Expression Of Mature Neuronal Markers Research Articles

Integrative Analysis of Long Isoform Sequencing and Functional Data Identifies Distinct Cortical Layer Neuronal Subtypes Derived from Human iPSCs.

Generation of human induced pluripotent stem cells (iPSCs) through reprogramming was a transformational change in the field of regenerative medicine that led to new possibilities for drug discovery and cell replacement therapy. Several protocols have been established to differentiate hiPSCs into neuronal lineages. However, low differentiation efficiency is one of the major drawbacks of these approaches. Here, we compared the efficiency of two methods of neuronal differentiation from iPSCs cultured in two different culture media, StemFlex Medium (SFM) and Essential 8 Medium (E8M). The results indicated that iPSCs cultured in E8M efficiently generated different types of neurons in a shorter time and without the growth of undifferentiated non-neuronal cells in the culture as compared to those generated from iPSCs in SFM. Furthermore, these neurons were validated as functional units immunocytochemically by confirming the expression of mature neuronal markers (i.e., NeuN, Beta tubulin, and Synapsin I), and whole-cell patch-clamp recordings. Long-read single-cell RNA sequencing confirms the presence of upper and deep layer cortical layer excitatory and inhibitory neuronal subtypes in addition to small populations of GABAergic neurons in day 30 neuronal cultures. Pathway analysis indicated that our protocol triggers the signaling transcriptional networks important for the process of neuronal differentiation in vivo.

Degree of differentiation impacts neurobiological signature and resistance to hypoxia of SH-SY5Y cells

Objective. SH-SY5Y cells are valuable neuronal in vitro models for studying patho-mechanisms and treatment targets in brain disorders due to their easy maintenance, rapid expansion, and low costs. However, the use of various degrees of differentiation hampers appreciation of results and may limit the translation of findings to neurons or the brain. Here, we studied the neurobiological signatures of SH-SY5Y cells in terms of morphology, expression of neuronal markers, and functionality at various degrees of differentiation, as well as their resistance to hypoxia. We compared these to neurons derived from human induced pluripotent stem cells (hiPSCs), a well-characterized neuronal in vitro model. Approach. We cultured SH-SY5Y cells and neurons derived from hiPSCs on glass coverslips or micro-electrode arrays. We studied expression of mature neuronal markers, electrophysiological activity, and sensitivity to hypoxia at various degrees of differentiation (one day up to three weeks) in SH-SY5Y cells. We used hiPSC derived neurons as a reference. Main results. Undifferentiated and shortly differentiated SH-SY5Y cells lacked neuronal characteristics. Expression of neuronal markers and formation of synaptic puncta increased during differentiation. Longer differentiation was associated with lower resistance to hypoxia. At three weeks of differentiation, MAP2 expression and vulnerability to hypoxia were similar to hiPSC-derived neurons, while the number of synaptic puncta and detected events were significantly lower. Our results show that at least three weeks of differentiation are necessary to obtain neurobiological signatures that are comparable to those of hiPSC-derived neurons, as well as similar sensitivities to metabolic stress. Significance. This indicates that extended differentiation protocols should be used to study neuronal characteristics and to model brain disorders with SH-SY5Y cells. We provided insights that may offer the basis for the utilization of SH-SY5Y cells as a more relevant neuronal model in the study of brain disorders.

Directly reprogrammed fragile X syndrome dorsal forebrain precursor cells generate cortical neurons exhibiting impaired neuronal maturation.

The neurodevelopmental disorder fragile X syndrome (FXS) is the most common monogenic cause of intellectual disability associated with autism spectrum disorder. Inaccessibility to developing human brain cells is a major barrier to studying FXS. Direct-to-neural precursor reprogramming provides a unique platform to investigate the developmental profile of FXS-associated phenotypes throughout neural precursor and neuron generation, at a temporal resolution not afforded by post-mortem tissue and in a patient-specific context not represented in rodent models. Direct reprogramming also circumvents the protracted culture times and low efficiency of current induced pluripotent stem cell strategies. We have developed a chemically modified mRNA (cmRNA) -based direct reprogramming protocol to generate dorsal forebrain precursors (hiDFPs) from FXS patient-derived fibroblasts, with subsequent differentiation to glutamatergic cortical neurons and astrocytes. We observed differential expression of mature neuronal markers suggesting impaired neuronal development and maturation in FXS- hiDFP-derived neurons compared to controls. FXS- hiDFP-derived cortical neurons exhibited dendritic growth and arborization deficits characterized by reduced neurite length and branching consistent with impaired neuronal maturation. Furthermore, FXS- hiDFP-derived neurons exhibited a significant decrease in the density of pre- and post- synaptic proteins and reduced glutamate-induced calcium activity, suggesting impaired excitatory synapse development and functional maturation. We also observed a reduced yield of FXS- hiDFP-derived neurons with a significant increase in FXS-affected astrocytes. This study represents the first reported derivation of FXS-affected cortical neurons following direct reprogramming of patient fibroblasts to dorsal forebrain precursors and subsequently neurons that recapitulate the key molecular hallmarks of FXS as it occurs in human tissue. We propose that direct to hiDFP reprogramming provides a unique platform for further study into the pathogenesis of FXS as well as the identification and screening of new drug targets for the treatment of FXS.

Enhancing Neurological Competence of Nanoencapsulated Cordyceps/Turmeric Extracts in Human Neuroblastoma SH-SY5Y Cells.

Neurological diseases, including Alzheimer's, Parkinson's diseases, and brain cancers, are reportedly caused by genetic aberration and cellular malfunction. Herbs with bioactive compounds that have anti-oxidant effects such as cordyceps and turmeric, are of interest to clinical applications due to their minimal adverse effects. The aim of study is to develop the nanoencapsulated cordyceps and turmeric extracts and investigate their capability to enhance the biological activity and improve neuronal function. Human neuroblastoma SH-SY5Y cells were utilized as a neuronal model to investigate the properties of nanoencapsulated cordyceps or turmeric extracts, called CMP and TEP, respectively. SH-SY5Y cells were treated with either CMP or TEP and examined the biological consequences, including neuronal maturation and neuronal function. The results showed that both CMP and TEP improved cellular uptake efficiency within 6h by 2.3 and 2.8 times, respectively. Besides, they were able to inhibit cellular proliferation of SH-SY5Y cells up to 153- and 218-fold changes, and increase the expression of mature neuronal markers (TUJ1, PAX6, and NESTIN). Upon the treatment of CMP and TEP, the expression of dopaminergic-specific genes (LMX1B, FOXA2, EN1, and NURR1), and the secretion level of dopamine were significantly improved up to 3.3-fold and 3.0-fold, respectively, while the expression of Alzheimer genes (PSEN1, PSEN2, and APP), and the secretion of amyloid precursor protein were significantly reduced by 32-fold and 108-fold, respectively. Importantly, the autophagy activity was upregulated by CMP and TEP at 6.3- and 5.5-fold changes, respectively. This finding suggested that the nanoencapsulated cordyceps and turmeric extracts accelerated neuronal maturation and alleviated neuronal pathology in human neural cells. This paves the way for nanotechnology-driven drug delivery systems that could potentially be used as an alternative medicine in the future for neurological diseases.

Accelerated Maturation of Non‐cycling Human Induced Pluripotent Stem Cell‐derived neurons for High Content Screening

Human induced pluripotent stem cell-derived (iPSC) neurons are increasingly being used as a platform for neurological disease modeling, high throughput/content screening and drug discovery. However, the differentiation and maturation of iPSC-derived neurons is not standardized and varies in methodology (i.e., timing) limiting their usefulness in automated drug screening platforms. To generate mature iPS-derived neurons for high content screening, we derived neuronal progenitor cells (NPCs) from the normal iPSC control lines, 7545-5b and 7753, using monolayer derivation methods. We differentiated these NPCs under environmental oxygen (21%) conditions and discerned that mature neuronal markers were predominantly expressed by 12 weeks post-differentiation. When we differentiated and matured 7753- and 7545-derived neuronal cultures under physiological (5%) oxygen levels, however, iPS neuronal maturation was markedly accelerated with increased gene and protein expression of mature neuronal markers (i.e., calretinin, calbindin, NeuN) being seen at 6 weeks post differentiation. Further modifications of the underlying extracellular matrix (i.e., poly-D-ornithine, laminin), and medium composition and transition shifted the expression of mature neuronal markers to 4 weeks post-differentiation. This coincided with the cessation of the neurogenesis burst which occurred between weeks 2 and 3 post-differentiation. qPCR microarray and western blot data as well as single cell analyses showed that 4 week iPS neuronal cultures were non-cycling with decreased expression of cyclin D1 and Ki67. Further optimization of cell seeding density and incubations ameliorated edge effects associated with prolonged cell culturing. Taken together, we have developed conditions for the derivation of a mature iPSC-derived neurons in 4 weeks, which are amenable to high throughput and high content screening applications.

Pulsed electromagnetic fields synergize with graphene to enhance dental pulp stem cell-derived neurogenesis by selectively targeting TRPC1 channels

Conventional root canal treatment replaces the infected pulp with defined materials. Alternative cell-based tissue engineering strategies aim to regenerate a fully functional pulp within the root canal. Despite recent advances in this area, however, the regeneration of an innervated pulp remains a major challenge in the field. Both graphene (2DG) and pulsed electromagnetic fields (PEMFs) independently have been shown to promote diverse cellular developmental programs. The present study showed that 2DG promoted the neurogenic induction of human dental pulp stem cells (hDPSCs) by upregulating and accelerating the expression of mature neuronal markers. Notably, 2DG induced the highest expression of transient receptor potential canonical cation channel type 1 (TRPC1) during early neurogenesis. As brief PEMF exposure promotes in vitro differentiation by activating a TRPC1-mitochondrial axis, an opportunity to combine 2DG with developmentally targeted PEMF exposure for synergistic effects was realizable. Neurogenic gene expression, neurotransmitter release, and reactive oxygen species (ROS) production were greatly enhanced by a brief (10 min) and low amplitude (2 mT) PEMF exposure timed to coincide with the highest TRPC1 expression from hDPSCs on 2DG. In contrast, hDPSCs on glass were less responsive to PEMF exposure. The capacity of PEMFs to promote neurogenesis was precluded by the administration of penicillin/streptomycin, mirroring previous studies demonstrating that aminoglycoside antibiotics block TRPC1-mediated calcium entry and verifying the contribution of TRPC1 in this form of magnetoreception. Hence, graphene created a more conducive environment for subsequent PEMF-stimulated neurogenic induction of hDPSCs through their mutual capacity to activate TRPC1with subsequent ROS production.

Refinement of a differentiation protocol using neuroblastoma SH-SY5Y cells for use in neurotoxicology research

Since most models used to study neuronal dysfunction display disadvantages and ethical concerns, a fast and reproducible in vitro model to study mitochondria-related neurodegeneration is required. Here, we optimized and characterized a 3-day retinoic acid-based protocol to differentiate the SH-SY5Y cell line into a neuronal-like phenotype and investigated alterations in mitochondrial physiology and distribution. Differentiation was associated with p21-linked cell cycle arrest and an increase in cell mass and area, possibly associated with the development of neurite-like extensions. Notably, increased expression of mature neuronal markers (neuronal-specific nuclear protein, microtubule-associated protein 2, βIII tubulin and enolase 2) was observed in differentiated cells. Moreover, increased mitochondrial content and maximal area per cell suggests mitochondrial remodeling. To demonstrate that this model is appropriate to study mitochondrial dysfunction, cells were treated for 6 h with mitochondrial toxicants (rotenone, antimycin A, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) and 6-hydroxydopamine (6-OHDA)). Differentiated cells were more susceptible to increasing concentrations of FCCP, antimycin A, and rotenone, while 6-OHDA showed a distinct dose-dependent neurotoxicity pattern. Even though differentiated cells did not exhibit a fully mature/differentiated neuronal phenotype, the protocol developed can be used to study neurotoxicity processes, mitochondrial dynamics, and bioenergetic impairment, representing an alternative to study mitochondrial impairment-related pathologies in vitro.

Valproic acid promotes mature neuronal differentiation of adipose tissue-derived stem cells through iNOS–NO–sGC signaling pathway

Valproic acid (VPA) remarkably promotes the differentiation of adipose tissue-derived stem cells (ASCs) to mature neuronal cells, enabling neuronal induction within only three days. Here, we investigated the involvement of NO-signaling in the VPA-promoted neuronal differentiation of ASCs as a possible mechanism. Cultured rat ASCs were differentiated to matured neuronal cells rich in dendrites and expressing βIII-tubulin protein, a neuronal marker, by treatments with VPA at 2 mM for 3 days and subsequently with the neuronal induction medium (NIM) containing cAMP-elevating agents for 2 h. Increased intracellular NO was detected in neuronal cells differentiated from ASCs treated with VPA by a fluorescence NO-specific probe, diaminofluorescein-FM diacetate. However, a NO donor (NOC18) increased the incidence of neuronal cells only to a lesser extent than VPA, indicating the insufficiency of exogenous NO. RT-PCR analysis of ASCs treated with VPA showed increased mRNA expression of inducible nitric oxide synthase (iNOS) with the acetylation of its associated histone H3K9. iNOS inhibitors (1400 W and dexamethasone) or a soluble guanylate cyclase (sGC) inhibitor (ODQ) decreased the incidence of neuronal cells differentiated from ASCs treated with VPA. These inhibitors also decreased the mRNA expression of mature neuronal markers, neurofilament medium polypeptide (NeFM) and microtubule-associated protein 2 (MAP2), as well as βIII-tubulin (TUBB3), to various extents. It was considered from these results that VPA promoted mature neuronal differentiation of ASCs through the iNOS–NO–sGC signaling pathway. This provided insights into the regulated neuronal differentiation of ASCs in clinical applications.

Rapid and stable changes in maturation-related phenotypes of the adult hippocampal neurons by electroconvulsive treatment

Electroconvulsive therapy (ECT) is a highly effective and fast-acting treatment for depression. Despite a long history of clinical use, its mechanism of action remains poorly understood. Recently, a novel cellular mechanism of antidepressant action has been proposed: the phenotype of mature brain neurons is transformed to immature-like one by antidepressant drug treatments. We show here that electroconvulsive stimulation (ECS), an animal model of ECT, causes profound changes in maturation-related phenotypes of neurons in the hippocampal dentate gyrus of adult mice. Single ECS immediately reduced expression of mature neuronal markers in almost entire population of dentate granule cells. After ECS treatments, granule cells showed some of physiological properties characteristic of immature granule cells such as higher somatic intrinsic excitability and smaller frequency facilitation at the detate-to-CA3 synapse. The rapid downregulation of maturation markers was suppressed by antagonizing glutamate NMDA receptors, but not by perturbing the serotonergic system. While single ECS caused short-lasting effects, repeated ECS induced stable changes in the maturation-related phenotypes lasting more than 2 weeks along with enhancement of synaptic excitation of granule cells. Augmentation of synaptic inhibition or blockade of NMDA receptors after repeated ECS facilitated regaining the initial mature phenotype, suggesting a role for endogenous neuronal excitation in maintaining the altered maturation-related phenotype probably via NMDA receptor activation. These results suggest that brief neuronal activation by ECS induces “dematuration” of the mature granule cells and that enhanced endogenous excitability is likely to support maintenance of such a demature state. The global increase in neuronal excitability accompanying this process may be relevant to the high efficacy of ECT.

Development of biomimetic micro-patterned device incorporated with neurotrophic gradient and supportive Schwann cells for the applications in neural tissue engineering

In these years, the artificial nerve guidance conduit (NGC) has been developed as an alternative way to repair peripheral nerve injury. Unlike autologous nerve graft, the artificial NGC without proper stimulating factors and guidance cues still cannot obtain satisfactory prognosis for clinical patients. In this study, a biodegradable polymer-based implantable device has been developed and characterized. By incorporating three stimulating factors: (1) micro-patterned surface that can directionally guide the axon as physical cue; (2) neurotrophic gradient membrane that can continually attract axon outgrowth from the proximal to distal stump as chemical cue; (3) Schwann cells (SCs) that can support the growth of neurite and form myelin sheath around axon as biological cue, we expect that this construct can be used as a promising NGC for peripheral nerve regeneration. The results showed that the micro-patterned surface with specific dimension of channels and chambers can be precisely fabricated by laser ablation. Attachment and directional extension of differentiated neural stem cells (NSCs) were observed in micro-channels. The gradient distribution of nerve growth factor 7S on gelatin membrane was successfully achieved. Significant improvement in neurite length and increase in neuronal gene expressions were also noticed in higher concentration region. When co-culturing with SCs, NSCs can differentiate toward neuronal cells with strong expression of mature neuronal markers: βIII tubulin and microtubule-associated protein-2 (Map 2). Meanwhile, myelin basic protein was also observed, suggesting that SCs can provide biological support to neuronal cells in vitro. In the future, this advanced artificial NGC may be used as implantable prosthesis for the treatment of peripheral nerve injury with better functional recovery.

M18 Spatio-temporal Characterisation Of Human Fetal Neural Stem Cells For Cell Replacement Therapies

Human fetal neural stem cells (hfNSC) hold great potential for cell therapy strategies but further studies are still needed for a proper understanding of these cells. In the present work we characterised hfNSC isolated from cortex, whole ganglionic eminence (WGE), cerebellum and forebrain at several developmental stages (6–12 weeks post conception (wpc)). We showed that tissue obtained from younger fetuses survived significantly better than older ones after dissociation, due to the presence of a higher number Nestin positive neural precursors. These results suggested the presence of more differentiated cells in samples obtained from older fetuses. In agreement, an increased expression of mature neuronal markers was observed in samples from older fetal brains. Interestingly, the proliferation ratios were not different between developmental stages. A common feature was observed for all the cultures studied independently of the brain area and the developmental stage; they went through 3–4 initial critical passages characterised by high percentages of cell death and substantial variability in the proliferation ratio. After this critical period, they stabilised, reduced cell death and maintained a constant expansion potential. Forebrain hfNSC were the most readily expandable and cerebellar cells the ones that proliferated least. Finally, we analysed the regional identity and differentiation potential of cortical and WGE derived hfNSC along passages. The expression of mature region specific markers after 3 and 6 passages was down-regulated. Interestingly, cortical cultures acquire a ventral phenotype along passages. Nevertheless, they both keep the multipotency after 6 passages and the potential to differentiate to mature region specific neurons. In conclusion, here we show that hfNSCs can be expanded in vitro and may be suitable for cell therapy in HD.

Neural differentiation of mouse embryonic and mesenchymal stem cells in a simple medium containing synthetic serum replacement

Neural differentiation of embryonic and adult stem cells has been reported previously. Several studies have used different proportions of serum or a cocktail of growth and differentiation factors for this purpose. In the present study, we examined neural differentiation of mouse embryonic stem (ES) cells in KoSR-containing media. We also investigated neural differentiation of mouse adipose tissue-derived stem cells (ADSCs) in a medium containing KoSR, a synthetic serum replacement, and compared it with neural differentiation in low-serum condition. Meanwhile, effect of β-ME on neural differentiation was investigated in both conditions. As revealed by RT-PCR and immunocytochemistry analyses, KoSR-containing medium induced neural differentiation of mouse ES cells. Moreover, under the culture conditions we tested, ADSCs were differentiated to neuron-like cells and expressed some neuronal markers. Low concentration of β-ME improved neuron-like differentiation of the ADSCs in the 4% FBS-supplemented medium, while addition of β-ME in KoSR condition decreased neural differentiation. KoSR-containing medium without any additional factor improved generation of neuron-like cells, upregulated the expression of mature neuronal markers and led to the formation of cytoplasmic processes. In summary, our findings are indicating that mouse embryonic and mesenchymal stem cells are capable of neural development in KoSR-containing media.

Feline bone marrow-derived mesenchymal stromal cells (MSCs) show similar phenotype and functions with regards to neuronal differentiation as human MSCs

Mesenchymal stromal cells (MSCs) show promise for treatment of a variety of neurological and other disorders. Cat has a high degree of linkage with the human genome and has been used as a model for analysis of neurological disorders such as stroke, Alzheimer's disease and motor disorders. The present study was designed to characterize bone marrow-derived MSCs from cats and to investigate the capacity to generate functional peptidergic neurons. MSCs were expanded with cells from the femurs of cats and then characterized by phenotype and function. Phenotypically, feline and human MSCs shared surface markers, and lacked hematopoietic markers, with similar morphology. As compared to a subset of human MSCs, feline MSCs showed no evidence of the major histocompatibility class II. Since the literature suggested Stro-1 as an indicator of pluripotency, we compared early and late passages feline MSCs and found its expression in >90% of the cells. However, the early passage cells showed two distinct populations of Stro-1-expressing cells. At passage 5, the MSCs were more homogeneous with regards to Stro-1 expression. The passage 5 MSCs differentiated to osteogenic and adipogenic cells, and generated neurons with electrophysiological properties. This correlated with the expression of mature neuronal markers with concomitant decrease in stem cell-associated genes. At day 12 induction, the cells were positive for MAP2, Neuronal Nuclei, tubulin βIII, Tau and synaptophysin. This correlated with electrophysiological maturity as presented by excitatory postsynaptic potentials (EPSPs). The findings indicate that the cat may constitute a promising biomedical model for evaluation of novel therapies such as stem cell therapy in such neurological disorders as Alzheimer's disease and stroke.

Early Natural Stimulation through Environmental Enrichment Accelerates Neuronal Development in the Mouse Dentate Gyrus

The dentate gyrus is the primary afferent into the hippocampal formation, with important functions in learning and memory. Granule cells, the principle neuronal type in the dentate gyrus, are mostly formed postnatally, in a process that continues into adulthood. External stimuli, including environmental enrichment, voluntary exercise and learning, have been shown to significantly accelerate the generation and maturation of dentate granule cells in adult rodents. Whether, and to what extent, such environmental stimuli regulate the development and maturation of dentate granule cells during early postnatal development is largely unknown. Furthermore, whether natural stimuli affect the synaptic properties of granule cells had been investigated neither in newborn neurons of the adult nor during early development. To examine the effect of natural sensory stimulation on the dentate gyrus, we reared newborn mice in an enriched environment (EE). Using immunohistochemistry, we showed that dentate granule cells from EE-reared mice exhibited earlier morphological maturation, manifested as faster peaking of doublecortin expression and elevated expression of mature neuronal markers (including NeuN, calbindin and MAP2) at the end of the second postnatal week. Also at the end of the second postnatal week, we found increased density of dendritic spines across the entire dentate gyrus, together with elevated levels of postsynaptic scaffold (post-synaptic density 95) and receptor proteins (GluR2 and GABAARγ2) of excitatory and inhibitory synapses. Furthermore, dentate granule cells of P14 EE-reared mice had lower input resistances and increased glutamatergic and GABAergic synaptic inputs. Together, our results demonstrate that EE-rearing promotes morphological and electrophysiological maturation of dentate granule cells, underscoring the importance of natural environmental stimulation on development of the dentate gyrus.

P78. Effects of hypoxia on neural differentiation of mouse embryonic stem cells

“Hypoxia” is one of the important environmental factors for early development of mammals, since embryogenesis proceeds under the low oxygen environment (~3%) until the vascular system is developed. It is known that mice deficient in the hypoxia-regulated transcription factor, HIF1a, show retarded development and failure of neural tube closure (Iyer et al., 1998). These suggest that hypoxia plays an important role in proper neural development. SFEB (Serum-free floating culture of embryoid body-like aggregates) culture is known to be an efficient method for the induction of neural tissue from mouse ES cells (Eiraku, Matsuo-Takasaki et al., 2008). To investigate the effect of hypoxia on neural differentiation, we have applied hypoxic condition to SFEB. After 4 days of SFEB culture, expression level of the undifferentiated ES cell markers such as Nanog, Rex-1 and E-cadherin was dramatically decreased in hypoxia (3% O2) compared with normoxia (20% O2). On the other hand, expression of neural ectoderm marker, N-cadherin was detected through the whole ES cell aggregates when cultured under hypoxic conditions but not when cultured under normoxic conditions. Between 8 and 11 days culture, the expression of neural progenitor markers (Pax6 and Nestin) was rapidly decreased in hypoxic condition. Finally, in hypoxic conditions, almost 100% of the cells in ES cell aggregates have differentiated into post-mitotic neuron (TuJ1-positive cells) by day 11. In accordance with this result, expression of mature neuronal markers, such as Map2 and Tbr1 was significantly increased in hypoxia. These results raised the possibility that hypoxia promotes commitment of ES cells into neural cells and accelerates neuronal differentiation. In our preliminary experiment, we have found that HIF1-a is induced within 24 h after starting low oxygen stimulus. We are now under investigating the biological function of Hif1-a in promotion of neural differentiation.

NRSF downregulation induces neuronal differentiation in mouse embryonic stem cells

Differentiation of embryonic stem (ES) cells into neurons is accompanied by global changes in transcriptional programs. One transcription factor that has been shown to be involved in neuronal differentiation is neuron restrictive silencing factor/RE1-silencing transcription factor (NRSF/REST). NRSF is a transcriptional repressor that silences the transcription of a large number of neuronal genes by binding to a 21-bp consensus DNA sequence, the RE1 binding site/neuron-restrictive silencer elements (RE1/NRSE), present in the regulatory regions of neuronal genes. The goal of the current study was to examine the role of NRSF during differentiation of ES cells into neurons. To do this, ShRNA construct was used to downregulate NRSF in undifferentiated ES cells. Our results show that although control ES cells required induction by retinoic acid (RA) to differentiate efficiently into neurons, downregulation of NRSF was sufficient to drive the ES cells down the neuronal lineage even in the absence of RA. This downregulation also led to increased expression of mature neuronal markers, and concomitantly decreased glial fibrillary acidic protein (GFAP) expression. The results suggest that NRSF downregulation increases the population of mature neurons at the expense of GFAP-positive cells.

Adult human olfactory neural progenitors cultured in defined medium

Neurosphere-forming cells (NSFCs) derived from primary cultures of adult human olfactory epithelium were established in minimum essential medium (MEM) with Hanks balanced salts and 10% heat-inactivated fetal bovine serum (FBS). A totally defined medium (DM) was employed to examine their proliferation, lineage restriction and differentiation. DMEM/F12 (DF) was found to support NSFCs and served as the base medium for this study. NSFCs were adapted to the DM through serial serum reductions at successive feedings. NSFCs in DF supplemented with N2, B27 or insulin attained saturation density and formed extensive processes. Immunolocalization of lineage specific markers [i.e., nestin, β-tubulin III, peripherin, neural cell adhesion molecule, A2B5, O4, microtubule-associated-protein-2 (MAP2) and glial fibrillary acidic protein], as well as 3-[4,5-dimtheylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide and ornithine decarboxylase assays were employed to characterize the NSFCs. The effects of trophic factors including epidermal growth factor (EGF), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), neurotrophic factors (NT-3), and basic fibroblast growth factor (bFGF) were evaluated. With the reduction of serum and the addition N2, B27, and other nutrients, there was a change in lineage restriction including an increase the expression of A2B5 and other glial markers as well as the expression of mature neuronal markers with a simultaneous reduction of nestin reactivity. NSFCs proliferated and maintained their pluripotency for over a year in the DM. Further studies will determine the utility of NSFCs for cell replacement therapy.