An in vitro pathway from embryonic stem cells to neurons and glia

Mouse embryonic stem (ES) cells can be induced to differentiate into neurons and glia in vitro. Induction protocols are straightforward and involve culture in the presence of retinoic acid. They result in an efficient conversion of undifferentiated ES cells to neural cells. Mature neurons produced have the key physiological, morphological and molecular properties of primary cultured neurons derived from the central nervous system. Most significantly, they form functional chemical synapses that utilize either glutamate, GABA or glycine as neurotransmitters. ES cell-derived glial cells also correspond well with their normal counterparts. During induction, ES cells undergo a series of developmental steps that resemble key stages in the early mouse embryo. This supports the hypothesis that the in vitro pathway is a valid model of the normal developmental pathway leading to neurons and glia. The in vitro system combines three experimental strengths. It is suitable for genetic manipulation, affords large numbers of cells and allows precise manipulation of the culture environment. It is thus suitable for a wide variety of mechanistic studies in the areas of neural development and cell biology.     

胶质细胞源性神经营养因子体外对脊髓运动神经元的作用

目的 :观察不同浓度的胶质细胞源性神经营养因子 (GDNF) ,对大鼠胚胎脊髓运动神经元生长活性的作用。方法 :取大鼠胚胎脊髓腹侧组织体外分离 ,进行原代细胞培养 ,应用抗神经微丝单克隆抗体 (mAb)SMI32进行运动神经元的免疫细胞化学染色 ,从细胞形态学及应用MTT比色法 ,研究GDNF对大鼠脊髓运动神经元的影响。结果 :GDNF能明显促进体外培养的大鼠脊髓运动神经元存活及突起的生长 (P <0 .0 5 ) ,且具有剂量依赖的趋势。结论 :不同浓度的GDNF对体外培养的大鼠胚胎脊髓运动神经元 ,有不同程度的促生长作用     

Downregulation of VAPB expression in motor neurons derived from induced pluripotent stem cells of ALS8 patients

Amyotrophic lateral sclerosis (ALS) is an incurable neuromuscular disease that leads to a profound loss of life quality and premature death. Around 10% of the cases are inherited and ALS8 is an autosomal dominant form of familial ALS caused by mutations in the vamp-associated protein B/C (VAPB) gene. The VAPB protein is involved in many cellular processes and it likely contributes to the pathogenesis of other forms of ALS besides ALS8. A number of successful drug tests in ALS animal models could not be translated to humans underscoring the need for novel approaches. The induced pluripotent stem cells (iPSC) technology brings new hope, since it can be used to model and investigate diseases in vitro. Here we present an additional tool to study ALS based on ALS8-iPSC. Fibroblasts from ALS8 patients and their non-carrier siblings were successfully reprogrammed to a pluripotent state and differentiated into motor neurons. We show for the first time that VAPB protein levels are reduced in ALS8-derived motor neurons but, in contrast to over-expression systems, cytoplasmic aggregates could not be identified. Our results suggest that optimal levels of VAPB may play a central role in the pathogenesis of ALS8, in agreement with the observed reduction of VAPB in sporadic ALS.     

Stage-dependent Olig2 expression in motor neurons and oligodendrocytes differentiated from embryonic stem cells

Although embryonic stem (ES) cells are capable of forming any cell type in the body, the mechanisms that control cell type-specific differentiation are largely unknown. In the present study, we examined the process of differentiation to motor neurons and oligodendrocytes from mouse (Olig2GFP) ES cells. Mouse ES cells undergo a sequential process of differentiation over a 3-week period to generate motor neurons and oligodendrocytes. At day 7 of differentiation, Olig2-expressing cells are biased to a neuronal lineage. However, further differentiation (day 32) resulted in the majority of Olig2-expressing cells exhibiting an oligodendrocyte phenotype as well as a reduced ability to make motor neurons. Exposure of human ES cells to Sonic hedgehog (Shh) likewise resulted in enhanced motor neuron differentiation. Our results establish the requirements for directing ES cells to become motor neurons and oligodendrocytes and show that ES cell-derived Olig2 + cells can give rise to both motor neurons and oligodendrocytes, depending on the time at which differentiation is initiated.     

AIF translocates to the nucleus in the spinal motor neurons in a mouse model of ALS

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by selective loss of motor neurons in the brain stem and the spinal cords. One of the causes for the familial ALS has been attributed to the mutations in copper-zinc superoxide dismutase (SOD1). Although the toxic function of the mutant enzyme has not been fully understood, the final cell death pathway has been suggested as caspase-dependent. In the present study, we present evidence that the activation of apoptosis inducing factor (AIF) may play a role to induce motor neuron death during ALS pathogenesis. In the spinal cord of SOD1 G93A transgenic mice, expression of AIF was detected in the motor neurons and astrocytes. The level of AIF expression increased as the disease progressed. In the symptomatic SOD1 G93A transgenic mice, AIF released from the mitochondria and translocated into the nucleus in the motor neurons as evidenced by confocal microscopy and biochemical analysis. These results suggest that AIF may play a role to induce motor neuron death in a mouse model of ALS.     

Generation of motor neurons by coculture of retinoic acid-pretreated embryonic stem cells with chicken notochords

Understanding neuroectoderm formation and its subsequent diversification to functional neural subtypes remains elusive. We have shown here for the first time that embryonic stem cells (ESCs) can differentiate into neurons and motor neurons (MNs) by using a coculture embryonic notochord model in vitro. Mouse ESCs were induced to form neural precursors via timed exposure to retinoic acid (RA) using the 4-/4+ RA protocol. These cells were then cocultured with alginate bead-encapsulated notochords isolated from Hamburger and Hamilton stage 6-10 chick embryos. The use of notochord alone was not able to induce neural differentiation from ESCs, and, therefore, notochord does not possess neural inducing activity. Hence, the most successful neuronal cells and MN differentiation was only observed following the coculture of RA-pretreated ESCs with notochord. This resulted in a significantly greater number of cells expressing microtubule-associated protein-2 (MAP2), HB9, choline acetyltransferase (ChAT) and MN-specific genes. While further characterization of these differentiated cells will be essential before transplantation studies commence, these data illustrate the effectiveness of embryonic notochord coculture in providing valuable molecular cues for directed differentiation of ESCs toward an MN lineage.     

大鼠脊髓源神经干细胞与运动神经元的差异蛋白质组学

目的 探讨大鼠脊髓源神经干细胞与运动神经元的差异蛋白质组学,寻找出重要的差异蛋白质.方法 采用双向电泳分离两种细胞的蛋白质,用DeCyder软件分析蛋白表达差异,并采用质谱(HPLC-ESI-MS/MS)进行鉴定.结果 脊髓源神经干细胞与运动神经元蛋白质凝胶分析获得1 300余个清晰的蛋白质斑点,在比国产分析的87个差异点中,初步鉴定出44个差异表达蛋白,其中24个在神经干细胞高表达,20个在运动神经元高表达.结论 脊髓源神经干细胞与运动神经元有各自特定的蛋白质表达,某些差异蛋白可能在神经干细胞向运动神经元分化中发挥关键作用.     

Cripto as a target for improving embryonic stem cell-based therapy in Parkinson's disease

Embryonic stem (ES) cells have been suggested as candidate therapeutic tools for cell replacement therapy in neurodegenerative disorders. However, limitations for the use of these cells lie in our restricted knowledge of the molecular mechanisms involved in their specialized differentiation and in the risk of tumor formation. Recent findings suggest that the EGF-CFC protein Cripto is a key player in the signaling pathways controlling neural induction in ES cells. Here we show that in vitro differentiation of Cripto(-/-) ES cells results in increased dopaminergic differentiation and that, upon transplantation into Parkinsonian rats, they result in behavioral and anatomical recovery with no tumor formation. The use of knockout ES cells that can generate dopamine cells while eliminating tumor risk holds enormous potential for cell replacement therapy in Parkinson's disease.     

High-purity lineage selection of embryonic stem cell-derived neurons

The derivation of somatic cell types from pluripotent and self-renewing embryonic stem (ES) cells offers attractive prospects for basic research, compound development, and regenerative medicine. A key prerequisite for biomedical applications of ES cells is the ability to differentiate and isolate defined somatic cell populations at high purity. In this study, we explore the potential of the Talpha1- enhanced green fluorescent protein (EGFP) transgene and polysialic acid (PSA)-neural cell adhesion molecule (NCAM) as lineage selection markers for the derivation of ES cell-derived neurons. Upon controlled in vitro differentiation, ES cells engineered to express EGFP under control of the Talpha1-tubulin promoter exhibited exclusive transgene expression in neurons. Similarly, PSA-NCAM expression during the early stages of ES cell differentiation was restricted to neuronal progeny. Talpha1- EGFP- and PSA-NCAM-positive neurons comprised both inhibitory and excitatory phenotypes. Compared to Talpha1-EGFP, the expression of PSA-NCAM was initiated at slightly earlier stages of neural differentiation. FACSorting of Talpha1-EGFP-positive cells and immunopanning of PSA-NCAMexpressing cells yielded neuronal populations at purities up to 99.6% and 96.9%, respectively. These findings depict Talpha1-EGFP and PSA-NCAM as suitable markers for high-purity selection of early ES cell-derived neurons.     

胚胎大鼠脊髓运动神经元的体外培养及鉴定

本实验建立了体外胚胎大鼠脊髓运动神经元的分离培养,并进行鉴定。取孕15d SD大鼠胚胎的脊髓腹侧组织,用胰蛋白酶和胶原酶消化分离成单细胞悬液,进行原代细胞培养,应用神经元特异性的烯醇化酶(NSE)抗体和抗神经微丝抗体-SMI32单克隆抗体对培养细胞进行鉴定,同时行尼氏染色。结果分离培养的细胞鉴定为脊髓运动神经元(SMN),SMN在体外适宜的条件下可存活4周左右,可作为神经科学研究的一种手段。     

Directed differentiation of ventral spinal progenitors and motor neurons from human embryonic stem cells by small molecules

Specification of distinct cell types from human embryonic stem cells (hESCs) is key to the potential application of these na?ve pluripotent cells in regenerative medicine. Determination of the nontarget differentiated populations, which is lacking in the field, is also crucial. Here, we show an efficient differentiation of motor neurons ( approximately 50%) by a simple sequential application of retinoid acid and sonic hedgehog (SHH) in a chemically defined suspension culture. We also discovered that purmorphamine, a small molecule that activates the SHH pathway, could replace SHH for the generation of motor neurons. Immunocytochemical characterization indicated that cells differentiated from hESCs were nearly completely restricted to the ventral spinal progenitor fate (NKX2.2+, Irx3+, and Pax7-), with the exception of motor neurons (HB9+) and their progenitors (Olig2+). Thus, the directed neural differentiation system with small molecules, even without further purification, will facilitate basic and translational studies using human motoneurons at a minimal cost.     

Differentiation of postnatal neural stem cells into glia and functional neurons on laminin-coated polymeric substrates

A series of polymeric biomaterials, including poly(methyl acrylate), chitosan, poly(ethyl acrylate) (PEA), poly(hydroxyethyl acrylate) (PHEA), and a series of random copolymers containing ethyl acrylate, hydroxyethyl acrylate, and methyl acrylate were tested in vitro as culture substrates and compared for their effect on the differentiation of neural stem cells (NSCs) obtained from the subventricular zone of postnatal rats. Immunocytochemical assay for specific markers and scanning electron microscopy techniques were employed to determine the adhesion of the cultured NSCs to the different biomaterials and the respective neuronal differentiation. The functional properties and the membrane excitability of differentiated NSCs were investigated using a patch-clamp. The results show that the substrate's surface chemistry influences cell attachment and neuronal differentiation, probably through its influence on adsorbed laminin, and that copolymers based on PEA and PHEA in a narrow composition window are suitable substrates to promote cell attachment and differentiation of adult NSCs into functional neurons and glia.     

The abnormal processing of TDP-43 is not an upstream event of reduced ADAR2 activity in ALS motor neurons

TDP-43 pathology in motor neurons is a hallmark of ALS. In addition, the reduced expression of an RNA editing enzyme, adenosine deaminase acting on RNA 2 (ADAR2), increases the expression of GluA2 with an unedited Q/R site in the motor neurons of patients with sporadic ALS. As the occurrence of these two disease-specific abnormalities in the same motor neurons suggests a molecular link between them, we examined the effects of altered TDP-43 processing on ADAR2 activity in TetHeLaG2m and Neuro2a cells. We found that ADAR2 activity did not consistently change due to the overexpression or knockdown of TDP-43 or the expression of abnormal TDP-43, including caspase-3-cleaved fragments, truncated TDP-43 lacking either nuclear localization or export signals and ALS-linked TDP-43 mutants. These results suggest that the abnormal processing of TDP-43 is not an upstream event of inefficient GluA2 Q/R site editing in the motor neurons of sporadic ALS patients.     

Persistent dopamine functions of neurons derived from embryonic stem cells in a rodent model of Parkinson disease

The derivation of dopamine neurons is one of the best examples of the clinical potential of embryonic stem (ES) cells, but the long-term function of the grafted neurons has not been established. Here, we show that, after transplantation into an animal model, neurons derived from mouse ES cells survived for over 32 weeks, maintained midbrain markers, and had sustained behavioral effects. Microdialysis in grafted animals showed that dopamine (DA) release was induced by depolarization and pharmacological stimulants. Positron emission tomography measured the expression of presynaptic dopamine transporters in the graft and also showed that the number of postsynaptic DA D(2) receptors was normalized in the host striatum. These data suggest that ES cell-derived neurons show DA release and reuptake and stimulate appropriate postsynaptic responses for long periods after implantation. This work supports continued interest in ES cells as a source of functional DA neurons.     

Force measurements of human embryonic stem cell-derived cardiomyocytes in an in vitro transplantation model

Human embryonic stem cell (hESC)-derived cardiomyocytes have been suggested for cardiac cell replacement therapy. However, there are no data on loaded contractions developed by these cells and the regulation thereof. We developed a novel in vitro transplantation model in which beating cardiomyocytes derived from hESCs (line H1) were isolated and transplanted onto noncontractile, ischemically damaged ventricular slices of murine hearts. After 2-3 days, transplanted cells started to integrate mechanically into the existing matrix, resulting in spontaneous movements of the whole preparation. Preparations showed a length-dependent increase of active tension. In transplanted early beating hESC-derived cardiomyocytes, frequency modulation by field stimulation was limited to a small range around their spontaneous beating rate. Our data demonstrate that this novel in vitro transplantation model is well suited to assess the mechanical properties and functional integration of cells suggested for cardiac replacement strategies.     

The effect of multivalent Sonic hedgehog on differentiation of human embryonic stem cells into dopaminergic and GABAergic neurons

Stem cell differentiation is regulated by complex repertoires of signaling ligands which often use multivalent interactions, where multiple ligands tethered to one entity interact with multiple cellular receptors to yield oligomeric complexes. One such ligand is Sonic hedgehog (Shh), whose posttranslational lipid modifications and assembly into multimers enhance its biological potency, potentially through receptor clustering. Investigations of Shh typically utilize recombinant, monomeric protein, and thus the impact of multivalency on ligand potency is unexplored. Among its many activities, Shh is required for ventralization of the midbrain and forebrain and is therefore critical for the development of midbrain dopaminergic (mDA) and forebrain gamma-aminobutyric acid (GABA) inhibitory neurons. We have designed multivalent biomaterials presenting Shh in defined spatial arrangements and investigated the role of Shh valency in ventral specification of human embryonic stem cells (hESCs) into these therapeutically relevant cell types. Multivalent Shh conjugates with optimal valencies, compared to the monomeric Shh, increased the percentages of neurons belonging to mDA or forebrain GABAergic fates from 33% to 60% or 52% to 86%, respectively. Thus, multivalent Shh bioconjugates can enhance neuronal lineage commitment of pluripotent stem cells and thereby facilitate efficient derivation of neurons that could be used to treat Parkinson's and epilepsy patients.     

Astrocyte-derived factors instruct differentiation of embryonic stem cells into neurons

Pluripotent embryonic stem (ES) cells may differentiate into neurons in vitro. This is valuable in the study of neurogenesis and in the generation of donor cells for neuronal transplantation. Here we show that astrocyte-derived factors instruct mouse and primate ES cells to differentiate into neurons. Cultured in astrocyte-conditioned medium (ACM) under free-floating conditions, within 4 days, colonies of undifferentiated mouse ES cells give rise to floating spheres of concentric stratiform structure with a periphery of neural stem cells, which are termed Neural Stem Spheres. Culturing the spheres on an adhesive substrate in ACM promotes neurogenesis, and cells in the spheres differentiate into neurons within 5 days, including dopaminergic neurons. In contrast, neither astrocytes nor oligodendrocytes are formed. The procedure developed for mouse ES cells can be applied to monkey ES cells. This neurogenesis pathway provides a new insight into mechanisms of specification of cell fates in early development and also provides a simple procedure for fast and efficient generation of a vast number of neural stem cells and neurons.