干细胞治疗感音神经性耳聋

治疗内耳疾病的主要困难之一是找到耳蜗毛细胞或者螺旋神经元丢失所导致的听力损失的治疗方法。本文讨论使用干细胞替代感觉细胞丢失为目的的几个治疗策略。作者最近在成年内耳中发现了可以分化为毛细胞的干细胞,发现了胚胎干细胞可在体外转化为毛细胞并表达毛细胞标记物。在动物模型中,成年内耳干细胞、神经干细胞和胚胎干细胞来源的前体细胞可分化成为毛细胞和神经细胞。本文将讨论使用干细胞再生损伤毛细胞的不同方法,介绍几种可行的动物模型,并讨论发展基于干细胞的细胞替代疗法治疗内耳损伤中存在的困难。     

Retinal stem/progenitor properties of iris pigment epithelial cells

Neural stem cells/progenitors that give rise to neurons and glia have been identified in different regions of the brain, including the embryonic retina and ciliary epithelium of the adult eye. Here, we first demonstrate the characterization of neural stem/progenitors in postnatal iris pigment epithelial (IPE) cells. Pure isolated IPE cells could form spheres that contained cells expressing retinal progenitor markers in non-adherent culture. The spheres grew by cell proliferation, as indicated by bromodeoxyuridine incorporation. When attached to laminin, the spheres forming IPE derived cells were able to exhibit neural phenotypes, including retinal-specific neurons. When co-cultured with embryonic retinal cells, or grafted into embryonic retina in vivo, the IPE cells could also display the phenotypes of photoreceptor neurons and Muller glia. Our results suggest that the IPE derived cells have retinal stem/progenitor properties and neurogenic potential without gene transfer, thereby providing a novel potential source for both basic stem cell biology and therapeutic applications for retinal diseases.     

Development and evolution of cerebellar neural circuits

The cerebellum controls smooth and skillful movements and it is also involved in higher cognitive and emotional functions. The cerebellum is derived from the dorsal part of the anterior hindbrain and contains two groups of cerebellar neurons: glutamatergic and gamma-aminobutyric acid (GABA)ergic neurons. Purkinje cells are GABAergic and granule cells are glutamatergic. Granule and Purkinje cells receive input from outside of the cerebellum from mossy and climbing fibers. Genetic analysis of mice and zebrafish has revealed genetic cascades that control the development of the cerebellum and cerebellar neural circuits. During early neurogenesis, rostrocaudal patterning by intrinsic and extrinsic factors, such as Otx2, Gbx2 and Fgf8, plays an important role in the positioning and formation of the cerebellar primordium. The cerebellar glutamatergic neurons are derived from progenitors in the cerebellar rhombic lip, which express the proneural gene Atoh1. The GABAergic neurons are derived from progenitors in the ventricular zone, which express the proneural gene Ptf1a. The mossy and climbing fiber neurons originate from progenitors in the hindbrain rhombic lip that express Atoh1 or Ptf1a. Purkinje cells exhibit mediolateral compartmentalization determined on the birthdate of Purkinje cells, and linked to the precise neural circuitry formation. Recent studies have shown that anatomy and development of the cerebellum is conserved between mammals and bony fish (teleost species). In this review, we describe the development of cerebellar neurons and neural circuitry, and discuss their evolution by comparing developmental processes of mammalian and teleost cerebellum.     

Integration and Long Distance Axonal Regeneration in the Central Nervous System from Transplanted Primitive Neural Stem Cells

Spinal cord injury (SCI) results in devastating motor and sensory deficits secondary to disrupted neuronal circuits and poor regenerative potential. Efforts to promote regeneration through cell extrinsic and intrinsic manipulations have met with limited success. Stem cells represent an as yet unrealized therapy in SCI. Recently, we identified novel culture methods to induce and maintain primitive neural stem cells (pNSCs) from human embryonic stem cells. We tested whether transplanted human pNSCs can integrate into the CNS of the developing chick neural tube and injured adult rat spinal cord. Following injection of pNSCs into the developing chick CNS, pNSCs integrated into the dorsal aspects of the neural tube, forming cell clusters that spontaneously differentiated into neurons. Furthermore, following transplantation of pNSCs into the lesioned rat spinal cord, grafted pNSCs survived, differentiated into neurons, and extended long distance axons through the scar tissue at the graft-host interface and into the host spinal cord to form terminal-like structures near host spinal neurons. Together, these findings suggest that pNSCs derived from human embryonic stem cells differentiate into neuronal cell types with the potential to extend axons that associate with circuits of the CNS and, more importantly, provide new insights into CNS integration and axonal regeneration, offering hope for repair in SCI.     

Cerebellar stem cells do not produce neurons and astrocytes in adult mouse

Although previous studies implied that cerebellar stem cells exist in some adult mammals, little is known about whether these stem cells can produce new neurons and astrocytes. In this study by bromodeoxyuridine (BrdU) intraperitoneal (i.p.) injection, we found that there are abundant BrdU⁺ cells in adult mouse cerebellum, and their quantity and density decreases significantly over time. We also found cell proliferation rate is diversified in different cerebellar regions. Among these BrdU⁺ cells, very few are mash1⁺ or nestin⁺ stem cells, and the vast majority of cerebellar stem cells are quiescent. Data obtained by in vivo retrovirus injection indicate that stem cells do not produce neurons and astrocytes in adult mouse cerebellum. Instead, some cells labeled by retrovirus are Iba1⁺ microglia. These results indicate that very few stem cells exist in adult mouse cerebellum, and none of these stem cells contribute to neurogenesis and astrogenesis under physiological condition.     

Morphogenetic and cellular movements that shape the mouse cerebellum; insights from genetic fate mapping

We used the cerebellum as a model to study the morphogenetic and cellular processes underlying the formation of elaborate brain structures from a simple neural tube, using an inducible genetic fate mapping approach in mouse. We demonstrate how a 90 degrees rotation between embryonic days 9 and 12 converts the rostral-caudal axis of dorsal rhombomere 1 into the medial-lateral axis of the wing-like bilateral cerebellar primordium. With the appropriate use of promoters, we marked specific medial-lateral domains of the cerebellar primordium and derived a positional fate map of the murine cerebellum. We show that the adult medial cerebellum is produced by expansion, rather than fusion, of the thin medial primordium. Furthermore, ventricular-derived cells maintain their original medial-lateral coordinates into the adult, whereas rhombic lip-derived granule cells undergo lateral to medial posterior transverse migrations during foliation. Thus, we show that progressive changes in the axes of the cerebellum underlie its genesis.     

Development of the cerebellum and cerebellar neural circuits

The cerebellum, a structure derived from the dorsal part of the most anterior hindbrain, is important for integrating sensory perception and motor control. While the structure and development of the cerebellum have been analyzed most extensively in mammals,recent studies have shown that the anatomy and development of the cerebellum is conserved between mammals and bony fish (teleost) species, including zebrafish. In the mammalian and teleost cerebellum,Purkinje and granule cells serve, respectively, as the major GABAergic and glutamatergic neurons. Purkinje cells originate in the ventricular zone (VZ), and receive inputs from climbing fibers. Granule cells originate in the upper rhombic lip (URL) and receive inputs from mossy fibers. Thus, the teleost cerebellum shares many features with the cerebellum of other vertebrates, and isa good model system for studying cerebellar function and development. The teleost cerebellum also has features that are specific to teleosts or have not been elucidated in mammals, including eurydendroid cells and adult neurogenesis. Furthermore, the neural circuitry in part of the optic tectum and the dorsal hindbrain closely resembles the circuitry of the teleost cerebellum; hence,these are called cerebellum-like structures. Here we describe the anatomy and development of cerebellar neurons and their circuitry, and discuss the possible roles of the cerebellum and cerebellum-like structures in behavior and higher cognitive functions. We also consider the potential use of genetics and novel techniques for studying the cerebellum in zebrafish.     

Generation of functional neurons and glia from multipotent adult mouse germ-line stem cells

Recently, we reported the successful establishment of multipotent adult germ-line stem cells (maGSCs) from cultured adult mouse spermatogonial stem cells. Similar to embryonic stem cells, maGSCs are able to self-renew and differentiate into derivatives of all three germ layers. These properties make maGSCs a potential cell source for the treatment of neural degenerative diseases. In this study, we describe the generation of maGSC-derived proliferating neural precursor cells using growth factor-mediated neural lineage induction. The neural precursors were positive for nestin and Sox1 and could be continuously expanded. Upon further differentiation, they formed functional neurons and glial cells, as demonstrated by expression of lineage-restricted genes and proteins and by electrophysiological properties. Characterization of maGSC-derived neurons revealed the generation of specific subtypes, including GABAergic, glutamatergic, serotonergic, and dopaminergic neurons. Electrophysiological analysis revealed passive and active membrane properties and postsynaptic currents, indicating their functional maturation. Functional networks formed at later stages of differentiation, as evidenced by synaptic transmission of spontaneous neuronal activity. In conclusion, our data demonstrate that maGSCs may be used as a new stem cell source for basic research and biomedical applications.     

Localization of the type 1 corticotropin releasing factor receptor (CRF-R1) in the embryonic mouse cerebellum

Corticotropin releasing factor (CRF) is present in the adult, as well as in the embryonic and postnatal rodent cerebellum. Further, the distribution of the type 1 CRF receptor has been described in adult and postnatal animals. The focus of the present study is to determine the distribution and cellular relationships of the type 1 CRF receptor (CRF-R1) during embryonic development of the cerebellum. Between embryonic day (E)11 and E12, CRF-R1 immunoreactive puncta are uniformly distributed in the ventricular zone, the site of origin of Purkinje cells, nuclear neurons, and GABAergic interneurons, as well as the germinal trigone, the birthplace of the precursors of granule cells. Between E13 and 18, the distribution of immunolabeled puncta decreases in both the ventricular zone and the germinal trigone and increases in the intermediate zone, as well as in the dorsal aspect of the cerebellar plate. Between E14 and 18, antibodies that label specific populations of cerebellar neurons were combined with the antibody for the receptor to determine the cellular elements that expressed CRF-R1. At E14, CRF-R1 immunoreactivity is co-localized in neurons immunolabeled with PAX-2, an antibody that is specific for GABAergic interneurons. These neurons continue to express CRF-R1 as they migrate dorsally toward the cerebellar surface. Between E16 and 18, Purkinje cells, immunolabeled with calbindin, near the dorsal surface of the cerebellum express CRF-R1 in their cell bodies and apical processes. CRF has been shown to have a depolarizing effect on adult and postnatal Purkinje cells. Further, CRF has been shown to contribute to excitability of hippocampal neurons during embryonic development by binding to CRF-R1; depolarization induced excitability appears to be critical for cell survival. The location of the type one CRF receptor and the presence of its primary ligand, CRF, in the germinal zones of the cerebellum and in migrating neurons suggest that this receptor/ligand interaction could be important in the regulation of neuronal survival through cellular mechanisms that lead to depolarization of embryonic cerebellar neurons.     

Genetic selection of sox1GFP-expressing neural precursors removes residual tumorigenic pluripotent stem cells and attenuates tumor formation after transplantation

Because of their ability to proliferate and to differentiate into diverse cell types, embryonic stem (ES) cells are a potential source of cells for transplantation therapy of various diseases, including Parkinson's disease. A critical issue for this potential therapy is the elimination of undifferentiated cells that, even in low numbers, could result in teratoma formation in the host brain. We hypothesize that an efficient solution would consist of purifying the desired cell types, such as neural precursors, prior to transplantation. To test this hypothesis, we differentiated sox1-green fluorescent protein (GFP) knock-in ES cells in vitro, purified neural precursor cells by fluorescence-activated cell sorting (FACS), and characterized the purified cells in vitro as well as in vivo. Immunocytofluorescence and RT-PCR analyses showed that this genetic purification procedure efficiently removed undifferentiated pluripotent stem cells. Furthermore, when differentiated into mature neurons in vitro, the purified GFP+ cell population generated enriched neuronal populations, whereas the GFP- population generated much fewer neurons. When treated with dopaminergic inducing signals such as sonic hedgehog (SHH) and fibroblast growth factor-8 (FGF8), FACS-purified neural precursor cells responded to these molecules and generated dopaminergic neurons as well as other neural subtypes. When transplanted, the GFP+ cell population generated well contained grafts containing dopaminergic neurons, whereas the GFP- population generated significantly larger grafts (about 20-fold) and frequent tumor-related deaths in the transplanted animals. Taken together, our results demonstrate that genetic purification of neural precursor cells using FACS isolation can effectively remove unwanted proliferating cell types and avoid tumor formation after transplantation.     

Transplanted neurons form both normal and ectopic projections in the adult brain

Transplantation of embryonic or stem cell derived neurons has been proposed as a potential therapy for several neurological diseases. Previous studies reported that transplanted embryonic neurons extended long-distance projections through the adult brain exclusively to appropriate targets. We transplanted E14 lateral ganglionic eminence (LGE) and E15 cortical precursors from embryonic mice into the intact adult brain and analyzed the projections formed by transplanted neurons. In contrast to previous studies, we found that transplanted embryonic neurons formed distinct long-distance projections to both appropriate and ectopic targets. LGE neurons transplanted into the adult striatum formed projections not only to the substantia nigra, a normal target, but also to the claustrum and through all layers of fronto-orbital cortex, regions that do not normally receive striatal input. In some cases, inappropriate projections outnumbered appropriate projections. To examine the relationship between the donor cells and host brain in establishing the pattern of projections, we transplanted cortical precursors into the adult striatum. Despite their heterotopic location, cortical precursors not only predominantly formed projections appropriate for cortical neurons, but they also formed projections to inappropriate targets. Transplantation of GFP-expressing cells into beta-galactosidase-expressing mice confirmed that the axonal projections were not created by the fusion of donor and host cells. These results suggest that repairing the brain using transplantation may be more complicated than previously expected, because exuberant ectopic projections could result in brain dysfunction. Understanding the signals regulating axonal extension in the adult brain will be necessary to harness stem cells or embryonic neurons for effective neuronal-replacement therapies.     

Derivation of neural precursor cells from human ES cells at 3% O(2) is efficient, enhances survival and presents no barrier to regional specification and functional differentiation

In vitro stem cell systems traditionally employ oxygen levels that are far removed from the in vivo situation. This study investigates whether an ambient environment containing a physiological oxygen level of 3% (normoxia) enables the generation of neural precursor cells (NPCs) from human embryonic stem cells (hESCs) and whether the resultant NPCs can undergo regional specification and functional maturation. We report robust and efficient neural conversion at 3% O(2), demonstration of tri-lineage potential of resultant NPCs and the subsequent electrophysiological maturation of neurons. We also show that NPCs derived under 3% O(2) can be differentiated long term in the absence of neurotrophins and can be readily specified into both spinal motor neurons and midbrain dopaminergic neurons. Finally, modelling the oxygen stress that occurs during transplantation, we demonstrate that in vitro transfer of NPCs from a 20 to 3% O(2) environment results in significant cell death, while maintenance in 3% O(2) is protective. Together these findings support 3% O(2) as a physiologically relevant system to study stem cell-derived neuronal differentiation and function as well as to model neuronal injury.     

Somatic plasticity of neural stem cells: Fact or fancy?

Several studies have described the potential for embryonic and adult neural stem cells to differentiate into non-neural cells such as muscle and blood, tissues that are derived from non-neuroectodermal germ layers. This raised the exciting possibility that these cells possessed a broader range of differentiation potential than originally thought and raised interesting prospects for possible transplantation utilization. However, a number of recent reports have raised questions about whether the phenomena observed actually represented true somatic plasticity. In this review, we critically analyze these studies with the aim of providing some criteria by which future studies that address this important problem may be evaluated.     

神经干细胞脑内移植后的存活、迁移和分化

从胚胎或成体大鼠脑组织、人胚脑组织均能分离到神经干细胞 ,将它们进行体外原代培养扩增或永生化后植入脑内 ,均能观察到其在脑内的迁移和分化现象。其分化能力主要取决于移植部位的脑内微环境 ,但这种影响作用是相对的。同时 ,体外培养环境如培养时间和细胞融合程度、维甲酸类诱导分化剂处理、NGF转导处理再移植或与嗜铬细胞 (分泌NGF)共移植等 ,也能决定神经干细胞脑内移植后向神经元方向分化的能力。神经干细胞移植为中枢神经系统功能重建和神经再生带来新的希望。     

体外诱导人骨髓间充质干细胞向多巴胺神经元分化的研究

通过体外诱导人骨髓间充质干细胞(bone marrow mesenchymal stemcells,BMSCs)向多巴胺(dopamine,DA)神经元分化,探讨人BMSCs来源的DA神经元的功能特征及其分化机制,为临床上细胞移植替代治疗诸如帕金森氏病(parkinson’sdisease,PD)等神经精神性疾病提供一种理想的细胞来源。通过密度梯度离心获取人骨髓中的单个核细胞,贴壁培养纯化BMSCs。50μmol/L脑源性神经营养因子(brain derivedneurotrophy factor,BDNF),10μmol/Lforskolin(FSK)和10μmol/LDA联合对BMSCs进行诱导。电子显微镜观察诱导2周后细胞是否具有神经元的超微结构特点;免疫细胞化学染色和RT-PCR检测DA神经元分化过程中的标志物酪氨酸羟化酶(tyrosine hydroxylase,TH)的表达以及转录因子Nurr1、Ptx3和Lmx1b的表达;高效液相色谱(highperformance liquid chromatogram,HPLC)检测诱导2周后的细胞多巴胺的释放水平。结果表明,诱导2周后,电镜下细胞胞浆中有大量密集的呈扁平囊状的粗面内质网及其间的一些游离核糖体以及神经微丝的形成。RT-PCR结果显示NSE(neuron specificenolase)、Nurr1、Ptx3、Lmx1b和TH的mRNA均有表达;免疫细胞化学染色结果表明诱导2周后TH阳性细胞(24·80±3·36)%的表达较诱导3d后(3·77±1·77)%明显提高(P<0·01);HPLC检测到诱导2周后的细胞DA释放水平[(1·22±0·36)μg/mL(n=6)]高于未经诱导的细胞[(0·75±0·22)μg/mL(n=6)(t=-2·79,P=0·038)]。由此得出,BDNF、FSK和DA可以在体外诱导人BMSCs向DA神经元分化,并具有DA神经元的功能特征,是临床用于治疗神经精神性疾病的理想细胞来源。     

A method for deriving homogenous population of oligodendrocytes from mouse embryonic stem cells

There is a pressing need for new therapeutics for the generation and transplantation of oligodendrocyte to the white matter to help replace and render injured cells that are lost in demyelinating disease. There are a few protocols describing a homogenous derivation of non-manipulated mouse embryonic stem cells to oligodendrocytes (ES-OL). Moreover, protocols that are successful in producing ES-OL do so with low efficiency. Therefore, we describe clear methodology for differentiation of mouse ES cells to oligodendrocyte to a high degree of homogenity and reproducibility in vitro. In addition, taking advantage of three defined media, we can generate a defined ES to oligodendrocyte lineage while selecting against neurons and astrocytes. More specifically, (1) Glial stem cell defining media (GSCDM), supplemented with appropriate combination of SHH and RA support pro-oligodendrocyte developing neural spheres from ES cells, (2) Oligodendrocyte differentiating media, induces lineage selection of oligodendrocytes progenitors from neural stem cells, and (3) Oligodendrocyte maturation media, supports oligodendrocytes progenitor maturation. Moreover, the ES cell derived oligodendrocytes display mature properites in the prescence of rat dorsal root gangila in vitro. Thus confirming thier potential for use to invesitgate developmental pathways and future potential use of cells in transplantation towards myelin repair.     

Behavior and Differentiation of the Neural Stem Cells in vivo

We studied the behavior and differentiation of human and rat neural stem cells after transplantation in the adult rat brain without immunosuppression. The rat stem cells were isolated from the presumptive neocortex of 15-day-old embryos. The human cells were isolated from the ventricular brain zone of 9-week-old embryos and cultivated for two weeks before transplantation. The results of histomorphological studies suggest that the microenvironment factors did not suppress the growth or development of transplanted stem cells. Both rat and human embryonic multipotent neural cells showed similar behavior and differentiation into neurons and glial cells. After transplantation, they continued to mitotically divide and migrated from the graft area to the surrounding tissue of a recipient brain. The presumptive glial cells migrated preferentially along the capillaries and fibrous structures of the recipient brain. Similar behavior of the rat and human neural stem cells in the microenvironment of the recipient adult rat brain and the absence of immune reaction suggest that the transplantation into the rat brain may serve as a model for studying the developmental biology of the human stem cells.     

Interplay between Hsp90 and TPR domain-containing proteins in steroidogenic signaling

EGFL7 drives the formation of neurons from neural stem cells. In the embryonic and adult brain this process is essential for neurogenesis and homeostasis of the nervous system. The function of adult neurogenesis is not fully understood but maybe it supports life-long learning and brain repair after injuries such as stroke. The transition of neural stem cells into mature neurons is tightly regulated. One of the essential signaling pathways governing this process is the Notch pathway, which controls metazoan development. In a recent publication, we identified a novel non-canonical Notch ligand, EGFL7, and described its impact on neural stem cells.¹ We explored the molecular mechanisms, which this molecule affects to regulate the self-renewal capacity of neural stem cells and to promote their differentiation into neurons. In this review, we discuss the implications of our findings for adult neurogenesis and illustrate the potential of EGFL7 to serve as an agent to increase neurogenesis and the self-renewal potential of the brain.