Developmental expression of glial markers in ependymocytes of the rat subcommissural organ: role of the environment

Summary The rat subcommissural organ (SCO), principally composed of modified ependymocytes (a type of glial cell), is a suitable model for the in vivo study of glial differentiation. An immunohistochemical study of the ontogenesis of rat SCO-ependymocytes from embryonic day 13 to postnatal day 10 shows that these cells express transitory glial fibrillary acidic protein (GFAP) from embryonic day 19 until postnatal day 3. However, S100 protein (S100) is never expressed in the SCO-cells, contrasting with the ventricle-lining cells of the third ventricle, which contain S100 as early as embryonic day 17. Environmental factors could be responsible for the repression of GFAP and S100 in adult rats, because GFAP and S100 are observed in ependymocytes of SCO 3 months after being grafted from newborn rat into the fourth ventricle of an adult rat. Neuronal factors might be involved in the control of the expression of S100, since after the destruction of serotonin innervation by neurotoxin at birth, S100 can be observed in some SCO-ependymocytes of adult rats. On the other hand, GFAP expression is apparently not affected by serotomin denervation, suggesting the existence of several factors involved in the differentiation of SCO-cells.     

Study of effect of serotonin on histogenesis of rat embryonal neocortex at model of ectopic neurotransplantation

A comparative study has been performed of dynamics of development of ectopic transplants of embryonal (E14) neocortex anlages obtained from intact rats and from the rats administered with serotonin inhibitor para-chlorophenylalanine (PCPA) at the 11th day of pregnancy as well as after incubation of such anlages in the serotonin-containing medium. The goal of this work was to elucidate effect of serotonin on division and differentiation of embryonal neocortex cells. Study of degeneration, mitotic activity, and differentiation (by using immunohistochemical detection of nerve cell nuclear protein--NeuN) of transplanted cells has allowed establishing that serotonin promotes survival and differentiation of transplant neuroepithelial cells as well as participates in regulation of their proliferation. It is suggested that serotonin produces stimulation effect on the cell cycle rate of transplantated cells to thereby accelerate neuronal differentiation.     

Origin of evoked potentials recorded in neocortical grafts

This study compared evoked potentials (EP) recorded from the neocortical vibrissae representation region of adult rats (the barrelfield) with those occurring in neocortical grafts 4–5 months after transplant. Epidural recording showed that displacing the vibrassae led to shaping of EP in the intact barrelfield beginning with a positive followed by a negative component. The EP recorded from the surface of the graft occurred with the same latency but consisted of a negative component only. In conclusion, coordinated "population" discharges of grafted neurons do generate local field poentials, but owing to the diffuse neuronal organization of the grafts, these potentials are difficult to distinguish from EP resulting from passive current from adjacent neocortical areas and subcortical structures of the host brain.Institute of Biological Physics, Academy of Sciences of the USSR, Pushchino, Moscow Region. Institute of Neurobiology and Brain Research, Academy of Sciences of the GDR, Magdeburg. Translated from Neirofiziologiya, Vol. 21, No. 4, pp. 490–497, July–August, 1989.     

大鼠胚胎神经干细胞纹状体内移植后特性

观察大鼠胚胎神经干细胞移植入成年大鼠纹状体后的存活、迁移和分化状况。自14天胎鼠脑室下区分离获得神经干细胞,利用无血清培养基培养扩增并进行鉴定。经4～5代的扩增后,以BrdU标记的神经干细胞通过脑立体定位注射移植入成年大鼠纹状体内,然后分别于移植后2周、4周、6周和8周时做脑冰冻切片,通过免疫组织化学和免疫荧光方法检测移植细胞的数量、定位和分化情况。8周后移植细胞的检出率约16%;移植细胞向周围宿主组织有广泛的迁移表现,尤以沿着白质束向头尾方向的迁移最为显著,最远向后侧达到内囊;纹状体中移植细胞主要分化为神经元和星形胶质细胞。星形胶质细胞数量最多,主要位于移植区与宿主组织临界部位,而神经元处于移植区中央。培养的大鼠胚胎神经干细胞可以作为移植替代治疗神经退行性疾病研究的供体细胞源,而移植中的迁移现象值得注意。     

Localization of putative stem cells and four cell populations with different differentiation degree in mouse mammary anlagen

The precise localization of putative stem cells and other cells within the embryonic mammary gland would help to elucidate the molecular pathways that govern normal mammary development. The ultrastructural appearances and the antigen of Sca-1 were considered together as putative stem cell markers, and the antigens of cytokeratin, CD10, Muc-1 and CD34 as differentiation markers. Electron microscopy was performed to reveal the ultrastructure of cells in different site of the mammary anlagen. An immunofluorescence system was used to reveal the whole structure profile of the mammary anlagen using the anti-cytokeratin antibody to localize specific types of cell populations such as Sca-1, CD10, Muc-1 and CD34 positive cells within the anlagen, which distribute near the inside edge, distribute uniformly, distribute in the central region and distribute in the site of mesenchyme around the neck of the anlagen, respectively. We also observed under electron microscope that some pale cells like putative stem cells reported by prevenient scholars, which are mainly distributed in the Sca-1⁺ cell population near the inside edge of the anlagen, have pale-stained nucleoplasm and cytoplasm, sparse organelles clustered close to their nucleus and have a lack of rough endoplasmic reticulums and cell polarization. The results indicate that the putative stem cells are localized near the edge of the mammary anlagen; the cell populations with different differentiation degree were localized in the central part and around the edge within the anlagen.     

Transplantation of porcine embryonic stem cells and their derived neuronal progenitors in a spinal cord injury rat model

Background aimsThe purpose of this study was to investigate therapeutic potential of green fluorescent protein expressing porcine embryonic stem (pES/GFP⁺) cells in A rat model of spinal cord injury (SCI).MethodsUndifferentiated pES/GFP⁺ cells and their neuronal differentiation derivatives were transplanted into the contused spinal cord of the Long Evans rat, and in situ development of the cells was determined by using a live animal fluorescence optical imaging system every 15 days. After pES/GFP⁺ cell transplantation, the behavior functional recovery of the SCI rats was assessed with the Basso, Beattie, and Bresnahan Locomotor Rating Scale (BBB scale), and the growth and differentiation of the grafted pES/GFP⁺ cells in the SCI rats were analyzed by immunohistochemical staining.ResultsThe relative green fluorescent protein expression level was decreased for 3 months after transplantation. The pES/GFP⁺-derived cells positively stained with neural specific antibodies of anti-NFL, anti-MBP, anti-SYP and anti-Tuj 1 were detected at the transplanted position. The SCI rats grafted with the D18 neuronal progenitors showed a significant functional recovery of hindlimbs and exhibited the highest BBB scale score of 15.20 ± 1.43 at week 24. The SCI rats treated with pES/GFP⁺-derived neural progenitors demonstrated a better functional recovery.ConclusionsTransplantation of porcine embryonic stem (pES)-derived D18 neuronal progenitors has treatment potential for SCI, and functional behavior improvement of grafted pES-derived cells in SCI model rats suggests the potential for further application of pES cells in the study of replacement medicine and functionally degenerative pathologies.     

Plasticity of endothelial cells during arterial-venous differentiation in the avian embryo

Remodeling of the primary vascular system of the embryo into arteries and veins has long been thought to depend largely on the influence of hemodynamic forces. This view was recently challenged by the discovery of several molecules specifically expressed by arterial or venous endothelial cells. We here analysed the expression of neuropilin-1 and TIE2, two transmembrane receptors known to play a role in vascular development. In birds, neuropilin-1 was expressed by arterial endothelium and wall cells, but absent from veins. TIE2 was strongly expressed in embryonic veins, but only weakly transcribed in most arteries. To examine whether endothelial cells are committed to an arterial or venous fate once they express these specific receptors, we constructed quail-chick chimeras. The dorsal aorta, carotid artery and the cardinal and jugular veins were isolated together with the vessel wall from quail embryos between embryonic day 2 to 15 and grafted into the coelom of chick hosts. Until embryonic day 7, all grafts yielded endothelial cells that colonized both host arteries and veins. After embryonic day 7, endothelial plasticity was progressively lost and from embryonic day 11 grafts of arteries yielded endothelial cells that colonized only chick arteries and rarely reached the host veins, while grafts of jugular veins colonized mainly host veins. When isolated from the vessel wall, quail aortic endothelial cells from embryonic day 11 embryos were able to colonize both host arteries and veins. Our results show that despite the expression of arterial or venous markers the endothelium remains plastic with regard to arterial-venous differentiation until late in embryonic development and point to a role for the vessel wall in endothelial plasticity and vessel identity.     

Study of effect of embryonic anlage allografts of the rat spinal cord on growth of regenerating fibers of the recipient nerve

A comparative study of the effect of tissue and suspension allografts of an embryonic spinal cord on regeneration of nerve fibers of impaired (by application of a ligature) sciatic nerve in rats was conducted. It was demonstrated that unlike tissue grafts that reach a large volume 21 and 60 days after transplantation, suspension grafts do not inhibit the growth of axons of the recipient to the periphery. It was established that introduction of a suspension of dissociated cells of the spinal cord embryonic anlages (but not fragments of these anlages) into the impaired sciatic nerve in rats results in an increase in the amount of myelinated regenerating nerve fibers of the recipient 60 days after the operation.     

Phase Relations between Host and Grafted SCN Depend on Graft Location in Rats

Fetal SCN grafts into intact rats were used as a model to study coupling among circadian oscillators. The phase relation in metabolic activity was analyzed between host SCN and fetal SCN grafted into the third (n = 24) or lateral (n = 18) ventricles. Host and third ventricle SCN grafts showed metabolic activity rhythms with peak values at CT 6, as extrapolated from drinking rhythmicity. In lateral ventricle grafted animals only the host SCN showed rhythmicity, although peak values oc curred as indicated by the drinking rhythms at CT 9. The present data suggest asymmetrical communication between host and grafted SCN depending on graft location, which may be related to different types of signal transmission.     

SENSITIVITY OF EARLY EMBRYONIC THYMIC LYMPHOID DEVELOPMENT TO 3H-THYMIDINE OF HIGH SPECIFIC ACTIVITY

The pulse technique, using high specific activity ³H-TdR to selectively kill cells in cell cycle, was applied to the thymic anlagen of chick embryos. With optimal specific and total ³H-TdR activities and pulse times of 2–4 hr the subsequent lymphoid development in organ culture of the thymic anlagen of 10-day-old chick embryos could be almost completely inhibited. The most important effect of the ³H-TdR was on the lymphoid precursor cells of the anlagen. The thymic epithelium appeared more resistant to ³H-TdR and allowed a lymphoid development of pulsed anlagen grafted to the chorioallantoic membrane of chick embryos when new lymphoid precursor cells were provided. The lymphoid precursor cells of the thymic anlagen of 10-day-old chick embryos therefore appeared to be in cell cycle with short generation time. The thymic anlagen of 8-, 9- and 10-day-old but not 7-day-old embryos showed a lymphoid development in organ culture. They did not differ with respect to the sensitivity to hot pulses of ³H-TdR. Thus no evidence of a lag in the onset of lymphoid precursor cell proliferation during the development of the early embryonic chick thymus was noted.     

Expression of neuronal markers suggests heterogeneity of chick sympathoadrenal cells prior to invasion of the adrenal anlagen

We have analyzed the distribution of neural crest-derived precursors and the expression of catecholaminergic and neuronal markers in developing adrenal tissue of chick embryos. Undifferentiated neural crest cells are found in presumptive adrenal regions from embryonic day 3 (E3) onward. An increasing proportion of cells expressing tyrosine hydroxylase (TH) mRNA indicates catecholaminergic differentiation of precursors not only in primary sympathetic ganglia, but also in presumptive adrenal regions. Whereas precursors and differentiating cells show mesenchymal distribution until E5, discrete adrenal anlagen form during E6. Even during E5, catecholaminergic cells with low or undetectable neurofilament M (NF-M) mRNA expression prevail in positions at which adrenal anlagen become distinct during E6. The predominance of TH-positive and NF-M-negative cells is maintained throughout embryogenesis in adrenal tissue. RNA encoding SCG10, a pan-neuronal marker like NF-M, is strongly expressed throughout adrenal anlagen during E6 but is found at reduced levels in chromaffin cells compared with neuronal cells at E15. Two additional neuronal markers, synaptotagmin 1 and neurexin 1, are expressed at low to undetectable levels in developing chromaffin cells throughout embryogenesis. The developmental regulation of neuronal markers shows at least three different patterns among the four mRNAs analyzed. Importantly, there is no generalized downregulation of neuronal markers in developing adrenal anlagen. Thus, our observations question the classical concept of chromaffin differentiation from a common sympathoadrenal progenitor expressing neuronal properties and suggest alternative models with changing instructive signals or separate progenitor populations for sympathetic neuronal and chromaffin endocrine cells.Chaya Kalcheim and Klaus Unsicker are supported by the Deutsche Forschungsgemeinschaft (SFB 488)     

Carbon nanotubes promote neuron differentiation from human embryonic stem cells

Human embryonic stem cells (hESCs) hold great promise for regenerative medicine and transplantation therapy due to their self-renewal and pluripotent properties. We report that 2D thin film scaffolds composed of biocompatible polymer grafted carbon nanotubes (CNTs), can selectively differentiate human embryonic stem cells into neuron cells while maintaining excellent cell viability. According to fluorescence image analysis, neuron differentiation efficiency of poly(acrylic acid) grafted CNT thin films is significant greater than that on poly(acrylic acid) thin films. When compared with the conventional poly-l-ornithine surfaces, a standard substratum commonly used for neuron culture, this new type thin film scaffold shows enhanced neuron differentiation. No noticeable cytotoxic effect difference has been detected between these two surfaces. The surface analysis and cell adhesion study have suggested that CNT-based surfaces can enhance protein adsorption and cell attachment. This finding indicates that CNT-based materials are excellent candidates for hESCs’ neuron differentiation.     

Incorporation of neurons of transplants of embryonal rat neocortex in the achievement of sensory function of the cerebral cortex of the recipient

The barrelfield of the adult rats was removed by suction and embryonic tissue of the somatosensory neocortex was transplanted into the cavity. Spontaneous and evoked activity of the grafted neurones was investigated extracellularly 2-3 months after the grafting. The light microscopy of the grafts revealed the presence of normal neuronal cells, but their distribution was diffuse, and they were not organized into barrels as in intact neocortex. The background activity of grafted neurones depended upon the level of the recipient's anaesthesia. The response types of the grafted neurones to vibrissae deflection and to tactile stimulation of the host body surfaces, their latencies and lability did not differ from such of the intact somatosensory cortex, but the receptive fields of the grafted neurones were larger. There was also substantial convergence of inputs from other surfaces upon the grafted neurones. The effectiveness of stimulation of the various skin areas was determined by the proximity of their neocortical representations to the graft.     

Islet cell engraftment and control of diabetes in rats after transplantation of pig pancreatic anlagen

The insufficient supply of tissue, loss posttransplantation, and limited potential for expansion of beta-cells restrict the use of islet allotransplantation for diabetes. A way to overcome the supply and expansion problems is to xenotransplant embryonic tissue. We have shown that whole rat pancreatic anlagen isotransplanted into the omentum of rats, or xenotransplanted into costimulatory blocked mice, undergo growth and differentiate into islets surrounded by stoma without exocrine tissue. Isotransplants normalize glucose tolerance in diabetic hosts. Here, we show that embryonic day 29 porcine pancreas transplanted into the omentum of adult diabetic rats undergoes endocrine tissue differentiation over 20 wk and normalizes body weights and glucose tolerance. Unlike rat-to-rodent transplants, individual alpha- and beta-cells engraft without a stromal component, and no immunosuppression is required for pig-to-rat transplants. Herein is described a novel means to effect the xenotransplantation of individual islet cells across a highly disparate barrier.     

Phase Relations between Host and Grafted SCN Depend on Graft Location in Rats

Fetal SCN grafts into intact rats were used as a model to study coupling among circadian oscillators. The phase relation in metabolic activity was analyzed between host SCN and fetal SCN grafted into the third ( n = 24) or lateral ( n = 18) ventricles. Host and third ventricle SCN grafts showed metabolic activity rhythms with peak values at CT 6, as extrapolated from drinking rhythmicity. In lateral ventricle grafted animals only the host SCN showed rhythmicity, although peak values oc curred as indicated by the drinking rhythms at CT 9. The present data suggest asymmetrical communication between host and grafted SCN depending on graft location, which may be related to different types of signal transmission.     

Functional integration of grafted neural stem cell-derived dopaminergic neurons monitored by optogenetics in an in vitro Parkinson model

Intrastriatal grafts of stem cell-derived dopamine (DA) neurons induce behavioral recovery in animal models of Parkinson''s disease (PD), but how they functionally integrate in host neural circuitries is poorly understood. Here, Wnt5a-overexpressing neural stem cells derived from embryonic ventral mesencephalon of tyrosine hydroxylase-GFP transgenic mice were expanded as neurospheres and transplanted into organotypic cultures of wild type mouse striatum. Differentiated GFP-labeled DA neurons in the grafts exhibited mature neuronal properties, including spontaneous firing of action potentials, presence of post-synaptic currents, and functional expression of DA D₂ autoreceptors. These properties resembled those recorded from identical cells in acute slices of intrastriatal grafts in the 6-hydroxy-DA-induced mouse PD model and from DA neurons in intact substantia nigra. Optogenetic activation or inhibition of grafted cells and host neurons using channelrhodopsin-2 (ChR2) and halorhodopsin (NpHR), respectively, revealed complex, bi-directional synaptic interactions between grafted cells and host neurons and extensive synaptic connectivity within the graft. Our data demonstrate for the first time using optogenetics that ectopically grafted stem cell-derived DA neurons become functionally integrated in the DA-denervated striatum. Further optogenetic dissection of the synaptic wiring between grafted and host neurons will be crucial to clarify the cellular and synaptic mechanisms underlying behavioral recovery as well as adverse effects following stem cell-based DA cell replacement strategies in PD.     

Induction of Secondary Embryos by Intra- and Interspecific Grafts of Center Cells under the blastopore in Horseshoe Crabs

When the cell mass (center cells) of the early gastrulae in both American and Asian horseshoe crabs was grafted into the embryo of the homologous species, secondary embryos were formed as a result of these grafts. Secondary embryos were also formed in a similar way when the center cells of heterologous embryos were grafted between the American and Asian horseshoe crab embryos. The characteristics of the secondary embryos were similar to the host embryos in both cases, indicating that the center cells played the roles same as those by the amphibian organizer. The homogenate of center cells also induced the formation of secondary embryos. This is the first published study in which secondary embryos of horseshoe crabs have been induced by grafting. The fact may mean that this type of embryonic induction is widespread in the animal kingdom.     

Effect of serotonin on the formation of neurons producing gonadotropin-releasing hormone in Wistar rats

The effect of serotonin on the formation of neurons producing gonadotropin-releasing hormone (GnRH) during embryogenesis of Wistar rats was studied. The neurons producing GnRH were detected immunocytochemically on days 18 and 21 of embryonic development and on day 15 of postnatal development of rats with normal serotonin metabolism and rats in which the synthesis of serotonin was inhibited by p-chlorophenylalanine. The total number of GnRH neurons in serotonin deficiency was larger than in the case of its normal metabolism at all developmental stages studied. This is an indirect evidence for the inhibitory effect of serotonin on the formation of GnRH neurons. To confirm the morphogenetic effect of serotonin, we studied the rate of formation of GnRH neurons by injecting bromodeoxyuridine in the formation period of these neurons. It was found that serotonin deficiency had no effect on the time of formation of GnRH neurons: over 97% of neurons formed on days 11 to 15 of embryonic development both in the experimental and control groups. Note that, in serotonin deficiency, the maximum number of GnRH neurons formed one day later than in the normal state. Thus, serotonin inhibits the proliferation of GnRH neuron progenitor cells and thereby has a morphogenetic effect on the development of these neurons.     

Intrastriatal grafts of mesenchymal stem cells in adult intact rats can elevate tyrosine hydroxylase expression and dopamine levels

MSCs (mesenchymal stem cells) derived from the bone marrow have shown to be a promising source of stem cells in a therapeutic strategy of neurodegenerative disorder. Also, MSCs can enhance the TH (tyrosine hydroxylase) expression and DA (dopamine) content in catecholaminergic cells by in vitro co‐culture system. In the present study, we investigated the effect of intrastriatal grafts of MSCs on TH protein and gene levels and DA content in adult intact rats. When MSCs were transplanted into the striatum of normal rats, the grafted striatum not only had significantly higher TH protein and mRNA levels, but also significantly higher DA content than the untransplanted striatum. Meanwhile, the grafted MSCs differentiated into neurons, astrocytes and oligodendrocytes; however, TH‐positive cells could not be detected in our study. These experimental results offer further evidence that MSCs are a promising candidate for treating neurodegenerative diseases such as Parkinson's disease.