Cord Cells Research Articles

Nanofiber mat spinal cord dressing-released glutamate impairs blood-spinal cord barrier

An excessive glutamate level can result in excitotoxic damage and death of central nervous system (CNS) cells, and is involved in the pathogenesis of many CNS diseases. It may also be related to a failure of the blood-spinal cord barrier (BSCB). This study was aimed at examining the effects of extended administration of monosodium glutamate on the BSCB and spinal cord cells in adult male Wistar rats. The glutamate was delivered by subarachnoidal application of glutamate-carrying electrospun nanofiber mat dressing at the lumbar enlargement level. Half of the rats with the glutamate-loaded mat application were treated systemically with the histone deacetylase inhibitor valproic acid. A group of intact rats and a rat group with subarachnoidal application of an 'empty' (i.e., carrying no glutamate) nanofiber mat dressing served as controls. All the rats were euthanized three weeks later and lumbar fragments of their spinal cords were harvested for histological, immunohistochemical and ultrastructural studies. The samples from controls revealed normal parenchyma and BSCB morphology, whereas those from rats with the glutamate-loaded nanofiber mat dressing showed many intraparenchymal microhemorrhages of variable sizes. The capillaries in the vicinity of the glutamate-carrying dressing (in the meninges and white matter alike) were edematous and leaky, and their endothelial cells showed degenerative changes: extensive swelling, enhanced vacuo-lization and the presence of vascular intraluminal projections. However, endothelial tight junctions were generally well preserved. Some endothelial cells were dying by necrosis or apoptosis. The adjacent parenchyma showed astrogliosis with astrocytic hypertrophy and swelling of perivascular astrocytic feet. Neurons in the parenchyma revealed multiple symptoms of degeneration, including, inter alia, perikaryal, dendritic and axonal swelling, and destruction of organelles. All the damage symptoms were slightly less severe in the rats given valproic acid treatment, and were absent from both the intact rats and the rats with 'empty' nanofiber mat dressing. These results demonstrate that glutamate-loaded nanofiber mat dressing can locally create glutamate levels capable of damaging BSCB and that the resulting damage can be mitigated with concurrent systemic valproate treatment.

Clinical Application and Exploration on Steps Progressively Corrections and Gradual Shortening Combined with Total Spinal Osteotomy for Angular Kyphosis—Basic Research and Clinical Application

Introduction: This study is designed to explore the biomechanical principles of posterior bilateral pedicle screw on steps progressively and tight closure on every step for severe angular kyphosis and the indication conditions and clinical pathways of above new technique. Materials and Methods: A total of 90 patients have severe angular kyphosis, 37 males and 53 females, with an average age of 47 years. All patients were treated with posterior bilateral pedicle screw and step tight closure for gradual shortening and orthopedic technology. Results: Average intraoperative blood loss was 2089 ml. Average operation time was 326 minutes. Kyphotic angle changed from 90.1° averagely preoperatively to 41.6° averagely postoperatively with an improvement rate of 65%. The distance from C7 plumb line to posterior upper edge of the S1 vertebral body was averagely 5.2 mm postoperatively, with a correction rate of 73%. Neurological complications occurred in 2 cases, accounting for 6%. Non-neurological complications occurred in 2 cases, accounting for 6%. Anatomical parameters were significantly improved postoperatively (P < 0.05). All patients were averagely followed up for 28 months. Segments undergoing osteotomy achieved bone fusion, without loss of orthopedic angle. Conclusions: The orthopedic technology of bilateral pedicle screw and step tight closure designed by human physiology and biomechanical principles can protect spinal cord cells from injury to the utmost. To choose an appropriate osteotomy plane during pre-surgical planning can make the repair results closer to the normal function of the spine. Intraoperative nerve root protection, full release and bone graft fusion are effective safeguards to ensure kyphosis correction and to avoid spine lateral offset.

The synergistic effect on osteogenic differentiation of human mesenchymal stem cells by diode laser-treated stimulating human umbilical vein endothelial cells

Angiogenesis plays an important role in determining the biostimulation of bone regeneration, in either new bone or blood vessel formation. Human umbilical cord cells (HUVECs) are important effector cells in angiogenesis and are indispensable for osteogenesis and for their heterogeneity and plasticity. However, there are very few studies about the effects of HUVECs on diode laser-stimulated/regulated osteogenesis. In this study, we used diode laser as a model biostimulation to examine the role of HUVECs on laser-stimulated osteogenesis. Several bone formation-related proteins were also significantly up-regulated by the diode laser stimulation, indicating that HUVECs may participate in diode laser-stimulated osteogenesis. Interestingly, when human mesenchymal stem cells (hMSCs) cultured with HUVECs were diode laser-treated, the osteogenesis differentiation of the hMSCs was significantly promoted, indicating the important role of HUVECs in diode laser-enhanced osteogenesis. Adequately activated HUVECs are vital for the success of diode laser-stimulated hard-tissue regeneration. These findings provided valuable insights into the mechanism of diode laser-stimulated osteogenic differentiation, and a strategy to optimize the evaluation system for the in vitro osteogenesis capacity of laser treatment in periodontal repair.

Isolation and Identification of Human Umbilical Cord and Placenta-derived Stem Cells and Their Component Analysis

Clinical transplantation evidence has been indicated that umbilical cord blood (UCB) can be useful in the hematopoietic reconstitution in the children, but can not be well in the adult hematopoietic transplantation because of the low count. This study was to evaluate a new method for collecting stem cells from human placenta and umbilical cord, and to comparatively analyze the similarity and difference of quality and quantity of the cells. The UCB was collected, in same time the placental tissue was sterily collected; the umbilical placenta was collected by perfusing the blood (UPB) cord arterial and venous vascules with 0.9% saline; the mesenchymal stem cells (MSC) from same source of umbilical cord and placental tissues were isolated and cultured. The cell colony assay and flow cytometry were performed to determine the proliferation capacities and cell markers of UMSC and PMSC. The total nuclear cells (NC) and hematopoietic stem cells (CD34(+)) from UPB and UCB were (17.45 ± 16.86) × 10(8), (6.9 ± 4.61) × 10(8) and (2.97 ± 2.25) × 10(6), (1.91 ± 1.7) × 10(6), respectively. Furthermore, the UPB contained more early precursor of hematopoietic stem cells (CD33(+) CD34(-)) (6.2 ± 13.5) × 10(5), (0.2 ± 0.8) × 10(5) ; and high proportion of MSC to NC (25.21 ± 18.69, 0.05 ± 0.10)%, respectively in 62 samples. There were no difference of the MSC level in UPB and UCB, as well as in the morphology and cell markers. UPB has rich hematopoietic stem cells and mesenchymal stem cells. Placenta may offer another source for hematopoietic stem cells in research of hematopoietic stem cells and regeneration medicine.

Organization of the nitrergic neuronal system in the primitive bony fishes Polypterus senegalus and Erpetoichthys calabaricus (Actinopterygii: Cladistia).

Cladistians are a group of basal actinopterygian fishes that constitute a good model for studying primitive brain features, most likely present in the ancestral bony fishes. The analysis of the nitrergic neurons (with the enzyme nitric oxide synthase; NOS) has helped in understanding important aspects of brain organization in all vertebrates studied. We investigated the nitrergic system of two cladistian species by means of specific antibodies against NOS and NADPH-diaphorase (NADPH-d) histochemistry, which, with the exception of the primary olfactory and terminal nerve fibers, labeled only for NADPH-d, yielded identical results. Double immunohistochemistry was conducted for simultaneous detection of NOS with tyrosine hydroxylase, choline acetyltransferase, calbindin, calretinin, and serotonin, to establish accurately the localization of the nitrergic neurons and fibers and to assess possible interactions between these neuroactive substances. The pattern of distribution in both species showed only subtle differences in the density of labeled cells. Distinct groups of NOS-immunoreactive cells were observed in pallial and subpallial areas, paraventricular region, tuberal and retromammillary hypothalamic areas, posterior tubercle, prethalamic and thalamic areas, optic tectum, torus semicircularis, mesencephalic tegmentum, interpeduncular nucleus, superior and middle reticular nuclei, magnocellular vestibular nucleus, solitary tract nucleus, nucleus medianus magnocellularis, the spinal cord and amacrine cells in the retina. Large neurons in cranial nerve sensory ganglia were also labeled. The comparison of these results with those from other vertebrates, using a neuromeric analysis, reveals a conserved pattern of organization of the nitrergic system from this primitive fish group to amniotes, including mammals.

Progress in the molecular genetic mechanism of gonadoblastoma

Gonadoblastoma (GB), a rare in situ germ cell tumor derived from sex cord and germ cells, is closely associated with gonadal dysgenesis. About 80% of GB individuals exhibit 46, XY female phenotype while the others are 45, XY and 46, XX with disorders of sex development. Moreover, 35% of GB can eventually develop into malignant tumors, such as seminoma and dysgerminoma tumors. The molecular genetic mechanism of GB remains to be fully uncovered due to phenotypic and genetic heterogeneity. Increasing studies show that the formation of GB is closely related to genes regulating sexual differentiation and determination (e.g., SRY, WT1, SOX9, Foxl2, TSPY, etc), and is affected by the interaction of genetic and epigenetic regulation. Here we describe the clinical and pathological features, diagnosis and treatment of GB, and also summarize the molecular genetic and epigenetic mechanisms underlying the gonadal abnormalities that lead to GB. We analyze and construct the common gene regulatory networks related to the development of GB, and describe some obstacles and deficiencies in current studies to provide innovative perspectives on further studying the pathological and molecular mechanisms of GB.

MicroRNA-21 regulates cell proliferation and apoptosis in H₂O₂-stimulated rat spinal cord neurons.

Oxidative stress can alter the expression level of microRNAs (miRNAs) and has a role in oxidative damage generated by reactive oxygen species (ROS). While previous studies have demonstrated that miR‑146a, miR‑21 and miR‑150 are essential for ROS production in heart disease, the role of these miRNAs in spinal cord injuries has not yet been examined. The present study focused on examining the role of miR‑146a, miR‑21 and miR‑150 during H2O2 stimulation in rat neuronal spinal cord (RN‑sc) cells. RN‑sc cells were treated with H2O2, and cells were harvested for reverse transcription quantitative polymerase chain reaction (RT‑qPCR) to detect the expression levels of miR‑146a, miR‑21 and miR‑150. The results demonstrated that miR‑146a, miR‑21 and miR‑150 expression was upregulated during H2O2 treatment. T-cell death and apoptosis were investigated using an MTT assay and flow cytometric analysis, respectively. Following miR‑21 silencing, H2O2‑induced cell death and apoptosis were reduced in RN‑sc cells, while miR‑150 silencing had no effect. Furthermore, Smad7 was identified as a direct target of miR‑21 using a Luciferase reporter assay, RT-qPCR and western blot analysis. In addition, while H2O2 downregulated Smad7 protein expression, this was reversed by inhibiting miR‑21 expression. Based on previous studies, it was predicted that miR‑21 has a role in ROS production through regulating Smad7 in rat spinal cord neurons.

Deep-sequencing of the T-cell receptor repertoire in patients with haplo-cord and matched-donor transplants.

Haplo-cord transplant has emerged as a feasible and reliable approach for haematopoietic stem cell transplant in patients who are unable to find matched-donor. This approach provides fast myeloid recovery, low incidence of graft vs host disease (GVHD) and favorable graft versus leukemia (GVL) effects. T cell recovery plays an important role in preventing infectious complications; it also mediates the GVHD and the GVL effects. Here, we utilized a novel RNA-based sequencing approach to quantitatively characterize the T cell receptor (TCRs) repertoire in patients underwent haplo-cord transplant in comparison with those underwent matched-donor transplant. Our study shows that higher percentage of cord cells early post transplant were associated with significantly higher TCR diversity. TCR diversity was significantly lower in patients with GVHD and in relapsed patients. A larger cohort study is needed to validate these data and to provide useful information on the specific TCR clones correlated with clinical outcome.

Evidence for a Role of the Transcriptional Regulator Maid in Tumorigenesis and Aging.

Maid is a helix-loop-helix protein that is involved in cell proliferation. In order to further elucidate its physiological functions, we studied Maid activity in two small fish model systems. We found that Maid expression was greatest in zebrafish liver and that it increased following partial hepatectomy. Maid levels were also high in hepatic preneoplastic foci induced by treatment of zebrafish with diethylnitrosamine (DEN), but low in hepatocellular carcinomas (HCC), mixed tumors, and cholangiocarcinomas developing in these animals. In DEN-treated transgenic medaka overexpressing Maid, hepatic BrdU uptake and proliferation were reduced. After successive breedings, Maid transgenic medaka exhibited decreased movement and a higher incidence of abnormal spine curvature, possibly due to the senescence of spinal cord cells. Taken together, our results suggest that Maid levels can influence the progression of liver cancer. In conclusion, we found that Maid is important regulator of hepatocarconogenesis and aging.

ROBO2 restricts the nephrogenic field and regulates Wolffian duct–nephrogenic cord separation

ROBO2 plays a key role in regulating ureteric bud (UB) formation in the embryo, with mutations in humans and mice leading to supernumerary kidneys. Previous studies have established that the number and position of UB outgrowths is determined by the domain of metanephric mesenchymal Gdnf expression, which is expanded anteriorly in Robo2 mouse mutants. To clarify how this phenotype arises, we used high-resolution 3D imaging to reveal an increase in the number of nephrogenic cord cells, leading to extension of the metanephric mesenchyme field in Robo2-null mouse embryos. Ex vivo experiments suggested a dependence of this effect on proliferative signals from the Wolffian duct. Loss of Robo2 resulted in a failure of the normal separation of the mesenchyme from the Wolffian duct/ureteric epithelium, suggesting that aberrant juxtaposition of these two compartments in Robo2-null mice exposes the mesenchyme to abnormally high levels of proliferative stimuli. Our data suggest a new model in which SLIT-ROBO signalling acts not by attenuating Gdnf expression or activity, but instead by limiting epithelial/mesenchymal interactions in the nascent metanephros and restricting the extent of the nephrogenic field. These insights illuminate the aetiology of multiplex kidney formation in human individuals with ROBO2 mutations.

Temporal kinetics of macrophage polarization in the injured rat spinal cord

Local activated macrophages derived from infiltrating monocytes play an important role in the damage and repair process of spinal cord injury (SCI). The present study investigates the dynamic change of classically activated proinflammatory (M1) and alternatively activated anti-inflammatory (M2) cells in a rat model with contusive SCI by flow cytometry (FCM) and immunohistochemistry. The macrophage subsets were immunophenotyped by using antibodies against cluster of differentiation (CD)-68, C-C chemokine receptor type 7 (CCR7), CD163, and arginase 1 (Arg1). The CD68(+) CD163(-) and CD68(+) CCR7(+) cells were determined to be M1 subsets, whereas the CD68(+) CD163(+) and CD68(+) Arg1(+) cell subpopulations represented M2 cells. The subsets of macrophages in the injured spinal cord at 1, 3, 5, 7, 14, and 28 days postinjury (dpi) were examined. In the sham-opened spinal cord, few M1 or M2 cells were found. After SCI, the phenotypes of both M1 and M2 cells were rapidly induced. However, M1 cells were detected and maintained at a high level for up to 28 dpi (the longest time evaluated in this study). In contrast, M2 cells were transiently detected at high levels before 7 dpi and returned to preinjury levels at 14 dpi. These results indicate that M1 cell response is rapidly induced and sustained, whereas M2 induction is transient after SCI in rat. Increasing the fraction of M2 cells and prolonging their residence time in the injured local microenvironment is a promising strategy for the repair of SCI.

Stem Cell Research and Regenerative Therapy: Towards A Bright Future

Submit Manuscript | http://medcraveonline.com J Stem Cell Res Ther 2015, 1(1): 00002 cell scientists, by using bone marrow or umbilical cord cells to establish neurons or pancreatic cells and using brain cells to give rise to other cell types. That was exciting enough to attract more interest in terms of finance, (life)-time and energy investments towards stem cell research and regeneration with the promise of greater scientific achievements and a better control over stem cell proliferation and commitment. However, as science is always about, restrictions and undesired results were unavoidable aching points which attenuate the enormous progress in the field of stem cells and regenerative medicine.

The effect dmso on differentiation into hepatocytes-like cell of umbilical cord stem cells

Recent studies indicated that DMSO can induce differentiation of mesenchymal stem cells derived from bone marrow, adipose tissue, umbilical cord blood, umbilical cord tissues into hepatocyte-like cells. However, the effect of DMSO is not similar in reports. In this study, umbilical cord cells were isolated and analyzed the expression of mesenchymal stem cells specific markers then they were exposured to DMSO with three concentrations: 0.01%, 0.1%, 1% within 3 days. Expression of specific markers was analyzed by RT-PCR after 10 days, 20 days, 30 days to investigate the differentiation level. The result showed that the isolated cells were positive with mesenchymal stem cells specific markers, such as: CD73, CD90, CD105. This expression is stable after 20 times of subculture. At the concentration 1% of DMSO, in the day 20-30th of in vitro culture, isolated cells expressed HNF4α gene, a transcription regulation factor in hepatocyte production. Conclusion: In the early part of this study, the umbilical cord mesenchymal stem cells expressed hepatic specific marker after DMSO treatment. It is necessary to investigate the hepatic functions of these cells in a longer in vitro culture.

Toward a Broader View of Ube3a in a Mouse Model of Angelman Syndrome: Expression in Brain, Spinal Cord, Sciatic Nerve and Glial Cells.

Angelman Syndrome (AS) is a devastating neurodevelopmental disorder characterized by developmental delay, speech impairment, movement disorder, sleep disorders and refractory epilepsy. AS is caused by loss of the Ube3a protein encoded for by the imprinted Ube3a gene. Ube3a is expressed nearly exclusively from the maternal chromosome in mature neurons. While imprinting in neurons of the brain has been well described, the imprinting and expression of Ube3a in other neural tissues remains relatively unexplored. Moreover, given the overwhelming deficits in brain function in AS patients, the possibility of disrupted Ube3a expression in the infratentorial nervous system and its consequent disability have been largely ignored. We evaluated the imprinting status of Ube3a in the spinal cord and sciatic nerve and show that it is also imprinted in these neural tissues. Furthermore, a growing body of clinical and radiological evidence has suggested that myelin dysfunction may contribute to morbidity in many neurodevelopmental syndromes. However, findings regarding Ube3a expression in non-neuronal cells of the brain have varied. Utilizing enriched primary cultures of oligodendrocytes and astrocytes, we show that Ube3a is expressed, but not imprinted in these cell types. Unlike many other neurodevelopmental disorders, AS symptoms do not become apparent until roughly 6 to 12 months of age. To determine the temporal expression pattern and silencing, we analyzed Ube3a expression in AS mice at several time points. We confirm relaxed imprinting of Ube3a in neurons of the postnatal developing cortex, but not in structures in which neurogenesis and migration are more complete. This furthers the hypothesis that the apparently normal window of development in AS patients is supported by an incompletely silenced paternal allele in developing neurons, resulting in a relative preservation of Ube3a expression during this crucial epoch of early development.

Critical role for prokineticin 2 in CNS autoimmunity.

Objective:To investigate the potential role of prokineticin 2 (PK2), a bioactive peptide involved in multiple biological functions including immune modulation, in CNS autoimmune demyelinating disease.Methods:We investigated the expression of PK2 in mice with experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis (MS), and in patients with relapsing-remitting MS. We evaluated the biological effects of PK2 on expression of EAE and on development of T-cell response against myelin by blocking PK2 in vivo with PK2 receptor antagonists. We treated with PK2 immune cells activated against myelin antigen to explore the immune-modulating effects of this peptide in vitro.Results:Pk2 messenger RNA was upregulated in spinal cord and lymph node cells (LNCs) of mice with EAE. PK2 protein was expressed in EAE inflammatory infiltrates and was increased in sera during EAE. In patients with relapsing-remitting MS, transcripts for PK2 were significantly increased in peripheral blood mononuclear cells compared with healthy controls, and PK2 serum concentrations were significantly higher. A PK2 receptor antagonist prevented or attenuated established EAE in chronic and relapsing-remitting models, reduced CNS inflammation and demyelination, and decreased the production of interferon (IFN)-γ and interleukin (IL)-17A cytokines in LNCs while increasing IL-10. PK2 in vitro increased IFN-γ and IL-17A and reduced IL-10 in splenocytes activated against myelin antigen.Conclusion:These data suggest that PK2 is a critical immune regulator in CNS autoimmune demyelination and may represent a new target for therapy.

Neural Stem/Progenitor Cells of the Spinal Cord are Activated in Response to Tail Loss in the Leopard Gecko

As for many lizards, the leopard gecko (Eublepharis macularius) is able to self‐detach its tail as an anti‐predation strategy and subsequently regenerate a replacement. The replacement tail includes a regenerated spinal cord. We hypothesize that endogenous neural stem progenitor cells (NSPCs) are present in the original spinal cord and that these cells are activated during regeneration. As many stem cells are slow cycling, we used a Bromodeoxyuridine (BrdU) pulse‐chase experiment to identify label‐retaining populations in the original spinal cord. We also conducted a spatiotemporal characterization of spinal cord cells before and during regeneration using a panel of common NSPC markers (including Sox2, Sox9, Vimentin, βIII‐Tubulin and Musashi‐1, and the proliferation marker PCNA). BrdU retaining cells were still present after a 20‐week chase. Prior to injury, expression of NSPC markers is limited, but co‐localization of BrdU and Sox2 in ependymal cells was observed. Following tail loss, ependymal cells in the original stump of the tail become highly proliferative and express our panel of NSPC markers. Outgrowth of the new tail is matched by proliferating, NSPC marker‐positive ependymal cells. These cells appear to contribute to regenerating spinal cord. We conclude that ependymal cells are a likely source of endogenous NSPCs in the gecko and that they are activated following tail loss.NSERC Grant: 400358

NFATc1 deficiency in T cells protects mice from experimental autoimmune encephalomyelitis.

NFATc1 is a member of the nuclear factor of activated T cells (NFAT) family of transcription factors. NFAT is activated upon T‐cell receptor activation followed by intracytoplasmatic calcium influx where calmodulin, a calcium sensor protein, activates the phosphatase calcineurin that dephosphorylates NFAT proteins and results in NFAT nuclear import. Here, we show the analysis of conditional NFATc1‐deficient mice bearing a deletion of NFATc1 in CD4+ and CD8+ T cells. NFATc1‐deficient CD4+ T cells polarized under Th17 conditions express reduced levels of the Th17‐associated transcription factor RORγT (where ROR is RAR‐related orphan receptor) as well as the Th17‐associated cytokines IL‐17A, IL‐17F, IL‐21, and IL‐10. In the murine model of experimental EAE, we found a strong reduction of the disease outcome in conditional NFATc1‐deficient mice, as compared with control littermates. This was accompanied by a diminished inflammation in the brain and spinal cord and reduced IL‐17A and IFN‐γ expression by antigen‐specific spleen, spinal cord, and brain cells. Altogether, these results reveal an important role of NFATc1 in inducing Th17‐cell responses and IFN‐γ, both being relevant for the EAE development.

Different expression of hematopoietic-supporting genes in cord, placental and bone marrow mesenchymal stromal cells

Different expression of hematopoietic-supporting genes in cord, placental and bone marrow mesenchymal stromal cells

Integration of donor mesenchymal stem cell-derived neuron-like cells into host neural network after rat spinal cord transection

Functional deficits following spinal cord injury (SCI) primarily attribute to loss of neural connectivity. We therefore tested if novel tissue engineering approaches could enable neural network repair that facilitates functional recovery after spinal cord transection (SCT). Rat bone marrow-derived mesenchymal stem cells (MSCs), genetically engineered to overexpress TrkC, receptor of neurotrophin-3 (NT-3), were pre-differentiated into cells carrying neuronal features via co-culture with NT-3 overproducing Schwann cells in 3-dimensional gelatin sponge (GS) scaffold for 14 days in vitro. Intra-GS formation of MSC assemblies emulating neural network (MSC-GS) were verified morphologically via electron microscopy (EM) and functionally by whole-cell patch clamp recording of spontaneous post-synaptic currents. The differentiated MSCs still partially maintained prototypic property with the expression of some mesodermal cytokines. MSC-GS or GS was then grafted acutely into a 2 mm-wide transection gap in the T9-T10 spinal cord segments of adult rats. Eight weeks later, hindlimb function of the MSC-GS-treated SCT rats was significantly improved relative to controls receiving the GS or lesion only as indicated by BBB score. The MSC-GS transplantation also significantly recovered cortical motor evoked potential (CMEP). Histologically, MSC-derived neuron-like cells maintained their synapse-like structures in vivo; they additionally formed similar connections with host neurites (i.e., mostly serotonergic fibers plus a few corticospinal axons; validated by double-labeled immuno-EM). Moreover, motor cortex electrical stimulation triggered c-fos expression in the grafted and lumbar spinal cord cells of the treated rats only. Our data suggest that MSC-derived neuron-like cells resulting from NT-3-TrkC-induced differentiation can partially integrate into transected spinal cord and this strategy should be further investigated for reconstructing disrupted neural circuits.

Isolation and culture of spinal cord motor neurons.

Isolated spinal motoneurons are a powerful tool for studying basic mechanisms of neurite growth and survival. Since motoneurons are a minor population of developing spinal cord cells, they need to be purified and enriched to separate them from non-neuronal cells. Therefore, the particular feature of embryonic motoneurons to express the low affinity neurotrophin receptor p75(NTR) is used to separate the motoneurons from other contaminating cells. Two ways are described to isolate embryonic motoneurons: the basic protocol taking advantage of the ability of p75(NTR) to bind lectin, and an alternative method using an antibody against p75(NTR) for a panning procedure. These protocols comprise suggestions for the cultivation of the isolated motoneurons for experiments regarding neural outgrowth and survival as well as instruction for the preparation of proteins of the cells.