Ora Dillon-Carter

Bone marrow grafts restore cerebral blood flow and blood brain barrier in stroke rats

We monitored alterations in cerebral blood flow (CBF) and blood-brain barrier (BBB) permeability following middle cerebral artery occlusion (MCAo) and intrastriatal transplantation of mouse bone marrow stromal cells (BMSCs) or saline infusion in adult Sprague-Dawley rats. Laser Doppler and Evans Blue assay revealed that BMSC grafts dose-dependently restored CBF and BBB to near normal levels at a much earlier period (Days 4-5 post-MCAo) in transplanted stroke animals compared to stroke animals that received saline infusion (Days 11-14 post-MCAo). Xenografted BMSCs survived in the absence of immunosuppression, and elevated levels of transforming growth factor-beta superfamily of neurotrophic factors were detected in transplanted stroke animals. These data suggest that early restoration of CBF and BBB following transplantation of BMSCs could mediate the reported functional outcomes in stroke animals.

Stable Expression of hrGFP by Mouse Embryonic Stem Cells: Promoter Activity in the Undifferentiated State and During Dopaminergic Neural Differentiation

Three promoters, cellular polypeptide chain elongation factor 1 alpha (EF1), cytomegalovirus (CMV), and Rous sarcoma virus (RSV) were examined for stable transgene expression in mouse embryonic stem (ES) cells and their progeny during dopaminergic neural differentiation. In undifferentiated ES cells the EF1 promoter was highly effective, while CMV had moderate activity. After 3 months in culture, expression of humanized renilla green fluorescent protein (hrGFP) was unchanged for the EF1 promoter and decreased for CMV. At the nestin‐positive stage of differentiation, hrGFP and nestin were colocalized in about 20% of cells for EF1, in contrast to 80% of cells for the CMV promoter. In tyrosine hydroxylase (TH)‐positive neurons neither the EF1 nor CMV promoter were effective. The RSV promoter was inactive in undifferentiated, nestin‐positive, and TH‐positive cells. Thus, EF1 and CMV are effective promoters for transgene expression in undifferentiated ES cells and nestin‐positive neural precursors.

Properties of [3H]AMPA binding in postmortem human brain from psychotic subjects and controls : increases in caudate nucleus associated with suicide

[3H]AMPA binding, a measure of the non-NMDA excitatory amino acid receptors, was measured in the frontal cortex, caudate nucleus, and nucleus accumbens of postmortem human brain tissue samples. In normal human frontal cortex, the binding data were best fit by a two-site model, with Kd values of 137 nM and 11.3 microM, and Bmax values of 2780 fmol/mg protein and 67.6 pmol/mg protein, respectively. Binding was linearly related to protein concentration and was strongly inhibited by glutamic acid and quisqualic acid. Binding was partially inhibited by kainic acid and glutamic acid diethyl ester and only slightly inhibited by N-methyl-D-aspartic acid. AMPA binding was not inhibited by neuroleptic drugs, in vitro. Freezing and storage did not result in a loss of AMPA binding, and there tended to be an increase in AMPA binding with extended freezer storage. When tissue frozen intact was compared to tissue frozen as a homogenate, the high-affinity binding parameters were unchanged, but there was an increase in the affinity and Bmax of the low-affinity site for the tissue frozen intact. Thus it appears that only the high-affinity site can be measured accurately in tissue frozen intact. AMPA binding was not significantly altered by premortem neuroleptic administration, age, postmortem delay, or by moderate durations of freezer storage. No differences in AMPA binding were found in psychotic subjects compared to normal controls. There was, however, a pronounced increase in total AMPA binding in the caudate nucleus in subjects that had committed suicide.

Trophic factor secreting kidney cell lines: in vitro characterization and functional effects following transplantation in ischemic rats.

Several kidney cell lines were investigated for their ability to produce glial cell line-derived neurotrophic factor (GDNF). Cell line-conditioned medium was analyzed using ELISA and two cell lines were identified which produce GDNF in physiologically active concentrations. ELISA analyses revealed that conditioned medium from these two cell lines also contained PDGF, bFGF, TGFbeta1 and TGFbeta2. Both of these cell lines were then transplanted into the striatal penumbra of rats, 1 h following middle cerebral artery occlusion. Behavioral testing revealed that both cell lines reduced the deficit associated with cerebral ischemia and reduced the infarct volume relative to controls. Reduction of infarct volume was likely achieved by the action of GDNF and/or other growth factors produced by the cells.

Intracerebral xenografts of mouse bone marrow cells in adult rats facilitate restoration of cerebral blood flow and blood-brain barrier.

We examined in the present study alterations in cerebral blood flow (CBF) and blood-brain barrier (BBB) permeability following intrastriatal transplantation of mouse bone marrow stromal cells (BMSCs) or saline infusion in adult Sprague-Dawley rats. Laser Doppler revealed that transplanted animals exhibited near normal cerebral blood flow (CBF, 150 perfusion units) at a much earlier period post-transplantation (day 4) compared to animals that received saline infusion (day 12) (ps<0.05). Similarly, Evans Blue assay demonstrated that transplanted animals exhibited near complete BBB reconstitution at day 5 post-transplantation, whereas animals that received saline infusion continued to display a compromised BBB up to 11 days post-transplantation. Transplanted animals displayed a cell dose-dependent CBF and BBB restoration. Enzyme-linked immunosorbent assay (ELISA) of transplanted BMSCs revealed elevated levels of transforming growth factor-beta superfamily of neurotrophic factors. Moreover, despite the absence of immunosuppression in this cross-species transplantation, at least in the acute phase (12 days post-transplantation), surviving xenografts were detected during periods of restored CBF and BBB permeability. These observations suggest that restoration of CBF and BBB permeability accompanies the reported functional outcomes associated with intracerebral transplantation of BMSCs.

A truncated SV40 large T antigen lacking the p53 binding domain overcomes p53-induced growth arrest and immortalizes primary mesencephalic cells.

Abstract As an alternative to primary fetal tissue, immortalized central nervous system (CNS)-derived cell lines are useful for in vitro CNS model systems and for gene manipulation with potential clinical use in neural transplantation. However, obtaining immortalized cells with a desired phenotype is unpredictable, because the molecular mechanisms of growth and differentiation of CNS cells are poorly understood. The SV40 large T antigen is commonly used to immortalize mammalian cells, but it interferes with multiple cell-cycle components, including p53, p300, and retinoblastoma protein, and usually produces cells with undifferentiated phenotypes. In order to increase the phenotypic repertoire of immortalized CNS cells and to address the molecular mechanisms underlying immortalization and differentiation, we constructed an expression vector containing a truncated SV40 large T gene that encodes only the amino-terminal 155 amino acids (T155), which lacks the p53-binding domain. Constructs were first transfected into a p53-temperature-sensitive cell line, T64-7B. Colonies expressing T155 proliferated at the growth-restrictive temperature. T155 was then transfected into primary cultures from embryonic day-14 rat mesencephalon. Two clonal cell lines were derived, AF-5 and AC-10, which co-expressed T155 and mature neuronal and astrocytic markers. Thus, the amino-terminal portion of SV40 large T is sufficient to: (1) overcome p53-mediated growth arrest despite the absence of a p53-binding region, and (2) immortalize primary CNS cells expressing mature markers while actively dividing. T155 and T155-transfectants may be useful for further studies of cell-cycle mechanisms and phenotyic expression in CNS cells or for further gene manipulation to produce cells with specific properties.

Transduction of Human GAD67 cDNA into Immortalized Striatal Cell Lines Using an Epstein–Barr Virus-Based Plasmid Vector Increases GABA Content

The M213-20 and M213-1L cell lines were immortalized from rat striatum using the tsA58 allele of the SV40 large T antigen, contain the GAD enzyme, and produce GABA (Giordano et al., 1994, Exp. Neurol. 124:395-400). Cell lines that produce large amounts of GABA may be useful for transplantation into the brain in conditions such as Huntingtons disease or epilepsy, where localized application of GABA may be of therapeutic value. We have explored the potential use of the pREP10 plasmid vector, which replicates episomally, to increase GAD expression and GABA production in M213-20 and M213-1L cells. Human GAD(67) cDNA was transfected into M213-20 and M213-1L, and subclones were isolated with hygromycin selection. Immunochemical studies showed increased GAD(67) expression compared to the parent M213-20 and M213-1L cell lines. Staining for the EBNA antigen and Southern blots demonstrated that the pREP10 plasmid was stably maintained in the cells for at least 12-15 months in culture. Several clones were isolated in which GABA concentrations were increased by as much as 4-fold (M213-1L) or 44-fold (M213-20) compared to the parent cell lines or 12-fold (M213-1L) and 94-fold (M213-20) greater than rat striatal tissue (1.678 +/- 0.4 micromol/g prot). The ability of these cells to continue to produce large amounts of GABA while being maintained in culture for extended periods suggests that similar methods might be used with human cell lines to produce cells that can be transplanted into the brain to deliver GABA for therapeutic purposes.

Transforming Growth Factors β1 and β2 in the Cerebrospinal Fluid of Chronic Schizophrenic Patients

Transforming growth factor betas (TGFβs) are potent immunosuppressive molecules released in the brain after injury. We hypothesized that TGFβ levels in cerebrospinal fluid (CSF) of schizophrenic patients would be altered because TGFβ can influence neural cell adhesion moelecule (N-CAM) expression in vitro. The levels of TGFβ1 and β2 in CSF of patients with schizophrenia and normal controls measured by ELISA showed no differences. There was evidence that the stability of TGFβ in CSF may be altered in schizophrenia. For a limited sample, TGFβ1 and N-CAM concentrations were significantly correlated in normal patients (r = 0.98) but not in schizophrenics. The results do not support an active neurodegeneration or anti-inflammatory response in the central nervous system, which is reflected in the CSF of chronic schizophrenics.

T155g-immortalized kidney cells produce growth factors and reduce sequelae of cerebral ischemia.

Fetal rat kidney cells produce high levels of glial-derived neurotrophic factor (GDNF) and exert neuroprotective effects when transplanted into the brain in animal models of Parkinsons disease and stroke. The purpose of the present experiment was to produce kidney cell lines that secrete GDNF. Genes encoding two truncated N-terminal fragments of SV40 large T antigen, T155g and T155c, which does not code for small t antigen, were used. T155g was transduced into E17 cultured fetal Sprague-Dawley rat kidney cortex cells using a plasmid vector, and T155c was transduced with a plasmid and a retroviral vector. Sixteen clones were isolated from cultures transfected with the T155g-expressing plasmid. No cell lines were obtained with T155c. Four clones produced GDNF at physiological concentrations ranging from 55 to 93 pg/ml of medium. These four clones were transplanted into the ischemic core or penumbra of rats that had undergone middle cerebral artery occlusion (MCAO). Three of the four clones reduced the volume of infarction and the behavioral abnormalities normally resulting from MCAO. Blocking experiments with antibodies to GDNF and platelet-derived growth factor (PDGF) suggested that these growth factors contributed only minimally to the reduction in infarct volume and behavioral abnormality. These cell lines may be useful for intracerebral transplantation in animal models of brain injury, stroke, or Parkinsons disease.

In vitro properties of a newly established medulloblastoma cell line, MCD-1

Medulloblastomas are poorly differentiated brain tumors believed to arise from primitive pleuripotential stem cells, and tend to express mixed neuronal and glial properties. In the present study, we examined immunohistochemical and neurotransmitter phenotypic properties in a newly established medulloblastoma cell line, MCD-1. MCD-1 cells were immortal, not contact-inhibited, but did not grow in soft agar. Immunohistochemical studies showed positive staining for neurofilament protein (NF), neuron-specific enolase (NSE), synaptophysin, MAP 2, tau, NCAM 180, vimentin, and S-100 protein. The cells expressed specific uptake of glutamate, serotonin, and choline, but not GABA or dopamine. A significant increase in process extension was seen in response to agents that enhance intracellular cyclic AMP, especially 3-isobutyl-1-methylxanthine (IBMX). Process formation induced by IBMX was associated with a decrease in cell proliferation as evidenced by a reduction in numbers of cells incorporating 5-bromo-2-deoxyuridine (BrdU). No increase in process extension was observed following exposure to NGF or retinoic acid. MCD-1 cells were shown to produce transforming growth factor beta (TGF beta), and were immunopositive for mutant p53. Transfection assays with the PG13-Luc reporter plasmid, which contains a p53-responsive enhancer element and a luciferase reporter gene, suggested MCD-1 cells are deficient in wild-type p53 and do not activate p53 on treatment with the anticancer agent adriamycin. The MCD-1 cell line is suggested to represent an abnormally differentiated cell type, which has some properties consistent with a multipotent neuronal phenotype while retaining some properties of immature cells of a glial lineage. The MCD-1 cell line can be used to provide a model of a medulloblastoma cell line that is resistant to growth-controlling and anticancer agents.