The location and distribution of neural crest-derived Schwann cells in developing peripheral nerves in the chick forelimb.

The location and distribution of neural crest-derived Schwann cells during development of the peripheral nerves of chick forelimbs were examined using chick-quail chimeras. Neural crest cells were labeled by transplantation of the dorsal part of the neural tube from a quail donor to a chick host at levels of the neural tube destined to give rise to brachial innervation. The ventral roots, spinal nerves, and peripheral nerves innervating the chick forelimb were examined for the presence of quail-derived neural crest cells at several stages of embryonic development. These quail cells are likely to be Schwann cells or their precursors. Quail-derived Schwann cells were present in ventral roots and spinal nerves, and were distributed along previously described neural crest migratory pathways or along the peripheral nerve fibers at all stages of development examined. During early stages of wing innervation, quail-derived Schwann cells were not evenly distributed, but were concentrated in the ventral root and at the brachial plexus. The density of neural crest-derived Schwann cells decreased distal to the plexus, and no Schwann cells were ever seen in advance of the growing nerve front. When the characteristic peripheral nerve branching pattern was first formed, Schwann cells were clustered where muscle nerves diverged from common nerve trunks. In still older embryos, neural crest-derived Schwann cells were evenly distributed along the length of the peripheral nerves from the ventral root to the distal nerve terminations within the musculature of the forelimb. These observations indicate that Schwann cells accompany axons into the developing limb, but they do not appear to lead or direct axons to their targets. The transient clustering of neural crest-derived Schwann cells in the ventral root and at places where axon trajectories diverge from one another may reflect a response to some environmental feature within these regions.     

Developmental potential of neural crest-derived cells migrating from segments of developing quail bowel back-grafted into younger chick host embryos

The technique of back-transplantation was used to investigate the developmental potential of neural crest-derived cells that have migrated to and colonized the avian bowel. Segments of quail bowel (removed at E4) were grafted between the somites and neural tube of younger (E2) chick host embryos. Grafts were placed at a truncal level, adjacent to somites 14-24. Initial experiments, done in vitro, confirmed that crest-derived cells are capable of migrating out of segments of foregut explanted at E4. The foregut, which at E4 has been colonized by cells derived from the vagal crest, served as the donor tissue. Comparative observations were made following grafts of control tissues, which included hindgut, lung primordia, mesonephros and limb bud. Additional experiments were done with chimeric bowel in which only the crest-derived cells were of quail origin. Targets in the host embryos colonized by crest-derived cells from the foregut grafts included the neural tube, spinal roots and ganglia, peripheral nerves, sympathetic ganglia and the adrenals, but not the gut. Donor cells in these target organs were immunostained by the monoclonal antibody, NC-1, indicating that they were crest-derived and developing along neural or glial lineages. Some of the crest-derived cells (NC-1-immunoreactive) that left the bowel and reached sympathetic ganglia, but not peripheral nerves or dorsal root ganglia, co-expressed tyrosine hydroxylase immunoreactivity, a neural characteristic never expressed by crest-derived cells in the avian gut. None of the cells leaving enteric back-grafts produced pigment. Cells of mesodermal origin were also found to leave donor explants and aggregate in dermis and feather germs near the grafts. These observations indicate that crest-derived cells, having previously migrated to the bowel, retain the ability to migrate to distant sites in a younger embryo. The routes taken by these cells appear to reflect, not their previous migratory experience, but the level of the host embryo into which the graft is placed. Some of the population of crest-derived cells that leave the back-transplanted gut remain capable of expressing phenotypes that they do not express within the bowel in situ, but which are appropriate for the site in the host embryo to which they migrate.     

Expression of cholinesterase in the Schwann cells of peripheral nerves during development

In peripheral nerves of mouse embryos Schwann cells exhibit a high activity of unspecific cholinesterase. At first (day 12 of embryonic development) this enzyme occurs in the nuclear envelope and in the granular endoplasmic reticulum. Thus, it is possible to differentiate between Schwann cells and fibroblasts which lack cholinesterase. Later on (day 16) the cholinesterase has shifted to the cell membrane of the Schwann cells. However, only that part of the plasmalemma which encircles single axons and the mesaxons exhibits an irregular deposition of the reaction end product. In newborns the first loops of the just formed myelin sheath are still stained. With maturation of the myelin sheath the enzyme activity disappears. The functional role of cholinesterase is unclear. Possible roles are discussed. The expression of cholinesterase in Schwann cells depends on the integrity of the axons. After a few hours, the cultivation of amputated limbs results in a reduction of the enzyme activity. After 1 day in culture cholinesterase disappears totally.     

An efficient system for selection and culture of Schwann cells from adult rat peripheral nerves

Schwann cells (SCs), the supporting cells of the peripheral nerves, are indispensable for regenerating the peripheral and central nervous system. Copious preparation of these cells in a well-defined manner is to be a privileged position. SCs cultivation is overwhelmed by contaminating fibroblasts which are often outgrowing as the predominant cell type in an in vitro culture. This study introduces a technically simple and efficient procedure for SCs isolation and enrichment based on implementing recombinant and defined supplements. Collected adult rat sciatic nerves were cultured for 10 days as in vitro predegeneration. After dissociation and plating, the medium changed to knockout serum replacement supplemented DMDM/F12 medium containing various growth factors. The whole procedure took 3 weeks and SCs purity was then evaluated through implementing specific cytoplasmic and membranous markers. The viability of enriched SCs were evaluated by MTT assay. Within 10 days, over 99 % homogenous SCs were achieved and confirmed through immunofluorescence staining and flow-cytometry for P75^NTR and S100 markers, respectively. MTT data revealed that the viability and metabolic activities of purified SCs were increased in expansion medium. This study provides a technically easy and efficient method with the benefits of not utilizing bovine serum or other animal products for SCs isolation and enrichment.     

The instability of the neural crest phenotypes: Schwann cells can differentiate into myofibroblasts

In the vertebrate embryo, the neural crest cells (NCCs) that migrate out from the neural primordium yield multiple phenotypes, including melanocytes, peripheral neurones and glia and, in the head, cartilage, bone, connective cells and myofibroblasts / vascular smooth muscle cells (SMCs). The differentiation of pluripotent NCCs is mainly directed by local growth factors. Even at postmigratory stages, NC-derived cells exhibit some fate plasticity. Thus, we reported earlier that pigment cells and Schwann cells are able in vitro to interconvert in the presence of endothelin 3 (ET3). Here, we further investigated the capacity of Schwann cells to reprogram their phenotype. We show that purified quail Schwann cells in dissociated cultures produce alpha smooth muscle actin ((alpha)SMA)-expressing myofibroblasts through the generation of a pluripotent progeny. This transdifferentiation took place independently of ET3, but was promoted by transforming growth factor beta1 (TGF(beta)1). Moreover, when implanted into chick embryos, the Schwann cells were found to contribute with host cephalic NCCs to perivascular SMCs. These data provided the first evidence for the acquisition of an NC-derived mesenchymal fate by Schwann cells and further demonstrate that the differentiation state of NC-derived cells is unstable and capable of reprogramming. The high plasticity of Schwann cells evidenced here also suggests that, as in the CNS, glial cells of the PNS may function as NC stem cells in particular circumstances such as repair.     

The effects of embryonic retinal neurons on neural crest cell differentiation into Schwann cells

Amongst the many cell types that differentiate from migratory neural crest cells are the Schwann cells of the peripheral nervous system. While it has been demonstrated that Schwann cells will not fully differentiate unless in contact with neurons, the factors that cause neural crest cells to enter the differentiative pathway that leads to Schwann cells are unknown. In a previous paper (Development 105: 251, 1989), we have demonstrated that a proportion of morphologically undifferentiated neural crest cells express the Schwann cell markers 217c and NGF receptor, and later, as they acquire the bipolar morphology typical of Schwann cells in culture, express S-100 and laminin. In the present study, we have grown axons from embryonic retina on neural crest cultures to see whether this has an effect on the differentiation of neural crest cells into Schwann cells. After 4 to 6 days of co-culture, many more cells had acquired bipolar morphology and S-100 staining than in controls with no retinal explant, and most of these cells were within 200 microns of an axon, though not necessarily in contact with axons. However, the number of cells expressing the earliest Schwann cell markers 217c and NGF receptor was not affected by the presence of axons. We conclude that axons produce a factor, which is probably diffusible, and which makes immature Schwann cells differentiate. The factor does not, however, influence the entry of neural crest cells into the earliest stages of the Schwann cell differentiative pathway.     

Transplantation of neural stem cells into the spinal cord after injury

Thanks to advances in the stem cell biology of the central nervous system (CNS), the previously inconceivable regeneration of the damaged CNS is approaching reality. The availability of signals to induce the appropriate differentiation of the transplanted and/or endogenous neural stem cells (NSCs) as well as the timing of the transplantation are important for successful functional recovery of the damaged CNS. Because the immediately post-traumatic microenvironment of the spinal cord is in an acute inflammatory stage, it is not favorable for the survival and differentiation of NSC transplants. On the other hand, in the chronic stage after injury, glial scars form in the injured site that inhibit the regeneration of neuronal axons. Thus, we believe that the optimal timing of transplantation is 1-2 weeks after injury.     

Failure of Schwann cells as supporting cells for adult neural progenitor cell grafts in the acutely injured spinal cord

Adult neural progenitor cells (NPC) co-grafted with fibroblasts replace cystic lesion defects and promote cell-contact-mediated axonal regeneration in the acutely injured spinal cord. Fibroblasts are required as a platform to maintain NPC within the lesion; however, they are suspected to create an inhospitable milieu for regenerating central nervous system (CNS) axons. Therefore, we thought to replace fibroblasts by primary Schwann cells, which might serve as a superior scaffold to maintain NPC within the lesion and might further enhance axon regrowth and remyelination following spinal cord injury. Adult rats underwent a cervical dorsal column transection immediately followed by transplantation of either NPC/Schwann cell or NPC/Schwann cell/fibroblast co-grafts. Animals receiving Schwann cell or fibroblast grafts alone, or Schwann cell/fibroblast co-grafts served as controls. At 3 weeks after injury/transplantation, histological analysis revealed that only fibroblast-containing grafts were able to replace the cystic lesion defect. In both co-cultures and co-grafts, Schwann cells and NPC were segregated. Almost all NPC migrated out of the graft into the adjacent host spinal cord. As a consequence, only peripheral-type myelin, but no CNS-type myelin, was detected within co-grafts containing NPC/Schwann cells. Corticospinal axon regeneration into Schwann-cell-containing co-grafts was reduced. Taken together, Schwann cells within NPC grafts contribute to remyelination. However, Schwann cells fail as a supporting platform to maintain NPC within the graft and impair CNS axon regeneration; this makes them an unfavorable candidate to support/augment NPC grafts following spinal cord injury.This work was supported by the Institute International de Recherche en Paraplégie Geneva, on behalf of an anonymous donation, and ReForM-Program, University of Regensburg, School of Medicine.     

Callosal transfer of impulses originating from superficial and deep nerves of the cat forelimb.

1. Experiments were performed in 18 chloralose-anaesthetized, curarized cats in order to study the callosal transfer of somatic information originated in exteroceptive and proprioceptive receptors. Several cutaneous and deep nerves of the forelimb were prepared and stimulated with graded intensities, so as to activate selectively afferent fibres pertaining to the different groups of Lloyd's classification. Simultaneous records were taken (and averaged on-line by means of a multichannel analyzer) from the distal end of a cut dorsal rootlet (C7-C8), from the cerebral cortex (SI, SII or area 3a, according to the experiment) and from the somesthetic callosal region (SCR). 2. The low-threshold afferent fibres (Group II) of cutaneous origin were found to have a wide projection to the SCR, with the maximal density in its middle portion. Some of the fastest corticocallosal impulses are relayed monosynaptically at cortical level. Plots of the amplitude of cortical and callosal responses as a function of stimulus strength showed that both central responses have the same threshold and exhibit a parallel, sharply-rising amplitude increase, thus suggesting that the cortico-callosal re-transmission system for afferent impulses of cutaneous origin is very powerful in nature. Impulses elicited in afferent fibres of higher threshold (Group III) do not enhance the cortical and callosal positive waves provoked by Group II afferent volleys. 3. Afferent fibres of deep origin were also found to send a wide projection to the SCR, although less substantial than that of cutaneous fibres. Stimulation of the deep radial nerve elicited mass responses in the whole SCR, provided the strength of stimuli was high enough to engage the Group II fibres. Only in the central portion of the SCR were small potentials recorded in response to pure Group I volleys of DRN. Experiments performed with selective stimulation of pure muscular branches of forelimb deep nerves as well as of articular and mixed (muscular and articular) branches gave evidence making it possible to ascertain the origin of deep afferent fibres projecting to the SCR. Stimulation of the forelimb muscular branches with strength provoking full activation of Group I afferent and additional engagement of those of Group II, did not provoke mass responses in the whole extent of the SCR. In order to obtain callosal potentials upon stimulation of pure muscular nerves, it was necessary to increase the stimulus strength at or above the threshold for Group III fibres. On the contrary, the same callosal foci unresponsive to Group I and II muscular afferent volleys exhibited clear-cut responses to stimulation of the lowest-threshold Group I and/or Group II afferents of articular and mixed nerves. From the results it might be inferred that only proprioceptive information originating from articular receptors and from extrafusal muscular afferents has access to the callosal interhemispheric transfer.     

人胎儿脊髓神经干细胞的分离培养

本文旨在探讨是否能够从低温保存的流产儿分离培养出脊髓神经干细胞。将14周流产儿在4℃下保存,2、6和12h后取脊髓,将颈段、胸段、腰骶段分别进行无血清培养,并用胎牛血清诱导分化。用克隆培养的方法验证培养细胞的干细胞特性;用免疫荧光细胞化学的方法检测神经干细胞标志nestin及干细胞诱导分化后神经元标志MAP2、星形胶质细胞标志GFAP、胆碱能标志ChAT,并比较不同时间点以及不同部位分离的神经T细胞的差异。在各个时间点,从颈段、胸段、腰骶段脊髓均分离培养出具有连续增殖能力的神经球,其中腰骶段分离出的神经球数量最多,12h组各段分离出的神经球较2、6h组显著减少。各段培养中的神经球均为nestin阳性,诱导分化后均能够产生GFAP阳性星形胶质细胞、MAP2阳性神经元以及ChAT阳性胆碱能神经元。各段培养中的神经干细胞的克隆形成能力相似。以上结果表明,从低温保存的人胎儿能够分离培养出脊髓神经干细胞,这为基础研究以及未来治疗应用提供了新的细胞来源。     

Countercurrent distribution of lymphocytes from human peripheral blood in an aqueous two-phase system. I. Separation into subsets of lymphocytes bearing distinctive markers

Lymphocytes from human peripheral blood have been separated by countercurrent distribution in a charged aqueous two-phase system composed of Dextran T 500 and polyethylene glycol 6000 with a cell yield of 59–88% and viability above 90%. A highly reproducible partition pattern was seen with four distinct peaks. Lymphocytes with surface membrane immunoglobulin (SmIg) were located in the first part of the distribution corresponding mainly to peak I. T lymphocytes as detected by E rosetting and α-naphthyl acetate esterase (ANAE) staining showed a broad distribution with a maximum in peaks II and III. ANAE-negative lymphocytes were seen in both extremes of the distribution, corresponding to B cells in the first part and to a population of E^? and SmIg^? lymphocytes in the last part. Monocytes were present in all fractions with some enrichment in peaks II–IV. Lymphocytes with low-affinity Fc receptors were found in B-cell-containing fractions in the first part of the distribution, but also in the last part. Lymphocytes with high-affinity Fc receptors were detected mainly in peak IV. It is thus demonstrated that peripheral blood lymphocytes can be fractionated into subpopulations enriched in cells with characteristic markers.     

Tracing of sensory neurones and spinal motoneurones of the pigeon by injection of fluorescent dyes into peripheral nerves

The projection of peripheral sensory and motor nerves was investigated in the pigeon (Columba livia) by means of retrogradely transported fluorescent dyes. Two combinations of fluorescent tracers were used that could be identified within the same cell when excited by light of 405 nm: 1) Propidium iodide and Bisbenzimide, which label the cytoplasm orange and the nucleus blue, respectively; 2) Fast Blue, which labels the cytoplasm blue, and Nuclear Yellow, which labels the nucleus (especially the nucleolar ring) yellow. The presence of the tracers in a given cell was confirmed microspectrophotometrically. Following injection of the tracers into peripheral nerves, labelled sensory neurones were seen in the dorsal root ganglia and motoneurones of the spinal cord. The peroneal and tibial nerves projected to L2-L5 and L2-L7, respectively, whereas the median and ulnar nerves projected to C12-Th2 and C13-Th1. Double-labelled sensory neurones were observed when both peroneal and tibial, or median and ulnar nerves were injected with different tracers. This indicates that some sensory neurones possess peripheral processes that dichotomize to pass down two different peripheral nerves. Double labelling was never seen in motoneurones, or in sensory neurones after tracer injection into the sciatic and femoral nerves.     

P2X7 purinoceptors contribute to the death of Schwann cells transplanted into the spinal cord

The potential to use Schwann cells (SCs) in neural repair for patients suffering from neurotrauma and neurodegenerative diseases is well recognized. However, significant cell death after transplantation hinders the clinical translation of SC-based therapies. Various factors may contribute to the death of transplanted cells. It is known that prolonged activation of P2X7 purinoceptors (P2X7R) can lead to death of certain types of cells. In this study, we show that rat SCs express P2X7R and exposure of cultured SCs to high concentrations of ATP (3–5 mM) or a P2X7R agonist, 2′(3′)-O-(4-benzoylbenzoyl)ATP (BzATP) induced significant cell death rapidly. High concentrations of ATP and BzATP increased ethidium uptake by SCs, indicating increased membrane permeability to large molecules, a typical feature of prolonged P2X7R activation. SC death, as well as ethidium uptake, induced by ATP was blocked by an irreversible P2X7R antagonist oxidized ATP (oxATP) or a reversible P2X7R antagonist A438079. oxATP also significantly inhibits the increase of intracellular free calcium induced by minimolar ATP concentrations. Furthermore, ATP did not cause death of SCs isolated from P2X7R-knockout mice. All these results suggest that P2X7R is responsible for ATP-induced SC death in vitro. When rat SCs were treated with oxATP before transplantation into uninjured rat spinal cord, 35% more SCs survived than untreated SCs 1 week after transplantation. Moreover, 58% more SCs isolated from P2X7R-knockout mice survived after being transplanted into rat spinal cord than SCs from wild-type mice. This further confirms that P2X7R is involved in the death of transplanted SCs. These results indicate that targeting P2X7R on SCs could be a potential strategy to improve the survival of transplanted cells. As many other types of cells, including neural stem cells, also express P2X7R, deactivating P2X7R may improve the survival of other types of transplanted cells.