Fetal Neural Cells Research Articles

Neonatal desensitization allows long-term survival of neural xenotransplants without immunosuppression

Preclinical development of human cells for potential therapeutic application in neurodegenerative diseases requires that their long-term survival, stability and functional efficacy be studied in animal models of human disease. Here we describe a strategy for long-term immune protection of human fetal and stem cell-derived neural cells transplanted into the adult rat brain, by desensitizing the host rat to similar cells in the neonatal period, without the need for additional immunosuppression.

Blood–Brain Barrier Promotes Differentiation of Human Fetal Neural Precursor Cells

In the stem cell niche, neural stem cells (NSCs) are in close contact with the specialized blood-brain barrier (BBB) endothelial cells (ECs) that modulate their proliferation and differentiation behavior. NSCs are also an attractive source for cell transplantation and neural tissue repair after central nervous system injury. After systemic grafting, they are confronted with the BBB before they can enter the brain parenchyma. We investigated the interactions of human fetal neural precursor cells (hfNPCs) with human brain ECs in an in vitro model using primary cultures. We demonstrated that hfNPCs efficiently differentiate to neurons, astrocytes, and oligodendrocytes and move to the subendothelial space of human BBB endothelium, but not to pulmonary artery ECs. Effective differentiation was found to be dependent on the chemokine CCL2/MCP-1, but not on CXCL8/IL-8. Our findings suggest that neural precursor cells specifically interact with the BBB endothelium and differentiate in the subendothelial niche into astrocytes, neurons, and oligodendrocytes, under the influence of the chemokine CCL2/MCP-1.

Labeling of human neural precursor cells using ferromagnetic nanoparticles

Fetal human neural precursor cells (NPCs) are unique with respect to their capacity to proliferate and to preserve their potential to differentiate into neurons and glia. Human mesencephalic neural precursor cells (hmNPCs) provide a source for dopaminergic neurons. Preclinical and clinical research will benefit from reliable in vivo tracking of transplanted cells. Here, we investigate the potency of very small superparamagnetic iron oxide particles (VSOPs) to label hmNPCs, the effect of VSOPs on survival, proliferation, and differentiation of hmNPCs, and the sensitivity of 1.5T magnetic resonance imaging (MRI) to detect labeled cells in living rats following transplantation. When incubated with VSOPs at 1.5 mM, >95% of hmNPCs incorporated VSOPs without detectable impact on cell viability (>90%) or proliferative capacity, as measured by the expression of proliferating cell nuclear antigen (PCNA) and cell cycle distribution. Labeled hmNPCs differentiate into neurons (>30%) and glia with no detectable difference compared to nonlabeled cells. Following transplantation into rat striata, marked paramagnetic signal changes were detected for as long as three months postsurgery using MRI, corresponding to the histologically-identified graft. Our data indicate that hmNPCs can be labeled with VSOPs without impairment of viability, proliferation, or multipotency. Labeled, transplanted cells are detectable in vivo using 1.5T MRI.

Combined use of BDNF, ascorbic acid, low oxygen, and prolonged differentiation time generates tyrosine hydroxylase-expressing neurons after long-term in vitro expansion of human fetal midbrain precursor cells

Freshly isolated fetal midbrain neural precursor cells (NPCs) that maintain the potential to differentiate into dopamine (DA) neurons represent a valuable source for cell therapy in Parkinson's disease. However, it is poorly understood why midbrain NPCs lose their dopaminergic differentiation potential after long-term culture. Here we report that human fetal midbrain NPCs can be extensively proliferated with fibroblast growth factor 2 (FGF-2), epidermal growth factor (EGF), and leukemia inhibitory factor (LIF) and efficiently differentiated into tyrosine hydroxylase-immunoreactive (TH-ir) neurons. We tested differentiation conditions including the use of low oxygen, ascorbic acid, and prolonged in vitro differentiation time which resulted in a 10-fold increase in the number of MAP2-positive neurons (up to 40–50% of total cells as compared to controls). Under these conditions TH-ir cells constituted 4.3±0.5% of the neuronal population and displayed immature morphologies. Notably, the use of brain-derived neurotrophic factor (BDNF) further increased the proportion of TH-ir neurons (up to 15% of total neurons). In contrast to previous reports, our findings demonstrate that long-term expanded fetal NPCs can generate TH-expressing cells under the appropriate culture conditions and without genetic manipulations.

In vivo gamma imaging of the secondary tumors of transplanted human fetal striatum neural stem cells-derived primary tumor cells

This study describes gamma-imaging of the secondary tumors from the transplanted human fetal striatum neural stem cells-derived primary tumor cells in nude mice. The subcutaneous primary tumors were detected to express integrin alphavbeta3, and the corresponding cells were isolated and enriched in vitro, then transplanted to the nude mice. The technetium-99m-labeled Arg-Gly-Asp peptide, with high affinity to integrin alphavbeta3, was prepared for biodistribution and gamma-imaging. The secondary tumors were readily visualized at 1-h postinjection, and the tumor uptake of radiotracer was similar to that of positive control animals transplanted with U87MG human glioma cells. The tumor specificity of radiotracer was demonstrated by blocking experiment. We concluded that gamma-imaging is a promising approach in imaging the tumorigenesis of transplanted stem cells in vivo.

A multitarget basal ganglia dopaminergic and GABAergic transplantation strategy enhances behavioural recovery in parkinsonian rats

The current transplantation paradigm for Parkinson's disease that places foetal dopaminergic cells in the striatum neither normalizes neuronal activity in basal ganglia structures such as the substantia nigra (SN) and subthalamic nucleus (STN) nor leads to complete functional recovery. It was hypothesized that restoration of parkinsonian deficits requires inhibition of the pathological overactivity of the STN and SN in addition to restoration of dopaminergic activity in the striatum. To achieve inhibition, a multitargeted basal ganglia transplantation strategy using GABAergic cells derived from either foetal striatal primordia (FSP) cells or human neural precursor cells (hNPCs) expanded in suspension bioreactors was investigated. In hemiparkinsonian rats, transplantation of foetal rat dopaminergic cells in the striatum in conjunction with GABAergic grafts in the STN and/or SN promoted significant improvement in forelimb akinesia and motor function compared to transplantation of intrastriatal dopaminergic grafts alone or in conjunction with undifferentiated hNPCs. In culture, FSP cells exhibited neuronal electrophysiological properties. However, recordings from GABAergic hNPCs revealed limited ionic conductances and an inability to fire action potentials. Despite this, they were almost as efficacious as FSP cells in inducing functional recovery following transplantation, suggesting that such recovery may have been mediated by secretion of GABA rather than by functional integration into the host. Thus, restoration of dopaminergic activity to the striatum in concert with inhibition of the STN and SN by GABAergic grafts may be beneficial for improving clinical outcomes in patients with Parkinson's disease and potential clinical application of this strategy may be enhanced by the use of differentiated hNPCs.

Zuckerkandl's organ improves long-term survival and function of neural stem cell derived dopaminergic neurons in Parkinsonian rats

Transplantation of neural stem cells (NSC) derived dopamine (DA) neurons has emerged as an alternative approach to fetal neural cell transplantation in Parkinson's disease (PD). However, similar to fetal neural cell, survival of these neurons following transplantation is also limited due to limited striatal reinnervation (graft with dense neuronal core), limited host–graft interaction, poor axonal outgrowth, lack of continuous neurotrophic factors supply and principally an absence of cell adhesion molecules mediated appropriate developmental cues. In the present study, an attempt has been made to increase survival and function of NSC derived DA neurons, by co-grafting with Zuckerkandl's organ (a paraneural organ that expresses neurotrophic factors as well as cell adhesion molecules); to provide continuous NTF support and developmental cues to transplanted DA neurons in the rat model of PD. 24 weeks post transplantation, a significant number of surviving functional NSC derived DA neurons were observed in the co-transplanted group as evident by an increase in the number of tyrosine hydroxylase immunoreactive (TH-IR) neurons, TH-IR fiber density, TH-mRNA expression and TH-protein level at the transplantation site (striatum). Significant behavioral recovery (amphetamine induced stereotypy and locomotor activity) and neurochemical recovery (DA-D2 receptor binding and DA and DOPAC levels at the transplant site) were also observed in the NSC + ZKO co-transplanted group as compared to the NSC or ZKO alone transplanted group. In vivo results were further substantiated by in vitro studies, which suggest that ZKO increases the NSC derived DA neuronal survival, differentiation, DA release and neurite outgrowth as well as protects against 6-OHDA toxicity in co-culture condition. The present study suggests that long-term and continuous NTF support provided by ZKO to the transplanted NSC derived DA neurons, helped in their better survival, axonal arborization and integration with host cells, leading to long-term functional restoration in the rat model of PD.

The search for a curative cell therapy in Parkinson's disease

Parkinson's disease (PD) is a neurodegenerative disorder, characterised by the progressive loss of dopaminergic neurons in the substantia nigra, and typically treated by dopamine replacement. This treatment, although very effective in the early stages of the disease, is not curative and has side-effects. As such there has been a search for a more definitive treatment for this condition, which has mainly concentrated on replacing the lost neurons with neural grafts. Possible cell sources for replacement range from autologous grafts of dopamine secreting cells to allografts of fetal ventral mesencephalon and neural precursor cells derived from fetal tissue or embryonic stem cells. Some of these cells have been the subject of clinical trials, which to date have produced disparate outcomes. Therefore, whilst cell therapies remain a promising treatment for PD, there is need for further refinement of the techniques involved in this experimental procedure, before any new trials in patients are undertaken.

Spinal GABAergic Transplants Attenuate Mechanical Allodynia in a Rat Model of Neuropathic Pain

Injury to the spinal cord or peripheral nerves can lead to the development of allodynia due to the loss of inhibitory tone involved in spinal sensory function. The potential of intraspinal transplants of GABAergic cells to restore inhibitory tone and thus decrease pain behaviors in a rat model of neuropathic pain was investigated. Allodynia of the left hind paw was induced in rats by unilateral L5- 6 spinal nerve root ligation. Mechanical sensitivity was assessed using von Frey filaments. Postinjury, transgenic fetal green fluorescent protein mouse GABAergic cells or human neural precursor cells (HNPCs) expanded in suspension bioreactors and differentiated into a GABAergic phenotype were transplanted into the spinal cord. Control rats received undifferentiated HNPCs or cell suspension medium only. Animals that received either fetal mouse GABAergic cell or differentiated GABAergic HNPC intraspinal transplants demonstrated a significant increase in paw withdrawal thresholds at 1 week post-transplantation that was sustained for 6 weeks. Transplanted fetal mouse GABAergic cells demonstrated immunoreactivity for glutamic acid decarboxylase and GABA that colocalized with green fluorescent protein. Intraspinally transplanted differentiated GABAergic HNPCs demonstrated immunoreactivity for GABA and beta-III tubulin. In contrast, intraspinal transplantation of undifferentiated HNPCs, which predominantly differentiated into astrocytes, or cell suspension medium did not affect any behavioral recovery. Intraspinally transplanted GABAergic cells can reduce allodynia in a rat model of neuropathic pain. In addition, HNPCs expanded in a standardized fashion in suspension bioreactors and differentiated into a GABAergic phenotype may be an alternative to fetal cells for cell-based therapies to treat chronic pain syndromes.

Dexamethasone blocks astroglial differentiation from neural precursor cells

In previous studies, we demonstrated functional neuronal and dopaminergic differentiation of fetal mesencephalic neural precursor cells. The major factors for orienting their progeny towards a dopaminergic phenotype are forskolin and interleukin-1beta. Here, we investigated the effects of dexamethasone (10 microM) on neuronal and glial differentiation. Exposure of mesencephalic neural precursor cells to dexamethasone significantly reduces the amount of glial fibrillary acidic protein astroglia, but not of galactocerebrosidase C oligodendroglia, MAP2ab neurons and tyrosine hydroxylase dopaminergic cells. Presuming a possible involvement of the nuclear factor-kappaB pathway, we examined the effects of wortmannin (phosphatidylinositol 3'-kinase inhibitor) and SN50 (nuclear factor-kappaB inhibitor) on gliogenesis. Both wortmannin and SN50 mimicked the effects of dexamethasone suggesting that dexamethasone specifically blocks astroglial differentiation from mesencephalic neural precursor cells most likely via inhibition of the nuclear factor-kappaB pathway.

Cocaine Alters Proliferation, Migration, and Differentiation of Human Fetal Brain-Derived Neural Precursor Cells

Maternal use of cocaine during pregnancy is associated with sustained morphological brain abnormalities and sustained cognitive deficits in the offspring. Here, we use a cell culture model of highly enriched human fetal brain-derived neural precursor cells (NPCs) to assess the effects of cocaine treatment on their proliferation, migration, and differentiation. Our data show that cocaine treatment markedly inhibited the proliferation of NPCs, a phenomenon that was associated with cell cycle arrest, possibly because of increased expression of the cyclin-dependent kinase inhibitor p21. In addition, treatment of NPCs with cocaine inhibited their migratory response to CXCL12 (stromal cell-derived factor-1alpha), a finding that correlated with cocaine-induced down-regulation of CXCR4 on NPCs. Finally, these data demonstrated that NPCs exposed to cocaine underwent differentiation into cells expressing neuronal markers that was associated with an inhibition of SOX2 (SRY-related HMG-box gene 2), a transcription factor that inhibits NPC differentiation. Taken together, these results point to several cellular mechanisms whereby exposure of human neural stem cells to cocaine in utero could contribute to subsequent neurodevelopmental and neurocognitive deficits.

A Fine Differentiation: Chlorpyrifos and Neuronal Development

Although chlorpyrifos has been restricted for use in the home, it is still permitted for agriculture and remains the most widely used organophosphate pesticide in the world. Animal studies and in vitro models have indicated that chlorpyrifos has direct and indirect effects on fetal and neonatal neural cell replication and differentiation. These effects include cholinesterase inhibition as well as immediate and delayed-onset changes in synapse formation, neurotransmitter release, neurotransmitter receptor expression, and intracellular signaling. Moreover, chlorpyrifos can exert simultaneous, opposite effects on axonal and dendritic growth. Now researchers from Duke University Medical Center show that chlorpyrifos has direct effects on the differentiation that determines the phenotypic fate of developing neuronotypic cells [EHP 114:667–672; Jameson et al.].

Massively Parallel Signature Sequencing Profiling of Fetal Human Neural Precursor Cells

We have examined gene expression in multipotent neural precursor cells (NPCs) derived from human fetal (f) brain tissue and compared its expression profiles with embryonic stem (ESC) cells, embryoid body cell (EBC), and astrocyte precursors using the technique of massively parallel signature sequencing (MPSS). Gene expression profiles show that fNPCs express core neural stem cells markers and share expression profiles with astrocyte precursor cells (APCs) rather than ESC or EBC. Gene expression analysis shows that fNPCs differ from other adult stem and progenitor cells in their marker expression and activation of specific functional networks such as the transforming growth factorbeta (TGFbeta) and Notch signaling pathways. In addition, our results allow us to identify novel genes expressed in fNPCs and provide a detailed profile of fNPCs.

Chemokine receptor CCR5 expression in in vitro differentiating human fetal neural stem/progenitor and glioblastoma cells

We have studied the expression of CC-chemokine receptor 5 (CCR5) at the protein level in human fetal neural stem/progenitor and glioblastoma cells in differentiation, using immunocytochemistry, routine fluorescence microscopy and confocal laser microscopy analysis. Neural stem/progenitor cells were isolated from the brain of 18–21 weeks old fetuses aborted due to medical indications, and propagated in vitro as neurospheres. Glioblastoma cells were isolated from tumour biopsies and propagated in vitro as spheres according to the same methods as fetal neural cells. Two stem/progenitor cell neurosphere and two glioblastoma spheroid cultures were initiated to differentiate using RA and cAMP. The cells were fixed and analyzed immunocytochemically on the 1st, 3rd, and 8th days of the differentiation. The expression of CCR5 was localized mainly in the cell nuclei, and was usually much weaker, if at all, in cytoplasm. Confocal laser microscopy analysis confirmed the same location. The expression of CCR5 was the highest one on the 3rd day of differentiation in all cultures, but showed also distinct differences between cultures, and in normal fetal differentiated stem/progenitor cells the expression of CCR5 was much weaker than in differentiated glioblastoma spheric cells.

Signalling pathway in the induction of neurite outgrowth in human mesenchymal stem cells

Recent in vivo transplantation studies have shown that mesenchymal stem cells (MSCs) were able to differentiate into mesoderm-derived cell types as well as cells with neuroectodermal characteristics, suggesting that transdifferentiation occurs in the mammalian system. We have reported an immortalized line of human MSCs (hMSCs), KP-hMSCs, which expresses CD29, CD44, CD90, and CD105, and complies with the characteristics shared by mere hMSCs. In a current experiment, we further demonstrated that expanded KP-hMSCs exhibited markers of neuroepithelial or neural precursor cells, such as Nestin, Musashi-1, Vimentin, NCAM, Pax-6, and Sox-9. KP-hMSCs simultaneously expressed proteins of the neuronal, astrocyte, and oligodendrocyte lineages during culture expansion; in addition, they initiated neurite outgrowth and eradicated protein expressions of astrocyte and oligodendrocyte lineages in response to the elevated signaling of the cAMP–PKA pathway after serum depletion in a defined neural induction medium. From the current results, KP-hMSCs may be used to elucidate molecular signaling on the neural differentiation of adult human non-neural tissues. We also presented evidence for the possibility that adult MSCs and fetal neuroepithelial or neural precursor cells both provide for the continual maintenance and repair of the postnatal neural tissues and may derive from the same origin or have one deriving from the other.

DNA Microarrays Reveal Relationship of Ewing Family Tumors to Both Endothelial and Fetal Neural Crest-Derived Cells and Define Novel Targets

Ewing family tumors (EFTs) are small round blue cell tumors that show features of neuroectodermal differentiation. However, the histogenetic origin of EFTs is still a matter of debate. We used high-density DNA microarrays for the identification of EFT-specific gene expression profiles in comparison with normal tissues of diverse origin. We identified 37 genes that are up-regulated in EFTs compared with normal tissues and validated expression of these genes in EFTs by both conventional and quantitative reverse transcription-polymerase chain reaction. The expression pattern of EFT-associated genes in normal tissues indicated a high similarity between EFTs and fetal and neuronal as well as endothelial tissues and supports the concept that a primitive neural crest-derived progenitor at the transition to mesenchymal and endothelial differentiation is transformed in EFTs. EFT-associated genes could be used for molecular discrimination between EFTs and other small round blue cell tumors and clearly identified a cell line (SK-N-MC) that was initially established as neuroblastoma as being an EFT. Ectopic expression of the EFT-specific EWS-FLI1 fusion protein in human embryonic kidney (HEK293) cells was not sufficient to induce the complete EFT-specific gene expression signature, suggesting that the EFT-specific gene expression profile is not just a consequence of EWS-FLI1 expression but depends on the histogenetic background of the EFT stem cell.

516. Vestibular Hair Cell Regeneration and Re-Establishment of Balance Function Induced by Math1 Gene Delivery

Due to their self-renewal and multi-potency, stem cells represent an attractive source for cell replacement therapy in neurological disorders. Genetic manipulation of these cells may allow controlled release of therapeutic proteins, suppress immune rejection, or produce essential neurotransmitters. Furthermore, when the expression cassette is incorporated into the host genome ex vivo, this technique also may be used as a method to trace cells following implantation into tissues of interest. We explored the possibility of transducing pluripotent fetal rat cortical neural progenitor cells using lentiviral vectors encoding either the green fluorescent protein (GFP) or neurotrophic factors (NT-3, BDNF, GDNF and CNTF) under control of the CMV promoter and the Woodchuck post-transcriptional regulatory element. Following isolation and expansion of the cells at clonal density on poly-ornithine-fibronectin-coated dishes in the presence of bFGF, cells were collected, infected and replated on the dishes. Staining of these cells for neural markers (such as nestin, GFAP, Tuj-1 and RIP) after transduction did not reveal any significant difference from non-transduced cells. However, when they were transduced with a vector encoding CNTF, cells started expressing GFAP. Cells continued to express the transgene, including when bFGF was withdrawn and when cells started to differentiate into GFAP positive cells. Following delayed (1 week) implantation into the lesion site of the moderately contused spinal cord, transduced cells survived well up to 4 weeks post-implantation (the longest time point currently examined). Migration of the cells was mainly restricted to white matter on either side of the lesion. Currently, the therapeutic and axonal growth stimulating properties of the implanted cells are being investigated in injured rats.

Treatment of spinal cord injury by transplantation of fetal neural precursor cells engineered to express BMP inhibitor

Spontaneous recovery after spinal cord injury is limited. Transplantation of neural precursor cells (NPCs) into lesioned adult rat spinal cord results in only partial functional recovery, and most transplanted cells tend to differentiate predominantly into astrocytes. In order to improve functional recovery after transplantation, it is important that transplanted neural precursor cells appropriately differentiate into cell lineages required for spinal cord regeneration. In order to modulate the fate of transplanted cells, we advocate transplanting gene-modified neural precursor cells. We demonstrate that gene modification to inhibit bone morphogenetic protein (BMP) signaling by noggin expression promoted differentiation of neural precursor cells into neurons and oligodendrocytes, in addition to astrocytes after transplantation. Furthermore, functional recovery of the recipient mice with spinal cord injury was observed when noggin-expressing neural precursor cells were transplanted. These observations suggest that gene-modified neural precursor cells that express molecules involved in cell fate modulation could improve central nervous system (CNS) regeneration.

Nuclear transfer of adult bone marrow mesenchymal stem cells: developmental totipotency of tissue-specific stem cells from an adult mammal.

Recent studies have demonstrated that somatic stem cells have a flexible potential greater than previously expected when they are transplanted into different tissues. On the other hand, recent studies also have revealed that these potentials might occur because of spontaneous cell fusion with recipient cells. The nuclei of somatic cells could have been reprogrammed when they were artificially or spontaneously fused with mouse embryonic stem (ES) cells. The resultant hybrid cells acquired a developmental pluripotency that the original somatic cells did not have but that ES cells did. LaBarge and Blau (Cell 2002; 111:589-601) demonstrated that adult bone marrow-derived cells contributed to muscle tissue in a stepwise biological progression. This means that bone marrow-derived cells became satellite cells of mononucleate muscle stem cells after the first irradiation-induced damage to the mouse, and after the second irradiation-induced damage, multinucleate myofibers appeared from the bone marrow-derived cells. Considered together, the differentiation potential of the somatic stem cell nucleus itself remains unclear. Although the pluripotency of somatic stem cell populations has been evaluated, the developmental totipotency of the nuclei of somatic stem cells, whether or not they fused with other cells, has not been shown, except in only one study concerning fetal neural cells (never in adult stem cells). Here, we showed the developmental totipotency of adult bovine mesenchymal stem cells by nuclear transfer.

Restorative potential of cholinergic rich transplants in cholchicine induced lesioned rats: a comparative study of single and multiple micro-transplantation approach

Restorative potential of fetal neural transplantation in colchicine induced neurodegeneration was studied in rats; where colchicine (2.5mg per site) was administered bilaterally into the hippocampus followed by bilateral infusions of fetal neural cell suspension rich in cholinergic neurons as single macro- or multiple micro-transplants in the hippocampal region 3 weeks post-colchicine (2.5mg per site) lesion. Animals were studied for neuro behavioural and neurochemical recovery at 4 and 24 weeks post-transplantation and electrophysiological (single cell recording) and immunohistochemical parameters, choline acetyl transferase (ChAT) expression was studied in hippocampus at 24 weeks post-transplantation. Colchicine lesioned rats receiving single macro- or multiple micro-transplants exhibited significant restoration in cognitive dysfunction caused by colchicine after 4 weeks of transplantation which remain persistent in multiple micro-transplanted group upto 24 weeks post-transplantation, whereas, single macro-transplanted animals did not exhibit any significant recovery. Neurochemical studies revealed significant restoration in acetylcholine esterase activity and cholinergic (muscarinic) receptor binding after 24 weeks post-transplantation as compared to 4 weeks post-transplantation in multiple micro-transplanted group. Single cell recording studied at 24 weeks post-transplantation exhibited significant restoration in firing rates when compared with lesioned group. The viability of cholinergic fibre at transplanted sites has further been confirmed by increase in ChAT immuno positivity in hippocampal region using monoclonal antibody and quantified using image analyser Leica Qwin 500 software. The results suggest that intra-hippocampal multiple site cholinergic rich transplants provide better and long term restoration in the cholinergic deficits induced by colchicine lesion as compared to single site macro-transplantation.