Pluripotent stem cells in neuropsychiatric disorders.

Neuropsychiatric disorders place an enormous medical burden on patients across all social and economic ranks. The current understanding of the molecular and cellular causes of neuropsychiatric disease remains limited, which leads to a lack of targeted therapies. Human-induced pluripotent stem cell (iPSC) technology offers a novel platform for modeling the genetic contribution to mental disorders and yields access to patient-specific cells for drug discovery and personalized medicine. Here, we review recent progress in using iPSC technology to model and potentially treat neuropsychiatric disorders by focusing on the most prevalent conditions in psychiatry, including depression, anxiety disorders, bipolar disorder and schizophrenia.

Differentiation of embryonic stem cell lines generated from adult somatic cells by nuclear transfer.

Embryonic stem (ES) cells are fully pluripotent in that they can differentiate into all cell types, including gametes. We have derived 35 ES cell lines via nuclear transfer (ntES cell lines) from adult mouse somatic cells of inbred, hybrid, and mutant strains. ntES cells contributed to an extensive variety of cell types, including dopaminergic and serotonergic neurons in vitro and germ cells in vivo. Cloning by transfer of ntES cell nuclei could result in normal development of fertile adults. These studies demonstrate the full pluripotency of ntES cells.

Transplantation of expanded mesencephalic precursors leads to recovery in parkinsonian rats.

In vitro expansion of central nervous system (CNS) precursors might overcome the limited availability of dopaminergic neurons in transplantation for Parkinson's disease, but generating dopaminergic neurons from in vitro dividing precursors has proven difficult. Here a three-dimensional cell differentiation system was used to convert precursor cells derived from E12 rat ventral mesencephalon into dopaminergic neurons. We demonstrate that CNS precursor cell populations expanded in vitro can efficiently differentiate into dopaminergic neurons, survive intrastriatal transplantation and induce functional recovery in hemiparkinsonian rats. The numerical expansion of primary CNS precursor cells is a new approach that could improve both the ethical and the technical outlook for the use of human fetal tissue in clinical transplantation.

Efficient generation of midbrain and hindbrain neurons from mouse embryonic stem cells.

Embryonic stem (ES) cells are clonal cell lines derived from the inner cell mass of the developing blastocyst that can proliferate extensively in vitro and are capable of adopting all the cell fates in a developing embryo. Clinical interest in the use of ES cells has been stimulated by studies showing that isolated human cells with ES properties from the inner cell mass or developing germ cells can provide a source of somatic precursors. Previous studies have defined in vitro conditions for promoting the development of specific somatic fates, specifically, hematopoietic, mesodermal, and neurectodermal. In this study, we present a method for obtaining dopaminergic (DA) and serotonergic neurons in high yield from mouse ES cells in vitro. Furthermore, we demonstrate that the ES cells can be obtained in unlimited numbers and that these neuron types are generated efficiently. We generated CNS progenitor populations from ES cells, expanded these cells and promoted their differentiation into dopaminergic and serotonergic neurons in the presence of mitogen and specific signaling molecules. The differentiation and maturation of neuronal cells was completed after mitogen withdrawal from the growth medium. This experimental system provides a powerful tool for analyzing the molecular mechanisms controlling the functions of these neurons in vitro and in vivo, and potentially for understanding and treating neurodegenerative and psychiatric diseases.

Enhanced proliferation, survival, and dopaminergic differentiation of CNS precursors in lowered oxygen.

Standard cell culture systems impose environmental oxygen (O(2)) levels of 20%, whereas actual tissue O(2) levels in both developing and adult brain are an order of magnitude lower. To address whether proliferation and differentiation of CNS precursors in vitro are influenced by the O(2) environment, we analyzed embryonic day 12 rat mesencephalic precursor cells in traditional cultures with 20% O(2) and in lowered O(2) (3 +/- 2%). Proliferation was promoted and apoptosis was reduced when cells were grown in lowered O(2), yielding greater numbers of precursors. The differentiation of precursor cells into neurons with specific neurotransmitter phenotypes was also significantly altered. The percentage of neurons of dopaminergic phenotype increased to 56% in lowered O(2) compared with 18% in 20% O(2). Together, the increases in total cell number and percentage of dopaminergic neurons resulted in a ninefold net increase in dopamine neuron yield. Differential gene expression analysis revealed more abundant messages for FGF8, engrailed-1, and erythropoietin in lowered O(2). Erythropoietin supplementation of 20% O(2) cultures partially mimicked increased dopaminergic differentiation characteristic of CNS precursors cultured in lowered O(2). These data demonstrate increased proliferation, reduced cell death, and enhanced dopamine neuron generation in lowered O(2), making this method an important advance in the ex vivo generation of specific neurons for brain repair.

NGF increases neuritic complexity of cholinergic interneurons in organotypic cultures of neonatal rat striatum.

The influence of NGF on cholinergic interneurons in organotypic roller tube cultures of 4 day postnatal rat striatum was examined after 13 to 16 days in vitro. Cultures were divided into four groups. The medium of the NGF treated group was supplemented with 5 ng/ml NGF, whereas control groups were cultured either without NGF, by adding 20 ng/ml neutralising anti-NGF antibody, or by adding both NGF and anti-NGF antibody to the medium. Two different cell populations were identified by an image analysis system which measured acetylcholinesterase staining intensity. It was demonstrated that NGF promotes survival of the large, intensely stained population. Eighty computer-assisted reconstructions of intensely stained cells, 20 for each treatment group, were performed in a random order by means of a neuron tracing system. Axons and dendrites were analysed separately. NGF enhanced complexity of neuritic, predominantly axonal trees by increasing the number of axonal segments by 91% to 100% (P < 0.01), the number of dendritic segments by 33% to 63% (P = 0.09 to P < 0.01), maximal axonal branch order by 37% to 50% (P < 0.05), and maximal dendritic branch order by 22% to 37% (P < 0.05). Further evidence of more complex neuritic trees was given by Sholl concentric sphere analysis. Anti-NGF antibody could block all these effects. General rules of branching architecture were not affected by NGF treatment as shown by analysing mean segment length in relation to the branch order, branch point exit angles, total tortuosity, Rall's ratio, and tapering of neuritic trees.

Comparison of the topology and growth rules of motoneuronal dendrites.

The complexity, shape, and branching modes of the dendrites of spinal motoneurons were compared in cat, rat, and frog using topological analysis and growth models. The complexity of motoneuronal dendrites, measured as the mean number of terminal segments, varied significantly among samples and was related to contractile properties of innervated motor units. Despite this variation, all mature motoneurons having a mean number of terminal segments per dendrite greater than ten (up to 24.3) exhibited a narrow range of values of coefficients describing the symmetry of tree shapes (0.42-0.47). This implies low variability in the topological shape of motoneuronal dendrites of different animals. This similarity of tree shapes proved to be a result of the similarity of growth rules. The growth of the dendrites could be described to a first approximation by a two-parameter (Q and S) model called the QS model and by a multitype Markovian model. The estimation of parameters of the QS model, in which parameter Q is related to the probability of branching of intermediate segments, revealed that Q was equal or close to 0, implying that branching of dendrites is restricted to terminal segments. The estimates of the parameter S, which describes whether the probability of branching increases (S < 0) or decreases (S > 0) exponentially with segment order, were positive. This was in agreement with the results of estimation of probabilities of branching provided by the Markovian model, which showed that the branching probabilities decreased with segment order in an exponential manner in most of the neurons studied. The QS and Markovian models involve different assumptions about the sequence and timing of branching events, and selection of the best model can provide insight into details of dendritic outgrowth. Extensive simulation of tree outgrowth using a Markovian model revealed significant differences between stimulated trees and real dendrites, particularly with regard to variability of the number of terminals and to symmetry. In contrast, the QS model provided a good fit to the mean values and standard deviations of basic topological parameters. This model is adequate to describe the shape of mature motoneuronal dendrites. It implies that dendritic branches have many opportunities to bifurcate during the whole time of development and that bifurcating potency of a branch is a function of the number and position of other branches of that dendrite. Combined with analysis of metrical properties such as lengths of segments, the QS model can assist in a quantitative analysis of development and plasticity.

A mathematical model for the estimation of human embryonic and fetal age.

Precise determination of donor age in human embryonic and fetal tissue is crucial for cell transplantation due to the existence of distinct time windows within which successful grafting is possible. This study demonstrates that between 4-12 wk postconception embryonic and fetal age can be estimated based on various morphometric parameters measured on a routine basis in suction abortion material. The greatest length, the neck-rump length, the foot length, and the proximal and distal arm and leg length were correlated with the anamnestic and ultrasonographically estimated age. Multivariate regression analyses showed a linear correlation between age and the logarithmic value of the various morphometric parameters. The best correlation was found for a mathematical model combining the limb parameters (r = 0.904; p < 0.001; n = 37). A prospective follow-up study (n = 40) was carried out to test the validity of the mathematical model. A high correlation was found between the calculated age and the estimated age based on anamnestic data (r = 0.749, p < 0.001). Outliers due to errors in the anamnestic data were readily identified by comparing anamnestic with calculated age. This method allows determination of embryonic and fetal age within and beyond the age group of the Carnegie classification and may, therefore, be useful for the needs of experimental and clinical cell transplantation.

Long-term survival of dopaminergic neurones in free-floating roller tube cultures of human fetal ventral mesencephalon.

Transplantation of human fetal ventral mesencephalon (VM) to Parkinsonian patients has shown beneficial effects in several clinical trials. However, further improvements in the transplantation technique are needed. Delayed surgery, i.e., the in vitro maintenance of the tissue prior to transplantation would present several advantages. The roller tube technique as initially described by Gähwiler (1981) was modified in several aspects for the long-term maintenance of dopaminergic neurones of human fetal VM. Tissue cultures were maintained free-floating in the medium for up to 42 days. The human fetal material was obtained from legal induced suction abortions. The embryonic age ranged from 5 to 12 weeks post-conception. Identification of VM was possible in 43% of the cases. Neurones in cultures were demonstrated by means of immunohistochemistry for tyrosine hydroxylase (TH) and gamma-amino butyric acid (GABA), by electron microscopy and by hybridisation histochemistry using a TH-mRNA-sensitive probe. A high variability in the number of TH-positive cells in individual cultures derived from the same embryo was observed. In 20 microns frozen sections of such tissue cultures the mean +/- SEM of TH-positive cells was 6.5 +/- 1.2/0.1 mm2 (n = 79; range: 0-73). The technique described insures the growth of long-term cultures of human fetal VM.

Noninvasive dopamine determination by reversed phase HPLC in the medium of free-floating roller tube cultures of rat fetal ventral mesencephalon: a tool to assess dopaminergic tissue prior to grafting.

The low availability of dopamine containing neurons for grafting in Parkinson's disease is a general problem. Free-floating roller tube (FFRT) cultures allow storage of fetal mesencephalic tissue prior to transplantation. Preoperative functional testing permits to select an optimized set of individual cultures for transplantation. Rat fetal ventral mesencephali (E13) were dissected out and divided into four equally sized pieces each and individually prepared as FFRT cultures. After 4, 8, 12, and 16 days in vitro (DIV) the medium of each culture was collected during routine medium change and immediately stabilized. Dopamine was extracted and probes were determined with reversed phase HPLC using electro-chemical detection. After 16 DIV cultures were fixed and cell counts performed in tyrosine hydroxylase (TH)-immunostained serial sections. The mean dopamine content +/- SEM In culture conditioned media was at 4 DIV: 21 +/- 2 pg, n = 38; at 8 DIV: 37 +/- 4 pg, n = 40; at 12 DIV: 52 +/- 7 pg, n = 38; and at 16 DIV: 39 +/- 5 pg, n = 38. In all cultures devoid of dopamine after 4 and 8 DIV (12.5%) levels remained below detectability at 12 and 16 DIV. Cultures derived from the rostral mesencephalon showed significantly higher dopamine values than those from the caudal mesencephalon at 12 DIV. The mean number of TH-immunoreactive (-ir) cells/culture +/- SEM after 16 DIV was 556 +/- 51, n = 40. The correlation between TH-ir cell number (CN) and dopamine content of rostrally derived cultures at 16 DIV was: CN = 7.4 (dopamine [pg]) + 248; R = 0.75; n = 19; p < 0.001. No dopamine was present in cultures without TH-ir cells. These results demonstrate that sequential noninvasive screening of dopamine in single cultures is feasible and that the dopamine content is correlated to the number of surviving TH-ir cells. This permits to select cultures rich in dopaminergic neurons for transplantation.

Medicaid nursing facility reimbursement methods: 1979-1997.

This article describes state Medicaid nursing facility reimbursement methods and rates in 1979-1997, using data derived from telephone surveys of state Medicaid reimbursement. The 1980s saw shifts toward prospective methodology. The late 1980s and early 1990s were characterized by adoption of casemix methods. The early 1990s also saw fewer changes in methodology with a hiatus in the mid-1990s followed recently by renewed changes to methodology. Medicaid per diem rates have increased faster than inflation but less rapidly than general health costs. The repeal of the Boren Amendment may now allow states to institute greater cost controls or moratoria on rate increases. Despite states' tendencies to follow one another's examples, Medicaid reimbursement remains diverse nationally, with wide differences in policies and rates.

Follow-up of rapists treated in a forensic psychiatric hospital.

Current research examining the effectiveness of sex offender treatment is encouraging. Despite this optimism, research focusing on men who sexually assault adult women is less optimistic. This study examines the preliminary results of 74 rapists admitted into the Phoenix program, a voluntary sex offender treatment program in a forensic psychiatric hospital. Patients were categorized according to whether they completed treatment or not. Treated rapists (n = 36) reoffended at a substantially lower rate than untreated rapists who were assessed but not treated (n = 38). Statistically significant differences in reoffense rates were found for violent, nonsexual offenses as well as nonviolent, nonsexual offenses. Although nearly twice as many treatment noncompleters reoffended sexually, the difference between the two groups was not statistically significant. Mean follow-up time was well over four years for both groups. The implications of these findings are discussed.

Simulation of the surface EMG of an active muscle.

A model of the motoneuronal (MN) pool-muscle system was developed. The model consists of four modules: (1) the input to the MN pool, (2) the MN pool, (3) the muscle and (4) the surface electromyogram (EMG). A control parameter activates the input fibers and determines the activity level of the muscle. A single compartment model with a homogenous membrane was used to model the MNs. The trajectory between spikes is determined by two voltage-gated K(+)- and two voltage-gated Ca(2+)-channels. The size of the MNs is adjusted by the size of the leakage conductance. The model muscle is of circular cross section and with parallel fibers. The motor unit (MU) territories are of circular shape and their area is proportional to the MU contraction force. Action potentials propagated along the muscle fibers are approximated by a dipole with a current source and current sink. The potential evoked by the dipole at the recording site is computed. The surface EMG is obtained by summing up (1) the potentials of all fibers of the MU and (2) the MU action potentials of all active MUs. Numerical results show that the MUs are recruited with increasing contraction force and that the active MUs modulate their firing frequency similar as in real muscles. The model will be used for investigation of the motor system in man.

Human ESC-derived neural rosettes and neural stem cell progression.

Neural stem cells (NSCs) are defined by their ability to self-renew while retaining differentiation potential toward the three main central nervous system (CNS) lineages: neurons, astrocytes, and oligodendrocytes. A less appreciated fact about isolated NSCs is their narrow repertoire for generating specific neuron types, which are generally limited to a few region-specific subtypes such as GABAergic and glutamatergic neurons. Recent studies in human embryonic stem cells have identified a novel neural stem cell stage at which cells exhibit plasticity toward generating a broad range of neuron types in response to appropriate developmental signals. Such rosette-stage NSCs (R-NSCs) are also distinct from other NSC populations by their specific cytoarchitecture, gene expression, and extrinsic growth requirements. Here, we discuss the properties of R-NSCs within the context of NSC biology and define some of the key questions for future investigation. R-NSCs may represent the first example of a NSC population capable of recreating the full cellular diversity of the developing CNS, with implications for both basic stem cell biology and translational applications in regenerative medicine and drug discovery.

Culture of substantia nigra neurons.

Primary cultures of nigral tissue are widely used as a model system to assay effects of trophic and toxic agents on dopaminergic neurons. Cultured dopaminergic neurons have been successfully transplanted in animals and led to behavioral improvement in animal models of Parkinson's disease. Cell cultures have also been used to study the development of substantia nigra, allowing investigators to identify early inductive events important for nigral development and to study dopaminergic differentiation and target innervation. This unit provides simple and reliable culture protocols for these applications. The first approach presented is the preparation of dissociated nigral cell cultures, the later steps of which can be used as a simple and efficient assay for testing growth factors. A second approach is the preparation of free-floating roller tube cultures, which may be used as a tool for neural transplantation and to study more complex developmental events. A third approach is the production of organotypic cultures using chicken plasma as a matrix. Organotypic cultures can maintain the in vivo cytoarchitecture of a host region in vitro.

Serum testosterone in adult sex offenders: a comparison between Caucasians and North American Indians.

Patients admitted to the Phoenix Program for sex offender treatment at Alberta Hospital Edmonton were separated by family history into a group of North American Indians and a group of Caucasians, with respective sample sizes of 53 and 192 after range matching the Caucasian to the North American Indian sample on a number of demographic variables. Controlling for body mass index and age, the two groups were equivalent in terms of 12 basic blood chemistry variables and 5 of 6 endocrine measures. Serum testosterone did differ significantly (p < .0005, covariate adjusted means of 22.3 and 26.5 nmol/L, respectively, for Caucasians and North American Indians). Further research will be required to establish the generality of this result and to ascertain the etiology.

Ascorbic acid increases the yield of dopaminergic neurons derived from basic fibroblast growth factor expanded mesencephalic precursors.

CNS precursors derived from E12 rat mesencephalon proliferate in the presence of basic fibroblast growth factor and differentiate in vitro into functional dopaminergic neurons, which upon transplantation alleviate behavioral symptoms in a rat model of Parkinson's disease. Here we show that the efficiency of dopaminergic differentiation decreases in the mesencephalic precursors that were proliferated or passaged for extended periods in vitro. Ascorbic acid treatment restored dopaminergic differentiation in these precursors and led to a greater than 10-fold increase in dopamine neuron yield compared with untreated cultures. The effect of ascorbic acid was stereospecific and could not be mimicked by any other antioxidants. The expression of sodium-dependent vitamin C transporter, a recently identified stereospecific ascorbic acid transporter, was maintained in mesencephalic precursors for extended in vitro periods. Pre-treatment of in vitro expanded mesencephalic precursors with ascorbic acid might facilitate the large-scale generation of dopaminergic neurons for clinical transplantation.