Dividing Olig2-expressing progenitor cells derived from ES cells

G-Olig2 is a knock-in ES cell line with GFP inserted into the Olig2 gene so that ES cell-derived neural cells that express Olig2 also express GFP. This tool allows visualization of the subset of cells that differentiate along the Olig2-expressing pathway. By manipulating culture conditions, it is possible to induce Olig2 expression in rapidly dividing cells. These cells have many of the features of glial progenitor cells but, unlike other glial progenitors, are able to divide rapidly for at least 1 month while still expressing Olig2. Even after 1-month expansion, the cells differentiate readily into astrocyte-like and oligodendrocyte-like cells when switched to serum-containing medium. Cellular memory is the property whereby cells remain specified to a particular lineage or pathway while undergoing division. ES cell-derived neural cells show cellular memory for a glial progenitor phenotype and thus provide a new and tractable model for this basic feature of neural development.     

Region-specific differentiation of embryonic stem cell-derived neural progenitor transplants into the adult mouse hippocampus following seizures

Embryonic stem (ES) cells can generate neural progenitors and neurons in vitro and incorporate into the adult central nervous system (CNS) following transplantation, suggesting their therapeutic potential for treating neurological disorders. However, our understanding of the conditions that direct ES-derived neural progenitor (ESNP) migration and differentiation within different regions of the adult CNS is incomplete. Rodents treated with the chemoconvulsant kainic acid (KA) experience seizures and display hippocampal sclerosis, as well as enhanced hippocampal neurogenesis, similar to pathological findings in patients with temporal lobe epilepsy (TLE). To examine the potential for ESNPs to incorporate into the adult hippocampus and differentiate into hippocampal neurons or glia following seizure-induced damage, we compared the fates of ESNPs after they were transplanted into the CA3 region or fimbria 1 week following KA-induced seizures. After 4-8 weeks, ESNPs grafted into the CA3 region had migrated to the dentate gyrus (DG), where a small subset adopted neural stem cell fates and continued to proliferate, based on bromodeoxyuridine uptake. Others differentiated into neuroblasts or dentate granule neurons. In contrast, most ESNPs transplanted into the fimbria migrated extensively along existing fiber tracts and differentiated into oligodendrocytes or astrocytes. Hippocampal grafts in mice not subjected to seizures displayed a marked tendency to form tumors, and this effect was more pronounced in the DG than in the fimbria. Taken together, these data suggest that seizures induce molecular changes in the CA3 region and DG that promote region-specific neural differentiation and suppress tumor formation.     

Myelinating cocultures of rodent stem cell line‐derived neurons and immortalized Schwann cells

Myelination is one of the most remarkable biological events in the neuron–glia interactions for the development of the mammalian nervous system. To elucidate molecular mechanisms of cell‐to‐cell interactions in myelin synthesis in vitro, establishment of the myelinating system in cocultures of continuous neuronal and glial cell lines are desirable. In the present study, we performed co‐culture experiments using rat neural stem cell‐derived neurons or mouse embryonic stem (ES) cell‐derived motoneurons with immortalized rat IFRS1 Schwann cells to establish myelinating cultures between these cell lines. Differentiated neurons derived from an adult rat neural stem cell line 1464R or motoneurons derived from a mouse ES cell line NCH4.3, were mixed with IFRS1 Schwann cells, plated, and maintained in serum‐free F12 medium with B27 supplement, ascorbic acid, and glial cell line‐derived neurotrophic factor. Myelin formation was demonstrated by electron microscopy at 4 weeks in cocultures of 1464R‐derived neurons or NCH4.3‐derived motoneurons with IFRS1 Schwann cells. These in vitro coculture systems utilizing the rodent stable stem and Schwann cell lines can be useful in studies of peripheral nerve development and regeneration.     

Differentiation and functional incorporation of embryonic stem cell-derived GABAergic interneurons in the dentate gyrus of mice with temporal lobe epilepsy

Cell therapies for neurological disorders require an extensive knowledge of disease-associated neuropathology and procedures for generating neurons for transplantation. In many patients with severe acquired temporal lobe epilepsy (TLE), the dentate gyrus exhibits sclerosis and GABAergic interneuron degeneration. Mounting evidence suggests that therapeutic benefits can be obtained by transplanting fetal GABAergic progenitors into the dentate gyrus in rodents with TLE, but the scarcity of human fetal cells limits applicability in patient populations. In contrast, virtually limitless quantities of neural progenitors can be obtained from embryonic stem (ES) cells. ES cell-based therapies for neurological repair in TLE require evidence that the transplanted neurons integrate functionally and replace cell types that degenerate. To address these issues, we transplanted mouse ES cell-derived neural progenitors (ESNPs) with ventral forebrain identities into the hilus of the dentate gyrus of mice with TLE and evaluated graft differentiation, mossy fiber sprouting, cellular morphology, and electrophysiological properties of the transplanted neurons. In addition, we compared electrophysiological properties of the transplanted neurons with endogenous hilar interneurons in mice without TLE. The majority of transplanted ESNPs differentiated into GABAergic interneuron subtypes expressing calcium-binding proteins parvalbumin, calbindin, or calretinin. Global suppression of mossy fiber sprouting was not observed; however, ESNP-derived neurons formed dense axonal arborizations in the inner molecular layer and throughout the hilus. Whole-cell hippocampal slice electrophysiological recordings and morphological analyses of the transplanted neurons identified five basic types; most with strong after-hyperpolarizations and smooth or sparsely spiny dendritic morphologies resembling endogenous hippocampal interneurons. Moreover, intracellular recordings of spontaneous EPSCs indicated that the new cells functionally integrate into epileptic hippocampal circuitry.     

Transplantation of neural cells derived from retinoic acid-treated cynomolgus monkey embryonic stem cells successfully improved motor function of hemiplegic mice with experimental brain injury

We induced neural cells by treating cynomolgus monkey embryonic stem (ES) cells with retinoic acid. The treated cells mainly expressed betaIIItubulin. They further differentiated into neurons expressing neurofilament middle chain (NFM) in elongated axons. Half of the cells differentiated into Islet1+ motoneurons in vitro. The monkey ES-derived neural cells were transplanted to hemiplegic mice with experimental brain injury mimicking stroke. The neural cells that had grafted into periventricular area of the mice distributed extensively over the injured cortex. Some of the transplanted cells expressed the neural stem/progenitor marker nestin 2 days after transplantation. The cells expressed markers characteristic of mature motoneurons 28 days after transplantation. Mice with the neural cell graft gradually recovered motor function, whereas control animals remained hemiplegic. This is the first demonstration that neural cells derived from nonhuman primate ES cells have the ability to restore motor function in an animal model of brain injury.     

Stable expression of a neuronal dopaminergic progenitor phenotype in cell lines derived from human amniotic fluid cells

Cells from human amniotic fluid derived from the fetus are considered a source of multipotent cells. Their properties have not been fully exploited, partially because unlike other embryonic sources such as embryonic stem (ES) cells, cell lines from amniocentesis samples have not been generated. We have established and characterized the properties of eight individual cell lines. Flow cytometry using several cell surface markers showed that all cell lines generated consisted of homogeneous populations that lack HLAII antigenicity. Using a combination of immunocytochemistry, Western blotting, and RT-PCR, we found weak expression of Oct4 and nestin and strong expression of tubulin-betaIII, MAP2, and tau. Specific markers for cholinergic, (nor)adrenergic, and GABAergic neurons or glia were weakly expressed or absent, whereas expression of factors implicated in early induction of dopaminergic neurons, TGF-beta3 and beta-catenin were present. Further analysis showed strong expression of EN-1, c-RET, PTX3, and NURR1 essential for induction and survival of midbrain dopaminergic neurons, TH, AADC, and VMAT2 components of dopamine synthesis and secretion, and syntaxin1A and SNAP-25 necessary for neurotransmitter exocytosis. This phenotype was retained throughout passages and up to the current passage 36. Expression of neuronal and dopaminergic markers in individual AF cell lines was comparable to expression in neurons induced from ES cells and in IMR-32 and SH-SY5Y neuroblastomas. Our data show that cell lines can be derived from subcultures of amniocentesis, and are primarily composed of a population of progenitors with a phenotype similar to that of committed mesencephalic dopaminergic neurons.     

Intraventricular transplantation of embryonic stem cell-derived neural stem cells in intracerebral hemorrhage rats

In the present study, we attempted to explore cell transplantation therapy for intracerebral hemorrhage (ICH) using embryonic stem (ES) cells. Collagenase-induced ICH rats were used as model animals. Mouse ES cells were differentiated into nestin-positive neural stem cells in vitro by alltrans retinoic acid (ATRA). ATRA-treated ES cells (10(5)) were transplanted into the lateral ventricle in the hemisphere contralateral to the hemorrhage 7 days after collagenase infusion. Twenty-eight days after transplantation, ES-derived neurons and astrocytes were observed around the hematoma cavities of the brain in all of the ten rats receiving grafts. Graft-derived neurons were found in the subependymal area of the lateral ventricle as cellular nodules. Although one of the ten rats receiving grafts showed uncontrolled growth of astroglia derived from the ES cells, intraventricular transplantation of ATRA-treated ES cells is an effective delivery system of neuronal lineage-committed progenitor cells toward the site of ICH.     

QKI expression is regulated during neuron-glial cell fate decisions

QKI proteins are expressed by differentiated glia and have been implicated as regulators of myelination, but are also thought to function during early neural development. This study shows that QKI proteins are expressed in neural progenitors of the ventricular zone (vz) during murine CNS development, but that their expression is down-regulated during neuronal differentiation. By contrast, neural progenitors located in specific subdomains of the vz maintain expression of QKI proteins as they differentiate and migrate away into the emerging nervous system. These QKI⁺ cells have characteristics consistent with the acquisition of a glial rather than neuronal fate; they express nestin, incorporate BrdU, fail to express neuronal markers, and similar QKI⁺ cells are found in the postnatal subventricular zone, a known area of gliogenesis. In vitro, neural progenitor cells also down-regulate QKI expression as they differentiate into neurons, but not if they differentiate into glia. Furthermore, neural progenitors in strictly delineated subdomains of the vz dramatically up-regulate expression of the QKI-5 isoform prior to the emergence of QKI⁺ cells from these regions. Taken together, these data indicate that (1) glia are generated from subsets of neural progenitors found in specific, identifiable subdomains of the vz (2) QKI expression is regulated as neural progenitors undergo the neuron-glial cell fate decision and (3) QKI expression is a characteristic of glial progenitors. J. Neurosci. Res. 54:46–57, 1998. © 1998 Wiley-Liss, Inc.     

Isolation and characterization of neural precursor cells from the Sox1-GFP reporter mouse

We have made use of a reporter mouse line in which enhanced green fluorescence protein (GFP) is inserted into the Sox1 locus. We show that the GFP reporter is coexpressed with the Sox1 protein as well as with other known markers for neural stem and progenitor cells, and can be used to identify and isolate these cells by fluorescence-activated cell sorting (FACS) from the developing or adult brain and from neurosphere cultures. All neurosphere-forming cells with the capacity for multipotency and self-renewal reside in the Sox1-GFP-expressing population. Thus, the Sox1-GFP reporter system is highly useful for identification, isolation and characterization of neural stem and progenitor cells, as well as for the validation of alternative means for isolating neural stem and progenitor cells. Further, transplantation experiments show that Sox1-GFP cells isolated from the foetal brain give rise to neurons and glia in vivo, and that many of the neurons display phenotypic characteristics appropriate for the developing brain region from which the Sox1-GFP precursors were derived. On the other hand, Sox1-GFP cells isolated from the adult subventricular zone or expanded neurosphere cultures gave rise almost exclusively to glial cells following transplantation. Thus, not all Sox1-GFP cells possess the same capacity for neuronal differentiation in vivo.     

Primate embryonic stem cell-derived neuronal progenitors transplanted into ischemic brain.

Transplantation of stem cells has the possibility of restoring neural functions after stroke damage. Therefore, we transplanted neuronal progenitors generated from monkey embryonic stem (ES) cells into the ischemic mouse brain to test this possibility. Monkey ES cells were caused to differentiate into neuronal progenitors by the stromal cell-derived inducing activity method. Focal cerebral ischemia was induced by occluding the middle cerebral artery by the intraluminal filament technique. The donor cells were transplanted into the ischemic lateral striatum at 24 h after the start of reperfusion. The cells transplanted into the ischemic brain became located widely around the ischemic area, and, moreover, the transplanted cells differentiated into various types of neurons and glial cells. Furthermore, at 28 days after the transplantation, over 10 times more cells in the graft were labeled with Fluorogold (FG) by stereotactic focal injection of FG into the anterior thalamus and substantia nigra on the grafted side when compared with the number at 14 days. From these results we confirmed the survival and differentiation of, as well as network formation by, monkey ES-cell-derived neuronal progenitors transplanted into the ischemic mouse brain.     

Identification of self-replicating multipotent progenitors in the embryonic nervous system by high Notch activity and Hes5 expression

Discrimination of neural stem cells from other progenitors in the developing mammalian brain has been hampered by the lack of specific markers. Identifying the progenitor pools and signalling pathways that guide mammalian neurogenesis are central to understanding the complex mechanisms that govern development of the nervous system. Notch signalling plays a pivotal role in the development of the mammalian nervous system by maintaining multipotent neural stem cells and regulating their fate. In order to identify putative neural stem cells in situ, we generated transgenic mice that express Green Fluorescent Protein (GFP) and report Notch signalling activity in the developing CNS. Here we show the subdivision of progenitors within the neural tube of these mice. We purify progenitors from the neural tube and show that cells with the highest levels of Notch-reporter activity have self-renewal capability and multipotency, whereas those lacking Hes5 expression do not form neurospheres in vitro. Using marker protein co-expression and cell sorting, we show that both neuroepithelial cells as well as some radial glia at all axial levels of the embryonic neural tube display active Notch signalling. However, Tbr2-positive basal progenitors of the developing telencephalon and differentiating Islet1/2- and Lim1-positive motor neurons outside the ventricular zone do not express Hes5-GFP. Quantitative analysis showed that Hes5 expression correlates better with neural stem cell potential than expression of the related gene Hes1. Thus, Notch activity through Hes5 identifies multipotent progenitors with stem cell properties and subdivides the different progenitors into defined pools.     

Distinct efficacy of pre-differentiated versus intact fetal mesencephalon-derived human neural progenitor cells in alleviating rat model of Parkinson’s disease

Neural progenitor cells have shown the effectiveness in the treatment of Parkinson's disease, but the therapeutic efficacy remains variable. One of important factors that determine the efficacy is the necessity of pre-differentiation of progenitor cells into dopaminergic neurons before transplantation. This study therefore investigated the therapeutic efficacy of mesencephalon-derived human neural progenitor cells with or without the pre-differentiation in alleviating a rat model of Parkinson's disease. We found that a combination of 50 ng/ml fibroblast growth factor 8, 10 ng/ml glial cell line-derived neurotrophic factor and 10 microM forskolin facilitated the differentiation of human fetal mesencephalic progenitor cells into dopaminergic neurons in vitro. More importantly, after transplanted into the striatum of parkinsonian rats, only pre-differentiated grafts resulted in an elevated production of dopamine in the transplanted site and the amelioration of behavioral impairments of the parkinsonian rats. Unlike pre-differentiated progenitors, grafted intact progenitors rarely differentiated into dopaminergic neurons in vivo and emigrated actively away from the transplanted site. These data demonstrates the importance of pre-differentiation of human progenitor cells before transplantation in enhancing therapeutic potency for Parkinson's disease.     

Context-dependent neuronal differentiation and germ layer induction of Smad4-/- and Cripto-/- embryonic stem cells

Activation of transforming growth factor-beta (TGF-beta) receptors typically elicits mesodermal development, whereas inhibition of this pathway induces neural fates. In vitro differentiated mouse embryonic stem (ES) cells with deletion of the TGF-beta pathway-related factors Smad4 or Cripto exhibited increased numbers of neurons. Cripto-/- ES cells developed into neuroecto-/epidermal cell types, while Smad4-/- cells also displayed mesodermal differentiation. ES cell differentiation into catecholaminergic neurons showed that these ES cells retained their ability to develop into dopaminergic and serotonergic neurons with typical expression patterns of midbrain and hindbrain genes. In vivo, transplanted ES cells to the mouse striatum became small neuronal grafts, or large grafts with cell types from all germ layers independent of their ES cell genotype. This demonstrates that Smad4-/- and Cripto-/- ES cells favor a neural fate in vitro, but also express the mesodermal phenotype, implying that deletion of either Smad4 or Cripto is not sufficient to block nonneuronal tissue formation.     

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.     

Quiescent neural cells regain multipotent stem cell characteristics influenced by adult neural stem cells in co-culture

The source of cells participating in central nervous system (CNS) tissue repair and regeneration is poorly defined. One possible source is quiescent neural cells that can persist in CNS in the form of dormant progenitors or highly specialized cell types. Under appropriate conditions, these quiescent cells may be capable of re-entering the mitotic cell cycle and contributing to the stem cell pool. The aim of this study was to determine whether in vitro differentiated neural stem cells (NSC) can regain their multipotent-like stem cell characteristics in co-culture with NSC. To this end, we induced neural differentiation by plating NSC, derived from the periventricular subependymal zone (SEZ) of ROSA26 transgenic mice in Neurobasal A/B27 medium in the absence of bFGF. Under these conditions, NSC differentiated into neurons, glia, and oligodendrocytes. While the level of Nestin expression was downregulated, persistence of dormant progenitors could not be ruled out. However, further addition of bFGF or bFGF/EGF with conditioned medium derived from adult NSC did not induce any noticeable cell proliferation. In another experiment, differentiated neural cells were cultured with adult NSC, isolated from the hippocampus of Balb/c mice, in the presence bFGF. This resulted in proliferating colonies of ROSA26 derived cells that mimicked NSC in their morphology, growth kinetics, and expressed NSC marker proteins. The average nuclear area and DAPI fluorescence intensity of these cells were similar to that of NSC grown alone. We conclude that reactivation of quiescent neural cells can be initiated by NSC-associated short-range cues but not by cell fusion.     

Culture method for the induction of neurospheres from mouse embryonic stem cells by coculture with PA6 stromal cells

Embryonic stem (ES) cells proliferate and maintain their pluripotency for over 1 year in vitro and may therefore provide a sufficient source for cell therapies. However, most of the previously reported methods for obtaining a source for cell therapies have not been simple. We describe here a novel method for induction of neurospheres from mouse ES cells by coculturing on PA6 cells instead of the formation of embryoid bodies. The ES cells cocultured with the PA6 stromal cell line for at least 3 days were capable of differentiating into spheres. The cells in the spheres were all green fluorescent protein (GFP) positive, showing that they were derived from GFP-expressing D3-ES cells. The spheres contained nestin-positive cells. The number of spheres increased when they were cocultured with PA6 for a longer period. Sphere formation was observed even after 10 mechanical dissociations and subculturings, showing its self-renewal ability. The cells differentiated into microtubule-associated protein-2 (MAP2)-positive neuronal cells and glial fibrillary acidic protein (GFAP)-positive glial cells. gamma-Aminobutyric acid-positive cells and tyrosine hydroxylase-positive cells were also observed in the spheres. The percentages of the MAP2- or GFAP-positive cells in the sphere changed according to the period of coculture on PA6 cells. At an early stage of coculture, more neurons were generated and, at a later period, more glial cells were generated. These results suggested that neurosphere could be generated from ES cells by coculturing with PA6, and that these cells resembled neural stem cells derived from mouse fetal brain tissue.     

Long‐term fate of grafted hippocampal neural progenitor cells following ischemic injury

The adult CNS has a very limited capacity to regenerate neurons after insult. To overcome this limitation, the transplantation of neural progenitor cells (NPCs) has developed into a key strategy for neuronal replacement. This study assesses the long‐term survival, migration, differentiation, and functional outcome of NPCs transplanted into the ischemic murine brain. Hippocampal neural progenitors were isolated from FVB‐Cg‐Tg(GFPU)5Nagy/J transgenic mice expressing green fluorescent protein (GFP). Syngeneic GFP‐positive NPCs were stereotactically transplanted into the hippocampus of FVB mice following a transient global cerebral ischemia model. Behavioral tests revealed that ischemia/reperfusion induced spatial learning disturbances in the experimental animals. The NPC transplantation promoted cognitive function recovery after ischemic injury. To study the long‐term fate of grafted GFP‐positive NPCs in a host brain, immunohistochemical approaches were applied. Confocal microscopy revealed that grafted cells survived in the recipient tissue for 90 days following transplantation and differentiated into mature neurons with extensive dendritic trees and apparent spines. Immunoelectron microscopy confirmed the formation of synapses between the transplanted GFP‐positive cells and host neurons that may be one of the factors underlying cognitive function recovery. Repair and functional recovery following brain damage represent a major challenge for current clinical and basic research. Our results provide insight into the therapeutic potential of transplanted hippocampal progenitor cells following ischemic brain injury. © 2014 Wiley Periodicals, Inc.