Isolation of neuronal precursors from differentiating P19 embryonal carcinoma cells by neuronal T alpha 1-promoter-driven GFP.

The induction of pluripotent P19 embryonal carcinoma (EC) cells with retinoic acid results in their differentiation into cells that resemble neurons, glia, and fibroblasts. To isolate and enrich the developing neurons from heterogeneously differentiating P19 EC cells, we used a recently introduced protocol combining the expression of green fluorescent protein (GFP) driven by a tissue-specific promoter and fluorescence-activated cell sorting. Cells were transfected with the gene for GFP, which is under the control of the neuronal T alpha 1 tubulin promoter. After four days of retinoic acid treatment, GFP was specifically detected in cells undergoing neuronal differentiation. Sorting of fluorescent differentiating P19 EC transfectants yielded populations highly enriched in neuronal precursors and neurons. Immunoreactivity for nestin and neurofilament was observed in 80 and 25% of the sorted cell population, respectively. These results demonstrate that differentiated neuronal precursor cells can be efficiently isolated from differentiating pluripotent embryonic cells in vitro, suggesting that this method can reproducibly provide homogeneous materials for further studies on neurogenesis.     

CNS stem and progenitor cell differentiation into functional neuronal circuits in three-dimensional collagen gels

The mammalian central nervous system (CNS) has little capacity for self-repair after injury, and neurons are not capable of proliferating. Therefore, neural tissue engineering that combines neural stem and progenitor cells and biologically derived polymer scaffolds may revolutionize the medical approach to the treatment of damaged CNS tissues. Neural stem and progenitor cells isolated from embryonic rat cortical or subcortical neuroepithelium were dispersed within type I collagen, and the cell-collagen constructs were cultured in serum-free medium containing basic fibroblast growth factor. The collagen-entrapped stem and progenitors actively expanded and efficiently generated neurons, which developed neuronal polarity, neurotransmitters, ion channels/receptors, and excitability. Ca2+ imaging showed that differentiation from BrdU+/TuJ1- to BrdU-/TuJ1+ cells was accompanied by a shift in expression of functional receptors for neurotransmitters from cholinergic and purinergic to predominantly GABAergic and glutamatergic. Spontaneous postsynaptic currents were recorded by patch-clamping from precursor cell-derived neurons and these currents were partially blocked by 10-microM bicuculline, and completely blocked by additional 10 microM of the kainate receptor antagonist CNQX, indicating an appearance of both GABAergic and glutamatergic synaptic activities. Staining with endocytotic marker FM1-43 demonstrated active synaptic vesicle recycling occurring among collagen-entrapped neurons. These results show that neural stem and progenitor cells cultured in 3D collagen gels recapitulate CNS stem cell development; this is the first demonstration of CNS stem and progenitor cell-derived functional synapse and neuronal network formation in a 3D matrix. The proliferative capacity and neuronal differentiating potential of neural progenitors in 3D collagen gels suggest their potential use in attempts to promote neuronal regeneration in vivo.     

Isolation of neuronal precursors from differentiating P19 embryonal carcinoma cells by neuronal Tα1-promoter-driven GFP

The induction of pluripotent P19 embryonal carcinoma (EC) cells with retinoic acid results in their differentiation into cells that resemble neurons, glia, and fibroblasts. To isolate and enrich the developing neurons from heterogeneously differentiating P19 EC cells, we used a recently introduced protocol combining the expression of green fluorescent protein (GFP) driven by a tissue-specific promoter and fluorescence-activated cell sorting. Cells were transfected with the gene for GFP, which is under the control of the neuronal Tα1 tubulin promoter. After four days of retinoic acid treatment, GFP was specifically detected in cells undergoing neuronal differentiation. Sorting of fluorescent differentiating P19 EC transfectants yielded populations highly enriched in neuronal precursors and neurons. Immunoreactivity for nestin and neurofilament was observed in 80 and 25% of the sorted cell population, respectively. These results demonstrate that differentiated neuronal precursor cells can be efficiently isolated from differentiating pluripotent embryonic cells in vitro, suggesting that this method can reproducibly provide homogeneous materials for further studies on neurogenesis.     

Platelet-derived growth factor receptor-alpha in ventricular zone cells and in developing neurons.

Cells in the early neuroepithelium differentiate and give rise to all cells in the central nervous system (CNS). The ways from a multipotent CNS stem cell to specialized neurons and glia are not fully understood. Using immunohistochemistry we found that neuroepithelial cells express the platelet-derived growth factor receptor-alpha (PDGFR-alpha) in the neural plate at embryonic day 8.5 and onwards in the neural tube. The protein was polarized to ventricular endfeet. Furthermore, PDGFR-alpha expression was localized to cells undergoing early neuronal development. We also found PDGFR-alpha expression in developing granule cells in the postnatal cerebellum, in Purkinje cells in the adult cerebellum and on processes of developing dorsal root ganglion cells. Previous reports mainly describe PDGFR-alpha expression in oligodendrocyte precursors and glial cells. We believe, in line with a few previous reports, that the PDGFR-alpha in addition marks a pool of undifferentiated cells, which are able to differentiate into neurons.     

effects of NGF and dibutyryl cAMP on neuronal differentiation of embryonal carcinoma cells

The P19S18O1A1 embryonal carcinoma cell line is capable of neuronal differentiation and is therefore useful in studying neuronal development and the influence of growth modulators on neuronal differentiation. We report here on the effects of nerve growth factor (NGF) and dibutyryl cyclic adenosine monophosphate (db cAMP), individually and combined, on differentiation of P19S18O1A1 cells. NGF alone did not induce any significant neuron-like changes in cultures exposed to NGF for as long as 12 days. Treatment with db cAMP resulted in changes in a significant population of the cells, including development of a neuron-like morphology, seen at both the light and electron microscopic level, loss of stage-specific embryonic antigen expression and the appearance of two neuronal markers, neurofilament protein and neuron-specific enolase. These changes were similar to changes seen when embryonal carcinoma (EC) cells are treated with retinoic acid. NGF in combination with dibutyryl cyclic adenosine monophosphate brought about similar changes as dibutyryl cyclic adenosine monophosphate alone, and was therefore not synergistic for induction of neuronal properties. In retinoic acid-treated cultures, the neuron-like cells had ultrastructural features very similar to neurons in non-tumorous, normal tissue, with typical organelles, such as one nucleolus, neurotubules and neurofilaments, while db cAMP-treated EC cells showed similar findings at the electronmicroscopic level. The results suggest that db cAMP can induce the neuronal phenotype in EC cells alone without pre-treatment with retinoic acid.     

Sources of neuronal material for implantation

The adult brain is an organ that does not have the natural ability to replace cells that have been lost through damage. Possible human interventions to rectify this situation include transplanting either developing neural tissue into the damaged host brain or transplantation of neural stem cells (cells that have the capacity to proliferate into neural cells and self‐replicate) into the damaged area. Fetal or embryonic stem cells can be extracted and differentiated in vitro into the specific desired progeny (e.g. neurons). The neuronal stem cells themselves can be extracted from fetuses and multiplied in culture and then transplanted into the damaged brain. There is the possibility of de‐differentiation, in which cells of one type can be converted into a different cell type; for example, a differentiated blood cell could be de‐differentiated back to its own hemopoietic stem cell and that stem cell could be converted into a neuronal stem cell which could then be differentiated into a neuron. It is probable that methods of generating large numbers of committed stem cells to treat conditions such as Alzheimer's disease will soon be increasingly common.     

Platelet-Derived Growth Factor Receptor-α in Ventricular Zone Cells and in Developing Neurons

Cells in the early neuroepithelium differentiate and give rise to all cells in the central nervous system (CNS). The ways from a multipotent CNS stem cell to specialized neurons and glia are not fully understood. Using immunohistochemistry we found that neuroepithelial cells express the platelet-derived growth factor receptor-α (PDGFR-α) in the neural plate at embryonic day 8.5 and onwards in the neural tube. The protein was polarized to ventricular endfeet. Furthermore, PDGFR-α expression was localized to cells undergoing early neuronal development. We also found PDGFR-α expression in developing granule cells in the postnatal cerebellum, in Purkinje cells in the adult cerebellum and on processes of developing dorsal root ganglion cells. Previous reports mainly describe PDGFR-α expression in oligodendrocyte precursors and glial cells. We believe, in line with a few previous reports, that the PDGFR-α in addition marks a pool of undifferentiated cells, which are able to differentiate into neurons.     

Expression of necdin, an embryonal carcinoma-derived nuclear protein, in developing mouse brain.

Necdin is a polypeptide sequence encoded by neural differentiation-specific mRNA derived from embryonal carcinoma cells. We have examined the expression of necdin and its mRNA in cultured cells and mouse brain by Northern blot analysis and immunohistochemistry. Among various established cell lines including neuroblastoma and glioma cells, only differentiated embryonal carcinoma cells (P19 and F9) expressed necdin mRNA. Necdin immunoreactivity was localized in the nuclei of differentiated neurons derived from P19 cells. Necdin mRNA was detected throughout brain regions of adult mouse; the relative abundances in the hypothalamus and midbrain were the highest, whereas those in the olfactory bulb and cerebellum were the lowest. In developing mouse brain, necdin mRNA was expressed during early periods of neuronal generation and differentiation, and the peak levels were attained during postnatal days 1-4. Necdin immunoreactivity was not detected in the neural stem cells on embryonic day 10, but was concentrated in the nuclei of brain cells, mostly neurons, at advanced stages of differentiation. The majority of differentiated neurons in the brain had necdin-immunoreactive nuclei on postnatal day 33. Thus, necdin may represent a valuable molecular marker for differentiated neurons both in vitro and in vivo.     

A method for generating high-yield enriched neuronal cultures from P19 embryonal carcinoma cells

P19 embryonal carcinoma (EC) cells are an invaluable tool for approximating the mechanisms that govern neuronal differentiation but with an enormous degree of simplification and have primarily been used to model the early stages of neurogenesis. However, they are often cultured under conditions that promote unrestricted non-neuronal growth that compromises neuronal viability. In this study we report an improved method to differentiate P19 EC cells that gives rise to high yields of functionally and morphologically mature neurons while significantly reducing the over-growth of non-neuronal cells in the cultures. In this protocol, P19 EC cells are induced in Minimum Essential Medium alpha supplemented with all-trans retinoic acid (RA) and 2.5% serum, and cultured as a monolayer. After RA-induction, cells are cultured on Matrigel coated-plates using defined media comprised of Neurobasal-A medium temporally supplemented with N2 and then B-27 for the remaining culture period. By treating the culture with Cytosine β-d-arabinofuranoside and 2′-Deoxycytidine for five days, the cultures are reliably promoted toward the neuronal differentiation vs non-neuronal differentiation, this accounting for a progressive neuronal enrichment of the cultures reaching 56% after 20 days of culture. P19-derived neural progenitor cells progressively expressed neuronal markers such as NeuN, Calretinin, Calbindin and Synapsin I in close resemblance to that occurring in vivo in the central nervous system (CNS). Furthermore, RA-induced P19 EC cells progressively acquired functional neuronal traits and after approximately 3 weeks in culture revealed mature neurophysiological properties, characteristics of CNS neurons. This protocol allows for a more specific assessment of the neuronal differentiation processes in vitro.     

Differentiation of radial glia-like cells from embryonic stem cells

Radial glial cells play important roles in neural development. They provide support and guidance for neuronal migration and give rise to neurons and glia. In vitro, neurons, astrocytes, and oligodendrocytes can be generated from neural and embryonic stem cells, but the generation of radial glial cells from these stem cells has not yet been reported. Since the differentiation of radial glial cells is indispensable during brain development, we hypothesize that stem cells also generate radial glial cells during in vitro neural differentiation. To test this hypothesis, we utilized five different clones of mouse embryonic (ES) and embryonal carcinoma (EC) stem cell lines to investigate the differentiation of radial glial cells during in vitro neural differentiation. Here, we demonstrate that radial glia-like cells can be generated from ES/EC cell lines. These ES/EC cell-derived radial glia-like cells are similar in morphology to radial glial cells in vivo, i.e., they are bipolar with an unbranched long process and a short process. They also express several cytoskeletal markers, such as nestin, RC2, and/or GFAP, that are characteristics of radial glial cells in vivo. The processes of these in vitro generated radial glia-like cells are organized into parallel arrays that resemble the radial glial scaffolds in neocortical development. Since radial glia-like cells were observed in all five clones of ES/EC cells tested, we suggest that the differentiation of radial glial cells may be a common pathway during in vitro neural differentiation of ES cells. This novel in vitro model system should facilitate the investigation of regulation of radial glial cell differentiation and its biological function.     

Modulated generation of neuronal cells from bone marrow by expansion and mobilization of circulating stem cells with in vivo cytokine treatment

The aim of the present study is to determine whether the expansion and mobilization of circulating bone marrow (BM) stem cells by in vivo treatment with granulocyte-colony stimulating factor (G-CSF) and stem cell factor (SCF) increase the amount of BM-derived neuronal cells in mouse brain. The presence of BM-derived cells in the brain was traced by transplanting into lethally irradiated adults and newborns adult BM from transgenic mice that ubiquitously expressed enhanced green fluorescent protein (GFP). GFP+ and Y-chromosome+ donor-derived cells were present in several brain areas of all treated mice (cortical and subcortical areas, cerebellum, olfactory bulb). The presence of GFP+ cells expressing nuclear neural specific antigen (NeuN), neurofilament, and beta-III tubulin in cortical forebrain and olfactory bulb (OB) was higher in G-CSF-SCF treated groups (P < 0.05, analysis of variance, Fisher post hoc). We observed that overall the amount of double positive cells was higher in animals treated at birth than in adults and in OB than in forebrain areas (P < 0.05). Temporal cortical areas of cytokine-treated adult animals revealed a mean threefold increase in the number of GFP+ cells expressing the nuclear neural specific antigen (211 +/- 86 GFP+NeuN+/mm(3) in G-CSF + SCF treated mice and 66 +/- 33 GFP+NeuN+/mm(3) in control animals). GFP+ cells coexpressing neuronal markers contain only one nucleus and have a DNA index (a measure of DNA ploidy) identical to that of surrounding neurons, thus excluding donor cell fusion with endogenous cells as a relevant phenomenon under these experimental conditions. Our results indicate that G-CSF and SCF administration modulates the availability of GFP+ cells in the brain and enhances their capacity to acquire neuronal characteristics. Cytokine stimulation of autologous stem cells might be seen as a new strategy for neuronal repair in neurodegenerative diseases.     

Neural stem cell differentiation in a cell-collagen-bioreactor culture system

Neural stem cells and neural progenitors (NSCs/NPs) are capable of self-renewal and can give rise to both neurons and glia. Such cells have been isolated from the embryonic brain and immobilized in three dimensional collagen gels. The collagen-entrapped NSCs/NPs recapitulate CNS stem cell development and form functional synapses and neuronal circuits. However, the cell-collagen constructs from static conditions contain hypoxic, necrotic cores and the cells are short-lived. In the present study, NSCs/NPs isolated from embryonic day 13 rat cortical neuroepithelium are immobilized in type I collagen gels and cultured in NASA-designed rotating wall vessel (RWV) bioreactors for up to 9 weeks. Initially, during the first 2 weeks of culture, a lag phase of cellular growth and differentiation is observed in the RWV bioreactors. Accelerated growth and differentiation, with the cells beginning to form large aggregates (approximately 1 mm in diameter) without death cores, begins during the third week. The collagen-entrapped NSCs/NPs cultured in RWV show active neuronal generation followed by astrocyte production. After 6 weeks in rotary culture, the cell-collagen constructs contain over 10 fold greater nestin+ and GFAP+ cells and two-fold more TuJ1 gene expression than those found in static cultures. In addition, TuJ1+ neurons in RWV culture give rise to extensive neurite outgrowth and considerably more synapsin I+ pre-synaptic puncta surrounding MAP2+ cell bodies and dendrites. These results strongly suggest that the cell-collagen-bioreactor culture system supports long-term NSC/NP growth and differentiation, and RWV bioreactors can be useful in generating neural tissue like constructs, which may have the potential for cell replacement therapy.     

Hepatocyte growth factor promotes neuronal differentiation of neural stem cells derived from embryonic stem cells

We previously reported that hepatocyte growth factor (HGF) promoted proliferation of neurospheres and neuronal differentiation of neural stem cells (NSCs) derived from mouse embryonic brain. In this study, spheres from mouse embryonic stem (ES) cells were generated by floating culture following co-culture on PA6 stromal cells. In contrast to the behavior of the neurospheres derived from embryonic brain, addition of HGF to the growth medium of the floating cultures decreased the number of spheres derived from ES cells. When spheres were stained using a MAP-2 antibody, more MAP-2-positive cells were observed in spheres cultured with HGF. When HGF was added to the growth and/or differentiation medium, more MAP-2-positive cells were also obtained. These results suggest that HGF promotes neuronal differentiation of NSCs derived from ES cells.     

Receptor for advanced glycation end products (RAGE) mediates neuronal differentiation and neurite outgrowth

The receptor for advanced glycation end products (RAGE) plays a crucial role in several disease processes, such as diabetes, inflammation, and neurodegeneration. In this article we report multiple roles of RAGE in neuronal differentiation and neurite outgrowth. In retinoic-induced P19 embryonic carcinoma stem cells, silencing the expression of RAGE by RNA interference (RNAi) blocked differentiation of the P19 cells into neuronal cells and enhanced the formation of vimentin-positive fibroblast-like cells. RAGE knockdown inhibited retinoic acid-induced activation and blocked nuclear translocation of NF-kappaB, suggesting RAGE regulates activation of NF-kappaB. RAGE was also shown to be involved in survival of P19 cells during retinoic acid differentiation. Additionally, knockdown of RAGE strongly inhibited neurite outgrowth in retinoic acid-differentiated P19 cells, indicating that RAGE is required for neurite outgrowth of differentiated P19 cells. Retinoic acid-treated P19 cells activated GTPases, Rac1, and Cdc42. This activation of the GTPases was inhibited in RAGE-knockdown cells. In primary cerebellar granule neurons, the knockdown of RAGE also inhibited neurite outgrowth. In these cells, overexpression of dominant-negative forms of Rac1 and Cdc42 inhibited neurite outgrowth, whereas overexpression of constitutively active forms of Rac1 and Cdc42 in RAGE-deficient neurons restored neurite outgrowth, indicating that RAGE mediated neurite outgrowth through the Rac1/Cdc42 pathway. This is the first report on the role of RAGE in cell lines and primary neurons, as determined by RNAi knockdown.     

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.