Molecular manipulation targeting regulation of dopaminergic differentiation and proliferation of neural stem cells or pluripotent stem cells

Parkinson's disease (PD) is a severe deliberating neurological disease caused by progressive degenerative death of dopaminergic neurons in the substantia nigra of midbrain. While cell replacement strategy by transplantation of neural stem cells and inducement of dopaminergic neurons is recommended for the treatment of PD, understanding the differentiation mechanism and controlled proliferation of grafted stem cells remain major concerns in their clinical application. Here we review recent studies on molecular signaling pathways in regulation of dopaminergic differentiation and proliferation of stem cells, particularly Wnt/beta-catenin signaling in stimulating formation of the dopaminergic phenotype, Notch signaling in inhibiting stem cell differentiation, and Sonic hedgehog functioning in neural stem cell proliferation and neuronal cell production. Activation of oncogenes involved in uncontrolled proliferation or tumorigenicity of stem cells is also discussed. It is proposed that a selective molecular manipulation targeting strategy will greatly benefit cell replacement therapy for PD by effectively promoting dopaminergic neuronal cell generation and reducing risk of tumorigenicity of in vivo stem cell applications.     

Dopamine neurons from embryonic stem cells.

Embryonic stem (ES) cells are viewed as a potential cell source for transplantation therapy in Parkinson's disease, with major advantages with respect to fetal cells regarding availability and standardization. Both in terms of proliferation and differentiation potential ES cells appear more promising than adult neural stem cells for replacement therapies. However, other cell-based therapies may take advantage of the special characteristics of neural stem cells. For dopamine neuronal replacement therapy, the first requisite is to generate neurons that have a correct and stable midbrain dopamine phenotype. Inductive protocols that mimick sequential developmental events have been recently developed for human ES cells. Genetic engineering techniques can also be used to induce or enhance phenotypic specification. Finally, nuclear transfer technology could provide insights into disease and individual specific mechanisms, helping to develop and test therapies rather than for direct therapeutic application.

Neurología 2004;19(Supl 2):73-76

     

Generation of dopaminergic neurons from embryonic stem cells

Neuronal transplantation is considered to be a promising therapeutic approach to neurodegenerative diseases. In addition to fetal tissues and neural stem cells, embryonic stem cells are good candidates for the creation of neurons. We have recently identified a stromal cell-derived inducing activity that promotes neural differentiation of mouse embryoric stem cells. This activity accumulated on the surface of PA6 stromal cells and induced efficient neuronal differentiation of co-cultured embryonic stem cells under serum-free conditions without the use of either retinoic acid or embryoid bodies. A high proportion of tyrosine hydroxylase-positive neurons producing dopamine are obtained. Induction of neurons with stromal cell-derived inducing activity may be a useful new method for basic neuroscience research and therapeutic applications, including cell transplantation therapy for Parkinson's disease.     

Adult human bone marrow stromal spheres express neuronal traits in vitro and in a rat model of Parkinson's disease

Adult human bone marrow stromal cells (hMSCs) grown in suspension culture gave rise to spheres of neural progenitor (NP) cells, capable of expressing both dopaminergic (DA) and GABAergic (GABA) traits. After transplantation into the Parkinsonian rat, human NPs and neurons were present at 2 weeks. Although no DA neurons appeared to survive transplantation, there were abundant GABA neurons present in the graft. By 4 weeks, however, all cells had died. Finding ways to prolong survival and promote the appropriate neurotransmitter phenotype is essential if hMSCs are to be clinically useful.     

BDNF and extracellular matrix regulate differentiation of mice neurosphere‐derived cells into a GABAergic neuronal phenotype

Differentiation of neurosphere‐derived cells is regulated by extracellular cues, namely, growth factors and proteins of the extracellular matrix (ECM). In this study we analyzed the influence of nerve growth factor (NGF), brain‐derived neurotrophic factor (BDNF), retinoic acid plus potassium chloride (RA‐KCl), and the nonsynthetic ECMs laminin (LN) and fibronectin (FN) versus the synthetic adhesion substrate poly‐L ‐lysine (PLL) in the in vitro differentiation of postnatal neurosphere cells. BDNF increased the number of differentiated neurons and decreased the number of neuronal precursors (nestin‐positive cells) compared with NGF or RA‐KCl. Moreover, cells treated with BDNF plus B27 supplement acquired a γ‐aminobutyric acid (GABA)–ergic phenotype and showed increased survival. No significant differences were found in the number of differentiated neurons in the presence of the ECMs alone. Nevertheless, FN or PLL in combination with BDNF promoted the acquisition of a GABAergic phenotype. The results obtained in this study highlight the importance of growth factors and ECM proteins for the potential of neurosphere cells to differentiate into neurons. © 2009 Wiley‐Liss, Inc.     

Depolarization promotes GAD 65-mediated GABA synthesis by a post-translational mechanism in neural stem cell-derived neurons

Neuronal activity regulates neurogenesis and neuronal differentiation in the mammalian brain. The commencement of neurotransmitter expression establishes the neuronal phenotype and enables the formation of functional connectivity between neurons. In addition, release of neurotransmitters from differentiating neurons may modulate the behaviour of neural precursors. Here, we show that neuronal activity regulates γ-aminobutyric acid (GABA) expression in neurons generated from stem cells of the striatum and adult subventricular zone (SVZ). Differentiating neurons display spontaneous Ca²⁺ events, which are voltage-gated calcium channel (VGCC) dependent. Depolarization increases both the frequency of Ca²⁺ transients and the amount of Ca²⁺ influx in differentiating neurons. We show that depolarization-dependent GABA expression is regulated by the amplitude and not by the frequency of Ca²⁺ influx. Brief activation of VGCCs leads to Ca²⁺ influx that in turn promotes a rapid expression of GABA. Depolarization-dependent GABA expression does not require changes in gene expression. Instead, it involves cAMP-dependent protein kinase (PKA) and Ca²⁺ and phospholipid-dependent protein kinase (PKC) signalling. Activity increases the number of glutamic acid decarboxylase (GAD) 65-immunoreactive neurons in a PKA-dependent manner, without altering the expression of GAD 65, suggesting that depolarization promotes recruitment of GAD 65 by a post-translational mechanism. In line with this, depolarization does not permanently increase the expression of GABA in neurons derived from neural stem cells of the embryonic striatum, cortex and adult SVZ. Thus, neuronal activity does not merely accelerate neuronal differentiation but it may alter the mechanism of GABA synthesis in newly generated neurons.     

Receptor for advanced glycation end products (RAGE) mediates neuronal differentiation and neurite outgrowth

The receptor for advanced glycation end products (RAGE) plays a crucial role in several disease processes, such as diabetes, inflammation, and neurodegeneration. In this article we report multiple roles of RAGE in neuronal differentiation and neurite outgrowth. In retinoic-induced P19 embryonic carcinoma stem cells, silencing the expression of RAGE by RNA interference (RNAi) blocked differentiation of the P19 cells into neuronal cells and enhanced the formation of vimentin-positive fibroblast-like cells. RAGE knockdown inhibited retinoic acid-induced activation and blocked nuclear translocation of NF-kappaB, suggesting RAGE regulates activation of NF-kappaB. RAGE was also shown to be involved in survival of P19 cells during retinoic acid differentiation. Additionally, knockdown of RAGE strongly inhibited neurite outgrowth in retinoic acid-differentiated P19 cells, indicating that RAGE is required for neurite outgrowth of differentiated P19 cells. Retinoic acid-treated P19 cells activated GTPases, Rac1, and Cdc42. This activation of the GTPases was inhibited in RAGE-knockdown cells. In primary cerebellar granule neurons, the knockdown of RAGE also inhibited neurite outgrowth. In these cells, overexpression of dominant-negative forms of Rac1 and Cdc42 inhibited neurite outgrowth, whereas overexpression of constitutively active forms of Rac1 and Cdc42 in RAGE-deficient neurons restored neurite outgrowth, indicating that RAGE mediated neurite outgrowth through the Rac1/Cdc42 pathway. This is the first report on the role of RAGE in cell lines and primary neurons, as determined by RNAi knockdown.     

Stage-specific and opposing roles of BDNF, NT-3 and bFGF in differentiation of purified callosal projection neurons toward cellular repair of complex circuitry

Cellular repair of neuronal circuitry affected by neurodegenerative disease or injury may be approached in the adult neocortex via transplantation of neural precursors ("neural stem cells") or via molecular manipulation and recruitment of new neurons from endogenous precursors in situ. A major challenge for potential future approaches to neuronal replacement will be to specifically direct and control progressive differentiation, axonal projection and connectivity of neural precursors along a specific neuronal lineage. This goal will require a progressively more detailed understanding of the molecular controls over morphologic differentiation of specific neuronal lineages, including neurite outgrowth and elongation, in order to accurately permit and direct proper neuronal integration and connectivity. Here, we investigate controls over the morphologic differentiation of a specific prototypical lineage of cortical neurons: callosal projection neurons (CPN). We highly enriched CPN to an essentially pure population, and cultured them at three distinct stages of development from embryonic and postnatal mouse cortex by retrograde fluorescence labelling, followed by fluorescence-activated cell sorting. We find that specific peptide growth factors exert direct stage-specific positive and negative effects over the morphologic differentiation and process outgrowth of CPN. These effects are distinct from the effects of these growth factors on CPN survival [Catapano et al. (2001)J. Neurosci., 21, 8863-8872]. These data may be critical for the future goal of directing lineage-specific neuronal differentiation of transplanted or endogenous precursors/"stem cells" toward cellular repair of complex cortical circuitry.     

The Specification of Neuronal Fate: A Common Precursor for Neurotransmitter Subtypes in the Rat Cerebral Cortex In Vitro

Neurotransmitter choice is a crucial step in neural development. In the cerebral cortex, pyramidal neurons use the excitatory neurotransmitter glutamate, whereas non-pyramidal cells use the inhibitory neurotransmitter GABA. We are interested in how these two neuronal types are generated. We labelled precursor cells from embryonic rat cerebral cortex with a retroviral vector in dissociated cell cultures, and examined the neurotransmitter phenotype of their progeny immunohistochemically after 2 weeks in vitro. We discovered, first, that precursor cells in culture generate glutamatergic and GABAergic neurons in proportions similar to those in vivo. Second, we found that neuronal precursor cells gave rise to both GABAergic and glutamatergic neurons. These results suggest that neuronal precursor cells in the cerebral cortex have the potential to generate both neuronal subtypes. Moreover, these data are consistent with a stochastic model of neurotransmitter specification.     

Role of gamma-aminobutyric acid in early neuronal development: studies with an embryonic neuroectodermal stem cell clone

gamma-Aminobutyric acid (GABA) has been known to function as an autocrine/paracrine signal molecule in addition to its well-known inhibitory neurotransmitter function. Studies on the developing brain and on primary brain cell cultures provided evidence for a variety of GABA functions in periods preceding the formation of synapses. The exact role of GABA in the early neural development, however, is still not well understood. In this study, one-cell-derived NE-4C neuroectodermal stem cells were induced to form neurons and astrocytes in vitro, and the role of GABA was investigated in defined phases of neurogenesis. Noninduced NE-4C cells contained GABA, expressed GABA(A)R alpha subunits, and carried functional GABA(A) ion channels. A moderate cytoplasmic GABA content was detected during the entire period of differentiation. By the time of the formation of differentiated neurons, neuron-like cells with both high and low GABA content were clearly distinguishable. HPLC analysis indicated that NE-4C cells released GABA into their fluid environment during all stages of neuronal development. By using the patch-clamp technique, GABA-evoked currents were recorded during the entire proliferation/differentiation period, whereas a GABA-evoked increase in intracellular Ca(2+) was detected only during the maturation of postmitotic neuronal precursors. Bicuculline blocked both the ion currents and the [Ca(2+)](i) increase in response to GABA. Neuron formation was facilitated by GABA through GABA(A) ion channels during postmitotic differentiation, but not earlier during the phases of cell fate commitment. Although the data clearly demonstrate an early responsiveness to GABA, understanding the significance of GABA influence in early neural cell fate decisions will require further investigation.     

New neurons in the vestibular nuclei complex after unilateral vestibular neurectomy in the adult cat

Recent findings revealed a reactive neurogenesis after lesions and in several models of disease. After unilateral vestibular neurectomy (UVN), we previously reported gamma-aminobutyric acid (GABA)ergic neurons are upregulated in the vestibular nuclei (VN) in the adult cat. Here, we ask whether this upregulation of GABAergic neurons resulted from a reactive neurogenesis. To determine the time course of cell proliferation in response to UVN, 5-bromo-2'-deoxyuridine (BrdU) was injected 3 h, 1, 3, 7, 15 and 30 days after UVN. We investigated the survival and differentiation in UVN cats injected with BrdU at 3 days and perfused 30 days after UVN. Results show a high number of BrdU-immunoreactive nuclei in the deafferented VN with a peak at 3 days after UVN and a decrease at 30 days. Most of the newly generated cells survived up to 1 month after UVN and gave rise to a variety of cell types. Confocal analysis revealed three cell lineages: microglial cells (OX 42/BrdU-immunoreactive cells); astrocytes [glial fibrillary acidic protein (GFAP)/BrdU-immunoreactive cells]; and neurons (NeuN/BrdU-immunoreactive cells). That UVN induced new neurons was confirmed by an additional marker (nestin) expressed by neural precursor cells. We show that most of the newly generated neurons have a GABAergic phenotype [glutamate decarboxylase (GAD)-67/BrdU-immunoreactive cells]. Morphological analysis showed two subtypes of GABAergic neurons: medium and small (30 vs. 10 microm, respectively). This is the first report of reactive neurogenesis in the deafferented VN in the adult mammalian CNS.     

Interplay of leukemia inhibitory factor and retinoic acid on neural differentiation of mouse embryonic stem cells

Embryonic stem (ES) cells have great potential for cell replacement in neurodegenerative disorders. Implantation of these cells into the brain, however, requires their prior differentiation. We examined the interplay between leukemia inhibitory factor (LIF) and retinoic acid (RA) on neural differentiation of mouse ES (mES) cells. Mouse embryonic stem cells were allowed to form cell aggregates, the so-called embryoid bodies (EBs), in the absence or presence of LIF. In the absence of LIF, mES cells downregulated the expression of the undifferentiated mES cell marker Oct-3/4, and increased mRNA levels of two neural precursor markers, Sox-1 and Nestin, as well as the neuronal marker beta-tubulin III. This neuronal differentiation was enhanced by treating EBs with RA. Moreover, RA irreversibly increased the number of postmitotic neurons in culture, as shown by the reduction of proliferating mES cells and the increase in beta-tubulin III-positive cells 6 days after RA removal, which in turn affected mES cell viability. The addition of LIF during EBs formation, however, blocked completely this neuronal differentiation. Our findings also showed that pre-differentiation of mES cells in vitro avoided the teratocarcinoma formation observed when proliferating mES cells were grafted into the brain. In addition, mES cells pre-differentiated with RA in culture showed a reduction in proliferation and the presence of neural phenotypes after grafting. In conclusion, the present results indicate that RA enhances neuronal differentiation of mES cells in the absence of LIF, although it compromises cell viability and transplantation.     

神经干细胞移植治疗颞叶癫痫的近期效果研究

目的探讨神经干细胞移植治疗颞叶癫痫的疗效。方法首先建立大鼠的海人酸颞叶癫痫模型。然后从胚胎大鼠的海马中分离培养出神经干细胞,将预诱导的、向氨基丁酸能神经元方向分化的胚胎神经干细胞移植入颞叶癫痫模型的海马内,在移植后1w、2w、3w和4w分别进行移植癫痫大鼠的癫痫最低阂值的测定、海马中氨基丁酸的测定和移植的神经干细胞在体内分化情况的鉴定。结果移植后的癫痫最低阈值和海马中的氨基丁酸逐渐增加,接近但未达到正常值,预诱导的神经干细胞全部分化为氨基丁酸能神经元。结论神经干细胞移植治疗颞叶癫痫具有一定效果。     

Differentiation of engrafted neuronal-restricted precursor cells is inhibited in the traumatically injured spinal cord

Differentiation of pluripotent neural stem cells engrafted into the adult normal and injured spinal cord is restricted to the glial lineage, suggesting that in vitro induction toward a neuronal lineage prior to transplantation and/or modification of the host environment may be necessary to initiate and increase the differentiation of neurons. In the present study, we investigated the differentiation of neuronal-restricted precursors (NRPs) grafted into the normal and contused adult rat spinal cord. NRPs proliferated through multiple passages in the presence of FGF2 and NT3 and differentiated into only neurons in vitro in the presence of retinoic acid and the absence of FGF2. Differentiated NRPs expressed GABA, glycine, glutamate, and ChAT. Two weeks to 2 months after engraftment of undifferentiated NRPs into adult normal spinal cord, large numbers of surviving cells were seen in all of the animals. The majority differentiated into betaIII-tubulin-positive neurons. Some transplanted NRPs expressed GABA and small numbers were glutamate- and ChAT-positive. NRPs were also transplanted into the epicenter of the contused adult rat spinal cord. Two weeks to 2 months after transplantation, some engrafted NRPs remained undifferentiated nestin-positive cells. Small numbers were MAP2- or betaIII-tubulin-positive neurons. However, the expression of GABA, glutamate, or ChAT was not observed. These results show that NRPs can differentiate into different types of neurons in the normal adult rat spinal cord, but that such differentiation is inhibited in the injured spinal cord. Manipulation of the microenvironment in the injured spinal cord will likely be necessary to facilitate neuronal replacement.     

维甲酸诱导胚胎大鼠脑海马神经干细胞向神经元的分化

背景：目前认为直接进行神经干细胞移植后细胞虽能存活,但是只分化成神经胶质细胞,并不能分化成有功能的神经元。 目的：探讨维甲酸诱导对胚胎大鼠脑海马神经干细胞向神经元分化的作用。 设计、时间及地点：细胞学体外实验,于2008-09/2009-02在辽宁医学院科技实验楼完成。 材料：胚龄13.5 d的SD大鼠由辽宁医学院实验动物中心提供。 方法：分离胎鼠脑海马组织,胰蛋白酶消化法体外培养获得神经干细胞。将原代和传代细胞以1×107 L-1接种到培养孔中,分别进行常规贴壁分化培养和维甲酸诱导分化培养。 主要观察指标：神经干细胞的鉴定,光镜及免疫组化检测神经干细胞诱导分化结果。 结果：免疫组织化学检测结果显示,原代和传代后得到的神经干细胞团均呈巢蛋白阳性。常规贴壁分化培养7 d后,神经元多呈椭圆型和近似三角形,胞体大,细胞边缘清楚,胞体上有多个突起;而维甲酸诱导分化培养后,神经元数量增加,形态清楚,但细胞胞体上突起较少。与常规贴壁分化培养比较,维甲酸诱导分化培养后神经干细胞向神经元分化率明显升高(P < 0.01),神经干细胞向神经胶质细胞分化率明显降低(P < 0.01)。 结论：从大鼠胚胎脑海马组织中分离得到可自我复制和多向分化的神经干细胞,维甲酸体外诱导后可以增加其向神经元方向分化的比例。     

脂肪源性γ-氨基丁酸能神经元移植治疗帕金森病大鼠的疗效观察

目的 研究脂肪来源的间充质干细胞诱导为γ-氨基丁酸(GABA)能神经元的方法 ,探讨GABA能神经元移植治疗帕金森病模型大鼠的疗效. 方法 取大鼠脂肪组织.利用本单位自行配制的神经干细胞培养基诱导分化为神经干细胞,利用GABA能神经元定向诱导培养基对神经干细胞进行二次定向诱导,并对其进行特异性鉴定.将诱导成功的神经干细胞、GABA能神经元分别移植入帕金森病大鼠模型的丘脑底核,在移植后2周、4周、8周观察大鼠行为学变化情况.结果 体外扩增的脂肪间充质干细胞经过神经干细胞培养基培养后,细胞定向诱导并表达巢蛋白、神经元烯醇化酶(NSE)等神经干细胞标志.经GABA能神经元定向分化培养基二次诱导后,免疫荧光鉴定细胞GAD65阳性.立体定向移植细胞4周后,神经干细胞组与GABA能神经元组的大鼠行为学均得到改善,且GABA能神经元组的疗效更加显著. 结论 脂防来源的间充质干细胞可诱导分化为GABA能神经元,将其移植人大鼠的丘脑底核可以明显改善帕金森病大鼠的运动功能.     

Neurogenic neuroepithelial and radial glial cells generated from six human embryonic stem cell lines in serum-free suspension and adherent cultures

The great potential of human embryonic stem (hES) cells offers the opportunity both for studying basic developmental processes in vitro as well as for drug screening, modeling diseases, or future cell therapy. Defining protocols for the generation of human neural progenies represents a most important prerequisite. Here, we have used six hES cell lines to evaluate defined conditions for neural differentiation in suspension and adherent culture systems. Our protocol does not require fetal serum, feeder cells, or retinoic acid at any step, to induce neural fate decisions in hES cells. We monitored neurogenesis in differentiating cultures using morphological (including on-line follow up), immunocytochemical, and RT-PCR assays. For each hES cell line, in suspension or adherent culture, the same longitudinal progression of neural differentiation occurs. We showed the dynamic transitions from hES cells to neuroepithelial (NE) cells, to radial glial (RG) cells, and to neurons. Thus, 7 days after neural induction the majority of cells were NE, expressing nestin, Sox1, and Pax6. During neural proliferation and differentiation, NE cells transformed in RG cells, which acquired vimentin, BLBP, GLAST, and GFAP, proliferated and formed radial scaffolds. gamma-Aminobutyric acid (GABA)-positive and glutamate positive neurons, few oligodendrocyte progenitors and astrocytes were formed in our conditions and timing. Our system successfully generates human RG cells and could be an effective source for neuronal replacement, since RG cells predominantly generate neurons and provide them with support and guidance.     

Transplantation of embryonic stem cells overexpressing Bcl-2 promotes functional recovery after transient cerebral ischemia

The study tested the hypothesis that transplantation of embryonic stem (ES) cells into rat cortex after a severe focal ischemia would promote structural repair and functional recovery. Overexpression of the human anti-apoptotic gene bcl-2 in ES cells was tested for increasing survival and differentiation of transplanted cells and promoting functional benefits. Mouse ES cells, pretreated with retinoic acid to induce differentiation down neural lineages, were transplanted into the post-infarct brain cavity of adult rats 7 days after 2-h occlusion of the middle cerebral artery (MCA). Over 1-8 weeks after transplantation, the lesion cavity filled with ES cell-derived cells that expressed markers for neurons, astrocytes, oligodendrocytes, and endothelial cells. ES cell-derived neurons exhibited dendrite outgrowth and formed a neuropil. ES cell-transplanted animals exhibited enhanced functional recovery on neurological and behavioral tests, compared to control animals injected with adult mouse cortical cells or vehicle. Furthermore, transplantation with ES cells overexpressing Bcl-2 further increased the survival of transplanted ES cells, neuronal differentiation, and functional outcome. This study supports that ES cell transplantation and gene modification may have values for enhancing recovery after stroke.     

神经干细胞和GABA能神经元治疗大鼠颞叶癫痫实验研究

目的探讨并比较神经干细胞(NSCs)和γ-氨基丁酸(GABA)能神经元移植治疗大鼠颞叶癫痫的疗效。方法取孕12 d SD大鼠胎鼠脑组织,分离培养NSCs并鉴定,取第3代NSCs定向诱导分化为GABA能神经元。48只SD大鼠随机分为4组,空白对照组、未移植组、NSCs移植组和GABA能神经元移植组,移植细胞用5-溴脱氧尿苷(BrdU)标记,在模型建立后的第4 d将上述两种细胞移植到癫痫大鼠右侧海马。分别在细胞移植后的4 w、8 w、12 w处死大鼠留取脑标本。常规HE染色和Nissl染色观察大鼠右侧海马的损伤与治疗情况并进行评价。结果 NSCs移植组和GABA能神经元移植组均于移植后第8 w时海马CA3(CA3)区神经元计数最多,组内比较时,与另外两个时间点之间的差异具有统计学意义(P<0.05)。进而取第8 w时间点进行组间比较,结果各组海马区神经元计数之间的差异均具有统计学意义(P<0.05)。结论两治疗组在移植后第8 w时海马区神经元计数最多,且NSC移植组的疗效优于GABA能神经元移植组。