National Human Neural Stem Cell Resource Publications

This paper considers embryo grading within a given infertility treatment and suggests an ethical approach to embryo donation for embryonic stem cell harvest. It is concluded that ethical considerations regarding human embryos do not necessarily preclude the use of certain embryos for biomedical research or transplantation. The argument is based on the following rationale: all embryos are not physiologically equal, some low-grade embryos will never be chosen for implantation, cells from low-grade embryos may be donated for transplantation or research, and embryonic stem cells can be harvested from low-grade embryos. This argument bears special importance at this time as embryos created by IVF are still the only source of embryonic stem cells, given the current controversy surrounding published studies of human somatic cell nuclear transfer.

Deciphering the factors that regulate human neural stem cells will greatly aid in their use as models of development and as therapeutic agents. The extracellular matrix (ECM) is a component of stem cell niches in vivo and regulates multiple functions in diverse cell types, yet little is known about its effects on human neural stem/precursor cells (NSPCs). We therefore plated human NSPCs on four different substrates (poly-L-ornithine, fibronectin, laminin, and matrigel) and compared their responses with those of mouse NSPCs. Compared with the other substrates, laminin matrices enhanced NSPC migration, expansion, differentiation into neurons and astrocytes, and elongation of neurites from NSPC-derived neurons. Laminin had a similar spectrum of effects on both human and mouse cells, highlighting the evolutionary conservation of NSPC regulation by this component of the ECM. Flow cytometry revealed that human NSPCs express on their cell surfaces the laminin-binding integrins alpha3, alpha6, alpha7, beta1, and beta4, and function-blocking antibodies to the alpha6 subunit confirmed a role for integrins in laminin-dependent migration of human NSPCs. These results define laminin and its integrin receptors as key regulators of human NSPCs.

Stem cells are quickly coming into focus of much biomedical research eventually aiming at the therapeutic applications for various disorders and trauma. It is important, however, to keep in mind the difference between the embryonic stem cells, somatic stem cells and somatic precursor cells when considering potential clinical applications. Here we provide the review of the current status of stem cell field and discuss the potential of therapeutic applications for blood and Immune system disorders, multiple sclerosis, hypoxic-ischemic brain injury and brain tumors. For the complimentary information about various stem cells and their properties we recommend consulting the National Institutes of Health stem cell resources (http://stemcells.nih.gov/info/basics).

As a novel neurotherapeutic strategy, stem cell transplantation has received considerable attention, yet little of this attention has been devoted to the probabilities of success of stem cell therapies for specific neurological disorders. Given the complexities of the cellular organization of the nervous system and the manner in which it is assembled during development, it is unlikely that a cellular replacement strategy will succeed for any but the simplest of neurological disorders in the near future. A general strategy for stem cell transplantation to prevent or minimize neurological disorders is much more likely to succeed. Two broad categories of neurological disease, inherited metabolic disorders and invasive brain tumors, are among the most likely candidates.

Greco CM, Berman RF, Martin RM, Tassone F, Schwartz PH, Chang A, Trapp BD, Iwahashi C, Brunberg J, Grigsby J, Hessl D, Becker EJ, Papazian J, Leehey MA, Hagerman RJ, Hagerman PJ.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is an adult-onset neurodegenerative disorder that affects carriers, principally males, of premutation alleles (55-200 CGG repeats) of the fragile X mental retardation 1 (FMR1) gene. Clinical features of FXTAS include progressive intention tremor and gait ataxia, accompanied by characteristic white matter abnormalities on MRI. The neuropathological hallmark of FXTAS is an intranuclear inclusion, present in both neurons and astrocytes throughout the CNS. Prior to the current work, the nature of the associations between inclusion loads and molecular measures (e.g. CGG repeat) was not defined. Post-mortem brain and spinal cord tissue has been examined for gross and microscopic pathology in a series of 11 FXTAS cases (males, age 67-87 years at the time of death). Quantitative counts of inclusion numbers were performed in various brain regions in both neurons and astrocytes. Inclusion counts were compared with specific molecular (CGG repeat, FMR1 mRNA level) and clinical (age of onset, age of death) parameters. In the current series, the three most prominent neuropathological characteristics are (i) significant cerebral and cerebellar white matter disease, (ii) associated astrocytic pathology with dramatically enlarged inclusion-bearing astrocytes prominent in cerebral white matter and (iii) the presence of intranuclear inclusions in both brain and spinal cord. The pattern of white matter pathology is distinct from that associated with hypertensive vascular disease and other diseases of white matter. Spongiosis was present in the middle cerebellar peduncles in seven of the eight cases in which those tissues were available for study. There is inclusion formation in cranial nerve nucleus XII and in autonomic neurons of the spinal cord. The most striking finding is the highly significant association between the number of CGG repeats and the numbers of intranuclear inclusions in both neurons and astrocytes, indicating that the CGG repeat is a powerful predictor of neurological involvement in males, both clinically (age of death) and neuropathologically (number of inclusions).

Arocena DG, Iwahashi CK, Won N, Beilina A, Ludwig AL, Tassone F, Schwartz PH, Hagerman PJ.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a neurodegenerative disorder that affects some adult carriers of pre-mutation alleles (55-200 CGG repeats) of the fragile X mental retardation 1 (FMR1) gene. FXTAS is thought to be caused by a toxic 'gain-of-function' of the expanded CGG-repeat FMR1 mRNA, which is found in the neuronal and astrocytic intranuclear inclusions associated with the disorder. Using a reporter construct with a FMR1 5' untranslated region harboring an expanded (premutation) CGG repeat, we have demonstrated that intranuclear inclusions can be formed in both primary neural progenitor cells and established neural cell lines. As with the inclusions found in post-mortem tissue, the inclusions induced by the expanded CGG repeat are alphaB-crystallin-positive; however, inclusions in culture are not associated with ubiquitin, indicating that incorporation of ubiquitinated proteins is a later event in the disease process. The absence of ubiquitinated proteins also argues against a model in which inclusion formation is due to a failure of the proteasomal degradative machinery. The presence of the expanded CGG repeat, as RNA, results in reduced cell viability as well as the disruption of the normal architecture of lamin A/C within the nucleus. This last observation, and the findings that lamin A/C is present in both the inclusions of FXTAS patients and the inclusions in cell culture, suggests that lamin A/C dysregulation may be a component of the pathogenesis of FXTAS; in particular, the Charcot-Marie-Tooth-type neuropathy associated with FXTAS may represent a functional laminopathy.

Despite the increasing importance of the pig as a large animal model, little is known about porcine neural precursor cells. To evaluate the markers expressed by these cells, brains were dissected from 60-day fetuses, enzymatically dissociated, and grown in the presence of epidermal growth factor, basic fibroblast growth factor, and platelet-derived growth factor. Porcine neural precursors could be grown as suspended spheres or adherent monolayers, depending on culture conditions. Expanded populations were banked or harvested for analysis using reverse transcription-polymerase chain reaction (RT-PCR), immunocytochemistry, microarrays, and flow cytometry, and results compared with data from analogous human forebrain progenitor cells. Cultured porcine neural precursors widely expressed neural cell adhesion molecule (NCAM), polysialic acid (PSA)-NCAM, vimentin, Ki-67, and Sox2. Minority subpopulations of cells expressed doublecortin, beta-III tubulin, synapsin I, glial fibrillary acidic protein (GFAP), and aquaporin 4 (AQP4) consistent with increased lineage restriction. A human microarray detected porcine transcripts for nogoA (RTN4) and stromal cell-derived factor 1 (SDF1), possibly cyclin D2 and Pbx1, but not CD133, Ki-67, nestin, or nucleostemin. Subsequent RT-PCR showed pig forebrain precursors to be positive for cyclin D2, nucleostemin, nogoA, Pbx1, vimentin, and a faint band for SDF1, whereas no signal was detected for CD133, fatty acid binding protein 7 (FABP7), or Ki-67. Human forebrain progenitor cells were positive for all the genes mentioned. This study shows that porcine neural precursors share many characteristics with their human counterparts and, thus, may be useful in porcine cell transplantation studies potentially leading to the application of this strategy in the setting of nervous system disease and injury.

This paper describes a gradient-generating microfluidic platform for optimizing proliferation and differentiation of neural stem cells (NSCs) in culture. Microfluidic technology has great potential to improve stem cell (SC) cultures, whose promise in cell-based therapies is limited by the inability to precisely control their behavior in culture. Compared to traditional culture tools, microfluidic platforms should provide much greater control over cell microenvironment and rapid optimization of media composition using relatively small numbers of cells. Our platform exposes cells to a concentration gradient of growth factors under continuous flow, thus minimizing autocrine and paracrine signaling. Human NSCs (hNSCs) from the developing cerebral cortex were cultured for more than 1 week in the microfluidic device while constantly exposed to a continuous gradient of a growth factor (GF) mixture containing epidermal growth factor (EGF), fibroblast growth factor 2 (FGF2) and platelet-derived growth factor (PDGF). Proliferation and differentiation of NSCs into astrocytes were monitored by time-lapse microscopy and immunocytochemistry. The NSCs remained healthy throughout the entire culture period, and importantly, proliferated and differentiated in a graded and proportional fashion that varied directly with GF concentration. These concentration-dependent cellular responses were quantitatively similar to those measured in control chambers built into the device and in parallel cultures using traditional 6-well plates. This gradient-generating microfluidic platform should be useful for a wide range of basic and applied studies on cultured cells, including SCs.

Schwartz PH, Tassone F, Greco CM, Nethercott HE, Ziaeian B, Hagerman RJ, Hagerman PJ.

BACKGROUND: Currently, there is no adequate animal model to study the detailed molecular biochemistry of fragile X syndrome, the leading heritable form of mental impairment. In this study, we sought to establish the use of immature neural cells derived from adult tissues as a novel model of fragile X syndrome that could be used to more fully understand the pathology of this neurogenetic disease. METHODS: By modifying published methods for the harvest of neural progenitor cells from the post-mortem human brain, neural cells were successfully harvested and grown from post-mortem brain tissue of a 25-year-old adult male with fragile X syndrome, and from brain tissue of a patient with no neurological disease. RESULTS: The cultured fragile X cells displayed many of the characteristics of neural progenitor cells, including nestin and CD133 expression, as well as the biochemical hallmarks of fragile X syndrome, including CGG repeat expansion and a lack of FMRP expression. CONCLUSION: The successful production of neural cells from an individual with fragile X syndrome opens a new avenue for the scientific study of the molecular basis of this disorder, as well as an approach for studying the efficacy of new therapeutic agents.

The goal of the present study was threefold: to determine whether viable human retinal progenitor cells (hRPCs) could be obtained from cadaveric retinal tissue, to evaluate marker expression by these cells, and to compare hRPCs to human brain progenitor cells (hBPCs). Retinas were dissected from post-mortem premature infants, enzymatically dissociated, and grown in the presence of epidermal growth factor and basic fibroblast growth factor. The cells grew as suspended spheres or adherent monolayers, depending on culture conditions. Expanded populations were banked or harvested for analysis by RT-PCR, immunocytochemistry, and flow cytometry. hBPCs derived from forebrain specimens from the same donors were grown and used for RT-PCR. Post-mortem human retinal specimens yielded viable cultures that grew to confluence repeatedly, although not beyond 3 months. Cultured hRPCs expressed a range of markers consistent with CNS progenitor cells, including nestin, vimentin, Sox2, Ki-67, GD2 ganglioside, and CD15 (Lewis X), as well as the tetraspanins CD9 and CD81, CD95 (Fas), and MHC class I antigens. No MHC class II expression was detected. hRPCs, but not hBPCs, expressed Dach1, Pax6, Six3, Six6, and recoverin. Minority subpopulations of hRPCs and hBPCs expressed doublecortin, β-III tubulin, and glial fibrillary acidic protein, which is consistent with increased lineage restriction in subsets of cultured cells. Viable progenitor cells can be cultured from the post-mortem retina of premature infants and exhibit a gene expression profile consistent with immature neuroepithelial cells. hRPCs can be distinguished from hBPC cultures by the expression of retinal specification genes and recoverin.

Fragile X syndrome, the most common form of mental impairment, is caused by expansion of a (CGG)n trinucleotide repeat element located in the 5' untranslated region of the fragile X mental retardation 1 (FMR1) gene. Repeat expansion is known to influence both transcription and translation; however, the mechanisms by which the CGG element exerts its effects are not known.

In the current work, we have utilized 5'-RLM-RACE to examine the influence of CGG repeat number on the utilization of transcription start sites in normal (n<55) and premutation (54<n<200) cell lines of both non-neural (lymphoblastoid) and neural (primary astrocyte) origin.

Our results demonstrate that, in both neural and non-neural cells, transcription of the FMR1 gene is initiated from several transcription start sites within a approximately 50 nt region that lies approximately 130 nt upstream of the CGG repeat element. For normal alleles, most transcripts initiate from the downstream-most start site, close to the single position identified previously. Surprisingly, as the size of the CGG repeat expands into the premutation range, initiation shifts to the upstream sites, suggesting that the CGG element may act as a downstream enhancer/modulator of transcription. The shift in start site selection for both neural and non-neural cells indicates that the effect is general. Furthermore, the correspondence between start site utilization and the degree of elevation of FMR1 mRNA suggests that a substantial fraction of the increased message in the premutation range may derive from the upstream start sites.

Human neural progenitor cells (hNPCs) can be recovered from postmortem human brains and used to study the molecular basis of neurogenesis. Human NPCs are being used to investigate the molecular basis of cell fate determination during stem cell divisions, based on comparison with the Drosophila model system. Drosophila neuroblasts and sensory organ precursors undergo well-defined asymmetric cell divisions (ACD), under the control of a genetically defined set of apical and basal determinants that are localized tightly and dynamically during division.

We show by indirect immunofluorescence, confocal microscopy, and time-lapse video-microscopy that LGN and AGS3, two human homologs of the Drosophila ACD determinant Pins, have distinct patterns of localization in hNPCs. When cells are grown under conditions favoring proliferation, LGN is distributed asymmetrically in a cell cycle-dependent manner; it localizes to one side of the dividing cell and segregates into one of the daughter cells. When the cells are grown under conditions favoring differentiation, LGN accumulates in double foci similar to those containing the mitotic apparatus protein NuMA, and in a pattern shown previously for LGN and NuMA in differentiated cells. AGS3, a slightly more distant Pins homolog than LGN, does not show asymmetric localization in these cells. The progenitor cell marker nestin also localizes asymmetrically in colcemid-treated hNPCs and colocalizes with LGN. The results suggest that hNPCs undergo ACD and that similar molecular pathways may underlie these divisions in Drosophila and human cells.

Schwartz, P.H., Bryant, P.J., Fuja, T.J., Su, H., O'Dowd, D.K., and Klassen, H.K.

Post-mortem human brain tissue represents a vast potential source of neural progenitor cells for use in basic research as well as therapeutic applications. Here we describe five human neural progenitor cell cultures derived from cortical tissue harvested from premature infants. Time-lapse videomicrography of the passaged cultures revealed them to be highly dynamic, with high motility and extensive, evanescent intercellular contacts. Karyotyping revealed normal chromosomal complements. Prior to differentiation, most of the cells were nestin, Sox2, vimentin, and/or GFAP positive, and a subpopulation was doublecortin positive. Multilineage potential of these cells was demonstrated after differentiation, with some subpopulations of cells expressing the neuronal markers beta-tubulin, MAP2ab, NeuN, FMRP, and Tau and others expressing the oligodendroglial marker O1. Still other cells expressed the classic glial marker glial fibrillary acidic protein (GFAP). RT-PCR confirmed nestin, SOX2, GFAP, and doublecortin expression and also showed epidermal growth factor receptor and nucleostemin expression during the expansion phase. Flow cytometry showed high levels of the neural stem cell markers CD133, CD44, CD81, CD184, CD90, and CD29. CD133 markedly decreased in high-passage, lineage-restricted cultures. Electrophysiological analysis after differentiation demonstrated that the majority of cells with neuronal morphology expressed voltage-gated sodium and potassium currents. These data suggest that post-mortem human brain tissue is an important source of neural progenitor cells that will be useful for analysis of neural differentiation and for transplantation studies.

Palmer, T.D., Schwartz, P.H., Taupain, P., Kaspar, B., Stein, S.A., and Gage, F.H.

NHNSCR Publications

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