AU4826200A - Lineage-restricted precursor cells isolated from mouse neural tube and mouse embryonic stem cells - Google Patents

Description

WO 00/68359 PCT/USOO/12446 LINEAGE-RESTRICTED PRECURSOR CELLS ISOLATED FROM MOUSE NEURAL TUBE AND MOUSE EMBRYONIC STEM CELLS Field of the Invention The present invention relates to multipotent 5 neuroepithelial (NEP) stem cells, and lineage-restricted, self-renewing precursor cells termed neuron-restricted precursors (NRPs) and glial-restricted precursors (GRPs) isolated from mouse neural tubes. Immunoselection against the cell surface markers E-NCAM and A2B5 were used to isolate 10 mouse NRPs and GRPs, respectively. The present invention also relates to lineage-restricted precursor cells isolated from mouse embryonic stem cells (ES cells) . As demonstrated herein, mouse ES cell-derived NRPs are E-NCAM immunoreactive, undergo self renewal in defined medium and differentiate into 15 multiple neuronal phenotypes in mass culture. Mouse ES cells also generate A2B5 immunoreactive cells that are similar to GRPs derived from mouse neural tubes and which differentiate into oligodendrocytes and astrocytes. The present invention also relates to methods for generating these lineage 20 restricted precursors in vitro from mouse neural tubes and cultured mouse ES cells. Background of the Invention Pluripotent stem cells in the central nervous system (CNS) generate differentiated neurons and glia either directly 25 or through the generation of lineage-restricted intermediate precursors (see e.g. Kilpatrick, T. J. and Bartlett, B. F. J. Neurosci. 1995 15:3653-3661; Kilpatrick, T. J. and Bartlett, B. F. Neuron 1993 10:255-265; Price et al. Brain Pathol. 1992 2:23-29; Reynolds et al. J. Neurosci. 1992 12:4565-4574; WO 00/68359 PCTIUSOO/12446 -2 Reynolds, B. A. and Weiss, S. Dev. Biol. 1996 175:1-13; Temple, S. and Davis, A. Development 1994 120:999-1008). Review of the relevant art indicates that different stem cells and lineage-restricted precursors with distinguishing 5 characteristics and abilities are obtained depending upon the species, the region of the central nervous system from which the cells were obtained, and age of the animal from which the cells were obtained. For example, multipotent neuroepithelial (NEP) cells 10 have been shown to be present in the spinal cord of rats at embryonic day 10.5. These rat NEP cells are self-renewing cells that can differentiate into neurons, oligodendrocytes, astrocytes (Kalyani et al. Dev. Biol. 1997 186:202-223), and peripheral nervous system (PNS) derivatives (Mujtaba et al. 15 Dev. Biol. 1998 200:1-15). Differentiation of rat NEP cells occurs via the generation of more restricted precursors. At embryonic day 13.5, two major types of lineage-restricted precursors are present in the rat: an E-NCAM immunoreactive neuronal precursor, termed NRP, that can generate multiple 20 kinds of neurons but not oligodendrocytes or astrocytes (Mayer-Proschel et al. Neuron 1997 19:773-785; Kalyani et al. J. Neurosci. 1998 18:7856-7868); and an A2B5 immunoreactive glial precursor termed GRP, that can generate oligodendrocytes and astrocytes, but cannot generate neurons (Rao, M. S. and 25 Mayer-Proschel, M. Dev. Biol. 1997 188:48-63; Mayer-Proschel et al. Neuron 1997 19:773-785; Rao et al. Proc. Natl. Acad. Sci. USA 1998 95:3996-4001). U.S. Patent 5,753,506 by Johe et al. describes a different multipotential stem cell isolated from adult rat 30 brain and regions of the embryonic rat brain including the cortex and striatum at embryonic day 14 and the hippocampus at embryonic day 16, as well as human fetal brain. This stem cell also undergoes self-renewal and can differentiate into neurons, astrocytes and oligodendrocytes upon culture in 35 appropriate differentiation conditions. Cells isolated from WO 00/68359 PCT/USOO/12446 -3 rat embryonic cortex and striatum at embryonic day 14 and rat embryonic hippocampus at embryonic day 16 were 70% pure. These cells can be maintained in the presence of FGF as well as EGF and/or TGF alpha. PDGF acts on these stem cells to 5 bias neuronal differentiation. Cattaneo and McKay (Nature 1990 347: 762) disclose a rat neuron-restricted precursor cell isolated from early embryonic brain, more specifically rat embryonic striatum primordia from embryonic day 13.5-14.5. A greater than 95% population of rat 10 striatal cells expressing nestin is disclosed. Gard et al. (Developmental Biology .1995 167:596) disclose a glial-restricted precursor cell isolated from the optic nerve of 4 day old rats which are bipotential, generating oligodendrocytes and A2B5*/GFAP* astrocytes. 15 U.S. Patent 5,411,883 by Boss et al. teaches cells isolated from ventral mesencephalon tissue from porcine or human embryos at Carnegie stages 15-18. The porcine precursor cells are demonstrated to differentiate into dopaminergic neurons. 20 The existence of spinal cord NRPs in chick embryos has been demonstrated by retroviral tracing (Leber et al. J. Neurosci. 1990 10:1688-1697; Leber, S.M. and Sanes, J.R. J. Neurosci. 1995 15:1236-1248). In addition, NRP cell lines that are E-NCAM 25 immunoreactive have been generated from human spinal cord cultures. Multipotent stem cells have also been identified in the mouse cortex in embryos between 12 and 18 days of gestation (Williams, B.P. and Price, J. Neuron 1995 14:1181-88); the 30 telencephalon and mesencephalon of mice at embryonic day 10 and the cerebrum of mice at embryonic day 17 (Kilpatrick, T. J. and Bartlett, B. F. J. Neurosci. 1995 15:3653-3661); adult mouse thoracic spinal cord (Weiss et al. J. Neurosci. 1996 16:7599-7609); and adult and embryonic forebrain of the mouse 35 (Weiss et al. Trends Neurosci. 1996 19:387-393).

WO 00/68359 PCT/USOO/12446 -4 An O-2A-like glial progenitor cell has also been identified in the optic nerve and cortex at late embryonic stages (reviewed in Collarini et al. J. Cell Sci. Suppl. 1991 15:117-123). U.S. Patent 5,688,692 by Jat discloses a method 5 for isolating a population of glial precursors from cortical cells derived from embryonic day 18 mice. Neuronally restricted precursor cells from the juvenile subventricular zone (svz) of mice have also been identified by clonal analysis in vivo and in vitro (Young, G.M. and 10 Levison, S.W. Dev. Neurosci. 1996 18:255-265). However, all of these cells differ significantly in growth factor requirements, culture conditions, and differentiation potential from rat NEP cells identified by Kalyani et al. (Dev. Biol. 1997 186:202-223) and rat GRP and 15 rat NRP cells identified by Mayer-Proschel et al. (Neuron 1997 19:773-785) and Kalyani et al. (J. Neurosci. 1998 18:7856 7868) and Rao, M. S. and Mayer-Proschel, M. (Dev. Biol. 1997 188:48-63), Mayer-Proschel et al. (Neuron 1997 19:773-785) and Rao et al. (Proc. Natl. Acad. Sci. USA 1998 95:3996-4001), 20 respectively. Sunmary of the Invention An object of the present invention is to provide an isolated, pure population of mouse neuron-restricted precursor cells derived from mouse neural tubes at embryonic day 12.0 25 or mouse embryonic stem cells by E-NCAM immunoreactivity. The mouse neuron-restricted precursor cells self-renew. Under differentiation conditions, these mouse NRP cells differentiate into neurons but not into glial cells. Another object of the present invention is to provide 30 methods for isolating pure populations of the mouse neuron restricted precursor cells. In one embodiment, a pure population of mouse neuron-restricted precursor cells is isolated by immunoselecting E-NCAM-immunoreactive cells from mouse E12.0 neural tube cells. In another embodiment mouse WO 00/68359 PCT/USOO/12446 -5 embryonic stem cells are incubated under differentiation inducing conditions so that the cells differentiate. E-NCAM immunoreactive cells are then selected from the differentiating cells. 5 Another object of the present invention is to provide an isolated, pure population of mouse glial-restricted precursor cells derived from mouse neural tubes at embryonic day 12.0 or mouse embryonic stem cells by A2B5 immunoreactivity. The mouse glial-restricted precursor cells 10 self-renew. Under differentiating conditions, these mouse GRP cells differentiate into oligodendrocytes and at least two distinct populations of astrocytes, but not into neurons. Another object of the present invention is to provide methods for isolating pure populations of the mouse glial 15 restricted precursor cells. In one embodiment, a pure population of mouse glial-restricted precursor cells is isolated by immunoselecting A2B5-immunoreactive cells from mouse E12.0 neural tube cells. In another embodiment, a pure population of mouse glial-restricted precursor cells is 20 isolated by incubating mouse embryonic stem cells under differentiation-inducing conditions so that the cells differentiate and immunoselecting A2B5-immunoreactive cells from the differentiating cells. Another object of the present invention is to provide 25 an isolated, pure population of mouse neuroepithelial stem cells derived from mouse neural tubes at embryonic day 8.5. These mouse neuroepithelial stem cells proliferate and self renew in adherent feeder-cell-independent culture medium containing effective amounts of fibroblast growth factor and 30 chick embryo extract. Under differentiating conditions, these mouse neuroepithelial stem cells differentiate into CNS neuronal cells, CNS glial cells including oligodendrocytes, A2B5-positive astrocytes, and A2B5-negative astrocytes, and neural crest stem cells.

WO 00/68359 PCT/USOO/12446 -6 Yet another object of the present invention is to provide a method of isolating a pure population of these mouse neuroepithelial stem cells. In this method, the neural tube from a mouse embryo is removed at embryonic day 8.5. Cells 5 from the neural tube are dissociated and plated in a feeder cell-independent culture on a substratum and in a medium containing fibroblast growth factor and chick embryo extract. The plated cells are then cultured at a temperature and in an atmosphere conducive to cell growth. 10 Detailed Description of the Invention The present invention relates to purified neuroepithelial (NEP) cells, neuroblasts and glioblasts isolated from the developing mouse spinal cord and methods for isolating pure populations of these cells from the developing mouse spinal 15 cord. The present invention also relates to pure populations of neuron- and glial-restricted precursor cells isolated from mouse embryonic stem (ES) cell cultures, as well as methods for isolating these cells from mouse ES cells. As demonstrated herein these cells exhibit characteristics 20 similar to those described for rat neuronal precursor cells and different from previously described mouse neurosphere cells. In the present invention, E-NCAM immunoreactivity is used to isolate pure populations of differentiated mouse ES cells which generate multiple neuronal phenotypes, but which 25 do not generate astrocytes or oligodendrocytes. A2B5 immunoreactivity is used to isolate pure populations of differentiated mouse ES which generate oligodendrocytes and astrocytes, but which do not generate neurons. Mouse NEP cells of the present invention are different 30 from classes of mouse stem cells described previously. The properties of mouse NEP cells of the present invention resemble those of rat NEP cells rather than other classes of stem cells isolated from other portions of the CNS and at different stages in development such as striatal embryonic WO 00/68359 PCT/USOO/12446 -7 cells described by Reynolds et al. (J. Neurosci 1992 12:4565 4574) , multipotential precursors from the mouse cerebrum described by Kilpatrick, T.J. and Bartlett, B.F. (J. Neurosci. 1995 15:3653-3661), and multipotential stem cells from the 5 adult mouse brain described by Gritti et al. (J. Neurosci 1996 16:1091-1100). For example, as demonstrated herein, mouse NEP cells of the present invention grow in adherent culture, do not express EGF-R immunoreactivity, survive and/or proliferate in EGF and 10 absolutely require FGF for both proliferation and survival. Further, PDGF, which has been shown to be an instructive molecule for stem cells derived from the cortical region in mice (see e.g. Williams et al. Neuron 1997 18:553-562 describing the role of PDGF in ventricular zone progenitor 15 cells), has no observable effect on the mouse NEP cells of the present invention, either alone or in combination with FGF. In these experiments, the antigenic properties, growth characteristics and differentiation potential of these early mouse NEP precursor cells were examined. Specifically, mouse 20 neural tubes at embryonic day 8.5 (E8.5) were isolated and cells were plated at low density to test the growth characteristics of NEP stem cells. NEP cells appeared homogeneous and were nestin immunoreactive. Virtually all cells incorporated BrdU over a 6 hour period indicating that 25 they were actively dividing cells when maintained in 25 ng/ml of FGF. NEP cells did not express any markers of early differentiation as tested by either immunocytochemistry or RT PCR. In particular, NEP cells did not express E-NCAM, S-111 tubulin or A2B5 immunoreactivity, thus indicating that they 30 were undifferentiated precursor cells. Nestin immunoreactive cells persisted around the ventricular zone at E 12.0, while -111 tubulin immunoreactive neurons appeared to differentiate at the ventricular margins. Growth factor dependence of E8.5 NEP cells was tested in 35 several growth factors at a variety of different WO 00/68359 PCT/USOO/12446 -8 concentrations. FGF was sufficient to maintain NEP cell proliferation. In contrast, NGF, PDGF and EGF were ineffective at any dose tested. Further, none of the growth factors tested acted synergistically with FGF to alter cell 5 division rates thus suggesting that EGF and PDGF are neither survival or proliferation factors for the mouse NEP cells of the present invention. The failure to see a response to EGF is indicative of these cells being a distinct population from both EGF dependent neurospheres and FGF/EGF dependent cells 10 isolated from other regions and later stages in the mouse (Reynolds et al. J. Neurosci. 1992 12:4565-4574; Reynolds, B.A. and Weiss, S. Dev. Biol. 1996 175:1-13; and Weiss et al. J. Neurosci. 1996 16:7599-7609). Differences of the E8.5 mouse NEP cells of the present 15 invention were confirmed by examination of EGF-R expression in cell culture. EGF-R is not detected in the mouse NEP cells of the present invention. In contrast, EGF-R was readily detected in whole brain, as well as on glial precursors, neurons and astrocytes. Identical results were obtained using 20 an antibody specific to mouse EGF-R. Accordingly, the cells of the present invention are clearly distinct from EGF dependent mouse neurospheres as well as FGF/EGF dependent mouse stem cells isolated at later stages of development. The E8.5 mouse NEP cells of the present invention can 25 also be maintained in defined medium with the addition of FGF for multiple passages (at least ten) and still be induced to differentiate by replating the cells on laminin or poly-L lysine and reducing FGF concentrations. NEP cells under these conditions readily differentiated into neurons, astrocytes and 30 oligodendrocytes. Thus, the mouse NEP cells are FGF dependent, grow in adherent culture and can generate CNS derivatives. In addition, these cells generate p75 immunoreactive crest cells, SMA immunoreactive smooth muscle cells and peripheral glial and neurons. PCR analysis also 35 confirmed an increase in the expression of p75 when NEP cells WO 00/68359 PCTIUSOO/12446 -9 of the present invention were induced to differentiate into neural crest by the addition of 10 ng/ml of BMP-4. Neural crest and its derivatives have not been generated from EGF dependent stem cells. 5 Thus, results from these experiments indicate that at least during early time periods of development, only FGF dependent pluripotent stem cells are present in the developing mouse spinal cord. Taken together with evidence that at later stages an EGF or EGF/FGF dependent cell is present, it 10 is believed that at least two different classes of stem cells exist. Since the NEP cells of the present invention do not express EGF-R immunoreactivity, it is believed that EGF-R can serve as a marker for selection between these two different populations of stem cells. 15 As also demonstrated herein, E-NCAM and A2B5 immunoreactivity can be used to isolate lineage-restricted precursor cells from the mouse neural tube. Differentiation in rat neural tubes has been shown to involve progressive restriction of cell fate (Mayer-Proschel 20 et al. Neuron 1997 19:773-785; Kalyani et al. J. Neurosci. 1998 18:7856-7868). Rat NEP cells generate more restricted multipotent precursors both in vitro and in vivo (Mayer Proschel et al. Neuron 1997 19:773-785; Rao et al. Proc. Natl Acad. Sci. USA 1998 95:3996-4001). Two such restricted 25 multipotent precursors have been described: an E-NCAM immunoreactive neuronal precursor and an A2B5 immunoreactive glial precursor (Rao, M.S. and Mayer-Proschel, M. Dev. Biol. 1997 188:48-63; Rao et al. Proc. Natl Acad. Sci. USA 1998 95:3996-4001). 30 To determine if similar neuronal precursors could be isolated from mouse neural tubes, cells from mouse neural tubes at embryonic day 12.0 (E12.0) were analyzed for the presence of E-NCAM immunoreactive cells. A significant proportion of E12.0 neural tubes cells were E-NCAM 35 immunoreactive (ranging from 70-80% in three independent WO 00/68359 PCTIUSOO/12446 - 10 experiments) . Immunoselected E-NCAM* cells divided in culture and expressed neuronal, but not glial, markers. Thus, based on their antigenic profile, E-NCAM immunoreactive cells appeared to be dividing neuron-restricted precursor cells. 5 To test the differentiation potential of E-NCAM immunoreactive cells, purified populations of E-NCAM* cells were analyzed in mass and clonal culture. E-NCAM* cells, when allowed to differentiate by addition of retinoic acid and reduction of FGF concentration, readily differentiated into 10 multiple kinds of neurons as assessed by RT-PCR and immunocytochemistry. Further, E-NCAM* cells failed to differentiate into either oligodendrocytes or astrocytes when grown under glial promoting conditions, indicating that these cells are restricted or limited in their differentiation 15 potential. Fura-2 Ca 2 ' imaging techniques were used to examine the ability of differentiated E-NCAM* cells to respond to neurotransmitters and elevated K*. E12.0 E-NCAM* cells were grown in culture for 10 days and allowed to differentiate. 20 They were then loaded with fura-2 and changes in internal Ca 2 , concentrations in response to stimulus application were monitored. Most cells (>95%) responded to external application of glutamate or acetylcholine. Smaller fractions of cells responded to dopamine, GABA or glycine. A 25 substantial fraction of the cells responded to more than one neurotransmitter, demonstrating heterogeneity in the neuronal population derived from E-NCAM* precursors. Further traces of internal Ca 2 . changes were plotted over time for two different cells from the same clonally derived culture. While both 30 cells responded to glutamate, acetylcholine and high K+, one of the cells also responded to dopamine, indicating that the receptor expression profile of cells in the same culture varies. Thus, E-NCAM immunoreactive cells isolated from E12.0 mouse neural tube are neuron-restricted precursor cells that 35 can differentiate into a heterogeneous population of neurons, WO 00/68359 PCT/USOO/12446 - 11 and closely resemble their rat counterparts in antigenic characteristics, differentiation properties and growth potential. Lineage-restricted glial precursors were also isolated 5 from the developing mouse spinal cord at E12.0 via A2B5 immunoreactivity. A2B5 immunoreactive cells were isolated by immunopanning and mass cultures of cells were tested for cell division by BrdU incorporation. A significant proportion of these cells divided in culture, thus indicating that these are 10 a mitotic population. The antigenic properties and differentiation potential of these cells was also examined. In contrast to rat glial restricted precursor cells, it was found that a small subset of the mouse A2B5 immunoreactive cells (<5%) were p-111 tubulin immunoreactive. This 15 population was also E-NCAM immunoreactive as determined by immunopanning experiments. Thus, unlike rat spinal cord cultures, the A2B5 epitope was not uniquely present on glial cells. To determine if the E-NCAM/S-111 tubulin negative subset 20 of A2B5 cells represented glial precursors, this population was examined by sequential panning. E-NCAM* immunoreactive cells were isolated and discarded and the E-NCAM- population was repanned to isolate A2B5 immunoreactive cells. Pooled cells were analyzed by PCR and immunocytochemistry. A2B5*/E 25 NCAM~ cells differentiated into oligodendrocytes and two kinds of astrocytes but failed to differentiate into neurons when grown under neuron promoting conditions thus indicating that these cells are limited in their differentiation potential. Accordingly, glial-restricted and neuron-restricted precursors 30 are both present in the developing mouse spinal cord and these precursor cells can be distinguished by E-NCAM and A2B5 immunoreactivity. Mouse ES cells have been shown to be capable of differentiating into neurons, astrocytes, and 35 oligodendrocytes. In addition, it has recently been WO 00/68359 PCT/USOO/12446 - 12 demonstrated that NEP-like cells can be harvested from ES cells, thereby bypassing the need to harvest NEP cells from fetal tissue. Accordingly, mouse ES cells cultures were analyzed to determine if more restricted neural precursors 5 that appear at developmentally later embryonic stages could be harvested in similar fashion. In these experiments, the ES-D3 cell line, which has been shown to be able to contribute to all cell lineages, was obtained from ATCC and grown in non-differentiating (as 10 aggregates in DMEM/F12, 10% FCS and LIF 10 ng/ml and as adherent cultures on fibronectin coated dishes in NEP basal medium) and differentiating conditions (as adherent cultures on poly-L-Lysine/laminin substrate in NEP basal medium). Undifferentiated ES cells did not express E-NCAM 15 immunoreactivity, but upon aggregation and treatment with retinoic acid (RA), a subset (approx. 5%) of cells began to express E-NCAM immunoreactivity. E-NCAM* cells were a mitotic population as assessed by BrdU incorporation. E-NCAM* cells co-expressed MAP2, a marker for neurons (DeCamilli et al. 20 Neuroscience 1984 11:817-846) and B-111 tubulin immunoreactivity, but did not express GFAP, GalC or 04 immunoreactivity. A subset of the E-NCAM cells were nestin immunoreactive, but none of the cells expressed GFAP, a glial marker. The co-expression of neuronal markers with E-NCAM and 25 the absence of glial markers indicate that ES cell derived E NCAM* immunoreactive cells are similar to E12.0 mouse neuronal restricted precursor cells. To further confirm the neuronal differentiation of ES cell derived E-NCAM cells, ES cells were induced to 30 differentiate and E-NCAM immunoreactive cells were selected by immunopanning. Cells were grown in mass or clonal culture in medium supplemented with FGF and NT-3. When mass cultures of E-NCAM cells were induced to differentiate by withdrawal of FGF and the addition of RA, cells became postmitotic, 35 elaborated extensive processes and synthesized multiple WO 00/68359 PCT/USOO/12446 - 13 neurotransmitters. Immunocytochemistry and PCR analysis showed the presence of various excitatory, inhibitory and cholinergic neurotransmitters after differentiation. E-NCAM immunoreactive cells failed to differentiate into either 5 oligodendrocytes or astrocytes when grown under glial promoting conditions, indicating that these cells are limited in their differentiation potential. Thus ES cells can be used as a source of neuron-restricted precursor cells. The differentiation potential of A2B5*/E-NCAM- cells was 10 tested by inducing ES cells to differentiate and depleting E NCAM immunoreactive cells by immunopanning. E-NCAM immunonegative cells were reselected for A2B5 immunoreactivity. This double selection procedure was utilized since, as described above, A2B5* immunoreactivity has 15 been detected on glial precursors, as well as on subsets of neurons, from mice. A2B5 immunopositive cells were grown in mass or clonal culture in medium supplemented with FGF and PDGF. When mass cultures of A2B5* cells were induced to differentiate by withdrawal of FGF, cells differentiated into 20 different glial populations. Two kinds of astrocytes could be identified as early as seven days after induction of differentiation. An A2B5*/GFAP- flat astrocyte which appeared similar to astrocytes previously characterized as Type I astrocytes and a A2B5*/GFAP* astrocyte which appeared similar 25 to type 2 astrocytes were detected in cultures. In addition, when cells were allowed to differentiate for longer time periods (10 days), oligodendrocyte differentiation was detected as assessed by GalC immunocytochemistry. A2B5 immunoreactive cells failed to differentiate into neurons when 30 grown under neuron promoting conditions, indicating that these cells are limited in their differentiation potential to glial lineages. The ability of ES cell derived A2B5 immunoreactive cells to differentiate into astrocytes and oligodendrocytes confirms their resemblance to E12.0 A2B5 derived GRP cells, 35 and indicates that like neuron-restricted precursors, glial WO 00/68359 PCTIUSOO/12446 - 14 restricted precursors can be isolated directly from differentiating ES cell cultures. Accordingly, provided in the present invention are pure populations of mouse neuroepithelial (NEP) cells isolated or 5 derived from mouse neural tubes at embryonic day 8.5 and mouse neuron- and glial-restricted precursor cells isolated or derived from mouse neural tubes at embryonic day 12.0 or mouse ES cells which exhibit similar characteristics to the rat NEP cells described by Kalyani et al. (Dev. Biol. 1997 186:202 10 223) and rat GRP and rat NRP cells identified by Mayer Proschel et al. (Neuron 1997 19:773-785) and.Kalyani et al. (J. Neurosci. 1998 18:7856-7868) and Rao, M. S. and Mayer Proschel, M. (Dev. Biol. 1997 188:48-63), Mayer-Proschel et al. (Neuron 1997 19:773-785) and Rao et al. (Proc. Natl. Acad. 15 Sci. USA 1998 95:3996-4001), respectively. For purposes of the present invention, by "pure" it is meant a population of cells in which greater than 95%, more preferably 99%, exhibit the same characteristics. For example, as demonstrated herein, the mouse NEP cells 20 of the present invention grow in adherent culture, do not exhibit EGF-R immunoreactivity, do not survive or proliferate in EGF and absolutely require FGF for both proliferation and survival. PDGF, an instructive molecule for cortical stem cells, had no observable effect either alone or in combination 25 with FGF. Under differentiating conditions, the mouse NEP cells differentiate into CNS neuronal cells, CNS glial cells including oligodendrocytes, A2B5-positive astrocytes, and A2B5-negative astrocytes, as well as neural crest stem cells. Like rat NRP cells, the mouse NRP cells are E-NCAM 30 immunoreactive. Further, E-NCAM appears to be expressed exclusively by the NRP cells at this stage in development as neither NEP cells nor glial precursors express E-NCAM epitopes at E12.0 stage of development. Both mouse and rat cells also proliferate in response to FGF-1 and 2 and can differentiate 35 into multiple classes of neurons. Some small differences in WO 00/68359 PCT/USOO/12446 - 15 proliferation and differentiation potential were noted as rat NRP cells appear more robust and survive over many more passages as compared to the more fragile mouse cell which could only be maintained for 10-12 passages. Further, rat NRP 5 cells appear to be more heterogeneous as assessed by Ca 2 . imaging (Kalyani et al. J. Neurosci. 1998 18:), while mouse cells show less heterogeneity and appear to mature more slowly. Mouse GRP cells of the present invention, like rat GRP 10 cells, generate oligodendrocytes and two kinds of astrocytes (A2B5*/GFAP* and A2B5*/GFAP~) . Like rat GRPs and unlike O-2A cells, mouse GRPs respond to CNTF by differentiating into astrocytes rather than oligodendrocytes. Also provided in the present invention are methods for 15 isolating pure populations of these mouse NEP from mouse neural tubes at embryonic day 8.5, as well as methods for isolating pure populations of the mouse NRP and GRP cells from mouse neural tubes at embryonic day 12.0 or from differentiating mouse embryonic stem cells. 20 To isolate pure populations of mouse NEP cells, the neural tube from a mouse embryo at embryonic day 8.5 is removed. To isolate pure populations of mouse NRP or GRP cells, the neural tube from a mouse embryo at embryonic day 12.0 is removed. In both methods, cells from the neural tube 25 are then dissociated in accordance with well known procedures such as is described in Example 2. The dissociated cells are then plated onto fibronectin coated cell culture plates in a medium containing fibroblast growth factor and chick embryo extract. The plated cells are then cultured at a temperature 30 and in an atmosphere known to be conducive to growth of most cells. Cells grown from the mouse neural tube removed at embryonic day 8.5 comprise a pure population of mouse NEP cells.

WO 00/68359 PCTIUSOO/12446 - 16 To isolate pure populations of mouse NRP cells, E-NCAM immunoreactive cells are selected from the cultured, plated cells obtained from the mouse neural tube removed at embryonic day 12.0 in accordance with well known E-NCAM immunoselection 5 procedures such as described in Example 4. To isolate pure populations of mouse GRP cells, cells which are not E-NCAM immunoreactive, but which are A2B5-immunoreactive cells are selected from the cultured, plated cells obtained from the mouse neural tube removed at embryonic day 12.0 in accordance 10 with well known A2B5 immunoselection procedures such as described in Example 4. The E-NCAM and A2B5 immunoselection procedures can also be used to isolated pure populations of mouse NRP and GRP cells from mouse ES cells which have been induced to 15 differentiated. In these methods, mouse ES cells are first plated onto dishes and exposed to conditions which induce differentiation. Various conditions which induce differentiation of ES cells have been described. In one embodiment, the ES cells are plated onto poly L-Lysine/laminin 20 double coated dishes as described in Example 3. The differentiating cells are then subjected to E-NCAM immunoselection to isolate NRP cells or A2B5 immunoselection to isolate GRP cells. Use of mouse ES cells to isolate pure populations of mouse NRP and GRP cells of the present 25 invention provides for a culture model for controlled, in vitro differentiation from a totipotent stem cell that can be readily grown and passaged over multiple generations. The mouse ES cell is amenable to perturbation, allowing the process of lineage restriction and differentiation to be 30 characterized in detail. The ability to isolate and grow purified populations of these mouse cells has multiple utilities. For example, these cells can be used in mouse models to develop new transplantation techniques, as well as therapeutically in 35 humans for transplantation in diseases characterized by WO 00/68359 PCT/USOO/12446 - 17 neuronal and/or glial degeneration. These cells are also useful in identifying new drugs which enhance survival and proliferation of these cells upon transplantation. The cells of the present invention can also be used in 5 the identification of genes specific to selected stages of development. In one embodiment, the cells can serve as a source of mRNA for generation of cDNA libraries that are specific to the stage development of the cells. The cells can also be used in the generation of cell 10 specific antibodies for use therapeutically and diagnostically. The following nonlimiting examples are provided to further illustrate the present invention. EXAMPLES 15 Example 1: Substrate preparation Fibronectin (Sigma) was diluted to a concentration of 20 pg/ml in distilled H 2 0 (Sigma). Fibronectin solution was applied to tissue culture dishes for a minimum of 4 hours. Laminin (Biomedical Technologies Inc.), used at a 20 concentration of 20 pg/ml, was dissolved in DPBS (Gibco-BRL). To prepare fibronectin-laminin double coated dishes, laminin was applied to fibronectin coated dishes and plates were incubated overnight at 40*. Excess laminin was withdrawn and the plates were rinsed with medium. 25 Example 2: Mouse cell cultures Mouse C57BI6 embryos were removed at embryonic day 8.5 and placed in a petridish containing DPBS (Gibco-BRL). The trunk segments of embryos were enzymatically treated with collagenase (Worthington; 10 mg/ml) and dispase (Boehringer 30 Mannheim; 20 mg/ml) for 10 minutes at room temperature. The enzyme solution was replaced with fresh medium with 10% chick embryo extract. The trunk segments were gently triturated to release the spinal cords from surrounding tissue. The spinal cords were then incubated in 0.05% trypsin solution for 5 35 minutes at 37 0 C. The cells were dissociated and plated in 35 WO 00/68359 PCT/USOO/12446 - 18 mm fibronectin coated dishes as mass cultures (5000 cells/dish). Cells were maintained at 37 0 C in 5% C0 2 /95% air in NEP basal medium and 10% CEE (Chicken Embryo Extract). The NEP basal medium used in all experiments described 5 herein is a chemically defined medium modified from that described by Stemple, D.L. and Anderson, D.J. (Cell 1992 71:973-985). The medium consists of DMEM-F12 (Gibco-BRL) supplemented with additives described by Bottenstein, J.E. and Sato, G. (Proc. Natl. Acad. Sci. USA. 1979 76:514-517) and 10 bFGF (25 ng/ml). The NEP complete medium is NEP basal medium with 10% CEE (Chicken Embryo Extract). Mouse C57B6 embryos were removed at embryonic day 12.0 and placed in a petridish containing DPBS. Spinal cords were mechanically dissected from surrounding connective tissue 15 using sharpened No. 5 forceps. Isolated spinal cords were incubated in 0.05% trypsin solution for 30 minutes. The trypsin solution was replaced with fresh medium. The spinal cords were gently triturated with a pasteur pipette to dissociate the cells. The dissociated cells were plated on 20 fibronectin/laminin coated dishes. Cells were maintained at 370 in 5% C0 2 /95% air in NEP complete medium. Differentiation into neural crest was promoted by adding BMP-4 (10 ng/ml) to the mouse NEP cells growing on fibronectin for a period of 4 days. The cells were then replated on 25 fibronectin/laminin coated dishes in neural crest medium [NEP basal medium supplemented with bFGF (10 ng/ml) , NGF (50 ng/ml), EGF (100 ng/ml)]. To promote maturation into Schwaan cells, dibutryl cAMP (5 pm/ml, Sigma) was added for an additional 7 days. To promote smooth muscle differentiation, 30 crest cells were grown in neural crest medium supplemented with 10% fetal bovine serum (Hyclone, Utah). Example 3: ES cell cultures Undifferentiated ES-D3 cells (ATCC) were grown as aggregates in suspension dishes (Nunc) in DMEM-F12 with 10% 35 FCS and LEF (Leukemia Inhibitory Factor, 10 ng/ml) for 4 days.

WO 00/68359 PCTIUSOO/12446 - 19 The medium was then changed to NEP basal medium and the cells were plated on fibronectin. The medium was changed every 2 days. Differentiation into NRPs and GRPs was induced by plating the cells on poly L-Lysine/laminin double coated 5 dishes. For long term neuronal differentiation FGF was withdrawn and Retinoic acid (Sigma) was added to the medium. Similarly for long term glial induction, cells were maintained in PDGF (10 ng/ml) and T3 (30 nm) with the withdrawal of FGF. Example 4: Immunopanning of E-NCAM* and A2B5* cells 10 E-NCAM* and A2B5* cells were purified from dissociated E 12.0 neural tube cells using a specific antibody capture assay (Wysocki, L. J., and Sato,V. L. Proc. Natl.Acad. Sci. USA 1978 75:2844-2848) with minor modifications. Specifically, the cells were trypsinized and 10 ml of cell suspension (Basal 15 medium and 20% FCS) was plated on a 100 mm petridish and incubated at 37 0 C in a 5% Co 2 humidified atmosphere for 2-3 hours for the maximum attachment of flat cells. The supernatant, containing an enriched population of neuronal and oligodendrocyte cells, was plated on dishes coated with either 20 E-NCAM antibody (5A5, Developmental Studies Hybridoma Bank (DSHB), 1: 1 dilution) or A2B5 antibody (ATCC, 1:1 dilution) to allow binding of all E-NCAM* and A2B5* cells. E-NCAM and A2B5 dishes were prepared by sequentially coating petridishes with an unlabeled antimouse IgM antibody (10 pg/ml) overnight, 25 rinsing 3x with DPBS, followed by incubation with E-NCAM or A2B5 hybridoma, supernatants for 1-3 hours at room temperature. Cells were allowed to bind to the dish for 1 hour at room temperature. The supernatant was discarded and the dish was washed 8x with DPBS. The bound cells were 30 mechanically scraped off and plated on fibronectin/laminin coated dishes in 1 ml of NEP basal medium either as mass (5000 cells/dish) or clonal (100 cells/dish) cultures. Growth factors were added every other day. In all cases, an aliquot of cells was tested for panning efficiency by 35 immunocytochemistry. In general, the panning efficiency was WO 00/68359 PCT/USOO/12446 - 20 greater than 90%. For E-NCAM panned OR NRP cultures, the NEP basal medium was supplemented with the following growth factors; NT3, PDGF, BDNF, and FGF (all at 10 ng/ml). For A2B5 panned or GRP cultures, the NEP basal medium was supplemented 5 with PDGF (10 ng/ml), and FGF (10 ng/ml). Example 5: Immunocytochemistry Staining procedures were in accordance with procedures described by Rao, M. S. and Mayer-Proschel, M. (Dev. Biol. 1997 188:48-63). Staining for the cell surface markers p75, 10 E-NCAM, and GalC was carried out in cultures of living cells. To stain cells with antibodies against cytoplasmic antigens, cultures were fixed with 2% formaldehyde for 20 minutes at room temperature. In general, dishes were incubated with primary antibody 15 for one hour followed by incubation with an appropriate secondary antibody for 30 minutes. Double labeling experiments were performed by simultaneously incubating cells in appropriate combinations of primary antibodies followed by non-crossreactive secondary antibodies. DAPI histochemistry 20 was performed as described by Kalyani et al.(Dev. Biol. 1997 186:202-223). DAPI (Sigma) staining was generally done after labeling had been completed. p75, E-NCAM, A2B5, and GalC were hybridoma supernatants obtained from DSHB. S-111 tubulin (Sigma), which stains neurons, nestin (monoclonal obtained 25 from DSHB; polyclonal obtained from Signal Pharmaceuticals), a marker for undifferentiated stem cells (Zimmerman et al. Neuron 1994 12(6): following 1388; Lendahl et al. Cell 1990 60:585-595; and Dahlstrand et al. Dev. Brain Res. 1995 84:109 129), MAP2 (Sigma), another neuronal marker were used as 30 described previously. Antibodies to glutamate and glycine were obtained from Signature Immunologicals and used as per manufacturer's recommendations. Antibodies to GFAP and GAD were obtained from Chemicon and used at 1:500 dilution.

WO 00/68359 PCT/USOO/12446 - 21 Example 6: BrdU incorporation To assess the proliferation of neuronal and oligodendrocyte precursor cells, 5 bromo-2'deoxyuridine (BrdU, Sigma) at a concentration of 10 ym was added to the cells for 5 24 hours. The cells were then fixed with 2% paraformaldehyde for 15 minutes at room temperature followed by 95% ethanol for 30 minutes at -20 0 C. Cells were then washed 3x with PBS and 5% goat serum, and permeabilized with 2N HCl for 10 minutes. Acid was removed by 3 washes with PBS and 5% goat serum and 10 the residual HCl was neutralized with 0.1 M sodium borate for 10 minutes. After rinsing with PBS cells were.incubated with anti-BrdU antibody (1: 100, Sigma) for 30 minutes at room temperature in buffer containing 0.5% TRITON X-100. The cells were then incubated with goat anti-mouse IgG (1:100, Jackson 15 Immunologicals)- for 30 minutes. After 3 washes with PBS, the cells were observed with a Zeiss Fluorescence microscope. Example 7: RNA extraction and cDNA synthesis Total RNA was isolated from cells or neural tubes by a modification of the guanidinium-isothiocyanate extraction 20 method (TRIzol, Gibco-BRl). cDNA was synthesized using 1-5 pg of total RNA in a 20 pl reaction. Superscript II (Gibco BRL), a modified Maloney murine leukemia virus reverse transcriptase and oligo (dT) 12-18 primers were used, and the Gibco-BRL protocol was followed. 25 Example 8: Polymerase Chain Reaction Aliquots of cDNA, equivalent to 1/20 of the above reaction, were used in a 50 yl reaction volume. PCR amplification was performed using Elongase polymerase (Gibco BRL). Primer sequences and cycling temperatures used for PCR 30 amplification of receptors are shown in the following Table. The reactions were run for 35 cycles and a 10 minute incubation at 72 0 C was added at the end to ensure complete extension.

WO 00/68359 PCT/USOO/12446 - 22 Gene Product Primers (5' to 3') SEQ ID (bp) Sense (top); NO: Antisense (bottom) p75 329 GCACATACTCAGACGAAGCCA 1 AGCAGCCAAGATGGAGCAATAGAC 2 ChAT 377 CTGAATACTGGCTGAATGACATG 3 AAATTAATGACAACATCCAAGAC 4 Is1-1 350 GCAGCATAGGCTTCAGCAAG 5 GTAGCAGGTCCGCAAGGTG 6 5 GAD 65 327 GAATCTTTTCTCCTGGTGGTG 7 GATCAAAAGCCCCGTACACAG 8 Calbindin28 276 GCAGAATCCCACCTGCAG 9 GTTGCTGGCATCGAAAGAG 10 Glutaminase 560 GCACAGACATGGTTGGGATACTAG 11 GCAGGGCTGTTCTGGAGTCG 12 Cyclophilin 302 CCACCGTGTTCTTCGACATC 13 GGTCCAGCATTTGCCATGG 14 EGF-R 205 GCTGGGGAAGAGGAGAGGAGA 15 ACGAGTGGTGGGCAGGTGTCTT 16 10 FGFR-1 764 TGGGAGCATCAACCACACCTACC 17 GCCCGAAGCAGCCCTCGCC 18 FGFR-4 672 ATCGGAGGCATTCGGCTGCG 19 AGAACTGCCGGGCCAAAGGG 20 PLP/DM20 505/400 GACATGAAGCTCTCACTGGCAC 21 CATACATTCTGGCATCAGCGC 22 MAP2 404 GAAGGAAAGGCACCACACTG 23 GCTGGCGATGGTGGTGGG 24 NF-M 186 GCCGAGCAGACCAAGGAGGCCATT 25 CTGGATGGTGTCCTGGTAGCTGCT 26 15 GFAP 346 TTGCAGACCTCACAGACGCTGCGT 27 CGGTTTTCTTCGCCCTCCAGCAAT 28 Example 9: Intracellular Ca 2 * Measurement Calcium imaging experiments were performed on clonal cultures of E-NCAM panned cells obtained from mouse E8.5 embryos as described above. Cells were loaded with 5 pm 20 Fura-2 AM (Sigma, Grynkiewics et al., 1985) plus pluronic F127 WO 00/68359 PCTIUSOO/12446 - 23 (Sigma, 80 pg/ml) in rat Ringer's solution (RR) at 23'C in the dark. Following the 20 minute incubation, cells were washed three times in RR and allowed to de-esterify for 30 minutes. Relative changes in intracellular Ca 2 * concentrations were 5 measured from the background-corrected ratio of fluorescence intensity by excitation at 340/380 nm. A response was defined as a minimum rise of 10% of the ratioed baseline value. A Zeiss-Attofluor imaging system and software (Atto Instruments Inc.) were used to acquire and analyze the data. Data points 10 were sampled at 1 Hz. Neurotransmitters (100 pM concentration) were made fresh in RR and delivered by bath exchange using a small volume loop injector (200 pl). RR consisted of (in mM) : 140 NaCl, 3 KCl, MgCl 2 , 2 CaCl 2 , 10 HEPES, and 10 glucose. Ascorbic acid (500 pM) was added to 15 dopamine solutions to prevent oxidation. Control application of 500 AM ascorbic acid had no effect. The pH of all solutions was adjusted to 7.4 with NaOH. High K* RR was made by substituting 50 mM K* for Na* in the normal RR.

Claims (8)

1. An isolated, pure population of mouse neuron restricted precursor cells derived from mouse neural tubes at embryonic day 12.0 or mouse embryonic stem cells by E-NCAM 5 immunoreactivity.

2. A method for isolating a pure population of the mouse neuron-restricted precursor cells of claim 1 comprising: (a) incubating mouse embryonic stem cells under differentiation-inducing conditions so that the cells 10 differentiate; and (b) isolating a pure population of mouse neuron restricted precursor cells by immunoselecting E-NCAM* immunoreactive cells from the differentiated cells.

3. A method for isolating a pure population of the 15 mouse neuron-restricted precursor cells of claim 1 comprising: (a) removing a neural tube from a mouse embryo at embryonic day 12.0; (b) dissociating cells from the neural tube; (c) plating the dissociated cells in a feeder-cell 20 independent culture on a substratum and in a medium containing fibroblast growth factor and chick embryo extract; (d) culturing the plated cells at a temperature and in an atmosphere conducive to cell growth; and (e) isolating a pure population of mouse neuron 25 restricted precursor cells by immunoselecting E-NCAM immunoreactive cells from the cultured, plated cells.

4. An isolated, pure population of mouse glial restricted precursor cells derived from mouse neural tubes at embryonic day 12.0 or mouse embryonic stem cells by A2B5* 30 immunoreactivity. WO 00/68359 PCT/USOO/12446 - 25

5. A method for isolating a pure population of the mouse glial-restricted precursor cells of claim 4 comprising: (a) incubating mouse embryonic stem cells under differentiation-inducing conditions so that the cells 5 differentiate; and (b) isolating a pure population of mouse glial-restricted precursor cells by immunoselecting A2B5-immunoreactive cells from the differentiated cells.

6. A method for isolating a pure population of the 10 mouse glial-restricted precursor cells of claim 4 comprising: (a) removing a neural tube from a mouse embryo at embryonic day 12.0; (b) dissociating cells from the neural tube; (c) plating the dissociated cells in a feeder-cell 15 independent culture on a substratum and in a medium containing fibroblast growth factor and chick embryo extract; (d) culturing the plated cells at a temperature and in an atmosphere conducive to cell growth; and (e) isolating a pure population of mouse glial-restricted 20 precursor cells by immunoselecting A2B5-immunoreactive cells from the cultured, plated cells.

7. An isolated, pure population of mouse neuroepithelial stem cells derived from mouse neural tubes at embryonic day 8.5 which proliferate and self renew in adherent 25 feeder-cell-independent culture medium containing fibroblast growth factor and chick embryo extract.

8. A method of isolating a pure population of the mouse neuroepithelial stem cells of claim 7 comprising: (a) removing a neural tube from a mouse embryo at 30 embryonic day 8.5; (b) dissociating cells from the neural tube; WO 00/68359 PCT/USOO/12446 - 26 (c) plating the dissociated cells in a feeder-cell independent culture on a substratum and in a medium containing fibroblast growth factor and chick embryo extract; and (d) culturing the plated cells at a temperature and in 5 an atmosphere conducive to cell growth to obtain a pure population of mouse neuroepithelial stem cells.