Bernadette Bellette

Multipotent stem cells from adult olfactory mucosa

Multipotent stem cells are thought to be responsible for the generation of new neurons in the adult brain. Neurogenesis also occurs in an accessible part of the nervous system, the olfactory mucosa. We show here that cells from human olfactory mucosa generate neurospheres that are multipotent in vitro and when transplanted into the chicken embryo. Cloned neurosphere cells show this multipotency. Multipotency was evident without prior culture in vitro: cells dissociated from adult rat olfactory mucosa generate leukocytes when transplanted into bone marrow–irradiated hosts, and cells dissociated from adult mouse olfactory epithelium generated numerous cell types when transplanted into the chicken embryo. It is unlikely that these results can be attributed to hematopoietic precursor contamination or cell fusion. These results demonstrate the existence of a multipotent stem‐like cell in the olfactory mucosa useful for autologous transplantation therapies and for cellular studies of disease. Developmental Dynamics 233:496–515, 2005.

Disease-specific, neurosphere-derived cells as models for brain disorders

SUMMARY There is a pressing need for patient-derived cell models of brain diseases that are relevant and robust enough to produce the large quantities of cells required for molecular and functional analyses. We describe here a new cell model based on patient-derived cells from the human olfactory mucosa, the organ of smell, which regenerates throughout life from neural stem cells. Olfactory mucosa biopsies were obtained from healthy controls and patients with either schizophrenia, a neurodevelopmental psychiatric disorder, or Parkinson’s disease, a neurodegenerative disease. Biopsies were dissociated and grown as neurospheres in defined medium. Neurosphere-derived cell lines were grown in serum-containing medium as adherent monolayers and stored frozen. By comparing 42 patient and control cell lines we demonstrated significant disease-specific alterations in gene expression, protein expression and cell function, including dysregulated neurodevelopmental pathways in schizophrenia and dysregulated mitochondrial function, oxidative stress and xenobiotic metabolism in Parkinson’s disease. The study has identified new candidate genes and cell pathways for future investigation. Fibroblasts from schizophrenia patients did not show these differences. Olfactory neurosphere-derived cells have many advantages over embryonic stem cells and induced pluripotent stem cells as models for brain diseases. They do not require genetic reprogramming and they can be obtained from adults with complex genetic diseases. They will be useful for understanding disease aetiology, for diagnostics and for drug discovery.

Variability in the Generation of Induced Pluripotent Stem Cells: Importance for Disease Modeling

In the field of disease modeling, induced pluripotent stem cells (iPSCs) have become an appealing choice, especially for diseases that do not have an animal model. They can be generated from patients with known clinical features and compared with cells from healthy controls to identify the biological bases of disease. This study was undertaken to determine the variability in iPSC lines derived from different individuals, with the aim of determining criteria for selecting iPSC lines for disease models. We generated and characterized 18 iPSC lines from eight donors and considered variability at three levels: (a) variability in the criteria that define iPSC lines as pluripotent cells, (b) variability in cell lines from different donors, and (c) variability in cell lines from the same donor. We found that variability in transgene expression and pluripotency marker levels did not prevent iPSCs from fulfilling all other criteria for pluripotency, including teratoma formation. We found low interindividual and interclonal variability in iPSCs that fulfilled the most stringent criteria for pluripotency, with very high correlation in their gene expression profiles. Interestingly, some cell lines exhibited reprogramming instability, spontaneously regressing from a fully to a partially reprogrammed state. This was associated with a low percentage of cells expressing the pluripotency marker stage‐specific embryonic antigen‐4. Our study shows that it is possible to define a similar “ground state” for each cell line as the basis for making patient versus control comparisons, an essential step in order to identify disease‐associated variability above individual and cell line variability.

A patient-derived stem cell model of hereditary spastic paraplegia with SPAST mutations

SUMMARY Hereditary spastic paraplegia (HSP) leads to progressive gait disturbances with lower limb muscle weakness and spasticity. Mutations in SPAST are a major cause of adult-onset, autosomal-dominant HSP. Spastin, the protein encoded by SPAST, is a microtubule-severing protein that is enriched in the distal axon of corticospinal motor neurons, which degenerate in HSP patients. Animal and cell models have identified functions of spastin and mutated spastin but these models lack the gene dosage, mutation variability and genetic background that characterize patients with the disease. In this study, this genetic variability is encompassed by comparing neural progenitor cells derived from biopsies of the olfactory mucosa from healthy controls with similar cells from HSP patients with SPAST mutations, in order to identify cell functions altered in HSP. Patient-derived cells were similar to control-derived cells in proliferation and multiple metabolic functions but had major dysregulation of gene expression, with 57% of all mRNA transcripts affected, including many associated with microtubule dynamics. Compared to control cells, patient-derived cells had 50% spastin, 50% acetylated α-tubulin and 150% stathmin, a microtubule-destabilizing enzyme. Patient-derived cells were smaller than control cells. They had altered intracellular distributions of peroxisomes and mitochondria and they had slower moving peroxisomes. These results suggest that patient-derived cells might compensate for reduced spastin, but their increased stathmin expression reduced stabilized microtubules and altered organelle trafficking. Sub-nanomolar concentrations of the microtubule-binding drugs, paclitaxel and vinblastine, increased acetylated α-tubulin levels in patient cells to control levels, indicating the utility of this cell model for screening other candidate compounds for drug therapies.

Low dose tubulin-binding drugs rescue peroxisome trafficking deficit in patient-derived stem cells in Hereditary Spastic Paraplegia

ABSTRACT Hereditary Spastic Paraplegia (HSP) is a genetically heterogeneous group of disorders, diagnosed by progressive gait disturbances with muscle weakness and spasticity, for which there are no treatments targeted at the underlying pathophysiology. Mutations in spastin are a common cause of HSP. Spastin is a microtubule-severing protein whose mutation in mouse causes defective axonal transport. In human patient-derived olfactory neurosphere-derived (ONS) cells, spastin mutations lead to lower levels of acetylated α-tubulin, a marker of stabilised microtubules, and to slower speed of peroxisome trafficking. Here we screened multiple concentrations of four tubulin-binding drugs for their ability to rescue levels of acetylated α-tubulin in patient-derived ONS cells. Drug doses that restored acetylated α-tubulin to levels in control-derived ONS cells were then selected for their ability to rescue peroxisome trafficking deficits. Automated microscopic screening identified very low doses of the four drugs (0.5 nM taxol, 0.5 nM vinblastine, 2 nM epothilone D, 10 µM noscapine) that rescued acetylated α-tubulin in patient-derived ONS cells. These same doses rescued peroxisome trafficking deficits, restoring peroxisome speeds to untreated control cell levels. These results demonstrate a novel approach for drug screening based on high throughput automated microscopy for acetylated α-tubulin followed by functional validation of microtubule-based peroxisome transport. From a clinical perspective, all the drugs tested are used clinically, but at much higher doses. Importantly, epothilone D and noscapine can enter the central nervous system, making them potential candidates for future clinical trials.

A patient-derived olfactory stem cell disease model for ataxia-telangiectasia

The autosomal recessive disorder ataxia-telangiectasia (A-T) is characterized by genome instability, cancer predisposition and neurodegeneration. Although the role of ataxia-telangiectasia mutated (ATM) protein, the protein defective in this syndrome, is well described in the response to DNA damage, its role in protecting the nervous system is less clear. We describe the establishment and characterization of patient-specific stem cells that have the potential to address this shortcoming. Olfactory neurosphere (ONS)-derived cells were generated from A-T patients, which expressed stem cell markers and exhibited A-T molecular and cellular characteristics that included hypersensitivity to radiation, defective radiation-induced signaling and cell cycle checkpoint defects. Introduction of full-length ATM cDNA into these cells corrected defects in the A-T cellular phenotype. Gene expression profiling and pathway analysis revealed defects in multiple cell signaling pathways associated with ATM function, with cell cycle, cell death and DNA damage response pathways being the most significantly dysregulated. A-T ONS cells were also capable of differentiating into neural progenitors, but they were defective in neurite formation, number of neurites and length of these neurites. Thus, ONS cells are a patient-derived neural stem cell model that recapitulate the phenotype of A-T, do not require genetic reprogramming, have the capacity to differentiate into neurons and have potential to delineate the neurological defect in these patients.

Nitric oxide regulates neurogenesis in adult olfactory epithelium in vitro

Nitric oxide regulates neurogenesis in the developing and adult brain. The olfactory epithelium is a site of neurogenesis in the adult and previous studies suggest a role for nitric oxide in this tissue during development. We investigated whether neuronal precursor proliferation and differentiation is regulated by nitric oxide using primary cultures of olfactory epithelial cells and an immortalized, clonal, neuronal precursor cell line derived from adult olfactory epithelium. In these cultures NOS inhibition reduced cell proliferation and stimulated neuronal differentiation, including expression of a voltage-dependent potassium conductance of the delayed rectifier type. In the neuronal precursor cell line, differentiation was associated with a significant decrease in nitric oxide release. In contrast, addition of nitric oxide stimulated proliferation and reduced neuronal differentiation. Nitric oxide regulated olfactory neurogenesis independently of added growth factors. Taken together these results indicate that nitric oxide levels can regulate cell proliferation and neuronal differentiation of olfactory precursor cells.

DNA methylation in schizophrenia in different patient-derived cell types

DNA methylation of gene promoter regions represses transcription and is a mechanism via which environmental risk factors could affect cells during development in individuals at risk for schizophrenia. We investigated DNA methylation in patient-derived cells that might shed light on early development in schizophrenia. Induced pluripotent stem cells may reflect a “ground state” upon which developmental and environmental influences would be minimal. Olfactory neurosphere-derived cells are an adult-derived neuro-ectodermal stem cell modified by developmental and environmental influences. Fibroblasts provide a non-neural control for life-long developmental and environmental influences. Genome-wide profiling of DNA methylation and gene expression was done in these three cell types from the same individuals. All cell types had distinct, statistically significant schizophrenia-associated differences in DNA methylation and linked gene expression, with Gene Ontology analysis showing that the differentially affected genes clustered in networks associated with cell growth, proliferation, and movement, functions known to be affected in schizophrenia patient-derived cells. Only five gene loci were differentially methylated in all three cell types. Understanding the role of epigenetics in cell function in the brain in schizophrenia is likely to be complicated by similar cell type differences in intrinsic and environmentally induced epigenetic regulation.Epigenetics: A genome-wide picture in patient-derived cellsSchizophrenia-associated differences in the DNA methylation status of patient-derived cells suggest it could affect early brain development. Mechanisms that control gene expression without altering the genetic code, such as DNA methylation, could explain how environmental risk factors contribute to schizophrenia in genetically susceptible individuals. Alan Mackay-Sim and colleagues from Griffith University, Australia, carried out genome-wide comparisons of DNA methylation in induced pluripotent stem (iPS) cells, olfactory neurosphere-derived cells and fibroblasts from patients and controls. Differences in the DNA methylation pattern between patient and control iPS cells, which could reflect what happens in the embryo, suggest a disease-associated effect very early on in development. Only five genes were differentially methylated in all three patient-derived cell types compared to controls. None of these genes has previously been associated with schizophrenia and may represent new targets for future research.DNA methylation of gene promoter regions represses transcription and is a mechanism via which environmental risk factors could affect cells during development in individuals at risk for schizophrenia. We investigated DNA methylation in patient-derived cells that might shed light on early development in schizophrenia. Induced pluripotent stem cells may reflect a “ground state” upon which developmental and environmental influences would be minimal. Olfactory neurosphere-derived cells are an adult-derived neuro-ectodermal stem cell modified by developmental and environmental influences. Fibroblasts provide a non-neural control for life-long developmental and environmental influences. Genome-wide profiling of DNA methylation and gene expression was done in these three cell types from the same individuals. All cell types had distinct, statistically significant schizophrenia-associated differences in DNA methylation and linked gene expression, with Gene Ontology analysis showing that the differentially affected genes clustered in networks associated with cell growth, proliferation, and movement, functions known to be affected in schizophrenia patient-derived cells. Only five gene loci were differentially methylated in all three cell types. Understanding the role of epigenetics in cell function in the brain in schizophrenia is likely to be complicated by similar cell type differences in intrinsic and environmentally induced epigenetic regulation.

USP9X deubiquitylating enzyme maintains RAPTOR protein levels, mTORC1 signalling and proliferation in neural progenitors

USP9X, is highly expressed in neural progenitors and, essential for neural development in mice. In humans, mutations in USP9X are associated with neurodevelopmental disorders. To understand USP9X’s role in neural progenitors, we studied the effects of altering its expression in both the human neural progenitor cell line, ReNcell VM, as well as neural stem and progenitor cells derived from Nestin-cre conditionally deleted Usp9x mice. Decreasing USP9X resulted in ReNcell VM cells arresting in G0 cell cycle phase, with a concomitant decrease in mTORC1 signalling, a major regulator of G0/G1 cell cycle progression. Decreased mTORC1 signalling was also observed in Usp9x-null neurospheres and embryonic mouse brains. Further analyses revealed, (i) the canonical mTORC1 protein, RAPTOR, physically associates with Usp9x in embryonic brains, (ii) RAPTOR protein level is directly proportional to USP9X, in both loss- and gain-of-function experiments in cultured cells and, (iii) USP9X deubiquitlyating activity opposes the proteasomal degradation of RAPTOR. EdU incorporation assays confirmed Usp9x maintains the proliferation of neural progenitors similar to Raptor-null and rapamycin-treated neurospheres. Interestingly, loss of Usp9x increased the number of sphere-forming cells consistent with enhanced neural stem cell self-renewal. To our knowledge, USP9X is the first deubiquitylating enzyme shown to stabilize RAPTOR.

Structural characterisation of high affinity Siglec-2 (CD22) ligands in complex with whole Burkitt's lymphoma (BL) Daudi cells by NMR spectroscopy.

Siglec-2 undergoes constitutive endocytosis and is a drug target for autoimmune diseases and B cell-derived malignancies, including hairy cell leukaemia, marginal zone lymphoma, chronic lymphocytic leukaemia and non-Hodgkin’s lymphoma (NHL). An alternative to current antibody-based therapies is the use of liposomal nanoparticles loaded with cytotoxic drugs and decorated with Siglec-2 ligands. We have recently designed the first Siglec-2 ligands (9-biphenylcarboxamido-4-meta-nitrophenyl-carboxamido-Neu5Acα2Me, 9-BPC-4-mNPC-Neu5Acα2Me) with simultaneous modifications at C-4 and C-9 position. In the current study we have used Saturation Transfer Difference (STD) NMR spectroscopy to monitor the binding of 9-BPC-4-mNPC-Neu5Acα2Me to Siglec-2 present on intact Burkitt’s lymphoma Daudi cells. Pre-treatment of cells with periodate resulted in significantly higher STD NMR signal intensities for 9-BPC-4-mNPC-Neu5Acα2Me as the cells were more susceptible to ligand binding because cis-binding on the cell surface was removed. Quantification of STD NMR effects led to a cell-derived binding epitope of 9-BPC-4-mNPC-Neu5Acα2Me that facilitated the design and synthesis of C-2, C-3, C-4 and C-9 tetra-substituted Siglec-2 ligands showing an 88-fold higher affinity compared to 9-BPC-Neu5Acα2Me. This is the first time a NMR-based binding study of high affinity Siglec-2 (CD22) ligands in complex with whole Burkitt’s lymphoma Daudi cells has been described that might open new avenues in developing tailored therapeutics and personalised medicine.