Technologies enabling autologous neural stem cell-based therapies for neurodegenerative disease and injury

The intrinsic abilities of mammalian neural stem cells (NSCs) to self-renew, migrate over large distances, and give rise to all primary neural cell types of the brain offer unprecedented opportunity for cell-based treatment of neurodegenerative diseases and injuries. This thesis discusses development of technologies in support of autologous NSC-based therapies, encompassing harvest of brain tissue biopsies from living human patients; isolation of NSCs from harvested tissue; efficient culture and expansion of NSCs in 3D polymeric microcapsule culture systems; optimization of microcapsules as carriers for efficient in vivo delivery of NSCs; genetic engineering of NSCs for drug-induced, enzymatic release of transplanted NSCs from microcapsules; genetic engineering for drug-induced differentiation of NSCs into specific therapeutic cell types; and synthesis of chitosan/iron-oxide nanoparticles for labeling of NSCs and in vivo tracking by cellular MRI.;Sub-millimeter scale tissue samples were harvested endoscopically from subventricular zone regions of living patient brains, secondary to neurosurgical procedures including endoscopic third ventriculostomy and ventriculoperitoneal shunt placement. On average, 12,000 +/- 3,000 NSCs were isolated per mm 3 of subventricular zone tissue, successfully demonstrated in 26 of 28 patients, ranging in age from one month to 68 years.;In order to achieve efficient expansion of isolated NSCs to clinically relevant numbers (e.g. hundreds of thousands of cells in Parkinson's disease and tens of millions of cells in multiple sclerosis), an extracellular matrix-inspired, microcapsule-based culture platform was developed. Initial culture experiments with murine NSCs yielded unprecedented expansion folds of 30x in 5 days, from initially minute NSC populations (154 +/- 15 NSCs per 450 mum diameter capsule). Within 7 days, NSCs expanded as almost perfectly homogenous populations, with 94.9% +/- 4.1% of cultured cells staining positive for Nestin, a marker for NSCs, 81.4 +/- 3.7% of cells staining positive for KI67, a proliferation marker, and 0% of cultured cells staining positive for GFAP, a marker indicative of undesired astrocytes.;The same microcapsules used for expansion were designed to contain NSCs beyond delivery to the brain, maintaining NSC phenotype and suppressing undesired astroglial differentiation during the acute phase of inflammation beyond surgical implantation. In vitro, >80% of encapsulated cells challenged with 0.1 % fetal calf serum over five days in culture showed persistent Nestin expression, compared to <20% under the same conditions outside of microcapsules, indicating that the microcapsule interior can preserve phenotype in the presence of serum concentrations at least an order of magnitude greater than those estimated to be present in cerebrospinal fluid (CSF) after surgical implantation.;In order to release transplanted NSCs on cue from microcapsules after the acute inflammatory response, NSCs were genetically engineered using the Tet-onRTM drug-inducible gene expression system to produce and secrete the enzyme alginase in response to the inducer drug doxycycline. Engineered NSCs, exposed to 1 mug/ml doxycycline, produced sufficient alginase to digest alginate, a structural component of the microcapsule wall, within 8 hours, effectively dissolving microcapsules and releasing encapsulated NSCs.;In order to direct differentiation of transplanted NSCs towards therapeutically valuable cell types (e.g. dopaminergic neurons in case of Parkinson's disease and oligodendrocytes in case of multiple sclerosis), NSCs were genetically engineered to inducibly express the proneural transcription factors NGN1 and Olig1 on demand. Induced expression of NGN1 yielded >90% neurons, induced expression of Olig1 yielded >80% oligodendrocytes, compared to neuron/oligodendrocyte yields <10% for GFP-expressing controls. NSCs with the capacity to inducibly express these transcription factors showed preservation of therapeutically valuable migratory capacity (average RMS migration rate of approximately 40 mum/hr before induction). Differentiating NSCs, however, showed largely arrested migration within 12 hours of induction for Olig1 cells and 36 hours of induction for NGN1 cells.;Finally, tracking of NSCs at the single cell level via high-resolution (11.7 T) cellular MRI, was made possible through development of contrast-enhancing, chitosan-functionalized ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles that are rapidly uptaken by NSCs. Chitosan, a positively charged derivative of chitin, promotes electrostatically-driven attachment of chitosan-USPIO nanoparticles to negatively charged domains on the outer leaflet of the cellular membrane, enhancing uptake by clathrin-mediated endocytosis (>10x increase in uptake efficiency relative to unmodified USPIO). Uptaken USPIOs remained in cells for at least 8 days due to charge-induced endosomal escape of nanoparticles into the cytosol.;In combination, all developed technologies offer a basis for clinical evaluation of autologous neural stem cell replacement therapies, the future of which promises to shift the present paradigm for treatment of neurodegenerative diseases and injuries.