonin on the inhibition of NSC's differentiation caused IL‐18 may attribute to the up‐regulation of these two major neurotrophic factors, BNDF and GNDF. The findings indicate that melatonin may play an important role promoting the survival of NSCs in neuroinflammatory diseases.     

FGF-2 Inhibits Apoptosis in Human Teratocarcinoma Cells during Differentiation on Collagen Substratum

Tera-2 is a human teratocarcinoma cell line, which is induced to differentiate into neuronal direction by retinoic acid. Once differentiated, the cells form an almost nondividing population that can be maintained for weeks under conventional culture conditions. If differentiation by retinoic acid is induced while the cells are growing on type I collagen or if the already-differentiated cells are transferred onto collagen, they survive only a few days unless the cultures are repeatedly supplied with FGF-2. Lack of this growth factor induces programmed cell death (apoptosis) detectable after 24–48 h, as marked by DNA cleavage and nuclear fragmentation. The undifferentiated stem cells survive and proliferate readily on collagen without addition of FGF-2. Tera-2 cells express two members of the FGF family, FGF-2 and FGF-4. The expression of both FGFs is turned off during differentiation on collagen substratum, whereas when cultivated on plain tissue culture dish, the expression of only FGF-4 becomes undetectable. The results indicate that signaling through cell surface FGF receptors is vital for the cells, and differentiation on collagen substratum results in complete extinction of the autocrine stimulatory loop.In vivo,such induction of growth factor dependency upon differentiation would result in apoptotic death of those cells which fail to find adequate conditions for continuing FGF stimulation.     

ETOH inhibits embryonic neural stem/precursor cell proliferation via PLD signaling

While a mother’s excessive alcohol consumption during pregnancy is known to have adverse effects on fetal neural development, little is known about the underlying mechanism of these effects. In order to investigate these mechanisms, we investigated the toxic effect of ethanol (ETOH) on neural stem/precursor cell (NSC) proliferation. In cultures of NSCs, phospholipase D (PLD) is activated following stimulation with epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2). Exposure of NSCs to ETOH suppresses cell proliferation, while it has no effect on cell death. Phosphatidic acid (PA), which is a signaling messenger produced by PLD, reverses ETOH inhibition of NSC proliferation. Blocking the PLD signal by 1-butanol suppresses the proliferation. ETOH-induced suppression of NSC proliferation and the protective effect of PA for ETOH-induced suppression are mediated through extracellular signal-regulated kinase signaling. These results indicate that exposure to ETOH impairs NSC proliferation by altering the PLD signaling pathway.     

人神经干细胞的体外生物学特性

本实验利用有丝分裂因子,体外诱导生成人神 经干细胞(NSCs),观察其生长特性并进行鉴定。取胎龄10-22周的大脑半球,分散细胞后种于添加表皮生长因子(EGF,20ng/ml)和/或碱性成纤维生长因子(bFGF,20ng/ml)的培养基中。利用免疫组织化学方法鉴定分化后的细胞类型。同时,进行细胞克隆分析、传代培养及端粒酶活性检测。结果显示:NSCs呈悬浮生长的干细胞球,其特异性抗原nestin阳性。NSCs具有增殖能力,可连续传代而不丢失其增殖和多分化潜能的干细胞特性。撤除EGF和bFGF的作用,细胞停止分裂,并分化为神经元、星形胶质细胞和少突胶质细胞。克隆分析显示NSCs生长呈密度依赖性。人NSCs表达较低的端粒酶水平,并随培养时间延长而下调。研究表明,利用有丝分裂因子,可在体外成功诱导生成人NSCs,其生长,分化受内外源因素的调节,相关的机制还有待阐明。     

Role of miRNA-146?in proliferation and differentiation of mouse neural stem cells

Neural stem cells (NSCs) have been defined as neural cells with the potential to self-renew and eventually generate all cell types of the nervous system. NSCs serve as an ideal cell type for nervous system repair. In the present study, miR-146 overexpression and predicted target (notch 1) were used to study proliferation and differentiation of mouse NSCs. shRNA were used to demonstrate the function of Notch 1 in proliferation of mouse NSCs and luciferase reporter assay was used to assess and confirm the binding sequence of 3′-UTR between Notch 1 and miR-146. Results showed that miR-146 overexpression and knockdown of notch 1 inhibited proliferation of mouse NSCs under serum-free cultural conditions and promoted spontaneous differentiation of mouse NSCs under contained serum cultural conditions respectively. Mouse NSCs spontaneously underwent differentiation into neurogenic cells with contained serum medium. However, when miR-146 was overexpressed, differentiation efficiency of glial cells from NSCs was increased, suggesting that Notch1 promoted NSC proliferation and repressed spontaneous differentiation of NSC in serum-free medium. In conclusion, our results demonstrate that miR-146 promoted spontaneous differentiation of NSCs, and this mechanism was influenced by miR-146, as well as its target (notch 1) and downstream gene.     

Evaluation of the differentiation status of neural stem cells based on cell morphology and the expression of Notch and Sox2

Neural stem cells (NSCs) isolated from a variety of sources are being developed as cellular therapies aimed at treating neurodegenerative diseases. During NSC culture and expansion it is important the cells do not differentiate prematurely because this may have an unfavorable effect on product quality and yield. In our study, we evaluated the use of Notch and Sox2 as markers for undifferentiated human and mouse NSCs. The expression of Notch2 and Sox2 during extensive-passage, low-oxygen culture and differentiation conditions were analyzed to confirm that the presence of these signature proteins directly correlates with the ability of NSCs to form new neurospheres and differentiate into multiple cell types. Using expression of Notch1, Notch2 and Sox2 as a reference, we then used flow cytometry to identify a specific morphological profile for undifferentiated murine and human NSCs. Our studies show that Notch and Sox2 expression, along with flow cytometry analysis, can be used to monitor the differentiation status of NSCs grown in culture for use in cellular therapies.     

Monitoring neural stem cell differentiation using PEDOT–PSS based MEA

Background

Transplantation is one potential clinical application of neural stem cells (NSCs). However, it is very difficult to monitor/control NSCs after transplantation and so provide effective treatment. Electrical measurement using a poly(3,4-ethylenedioxythiophene)–poly(styrenesulfonate) (PEDOT–PSS) modified microelectrode array (MEA) is a biocompatible, non-invasive, non-destructive approach to understanding cell conditions. This property makes continuous monitoring available for the evaluation/assessment of the development of cells such as NSCs.

Methods

A PEDOT–PSS modified MEA was used to monitor electrical signals during NSC development in a culture derived from rat embryo striatum in order to understand the NSC differentiation conditions.

Results

Electrical data indicated that NSCs with nerve growth factor (NGF) generate a cultured cortical neuron-like burst pattern while a random noise pattern was measured with epidermal growth factor (EGF) at 4 days in vitro (DIV) and a burst pattern was observed in both cases at 11 DIV indicating the successful monitoring of differentiation differences and developmental changes.

Conclusions

The electrical analysis of cell activity using a PEDOT–PSS modified MEA could indicate neural network formation by differentiated neurons. Changes in NSC differentiation could be monitored.

General significance

The method is based on non-invasive continuous measurement and so could prove a useful tool for the primary/preliminary evaluation of a pharmaceutical analysis. This article is part of a Special Issue entitled Organic Bioelectronics—Novel Applications in Biomedicine.     

Epidermal growth factor and three‐dimensional scaffolds provide conducive environment for differentiation of mouse embryonic stem cells into oocyte‐like cells

Three‐dimensional (3D) culture provides a biomimicry of the naive microenvironment that can support cell proliferation, differentiation, and regeneration. Some growth factors, such as epidermal growth factor (EGF), facilitate normal meiosis during oocyte maturation in vivo. In this study, a scaffold‐based 3D coculture system using purified alginate was applied to induce oocyte differentiation from mouse embryonic stem cells (mESCs). mESCs were induced to differentiate into oocyte‐like cells using embryoid body protocol in the two‐dimensional or 3D microenvironment in vitro. To increase the efficiency of the oocyte‐like cell differentiation from mESCs, we employed a coculture system using ovarian granulosa cells in the presence or absence of epidermal growth factor (+EGF or ?EGF) for 14 days and then the cells were assessed for germ cell differentiation, meiotic progression, and oocyte maturation markers. The cultures exposed to EGF in the alginate‐based 3D microenvironment showed the highest level of premeiotic (Oct4 and Mvh), meiotic (Scp1, Scp3, Stra8, and Rec8), and oocyte maturation (Gdf9, Cx37, and Zp2) marker genes (p < .05) in comparison to other groups. According to the gene‐expression patterns, we can conclude that alginate‐based 3D coculture system provided a highly efficient protocol for oocyte‐like cell differentiation from mESCs. The data showed that this culture system along with EGF improved the rate of in vitro oocyte‐like cell differentiation.     

Control of neural stem cell survival by electroactive polymer substrates

Stem cell function is regulated by intrinsic as well as microenvironmental factors, including chemical and mechanical signals. Conducting polymer-based cell culture substrates provide a powerful tool to control both chemical and physical stimuli sensed by stem cells. Here we show that polypyrrole (PPy), a commonly used conducting polymer, can be tailored to modulate survival and maintenance of rat fetal neural stem cells (NSCs). NSCs cultured on PPy substrates containing different counter ions, dodecylbenzenesulfonate (DBS), tosylate (TsO), perchlorate (ClO(4)) and chloride (Cl), showed a distinct correlation between PPy counter ion and cell viability. Specifically, NSC viability was high on PPy(DBS) but low on PPy containing TsO, ClO(4) and Cl. On PPy(DBS), NSC proliferation and differentiation was comparable to standard NSC culture on tissue culture polystyrene. Electrical reduction of PPy(DBS) created a switch for neural stem cell viability, with widespread cell death upon polymer reduction. Coating the PPy(DBS) films with a gel layer composed of a basement membrane matrix efficiently prevented loss of cell viability upon polymer reduction. Here we have defined conditions for the biocompatibility of PPy substrates with NSC culture, critical for the development of devices based on conducting polymers interfacing with NSCs.     

The frequency of neural stem cells in in vitro culture systems: insights from simple modeling

Neural stem cell (NSC) culture offers a renewable resource for cell replacement treatment of neurodegenerative diseases. In a NSC culture, the frequency of NSCs is estimated to be only a few percent, at most, while the majority of cells are neuroprogenitor cells (NPCs), the progeny of NSCs with a limited capability to proliferate. Here, the effects of several cell culture parameters on the cell composition of NSC cultures were illustrated over time using a simple model. The model shows thatvarious initial cell compositions in NSC cultures converge into a single steady state. The model also implies that the rate of increase in total cell number entirely depends on the self-renewal rate of NSCs, regardless of the proliferative capacity of NPCs. Furthermore, the model predicts that difference sin cell passage methods between neurosphere and monolayer cultures results in a change in the frequency of NSCs in culture.     

Survival of Neural Stem Cells Undergoing DNA Damage-Induced Astrocytic Differentiation in Self-Renewal-Promoting Conditions In Vitro

We recently reported that neural stem cells (NSCs) become senescent and commit to astrocytic differentiation upon X-ray irradiation. Surprisingly, under self-renewing culture conditions, some of these senescent cells undergo p53-independent apoptosis, which can be suppressed by caspase inhibition and BCL2 overexpression. Inhibition of apoptosis proved beneficial for astroglial differentiation efficiency; hence the toxicity of DNA damage on NSCs was specifically tested in context of the culture conditions. In this regard, self-renewal-promoting culture conditions proved incompatible with terminal astrocyte differentiation and impacted negatively on the viability of NSCs following DNA damage-induced cell cycle exit. On the contrary, a switch to differentiation-supporting conditions ablated apoptosis and conveyed tolerance to DNA damage. Thus, stem cell death has likely not originated from DNA break toxicity, while the potentially confounding effect of stem cell niche should always be taken in consideration in stem cell irradiation experiments.     

RGCC balances self‐renewal and neuronal differentiation of neural stem cells in the developing mammalian neocortex

During neocortical development, neural stem cells (NSCs) divide symmetrically to self‐renew at the early stage and then divide asymmetrically to generate post‐mitotic neurons. The molecular mechanisms regulating the balance between NSC self‐renewal and neurogenesis are not fully understood. Using mouse in utero electroporation (IUE) technique and in vitro human NSC differentiation models including cerebral organoids (hCOs), we show here that regulator of cell cycle (RGCC) modulates NSC self‐renewal and neuronal differentiation by affecting cell cycle regulation and spindle orientation. RGCC deficiency hampers normal cell cycle process and dysregulates the mitotic spindle, thus driving more cells to divide asymmetrically. These modulations diminish the NSC population and cause NSC pre‐differentiation that eventually leads to brain developmental malformation in hCOs. We further show that RGCC might regulate NSC spindle orientation by affecting the organization of centrosome and microtubules. Our results demonstrate that RGCC is essential to maintain the NSC pool during cortical development and suggest that RGCC defects could have etiological roles in human brain malformations.