Target‐specific forebrain projections and appropriate synaptic inputs of hESC‐derived dopamine neurons grafted to the midbrain of parkinsonian rats

Dopamine (DA) neurons derived from human embryonic stem cells (hESCs) are a promising unlimited source of cells for cell replacement therapy in Parkinson's disease (PD). A number of studies have demonstrated functionality of DA neurons originating from hESCs when grafted to the striatum of rodent and non‐human primate models of PD. However, several questions remain in regard to their axonal outgrowth potential and capacity to integrate into host circuitry. Here, ventral midbrain (VM) patterned hESC‐derived progenitors were grafted into the midbrain of 6‐hydroxydopamine‐lesioned rats, and analyzed at 6, 18, and 24 weeks for a time‐course evaluation of specificity and extent of graft‐derived fiber outgrowth as well as potential for functional recovery. To investigate synaptic integration of the transplanted cells, we used rabies‐based monosynaptic tracing to reveal the origin and extent of host presynaptic inputs to grafts at 6 weeks. The results reveal the capacity of grafted neurons to extend axonal projections toward appropriate forebrain target structures progressively over 24 weeks. The timing and extent of graft‐derived dopaminergic fibers innervating the dorsolateral striatum matched reduction in amphetamine‐induced rotational asymmetry in the animals where recovery could be observed. Monosynaptic tracing demonstrated that grafted cells integrate with host circuitry 6 weeks after transplantation, in a manner that is comparable with endogenous midbrain connectivity. Thus, we demonstrate that VM patterned hESC‐derived progenitors grafted to midbrain have the capacity to extensively innervate appropriate forebrain targets, integrate into the host circuitry and that functional recovery can be achieved when grafting fetal or hESC‐derived DA neurons to the midbrain.     

Zonisamide promotes survival of human-induced pluripotent stem cell-derived dopaminergic neurons in the striatum of female rats

The transplantation of dopaminergic (DA) progenitors derived from pluripotent stem cells improves the behavior of Parkinson's disease model animals. However, the survival of DA progenitors is low, and the final yield of DA neurons is only approximately 0.3%–2% the number of transplanted cells. Zonisamide (ZNS) increases the number of survived DA neurons upon the transplantation of mouse-induced pluripotent stem (iPS) cell-derived DA progenitors in the rat striatum. In this study, we induced DA progenitors from human iPS cells and transplanted them into the striatum of female rats with daily administration of ZNS. The number of survived DA neurons was evaluated 1 and 4 months after transplantation by immunohistochemistry, which revealed that the number of survived DA neurons was significantly increased with the administration of ZNS. To assess the mechanism of action of ZNS, we performed a gene expression analysis to compare the gene expression profiles in striatum treated with or without ZNS. The analysis revealed that the expression of SLIT-and NTRK-like protein 6 (SLITRK6) was upregulated in rat striatum treated with ZNS. In conclusion, ZNS promotes the survival of DA neurons after the transplantation of human-iPS cell-derived DA progenitors in the rat striatum. SLITRK6 is suggested to be involved in this supportive effect of ZNS by modulating the environment of the host brain.     

Human embryonic stem cells differentiate into oligodendrocytes in high purity and myelinate after spinal cord transplantation

Human embryonic stem cells (hESCs) demonstrate remarkable proliferative and developmental capacity. Clinical interest arises from their ability to provide an apparently unlimited cell supply for transplantation, and from the hope that they can be directed to desirable phenotypes in high purity. Here we present for the first time a method for obtaining oligodendrocytes and their progenitors in high yield from hESCs. We expanded hESCs, promoted their differentiation into oligodendroglial progenitors, amplified those progenitors, and then promoted oligodendroglial differentiation using positive selection and mechanical enrichment. Transplantation into the shiverer model of dysmyelination resulted in integration, differentiation into oligodendrocytes, and compact myelin formation, demonstrating that these cells display a functional phenotype. This differentiation protocol provides a means of generating human oligodendroglial lineage cells in high purity, for use in studies of lineage development, screening assays of oligodendroglial-specific compounds, and treating neurodegenerative diseases and traumatic injuries to the adult CNS.     

Generation of embryonic stem cells and transgenic mice expressing green fluorescence protein in midbrain dopaminergic neurons

We have generated embryonic stem (ES) cells and transgenic mice with green fluorescent protein (GFP) inserted into the Pitx3 locus via homologous recombination. In the central nervous system, Pitx3-directed GFP was visualized in dopaminergic (DA) neurons in the substantia nigra and ventral tegmental area. Live primary DA neurons can be isolated by fluorescence-activated cell sorting from these transgenic mouse embryos. In culture, Pitx3-GFP is coexpressed in a proportion of ES-derived DA neurons. Furthermore, ES cell-derived Pitx3-GFP expressing DA neurons responded to neurotrophic factors and were sensitive to DA-specific neurotoxin N-4-methyl-1, 2, 3, 6-tetrahydropyridine. We anticipate that the Pitx3-GFP ES cells could be used as a powerful model system for functional identification of molecules governing mDA neuron differentiation and for preclinical research including pharmaceutical drug screening and transplantation. The Pitx3 knock-in mice, on the other hand, could be used for purifying primary neurons for molecular studies associated with the midbrain-specific DA phenotype at a level not previously feasible. These mice would also provide a useful tool to study DA fate determination from embryo- or adult-derived neural stem cells.     

Stromal factors SDF1α, sFRP1, and VEGFD induce dopaminergic neuron differentiation of human pluripotent stem cells

Human embryonic stem cell (hESC)-derived dopaminergic (DA) neurons hold potential for treating Parkinson's disease (PD) through cell replacement therapy. Generation of DA neurons from hESCs has been achieved by coculture with the stromal cell line PA6, a source of stromal cell-derived inducing activity (SDIA). However, the factors produced by stromal cells that result in SDIA are largely undefined. We previously reported that medium conditioned by PA6 cells can generate functional DA neurons from NTera2 human embryonal carcinoma stem cells. Here we show that PA6-conditioned medium can induce DA neuronal differentiation in both NTera2 cells and the hESC I6 cell line. To identify the factor(s) responsible for SDIA, we used large-scale microarray analysis of gene expression combined with mass spectrometric analysis of PA6-conditioned medium (CM). The candidate factors, hepatocyte growth factor (HGF), stromal cell-derived factor-1 α (SDF1α), secreted frizzled-related protein 1 (sFRP1), and vascular endothelial growth factor D (VEGFD) were identified, and their concentrations in PA6 CM were established by immunoaffinity capillary electrophoresis. Upon addition of SDF1α, sFRP1, and VEGFD to the culture medium, we observed an increase in the number of cells expressing tyrosine hydroxylase (a marker for DA neurons) and βIII-tubulin (a marker for immature neurons) in both the NTera2 and I6 cell lines. These results indicate that SDF1α, sFRP1, and VEGFD are major components of SDIA and suggest the potential use of these defined factors to elicit DA differentiation of pluripotent human stem cells for therapeutic intervention in PD.     

Preferential sensitivity of human dopaminergic neurons to gp120-induced oxidative damage

The dopamine (DA)-rich midbrain is known to be a key target of human immunodeficiency virus (HIV)-1. Studies of simian immunodeficiency virus (SIV)-induced neuropathogenesis recently established that there is a major disruption within the nigrostriatal dopaminergic system characterized by marked depletion of dopaminergic neurons, microglial cell activation, and reactive astrocytes. Using a human mesencephalic neuronal/glial culture model, which contains dopaminergic neurons, microglia, and astrocytes, experiments were performed to characterize the damage to dopaminergic neurons induced by HIV-1 gp120. Functional impairment was assessed by DA uptake, and neurotoxicity was measured by apoptosis and oxidative damage. Through the use of this mesencephalic neuronal/glial culture model, we were able to identify the relative sensitivity of dopaminergic neurons to gp120-induced damage, manifested as reduced function (decreased DA uptake), morphological changes, and reduced viability. We also showed that gp120-induced oxidative damage is involved in this neuropathogenic process.     

Identification of midbrain floor plate radial glia-like cells as dopaminergic progenitors

The floor plate (FP), a signaling center and a structure rich in radial glia-like cells, has been traditionally thought to be devoid of neurons and neuronal progenitors. However, in the midbrain, the FP contains neurons of the dopaminergic (DA) lineage that require contact with radial glia-like cells for their induction. We, therefore, decided to explore the interaction relationship between radial glia and neurons during DA neurogenesis. Taking advantage of a novel FP radial glia-like cell culture system and retroviruses, DA neurons were lineage traced in vitro. In utero BrdU pulse-chases extensively labeled the midbrain FP and traced DA neurons both in vivo and in FP cultures. Moreover, from E9.5 to E13.5 the midbrain FP contained dividing cells only in the most apical part of the neuroepithelium, in cells identified as radial glia-like cells. We, therefore, hypothesized that midbrain FP radial glia-like cells could be DA progenitors and tested our hypothesis in vivo. Lineage tracing of DA progenitors with EGFP in Tis21-EGFP knock-in mice, and genetic fate mapping in GLAST::CreERT2/ZEG mice identified the neuroepithelium of the midbrain FP, and specifically, GLAST+ radial glia-like cells as DA progenitors. Combined, our experiments support the concept that the midbrain FP differs from other FP regions and demonstrate that FP radial glia-like cells in the midbrain are neurogenic and give rise to midbrain DA neurons.     

Dopaminergic properties and function after grafting of attached neural precursor cultures

Generation of dopaminergic (DA) neurons from multipotent embryonic progenitors represents a promising therapeutical strategy for Parkinson's disease (PD). Aim of the present study was the establishment of enhanced cell culture conditions, which optimize the use of midbrain progenitor cells in animal models of PD. In addition, the progenitor cells were characterized during expansion and differentiation according to morphological and electrophysiological criteria and compared to primary tissue. Here, we report that CNS precursors can be expanded in vitro up to 40-fold and afterwards be efficiently differentiated into DA neurons. After 4-5 days under differentiation conditions, more than 70% of the neurons were TH+, equivalent to 30% of the total cell population. Calcium imaging revealed the presence of calcium-permeable AMPA receptors in the differentiated precursors which are capable to contribute to many developmental processes. The overall survival rate, degree of reinnervation and the behavioral performance after transplantation of 4 days in-vitro-differentiated cells were similar to results after direct grafting of E14 ventral mesencephalic cells, whereas after shorter or longer differentiation periods, respectively, less effects were achieved. Compared to the amount of in-vitro-generated DA neurons, the survival rate was only 0.8%, indicating that these cells are very vulnerable. Our results suggest that expanded and differentiated DA precursors from attached cultures can survive microtransplantation and integrate within the striatum in terms of behavioral recovery. However, there is only a short time window during in vitro differentiation, in which enough cells are already differentiated towards a DA phenotype and simultaneously not too mature for implantation. However, additional factors and/or genetical manipulation of these expanded progenitors will be required to increase their in vivo survival in order to improve both the ethical and the technical outlook for the use of fetal tissue in clinical transplantation.     

Rapid differentiation of NT2 cells in Sertoli-NT2 cell tissue constructs grown in the rotating wall bioreactor

Cell replacement therapy is of great interest as a long-term treatment of neurodegenerative diseases such as Parkinson's disease (PD). We have previously shown that Sertoli cells (SC) provide neurotrophic support to transplants of dopaminergic fetal neurons and NT2N neurons, derived from the human clonal precursors cell line NTera2/D1 (NT2), which differentiate into dopaminergic NT2N neurons when exposed to retinoic acid. We have created SC-NT2 cell tissue constructs cultured in the high aspect ratio vessel (HARV) rotating wall bioreactor. Sertoli cells, NT2, and SC plus NT2 cells combined in starting ratios of 1:1, 1:2, 1:4 and 1:8 were cultured in the HARV in DMEM with 10% fetal bovine serum and 1% growth factor reduced Matrigel for 3 days, without retinoic acid. Conventional, non-HARV, cultures grown in the same culture medium were used as controls. The presence of tyrosine hydroxylase (TH) was assessed in all culture conditions. Sertoli-neuron-aggregated-cell (SNAC) tissue constructs grown at starting ratios of 1:1 to 1:4 contained a significant amount of TH after 3 days of culture in the HARV. No TH was detected in SC HARV cultures, or SC, NT2 or SC-NT2 conventional co-cultures. Quantitative stereology of immunolabled 1:4 SNAC revealed that approximately 9% of NT2 cells differentiate into TH-positive (TH+) NT2N neurons after 3 days of culture in the HARV, without retinoic acid. SNAC tissue constructs also released dopamine (DA) when stimulated with KCl, suggesting that TH-positive NT2N neurons in the SNAC adopted a functional dopaminergic phenotype. SNAC tissue constructs may be an important source of dopaminergic neurons for neuronal transplantation.     

Fibroblast growth factor-20 promotes the differentiation of Nurr1-overexpressing neural stem cells into tyrosine hydroxylase-positive neurons

Stem cells are currently considered as alternative cell resources for restorative transplantation strategies in Parkinson's disease. However, the mechanisms that induce differentiation of a stem cell toward the dopaminergic phenotype are still partly unknown thus hampering the production of dopaminergic neurons from stem cells. In the past, FGF-20 has been found to promote the survival of ventral mesencephalic (VM) dopaminergic (DA) neurons in culture. We hereby provide evidence that FGF-20, a growth factor of the FGF family, is expressed in the adult and 6-OHDA-lesioned striatum and substantia nigra, but is not expressed by VM glia or DA neurons, suggesting that FGF-20 may work on DA neurons in a paracrine- or target-derived manner. We also found that co-culture of Nurr1-NSCs with Schwann cells overexpressing FGF-20 induced the acquisition of a neuronal morphology by the NSCs and the expression of tyrosine hydroxylase (TH) as assessed by immunocytochemistry, cell ELISA, and Western blot analysis. RT-PCR showed, that both, Schwann cells and Nurr1-NSCs (differentiated or not), expressed the FGF-1 receptor suggesting that both direct and indirect actions of FGF-20 are possible. We show that differentiated Nurr1 cells retained both neuronal morphology and TH expression after transplantation into the striatum of 6-OHDA-lesioned postnatal or adult rats, but that neuritogenesis was only observed after postnatal grafts. Thus, our results suggest that FGF-20 promotes the differentiation of Nurr1-NSCs into TH-positive neurons and that additional factors are required for the efficient differentiation of DA neurons in the adult brain.     

The response of human and rat fetal ventral mesencephalon in culture to the brain-derived neurotrophic factor treatment

Brain-derived neurotrophic factor (BDNF) has been shown to increase the survival of dopaminergic neurons in rodent mesencephalic cultures. The mRNAs of BDNF and trk B receptor have been found to be expressed in the substantia nigra of rat. In this study, the action of BDNF was studied on the survival and transmitter-specific differentiation of dopaminergic neurons of fetal human CNS aged 9–10-week in vitro. Dopaminergic neuron viability and phenotypic expression were monitored by tyrosine hydroxylase (TH) immunohistochemistry and measurement of dopamine (DA) content with HPLC, respectively. After seven days of treatment with BDNF there were 2.2-fold greater number of TH⁺ neurons surviving than in untreated cultures. Although very low levels of DA were detectable in human tissue, considerable amounts of DA was found in the culture medium from around 13 days in vitro (DIV), indicating that DA in human fetal tissue tended to be synthesised and released into the incubation medium more readily than from cultured rat fetal tissue during the same period. The content of DA in the BDNF-treated cultures was approximately double that of untreated cultures after 7 days. In rat fetal tissue, the capacity of each TH⁺ neuron to produce DA was not changed in the BDNF-treated cultures (7 DIV) compared with control cultures, suggesting that BDNF does not up-regulate the production of DA but rather acts to reduce cell death rates. Ciliary neurotrophic factor (CNTF) treatment of rat mesencephalic culture failed to improve the period of survival of fetal dopaminergic neurons and had no effect on the production of DA in cultures. Taken together, our results suggest that BDNF has potent trophic effect on both rat and human fetal mesencephalic dopaminergic neurons in culture and has a potential application in the treatment of Parkinson's disease.     

IL-1β is released from the host brain following transplantation but does not compromise embryonic dopaminergic neuron survival

Poor survival of transplanted dopaminergic (DA) neurons remains a serious obstacle to the success of cell replacement therapy as an alternative to the current treatments for Parkinson's disease. We have examined the temporal release profile of an inflammatory cytokine, interleukin-1 beta (IL-1 beta) following transplantation of fetal mesencephalic tissue into the rat striatum. The amounts of IL-1 beta released in vivo when added to cultures of embryonic DA neurons, did not significantly reduce the survival of DA neurons in vitro, and inclusion of the naturally-occurring IL-1 receptor antagonist, IL-1ra, did not appear to affect the numbers of surviving DA neurons present after 5 days in vitro. Neither did inclusion of IL-1ra in cell suspensions during transplantation increase the survival of transplanted fetal DA neurons. Thus, although IL-1 beta is released following implantation of a neural transplant, we suggest that this pro-inflammatory cytokine does not play an active role in reducing survival of transplanted DA neurons, unlike other cytokines such as tumor necrosis factor alpha. Modulation of IL-1 beta activity, therefore, will not offer significant improvements to neural transplantation as a treatment for PD.     

IL-1 beta is released from the host brain following transplantation but does not compromise embryonic dopaminergic neuron survival

Poor survival of transplanted dopaminergic (DA) neurons remains a serious obstacle to the success of cell replacement therapy as an alternative to the current treatments for Parkinson's disease. We have examined the temporal release profile of an inflammatory cytokine, interleukin-1 beta (IL-1 beta) following transplantation of fetal mesencephalic tissue into the rat striatum. The amounts of IL-1 beta released in vivo when added to cultures of embryonic DA neurons, did not significantly reduce the survival of DA neurons in vitro, and inclusion of the naturally-occurring IL-1 receptor antagonist, IL-1ra, did not appear to affect the numbers of surviving DA neurons present after 5 days in vitro. Neither did inclusion of IL-1ra in cell suspensions during transplantation increase the survival of transplanted fetal DA neurons. Thus, although IL-1 beta is released following implantation of a neural transplant, we suggest that this pro-inflammatory cytokine does not play an active role in reducing survival of transplanted DA neurons, unlike other cytokines such as tumor necrosis factor alpha. Modulation of IL-1 beta activity, therefore, will not offer significant improvements to neural transplantation as a treatment for PD.     

Characterization of axon guidance cue sensitivity of human embryonic stem cell-derived dopaminergic neurons

Dopaminergic neurons derived from human embryonic stem cells will be useful in future transplantation studies of Parkinson's disease patients. As newly generated neurons must integrate and reconnect with host cells, the ability of hESC-derived neurons to respond to axon guidance cues will be critical. Both Netrin-1 and Slit-2 guide rodent embryonic dopaminergic (DA) neurons in vitro and in vivo, but very little is known about the response of hESC-derived DA neurons to any axonal guidance cues. Here we examined the ability of Netrin-1 and Slit-2 to affect human ESC DA axons in vitro. hESC DA neurons mature over time in culture with the developmental profile of DA neurons in vivo, including expression of the DA neuron markers FoxA2, En-1 and Nurr-1, and receptors for both Netrin and Slit. hESC DA neurons respond to exogenous Netrin-1 and Slit-2, showing an increased responsiveness to Netrin-1 as the neurons mature in culture. These responses were maintained in the presence of pro-inflammatory cytokines that might be encountered in the diseased brain. These studies are the first to evaluate and confirm that suitably matured human ES-derived DA neurons can respond appropriately to axon guidance cues.     

Neurogenic neuroepithelial and radial glial cells generated from six human embryonic stem cell lines in serum-free suspension and adherent cultures

The great potential of human embryonic stem (hES) cells offers the opportunity both for studying basic developmental processes in vitro as well as for drug screening, modeling diseases, or future cell therapy. Defining protocols for the generation of human neural progenies represents a most important prerequisite. Here, we have used six hES cell lines to evaluate defined conditions for neural differentiation in suspension and adherent culture systems. Our protocol does not require fetal serum, feeder cells, or retinoic acid at any step, to induce neural fate decisions in hES cells. We monitored neurogenesis in differentiating cultures using morphological (including on-line follow up), immunocytochemical, and RT-PCR assays. For each hES cell line, in suspension or adherent culture, the same longitudinal progression of neural differentiation occurs. We showed the dynamic transitions from hES cells to neuroepithelial (NE) cells, to radial glial (RG) cells, and to neurons. Thus, 7 days after neural induction the majority of cells were NE, expressing nestin, Sox1, and Pax6. During neural proliferation and differentiation, NE cells transformed in RG cells, which acquired vimentin, BLBP, GLAST, and GFAP, proliferated and formed radial scaffolds. gamma-Aminobutyric acid (GABA)-positive and glutamate positive neurons, few oligodendrocyte progenitors and astrocytes were formed in our conditions and timing. Our system successfully generates human RG cells and could be an effective source for neuronal replacement, since RG cells predominantly generate neurons and provide them with support and guidance.     

Induction of Dopaminergic Neurotransmitter Phenotype in Rat Embryonic -Cerebrocortex by the Synergistic Action of Neurotrophins and Dopamine

Neurotrophins, including nerve growth factor, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4/51 (NT-4/5), have been shown to enhance survival and differentiation of a variety of central neuronal populations, such as those with the dopaminergic, cholinergic, GABAergic phenotype during development. In this paper we present evidence that BDNF, NT-3 and NT-4/5 acting synergistically with dopamine (DA) can artificially induce the full dopaminergic phenotype in rat fetal cerebral cortex which normally has very few dopaminergic neurons in adulthood. Thus, BDNF/DA, NT-3/DA, NT-4/DA elicited a great increase in the number of tyrosine hydroxylase (TH)-immunoreactive cells, which was up to 57% of total neuronal population in cultures of fetal rat cortical cells. This stimulatory effect was not dependent on glial proliferation, or on addition of serum to the culture. Pharmacological studies showed that dopamine receptors D, and D2 were involved in this effect. The TH’cortical cells possessed other biochemical phenotypic features of dopaminergic neurons. Thus, high-affinity DA uptake was elevated in cortical cultures treated with neurotrophin/DA. Also DA and 3,4–dihydroxyphenlacetic acid production was detected (5.42 ± 1.24 and 13.72 ? 2.84 pmol/dish respectively, zero in controls). This shows the presence of functionally active TH, aromatic acid decarboxylase and monoamine oxidase. Neurotrophins/DA had no effect on noradrenergic phenotype expression by cortical fetal cells. Taken together, these results support the long-standing view that development of the central nervous system is determined not only by intrinsic genetic programmes, but also involves environmental influences such as the action of growth factors and extracellular neurotransmitters. In this case we report the effect of specific DA phenotype-inducing agents.     

Cell implantation therapies for Parkinson's disease using neural stem, transgenic or xenogeneic donor cells

A new therapeutic neurological and neurosurgical methodology involves cell implantation into the living brain in order to replace intrinsic neuronal systems, that do not spontaneously regenerate after injury, such as the dopaminergic (DA) system affected in Parkinson's disease (PD) and aging. Current clinical data indicate proof of principle for this cell implantation therapy for PD. Furthermore, the disease process does not appear to negatively affect the transplanted cells, although the patient's endogenous DA system degeneration continues. However, the optimal cells for replacement, such as highly specialized human fetal dopaminergic cells capable of repairing an entire degenerated nigro-striatal system, cannot be reliably obtained or generated in sufficient numbers for a standardized medically effective intervention. Xenogeneic and transgenic cell sources of analogous DA cells have shown great utility in animal models and some promise in early pilot studies in PD patients. The cell implantation treatment discipline, using cell fate committed fetal allo- or xenogeneic dopamine neurons and glia, is currently complemented by research on potential stem cell derived DA neurons. Understanding the cell biological principles and developing methodology necessary to generate functional DA progenitors is currently our focus for obtaining DA cells in sufficient quantities for the unmet cell transplantation need for patients with PD and related disorders.     

Redefining Parkinson’s Disease Research Using Induced Pluripotent Stem Cells

Parkinson's disease (PD) is a movement disorder associated with the degeneration of nigral dopaminergic (DA) neurons. One of the greatest obstacles for PD research is the lack of patient-specific nigral DA neurons for mechanistic studies and drug discovery. The advent of induced pluripotent stem cells (iPSCs) has overcome this seemingly intractable problem and changed PD research in many profound ways. In this review, we discuss recent development in the generation and analyses of patient-specific iPSC-derived midbrain DA neurons. Results from this novel platform of human cellular models of PD have offered a tantalizing glimpse of the promising future of PD research. With the development of the latest genomic modification technologies, dopaminergic neuron differentiation methodologies, and cell transplantation studies, PD research is poised to enter a new phase that utilizes the human model system to identify the unique vulnerabilities of human nigral DA neurons and disease-modifying therapies based on such mechanistic studies.     

Sonic hedgehog and FGF8: inadequate signals for the differentiation of a dopamine phenotype in mouse and human neurons in culture

Embryonic mouse striatal neurons and human neurons derived from the NT2/hNT stem cell line can be induced, in culture, to express the dopaminergic (DA) biosynthetic enzyme tyrosine hydroxylase (TH). The novel expression of TH in these cells is signaled by the synergistic interaction of factors present in the media, such as fibroblast growth factor 1 (FGF1) and one of several possible coactivators [DA, phorbol 12-myristate 13-acetate (TPA), isobutylmethylxanthine (IBMX), or forskolin]. Similarly, in vivo, it has recently been reported that the expression of TH in the developing midbrain is mediated by the synergy of FGF8 and the patterning molecule sonic hedgehog (Shh). In the present study, we examined whether the putative in vivo DA differentiation factors can similarly signal TH in our in vitro cell systems. We found that FGF8 and Shh induced TH expression in fewer than 2% of NT2/hNT cells and less than 5% of striatal neurons. The latter could be amplified to as much as 30% by increasing the concentration of growth factor 10-fold or by the addition of other competent coactivators (IBMX/forskolin, TPA, and DA). Additivity/inhibitor experiments indicated that FGF8 worked through traditional tyrosine kinase-initiated MAP/MEK signaling pathways. However, the Shh signal transduction cascade remained unclear. These data suggest that cues effective in vivo may be less successful in promoting the differentiation of a DA phenotype in mouse and human neurons in culture. Thus, our ability to generate DA neurons from different cell lines, for use in the treatment of Parkinson's disease, will depend on the identification of appropriate differentiation signals for each cell type under investigation.