成功的将胚胎干细胞培养成小脑神经元

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干细胞需要在特别的环境下给予刺激物，才能达到细胞分化(differentiation)与特异化(specialization)的目的，到目前为止科学家仍无法随心所欲的运用干细胞以获得想要分化的细胞。但是，洛克菲勒大学(Rockefeller University)的科学家成功的将胚胎干细胞移植到小鼠的脑部，并完整的分化成小脑颗粒神经细胞(granule neurons)，此研究发表于2月20日的PNAS期刊。

  目前干细胞的研究在减缓心脏疾病以及再生某些器官上已有进展，但是，对于一些脑部疾病，例如：阿兹海默症(Alzheimer’s disease)或帕金森氏症(Parkinson's disease)却仍无所获，很重要的原因可能是神经干细胞，特别是与脑部发育相关的干细胞，已停止分化及生长。以小脑为例，小脑主要负责学习、记忆以及感官感觉(sensory perseption)，然而，小脑病变却占了小儿科脑肿瘤比例的40%左右。

  Mary E. Hatten教授及博士后研究员Enrique Salero，穷尽其所有的知识让胚胎干细胞分化为成熟的小脑颗粒神经元，他们在细胞中加入了讯息分子以诱导特别的转录因子(transcription factors)能受到调控，使得细胞内的某些基因能被开启或关闭。然后再将这些新分化的细胞移植到新生小鼠的小脑皮层(cerebellar cortex)，一旦小鼠脑细胞开始生长，就会将这些移植的细胞合并到原始的颗粒细胞层，并伸展出突触能与其它的神经元交流，成为典型的颗粒细胞。

  Hatten教授表示：「这份研究成果令人感到十分振奋，因为这是首次看到细胞能移动到它要分化的区域，并且成功的伸展出突触。但是，目前还不确定这个看似成熟的小脑颗粒细胞，是否与人体脑部的颗粒细胞具有相同的功能，因此，研究人员接下来还需比对细胞内的基因组成，以观察此分化而得的颗粒神经元功能是否正常。」

（编译/陈瑞娟） (资料来源 : Bio.com)

部分英文原文：

Differentiation of ES cells into cerebellar neurons

Enrique Salero*, and Mary E. Hatten

Laboratory of Developmental Neurobiology, The Rockefeller University, 1230 York Avenue, New York, NY 10021-6399

Communicated by Torsten N. Wiesel, The Rockefeller University, New York, NY, December 15, 2006 (received for review September 15, 2006)

The neuronal circuits of the cerebellar cortex are essential for motor and sensory learning, associative memory formation, and the vestibular ocular reflex. In children and young adults, tumors of the granule cell, the medulloblastomas, represent 40% of brain tumors. We report the differentiation of E14 ES cells into mature granule neurons by sequential treatment with secreted factors (WNT1, FGF8, and RA) that initiate patterning in the cerebellar region of the neural tube, bone morphogenic proteins (BMP6/7 and GDF7) that induce early granule cell progenitor markers (MATH1, MEIS1, ZIC1), mitogens (SHH, JAG1) that control proliferation and induce additional granule cell markers (Cyclin D2, PAX2/6), and culture in glial-conditioned medium to induce markers of mature granule neurons (GABA6r), including ZIC2, a unique marker for granule neurons. Differentiated ES cells formed classic \"T-shaped\" granule cell axons in vitro, and implantation of differentiated Pde1c-Egfp-BAC transgenic ES cells into the external granule cell layer of neonatal mice resulted in the extension of parallel fibers, migration across the molecular layer, incorporation into the internal granule cell layer, and extension of short dendrites, typical of young granule cells forming synaptic connections with afferent mossy fibers. These results underscore the utility of treating ES cells with local, inductive signals that regulate CNS neuronal development in vivo as a strategy for cell replacement therapy of defined neuronal populations.


cerebellum | CNS development | granule neuron | stem cell

The cerebellar cortex is a remarkably simple laminar structure, with two principal neurons, the granule cell and the Purkinje cell, and a diverse set of interneurons, which modulate the output of the Purkinje cell to the cerebellar nuclei (1). The cerebellar circuitry coordinates movement and balance and functions in sensory discrimination (2) and cognitive processing (3). Long-term depression of parallel fiber synapses onto Purkinje has been assumed to control the simple vestibular ocular reflex. Recent studies on both simple functions and adaptive control of the cerebellum suggest that multiple plasticity mechanisms may contribute to cerebellum-dependent learning. Multiple plasticity mechanisms are probably important to encode memories over different time scales, to regulate the dynamics of movement, and to allow bidirectional changes in the amplitude of movements (4). Although the role of the cerebellum in learning and memory is becoming more complex, the remarkably simple architectonics of the cerebellum make it an attractive model system for developing cell replacement strategies.

Replacement therapy in the cerebellum, like that in other brain regions, depends on the ability to induce progenitor cells to differentiate into cells with specific cell fates. The pluripotent nature of mouse ES cells was formally demonstrated by their ability to contribute to all tissues of adult mice, including the germ line, after their injection into host blastocysts (5). In addition to their developmental potential in vivo, ES cells display a remarkable capacity to form differentiated cell types in culture. Recently, Sato et al. (6) demonstrated that activation of the canonical Wnt pathway could replace the requirement of mouse embryonic fibroblast-conditioned media in the maintenance of undifferentiated hES cells for short periods of time (5–7 days). In addition, Wichterle et al. (7) demonstrated that treating ES cells with the series of signals that induce specific cell populations during normal, in vivo development induces mouse ES cells to differentiate into spinal progenitor cells and subsequently into motor neurons. In the present study we have tested the signals that induce formation of the cerebellar territory (8–10) from rhombomere 1 (11), signals that dorsalize the neural tube specify (12) granule neurons (13), and mitogens that expand the pool of granule cell progenitors (GCPs) in the neonatal cerebellar cortex (14, 15). To monitor ES cell differentiation we measured expression of genes essential for granule neuron development, unique markers for GCPs and differentiated granule neurons, and markers for differentiating granule neurons. We also monitored the expression-specific markers for cerebellar Purkinje neurons (16) and markers for cerebellar astroglia.

During cerebellar development, granule neuron progenitors arise from the boundary of the mesencephalon and metencephalon in an area known as the rhombic lip. Recent studies reveal that the anterior rhombic lip, once thought to exclusively generate granule neurons of the cerebellar cortex (17), generates precursors of the cerebellar and precerebellar nuclei, which project afferent fibers to the cerebellar cortex or receive efferent fibers from the cerebellum. Progenitors of the Purkinje neuron arise from the ventricular zone of the cerebellar territory and express specific transcription factors (20).

Over the past 5 years, the Gene Expression Neuronal Database (GENSAT) Project has used Egfp-BAC transgenic mice to define patterns of CNS gene expression in the developing and adult mouse brain (28). The GENSAT Project has generated hundreds of Egfp-BAC transgenic lines and identified markers for cerebellar granule cells, including Pde1c for granule cells. Pde1c is expressed by GCPs very early in the program of development, commencing at E10, when the earliest GCP markers, including Math1 (18), Zic1,2 (19), and Meis1 (20), are expressed. Expression continues throughout the lifetime of the animal, making Pde1c a granule neuron marker that is expressed at all stages of life. To provide markers for ES differentiation into granule cells, we generated ES cells from Pde1c-Egfp-BAC transgenic mice. To test whether the local signals and transcription factors that establish the cerebellar primordium and specify neural fates in vivo can be harnessed in vitro to direct the differentiation of mouse ES cells into granule neurons, Purkinje cells, and cerebellar glial cells, we studied the influence of cerebellar \"organizer molecules,\" dorsal signals and proteins that expand the GCP cell population on ES cell differentiation.