Development of a Smart Wireless Multisensor Platform for an Optogenetic Brain Implant.

Implantable cell replacement therapies promise to completely restore the function of neural structures, possibly changing how we currently perceive the onset of neurodegenerative diseases. One of the major clinical hurdles for the routine implementation of stem cell therapies is poor cell retention and survival, demanding the need to better understand these mechanisms while providing precise and scalable approaches to monitor these cell-based therapies in both pre-clinical and clinical scenarios. This poses significant multidisciplinary challenges regarding planning, defining the methodology and requirements, prototyping and different stages of testing. Aiming toward an optogenetic neural stem cell implant controlled by a smart wireless electronic frontend, we show how an iterative development methodology coupled with a modular design philosophy can mitigate some of these challenges. In this study, we present a miniaturized, wireless-controlled, modular multisensor platform with fully interfaced electronics featuring three different modules: an impedance analyzer, a potentiostat and an optical stimulator. We show the application of the platform for electrical impedance spectroscopy-based cell monitoring, optical stimulation to induce dopamine release from optogenetically modified neurons and a potentiostat for cyclic voltammetry and amperometric detection of dopamine release. The multisensor platform is designed to be used as an opto-electric headstage for future in vivo animal experiments.

Nonhypoxic pharmacological stabilization of Hypoxia Inducible Factor 1α: Effects on dopaminergic differentiation of human neural stem cells.

Parkinson's disease is a neurodegenerative disease resulting in degeneration of midbrain dopaminergic neurons. Exploratory studies using human foetal tissue or predifferentiated stem cells have suggested that intracerebral transplantation of dopaminergic precursor cells may become an effective treatment for patients with Parkinson's disease. However, strategies for dopaminergic stem cell differentiation vary widely in efficiency, and better methods still need to be developed. Hypoxia Inducible Factor 1 (HIF-1) is a transcription factor involved in the regulation of genes important for cellular adaption to hypoxia and low glucose supply. HIF-1 is to a large degree regulated by the availability of oxygen as in its presence, the subunit HIF-1α is degraded by HIF prolyl hydroxylase enzymes (HPHs). Stabilization of HIF-1α through inhibition of HPHs has been shown to increase dopaminergic differentiation of stem cells and to protect dopaminergic neurons against neurotoxins. This study investigated the effects of noncompetitive (FG-0041) and competitive (Compound A and JNJ-42041935) HIF-1α stabilizing compounds on the dopaminergic differentiation of human neural stem cells. Treatment with all HPH inhibitors at high oxygen tension (20%) resulted in HIF-1α stabilization as assessed by immunocytochemistry for HIF-1α and detection of increased levels of vascular endothelial growth factor in the conditioned culture medium. Following 10 days of HIF-1α stabilization, the cultures displayed a slightly reduced proliferative activity and significantly increased relative levels of tyrosine hydroxylase-positive dopaminergic neurons. In conclusion, HIF-1α stabilization may represent a promising strategy for the generation of dopaminergic neurons intended for use in experimental in vitro studies and cell replacement therapies.

In vivo inhibition of the mitochondrial H+-ATP synthase in neurons promotes metabolic preconditioning.

A key transducer in energy conservation and signaling cell death is the mitochondrial H(+)-ATP synthase. The expression of the ATPase inhibitory factor 1 (IF1) is a strategy used by cancer cells to inhibit the activity of the H(+)-ATP synthase to generate a ROS signal that switches on cellular programs of survival. We have generated a mouse model expressing a mutant of human IF1 in brain neurons to assess the role of the H(+)-ATP synthase in cell death in vivo. The expression of hIF1 inhibits the activity of oxidative phosphorylation and mediates the shift of neurons to an enhanced aerobic glycolysis. Metabolic reprogramming induces brain preconditioning affording protection against quinolinic acid-induced excitotoxicity. Mechanistically, preconditioning involves the activation of the Akt/p70S6K and PARP repair pathways and Bcl-xL protection from cell death. Overall, our findings provide the first in vivo evidence highlighting the H(+)-ATP synthase as a target to prevent neuronal cell death.

Optimization of the magnetic labeling of human neural stem cells and MRI visualization in the hemiparkinsonian rat brain.

BACKGROUND: Magnetic resonance imaging is the ideal modality for non-invasive in vivo cell tracking allowing for longitudinal studies over time. Cells labeled with superparamagnetic iron oxide nanoparticles have been shown to induce sufficient contrast for in vivo magnetic resonance imaging enabling the in vivo analysis of the final location of the transplanted cells. For magnetic nanoparticles to be useful, a high internalization efficiency of the particles is required without compromising cell function, as well as validation of the magnetic nanoparticles behaviour inside the cells. RESULTS: In this work, we report the development, optimization and validation of an efficient procedure to label human neural stem cells with commercial nanoparticles in the absence of transfection agents. Magnetic nanoparticles used here do not affect cell viability, cell morphology, cell differentiation or cell cycle dynamics. Moreover, human neural stem cells progeny labeled with magnetic nanoparticles are easily and non-invasively detected long time after transplantation in a rat model of Parkinson's disease (up to 5 months post-grafting) by magnetic resonance imaging. CONCLUSIONS: These findings support the use of commercial MNPs to track cells for short- and mid-term periods after transplantation for studies of brain cell replacement therapy. Nevertheless, long-term MR images should be interpreted with caution due to the possibility that some MNPs may be expelled from the transplanted cells and internalized by host microglial cells.

Human midbrain precursors activate the expected developmental genetic program and differentiate long-term to functional A9 dopamine neurons in vitro. Enhancement by Bcl-X(L).

Understanding the molecular programs of the generation of human dopaminergic neurons (DAn) from their ventral mesencephalic (VM) precursors is of key importance for basic studies, progress in cell therapy, drug screening and pharmacology in the context of Parkinson's disease. The nature of human DAn precursors in vitro is poorly understood, their properties unstable, and their availability highly limited. Here we present positive evidence that human VM precursors retaining their genuine properties and long-term capacity to generate A9 type Substantia nigra human DAn (hVM1 model cell line) can be propagated in culture. During a one month differentiation, these cells activate all key genes needed to progress from pro-neural and pro-dopaminergic precursors to mature and functional DAn. For the first time, we demonstrate that gene cascades are correctly activated during differentiation, resulting in the generation of mature DAn. These DAn have morphological and functional properties undistinguishable from those generated by VM primary neuronal cultures. In addition, we have found that the forced expression of Bcl-X(L) induces an increase in the expression of key developmental genes (MSX1, NGN2), maintenance of PITX3 expression temporal profile, and also enhances genes involved in DAn long-term function, maintenance and survival (EN1, LMX1B, NURR1 and PITX3). As a result, Bcl-X(L) anticipates and enhances DAn generation.

Omnidirectional leaky opto-electrical fiber for optogenetic control of neurons in cell replacement therapy.

The pathophysiological progress of Parkinson's disease leads through degeneration of dopaminergic neurons in the substantia nigra to complete cell death and lack of dopamine in the striatum where it modulates motor functions. Transplantation of dopaminergic stem cell-derived neurons is a possible therapy to restore dopamine levels. We have previously presented multifunctional pyrolytic carbon coated leaky optoelectrical fibers (LOEFs) with laser ablated micro-optical windows (µOWs) as carriers for channelrhodopsin-2 modified optogenetically active neurons for light-induced on-demand dopamine release and amperometric real-time detection. To increase the dopamine release by stimulating a larger neuronal population with light, we present here a novel approach to generate µOWs through laser ablation around the entire circumference of optical fibers to obtain Omni-LOEFs. Cyclic voltammetric characterization of the pyrolytic carbon showed that despite the increased number of µOWs, the electrochemical properties were not deteriorated. Finally, we demonstrate that the current recorded during real-time detection of dopamine upon light-induced stimulation of neurons differentiated on Omni-LOEFs is significantly higher compared to recordings from the same number of cells seeded on LOEFs with µOWs only on one side. Moreover, by varying the cell seeding density, we show that the recorded current is proportional to the dimension of the cell population.

Embedded 3D Printing in Self-Healing Annealable Composites for Precise Patterning of Functionally Mature Human Neural Constructs.

Human in vitro models of neural tissue with tunable microenvironment and defined spatial arrangement are needed to facilitate studies of brain development and disease. Towards this end, embedded printing inside granular gels holds great promise as it allows precise patterning of extremely soft tissue constructs. However, granular printing support formulations are restricted to only a handful of materials. Therefore, there has been a need for novel materials that take advantage of versatile biomimicry of bulk hydrogels while providing high-fidelity support for embedded printing akin to granular gels. To address this need, Authors present a modular platform for bioengineering of neuronal networks via direct embedded 3D printing of human stem cells inside Self-Healing Annealable Particle-Extracellular matrix (SHAPE) composites. SHAPE composites consist of soft microgels immersed in viscous extracellular-matrix solution to enable precise and programmable patterning of human stem cells and consequent generation mature subtype-specific neurons that extend projections into the volume of the annealed support. The developed approach further allows multi-ink deposition, live spatial and temporal monitoring of oxygen levels, as well as creation of vascular-like channels. Due to its modularity and versatility, SHAPE biomanufacturing toolbox has potential to be used in applications beyond functional modeling of mechanically sensitive neural constructs.

In vitro and in vivo enhanced generation of human A9 dopamine neurons from neural stem cells by Bcl-XL.

Human neural stem cells derived from the ventral mesencephalon (VM) are powerful research tools and candidates for cell therapies in Parkinson disease. Previous studies with VM dopaminergic neuron (DAn) precursors indicated poor growth potential and unstable phenotypical properties. Using the model cell line hVM1 (human ventral mesencephalic neural stem cell line 1; a new human fetal VM stem cell line), we have found that Bcl-X(L) enhances the generation of DAn from VM human neural stem cells. Mechanistically, Bcl-X(L) not only exerts the expected antiapoptotic effect but also induces proneural (NGN2 and NEUROD1) and dopamine-related transcription factors, resulting in a high yield of DAn with the correct phenotype of substantia nigra pars compacta (SNpc). The expression of key genes directly involved in VM/SNpc dopaminergic patterning, differentiation, and maturation (EN1, LMX1B, PITX3, NURR1, VMAT2, GIRK2, and dopamine transporter) is thus enhanced by Bcl-X(L). These effects on neurogenesis occur in parallel to a decrease in glia generation. These in vitro Bcl-X(L) effects are paralleled in vivo, after transplantation in hemiparkinsonian rats, where hVM1-Bcl-X(L) cells survive, integrate, and differentiate into DAn, alleviating behavioral motor asymmetry. Bcl-X(L) then allows for human fetal VM stem cells to stably generate mature SNpc DAn both in vitro and in vivo and is thus proposed as a helpful factor for the development of cell therapies for neurodegenerative conditions, Parkinson disease in particular.

Stem cell therapy for human neurodegenerative disorders-how to make it work.

Recent progress shows that neurons suitable for transplantation can be generated from stem cells in culture, and that the adult brain produces new neurons from its own stem cells in response to injury. These findings raise hope for the development of stem cell therapies in human neurodegenerative disorders. Before clinical trials are initiated, we need to know much more about how to control stem cell proliferation and differentiation into specific phenotypes, induce their integration into existing neural and synaptic circuits, and optimize functional recovery in animal models closely resembling the human disease.

Multifactoriality of Parkinson's Disease as Explored Through Human Neural Stem Cells and Their Transplantation in Middle-Aged Parkinsonian Mice.

Parkinson's disease (PD) is an age-associated neurodegenerative disorder for which there is currently no cure. Cell replacement therapy is a potential treatment for PD; however, this therapy has more clinically beneficial outcomes in younger patients with less advanced PD. In this study, hVM1 clone 32 cells, a line of human neural stem cells, were characterized and subsequently transplanted in middle-aged Parkinsonian mice in order to examine cell replacement therapy as a treatment for PD. In vitro analyses revealed that these cells express standard dopamine-centered markers as well as others associated with mitochondrial and peroxisome function, as well as glucose and lipid metabolism. Four months after the transplantation of the hVM1 clone 32 cells, striatal expression of tyrosine hydroxylase was minimally reduced in all Parkinsonian mice but that of dopamine transporter was decreased to a greater extent in buffer compared to cell-treated mice. Behavioral tests showed marked differences between experimental groups, and cell transplant improved hyperactivity and gait alterations, while in the striatum, astroglial populations were increased in all groups due to age and a higher amount of microglia were found in Parkinsonian mice. In the motor cortex, nonphosphorylated neurofilament heavy was increased in all Parkinsonian mice. Overall, these findings demonstrate that hVM1 clone 32 cell transplant prevented motor and non-motor impairments and that PD is a complex disorder with many influencing factors, thus reinforcing the idea of novel targets for PD treatment that tend to be focused on dopamine and nigrostriatal damage.

Next generation human brain models: engineered flat brain organoids featuring gyrification.

Brain organoids are considered to be a highly promising in vitro model for the study of the human brain and, despite their various shortcomings, have already been used widely in neurobiological studies. Especially for drug screening applications, a highly reproducible protocol with simple tissue culture steps and consistent output, is required. Here we present an engineering approach that addresses several existing shortcomings of brain organoids. By culturing brain organoids with a polycaprolactone scaffold, we were able to modify their shape into a flat morphology. Engineered flat brain organoids (efBOs) possess advantageous diffusion conditions and thus their tissue is better supplied with oxygen and nutrients, preventing the formation of a necrotic tissue core. Moreover, the efBO protocol is highly simplified and allows to customize the organoid size directly from the start. By seeding cells onto 12 by 12 mm scaffolds, the brain organoid size can be significantly increased. In addition, we were able to observe folding reminiscent of gyrification around day 20, which was self-generated by the tissue. To our knowledge, this is the first study that reports intrinsically caused gyrification of neuronal tissue in vitro. We consider our efBO protocol as a next step towards the generation of a stable and reliable human brain model for drug screening applications and spatial patterning experiments.

Generation and properties of a new human ventral mesencephalic neural stem cell line.

Neural stem cells (NSCs) are powerful research tools for the design and discovery of new approaches to cell therapy in neurodegenerative diseases like Parkinson's disease. Several epigenetic and genetic strategies have been tested for long-term maintenance and expansion of these cells in vitro. Here we report the generation of a new stable cell line of human neural stem cells derived from ventral mesencephalon (hVM1) based on v-myc immortalization. The cells expressed neural stem cell and radial glia markers like nestin, vimentin and 3CB2 under proliferation conditions. After withdrawal of growth factors, proliferation and expression of v-myc were dramatically reduced and the cells differentiated into astrocytes, oligodendrocytes and neurons. hVM1 cells yield a large number of dopaminergic neurons (about 12% of total cells are TH+) after differentiation, which also produce dopamine. In addition to proneural genes (NGN2, MASH1), differentiated cells show expression of several genuine mesencephalic dopaminergic markers such as: LMX1A, LMX1B, GIRK2, ADH2, NURR1, PITX3, VMAT2 and DAT, indicating that they retain their regional identity. Our data indicate that this cell line and its clonal derivatives may constitute good candidates for the study of development and physiology of human dopaminergic neurons in vitro, and to develop tools for Parkinson's disease cell replacement preclinical research and drug testing.

Pyrolytic Carbon Nanograss Enhances Neurogenesis and Dopaminergic Differentiation of Human Midbrain Neural Stem Cells.

Advancements in research on the interaction of human neural stem cells (hNSCs) with nanotopographies and biomaterials are enhancing the ability to influence cell migration, proliferation, gene expression, and tailored differentiation toward desired phenotypes. Here, the fabrication of pyrolytic carbon nanograss (CNG) nanotopographies is reported and demonstrated that these can be employed as cell substrates boosting hNSCs differentiation into dopaminergic neurons (DAn), a long-time pursued goal in regenerative medicine based on cell replacement. In the near future, such structures can play a crucial role in the near future for stem-cell based cell replacement therapy (CRT) and bio-implants for Parkinson's disease (PD). The unique combination of randomly distributed nanograss topographies and biocompatible pyrolytic carbon material is optimized to provide suitable mechano-material cues for hNSCs adhesion, division, and DAn differentiation of midbrain hNSCs. The results show that in the presence of the biocoating poly-L-lysine (PLL), the CNG enhances hNSCs neurogenesis up to 2.3-fold and DAn differentiation up to 3.5-fold. Moreover, for the first time, consistent evidence is provided, that CNGs without any PLL coating are not only supporting cell survival but also lead to significantly enhanced neurogenesis and promote hNSCs to acquire dopaminergic phenotype compared to PLL coated topographies.

Lysosomal perturbations in human dopaminergic neurons derived from induced pluripotent stem cells with PARK2 mutation.

Mutations in the PARK2 gene encoding parkin, an E3 ubiquitin ligase, are associated with autosomal recessive early-onset Parkinson's disease (PD). While parkin has been implicated in the regulation of mitophagy and proteasomal degradation, the precise mechanism leading to neurodegeneration in both sporadic and familial PD upon parkin loss-of-function remains unknown. Cultures of isogenic induced pluripotent stem cell (iPSC) lines with and without PARK2 knockout (KO) enable mechanistic studies of the effect of parkin deficiency in human dopaminergic neurons. We used such cells to investigate the impact of PARK2 KO on the lysosomal compartment and found a clear link between parkin deficiency and lysosomal alterations. PARK2 KO neurons exhibited a perturbed lysosomal morphology with enlarged electron-lucent lysosomes and an increased lysosomal content, which was exacerbated by mitochondrial stress and could be ameliorated by antioxidant treatment. We also found decreased lysosomal enzyme activity and autophagic perturbations, suggesting an impairment of the autophagy-lysosomal pathway in parkin-deficient cells. Interestingly, activity of the GBA-encoded enzyme, ß-glucocerebrosidase, was increased, suggesting the existence of a compensatory mechanism. In conclusion, our data provide a unique characterization of the morphology, content, and function of lysosomes in PARK2 KO neurons and reveal an important new connection between mitochondrial dysfunction and lysosomal dysregulation in PD pathogenesis.

3D-Printed Soft Lithography for Complex Compartmentalized Microfluidic Neural Devices.

Compartmentalized microfluidic platforms are an invaluable tool in neuroscience research. However, harnessing the full potential of this technology remains hindered by the lack of a simple fabrication approach for the creation of intricate device architectures with high-aspect ratio features. Here, a hybrid additive manufacturing approach is presented for the fabrication of open-well compartmentalized neural devices that provides larger freedom of device design, removes the need for manual postprocessing, and allows an increase in the biocompatibility of the system. Suitability of the method for multimaterial integration allows to tailor the device architecture for the long-term maintenance of healthy human stem-cell derived neurons and astrocytes, spanning at least 40 days. Leveraging fast-prototyping capabilities at both micro and macroscale, a proof-of-principle human in vitro model of the nigrostriatal pathway is created. By presenting a route for novel materials and unique architectures in microfluidic systems, the method provides new possibilities in biological research beyond neuroscience applications.

Bimodal viral vectors and in vivo imaging reveal the fate of human neural stem cells in experimental glioma model.

Transplantation of genetically engineered cells into the CNS offers immense potential for the treatment of several neurological disorders. Monitoring expression levels of transgenes and following changes in cell function and distribution over time is critical in assessing therapeutic efficacy of such cells in vivo. We have engineered lentiviral vectors bearing fusions between different combinations of fluorescent and bioluminescent marker proteins and used bioluminescence imaging and intravital-scanning microscopy in real time to study the fate of human neural stem cells (hNSCs) at a cellular resolution in glioma-bearing brains in vivo. Using Renilla luciferase (Rluc)-DsRed2 or GFP-Rluc-expressing malignant human glioma model, transduced hNSCs were shown to migrate extensively toward gliomas, with hNSCs populating gliomas at 10 d after transplantation. Furthermore, transduced hNSCs survived longer in mice with gliomas than in normal brain, but did not modulate glioma progression in vivo. These studies demonstrate the utility of bimodal viral vectors and real-time imaging in evaluating fate of NSCs in diseased models and thus provide a platform for accelerating cell-based therapies for CNS disorders.

Enhanced dopaminergic differentiation of human neural stem cells by synergistic effect of Bcl-xL and reduced oxygen tension.

Neural stem cells constitute a promising source of cells for transplantation in Parkinson's disease, but a protocol for controlled dopaminergic differentiation is not yet available. Here we investigated the effect of the anti-apoptotic protein Bcl-x(L) and oxygen tension on dopaminergic differentiation and survival of a human ventral mesencephalic stem cell line (hVM1). hVM1 cells and a Bcl-x(L) over-expressing subline (hVMbcl-x(L)) were differentiated by sequential treatment with fibroblast growth factor-8, forskolin, sonic hedgehog, and glial cell line-derived neurotrophic factor. After 10 days at 20% oxygen, hVMbcl-x(L) cultures contained proportionally more tyrosine hydroxylase(TH)-positive cells than hVM1 control cultures. This difference was significantly potentiated from 11 +/- 0.8% to 17.2 +/- 0.2% of total cells when the oxygen tension was lowered to 3%. Immunocytochemistry and Q-PCR-analysis revealed expression of several dopaminergic markers besides of TH just as dopamine was detected in the culture medium by HPLC analysis. Although Bcl-x(L)-over-expression reduced cell death in the cultures, it did not alter the relative content of GABAergic, neurons, while the content of astroglial cells was reduced in hVMbcl-x(L) cell cultures compared with control. We conclude that Bcl-x(L) and lowered oxygen tension act in concert to enhance dopaminergic differentiation and survival of human neural stem cells.

Leaky Optoelectrical Fiber for Optogenetic Stimulation and Electrochemical Detection of Dopamine Exocytosis from Human Dopaminergic Neurons.

In Parkinson's disease, the degeneration of dopaminergic neurons in substantia nigra leads to a decrease in the physiological levels of dopamine in striatum. The existing dopaminergic therapies effectively alleviate the symptoms, albeit they do not revert the disease progression and result in significant adverse effects. Transplanting dopaminergic neurons derived from stem cells could restore dopamine levels without additional motor complications. However, the transplanted cells disperse in vivo and it is not possible to stimulate them on demand to modulate dopamine release to prevent dyskinesia. In order to address these issues, this paper presents a multifunctional leaky optoelectrical fiber for potential neuromodulation and as a cell substrate for application in combined optogenetic stem cell therapy. Pyrolytic carbon coated optical fibers are laser ablated to pattern micro-optical windows to permit light leakage over a large area. The pyrolytic carbon acts as an excellent electrode for the electrochemical detection of dopamine. Human neural stem cells are genetically modified to express the light sensitive opsin channelrhodopsin-2 and are differentiated into dopaminergic neurons on the leaky optoelectrical fiber. Finally, light leaking from the micro-optical windows is used to stimulate the dopaminergic neurons resulting in the release of dopamine that is detected in real-time using chronoamperometry.

Neuronal and Glial Differentiation of Human Neural Stem Cells Is Regulated by Amyloid Precursor Protein (APP) Levels.

Amyloid precursor protein (APP) is implicated in neural development as well as in the pathology of Alzheimer's disease (AD); however, its biological function still remains unclear. It has been reported that APP stimulates the proliferation and neuronal differentiation of neural stem cells (NSCs), while other studies suggest an important effect enhancing gliogenesis in NSCs. As expected, APP protein/mRNA is detected in hNS1 cells, a model cell line of human NSCs, both under proliferation and throughout the differentiation period. To investigate the potential function that APP plays in cell fate specification and differentiation of hNS1 cells, we transiently increased human APP levels in these cells and analyzed its cell intrinsic effects. Our data indicate that increased levels of APP induce early cell cycle exit and instructively direct hNS1 cell fate towards a glial phenotype, while decreasing neuronal differentiation. Since elevated APP levels also enhanced APP intracellular domain (AICD)-immunoreactivity, these effects could be, in part, mediated by the APP/AICD system. The AICD domain can play a potential role in signal transduction by its molecular interaction with different target genes such as GSK3B, whose expression was also increased in APP-overexpressing cells that, in turn, may contribute to promoting gliogenesis and inhibiting neurogenesis in NSCs. These data suggest an important action of APP in modulating hNSCs differentiation (probably in an AICD-GSK-3ß-dependent manner) and may thus be important for the future development of stem cell therapy strategies for the diseased mammalian brain.

Dopaminergic differentiation of human neural stem cells mediated by co-cultured rat striatal brain slices.

Properly committed neural stem cells constitute a promising source of cells for transplantation in Parkinson's disease, but a protocol for controlled dopaminergic differentiation is not yet available. To establish a setting for identification of secreted neural compounds promoting dopaminergic differentiation, we co-cultured cells from a human neural forebrain-derived stem cell line (hNS1) with rat striatal brain slices. In brief, coronal slices of neonatal rat striatum were cultured on semiporous membrane inserts placed in six-well trays overlying monolayers of hNS1 cells. After 12 days of co-culture, large numbers of tyrosine hydroxylase (TH)-immunoreactive, catecholaminergic cells could be found underneath individual striatal slices. Cell counting revealed that up to 25.3% (average 16.1%) of the total number of cells in these areas were TH-positive, contrasting a few TH-positive cells (<1%) in non-induced areas. The presence of dopamine in the conditioned culture medium was confirmed by HPLC analysis. Interestingly, not all striatal slice cultures induced TH-expression in underlying hNS1 cells. Common to TH-inductive cultures was, however, the presence of degenerating, necrotic areas, suggesting that factors released during striatal degeneration were responsible for the dopaminergic induction of the hNS1 cells. Ongoing experiments aim to identify such factors by comparing protein profiles of media conditioned by degenerating (necrotic) versus healthy striatal slice cultures.