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Biomedical subjects

A E Willing

Publications and source records attributed to A E Willing.

At least 19 recordsLinked to original sources
PubMed2006

Influence of retinoic acid and lithium on proliferation and dopaminergic potential of human NT2 cells.

Our laboratory is working with the human NTera2/D1 (NT2) cell line, which has properties similar to those of progenitor cells in the central nervous system (CNS). These neural-like precursor cells can differentiate into all three major lineages, neurons, astrocytes, and oligodendrocytes. The pure neuronal population, hNT neurons, possess characteristics of dopamine (DA) cells. First, we analyzed whether the retinoic acid (RA)-treated hNT neurons and the NT2 precursor cells expressed two transcription factors required for development of the midbrain DA neurons. We report that NT2 cells endogenously expressed Engrailed-1 and Ptx3, whereas RA-treated hNT neurons did not express Engrailed-1 or Ptx3. Next we examined the influence of lithium treatment on Engrailed-1 and Ptx3 as well as another critical transcription factor, Nurr1. Previous research has shown that lithium can mimic the Wnt pathway, which is important for the induction of these transcription factors. Finally, we investigated the effect of lithium treatment on the viability and proliferation of NT2 cells, because lithium has been shown to stimulate neurogenesis in adult neural precursors. Lithium treatment increased the viability and proliferation of NT2 cells. The expression of transcription factors essential for the induction and maintenance of the DA phenotype was not increased in NT2 after lithium treatment. We conclude that the NT2 cell line is an excellent in vitro model system for studying the influence of pharmalogical agents on proliferation, differentiation, and apoptosis of a human neural progenitor cell line.

Antineoplastic Agents↗
PubMed2005

The role of connexins in the differentiation of NT2 cells in Sertoli-NT2 cell tissue constructs grown in the rotating wall bioreactor.

Neural transplantation is developing as a successful treatment for neurodegenerative diseases such as Parkinson's disease. The human Ntera-2/D1 (NT2) cell line is an attractive alternative to the use of human fetal neurons as a cell source for transplantation. We have explored combining NT2 cells, as a neuronal source, and Sertoli cells, which may act as a graft facilitator to enhance neuronal survival and differentiation, and ameliorate the host immune response, into a tissue construct for use in cell replacement therapy for neurodegenerative disease. This Sertoli-NT2-aggregated cell (SNAC) tissue construct is formed in the high aspect ratio vessel (HARV) bioreactor. NT2 cells differentiate to dopaminergic NT2N neurons within the SNAC tissue construct without retinoic acid. We report here that the gap junction protein connexin 43 is decreased among differentiated NT2N neurons. Inhibition of connexin 43 with 18beta glycyrrhetinic acid and carbenoxolone, a glycyrrhetinic acid derivative, during formation of the SNAC tissue constructs disrupts the differentiation of NT2 cells. Therefore, connexin 43 is important in the differentiation of NT2 cells in the SNAC tissue construct.

Animals↗
PubMed2005

Behavioral alterations in Lewis rats following two-day continuous 3-nitropropionic acid administration.

The mitochondrial toxin, 3-nitropropionic acid (3-NP), produces motor dysfunction and striatal atrophy in rats. However, rat strain and method of administration may contribute to variability in the deficits caused by 3-NP toxicity. To evaluate this, changes in nocturnal spontaneous locomotor activity from chronic administration of 3-NP using an osmotic mini pump, were examined in the Lewis rats. Lewis rats were treated with 3-NP or saline for 2 days and behavior was tested daily for a 15 day period. Animals receiving 3-NP displayed significantly less spontaneous activity than animals in the saline group. 3-NP treated animals also weighed significantly less when compared to saline treated animals. These results demonstrate that even though there were no significant alterations in overt anatomical pathology, even short-term exposure to 3-NP produced significant effects. This short-term administration may present a potential paradigm for examination of sub-threshold neurotoxicity.

Animals↗
PubMed2005

Survival of rat or mouse ventral mesencephalon neurons after cotransplantation with rat sertoli cells in the mouse striatum.

Transplanting cells across species (xenotransplantation) for the treatment of Parkinson's disease has been considered an option to alleviate ethical concerns and shortage of tissues. However, using this approach leads to decreased cell survival; the xenografted cells are often rejected. Sertoli cells (SCs) are testis-derived cells that provide immunological protection to developing germ cells and can enhance survival of both allografted and xenografted cells. It is not clear whether these cells will maintain their immunosuppressive support of cografted cells if they are transplanted across species. In this study, we investigated the immune modulatory capacity of SCs and the feasibility of xenografting these cells alone or with allografted and xenografted neural tissue. Transplanting xenografts of rat SCs into the mouse striatum with either rat or mouse ventral mesencephalon prevented astrocytic infiltration of the graft site, although all transplants showed activated microglia within the core of the graft. Surviving tyrosine hydroxylase-positive neurons were observed in all conditions, but the size of the grafts was small at best. SCs were found at 1 and 2 weeks posttransplant. However, few SCs were found at 2 months posttransplant. Further investigation is under way to characterize the immune capabilities of SCs in a xenogeneic environment.

Animals↗
PubMed2004

Do hematopoietic cells exposed to a neurogenic environment mimic properties of endogenous neural precursors?

Hematopoietic progenitors are cells, which under challenging experimental conditions can develop unusual phenotypic properties, rather distant from their original mesodermal origin. As previously reported, cells derived from human umbilical cord blood (HUCB) or human bone marrow (BM) under certain in vivo or in vitro conditions can manifest neural features that resemble features of neural-derived cells, immunocytochemically and in some instances also morphologically. The present study explored how hematopoietic-derived cells would respond to neurogenic signals from the subventricular zone (SVZ) of adult and aged (6 and 16 months old) rats. The mononuclear fraction of HUCB cells was transplanted into the SVZ of immunosuppressed (single cyclosporin or three-drug treatment) animals. The triple-suppression paradigm allowed us to protect transplanted human cells within the brain and to explore further their phenotypic and migratory properties. One week after implantation, many surviving HUCB cells were located within the SVZ and the vertical limb of the rostral migratory stream (RMS). The migration of HUCB cells was restricted exclusively to the pathway leading to the olfactory bulb. In younger animals, grafted cells navigated almost halfway through the vertical limb, whereas, in the older animals, the migration was less pronounced. The overall cell survival was greater in younger animals than in older ones. Immunocytochemistry for surface CD antigen expression showed that many HUCB cells, either cultured or within the brain parenchyma, retained their hematopoietic identity. A few cells, identified by using human-specific antibodies (anti-human nuclei, or mitochondria) expressed nestin and doublecortin, markers of endogenous neural progenitors. Therefore, it is believed that the environment of the neurogenic SVZ, even in aged animals, was able to support survival, "neuralization," and migratory features of HUCB-derived cells.

Age Factors↗
PubMed2004

Green fluorescent protein bone marrow cells express hematopoietic and neural antigens in culture and migrate within the neonatal rat brain.

Finding a reliable source of alternative neural stem cells for treatment of various diseases and injuries affecting the central nervous system is a challenge. Numerous studies have shown that hematopoietic and nonhematopoietic progenitors derived from bone marrow (BM) under specific conditions are able to differentiate into cells of all three germ layers. Recently, it was reported that cultured, unfractionated (whole) adult BM cells form nestin-positive spheres that can later initiate neural differentiation (Kabos et al., 2002). The identity of the subpopulation of BM cells that contributes to neural differentiation remains unknown. We therefore analyzed the hematopoietic and neural features of cultured, unfractionated BM cells derived from a transgenic mouse that expresses green fluorescent protein (GFP) in all tissues. We also transplanted the BM cells into the subventricular zone (SVZ), a region known to support postnatal neurogenesis. After injection of BM cells into the neurogenic SVZ in neonatal rats, we found surviving GFP+ BM cells close to the injection site and in various brain regions, including corpus callosum and subcortical white matter. Many of the grafted cells were detected within the rostral migratory stream (RMS), moving toward the olfactory bulb (OB), and some cells reached the subependymal zone of the OB. Our in vitro experiments revealed that murine GFP+ BM cells retained their proliferation and differentiation potential and predominantly preserved their hematopoietic identity (CD45, CD90, CD133), although a few expressed neural antigens (nestin, glial fibrillary acdiic protein, TuJ1).

Animals↗
PubMed2003

Intravenous versus intrastriatal cord blood administration in a rodent model of stroke.

Human umbilical cord blood (hUCB) is a rich source of hematopoietic stem cells that have been used to reconstitute immune cells and blood lineages. Cells from another hematopoietic source, bone marrow, have been found to differentiate into neural cells and are effective in the treatment of stroke. In this study, we administered hUCB cells intravenously into the femoral vein or directly into the striatum and assessed which route of cell administration produced the greatest behavioral recovery in rats with permanent middle cerebral artery occlusion (MCAO). All animals were immunosuppressed with cyclosporine (CSA). When spontaneous activity was measured using the Digiscan automated system, it was found to be significantly less when hUCB was transplanted 24 hr after stroke compared with nontransplanted, stroked animals (P < 0.01). Furthermore, behavioral recovery was similar with both striatal and femoral hUCB delivery. This is in contrast to the step test, in which significant improvements were found only after femoral delivery of the hUCB cells. In the passive avoidance test, transplanted animals learned the task faster than nontransplanted animals (P < 0.05). Together, these results suggest that hUCB transplantation may be an effective treatment for brain injuries, such as stroke, or neurodegenerative disorders. In addition, intravenous delivery may be more effective than striatal delivery in producing long-term functional benefits to the stroked animal.

Animals↗
PubMed2002

Lack of NF-kappaB p50 exacerbates degeneration of hippocampal neurons after chemical exposure and impairs learning.

The roles of activated NF-kappaB subunits in the CNS remain to be discerned. Members of this family of transcription factors are essential to diverse physiological processes and can be activated by pathogens, stress, pharmacological agents, and trauma. We are particularly interested in long-term NF-kappaB activation and its involvement in neuroplastic changes in the brain resulting from acquisition of memory as well as injury. Here, we use lesioning by the limbic-specific neurotoxicant trimethyltin (TMT) as a model in which to examine activation of the NF-kappaB p50 subunit before, during, and after neuronal degeneration. Neurons in wild-type mice that survived TMT-induced injury contained activated p50 and did not label with Fluoro-Jade, a histochemical marker of degenerating neurons. Granule cells of the wild-type dentate gyrus subregion, an area particularly vulnerable to TMT-induced degeneration, contained less activated p50 protein than CA regions. We compared the extent of degeneration in wild-type and p50-null mice and found a fivefold increase in death of hippocampal neurons in mice lacking p50. The hippocampus is key to processes of learning and memory, and NF-kappaB has reported involvement in these processes. The enhanced hippocampal degeneration in p50-null mice prompted us to evaluate their basal learning abilities, and we discovered that difficulties in task acquisition were an additional consequence of p50 ablation. These results indicate that absence of p50 negatively modulates learning ability as well as hippocampal responsiveness to brain injury after a chemical-induced lesion.

Animals↗
PubMed2001

In vitro induction and in vivo expression of bcl-2 in the hNT neurons.

Bcl-2 encodes membrane-associated proteins that suppress programmed cell death in cells of various origins. Compelling evidence suggests that bcl-2 is also involved in neuronal differentiation and axonal regeneration. The human Neuro-Teratocarcinoma (hNT) neurons constitute a terminally differentiated human neuronal cell line that is derived from the Ntera-2/clone D1 (NT2) precursors upon retinoic acid (RA) treatment. After transplantation into the central nervous system (CNS), the hNT neurons survive, engraft, maintain their neuronal identity, and extend long neurite outgrowth. We were particularly interested in the intracellular determinants that confer these post-transplant characteristics to the hNT neurons. Thus, we asked whether the hNT neurons express bcl-2 after transplantation into the rat striatum and if RA induction of the neuronal lineage is mediated by bcl-2. The grafted hNT neurons were first identified using three different antibodies that recognize human-specific epitopes, anti-hMit, anti-hNuc, and NuMA. After a 1-month post-transplant survival time, NuMA immunostaining revealed that 12% of the hNT neurons survived the transplantation. These neurons extended long neuritic processes within the striatum, as demonstrated using the human-specific antibody against the midsize neurofilament subunit HO14. Importantly, we found that 85% of the implanted hNT neurons expressed bcl-2 and that the in vitro induction of the neuronal lineage from the NT2 precursors with RA resulted in an upregulation of bcl-2 expression. Together, these data suggest that the differentiation of the hNT neurons to a neuronal lineage could be mediated at least partially by bcl-2.

Animals↗
PubMed2001

Apoptosis in cultured hNT neurons.

Programmed cell death (apoptosis) is an important mechanism shaping the size of different cell populations within the developing nervous system. In our study we used the NT2/D1 clone originally established from the Ntera 2 cell line to investigate the baseline levels of apoptosis in cultured postmitotic hNT (NT2-N) neurons previously treated for 3, 4 or 5 weeks with retinoic acid (RA) and compared it with apoptosis in NT2 precursors unexposed to RA. First, we examined whether different lengths of exposure to RA might affect baseline apoptotic rate in differentiating hNT neurons. Second, we investigated whether cultured hNT neurons, previously shown to possess dopaminergic characteristics, would be preferentially affected by apoptosis. Using the terminal deoxynucleotidyl transferase (tdt)-labeling technique we found that the postmitotic hNT neuronal cells exposed to RA demonstrated significantly higher numbers of apoptotic cells (12.5-15.8%) in comparison to rapidly dividing NT2 precursor cell line (3.6-4.4%) at both studied (1 and 5 days in vitro, DIV) time points. Similar apoptotic nuclear morphology, including a variable extent of nuclear fragmentation was observed in all examined hNT cultures. On the other hand, the incidence of apoptotic nuclei was rare in cultures of NT2 precursors not subjected to RA treatment. Combined immunocytochemistry for tyrosine hydroxylase (TH) and Hoechst staining revealed dopaminergic hNT neurons destined to die. Our double-labeling studies have demonstrated that only a subset of TH-positive hNT cells had condensed chromatin after 1 (approx. 15%) and 5 (approx. 20%) DIV. NT2 precursors were not TH-positive. Collectively, our results demonstrated that exposure to differentiating agent RA triggers an apoptotic commitment in a subset of postmitotic hNT neurons. These results suggest that this cell line may serve as a model of neuronal development to test various pathogenic factors implicated in the etiology of Parkinson's disease (PD), as well as to screen numerous pharmacological treatments that may slow or prevent dopaminergic deterioration.

Antineoplastic Agents↗
PubMed2001

Comparison of calcium-binding proteins expressed in cultured hNT neurons and hNT neurons transplanted into the rat striatum.

An alternative source of cells for neural transplantation and brain repair that has many characteristics of immature neurons is the hNT neuron, derived from an embryonal human teratocarcinoma (NTera2) cell line that is terminally differentiated in vitro with retinoic acid. The majority of hNT neurons are GABAergic in cell culture. We have determined the calcium-binding protein (CBP) phenotypes of hNT neurons for three CBPs, calretinin (CR), calbindin D-28K (CB), and parvalbumin (PV), in cell culture and after transplantation into the rat striatum. In cell culture, 95% of all cell profiles were human nuclear matrix antigen (NuMA) positive. PV-positive hNT neurons constituted 50% of all neuron-like profiles, with CB+ and CR+ constituting 14 and 6% of cells, respectively. In contrast, when the striatal grafts were examined after 30 days survival using confocal microscopy, only 10% of hNT neurons immunopositive for NuMA were PV+; 19% were CB+/NuMA+, approximately the same percentage as was seen in vitro, and 82% of grafted hNT neurons were CR+. These results suggest that hNT neurons can be subdivided into at least three subpopulations based on the CBP phenotype that they express and that there is a CBP phenotypic shift following transplantation. Three related hypotheses are proposed to account for this phenotypic shift of hNT neurons after transplantation: (a) selective survival of the CR+ subpopulation of hNT neurons, (b) selective transitory quiescence of the transplanted PV+ cells due to transplantation stress, or (c) dedifferentiation of the hNT neurons following transplantation, which may allow them to respond to local environmental cues during the engraftment process.

Animals↗
PubMed2001

NF-kappaB p50 is increased in neurons surviving hippocampal injury.

Signal transduction pathways that lead to the modulation of genes related to survival and repair mechanisms are activated in neurons that survive injury. These protein kinase/phosphatase cascades converge on transcription factors, the DNA binding proteins that directly regulate gene expression. In this study we examined expression of the NF-kappaB p50 subunit in the rat hippocampus 7 days after injury caused by middle cerebral artery occlusion or trimethyltin treatment. We found increased levels of p50 in neurons throughout the hippocampus after both treatments, localized not only in cell bodies but also in processes. At the 7-day time point, Fluoro-Jade histochemistry revealed hippocampal neurodegeneration in trimethyltin-treated rats but not in those lesioned by middle cerebral artery occlusion. p50 was not expressed in Fluoro-Jade-positive degenerating cells, supporting the role of this transcriptional subunit in neurosurvival. Because phosphorylation of the inhibitor IkappaB protein by IkappaB kinase is the classic step in NF-kappaB activation, phospho-IkappaBalpha immunoreactivity was examined as an indication of IkappaB kinase activity. Levels of phospho-IkappaBalpha were increased in neurons throughout the hippocampus 7 days postinjury. Immunoblotting for phospho-IkappaBalpha demonstrated increased levels 1 day postinjury that remained elevated for at least 7 days. These data suggest that NF-kappaB signal transduction is involved in an adaptive response of neurons that survive injury.

Animals↗
PubMed2001

hNT neurons delay onset of motor deficits in a model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a degenerative motor neuron disease that manifests as a progressive muscular weakness leading to paralysis and death. Because of the diffuse nature of the motor neuron death, this disease is not considered a good candidate for treatment through neural transplantation. The purpose of this study was to show that transplantation of human neuron-like cells (hNT neurons) into the spinal cord of a transgenic ALS mouse model would improve motor deficits. The hNT neurons were transplanted bilaterally into L4-L5 spinal cord of the transgenic mice ( approximately 8 weeks of age), and the animals were evaluated on health and behavioral measures. The animals were perfused, and immunohistochemistry was performed to identify the transplanted cells. Transplantation of the hNT neurons into the spinal cord delayed the onset of motor behavioral symptoms. This was the first demonstration that even localized transplantation of neural cells directly into the parenchyma could improve motor function in an ALS model. Further study is needed to delineate the mechanism underlying these effects. This therapeutic approach has the potential to restore neural transmission, thereby improving quality of life for the ALS patient and possibly extend life expectancy.

Amyotrophic Lateral Sclerosis↗
PubMed2001

Intraspinal implantation of hNT neurons into SOD1 mice with apparent motor deficit.

INTRODUCTION: The aim of this study was to determine the effect of hNT neuron transplants on motor neuron function in SOD1 (G93A) mice when motor deficits were already apparent. METHOD: The hNT neurons were implanted into L(4)-L(5) segments of the ventral horn spinal cord of mice at 15-16 weeks of age: either G93A mice, transgenic mice carrying the normal allele for human SOD1 gene (hTg), or control wild type mice (wt). Behavioral tests (rotorod, beam balance, extension reflex, footprint) were performed prior to transplantation and at weekly intervals afterwards. RESULTS: HNT neuron transplantation in the SOD1 mice delayed disease progression for 3-4 weeks, although lifespan was not affected. CONCLUSION: These results suggest that hNT neuron transplantation may be a promising therapeutic strategy for ALS in the later phase of the neurodegeneration.

Animals↗
PubMed2001

Intravenous administration of human umbilical cord blood reduces behavioral deficits after stroke in rats.

BACKGROUND AND PURPOSE: Human umbilical cord blood cells (HUCBC) are rich in stem and progenitor cells. In this study we tested whether intravenously infused HUCBC enter brain, survive, differentiate, and improve neurological functional recovery after stroke in rats. In addition, we tested whether ischemic brain tissue extract selectively induces chemotaxis of HUCBC in vitro. METHODS: Adult male Wistar rats were subjected to transient (2-hour) middle cerebral artery occlusion (MCAO). Experimental groups were as follows: group 1, MCAO alone (n=5); group 2, 3x10(6) HUCBC injected into tail vein at 24 hours after MCAO (n=6) (animals of groups 1 and 2 were killed at 14 days after MCAO); group 3, MCAO alone (n=5); group 4, MCAO injected with PBS at 1 day after stroke (n=8); and group 5, 3x10(6) HUCBC injected into tail vein at 7 days after MCAO (n=5). Rats of groups 3, 4, and 5 were killed at 35 days after MCAO. Behavioral tests (rotarod and Modified Neurological Severity Score [mNSS]) were performed. Immunohistochemical staining was used to identify cells derived from HUCBC. Chemotactic activity of ischemia brain tissue extracts toward HUCBC at different time points was evaluated in vitro. RESULTS: Treatment at 24 hours after MCAO with HUCBC significantly improved functional recovery, as evidenced by the rotarod test and mNSS (P<0.05). Treatment at 7 days after MCAO with HUCBC significantly improved function only on the mNSS (P<0.05). Some HUCBC were reactive for the astrocyte marker glial fibrillary acidic protein and the neuronal markers NeuN and microtubule-associated protein 2. In vitro, significant HUCBC migration activity was present at 24 hours after MCAO (P<0.01) compared with normal brain tissue. CONCLUSIONS: Intravenously administered HUCBC enter brain, survive, migrate, and improve functional recovery after stroke. HUCBC transplantation may provide a cell source to treat stroke.

Animals↗
PubMed2001

Taurine increases rat survival and reduces striatal damage caused by 3-nitropropionic acid.

Taurine acts as an antioxidant protecting neurons from free radical-mediated cellular damage. 3-nitropropionic acid (3-NP) inhibits energy metabolism, initiating oxidative stress. With the objective to examine whether taurine can protect glia and neurons from damage produced by 3-NP, male Wistar and Sprague-Dawley rats were treated with either (1) saline, (2) taurine (3) 3-NP and saline, or (4) 3-NP and taurine for 4 days. Survival was determined and brains were processed immunohistochemically. Large striatal lesions and increased GFAP, SOD, and taurine immunoreactivity were detected in the 3-NP group when compared with control groups. In contrast, animals receiving 3-NP and taurine exhibited less GFAP, SOD, and taurine immunoreactivity, along with increased survival rates. Results indicate that taurine treatment after 3-NP administration protects the striatum from damage.

Animals↗
PubMed2000

Dopaminergic phenotype of hNT cells in vitro.

We investigated the catecholaminergic nature of cultured hNT neurons previously treated either for 4 or 5 weeks with retinoic acid (RA). There were significantly more tyrosine hydroxylase (TH)-positive neurons (60%) in cultures treated for 4 weeks with RA compared to 5 week-treated cultures (</=15%). Furthermore, numerous TH-positive hNT cells were also immunoreactive to dopamine transporter (DAT), dopamine receptor (D2) and aldehyde dehydrogenase (AHD-2), an enzyme exclusively expressed by dopaminergic (DA) ventral mesencephalic (VM) precursors. Thus this cell line has all the necessary cellular machinery to produce functional DA neurons and therefore is a good alternative tissue source to fetal VM.

Aldehyde Dehydrogenase↗