Developmental expression of cell type-specific markers in mouse cerebellar cells in vitro

The expression of several cell type-specific markers was studied by indirect immunofluorescence in discontinuous BSA gradient fractionated and unfractionated mouse cerebellar cells cultured for 3 days in vitro from embryonic day 13 through postnatal day 9. Cell surface antigen NS-4 and tetanus toxin receptors are present at all ages studied. Thy-1 first detected on neurons with large cell bodies (Purkinje and/or Golgi Type II.neurons) onpostnatal day 3, but absent from all neurons with small cell bodies (granule, basket, and stellate cells). At all ages Thy-1 antigen is absent from astro- and oligodendroglia, and fibroblasts or fibroblast-like cells. Fibroblast-like cells express fibronectin at all ages studied. Astroglia expressing glial fibrillary acidic (GFA) protein are first detectable in cultures from 16-day-old embryos. Their number increases at later ages. At all ages studied fluorescein conjugated Ricinus communis agglutinin 120 brightly labels the cell surfaces of fibroblastic cells and large neurons, less brightly those of astrocytes, bu not those of small neurons. Oligodendroglia become detectable in cerebellar cultures from 16- and 17-day-old embryos maintained in vitro for 3 days using antibodies to 04 antigen and bovine corpus callosum, respectively. At embryonic ages BSA step gradient procedures do not result in enrichment of particular cell types as recognized by the available markers. From birth onward, however, enrichment of cell populations was obtained corresponding to the ones characterized at postnatal day 6 as described in the companion paper (Schnitzer and Schachner 1981b).     

Thyroid hormone-dependent development of mouse cerebellar Purkinje cells in vitro

Using a well-defined medium with insulin, transferrin and selenium but without serum and albumin, we quantitatively determined the effect of thyroid hormones on the development of Purkinje cells in mouse cerebellar monolayer cultures. Addition of a thyroid hormone, T3 or T4, to the serum-free medium resulted in a highly elaborate dendritic development of Purkinje cells. The cultured Purkinje cells in the presence of T4 even showed similarities in shape and in synapse formation to normal Purkinje cells in vivo. Such effect of T4 on the dendritic arborization of Purkinje cells was dose dependent and significantly sensitive to a low dose of T4 even at 50 pM. The effect of T4 was confirmed by an inhibition experiment using amiodarone, which was reported to induce thyroid dysfunction. Furthermore, T4 affected not only Purkinje cell development but also the shape of other neural cells such as small interneurons (mainly granule cells) and astrocytes in cerebellar cultures. T4 induced development of both interneurons and astrocytes having long processes. These results indicate that thyroid hormones play a pivotal role in the development of mouse Purkinje cell dendrites acting on Purkinje cells directly and/or indirectly via the close interaction with interneurons and astrocytes.     

Characterization of different neuron populations in mouse statoacoustic ganglion cultures

Statoacoustic ganglion (SAG) cells were grown in primary culture and the appearance of different neuronal phenotypes was investigated. Analysis criteria were shape, size and staining for the immunocytochemical markers: neurofilament proteins (NF-200 kDa), neuron-specific enolase (NSE), calretinin, a calcium-binding protein and substance P, a neurotransmitter. Cultures were prepared from dissociated SAG cells of 13 gestation-day-old mouse embryos. Neurons were identified and counted after 7 days in vitro. At this stage, neurons were organized in small clusters forming an extensive network of neurites grown on a layer of fibroblasts and glia. Most neurons identified by NF or NSE immunoreactivity showed a typical adult-like bipolar profile. The diameters of the neurons were between 5.62 and 17.00 μm and displayed a normal distribution (mean: 10.6 μm). Two distinct subpopulations were identified by the expression of calretinin and substance P. Calretinin-immunoreactive neurons were large and very rare and had a mean diameter of 11.3 μm; the distribution of substance P was more extensive than that of calretinin and identified a population of small neurons with a mean diameter of 8.9 μm. The distributions of these two markers in SAG cultures were consistent with in vivo results. In conclusion, dissociated SAG cell cultures appear to be a suitable model for analyzing the development of the immunocytochemical and functional characteristics of the neurons of this inner ear ganglio.     

Experimental modification of postnatal cerebellar granule cell migration in vitro

Histotypic migration of [³H]thymidine pulse-labeled granule cell neurons in cerebellar folium explants was monitored in the presence of antibodies to cell adhesion molecules and quantified by automatic image analysis. When explants were cultured in the presence of monovalent antibody fragments to cell adhesion molecules L1 and N-CAM, an inhibition of cell migration of33.3 ± 4.4% and 13.9 ± 2.1%, respectively, was observed. In the presence of an equimolar mixture of monovalent antibody fragments to L1 antigen and N-CAM no additive effects in inhibition of cell migration were seen. Antibodies to the L2 carbohydrate epitope which is common to L1, N-CAM and other cell surface glycoproteins showed a similarly small effect on cell migration as antibodies to N-CAM. Monoclonal antibodies to cell surface antigen M2 and polyclonal antibodies to mouse liver membranes reacting with the surface of all cerebellar cell types did not alter the migratory behavior of granule cells. Cultivation of explants in the presence of neuraminidase, ganglioside binding toxins, as well as glycosaminoglycans and glycosaminoglycan degrading enzymes, also did not modify the extent of cell migration under the culture conditions used.     

体外分离培养小鼠肺间充质干细胞的生物学特性及其对肺损伤干预效果

背景：肺脏含有多种内源性干细胞，分别为来源于内胚层或中胚层的前体细胞，阐明肺组织间充质干细胞的特性有助于了解其在肺损伤中的生物学作用。 目的：观察体外分离培养的小鼠肺组织间充质干细胞生长状态、表面标志、分化功能等生物学特性，探讨其对肺损伤鼠的干预效果。 设计、时间及地点：细胞学体外观察与随机对照动物体内实验，于2005-10/2007-08在解放军军事医学科学院基础医学研究所细胞生物学实验室完成。 材料：清洁级三四周龄C57BL/6雄鼠，用于分离培养肺组织间充质干细胞；清洁级6~8周龄C57BL/6雌鼠20只，分为细胞移植组、模型对照组，10只/组。 方法：分离雄鼠肺组织，Ⅱ型胶原酶消化后，Ficoll法离心，收集界面层细胞，贴壁法分离培养肺组织间充质干细胞，当细胞接近70%~80%汇合时消化传代。细胞移植组、模型对照组小鼠按20 mg/kg腔注射马利兰抑制骨髓干细胞的迁移后，均建立博莱霉素诱导的肺纤维化模型，造模后细胞移植组尾静脉注入5×105个肺间充质干细胞，模型对照组气管内注射100 μL PBS。14 d后取材，制备肺组织石蜡切片。 主要观察指标：体外培养的肺组织间充质干细胞的生长状态、免疫表型、基因表达、分化潜能、集落形成能力以及静脉移植减轻肺损伤效果。 结果：小鼠肺组织来源的间充质干细胞为成纤维样细胞，在体外能够长期培养和快速扩增；其细胞免疫表型为Sca-1+，CD44+，CD29+，CD105+，CD54+，CD34-，CD45-，CD11b-，c-kit-，CD31-；不表达肺泡上皮细胞标志表面活性蛋白C、水通道蛋白5、clara细胞分泌蛋白，胚胎干细胞标志Oct-4和Nanog基因在培养扩增的细胞中持续表达；能够被诱导分化为脂肪细胞、成骨细胞和表达表面活性蛋白C的肺泡2型上皮样细胞；集落形成率约3%；经静脉输注后，细胞移植组肺损伤和纤维化程度较模型对照组减轻，Masson染色显示只有少量血管周围呈阳性，且经骨髓抑制处理后已接近正常肺组织。 结论：体外培养的小鼠肺间充质干细胞能够快速扩增，可向脂肪细胞、成骨细胞和肺泡上皮细胞方向分化；静脉移植后有助于肺组织修复、减轻肺损伤和纤维化程度，且骨髓抑制不影响肺间充质干细胞的保护作用。     

Developmental stage-specific changes in lectin binding to mouse cerebellar cells in vitro

Eleven fluorescein isothiocyanate-conjugated (FITC) lectins, each with distinct carbohydrate-binding properties, were used to assess cell surface glycoconjugates of embryonic and early postnatal cerebellar cells in vitro. Fluorescence staining of embryonic day 13 (E13) cerebellar cells with FITC Ricinus communis agglutinin diminished markedly between 24 and 72 hr in vitro. No staining of postnatal day 0 (P0) or postnatal day 7 (P7) cells was observed with FITC Ricinus communis agglutinin. A similar, but less pronounced decrease in FITC concanavalin A, FITC Lens culinaris, and FITC wheat germ agglutinin was observed between embryonic day 13 and birth. No specific staining of E13, P0, or P7 cultures was observed with FITC peanut agglutinin, FITC Dolichos bifloris agglutinin, FITC soybean agglutinin, FITC Wistaria floribundis agglutinin, FITC Phaseolus vulgaris agglutinin, FITC Limulus polyphemus agglutinin, or FITC Ulex europaeusI agglutinin. Similar results were obtained with 125I-lectin binding assays. Ricinus communis 125-I-agglutinin binding decreased dramatically between embryonic day 13 and birth. Less pronounced decreases were observed in 125I-concanavalin A and wheat germ 125I-agglutinin binding. Very low levels of soybean 125I-agglutinin or Ulex europaeusI 125I-agglutinin were bound by either embryonic or early postnatal cerebellar cells in vitro.     

Granule cell raphes in the cerebellar cortex of chicken and mouse

The cerebellar cortex of the chicken embryo contains parasagittal segments of Purkinje cells. At intermediate stages of development, cell-dense ribbons of migrating granule cells ("raphes") are found between the segments. The complementary pattern of granule cell raphes and Purkinje cell segments represents a basic scheme of cerebellar organization that coincides with the expression domains of various genes, such as cadherins, gene regulatory proteins, and ephrins and their receptors. We have recently found the raphe/segment pattern also in a mammalian species, the postnatal mouse. Like in the chicken, the parasagittal raphes of granule cells were observed at the boundaries of Purkinje cell segments that differentially express cadherins. The number and arrangement of the raphes in the different cerebellar lobules is roughly similar in both species. The raphe/segment pattern is thus more widely distributed in vertebrates than previously assumed.     

Taurine-induced increase of the Cl-conductance of cerebellar Purkinje cell dendrites in vitro

Whether taurine increases the Cl-conductance of cerebellar Purkinje cell dendrites was examined by intradendritic recording technique in vitro. Taurine-induced hyperpolarization was inverted to depolarization by lowering the concentration of external Cl⁻, and also by hyperpolarizing the dendritic membrane by intradendritic DC current injection. The reversal potential for the taurine action was found to be linearly related to the logarithmic concentrations of external Cl⁻, the slope being 59 mV for a 10-fold change of external Cl⁻ concentration. These results suggest that taurine increases a Cl-conductance for exerting its inhibitory action on cerebellar Purkinje cell dendrites. This finding may support the transmitter role of taurine in the mammalian cerebellum.     

Weaver mouse cerebellar granule neurons fail to migrate on wild-type astroglial processes in vitro

To study the regulation of glial-guided neuronal migration, we have analyzed the behavior of cerebellar granule neurons purified from the homozygous weaver (wv/wv) B6CBA-w mouse, an autosomal recessive genetic mutation that suffers a failure of granule cell migration along Bergmann glial processes (Rakic and Sidman, 1973a, b; Rezai and Yoon, 1972), on the processes of astroglia purified from homozygous normal B6CBA-Aw-J-wv (+/+) mouse cerebella. When co-cultured with normal astroglia, weaver granule neurons failed to form neuron-glia contacts characteristic of migrating neurons and impaired normal astroglial morphological differentiation. Normal astroglial cells co-cultured with weaver granule cells had enlarged cell somata with stunted processes and enlarged endfeet compared to normal astroglia co-cultured with normal granule cells. In contrast, normal neurons associated with weaver astroglia, forming tight appositions seen for migrating neurons in vivo, and enhanced weaver astroglial morphological differentiation. Weaver astroglia co-cultured with normal granule cells contained a more normal complement of glial filaments and had a smaller perikaryon with longer, more tapered processes than their counterparts co-cultured with weaver neurons. These results suggest, in agreement with the study of Goldowitz and Mullen (1982) on heterozygous mutant chimeras, that the granule neuron is a primary site of action of the weaver gene, and further support our previous findings that neuron-glia interactions regulate astroglial morphological differentiation (Hatten, 1985).     

Age related changes in Purkinje cell number in the cerebellar nodulus of the mouse

The cortex of the cerebellar nodulus of mice aged 6, 15, 22, 25, 28 and 31 months was examined in parasagittal sections using quantitative histological techniques. The number of Purkinje cells per mm declined from 11.2 +/- 0.8 or 6 months to 7.0 +/- 0.6 at 31 months. Granule cell density remained constant (2.58 x 10(6) per mm3) between 6 and 31 months of age. The granule cell to Purkinje cell ratio increased from 119 +/- 7 at 6 months of age to 173 +/- 7 at 31 months of age. The change in Purkinje cell number began between 15 and 22 months of age. These results do not differ significantly from those of a similar study of quantitative histological changes in the anterior lobes of the same cerebella. This suggests that loss of Purkinje cells occurs in a similar fashion throughout the cerebellum despite differences in afferent and efferent connections in different regions.     

Embryonic cerebellar astroglia in vitro

Three types of astroglia appear during cerebellar development--radial glia and Bergmann glia, which are thought to facilitate neuronal migration, and astrocytes, which are thought to compartmentalize mature granule neurons. Cells resembling Bergmann glia and astrocytes have been described in cultures of cerebellar cells harvested from early postnatal cerebellum. In this study, we have used cell-type specific antisera to visualize embryonic forms of cerebellar astroglia and their interaction with embryonic neurons in vitro. When cells were dissociated from mouse cerebellum on the thirteenth embryonic day (E13), 3 forms of cells were stained with antisera raised against purified glial filament protein ( AbGF ), all of which had more elongated processes and less complex shapes than astroglia from postnatal day 7. The vast majority of embryonic cerebellar neurons did not contact these immature forms of astroglia.     

Characterization of Ca2+-dependent and -independent aggregation mechanisms among mouse cerebellar cells

To gain insight into the cellular and molecular mechanisms underlying cell interactions in the early postnatal mouse cerebellum, Ca2+-dependent and -independent aggregation mechanisms were characterized using single cell suspensions under conditions that allow discrimination between the two mechanisms. When cerebellar cells were derived from newborn to 10-day-old mouse cerebellum, both mechanisms were active and showed no major change in activity during this time period. Mg2+ could not replace Ca2+ in the Ca2+-dependent mechanism. In contrast to the Ca2+-independent mechanisms, the Ca2+-dependent mechanism was inactive at low temperatures, suggesting a necessity for molecular rearrangement within the surface membrane during aggregation. Neuraminidase, chondroitinase, heparinase or hyaluronidase treatment of cells did not influence the aggregation of cells under Ca2+-dependent and -independent conditions. Chondroitin sulfate inhibited and hyaluronic acid stimulated the Ca2+-dependent mechanism, whereas chondroitin sulfate only slightly and hyaluronic acid strongly inhibited the Ca2+-independent one. Dextran sulfate slightly inhibited both mechanisms, whereas heparin and fucoidan, a complex sulfated carbohydrate, did not influence cell aggregation, while they strongly inhibited attachment of cells to laminin. The polycation poly-L-lysine slightly stimulated the Ca2+-independent mechanism, but inhibited the Ca2+-dependent one. Interestingly, chondroitin sulfate and hyaluronic acid strongly stimulated cell aggregation under conditions where both mechanisms were almost destroyed or inactive. Dextran sulfate showed only a small effect under these conditions. These observations indicate that different molecular mechanisms are active in cell-cell versus cell-extracellular matrix interactions and suggest a hitherto unknown complexity in molecular mechanisms during early postnatal cerebellar development.     

Neural populations in the human cerebellum: estimations from isolated cell nuclei

The possibility of stimulation of receptor structures with focused ultrasound (focused beam of high frequency mechanical waves) was investigated. Stimulation of single Pacinian corpuscle isolated from cat's mesentery resulted in receptor and action potentials. Stimulation of frog's ear labyrinth resulted in evoked potentials recorded from midbrain auditory area, their characteristics being much the same as those for responses to adequate sound stimuli. It is concluded that focused ultrasound is an advantageous agent for stimulation of various mechanoreceptors both isolated and, especially, located deep in the body. Some problems related to sensory specificity are discussed.     

Effects of Purkinje cell degeneration on the noradrenergic projection to mouse cerebellar cortex

We have examined the effects of a genetically programmed target cell death on the noradrenergic afferent projection to mouse cerebellar cortex. We have observed that the noradrenergic axon terminals originating in in locus coeruleus are maintained in the cerebellar cortex of the Purkinje cell degeneration (pcd) mutant mouse in spite of the absence of Purkinje cells, the targets for the noradrenergic projection. The number of noradrenergic terminals in the atrophic mutant cerebellar cortex is approximately normal as assessed by counts of fibers exhibiting catecholamine fluorescence and by measurement of high affinity uptake of tritium-labeled norepinephrine (NE) by synaptosomes prepared from cerebellar cortex. An increased density of NE fibers is observed which appears to be a consequence of reduced cerebellar mass in the mutant. Although the number of noradrenergic terminals is unaffected, morphological and biochemical alterations are observed in this system. The fibers are more brightly fluorescent in mutant than in normal mice and their pattern is less orderly. The content of the endogenous transmitter, NE, is increased from 150 to 170% whereas the activity of the rate-limiting enzyme tyrosine hydroxylase (TH) is reduced to about 60% of normal values. These changes appear to be permanent as they are still present in 6 month-old mutant animals, the oldest studied. No alterations in either NE content or TH activity are found in pcd/pcd hippocampus, another target for the locus coeruleus axons.     

小鼠胚胎干细胞体外向血管平滑肌细胞的定向分化*★

目的：血管重建手术在多数情况下以自体血管作为替代品，但来源有限。胚胎干细胞具有发育全能性，目前其定向诱导机制尚不明确。通过选择适当的诱导剂，探讨胚胎干细胞体外向血管平滑肌细胞分化的趋势。 方法：实验于2006-08/2007-02在南昌大学第二附属医院血液病研究所完成。①动物及细胞系：清洁级孕12.5 d昆明小白鼠1只，由南昌大学医学院动物中心提供，实验过程中对动物的处置符合动物伦理学标准。小鼠胚胎干细胞系129X/SvJ编号为SCRC-1018，由American Type Culture Collection提供。②实验方法：取孕12.5 d鼠胚胎，去除头部、内脏及四肢，将组织块剪碎，胰酶消化，分离培养胚胎成纤维细胞，传至3~5代时更换为含体积分数为0.15胎牛血清的DMEM高糖培养基，24 h后收集培养液，加入2.0 mmol/L L-谷氨酰胺，1×非必需氨基酸，0.1 mmol/L β-巯基乙醇，1 000 Ｕ/mL白血病抑制因子，此即为条件培养基。常规复苏小鼠胚胎干细胞系129X/SvJ，以1×106密度接种，传代时用差速贴壁法分离去除已分化的胚胎干细胞，加入条件培养基5 mL，经过悬滴-悬浮培养，构建拟胚体分化模型。设立3组，各组均置于明胶包被的T25培养瓶中，每瓶加入50个拟胚体，使其均匀分布于培养瓶底。诱导组7~10 d加入10-9 mol/L全反式维甲酸和3μg/L转化生长因子β1，10~21ｄ加入 20μg/L血小板源性生长因子。血清对照组仅加入去生长因子胎牛血清，全反式维甲酸组仅加入全反式维甲酸。诱导21ｄ的拟胚体，用胰蛋白酶和胶原酶Ⅱ联合消化为单个细胞，再加入20μg/L血小板源性生长因子继续诱导7ｄ。③实验评估：应用RT-PCR法检测诱导细胞血管平滑肌肌动蛋白、血管平滑肌肌球蛋白重链基因的表达。通过免疫细胞化学技术检测血管平滑肌肌动蛋白的表达来鉴定细胞性质。 结果：①胚胎干细胞生长及拟胚体诱导分化：胚胎干细胞在体外能自发形成拟胚体，经过不同生长因子的分阶段联合诱导，拟胚体贴壁后球体略摊开，周围出现大量的梭形细胞。②RT-PCR检测：拟胚体血管平滑肌肌动蛋白和血管平滑肌肌球蛋白重链基因均呈强阳性表达。③免疫细胞化学检测：荧光显微镜下，罗丹明染色细胞浆呈红色荧光，并可见肌丝结构；DAPI染色细胞核呈蓝色荧光。诱导组血管平滑肌肌动蛋白阳性率明显高于血清对照组、全反式维甲酸组（P < 0.05）。 结论：胚胎干细胞经维甲酸、转化生长因子β1以及血小板源性生长因子分阶段联合诱导后，可分化为血管平滑肌细胞且纯度较高。随着细胞纯度和活性等问题的进一步解决，胚胎干细胞将有可能成为血管组织工程的种子细胞。