Differentiation of human dopamine neurons from an embryonic carcinomal stem cell line

Previous studies from this laboratory have demonstrated that fibroblast growth factor 1 together with a number of co-activator molecules (dopamine, TPA, IBMX/forskolin), will induce the expression of the catecholamine biosynthetic enzyme tyrosine hydroxylase (TH) in 10% of human neurons (hNTs) derived from the NT2 cell line [10]. In the present study, we found that TH induction was increased to nearly 75% in hNTs when cells were permitted to age 2 weeks in culture prior to treatment with the differentiation cocktail. This high level of TH expression was sustained 7 days after removal of the differentiating agents from the media. Moreover, the induced TH present in these cells was enzymatically active, resulting in the production of low levels of dopamine (DA) and its metabolite DOPAC. These findings suggest that hNTs may provide an important tissue culture model for the study of factors regulating TH gene expression in human neurons. Moreover, hNTs may serve, in vivo, as a source of human DA neurons for use in transplantation therapies.     

The transcription factor Nurr1 in human NT2 cells and hNT neurons

Human, neuronally committed hNT or NT2-N cells, originally derived from the Ntera2/D1 (NT2) clone after exposure to retinoic acid (RA), represent a potentially important source of cells to treat neurodegenerative diseases. Our previous in vitro experiments showed that hNT cells possess immunocytochemically detectable markers typical of dopaminergic (DA) ventral mesencephalic (VM) neurons, including tyrosine hydroxylase (TH), dopamine transporter (DAT), dopamine receptor (D2), and aldehyde dehydrogenase (AHD-2). In the current study, we sought to examine whether Nurr1, an orphan receptor of the nuclear receptor superfamily shown to be essential for the development, differentiation and survival of midbrain DA neurons, would be expressed in 3, 4, or 5 week RA-induced hNT neurons and their NT2 precursors. Our immunocytochemical analyses indicate that NT2 cells as well as hNT neurons independent of the length of RA-driven differentiation were Nurr1-immunoreactive. RT-PCR analysis confirmed the expression of Nurr1-specific mRNA in both NT2 precursors and the hNT neurons. Furthermore, immunocytochemical co-expression of Nurr1 and TH was detected in hNT neurons. The findings of this study suggest that Nurr1 may be important during the development of hNT neurons and involved in their differentiation into the dopaminergic phenotype.     

TGF-beta promotes survival on mesencephalic dopaminergic neurons in cooperation with Shh and FGF-8

Impaired neuronal survival is a key event in the development of degenerative diseases, such as Parkinson's disease (PD). Here we show that transforming growth factor beta (TGF-beta) acts directly on rat E14 midbrain dopaminergic neurons in vitro, its survival-promoting effect being not mediated by BDNF, NT-3, or GDNF. Treatment with TGF-beta, sonic hedgehog (Shh), or fibroblast growth factor-8 (FGF8) significantly increased number of tyrosine hydroxylase (TH)-immunoreactive neurons after 7 days, whereas application of these factors added together further increased number of TH-positive neurons, compared to single-factor treatments. Neutralization of endogenous TGF-beta, Shh, or FGF8 significantly reduced number of dopaminergic neurons. TGF-beta treatment decreased number of apoptotic cells, having no effect on cell proliferation. Neutralization of TGF-beta in vivo during chick E6-10 resulted in reduced number of midbrain dopaminergic neurons. The results suggest that TGF-beta is required for survival of mesencephalic dopaminergic neurons acting in cooperation with Shh and FGF8.     

Apoptosis in cultured hNT neurons

Programmed cell death (apoptosis) is an important mechanism shaping the size of different cell populations within the developing nervous system. In our study we used the NT2/D1 clone originally established from the Ntera 2 cell line to investigate the baseline levels of apoptosis in cultured postmitotic hNT (NT2-N) neurons previously treated for 3, 4 or 5 weeks with retinoic acid (RA) and compared it with apoptosis in NT2 precursors unexposed to RA. First, we examined whether different lengths of exposure to RA might affect baseline apoptotic rate in differentiating hNT neurons. Second, we investigated whether cultured hNT neurons, previously shown to possess dopaminergic characteristics, would be preferentially affected by apoptosis. Using the terminal deoxynucleotidyl transferase (tdt)-labeling technique we found that the postmitotic hNT neuronal cells exposed to RA demonstrated significantly higher numbers of apoptotic cells (12.5-15.8%) in comparison to rapidly dividing NT2 precursor cell line (3.6-4.4%) at both studied (1 and 5 days in vitro, DIV) time points. Similar apoptotic nuclear morphology, including a variable extent of nuclear fragmentation was observed in all examined hNT cultures. On the other hand, the incidence of apoptotic nuclei was rare in cultures of NT2 precursors not subjected to RA treatment. Combined immunocytochemistry for tyrosine hydroxylase (TH) and Hoechst staining revealed dopaminergic hNT neurons destined to die. Our double-labeling studies have demonstrated that only a subset of TH-positive hNT cells had condensed chromatin after 1 (approx. 15%) and 5 (approx. 20%) DIV. NT2 precursors were not TH-positive. Collectively, our results demonstrated that exposure to differentiating agent RA triggers an apoptotic commitment in a subset of postmitotic hNT neurons. These results suggest that this cell line may serve as a model of neuronal development to test various pathogenic factors implicated in the etiology of Parkinson's disease (PD), as well as to screen numerous pharmacological treatments that may slow or prevent dopaminergic deterioration.     

In vitro and in vivo characterization of hNT neuron neurotransmitter phenotypes

The hNT neuron exhibits many characteristics of neuroepithelial precursor cells, making them an excellent model to study neuronal plasticity in vitro and in vivo. These cells express a number of neurotransmitters in vitro, including dopamine, gamma-aminobutyric acid and acetylcholine. However, there have been few reports of the neurotransmitters that hNT neurons express in vivo. The present study examined whether hNT neurons express the same neurotransmitters in vivo as they do in vitro. First, the expression of tyrosine hydroxylase (TH), glutamic acid decarboxylase (GAD), choline acetyltransferase (ChAT) and the human specific nuclear marker NuMA by hNT neurons was confirmed. Nineteen normal animals were then transplanted with 80,000 hNT neurons aimed at the striatum, hippocampus or cerebral cortex. Five additional animals received injections of medium. All animals received daily intraperitoneal injections of cyclosporine (10 mg/kg) and survived 30 days. Sections through the transplants were examined for NuMA-positive hNT neurons, and for the presence of the three neurotransmitter markers: TH, GAD and ChAT. The hNT neurons were found in the striatum and cortex. Of the hNT neurons found within the rat striatum, 33% were ChAT-positive. In the cortex, only 4% of the neurons expressed ChAT. No GAD-positive hNT neurons were detected at either site. No NuMA-positive neurons were found in the hippocampus. The implanted hNT neurons did not induce activation of astrocytes as determined by immunocytochemistry for glial fibrillary acidic protein (GFAP). Moreover, no hNT neuron was found to express GFAP in vivo. Together, these data suggest that the hNT neurons engraft in the new host tissue, maintain their neuronal identity and may be guided in differentiation according to local environmental cues.     

Generation of dopamine neurons from embryonic stem cells in the presence of the neuralizing activity of bone marrow stromal cells derived from adult mice

Stromal cell lines such as PA6 and MS5 have been employed for generating dopamine (DA) neurons from embryonic stem (ES) cells. The present study was designed to test whether bone marrow stromal cells (BMSC) derived from adult mice might be available as a feeder layer to produce DA cells efficiently from ES cells. When ES cells were grown on BMSC in the presence of fibroblast growth factor 8 (FGF8) and sonic hedgehog (SHH), about 40% of TuJ1-positive neurons expressed tyrosine hydroxylase (TH). Because these cells labeled with TH were negative for dopamine-beta-hydroxylasae (DBH), the marker for noradrenergic and adrenergic neurons, the TH-positive cells were most likely DA neurons. They indeed expressed midbrain DA neuron markers such as Nurr 1, Ptx-3, and c-ret and were capable of synthesizing and releasing DA in vitro. Furthermore, DA neurons differentiated from ES cells in this differentiation protocol survived transplantation in rats with 6-hydroxydopamine lesions and reversed the lesion-induced circling behavior. The data indicate that BMSC can facilitate an efficient induction of DA neurons from ES cells and that the generated DA neurons are biologically functional both in vitro and in vivo. Insofar as BMSC have recently been employed in autologous cell therapy for ischemic heart and arteriosclerotic limb diseases, the present study raises the possibility that autologous BMSC can be applied in future cell transplantation therapy in Parkinson's disease.     

Generation of tyrosine hydroxylase-producing neurons from precursors of the embryonic and adult forebrain

We have explored the plastic ability of neuronal precursors to acquire different identities by manipulating their surrounding environment. Specifically, we sought to identify potential signals involved in the specification of forebrain dopaminergic neurons. Here we describe culture conditions under which tyrosine hydroxylase (TH) expression is induced in neuronal precursors, which were derived directly from the embryonic striatum and adult subependyma (SE) of the lateral ventricle or generated from multipotent forebrain stem cells. TH was successfully induced in all of these cell types by 24 hr exposure to basic fibroblast growth factor (FGF2) and glial cell conditioned media (CM). The greatest magnitude of the inductive action was on embryonic striatal precursors. Although FGF2 alone induced limited TH expression in striatal cells (1.1 +/- 0.2% of neurons), these actions were potentiated 17.5-fold (19.6 +/- 1.5% of neurons) when FGF2 was coadministered with B49 glial cell line CM. Of these TH-immunoreactive cells, approximately 15% incorporated bromodeoxyuridine (BrdU), indicating that they were newly generated, and 95% coexpressed the neurotransmitter GABA. To investigate whether precursors of the adult forebrain subependyma were competent to respond to the instructive actions of FGF2+CM, they were first labeled in vivo with a pulse of BrdU. Although none of the cells expressed TH in control, 0.2% of total cells showed TH immunoreactivity in FGF2+CM-treated cultures. Under these same conditions only, in vitro-generated precursors from epidermal growth factor-responsive stem cells exhibited TH expression in 10% of their total neuronal progeny. Regulation of neurotransmitter phenotype in forebrain neuronal precursors, by the synergistic action of FGF2 and glial-derived diffusible factors, may represent a first step in understanding how these cells are generated in the embryonic and adult brain and opens the prospect for their manipulation in vitro and in vivo for therapeutic use.     

SHH and FGF8 play distinct roles during development of noradrenergic neurons in the locus coeruleus of the zebrafish

Several signaling pathways have been implicated in the development of dopaminergic and serotonergic neurons. Here, we analyzed the formation of noradrenergic (NAergic) cells in the locus coeruleus (LC) of zebrafish. In the sonic hedgehog (shh) mutant, cells positive for tyrosine hydroxylase, a marker for putative NAergic cells in the LC were reduced. Similarly, the inhibition of translation of all hh genes and the perturbation of Shh signaling by forskolin resulted in a decrease in the number of cells. Conversely, when SHH was overexpressed, an increase in number was observed. Thus, Shh is involved in maintaining the appropriate number of cells in the LC. While elevated levels of bone morphogenetic protein 4 (BMP4) did not attenuate tyrosine hydroxylase-positive cells, exogenous fibroblast growth factor 8 (FGF8) rescued NAergic neurons in the acerebellar (ace) mutant, providing direct in vivo evidence that Fgf8 is required for the induction of NAergic neurons in the LC.     

Differential connexin expression,gap junction intercellular coupling,and hemichannel formation in NT2/D1 human neural progenitors and terminally differentiated hNT neurons

Connexin-mediated gap junctions and open hemichannels in nonjunctional membranes represent two biologically relevant mechanisms by which neural progenitors can coordinate their response to changes in the extracellular environment. NT2/D1 cells are a teratocarcinoma progenitor line that can be induced to differentiate terminally into functional hNT neurons and NT-G nonneuronal cells. Clinical transplants of hNT neurons and experimental grafts of NT2/D1 progenitors or hNT neurons have been used in cell-replacement therapy in vivo. Previous studies have shown that NT2/D1 cells express connexin 43 (Cx43) and that NT2/D1 progenitors are capable of dye transfer. To determine whether NT2/D1 progenitors and differentiated hNT cultures express other connexins, Cx26, Cx30, Cx32, Cx36, Cx37, Cx43, and Cx46.6 mRNA and protein were analyzed. NT2/D1 progenitors express Cx30, Cx36, Cx37, and Cx43. hNT/NT-G cultures express Cx36, Cx37, and de novo Cx46.6. Cx26 and Cx32 were not expressed in NT2/D1 or hNT/NT-G cells. NT2/D1 progenitors formed functional gap junctions as assessed by dye coupling as well as open hemichannels in nonjunctional membranes as assessed by dye-uptake studies. Dye coupling was inhibited by the gap junction blocker 18alpha-glycyrrhetinic acid. Hemichannel activity was inhibited by the dual-specificity chloride channel/connexin hemichannel inhibitor flufenamic acid but not by the chloride channel inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. Both dye coupling and dye uptake were substantially reduced following differentiation of NT2/D1 progenitors. We conclude that the pattern of connexin expression in NT2/D1 cells changes over the course of differentiation corresponding with a reduction in biochemical coupling and hemichannel activity in differentiated cells.     

Intrastriatal and intranigral grafting of hNT neurons in the 6-OHDA rat model of Parkinson's disease

The clinical findings on neural transplantation for Parkinson's disease (PD) reported thus far are promising but many issues must be addressed before neural transplantation can be considered a routine therapeutic option for PD. The future of neural transplantation for the treatment of neurological disorders may rest in the discovery of a suitable alternative cell type for fetal tissue. One such alternative may be neurons derived from a human teratocarcinoma (hNT). hNT neurons have been shown to survive and integrate within the host brain following transplantation and provide functional recovery in animal models of stroke and Huntington's disease. In this study, we describe the transplantation of hNT neurons in the substantia nigra (SN) and striatum of the rat model for PD. Twenty-seven rats were grafted with one of three hNT neuronal products; hNT neurons, hNT-DA neurons, or lithium chloride (LiCl) pretreated hNT-DA neurons. Robust hNT grafts could be seen with anti-neural cell adhesion molecule and anti-neuron-specific enolase immunostaining. Immunostaining for tyrosine hydroxylase (TH) expression revealed no TH-immunoreactive (THir) neurons in any animals with hNT neuronal grafts. THir cells were observed in 43% of animals with hNT-DA neuronal grafts and all animals with LiCl pretreated hNT-DA neuronal grafts (100%). The number of THir neurons in these animals was low and not sufficient to produce significant functional recovery. In summary, this study has demonstrated that hNT neurons survive transplantation and express TH in the striatum and SN. Although hNT neurons are promising as an alternative to fetal tissue and may have potential clinical applications in the future, further improvements in enhancing TH expression are needed.     

In vitro induction and in vivo expression of bcl-2 in the hNT neurons.

Bcl-2 encodes membrane-associated proteins that suppress programmed cell death in cells of various origins. Compelling evidence suggests that bcl-2 is also involved in neuronal differentiation and axonal regeneration. The human Neuro-Teratocarcinoma (hNT) neurons constitute a terminally differentiated human neuronal cell line that is derived from the Ntera-2/clone D1 (NT2) precursors upon retinoic acid (RA) treatment. After transplantation into the central nervous system (CNS), the hNT neurons survive, engraft, maintain their neuronal identity, and extend long neurite outgrowth. We were particularly interested in the intracellular determinants that confer these post-transplant characteristics to the hNT neurons. Thus, we asked whether the hNT neurons express bcl-2 after transplantation into the rat striatum and if RA induction of the neuronal lineage is mediated by bcl-2. The grafted hNT neurons were first identified using three different antibodies that recognize human-specific epitopes, anti-hMit, anti-hNuc, and NuMA. After a 1-month post-transplant survival time, NuMA immunostaining revealed that 12% of the hNT neurons survived the transplantation. These neurons extended long neuritic processes within the striatum, as demonstrated using the human-specific antibody against the midsize neurofilament subunit HO14. Importantly, we found that 85% of the implanted hNT neurons expressed bcl-2 and that the in vitro induction of the neuronal lineage from the NT2 precursors with RA resulted in an upregulation of bcl-2 expression. Together, these data suggest that the differentiation of the hNT neurons to a neuronal lineage could be mediated at least partially by bcl-2.     

In vitro induction and in vivo expression of bcl-2 in the hNT neurons

Bcl-2 encodes membrane-associated proteins that suppress programmed cell death in cells of various origins. Compelling evidence suggests that bcl-2 is also involved in neuronal differentiation and axonal regeneration. The human Neuro-Teratocarcinoma (hNT) neurons constitute a terminally differentiated human neuronal cell line that is derived from the Ntera-2/clone D1 (NT2) precursors upon retinoic acid (RA) treatment. After transplantation into the central nervous system (CNS), the hNT neurons survive, engraft, maintain their neuronal identity, and extend long neurite outgrowth. We were particularly interested in the intracellular determinants that confer these post-transplant characteristics to the hNT neurons. Thus, we asked whether the hNT neurons express bcl-2 after transplantation into the rat striatum and if RA induction of the neuronal lineage is mediated by bcl-2. The grafted hNT neurons were first identified using three different antibodies that recognize human-specific epitopes, anti-hMit, anti-hNuc, and NuMA. After a 1-month post-transplant survival time, NuMA immunostaining revealed that 12% of the hNT neurons survived the transplantation. These neurons extended long neuritic processes within the striatum, as demonstrated using the human-specific antibody against the midsize neurofilament subunit HO14. Importantly, we found that 85% of the implanted hNT neurons expressed bcl-2 and that the in vitro induction of the neuronal lineage from the NT2 precursors with RA resulted in an upregulation of bcl-2 expression. Together, these data suggest that the differentiation of the hNT neurons to a neuronal lineage could be mediated at least partially by bcl-2.     

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.     

Identified postnatal mesolimbic dopamine neurons in culture: morphology and electrophysiology.

To examine the intrinsic properties of postnatal mesolimbic dopamine (DA) neurons, we dissociated the ventral tegmental area (VTA) from postnatal rats, enriched for DA neurons by microdissection or gradient purification, and grew the cells in culture. In these cultures, up to 50% of neurons were dopaminergic. DA neurons resembled their in vivo counterparts in soma shapes, and in showing two levels of tyrosine hydroxylase (TH) expression, axodendritic differentiation, two sizes of synaptic vesicles, nest-like synaptic arrangements with non-DA cells, and synaptic specializations. Electrophysiologically, however, they could not be distinguished from non-DA cells, which could be consistent with heterogeneity in cell properties. To examine a functional subset of VTA DA neurons, we retrogradely labeled VTA neurons projecting to the nucleus accumbens. These mesoaccumbens neurons were 86% TH positive, 56% cholecystokinin positive, and 0% neurotensin positive; they also displayed the soma shapes characteristic of DA neurons more generally and two levels of TH expression. Like their in vivo counterparts, mesoaccumbens cells generally fired single broad spikes that were triggered by slow depolarizations and had robust spike afterhyperpolarizations, low- and high-threshold Ca2+ spikes, rapid accommodation of firing, time-dependent anomalous rectification, and hyperpolarizing autoreceptor responses. Strikingly, the expression of these active properties did not change with time in culture. Mesoaccumbens DA cells could be identified by a distinctive subset of properties that made up an electrophysiological signature; however, unlike their in vivo counterparts, they were less often spontaneously active and never fired in bursts. These results suggest that most DA cell properties are intrinsic to the cells, including a significant heterogeneity that is maintained in postnatal culture; their level and mode of activity, however, appear to require afferent input. Culturing identified postnatal VTA DA neurons now makes possible examination of the impact of their individual properties on synaptic function.     

Evidence that adenosine A2 and dopamine autoreceptors antagonistically regulate tyrosine hydroxylase activity in rat striatal synaptosomes

Incubation of rat striatal synaptosomes with the adenosine receptor agonist 2-chloroadenosine (2-CADO) produced a concentration-dependent increase of dopamine (DA) synthesis (about 50% of control value). The effect was not additive with the stimulation produced by either 10 microM forskolin or 2 mM dibutyryl cyclic AMP. Pretreatment of striatal synaptosomes with 2-CADO produced an activation of tyrosine hydroxylase (TH) which withstood washing and lysing of the tissue. This activation was largely independent of the presence of Ca2+ ion in the preincubation medium and, when analyzed as a function of different concentrations of the pterin cofactor 6-methyl-5,6,7,8-tetrahydropterin (0.08-0.4 mM), it was associated with an apparent increase in the Vmax of the enzyme. Quinpirole, a selective D2 DA receptor agonist, reduced control synaptosomal DA synthesis and caused a persistent inhibition of TH activity. When added together with 2-CADO, quinpirole depressed the stimulation of DA synthesis and TH activity produced by the adenosine analog. The effect of quinpirole was stereospecifically antagonized by the D2 DA antagonist sulpiride. Quinpirole also inhibited the activation of TH elicited by a submaximal concentration of forskolin, but not that produced by dibutyryl cyclic AMP. The inhibitory effect of quinpirole on basal and 2-CADO-stimulated TH activities was mimicked by DA. These results indicate that presynaptic DA autoreceptors and adenosine A2 receptors interact antagonistically in controlling DA synthesis in rat striatal synaptosomes presumably by exerting opposite inputs on a presynaptic adenylate cyclase system.