Neural Stem/Progenitor Cells Differentiate In Vitro to Neurons by the Combined Action of Dibutyryl cAMP and Interferon-γ (original) (raw)
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PLoS ONE, 2011
Neural stem/progenitor cells (NSPCs) have great potential as a cell replacement therapy for spinal cord injury. However, poor control over transplant cell differentiation and survival remain major obstacles. In this study, we asked whether dibutyryl cyclic-AMP (dbcAMP), which was shown to induce up to 85% in vitro differentiation of NSPCs into neurons would enhance survival of transplanted NSPCs through prolonged exposure either in vitro or in vivo through the controlled release of dbcAMP encapsulated within poly(lactic-co-glycolic acid) (PLGA) microspheres and embedded within chitosan guidance channels. NSPCs, seeded in fibrin scaffolds within the channels, differentiated in vitro to betaIII-tubulin positive neurons by immunostaining and mRNA expression, in response to dbcAMP released from PLGA microspheres. After transplantation in spinal cord injured rats, the survival and differentiation of NSPCs was evaluated. Untreated NSPCs, NSPCs transplanted with dbcAMP-releasing microspheres, and NSPCs pre-differentiated with dbcAMP for 4 days in vitro were transplanted after rat spinal cord transection and assessed 2 and 6 weeks later. Interestingly, NSPC survival was highest in the dbcAMP pre-treated group, having approximately 80% survival at both time points, which is remarkable given that stem cell transplantation often results in less than 1% survival at similar times. Importantly, dbcAMP pre-treatment also resulted in the greatest number of in vivo NSPCs differentiated into neurons (3764%), followed by dbcAMP-microsphere treated NSPCs (27614%) and untreated NSPCs (1567%). The reverse trend was observed for NSPC-derived oligodendrocytes and astrocytes, with these populations being highest in untreated NSPCs. This combination strategy of stem cell-loaded chitosan channels implanted in a fully transected spinal cord resulted in extensive axonal regeneration into the injury site, with improved functional recovery after 6 weeks in animals implanted with pre-differentiated stem cells in chitosan channels.
Brain Pathology, 2006
Currently available effective treatments of the diseased or damaged central nervous system (CNS) are restricted to a limited pharmacological relief of symptoms or those given to avoid further damage. Therefore the search is on for treatments that can restore function in the CNS. During recent years replacement of damaged neurons by cell transplantation is being enthusiastically explored as a potential treatment for many neurodegenerative diseases, stroke and traumatic brain injury. Several references in both scientific journals and popular newspapers concerning different types of cultured stem cells, potentially exploitable to treat pathological conditions of the brain, raise important questions pertinent to the fundamental and realistic differences between grafts of primary neural cells and the transplantation of in vitro expanded neural stem cells (NSCs). Our aim is to review the available information on the grafting of different NSC types into the adult rodent brain, focusing on critical aspects for the development of clinical therapies to replace damaged neurons.
Stem Cells and Development, 2011
Neurons derived from human embryonic stem cells hold promise for the therapy of neurological diseases. Quality inspection of human embryonic stem cell-derived neurons has often been based on immunolabeling for neuronal markers. Here we put emphasis on their physiological properties. Electrophysiological measurements were carried out systematically at different stages of neuronal in vitro development, including the very early stage, neuroepithelial rosettes. Developing human neurons are able to generate action potentials (APs) as early as 10 days after the start of differentiation. Tyrosine hydroxylase (TH)-positive (putative dopaminergic, DA) neurons tend to aggregate into clumps, and their overall yield per coverslip is relatively low (8.3%) because of areas void of DA neurons. On the same in vitro day, neighboring neurons can be in very different stages of differentiation, including repetitive AP firing, single full-size AP, and abortive AP. Similarly, the basic electrophysiological parameters (resting membrane potential, input resistance, peak sodium, and peak potassium currents) are scattered in a wide range. Visual appearance of differentiating neurons, and number of primary and secondary dendrites cannot be used to predict the peak sodium current or AP firing properties of cultured neurons. Approximately 13% of neurons showed evidence of hyperpolarization-induced current (I h ), a characteristic of DA neurons; however, no neurons with repetitive APs showed I h . The electrophysiological measurements thus indicate that a standard DA differentiation (dibutyryl cyclic AMP-based) protocol, applied for 2-5 weeks, produces a heterogeneous ensemble of mostly immature neurons. The overall quality of human neurons under present conditions (survival factors were not used) begins to deteriorate after 12 days of differentiation.
Differentiated Neuronal Cell Lines as Donor Tissue for Transplantation into the CNS
Annals of the New York Academy of Sciences, 1987
We are investigating the use of specific neural cell lines for transplantation into the damaged central nervous system (CNS). Treatment of neuronal cell lines in vitro with various growth modulators and antimitotic drugs has been shown to render cells amitotic and to induce the normal neuronal phenotype.'-3 In this study, several mouse and human neuroblastomas and a rat pheochromocytoma were treated with either 0.5 pg/ml mitomycin C (mito) and lo-' M bromodeoxyuridine (Brdu) or 10 pg/ ml prostaglandin E I and 500 pg/ml dibutyryl cyclic adenosine monophosphate for examination of their neuronal properties before implantation into the lesioned CNS. The pheochromocytoma cells (PC12 cells) were first treated with 100 ng/ml nerve growth factor (NGF) to induce neuritic sprouting. Maximum neurite outgrowth occurred over 4 days, after which time PC12 cells were exposed to mito-Brdu for an additional 3 days. FIGURE la shows the normal appearance of mitotic PC12 cells. Cells are round with only a few anchoring extensions arising from some cells. When PC12 cells are treated as described, neuritic extensions can be quite long and branched. Significantly, removal of all drugs and NGF from the culture does not induce a reversion to the undifferentiated, mitotic state (FIG. Ib), and differentiated cells can be maintained in vitro for at least 1 month after drug removal. These cells show typical tyrosine hydroxylase immunocytochemical staining (FIG. lc) and produce 4.3 pg dopamine/mg protein as measured by high-performance liquid chromatography. N,AB-1 cells, a mouse neuroblastoma subclone of Neuro 2a, is an adrenergic line that extends bipolar, unbranched neurites for more than 50 pm when treated with either drug combination for 4 days. These cells become arrested in G,G, of the cell cycle as measured by flow cytometry of propidium iodide-stained DNA4 and show a significant change in cell surface glycoproteins. Living, control N,AB-1 cells bound fluorescein-labeled wheat germ agglutinin (FL-WGA) in a discontinuous pattern around the cell body (FIG. 2a) whereas differentiated cells showed an intense green fluorescence outlining both neurites and cell body completely (FIG. 2b). Differentiated aThis work was supported by Grants NS19711 (to M. F. D. Notter) and NS15109 (to D. M.
Neural stem cells and strategies for the regeneration of the central nervous system
Proceedings of the Japan Academy, Series B, 2010
The adult mammalian central nervous system (CNS), especially that of adult humans, is a representative example of organs that do not regenerate. However, increasing interest has focused on the development of innovative therapeutic methods that aim to regenerate damaged CNS tissue by taking advantage of recent advances in stem cell and neuroscience research. In fact, the recapitulation of normal neural development has become a vital strategy for CNS regeneration. Normal CNS development is initiated by the induction of stem cells in the CNS, i.e., neural stem cells (NSCs). Thus, the introduction or mobilization of NSCs could be expected to lead to CNS regeneration by recapitulating normal CNS development, in terms of the activation of the endogenous regenerative capacity and cell transplantation therapy. Here, the recent progress in basic stem cell biology, including the author's own studies, on the prospective identication of NSCs, the elucidation of the mechanisms of ontogenic changes in the dierentiation potential of NSCs, the induction of neural fate and NSCs from pluripotent stem cells, and their therapeutic applications are summarized. These lines of research will, hopefully, contribute to a basic understanding of the nature of NSCs, which should in turn lead to feasible strategies for the development of ideal ''stem cell therapies'' for the treatment of damaged brain and spinal cord tissue.
Working with Stem Cells, 2016
Oligodendrocyte progenitor cells (OPCs) may have beneficial effects in cell replacement therapy of neurodegenerative disease owing to their unique capability to differentiate into myelinogenic oligodendrocytes (OLs) in response to extrinsic signals. Therefore, it is of significance to establish an effective differentiation methodology to generate highly pure OPCs and OLs from some easily accessible stem cell sources. To achieve this goal, in this study, we present a rapid and efficient protocol for oligodendroglial lineage differentiation from mouse neural stem cells (NSCs), rat NSCs, or mouse embryonic stem cell-derived neuroepithelial stem cells. In a defined culture medium containing Smoothened Agonist, basic fibroblast growth factor, and platelet-derived growth factor-AA, OPCs could be generated from the above stem cells over a time course of 4-6 days, achieving a cell purity as high as ∼90%. In particular, these derived OPCs showed high expandability and could further differentiate into myelin basic protein-positive OLs within 3 days or alternatively into glial fibrillary acidic protein-positive astrocytes within 7 days. Furthermore, transplantation of rodent NSCderived OPCs into injured spinal cord indicated that it is a feasible strategy to treat spinal cord injury. Our results suggest a differentiation strategy for robust production of OPCs and OLs from rodent stem cells, which could provide an abundant OPC source for spinal cord injury.
Interferon-γ-induced neuronal differentiation of human umbilical cord blood-derived progenitors
Leukemia, 2009
Human umbilical cord blood (HUCB) provides a source of progenitors for cell therapy. We isolated and characterized an HUCB-derived population of progenitors (HUCBNP), differentiated toward neuronal phenotype by human neuroblastomaconditioning medium (CM) and nerve growth factor (NGF), which have been found to confer neuroprotection toward hypoxia-mediated neuronal injury. This study investigated whether interferon-c (IFN-c) contributes to HUCBNP differentiation. IFN-c was detected in the CM used for the induction of differentiation of HUCBNP and a neutralizing antibody of IFN-c significantly inhibited either IFN-c or CM-induced differentiation. Transcriptome analysis of CM-differentiated HUCBNP, identified 86 genes as highly upregulated, among them 25 were IFN-induced (such as 2 0 ,5 0 -oligoadenylate synthetase 1 and 2, IFN-induced protein and transmembrane proteins, STAT1 (IFNc-receptor signal transducer and activator of transcription) and chemokine C-X-C motif ligand 5). Treatment of HUCBNP with human recombinant IFN-c, inhibited cell proliferation in a dosedependent manner. IFN-c (1-100 ng/ml) enhanced neuronal differentiation, expressed by neurite outgrowths and increased expression of the neuronal markers b-tubulin III, microtubuleassociated protein 2, neuronal nuclei, neurofilament M and neuronal-specific enolase. IFN-c additively cooperated with NGF to induce the differentiation of HUCBNP. These data indicate that IFN-c promotes neuronal differentiation of HUCBderived progenitors, proposing its use in future protocols towards cell therapy.
Health Science Journal of Indonesia, 2018
Background: Using of neuron cells for in vitro neurobiology study is needed. Neuron cell can be obtained from a primary neuron or neuronal cell lines, depend on the aim of the study because both are not equivalent. Various methods are performed to obtain primary neurons from the cortical, hippocampal and whole brain of pre or neonatal rat. The limitations of neuron cells to proliferate so that is necessary to develop a method to isolate neuron progenitor cells (NPCs). The aim of the present study was to isolate NPCs from whole brain post-natal rat. Methods: Whole brain were obtained from neonates Sprague Dawley rat. There are 2 step to get NSC; first isolation by taking the brain into the 15 ml of tube with 1 ml of 0,05% trypsin EDTA for 400g brain (incubated in the 370C, 5% CO2 for 10 minutes), tirturation with adding 1 ml culture medium and 5 ml HBSS-glucose then filtered by 70μm pore size membrane and centrifuged 2000 rpm for 10 minutes. Second: remove of supernatant with a...
Transplantation of neural progenitors enhances production of endogenous cells in the impaired brain
Molecular Psychiatry, 2007
Grafting of neural progenitors has been shown to reverse a wide variety of neurobehavioral defects. While their role of replacing injured cells and restoring damaged circuitries has been shown, it is widely accepted that this cannot be the only mechanism, as therapy can occur even when an insufficient number of transplanted cells are found. We hypothesized that one major mechanism by which transplanted neural progenitors exert their therapeutic effect is by enhancing endogenous cells production. Consequently, in an allographic model of transplantation, prenatally heroin-exposed genetically heterogeneous (HS) mice were made defective in their hippocampal neurobehavioral function by exposing their mothers to heroin (10 mg kg À1 heroin on gestation days 9-18). Hippocampal damage was confirmed by deficient performance in the Morris maze (P < 0.009), and decreased production of endogenous cells in the dentate gyrus by 39% was observed. On postnatal day 35, they received an HS-derived neural progenitors transplant followed by repeated bromodeoxyuridine injections. The transplant returned endogenous cells production to normal levels (P < 0.006) and reversed the behavioral defects (P < 0.03), despite the fact that only 0.0334% of the transplanted neural progenitors survived and that they differentiated mainly to astrocytes. An immunological study demonstrated the presence of macrophages and T cells as a possible explanation for the paucity of the transplanted cells. This study suggests one mechanism for the therapeutic action of neural progenitors, the enhancement of the production of endogenous cells, pointing to future clinical applications in this direction by use of neural progenitors or by analogous cell-inducing techniques.