Fluvastatin Converts Human Macrophages into Foam Cells with Increased Inflammatory Response to Inactivated Mycobacterium tuberculosis H37Ra.

Cholesterol biosynthesis inhibitors (statins) protect hypercholesterolemic patients against developing active tuberculosis, suggesting that these drugs could help the host to control the pathogen at the initial stages of the disease. This work studies the effect of fluvastatin on the early response of healthy peripheral blood mononuclear cells (PBMCs) to inactivated Mycobacterium tuberculosis (Mtb) H37Ra. We found that in fluvastatin-treated PBMCs, most monocytes/macrophages became foamy cells that overproduced NLRP3 inflammasome components in the absence of immune stimulation, evidencing important cholesterol metabolism/immunity connections. When both fluvastatin-treated and untreated PBMCs were exposed to Mtb H37Ra, a small subset of macrophages captured large amounts of bacilli and died, concentrating the bacteria in necrotic areas. In fluvastatin-untreated cultures, most of the remaining macrophages became epithelioid cells that isolated these areas of cell death in granulomatous structures that barely produced IFNγ. By contrast, in fluvastatin-treated cultures, foamy macrophages surrounded the accumulated bacteria, degraded them, markedly activated caspase-1 and elicited a potent IFNγ/cytotoxic response. In rabbits immunized with the same bacteria, fluvastatin increased the tuberculin test response. We conclude that statins may enhance macrophage efficacy to control Mtb, with the help of adaptive immunity, offering a promising tool in the design of alternative therapies to fight tuberculosis.

Immunohistochemical Study of ASC Expression and Distribution in the Hippocampus of an Aged Murine Model of Alzheimer's Disease.

Neuroinflammation is involved in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease (AD), and is notably dependent on age. One important inflammatory pathway exerted by innate immune cells of the nervous system in response to danger signals is mediated by inflammasomes (IF) and leads to the generation of potent pro-inflammatory cytokines. The protein "apoptosis-associated speck-like protein containing a caspase recruitment domain" (ASC) modulates IF activation but has also other functions which are crucial in AD. We intended to characterize immunohistochemically ASC and pattern recognition receptors (PRR) of IF in the hippocampus (HP) of the transgenic mouse model Tg2576 (APP), in which amyloid-beta (Aß) pathology is directly dependent on age. We show in old-aged APP a significant amount of ASC in microglia and astrocytes associated withAß plaques, in the absence of PRR described by others in glial cells. In addition, APP developed foci with clusters of extracellular ASC granules not spatiallyrelated to Aß plaques, which density correlated with the advanced age of mice and AD development. Clusters were associated withspecific astrocytes characterized by their enlarged ring-shaped process terminals, ASC content, and frequent perivascular location. Their possible implication in ASC clearance and propagation of inflammation is discussed.

Liver Growth Factor "LGF" as a Therapeutic Agent for Alzheimer's Disease.

Alzheimer's disease (AD) is a progressive degenerative disorder and the most common cause of dementia in aging populations. Although the pathological hallmarks of AD are well defined, currently no effective therapy exists. Liver growth factor (LGF) is a hepatic albumin-bilirubin complex with activity as a tissue regenerating factor in several neurodegenerative disorders such as Parkinson's disease and Friedreich's ataxia. Our aim here was to analyze the potential therapeutic effect of LGF on the APPswe mouse model of AD. Twenty-month-old mice received intraperitoneal (i.p.) injections of 1.6 µg LGF or saline, twice a week during three weeks. Mice were sacrificed one week later, and the hippocampus and dorsal cortex were prepared for immunohistochemical and biochemical studies. LGF treatment reduced amyloid-ß (Aß) content, phospho-Tau/Tau ratio and the number of Aß plaques with diameter larger than 25 µm. LGF administration also modulated protein ubiquitination and HSP70 protein levels, reduced glial reactivity and inflammation, and the expression of the pro-apoptotic protein Bax. Because the administration of this factor also restored cognitive damage in APPswe mice, we propose LGF as a novel therapeutic tool that may be useful for the treatment of AD.

Liver Growth Factor Induces Glia-Associated Neuroprotection in an In Vitro Model of Parkinson´s Disease.

Parkinson's disease is a neurodegenerative disorder characterized by the progressive death of dopaminergic (DA) neurons in the substantia nigra (SN), which leads to a loss of the neurotransmitter dopamine in the basal ganglia. Current treatments relieve the symptoms of the disease, but none stop or delay neuronal degeneration. Liver growth factor (LGF) is an albumin-bilirubin complex that stimulates axonal growth in the striatum and protects DA neurons in the SN of 6-hydroxydopamine-lesioned rats. Our previous results suggested that these effects observed in vivo are mediated by microglia and/or astrocytes. To determine if these cells are LGF targets, E14 (embryos from Sprague Dawley rats of 14 days) rat mesencephalic glial cultures were used. Treatment with 100 pg/mL of LGF up-regulated the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinases 1/2 (ERK1/2) and the cyclic AMP response element binding protein (CREB) phosphorylation in glial cultures, and it increased the microglia marker Iba1 and tumor necrosis factor alpha (TNF-alpha) protein levels. The treatment of E14 midbrain neurons with a glial-conditioned medium from LGF-treated glial cultures (GCM-LGF) prevented the loss of DA neurons caused by 6-hydroxy-dopamine. This neuroprotective effect was not observed when GCM-LGF was applied in the presence of a blocking antibody of TNF-alpha activity. Altogether, our findings strongly suggest the involvement of microglia and TNF-alpha in the neuroprotective action of LGF on DA neurons observed in vitro.

RNA-Seq blood transcriptome profiling in familial attention deficit and hyperactivity disorder (ADHD).

We have carried an exploratory study by blood transcriptome to find RNA expression signatures in familial ADHD. Samples were collected from three cases with familial ADHD and their paired controls and evaluated by RNA-Seq. Transcriptome profiling identified 7 differentially expressed transcripts with a FDR <0.05 that were involved in pathways in Huntington's disease or axonal guidance signaling previously implicated in ADHD, and enriched for signal peptide, growth factor binding, and notably the lipid metabolism pathways. These findings show that blood transcriptome can have an associated signature and highlight a potential to use blood transcriptome to identify patterns of ADHD.

Ultrasmall, water dispersible, TWEEN80 modified Yb:Er:NaGd(WO4)2 nanoparticles with record upconversion ratiometric thermal sensitivity and their internalization by mesenchymal stem cells.

This work presents the synthesis by coprecipitation of diamond shaped Yb:Er:NaGd(WO4)2 crystalline nanoparticles (NPs) with diagonal dimensions in the 5-7 nm × 10-12 nm range which have been modified with TWEEN80 for their dispersion in water, and their interaction with mesenchymal stem cells (MSCs) proposed as cellular NP vehicles. These NPs belong to a large family of tetragonal Yb:Er:NaT(XO4)2 (T = Y, La, Gd, Lu; X = Mo, W) compounds with green (2H11/2 + 4S3/2 â†’ 4I15/2) Er-related upconversion (UC) efficiency comparable to that of Yb:Er:ß-NaYF4 reference compound, but with a ratiometric thermal sensitivity (S) 2.5-3.5 times larger than that of the fluoride. At the temperature range of interest for biomedical applications (∼293-317 K/20-44 °C) S = 108-118 × 10-4 K-1 for 20 at%Yb:5 at%Er:NaGd(WO4)2 NPs, being the largest values so far reported using the 2H11/2/4S3/2 Er intensity ratiometric method. Cultured MSCs, incubated with these water NP emulsions, internalize and accumulate the NPs enclosed in endosomes/lysosomes. Incubations with up to 10 µg of NPs per ml of culture medium maintain cellular metabolism at 72 h. A thermal assisted excitation path is discussed as responsible for the UC behavior of Yb:Er:NaT(XO4)2 compounds.

Liver Growth Factor (LGF) Upregulates Frataxin Protein Expression and Reduces Oxidative Stress in Friedreich's Ataxia Transgenic Mice.

Friedreich's ataxia (FA) is a severe disorder with autosomal recessive inheritance that is caused by the abnormal expansion of GAA repeat in intron 1 of FRDA gen. This alteration leads to a partial silencing of frataxin transcription, causing a multisystem disorder disease that includes neurological and non-neurological damage. Recent studies have proven the effectiveness of neurotrophic factors in a number of neurodegenerative diseases. Therefore, we intend to determine if liver growth factor (LGF), which has a demonstrated antioxidant and neuroprotective capability, could be a useful therapy for FA. To investigate the potential therapeutic activity of LGF we used transgenic mice of the FXNtm1MknTg (FXN)YG8Pook strain. In these mice, intraperitoneal administration of LGF (1.6 µg/mouse) exerted a neuroprotective effect on neurons of the lumbar spinal cord and improved cardiac hypertrophy. Both events could be the consequence of the increment in frataxin expression induced by LGF in spinal cord (1.34-fold) and heart (1.2-fold). LGF also upregulated by 2.6-fold mitochondrial chain complex IV expression in spinal cord, while in skeletal muscle it reduced the relation oxidized glutathione/reduced glutathione. Since LGF partially restores motor coordination, we propose LGF as a novel factor that may be useful in the treatment of FA.

Liver growth factor as a tissue regenerating factor in neurodegenerative diseases.

Liver growth factor (LGF) is a hepatic mitogen purified by our group in 1986. In the following years we demonstrated its activity both in "in vivo" and "in vitro" systems, stimulating hepatocytes mitogenesis as well as liver regeneration in several models of liver injury. Furthermore, we established its chemical composition (albumin-bilirubin complex) and its mitogenic actions in liver. From 2000 onwards we used LGF as a tissue regenerating factor in several models of extrahepatic diseases. The use of Liver growth factor as a neural tissue regenerator has been recently protected (Patent No US 2014/8,642,551 B2). LGF administration stimulates neurogenesis and neuron survival, promotes migration of newly generated neurons, and induces the outgrowth of striatal dopaminergic terminals in 6-hidroxydopamine-lesioned rats. Furthermore, LGF treatment raises striatal dopamine levels and protects dopaminergic neurons in hemiparkinsonian animals. LGF also stimulates survival of grafted foetal neural stem cells in the damaged striatum, reduces rotational behaviour and improves motor coordination. Interestingly, LGF also exerts a neuroprotective role both in an experimental model of cerebellar ataxia and in a model of Friedrich´s ataxia. Microglia seem to be the cellular target of LGF in the CNS. Moreover, the activity of the factor could be mediated by the stimulation of MAPK´s signalling pathway and by regulating critical proteins for cell survival, such as Bcl-2 and phospho-CREB. Since the factor shows neuroprotective and neurorestorative effects we propose LGF as a patented novel therapeutic tool that may be useful for the treatment of Parkinson´s disease and cerebellar ataxias. Currently, our studies have been extended to other neurological disorders such as Alzheimer's disease (Patent No: US 2014/0113859 A1).

Neuroprotective role of liver growth factor "LGF" in an experimental model of cerebellar ataxia.

Cerebellar ataxias (CA) comprise a heterogeneous group of neurodegenerative diseases characterized by a lack of motor coordination. They are caused by disturbances in the cerebellum and its associated circuitries, so the major therapeutic goal is to correct cerebellar dysfunction. Neurotrophic factors enhance the survival and differentiation of selected types of neurons. Liver growth factor (LGF) is a hepatic mitogen that shows biological activity in neuroregenerative therapies. We investigate the potential therapeutic activity of LGF in the 3-acetylpiridine (3-AP) rat model of CA. This model of CA consists in the lesion of the inferior olive-induced by 3-AP (40 mg/kg). Ataxic rats were treated with 5 µg/rat LGF or vehicle during 3 weeks, analyzing: (a) motor coordination by using the rota-rod test; and (b) the immunohistochemical and biochemical evolution of several parameters related with the olivo-cerebellar function. Motor coordination improved in 3-AP-lesioned rats that received LGF treatment. LGF up-regulated NeuN and Bcl-2 protein levels in the brainstem, and increased calbindin expression and the number of neurons receiving calbindin-positive projections in the cerebellum. LGF also reduced extracellular glutamate and GABA concentrations and microglia activation in the cerebellum. In view of these results, we propose LGF as a potential therapeutic agent in cerebellar ataxias.

Neuroprotective activity of peripherally administered liver growth factor in a rat model of Parkinson's disease.

Liver growth factor (LGF) is a hepatic mitogen purified some years ago that promotes proliferation of different cell types and the regeneration of damaged tissues, including brain tissue. Considering the possibility that LGF could be used as a therapeutic agent in Parkinson's disease, we analyzed its potential neuroregenerative and/or neuroprotective activity when peripherally administered to unilaterally 6-hydroxydopamine (6-OHDA)-lesioned rats. For these studies, rats subjected to nigrostriatal lesions were treated intraperitoneally twice a week with LGF (5 microg/rat) for 3 weeks. Animals were sacrificed 4 weeks after the last LGF treatment. The results show that LGF stimulates sprouting of tyrosine hydroxylase-positive terminals and increases tyrosine hydroxylase and dopamine transporter expression, as well as dopamine levels in the denervated striatum of 6-OHDA-lesioned rats. In this structure, LGF activates microglia and raises tumor necrosis factor-alpha protein levels, which have been reported to have a role in neuroregeneration and neuroprotection. Besides, LGF stimulates the phosphorylation of MAPK/ERK1/2 and CREB, and regulates the expression of proteins which are critical for cell survival such as Bcl2 and Akt. Because LGF partially protects dopamine neurons from 6-OHDA neurotoxicity in the substantia nigra, and reduces motor deficits in these animals, we propose LGF as a novel factor that may be useful in the treatment of Parkinson's disease.

Effects of intravenous administration of human umbilical cord blood stem cells in 3-acetylpyridine-lesioned rats.

Cerebellar ataxias include a heterogeneous group of infrequent diseases characterized by lack of motor coordination caused by disturbances in the cerebellum and its associated circuits. Current therapies are based on the use of drugs that correct some of the molecular processes involved in their pathogenesis. Although these treatments yielded promising results, there is not yet an effective therapy for these diseases. Cell replacement strategies using human umbilical cord blood mononuclear cells (HuUCBMCs) have emerged as a promising approach for restoration of function in neurodegenerative diseases. The aim of this work was to investigate the potential therapeutic activity of HuUCBMCs in the 3-acetylpyridine (3-AP) rat model of cerebellar ataxia. Intravenous administered HuUCBMCs reached the cerebellum and brain stem of 3-AP ataxic rats. Grafted cells reduced 3-AP-induced neuronal loss promoted the activation of microglia in the brain stem, and prevented the overexpression of GFAP elicited by 3-AP in the cerebellum. In addition, HuUCBMCs upregulated the expression of proteins that are critical for cell survival, such as phospho-Akt and Bcl-2, in the cerebellum and brain stem of 3-AP ataxic rats. As all these effects were accompanied by a temporal but significant improvement in motor coordination, HuUCBMCs grafts can be considered as an effective cell replacement therapy for cerebellar disorders.

Cell volume regulation modulates NLRP3 inflammasome activation.

Cell volume regulation is a primitive response to alterations in environmental osmolarity. The NLRP3 inflammasome is a multiprotein complex that senses pathogen- and danger-associated signals. Here, we report that, from fish to mammals, the basic mechanisms of cell swelling and regulatory volume decrease (RVD) are sensed via the NLRP3 inflammasome. We found that a decrease in extracellular osmolarity induced a K(+)-dependent conformational change of the preassembled NLRP3-inactive inflammasome during cell swelling, followed by activation of the NLRP3 inflammasome and caspase-1, which was controlled by transient receptor potential channels during RVD. Both mechanisms were necessary for interleukin-1ß processing. Increased extracellular osmolarity prevented caspase-1 activation by different known NLRP3 activators. Collectively, our data identify cell volume regulation as a basic conserved homeostatic mechanism associated with the formation of the NLRP3 inflammasome and reveal a mechanism for NLRP3 inflammasome activation.

Liver growth factor promotes the survival of grafted neural stem cells in a rat model of Parkinson's disease.

Neural stem cells (NSCs) with self-renewal and multilineage potential are considered good candidates for cell replacement of damaged nerve tissue. Several studies have focused on the ability of the neurotrophic factors coadministration to improve the efficiency of grafted NSCs. Liver growth factor (LGF) is an hepatic mitogen that promotes regeneration of damaged tissues, including brain tissue. It has neurogenic activity and has partially restored the nigrostriatal dopaminergic system in an experimental model of Parkinson's disease. Present results demonstrate that in the dopamine- depleted striatum of 6-hydroxydopamine-lesioned rats, grafted NSCs retained their ability to differentiate into neurons, astrocytes, and oligodendrocytes. NSCs also differentiated into microglia/macrophages and endothelial cells. Thus, 23 ± 5.6% of them were inmunoreactive for isolectin IB4, and a small population integrated into blood vessels, showing an endothelial-like morphology. Intrastriatal infusion of LGF promoted the viability of the implants, and favored their differentiation to an endothelial-like phenotype. Moreover, LGF infusion raised the expression of the anti-apoptotic protein Bcl-2 by 3.9 ± 0.9 fold without affecting the levels of the pro-apoptotic protein Bax. Since LGF-treated rats also showed a significant reduction in apomorphine-induced rotational behavior, our results suggest that administration of this factor might be a convenient treatment for Parkinson's disease cell replacement therapies based on NSCs transplantation.

Applications of human umbilical cord blood cells in central nervous system regeneration.

In recent decades, there has been considerable amount of information about embryonic stem cells (ES). The dilemma facing scientists interested in the development and use of human stem cells in replacement therapies is the source of these cells, i.e. the human embryo. There are many ethical and moral problems related to the use of these cells. Hematopoietic stem cells from umbilical cord blood have been proposed as an alternative source of embryonic stem cells. After exposure to different agents, these cells are able to express antigens of diverse cellular lineages, including the neural type. The In vitro manipulation of human umbilical cord blood (hUCB) cells has shown their stem capacity and plasticity. These cells are easily accessible, In vitro amplifiable, well tolerated by the host, and with more primitive molecular characteristics that give them great flexibility. Overall, these properties open a promising future for the use of hUCB in regenerative therapies for the Central Nervous System (CNS). This review will focus on the available literature concerning umbilical cord blood cells as a therapeutic tool for the treatment of neurodegenerative diseases.

Mobilization of neural stem cells and generation of new neurons in 6-OHDA-lesioned rats by intracerebroventricular infusion of liver growth factor.

Neural stem cells with self-renewal and multilineage potential persist in the subventricular zone of the adult mammalian forebrain. These cells remain relatively quiescent but, under certain conditions, can be stimulated, giving rise to new neurons. Liver growth factor (LGF) is a mitogen for liver cells that shows biological activity in extrahepatic sites and is useful for neuroregenerative therapies. The aim of this study was to investigate the potential neurogenic activity of LGF in the 6-hydroxydopamine rat model of Parkinson's disease. Proliferation was significantly increased in the subventricular zone and denervated striatum of rats receiving ICV LGF infusions, and 25% of the proliferating cells were doublecortin-positive neurons. Doublecortin-positive cells with the morphology of migrating neuroblasts were also observed in the dorsal and ventral regions of the striatum of LGF-infused animals. Moreover, some newly generated cells were neuronal nuclei-positive mature neurons. LGF also stimulated microglia and induced astrogliosis, both phenomena associated with generation and migration of new neurons in the adult brain. In summary, our study shows that LGF stimulates neurogenesis when applied intraventricularly in 6-hydroxydopamine-lesioned rats. Considering that this factor also promotes neuronal migration into damaged tissue, we propose LGF as a novel factor useful for neuronal replacement in neurodegenerative diseases.

Driving neural stem cells towards a desired phenotype.

Neural stem cells are defined as clonogenic cells with self-renewal capacity and the ability to generate all neural lineages. Cells with these characteristics have been isolated from the embryonic and adult Central Nervous System. Numerous reports show that extrinsic factors and intracellular mechanisms may trigger both endogenous and in vitro cultured neural stem cells to differentiate into desired cell outcomes. This plasticity opens new approaches for the use of neural stem cells as a source of cells for replacement therapy in damaged brain. In this review we present the evidence for the involvement of trophic factors, neurotransmitters, second messengers, aminoacids, and factors released by endothelial and glial cells, which have been reported to influence neural stem cells phenotypic choice in vitro and in vivo.

Kainic acid triggers oligodendrocyte precursor cell proliferation and neuronal differentiation from striatal neural stem cells.

Glutamate is an excitatory amino acid that serves important functions in mammalian brain development through alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/ kainate receptor stimulation. Neural stem cells with self-renewal and multilineage potential are a useful tool to study the signals involved in the regulation of brain development. We have investigated the role played by AMPA/kainate receptors during the differentiation of neural stem cells derived from fetal rat striatum. The application of 1 and 10 microM kainic acid increased significantly the phosphorylation of the cyclic AMP response element binding protein (CREB), raised bromodeoxyuridine incorporation in O4-positive oligodendrocyte precursors, and increased the number of O1-positive cells in the cultures. Increased CREB phosphorylation and proliferation were prevented by the AMPA receptor antagonist 4-4(4-aminophenyl)-1,2-dihydro-1-methyl-2-propylcarbamoyl-6,7-methylenedioxyphthalazine (SYM 2206) and by protein kinase A and protein kinase C inhibitors. Cultures treated with 100 microM kainic acid showed decreased proliferation, a lower proportion of O1-positive cells, and apoptosis of O4-positive cells. None of these effects were prevented by SYM 2206, suggesting that kainate receptors take part in these events. We conclude that AMPA receptor stimulation by kainic acid promotes the proliferation of oligodendrocyte precursors derived from neural stem cells through a mechanism that requires the activation of CREB by protein kinase A and C. In the neurons derived from these cells, either AMPA or kainate receptor stimulation produces neuritic growth and larger cell bodies.

Intrastriatal infusion of liver growth factor stimulates dopamine terminal sprouting and partially restores motor function in 6-hydroxydopamine-lesioned rats.

Liver growth factor (LGF) is a mitogen for liver cells that shows biological activity in extrahepatic sites and may be useful for neuroregenerative therapies. The aim of this work was to investigate the effects of the intrastriatal (IS) infusion of LGF in the 6-hydroxydopamine rat model of Parkinson's disease. Tyrosine hydroxylase-positive innervation was significantly increased in the dopamine-denervated striatum of rats receiving intrastriatal LGF infusions (160 ng/day/rat x 15 days) as compared with a vehicle-infused group. There was no evidence of dopaminergic neurogenesis in the striatum or substantia nigra in any experimental group at the times studied. However, in those animals undergoing IS-LGF infusion for 48 hr, we found a significant increase in both microglial proliferation and in the number of microglial cells that acquired the ameboid morphology. This is characteristic of activated microglia/macrophages that has been reported to play an important role in dopamine terminal sprouting. In summary, our study shows that IS infusion of LGF stimulates the outgrowth of tyrosine hydroxylase-positive terminals in the striatum of 6-hydroxydopamine-treated rats. As apomorphine-induced rotational behavior was also reduced in these animals, we propose LGF as a novel factor that, when delivered to the striatum, may be useful in the treatment of Parkinson's disease.

Tyrosine hydroxylase induction by basic fibroblast growth factor and cyclic AMP analogs in striatal neural stem cells: role of ERK1/ERK2 mitogen-activated protein kinase and protein kinase C.

Neural stem cells (NSC) with self-renewal and multilineage potential are considered good candidates for cell replacement of damaged nervous tissue. In vitro experimental conditions can differentiate these cells into specific neuronal phenotypes. In the present study, we describe the combined effect of basic fibroblast growth factor (bFGF) and dibutyryladenosine 3',5'-cyclic monophosphate (dbcAMP) on the differentiation of fetal rat striatal NSC into tyrosine hydroxylase-positive cells. Tyrosine hydroxylase induction was accompanied by the activation of ERK1/ERK2 mitogen-activated protein kinase and was inhibited by the ERK1/ERK2 pathway blocker PD98059, suggesting that ERK activation may be important for this process. In addition, protein kinase C (PKC) was shown to be required for tyrosine hydroxylase protein expression. The inhibition of PKC by staurosporin, as well as its downregulation, decreased the ability of bFGF+dbcAMP to generate tyrosine hydroxylase-positive cells. Moreover, the PKC activator phorbol 12-myristate 13-acetate (PMA) together with bFGF and dbcAMP led to a significant increase in phospho-ERK1/ERK2 levels, and the percentage of beta-tubulin III-positive cells that expressed tyrosine hydroxylase increased by 3.5-fold. PMA also promoted the phosphorylation of the cyclic AMP response element binding protein that might contribute to the increase in tyrosine hydroxylase-positive cells observed in bFGF+dbcAMP+PMA-treated cultures. From these results, we conclude that the manipulation in vitro of NSC from rat fetal striatum with bFGF, cyclic AMP analogs, and PKC activators promotes the generation of tyrosine hydroxylase-positive neurons.