Dataset Information

Pharmacological Transdifferentiation of Human Nasal Olfactory Stem Cells into Dopaminergic Neurons.

ABSTRACT: The discovery of novel drugs for neurodegenerative diseases has been a real challenge over the last decades. The development of patient- and/or disease-specific in vitro models represents a powerful strategy for the development and validation of lead candidates in preclinical settings. The implementation of a reliable platform modeling dopaminergic neurons will be an asset in the study of dopamine-associated pathologies such as Parkinson's disease. Disease models based on cell reprogramming strategies, using either human-induced pluripotent stem cells or transcription factor-mediated transdifferentiation, are among the most investigated strategies. However, multipotent adult stem cells remain of high interest to devise direct conversion protocols and establish in vitro models that could bypass certain limitations associated with reprogramming strategies. Here, we report the development of a six-step chemically defined protocol that drives the transdifferentiation of human nasal olfactory stem cells into dopaminergic neurons. Morphological changes were progressively accompanied by modifications matching transcript and protein dopaminergic signatures such as LIM homeobox transcription factor 1 alpha (LMX1A), LMX1B, and tyrosine hydroxylase (TH) expression, within 42 days of differentiation. Phenotypic changes were confirmed by the production of dopamine from differentiated neurons. This new strategy paves the way to develop more disease-relevant models by establishing reprogramming-free patient-specific dopaminergic cell models for drug screening and/or target validation for neurodegenerative diseases.

SUBMITTER: Chabrat A

PROVIDER: S-EPMC6545791 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Pharmacological Transdifferentiation of Human Nasal Olfactory Stem Cells into Dopaminergic Neurons.

Chabrat Audrey A Lacassagne Emmanuelle E Billiras Rodolphe R Landron Sophie S Pontisso-Mahout Amélie A Darville Hélène H Dupront Alain A Coge Francis F Schenker Esther E Piwnica David D Nivet Emmanuel E Féron François F Mannoury la Cour Clotilde C

Stem cells international 20190519

The discovery of novel drugs for neurodegenerative diseases has been a real challenge over the last decades. The development of patient- and/or disease-specific <i>in vitro</i> models represents a powerful strategy for the development and validation of lead candidates in preclinical settings. The implementation of a reliable platform modeling dopaminergic neurons will be an asset in the study of dopamine-associated pathologies such as Parkinson's disease. Disease models based on cell reprogrammi ...[more]

PMID: 31236114

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Project description:The olfactory neuroepithelium is located in the upper vault of the nasal cavity, lying on the olfactory cleft and projecting into the dorsal portion of the superior and middle turbinates beyond the mid-portion of the nasal septum. It is composed of a variety of cell types including olfactory sensory neurons, supporting glial-like cells, microvillar cells, and basal stem cells. The cells of the neuroepithelium are often intermingled with respiratory and metaplastic epithelial cells. Olfactory neurons undergo a constant self-renewal in the timespan of 2-3 months; they are directly exposed to the external environment, and thus they are vulnerable to physical and chemical injuries. The latter might induce metabolic perturbations and ultimately be the cause of cell death. However, the lifespan of olfactory neurons is biologically programmed, and for this reason, these cells have an accelerated metabolic cycle leading to an irreversible apoptosis. These characteristics make these cells suitable for research related to nerve cell degeneration and aging. Recent studies have shown that a non-invasive and painless olfactory brushing procedure allows an efficient sampling from the olfactory neuroepithelium. This approach allows to detect the pathologic prion protein in patients with sporadic Creutzfeldt-Jakob disease, using the real-time quaking-induced conversion assay. Investigating the expression of all the proteins associated to neurodegeneration in the cells of the olfactory mucosa is a novel approach toward understanding the pathogenesis of human neurodegenerative diseases. Our aim was to investigate the expression of α-synuclein, β-amyloid, tau, and TDP-43 in the olfactory neurons of normal subjects. We showed that these proteins that are involved in neurodegenerative diseases are expressed in olfactory neurons. These findings raise the question on whether a relationship exists between the mechanisms of protein aggregation that occur in the olfactory bulb during the early stage of the neurodegenerative process and the protein misfolding occurring in the olfactory neuroepithelium.

Project description:BackgroundOlfactory ecto-mesenchymal stem cells (OE-MSC) are mesenchymal stem cells derived from the lamina propria of the nasal mucosa. They display neurogenic and immunomodulatory properties and were shown to induce recovery in animal models of spinal cord trauma, hearing loss, Parkinsons's disease, amnesia, and peripheral nerve injury. As a step toward clinical practice, we sought to (i) devise a culture protocol that meets the requirements set by human health agencies and (ii) assess the efficacy of stem cells on neuron differentiation.MethodsNasal olfactory mucosa biopsies from three donors were used to design and validate the good manufacturing process for purifying stem cells. All processes and procedures were performed by expert staff from the cell therapy laboratory of the public hospital of Marseille (AP-HM), according to aseptic handling manipulations. Premises, materials and air were kept clean at all times to avoid cross-contamination, accidents, or even fatalities. Purified stem cells were cultivated for 24 or 48 h and conditioned media were collected before being added to the culture medium of the neuroblastoma cell line Neuro2a.ResultsCompared to the explant culture-based protocol, enzymatic digestion provides higher cell numbers more rapidly and is less prone to contamination. The use of platelet lysate in place of fetal calf serum is effective in promoting higher cell proliferation (the percentage of CFU-F progenitors is 15.5%), with the optimal percentage of platelet lysate being 10%. Cultured OE-MSCs do not show chromosomal rearrangement and, as expected, express the usual phenotypic markers of mesenchymal stem cells. When incorporated in standard culture medium, the conditioned medium of purified OE-MSCs promotes cell differentiation of Neuro2a neuroblastoma cells.ConclusionWe developed a safer and more efficient manufacturing process for clinical grade olfactory stem cells. With this protocol, human OE-MSCs will soon be used in a Phase I clinical based on their autologous transplantation in digital nerves with a neglected injury. However, further studies are required to unveil the underlying mechanisms of action.

Dataset Information

Pharmacological Transdifferentiation of Human Nasal Olfactory Stem Cells into Dopaminergic Neurons.

Publications

Pharmacological Transdifferentiation of Human Nasal Olfactory Stem Cells into Dopaminergic Neurons.

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