Influence of epidermal growth factor on the maturation of fetal rat brain cells in aggregate culture. An immunocytochemical study

Maturation of astrocytes, neurons, and oligodendrocytes was studied in serum-free aggregating cell cultures of fetal rat telencephalon by an immunocytochemical approach. Cell type-specific immunofluorescence staining was examined by using antibodies directed against glial fibrillary acidic protein (GFAP) and vimentin, two astroglial markers; neuron-specific enolase (NSE) and neurofilament (NF), two neuronal markers, and galactocerebroside (GC), an oligodendroglial marker. It was found that the cellular maturation in aggregates is characterized by distinct developmental increases in immunoreactivity for GFAP, vimentin, NSE, NF, and GC, and by a subsequent decrease of vimentin-positive structures in more differentiated cultures. These findings are in agreement with observations in vivo, and they corroborate previous biochemical studies of this histotypic culture system. Treatment of very immature cultures with a low dose of epidermal growth factor (EGF, 5 ng/ml) enhanced the developmental increase in GFAP, NSE, NF and GC immunoreactivity, suggesting an acceleration of neuronal and glial maturation. In addition, EGF was found to alter the cellular organization within the aggregates, presumably by influencing cell migration.     

Role for glial cells in regulating the functional expression of neuropeptide Y (NPY) neurons in aggregate cultures derived from dissociated fetal brain cells

A series of studies from our laboratory have established an aggregate culture system of fetal rat brain cells expressing neuropeptide Y (NPY) which can serve as a model to study the role of glia-neuron paracrine interactions in the developmental expression of NPY neurons. In this system, NPY production increases progressively with culture-age and it is induced by forskolin (FOR) and phorbol 12-myristate 13-acetate (PMA). We addressed the following question: Is the functional expression of the NPY neurons impaired in the absence of glial cells (particularly astrocytes) and if so, can secretory products of aggregates composed of the full complement of brain cells ( intact aggregates) restore the function of the impaired NPY neurons? Aggregates were generated from 17-day-old fetal rat cortex and maintained in serum-free medium for 13–15 days. Cytosine arabinoside (CA; doses of 0.5–8 μM) was added to the cultures on day 1 and the effectiveness in elimination of glial cells was verified on day 15 by measuring the incorporation of ³H thymidine into DNA and by immunostaining for the astrocyte marker glial fibrillary acidic protein (GFAP). Basal NPY production and FOR (10 μM) + PMA (20 nM) stimulated production of NPY on days 13–15 were taken as functional criteria. FOR + PMA induced ≈2-fold increase in NPY production in control cultures (no CA). CA inhibited both basal and FOR + PMA induced production of NPY and DNA synthesis in a dose-dependent manner: at 6 μM CA, basal NPY production was reduced by about 50%, FOR + PMA stimulated production of NPY and DNA synthesis were completely inhibited, and astrocytes were essentially eliminated. A competing dose of 100 μM deoxycytidine completely reversed the effects of 6 μM CA on DNA synthesis and basal and FOR + PMA stimulated production of NPY. Conditioned media derived from intact aggregates completely restored basal and partially (≈50%) restored FOR + PMA stimulated production of NPY in CA-treated aggregates. These results implicate secretory products of glial cells (astrocytes?) and/or glial-neuron paracrine interactions in regulating the developmental expression of NPY neurons in culture such that activation of the cAMP and protein kinase C pathways leads to increased production of NPY. © 1994 Wiley-Liss, Inc.     

Differentiation of human adult CD34+ stem cells into cells with a neural phenotype: role of astrocytes

It has recently been reported that adult hematopoietic stem cells can differentiate into neural cells, opening new frontiers in therapy for neurodegenerative diseases. In this study, adult human hematopoietic stem cells (HSCs) were isolated via magnetic bead sorting, using a specific CD34 antibody and cultured with human astrocyte culture conditioned medium (ACM). In order to evaluate their differentiation into neurons and/or astrocytes, ACM-treated cultures were probed for the expression of several neural markers. We observed morphological modifications and, after 20 days of treatment, cell morphology displayed extending processes. Immunocytochemistry, Western blotting and RT-PCR showed the expression of neuronal markers such as neurofilaments, neuron specific enolase (NSE) and NeuN in ACM-treated HSCs cultured in poly-L-lysine-coated dishes. On the contrary, when the same ACM-treated cells were grown on a plastic substrate, they expressed high levels of glial fibrillary acidic protein (GFAP), with only weak expression of neuronal markers. Nestin, a neural progenitor cell marker, was present in treated cells, regardless of the substrate. These results demonstrate that astrocytes can generate a suitable microenvironment for inducing HSCs to differentiate into neural cells. Therefore, adult bone marrow may represent a readily accessible source of cells for treating neurodegenerative diseases.     

Chronic Suppression of Bioelectric Activity and Cell Survival in Primary Cultures of Rat Cerebral Cortex: Biochemical Observations

Chronic suppression of spontaneously occurring bioelectric activity (BEA) has been shown to increase neuronal cell death in tissue culture, but may also affect astrocytes. We investigated this process in primary cultures of rat cerebral cortex by measuring the levels of NSE (neuron-specific enolase) and GFAP (glial fibrillary acidic protein) in relation to general tissue markers, including measurements for cell death and proliferation. In electrically active (control) cultures, the content of DNA, protein, and NSE became maximal between 21 and 28 days in vitro (DIV) and thereafter decreased, whereas the content of GFAP rose continuously up to 43 DIV. Chronic suppression of BEA by tetrodotoxin (TTX; from 6 DIV) decreased the content of DNA, total protein, and especially NSE. The content of GFAP was decreased in all culture series investigated, but with great temporal variations among culture series. Chronic TTX treatment (started at 6 DIV) increased the efflux of lactate dehydrogenase, a marker for cell lysis, between 12 and 21 DIV, but this efflux was mainly derived from the supporting glial cells with which the cerebral cortex cultures were cocultured. Chronic, but not acute (7 h) TTX treatment decreased total [3H]thymidine incorporation into DNA from 14 DIV; this appeared to be due to a reduced number of astrocytes. Chronic suppression of BEA with xylocaine from 6 DIV had similar effects on DNA-, protein-, and NSE-content as TTX, but led to an increased content of GFAP at 21 DIV. Chronic suppression of synaptic transmission with 10 mM Mg2+ and 0.2 mM Ca2+, starting at 6 DIV, increased the content of DNA, protein, and GFAP at 21 DIV, but NSE was still decreased. We conclude that chronic suppression of BEA in cerebral cortex cultures enhances neuronal cell death, whereas astrocytes are differentially affected, depending on the suppressing agent. As astrocytes may have a modulating effect on neuronal survival, their involvement should be regarded when studying the effects of chronic suppression of BEA on neuronal development.     

Vimentin-GFAP transition in primary dissociated cultures of rat embryo spinal cord

Primary dissociated cultures derived from 15-day-old rat embryo spinal cord with or without dorsal root ganglia (DRG) were grown on polylysine, Primaria and laminin substrates. On polylysine and Primaria substrates, spinal cord neurons formed aggregates connected by bundles of neurites in a distinctive pattern similar to that observed in cultures derived from embryonal rat brain and neonatal rat cerebellum. After 2 days in culture, the number of cells stained with GFAP antibodies progressively increased within the vimentin-positive monolayer surrounding the neuronal aggregates. These astrocytes had the typical appearance of astrocytes in primary dissociated cultures derived from late fetal or early neonatal murine brain, i.e. large flat or stellate cells with thick processes staining equally well with GFAP and vimentin antibodies. Astrocytes found within the neuronal aggregates in 4-5 day cultures were markedly different, i.e. small stellate cells with slender processes forming a delicate mesh throughout the aggregate. These GFAP-positive cells stained only weakly with vimentin antibodies. Spinal cord neurons formed aggregates on laminin substrates but failed to extend neurites and rapidly degenerated. The large flat cells in the surrounding monolayer gradually invaded the aggregates. These cells stained with both GFAP and vimentin antibodies. DRG neurons developed equally well on Primaria and laminin substrates, extending their neurites on the vimentin-positive flat cells forming the monolayer regardless of their reactivity with GFAP antibodies.     

Neuron–Glia Interaction in the Suprachiasmatic Nucleus: A Double Labeling Light and Electron Microscopic Immunocytochemical Study in the Rat

The morphological interactions between astroglial and neuronal elements were elucidated in the rat suprachiasmatic nucleus (SCN) by light and electron microscopic immunocytochemistry using antibodies against glial fibrillary acidic protein (GFAP), vasoactive intestinal peptide (VIP) and arginine-vasopressin (AVP). Throughout the SCN, particularly in its ventral portion, GFAP-like-immunoreactive (GFAP-LI) astroglial elements were found. These astrocytes displaying GFAP-like immunoreactivity occasionally contained fairly well-developed organelles. Some of these astrocytes were found as satellite cells in close contact with non-immunoreactive neuronal perikarya and processes. Around the neurons, GFAP-LI astroglial processes were also observed to cover some portions of presynaptic and postsynaptic elements. In addition, these astroglial elements were seen between two neuronal somata and pericytes of blood capillaries as glial endfeet. By double labeling immunoelectron microscopy using antibodies against GFAP/VIP and GFAP/AVP, some portions of VIP-like-immunoreactive or AVP-like-immunoreactive neuronal somata and processes were found to be engulfed by GFAP-LI astroglial processes. The possible functional roles of the morphological interactions between astroglial and neuronal elements are discussed.     

Serum-free culture of rat post-natal and fetal brainstem neurons

Serum-free medium is essential for cell culture studies in which complete control of the environment is required. Primary culture of post-natal brainstem neurons in defined medium has not been described in the literature, and successful culture of primary brainstem neurons is typically restricted to embryonic ages E14-E18. This study describes a method for culture of fetal and post-natal brainstem neurons using a serum-free culture medium. The culture system is based on Neurobasal medium supplemented with antioxidant-rich B27. Media and supplements are commercially available products from Life Technologies. Neuron survival was optimized by replacing glutamine with GlutaMaxI, by matching osmolality with neuronal age, and by using Hibernate medium to increase neuron survival during tissue dissociation. Fetal E14, E16, E20, and post-natal P3 and P6 cultures were examined after 4, 7, and 9 days in culture. Neuron and glial cells present in the cultures were identified using immunocytochemistry with antibodies raised against microtubule-associated protein 2 (MAP2) and glial fibrillary acidic protein (GFAP), respectively. Fetal E14 cultures had more bipolar neurons than multipolar neurons compared with developmentally older P6 cultures. Early fetal cultures had a higher percentage of neurons than late fetal and early post-natal cultures. Neuron survival was similar between 4 and 9 days in culture for all age groups tested. This is the first reliable, defined culture medium that supports brainstem neurons from late fetal and early post-natal stages of the rat for up to 6 days post-partum.     

Cell type-selective expression of green fluorescent protein and the calcium indicating protein,yellow cameleon,in rat cortical primary cultures

A cell type-specific green fluorescent protein (GFP) expression system in rat cortical primary cultures has been developed for the fluorescence labeling of brain cells. Lipid-mediated transfection (lipofection) was employed, allowing the establishment of a convenient efficient system for the analysis of individual cells. To achieve cell type-specific labeling, GFP expression vectors containing the rat neuron-specific enolase (NSE) gene promoter, human glial fibril acidic protein (GFAP) gene promoter, human elongation factor (EF-1alpha) gene promoter, or human cytomegalovirus (CMV) immediate early promoter were constructed, and their specificities examined. Vectors containing the CMV or GFAP promoter resulted primarily in GFP expression in astrocytes, while those containing the EF1-alpha or NSE promoter resulted primarily in GFP expression in neurons. This labeling system was applied to the morphological analysis of living neurons and to cell type-selective calcium imaging. Confocal microscopy revealed that individual GFP-expressing neurons had processes, which were longer than 500 microm and bore spine-like protrusions. A calcium-indicating GFP variant, yellow cameleon (YC2.1), was expressed in the same system, and cell type-selective calcium imaging performed. On pharmacological stimulation, YC2.1-expressing neurons responded to depolarizing stimuli, but not to the metabotropic glutamate receptor agonist, trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid (tACPD), while astrocytes responded only to tACPD.     

Immunocytochemical localization and developmental profile of neuron specific enolase (NSE) and non-neuronal enolase (NNE) in aggregating cell cultures of fetal rat brain

In aggregating cultures, neuron specific enolase (NSE) was first detected biochemically at 3 days. NSE levels increased with time in aggregate cultures and at 48 days reached a level which was 33% of that found in adult rat brain in vivo. The level of non-neuronal enolase (NNE) was essentially identical in aggregate cultures and normal rat brain. Immunocytochemically, NSE(+) cells first appeared at 10 days in vitro. Their number increased until 20 days in culture and then remained constant. When the immunocytochemical localization of NSE and NNE was compared in vibratome sections of 25 day aggregates, all identifiable neurons were NSE(+), NNE(−) and glial cells were NSE(−), NNE(+). In 1 μm thick epon sections of 30 day aggregates NSE antiserum stained neuronal cytoplasm intensely. Comparison of NSE staining in 1 μm thick epon sections with the same cell in an adjacently cut thin section provided conclusive evidence that NSE(+) cells were neurons and NSE(−) cells were glia. These results demonstrate that the three-dimensional organization of aggregate cells provides an excellent environment for neuronal differentiation and also emphasize the advantages of this culture system for multidisciplinary studies of brain development.     

成鼠骨髓基质细胞分化为神经元的体外研究

目的 :通过一定的培养条件 ,使骨髓基质细胞 (BMSC)分化为神经元和胶质细胞。方法 :以含有 EGF、b FGF的培养液培养 BMSC,经传代、换液去除杂质细胞 ,撤掉 EGF、 b FGF并加胶质细胞条件培养液及 BDNF,待细胞分化后进行形态学观察及 NSE、 GFAP染色。结果 :撤掉 EGF、 b FGF并加 BDNF,胶质细胞条件培养液后 2天可见分化细胞 ,NSE阳性细胞占细胞总数的 38.47± 3.2 7% ,GFAP阳性细胞占细胞总数的 5 0 .73± 3.2 6 % ,GFAP阳性细胞占细胞总数的 5 0 .73± 4.6 5 %。结论 :本实验通过 EGF、 b FGF及适宜的培养液成功对骨髓基质细胞进行定向 ,使其转化为神经干细胞并最终诱导其分化为神经元和胶质细胞     

Glial and neuronal differentiation in the human fetal brain 9–23 weeks of gestation

Nineteen human fetal brains ranging from 9-23 weeks of gestation were examined immunocytochemically for evidence of glial and neuronal differentiation. Radial glia were positive for vimentin and glial fibrillary acidic protein (GFAP) throughout the age range. S100-positive cells which were presumed to be astrocytes were present from 9 weeks; they were always more widespread in the cerebrum and the brainstem than GFAP-positive mature astrocytes, which could be detected with certainty only at 14 weeks. Carbonic anhydrase II (CA II)-positive oligodendrocytes were present in the brainstem in small numbers from 17 weeks. Neuronal fibre tracts in the cerebrum were positive for 160 kD phosphorylated neurofilament protein (BF10) from 9 weeks, but negative for 200 kD phosphorylated neurofilament protein (RT97) and for 70 and 200 kD non-phosphorylated neurofilament protein (NFP) whereas most tracts in the brainstem were positive for BF10 from 9 weeks and positive for the other neurofilament proteins from 14 weeks. Corticospinal tracts differed in remaining negative for neurofilament proteins other than BF10, which showed positive reaction throughout. Perikarya of differentiated neurons in all areas of the brain were neurofilament-negative but neuron specific enolase (NSE)-positive. Germinal eminence cells were focally vimentin-positive from 15 weeks, focally GFAP-positive from 17 weeks, and negative for all NFP and for NSE. The value of a short fixation time and pretreatment with trypsin in the immunocytochemical demonstration of GFAP is stressed.     

Region-specific immunolocalization of atrial natriuretic peptide in mixed fetal rat brain cell cultures.

In adult rodent CNS, atrial natriuretic peptide (ANP) has been localized by immunolabeling and nucleic acid hybridization techniques primarily to hypothalamic neurons and, to a lesser extent, to neurons of the telencephalon and brainstem. In canine brain, ANP immunoreactivity has been reported in neocortical and brainstem astrocytes. Yet, in a recent study using fetal rat tissue, ANP was detected by radioimmunoassay in predominantly neuronal, but not glial, cultures. In the present study ANP was detected by radioimmunoassay on in vitro day 8 in media derived from fetal rat diencephalic and rhombencephalic, but not telencephalic, cultures and in cell homogenates from all 3 regions surveyed. Using indirect immunofluorescence less than one cell per 400x field stained for ANP in the telencephalic cultures and ANP immunopositivity colocalized exclusively to neurons by concomitant anti-neurofilament immunolabeling. In diencephalic monolayers, approximately 1-3 cells per 400x field were ANP-positive; although most of these cells were neurons, small numbers of ANP-positive astrocytes were also detected using anti-glial fibrillary acidic protein (GFAP) immunolabeling. ANP-positive cells were most numerous in rhombencephalic cultures (5-10 cells per 400x field); as in diencephalic cultures, ANP immunoreactivity colocalized to both neurons and astrocytes in this region. In diencephalon and rhombencephalon, less than 1% of all ANP-positive cells were astrocytes and less than 1% of GFAP-positive astrocytes exhibited immunoreactive ANP. In fetal brain, neuronal and astrocytic ANP may play a role in fluid and electrolyte homeostasis. Alternatively, fetal brain cell ANP may subserve entirely different functions (e.g. as trophic factors) as has been suggested for other neuropeptides in the developing nervous system.     

Astrocytic shape and glial fibrillary acidic protein immunoreactivity are modified by estradiol in primary rat hypothalamic cultures

Primary cultures from fetal rat hypothalamus (embryonic day 15-16) were grown for 9 days in a serum-free medium and then fixed and immunostained for glial fibrillary acidic protein (GFAP). The majority of the GFAP-immunoreactive astrocytes were flat, polygonal, without processes and showed a low intensity immunoreactivity which was restricted to the perinuclear region. Elongated, process-bearing astrocytes, with an intense immunoreactivity in the soma and processes, were also observed in a smaller proportion (30%). Addition of estradiol (10(-12) to 10(-8) M) to the culture medium 24 h before GFAP immunostaining resulted in an increased proportion (59-69%) of process-bearing, intense immunoreactive cells. This effect was blocked by tamoxifen (10(-8) M). The total number of GFAP-immunoreactive astrocytes was not modified by estrogen or tamoxifen. These results indicate that estradiol may modulate the cell shape and the distribution of GFAP in astrocytes in culture and suggest that astrocytes can be a target for sex steroids during development of the central nervous system.     

Quantitative analysis of fetal rat brain neurons developing in primary cultures. I. Stereological study of the neuronal differentiation

An ultrastructural stereological analysis was performed to analyze the morphological differentiation of primary cultures of fetal rat brain neurons, growing for two weeks in a serum-free medium. The number of neurons and of gliofibrillary acidic protein (GFAP)-positive glial cells was estimated by light microscopy counting in the culture wells. These cultures provided a quasi-pure neuronal population, since the number of GFAP-positive glial cells was found to be 1% (day 7) and 2% (day 14) respectively of the total number of cultured cells. Cell counts and the stereological measurements were related to the surface area of the culture well. The neuronal differentiation was characterized by an increase in the plasma membrane surface area (x9) and volume (x8) of neurites, contrasting with the decrease in the perikarya surface area and volume. These primary stereological data were combined with the number of neurons to obtain parameters characterizing an average neuron. The increase in membrane surface area of an average neuron was found to be a linear function of time, 29 micron 2 and 445 micron 2 of new membrane being added per day of culture to perikarya and neurites respectively. The number of chemical synapses was also counted and compared to the changes in the plasma membrane surface area. After 7 days in vitro they increased in number more rapidly than the increase in the plasma membrane surface area of neurons.     

星形胶质细胞诱导成年骨髓基质细胞分化成神经元样细胞的实验研究

目的探讨星形胶质细胞能否在体外诱导成年大鼠骨髓基质细胞向神经元方向分化。方法采用新生鼠海马组织来源的星形胶质细胞与转染有绿色荧光蛋白基因的骨髓基质细胞(MSCs)进行共培养,并分成三组:共培养组、共培养 脑源性神经营养因子(BDNF)组、单纯碱性成纤维生长因子(bFGF) BDNF诱导分化组,在相差显微镜下每日观察、记录MSCs的诱导分化状况,并应用免疫荧光染色技术对分化后的MSCs进行鉴定,同时应用流式细胞术测定MSCs分化前后及星形胶质细胞的DNA含量。结果共培养第4天,部分MSCs已初步具备神经元形态:折光性强的锥形或圆形胞体及长的多极突起,免疫荧光染色呈微管相关蛋白(MAP-2ab)、神经元特异性烯醇化酶(NSE)阳性;DNA的含量测定结果显示诱导分化后第7天未发现有四倍体、六倍体细胞。结论(1)星形胶质细胞可以在体外诱导成年大鼠MSCs向神经元方向分化,这种分化并不是由细胞融合引起的。(2)星形胶质细胞具有调控神经元的分化、促进神经元成熟的功能。     

Rapid growth of astrocytic processes in N. magnocellularis following cochlea removal.

Removal of the cochlea or pharmacological blockade of eighth nerve activity in young postnatal chickens results in rapid transneuronal cell death and atrophy in neurons of n. magnocellularis. The present experiments were designed to examine the influence of afferent input on astrocyte structure in n. magnocellularis. Young chickens were subjected to unilateral cochlea removal. At times ranging from 5 minutes to 72 hours later, the brainstems were histologically processed with a polyclonal antibody against glial fibrillary acidic protein (GFAP). A second group of chick brainstems was impregnated by a Golgi method 6 hours after unilateral cochlea removal and impregnated three-dimensional reconstructions were made of glial cells in n. magnocellularis (NM). Analyses of GFAP positive processes in NM revealed an observable increase in the number of astrocytic processes at the borders of the nucleus within 30 minutes of cochlea removal and a twofold increase in GFAP + glial processes by 6 hours. A secondary increase in the number and density of GFAP + processes occurred between 24 and 72 hours following cochlea removal, during the period of axonal degeneration, and transneuronal cell atrophy and death. Analyses of astrocytes impregnated by the Golgi method revealed that individual glial cells had increased their total process length and the number of processes by approximately twofold by 6 hours after cochlea removal. These results suggest that the structure of astrocytes is rapidly and dramatically influenced by the level of excitatory activity in a neuronal system. Furthermore, the similarity of results obtained with GFAP immunohistochemistry and three-dimensional reconstruction of astrocytes provides evidence that the short-term changes observed following cochlea removal represent the actual growth of glial processes. We speculate that modulations in glial processes as a function of afferent activity may act to influence synaptic efficacy.     

Ontogeny of radial and other astroglial cells in murine cerebral cortex.

Three cell forms of astroglial lineage populate the prenatal and early postnatal murine cerebral wall. In the present review we consider the ontogeny of these cell forms with respect to histogenetic events of the perinatal period. Classic bipolar radial glial cells predominate prior to E17. The bipolar coexist with monopolar radial forms in the perinatal period. Both bipolar and monopolar radial forms coexist with multipolar astrocytes in the course of the first postnatal week and are ultimately succeeded by the multipolar cells. The shift from bipolar to monopolar radial forms is initially coincident with translocation of somata of bipolar cells from the ventricular zone to the upper intermediate zone and cortical strata. Arborization appears to occur both at the growing tips and along the shaft of the processes of both bipolar and monopolar radial cell types. As arborization continues, the processes of the monopolar radial cells come to resemble those of the multipolar astrocytes. Eventually the radial cells are fully transformed into the multipolar astrocytic forms. During this period of transition, radial processes in the cortex appear to be degenerating, suggesting that regressive processes contribute to the cytologic transformation. This sequence of transformations begins late in the period of neuronal migration and continues through the early stages of growth and differentiation in the murine cerebral cortex. The signals that induce these changes may arise from differentiating neurons within the cortex. These transformations occur at a time when radial glial fibers are no longer required as guides for neuronal migration, and the glial population assumes new roles related to the development and operation of cortical neuronal circuits.     

碱性成纤维生长因子等诱导间质干细胞分化为神经元样细胞的研究

目的　体外定向诱导成人骨髓间质干细胞 (MSC)分化为神经元样细胞。方法　采用Ficoll Paque液 (10 77g/L)离心分离成人MSC ,体外扩增 ,分别采用含碱性成纤维细胞生长因子 (bFGF)和叔丁对甲氧酚 (BHA)或硫代甘油等试剂的无血清DMEM诱导MSC分化为神经元。免疫组化鉴定神经元烯醇化酶 (NSE)、神经丝蛋白 (NF)、胶质纤维酸性蛋白 (GFAP)、巢蛋白 (nestin)的表达。结果　成人骨髓间质干细胞在体外扩增原代可获得 5× 10 5,10代可获得 2× 10 10 个细胞。加入bFGF和BHA等诱导剂或硫代甘油诱导后 ,MSC胞体收缩 ,突起伸出 ;免疫组化显示诱导出的神经元样细胞NSE、NF、nestin表达阳性 ,GFAP阴性。结论　成人骨髓间质干细胞在体外可以分化为神经元样细胞。     

In search of the central respiratory neurons: I. Dissociated cell cultures of respiratory areas from the upper medulla.

Dissociated cells from the areas of the nucleus ambiguus and the nucleus tractus solitarius obtained by tissue punch or block dissection from coronal slices of the medulla at the level of the obex were cultured from fetal rats at 18 to 21 days gestation. The dissociated neurons were plated either directly in vitrogen-coated 35 mm tissue culture dishes or in such dishes which had been seeded with subcultures of cortex- or medulla-derived astrocytes. After the astrocytes reached confluency and were treated with an antimitotic agent, dissociated nucleus ambiguus or nucleus tractus solitarius was plated at 0.5-1.0 x 10(6) cells per dish. Neurons grew well on monolayers of medullary or cortical astrocytes, but survived poorly on vitrogen-coated dishes without a cellular substrate. Rat medulla was preferred as the source of astrocytes. Tissue dissociation with papain rather than trypsin produced less cellular debris, and the neuronal yield from the tissue was higher. The neuronal population was heterogenous in morphology including small and large bipolar, pyramidal, and multipolar cells. Neurons sensitive to CO2 and/or low pH (Rigatto et al., J Neurosci Res 33:590-597, 1992) did not appear to have any definitive morphologic characteristics, but most were multipolar. These neurons stained well with antibodies to neuron-specific enolase and Fragment C of tetanus toxin, but not to choline acetyltransferase (ChAT). These findings suggest that neurons possibly responsible for the central regulation of respiration can be maintained for several weeks in dissociated cell culture, providing a system for neurotransmitter, electrophysiological, and morphological studies.