Differentiation Of Neural Precursor Cells Research Articles

Unfolded protein response, activated by OASIS family transcription factors, promotes astrocyte differentiation

OASIS is a member of the CREB/ATF family of transcription factors and modulates cell- or tissue-specific unfolded protein response signalling. Here we show that this modulation has a critical role in the differentiation of neural precursor cells into astrocytes. Cerebral cortices of mice specifically deficient in OASIS (Oasis(-/-)) contain fewer astrocytes and more neural precursor cells than those of wild-type mice during embryonic development. Furthermore, astrocyte differentiation is delayed in primary cultured Oasis(-/-) neural precursor cells. The transcription factor Gcm1, which is necessary for astrocyte differentiation in Drosophila, is revealed to be a target of OASIS. Introduction of Gcm1 into Oasis(-/-) neural precursor cells improves the delayed differentiation of neural precursor cells into astrocytes by accelerating demethylation of the Gfap promoter. Gcm1 expression is temporally controlled by the unfolded protein response through interactions between OASIS family members during astrocyte differentiation. Taken together, our findings demonstrate a novel mechanism by which OASIS and its associated family members are modulated by the unfolded protein response to finely control astrocyte differentiation.

In Vitro Characterization of Trophic Factor Expression in Neural Precursor Cells

In cellular transplantation strategies for repairing the injured central nervous system, interactions between transplanted neural precursor cells (NPCs) and host tissue remain incompletely understood. Although trophins may contribute to the benefits observed, little research has explored this possibility. Candidate trophic factors were identified, and primers were designed for these genes. Template RNA was isolated from 3 NPC sources, and also from bone marrow stromal cells (BMSCs) and embryonic fibroblasts as comparative controls. Quantitative polymerase chain reaction was performed to determine the effect of cell source, passaging, cellular differentiation, and environmental changes on trophin factor expression in NPCs. Results were analyzed with multivariate statistical analyses. NPCs, BMSCs, and fibroblasts each expressed trophic factors in unique patterns. Trophic factor expression was similar among NPCs whether harvested from rat or mouse, brain or spinal cord, or their time in culture. The expression of neurotrophin NT-3, NT-4/5, glial-derived neurotrophic factor, and insulin-like growth factor-1 decreased with time in culture. Induced differentiation of NPCs led to a marked and statistically significant increase in the expression of trophic factors. Culture conditions and environmental changes were also associated with significant changes in trophin expression. These results suggest that trophins could contribute to the benefits associated with transplantation of NPCs as well as BMSCs. Trophic factor expression changes with NPC differentiation and environmental conditions, which could have important implications with regard to their behavior after in vivo transplantation.

Chemokines influence the migration and fate of neural precursor cells from the young adult and middle-aged rat subventricular zone

We have previously demonstrated a role for the chemokines MCP-1, MIP-1α and GRO-α in directing subventricular zone (SVZ)-derived neural precursor cell migration towards the site of cell death in the adult rodent brain. However the influence of chemokines such as MCP-1, MIP-1α and GRO-α on the differentiation of adult neural precursor cells has not previously been investigated. Further, as the majority of neurological disorders and injuries occur during ageing, it is important to investigate the effect of chemokines on adult neural precursor cell cultures obtained from the ageing brain. This study therefore examined the effect of MCP-1, MIP-1α and GRO-α on SVZ-derived neural precursor cell differentiation in vitro, and assessed whether precursor cells from the middle-aged rat brain (13months old) follow the same migratory and differential profile as neural precursor cells obtained from the young adult rat brain (2months old). We observed that each of the chemokines examined generated differing effects in regards to neuronal or glial differentiation. Further, both MIP-1α and GRO-α increased total cell number, suggesting an effect on precursor cell proliferation and/or survival. In agreement with cultures obtained from young adult brains, SVZ-derived neural precursor cells cultured from the middle-aged brain exhibited chemotactic migration in response to a concentration gradient. These results indicate that the chemokines MCP-1, MIP-1α and GRO-α can influence both the migration and fate choice of SVZ-derived neural precursor cells, as well as promoting cell viability. While a response to each of these chemokines is maintained in the middle-aged brain, a distinct age-related alteration in differential fate can be identified.

There is More to a Lipid than just Being a Fat: Sphingolipid-Guided Differentiation of Oligodendroglial Lineage from Embryonic Stem Cells

Dr. Robert K. Yu's research showed for the first time that the composition of glycosphingolipids is tightly regulated during embryo development. Studies in our group showed that the glycosphingolipid precursor ceramide is also critical for stem cell differentiation and apoptosis. Our new studies suggest that ceramide and its derivative, sphingosine-1-phosphate (S1P), act synergistically on embryonic stem (ES) cell differentiation. When using neural precursor cells (NPCs) derived from ES cells for transplantation, residual pluripotent stem (rPS) cells pose a significant risk of tumor formation after stem cell transplantation. We show here that rPS cells did not express the S1P receptor S1P1, which left them vulnerable to ceramide or ceramide analog (N-oleoyl serinol or S18)-induced apoptosis. In contrast, ES cell-derived NPCs expressed S1P1 and were protected in the presence of S1P or its pro-drug analog FTY720. Consistent with previous studies, FTY720-treated NPCs differentiated predominantly toward oligodendroglial lineage as tested by the expression of the oligodendrocyte precursor cell (OPC) markers Olig2 and O4. As the consequence, a combined administration of S18 and FTY720 to differentiating ES cells eliminated rPS cells and promoted oligodendroglial differentiation. In addition, we show that this combination promoted differentiation of ES cell-derived NPCs toward oligodendroglial lineage in vivo after transplantation into mouse brain.

Acute Hepatocyte Growth Factor Treatment Induces Long-Term Neuroprotection and Stroke Recovery via Mechanisms Involving Neural Precursor Cell Proliferation and Differentiation

Hepatocyte growth factor (HGF) is an interesting candidate for acute stroke treatment as shown by continuous infusion or gene delivery protocols. However, little is known about HGF-mediated long-term effects. The present study therefore analyzed long-term effects of an acute intrastriatal HGF treatment (5 μg) after a 45-minute stroke, with regard to brain injury and neurologic recovery. Hepatocyte growth factor induced long-term neuroprotection as assessed by infarct volume and neuronal cell death analysis for as long as 4 weeks after stroke, which was associated with sustained neurologic recovery as evidenced by corner-turn and tight-rope tests. Analyzing underlying mechanisms of HGF-induced sustained neuroprotection, enhanced cell proliferation followed by increased neuronal differentiation of neural precursor cells (NPCs) was observed in the ischemic striatum of HGF-treated mice, which persisted for up to 4 weeks. In line with this, HGF promoted neurosphere formation as well as proliferation of NPC and decreased caspase-3-dependent hypoxic injury in vitro. Preservation of blood-brain barrier integrity 24 hours after stroke was furthermore noticed in animals receiving HGF, which was associated with the inhibition of matrix metalloproteases (MMP)-2 and MMP-9 at 4 and 24 hours, respectively. We suggest that sustained recruitment of proliferating cells together with improved neurovascular remodeling provides an explanation for HGF-induced long-term neuroprotection.

Wogonin induces differentiation and neurite outgrowth of neural precursor cells

Wogonin is a flavonoid isolated from Scutellaria baicalensis root, and has multiple pharmacological effects, including anti-inflammatory, anti-oxidant, and anti-cancer effects. It is also neuroprotective in the brain under many stress conditions, but wogonin does not elevate neuronal cell survival. Thus, the mechanisms controlling the neuroprotective effect of wogonin are not clear. Neural precursor cells (NPCs), present in the hippocampus and subventricular zone of adult brains, replace damaged cells. In this study we investigated the biological functions underlying the neuroprotective effect of wogonin on NPCs. We initially examined survival of NPCs but found it was slightly reduced at concentrations higher than 2 μg/ml. When we explored differentiation of NPCs into neuronal cells, the number of differentiated cells expressing neurofilaments was increased remarkably (fourfold) in the hippocampal NPCs treated with wogonin. Wogonin maximally elevated the expressions of presynaptic protein, synapsin I and postsynaptic protein (PSD95) at a concentration of 0.7 μg/ml. Differentiated cells containing longer neurites were significantly increased in cortical NPCs, primarily cultured from rat E14 embryonic brain. Wogonin also promoted differentiation of NPCs into mature neurons in vivo. When transplanted into the adult rat hippocampus, NPCs differentiated into cells expressing NeuN, the mature neuron marker, by 4 weeks after transplantation. These data indicate that wogonin induces differentiation of NPCs both in culture and in vivo, and suggest that facilitation of NPC differentiation is a biological activity by which wogonin protects neurons in damaged brain.

Differential Regulation of Proliferation and Differentiation in Neural Precursor Cells by the Jak Pathway

Neuronal precursor cells (NPCs) are temporally regulated and have the ability to proliferate and differentiate into mature neurons, oligodendrocytes, and astrocytes in the presence of growth factors (GFs). In the present study, the role of the Jak pathway in brain development was investigated in NPCs derived from neurosphere cultures using Jak2 and Jak3 small interfering RNAs and specific inhibitors. Jak2 inhibition profoundly decreased NPC proliferation, preventing further differentiation into neurons and glial cells. However, Jak3 inhibition induced neuronal differentiation accompanied by neurite growth. This phenomenon was due to the Jak3 inhibition-mediated induction of neurogenin (Ngn)2 and NeuroD in NPCs. Jak3 inhibition induced NPCs to differentiate into scattered neurons and increased the expression of Tuj1, microtubule associated protein 2 (MAP2), Olig2, and neuroglial protein (NG)2, but decreased glial fibrillary acidic protein (GFAP) expression, with predominant neurogenesis/polydendrogenesis compared with astrogliogenesis. Therefore, Jak2 may be important for NPC proliferation and maintenance, whereas knocking-down of Jak3 signaling is essential for NPC differentiation into neurons and oligodendrocytes but does not lead to astrocyte differentiation. These results suggest that NPC proliferation and differentiation are differentially regulated by the Jak pathway.

Transplantation of TAT-Bcl-x L-transduced neural precursor cells: Long-term neuroprotection after stroke

Neural precursor cells (NPC) are an interesting tool in experimental stroke research, but their therapeutic potential is limited due to poor long-term survival. We therefore in vitro transduced subventricular zone-(SVZ)-derived NPC with the anti-apoptotic fusion protein TAT-Bcl-x L and analyzed NPC survival, differentiation, and post-stroke functional deficits after experimental ischemia in mice. Survival of TAT-Bcl-x L-transduced NPC, which were injected at day 7 post-stroke into the ischemic striatum, was significantly increased at 4 weeks after stroke. Increased survival of NPC was associated with reduced infarct injury and decreased post-stroke functional deficits. Animals grafted with TAT-Bcl-x L-transduced NPC showed an increased number of immature cells expressing the neuronal marker doublecortin. Since mature neuronal differentiation of NPC was not observed, reduced post-stroke injury cannot be attributed to enhanced neuronal regeneration, but rather to indirect by-stander effects of grafted NPC. In line with this, NPC-mediated neuroprotection of cortical neurons in vitro was associated with increased secretion of growth factors. Thus, in vitro transduction of cultivated NPC with TAT-Bcl-x L results in enhanced resistance of transplanted NPC followed by long-term neuroprotection and ameliorated functional deficits after transient focal cerebral ischemia in mice.

Time-dependent fate of transplanted neural precursor cells in experimental autoimmune encephalomyelitis mice

Transplanted Neural Precursor Cells (NPCs) are capable of long-distance migration inside the inflamed CNS, but exhibit limited myelinating capacities in animal models of Multiple Sclerosis (MS). Inflammation seems to be both beneficial for the recruitment and migration of NPCs and restrictive for their terminal differentiation. In the present study, a set of transplantation experiments was applied in order to investigate the migratory potential, the differentiation pattern and long-term survival of NPCs in Experimental Autoimmune Encephalomyelitis (EAE) mice, the animal model of MS. The in vitro differentiation potential of NPCs in the presence of either pro- (TNFa, INFγ) or anti- (TGFb) inflammatory cytokines was also analyzed. According to the in vivo results obtained, at the acute phase of EAE only a small fraction of transplanted NPCs succeed to differentiate, whereas at chronic phase most of them followed a differentiation process to glial cell lineage along white matter tracts. However, this differentiation was not fully completed, since 8months after their transplantation a number of NPCs remained as pre-oligodendrocytes. Glial differentiation of NPCs was also found to be inhibited or promoted following their treatment with TNFa or TGFb respectively, in vitro. Our findings suggest that inflammation triggers migration whereas the anti-inflammatory component is a prerequisite for NPCs to follow glial differentiation thereby providing myelinating oligodendrocytes. It is speculated that the fine balance between the pro- and anti-inflammatory determinants in the CNS may be a key factor for transplanted NPCs to exhibit a better therapeutic effect in EAE and MS. This article is part of a Special Issue entitled "Interaction between repair, disease, & inflammation."

Pro‐gliogenic effect of IL‐1α in the differentiation of embryonic neural precursor cells in vitro

Inflammation is regarded as a main obstacle to brain regeneration. Major detrimental effects are attributed to microglial/macrophagic products, such as TNF-alpha and interleukin (IL)-6. The role of cytokines of the IL-1 family, particularly of IL-1alpha, in the modulation of neural precursor cell (NPC) properties is less characterized. IL-1alpha is one of the most abundant cytokines released upon acute stimulation of microglia with lipopolysaccharide and is down-regulated upon chronic stimulation. As we recently demonstrated, acutely activated microglia reduces NPC survival, prevent neuronal differentiation and promote glial differentiation. Chronically activated microglia are instead permissive to NPC survival and neuronal differentiation, and less effective in promoting astrocytic differentiation. We thus investigated whether IL-1alpha could contribute to the effects of acutely activated microglia on NPC. We found that NPC express functional IL-1 receptors and that exposure to recombinant IL-1alpha strongly enhances NPC differentiation into astrocytes, without affecting cell viability and neuronal differentiation. In the same conditions, recombinant IL-1beta has pro-gliogenic effects at concentrations 10-fold higher than those found in activated microglial conditioned media. Interestingly, immunodepletion of IL-1alpha in activated microglial conditioned media fails to revert microglial pro-gliogenic action and slightly enhances neuronal differentiation, revealing that other microglial-derived factors contribute to the modulation of NPC properties.

Induction of Manganese Superoxide Dismutase by Nuclear Translocation and Activation of SIRT1 Promotes Cell Survival in Chronic Heart Failure

Oxidative stress plays a pivotal role in chronic heart failure. SIRT1, an NAD(+)-dependent histone/protein deacetylase, promotes cell survival under oxidative stress when it is expressed in the nucleus. However, adult cardiomyocytes predominantly express SIRT1 in the cytoplasm, and its function has not been elucidated. The purpose of this study was to investigate the functional role of SIRT1 in the heart and the potential use of SIRT1 in therapy for heart failure. We investigated the subcellular localization of SIRT1 in cardiomyocytes and its impact on cell survival. SIRT1 accumulated in the nucleus of cardiomyocytes in the failing hearts of TO-2 hamsters, postmyocardial infarction rats, and a dilated cardiomyopathy patient but not in control healthy hearts. Nuclear but not cytoplasmic SIRT1-induced manganese superoxide dismutase (Mn-SOD), which was further enhanced by resveratrol, and increased the resistance of C2C12 myoblasts to oxidative stress. Resveratrol's enhancement of Mn-SOD levels depended on the level of nuclear SIRT1, and it suppressed the cell death induced by antimycin A or angiotensin II. The cell-protective effects of nuclear SIRT1 or resveratrol were canceled by the Mn-SOD small interfering RNA or SIRT1 small interfering RNA. The oral administration of resveratrol to TO-2 hamsters increased Mn-SOD levels in cardiomyocytes, suppressed fibrosis, preserved cardiac function, and significantly improved survival. Thus, Mn-SOD induced by resveratrol via nuclear SIRT1 reduced oxidative stress and participated in cardiomyocyte protection. SIRT1 activators such as resveratrol could be novel therapeutic tools for the treatment of chronic heart failure.

Calcium-Permeable AMPA Receptors Mediate Glutamatergic Signaling in Neural Precursor Cells of the Postnatal Olfactory Bulb

Neural precursor cells (NPCs) in the mammalian olfactory bulb give rise to local inhibitory neurons that integrate into existing circuitry throughout adult life. However, the functional properties of neurotransmitter receptors expressed by NPCs are not well understood. In this study, we use patch-clamp recording and calcium imaging to explore the properties of glutamate receptors expressed by NPCs in the olfactory bulb subependymal layer. We find that calcium-permeable AMPA receptors (AMPARs) are the major receptor type underlying glutamatergic signaling in olfactory bulb NPCs. We also show that when transmitter uptake is reduced, glutamate spillover from distant nerve terminals in the olfactory bulb can activate nonsynaptic NPC AMPARs and generate increases in intracellular calcium. Together, these results suggest that Ca(2+) influx via AMPARs may contribute to calcium-dependent processes that govern NPC differentiation and maturation.

Human cytomegalovirus infection inhibits the differentiation of human hippocampus neural precursor cells into astrocytes

HCMV is a major cause of congenital brain disease in humans, and its neuropathogenesis is not yet fully understood. The objective of the present study is to investigate the effect of human cytomegalovirus (HCMV) infection on human hippocampus neural precursor cell (NPCs) differentiation in vitro. Fetal hippocampus tissue was dissociated mechanically and then cultured in proliferation medium with EGF and bFGF. The identification and purity of the NPCs were confirmed by using immunofluorescence to detect the expression of the NPCs marker-Nestin. To drive NPCs differentiation, bFGF and EGF were withdrawn from the medium and replaced with FBS (10%). HCMV AD169 (MOI=5) was added into the differentiation medium at the onset of the differentiation. After 7 days of differentiation, in order to confirm whether NPCs are permissive for HCMV infection, immunofluorescence was used to stain for the presence of immediate early (IE) and late (pp65) HCMV proteins in the infected cells. The effects of HCMV infection on NPCs’ differentiation was observed by detecting the ratio of nestin and GFAP positive cells with confocal microscopy and immunofluorescence. The data showed that 95%±8% of the cells (passage 4-8) cultured were Nestin positive which suggested that majority of the cells were NPCs. On day 7 postinfection, most of the infected cells were IE and PP65 positive. The percentage of Nestin-positive cells were 93%±10% and 50%±19% (t=6.03, p<0.01) and those of GFAP-positive cells were 55±17% and 81%±11% (t=3.77, p<0.01) in HCMV treated and control groups respectively. These findings indicate that NPCs are HCMV permissive cells and HCMV (AD 169) infection suppresses the differentiation of Hippocampus-genetic human NPCs into astrocytes. These effects may provide part of the explanation for the abnormalities in brain development associated with congenital HCMV infection.

Matrigel supports survival and neuronal differentiation of grafted embryonic stem cell‐derived neural precursor cells

Cell replacement therapy holds great promise as a means of treating neurological disorders, including Parkinson's disease. However, one of the major obstacles to the success of this treatment is the low survival rate of grafted cells, which probably results from mechanical damage, acute inflammation, and immunological rejection. To overcome this problem, we investigated the effect of different types of extracellular matrix (ECM) on the survival and differentiation of embryonic stem (ES) cell-derived neural precursor cells (NPCs). We tested materials from natural sources, including collagen, ornithine/laminin, and growth factor-reduced Matrigel (gfrMG), as well as the synthetic biomaterial PuraMatrix, which consists of self-assembling polypeptides. GfrMG efficiently supported cell survival, migration, and neurite outgrowth in vitro and promoted proliferation of grafted cells in vivo, resulting in larger graft volume and an increase in the number of TH-positive dopaminergic neurons in the graft. GfrMG did not induce dopaminergic differentiation directly; rather, it reduced the invasion of pan-leukocytic CD45-positive cells into the graft. Insofar as the inflammatory or immune response in the host brain inhibits neuronal differentiation of grafted NPCs, gfrMG may increase the number of TH-positive cells by suppressing this effect. Thus, gfrMG appears to provide a suitable scaffold that supports survival and differentiation of NPCs. However, because it is derived from mouse sarcomas, a human-derived matrix or synthetic biomaterial must be developed for clinical applications.

Basic mechanisms of MCD in animal models

Epilepsy-associated glioneuronal malformations (malformations of cortical development [MCD]) include focal cortical dysplasias (FCD) and highly differentiated glioneuronal tumors, most frequently gangliogliomas. The neuropathological findings are variable but suggest aberrant proliferation, migration, and differentiation of neural precursor cells as essential pathogenetic elements. Recent advances in animal models for MCDs allow new insights in the molecular pathogenesis of these epilepsy-associated lesions. Novel approaches, presented here, comprise RNA interference strategies to generate and study experimental models of subcortical band heterotopia and study functional aspects of aberrantly shaped and positioned neurons. Exciting analyses address impaired NMDA receptor expression in FCD animal models compared to human FCDs and excitatory imbalances in MCD animal models such as lissencephaly gene ablated mice as well as in utero irradiated rats. An improved understanding of relevant pathomechanisms will advance the development of targeted treatment strategies for epilepsy-associated malformations.

Effects of FGF-2 and EGF removal on the differentiationof mouse neural precursor cells

Cell therapy for neurological disorders has advanced, and neural precursor cells (NPC) may become the ideal candidates for neural transplantation in a wide range of diseases. However, additional work has to be done to determine either the ideal culture environment for NPC expansion in vitro, without altering their plasticity, or the FGF-2 and EGF mechanisms of cell signaling in neurospheres growth, survival and differentiation. In this work we evaluated mouse neurospheres cultured with and without FGF-2 and EGF containing medium and showed that those growth factors are responsible for NPC proliferation. It is also demonstrated that endogenous production of growth factors shifts from FGF-2 to IGF-1/PDGFb upon EGF and FGF-2 withdrawal. Mouse NPC cultured in suspension showed different patterns of neuronal localization (core versus shell) for both EGF and FGF-2 withdrawal and control groups. Taken together, these results show that EGF and FGF-2 removal play an important role in NPC differentiation and may contribute to a better understanding of mechanisms of NPC differentiation. Our findings suggest that depriving NPC of growth factors prior to grafting might enhance their chance to effectively integrate into the host.

Reelin affects chain-migration and differentiation of neural precursor cells

In the adult mammalian brain, multipotential neural stem cells (NSC) persist throughout life in areas where neurogenesis is maintained. A distinctive trait of NSCs growing in vitro as neurospheres (NS), is their ability to self-renew, differentiate and migrate to sites of injury, such as gliomas. We have studied the role of Reelin, an extracellular matrix protein involved in brain development, in NSCs derived from normal newborn mice or from reeler, a natural mutant in which Reelin is not expressed. We show that the absence of Reelin negatively affects proliferation, NS-forming ability, and neuronal differentiation. Reeler NSCs are unable to migrate in chains, a migration mode typical of neural precursors homing to injury sites in adult CNS. All these effects are partially rescued by ectopic Reelin supplementation. Finally, we show that reeler NSCs fail to migrate in vivo towards gliomas. Overall, our results indicate that Reelin affects all major features of postnatal NSCs, and that it is required for the proper homing of NSCs to tumor sites in adult brain.

Selective induction of neocortical GABAergic neurons by the PDK1-Akt pathway through activation of Mash1

Extracellular stimuli regulate neuronal differentiation and subtype specification during brain development, although the intracellular signaling pathways that mediate these processes remain largely unclear. We now show that the PDK1-Akt pathway regulates differentiation of telencephalic neural precursor cells (NPCs). Active Akt promotes differentiation of NPC into gamma-aminobutyric acid-containing (GABAergic) but not glutamatergic neurons. Disruption of the Pdk1 gene or expression of dominant-negative forms of Akt suppresses insulin-like growth factor (IGF)-1 enhancement of NPC differentiation into neurons in vitro and production of neocortical GABAergic neurons in vivo. Furthermore, active Akt increased the protein levels and transactivation activity of Mash1, a proneural basic helix-loop-helix protein required for the generation of neocortical GABAergic neurons, and Mash1 was required for Akt-induced neuronal differentiation. These results have unveiled an unexpected role of the PDK1-Akt pathway: a key mediator of extracellular signals regulating the production of neocortical GABAergic neurons.

Differential Processing of Amyloid-β Precursor Protein Directs Human Embryonic Stem Cell Proliferation and Differentiation into Neuronal Precursor Cells

The amyloid-beta precursor protein (AbetaPP) is a ubiquitously expressed transmembrane protein whose cleavage product, the amyloid-beta (Abeta) protein, is deposited in amyloid plaques in neurodegenerative conditions such as Alzheimer disease, Down syndrome, and head injury. We recently reported that this protein, normally associated with neurodegenerative conditions, is expressed by human embryonic stem cells (hESCs). We now report that the differential processing of AbetaPP via secretase enzymes regulates the proliferation and differentiation of hESCs. hESCs endogenously produce amyloid-beta, which when added exogenously in soluble and fibrillar forms but not oligomeric forms markedly increased hESC proliferation. The inhibition of AbetaPP cleavage by beta-secretase inhibitors significantly suppressed hESC proliferation and promoted nestin expression, an early marker of neural precursor cell (NPC) formation. The induction of NPC differentiation via the non-amyloidogenic pathway was confirmed by the addition of secreted AbetaPPalpha, which suppressed hESC proliferation and promoted the formation of NPCs. Together these data suggest that differential processing of AbetaPP is normally required for embryonic neurogenesis.

Astrocyte Differentiation of Neural Precursor Cells is Enhanced by Retinoic Acid Through a Change in Epigenetic Modification

Neurons, astrocytes, and oligodendrocytes-the three major cell types that comprise the central nervous system-are generated from common multipotent neural precursor cells (NPCs). Members of the interleukin-6 family of cytokines, including leukemia inhibitory factor (LIF), induce astrocyte differentiation of NPCs by activating the transcription factor signal transducer and activator of transcription 3 (STAT3). We show here that retinoic acid (RA) facilitates LIF-induced astrocyte differentiation of NPCs. RA and LIF synergistically activate the promoter of gfap, which encodes the astrocytic marker glial fibrillary acidic protein, and a putative RA response element in the promoter was found to be critical for this activation. Histone H3 acetylation around the STAT-binding site in the gfap promoter was increased in NPCs treated with RA, allowing STAT3 to gain access to the promoter more efficiently. These results suggest that RA acts in concert with LIF to induce astrocyte differentiation of NPCs through an epigenetic mechanism that involves cross-talk between distinct signaling pathways.