WO1998048001A1 - Procedes de differenciation de cellules neurales souches - Google Patents

Procedes de differenciation de cellules neurales souches Download PDF

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WO1998048001A1
WO1998048001A1 PCT/US1998/008364 US9808364W WO9848001A1 WO 1998048001 A1 WO1998048001 A1 WO 1998048001A1 US 9808364 W US9808364 W US 9808364W WO 9848001 A1 WO9848001 A1 WO 9848001A1
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cells
cell
neural crest
neural
neurons
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PCT/US1998/008364
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WO1998048001A9 (fr
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David J. Anderson
Nirao M. Shah
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California Institute Of Technology
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Priority claimed from US08/846,028 external-priority patent/US6001654A/en
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Priority to AU72580/98A priority Critical patent/AU7258098A/en
Publication of WO1998048001A1 publication Critical patent/WO1998048001A1/fr
Publication of WO1998048001A9 publication Critical patent/WO1998048001A9/fr

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Definitions

  • the invention relates to methods for the differentiation of mammalian multipotent neural stem cells to neurons or smooth muscle cells.
  • the neural crest is a transient embryonic precursor population, whose derivatives include cells having widely different mo ⁇ hologies, characteristics and functions. These derivatives include the neurons and glia of the entire peripheral nervous system, melanocytes, cartilage and connective tissue of the head and neck, stroma of various secretory glands and cells in the outflow tract of the heart (for review, see Anderson, D.J. (1989) Neuron 3: 1- 12).
  • the neural crest generates most of the peripheral nervous system (PNS), skin melanocytes, and mesectodermal derivatives such as smooth muscle (SM) cells, bone, and cartilage Horstadius, S. , London; Oxford University Press (1950); Le Douarin et al. (1994) Curr. Opin. Genet.
  • culture conditions which allow the growth and self-renewal of mammalian neural crest stem cells are desirable so that the particulars of the development of these mammalian stem cells may be ascertained.
  • This is desirable because a number of tumors of neural crest derivatives exist in mammals, particularly humans. Knowledge of mammalian neural crest stem cell development is therefore needed to understand these disorders in humans.
  • the ability to isolate and grow mammalian neural crest stem cells in vitro allows for the possibility of using said stem cells to treat peripheral neurological disorders in mammals, particularly humans.
  • the ability to preferentially differentiate neural stem cells allows for a number of treatments which require the growth or regeneration of damaged, injured or deficient neurons or smooth muscle cells. It is therefore an object of the invention to provide methods utilizing growth factors from the TGF- ⁇ superfamily to preferentially differentiate neural stem cells to neurons and/or smooth muscle cells.
  • the invention includes a method for producing a population of mammalian neurons and/or smooth muscle cells comprising contacting at least one mammalian neural stem cell with a culture medium containing one or more growth factors from the TGF- ⁇ superfamily.
  • the growth factor may be contained within an extract from mammalian tissue or may be substantially pure.
  • the growth factor or combination of growth factors may administered to cells in vivo or ex vivo.
  • the growth factors include the group of biologically active polypeptides which control the growth and differentiation of responsive cells. Examples of these growth factors include but are not limited to; the TGF- ⁇ series of growth factors as exemplified by TGF- ⁇ l-TGF- ⁇ 4, the BMP series of growth factors as exemplified by BMP-2 and BMP-4.
  • Figure 1 A depicts the migration of rat neural crest cells from the neural tube.
  • Figure IB demonstrates the expression of LNGFR and nestin by neural crest cells.
  • Figures 1C and ID show the FACS profile from neural crest cells stained with anti-LNGFR (ID) and a control showing the background staining of the secondary antibody (1C).
  • Figure 2 demonstrates the clonal expansion of LNGFR + , nestin + rat neural crest cells.
  • Figure 3 is a flow chart summarizing experiments demonstrating the multipotency of mammalian neural crest cells.
  • Figure 4 demonstrates the expression of neuronal traits in clones derived from LNGFR + founder cells.
  • Figure 5 demonstrates the expression of Schwann cell phenotype by neural crest-derived glia.
  • Figure 6 shows the expression of peripherin, GFAP, and O 4 in a clone derived from a LNGFR + founder cell.
  • Figure 7 is a flow chart summarizing experiments demonstrating the self- renewal of mammalian neural crest cells.
  • Figure 8 demonstrates the self-renewal of multipotent neural crest cells.
  • Figure 9 demonstrates the multipotency of secondary founder cells.
  • Figure 10 provides a flow chart summarizing experiments demonstrating the substrate effect on the fate of mammalian neural crest cells.
  • Figure 11 demonstrates that the neuronal differentiation of multipotent neural crest cells is affected by their substrate.
  • Figure 12 summarizes the percentage of different clone types which result when founder cells are grown on either FN or FN/PDL substrates.
  • Figure 13 provides a flow chart summarizing experiments demonstrating the instructive effect of the substrate on neural crest cell fate.
  • Figure 14 summarizes the percentage of the different clone types which result when founder cells are treated with a PDL lysine overlay at 48 hours (panel A) or day 5 (panel B).
  • Figure 15 demonstrates the genetic-engineering of a multipotent neural stem cell.
  • Panel A depicts the expression of E. coli ⁇ -galactosidase (lacZ) in neural crest stem cells following infection with a lacZ-containing retrovirus. ⁇ -galactosidase + cells are indicated by the solid arrows.
  • Panel B depicts neural crest stem cells in phase contrast, in the same microscopic field as shown in Panel A. Cells which do not express ⁇ -galactosidase are indicated by open arrows.
  • Figure 16 demonstrates the specificity of a supernatant from a hybridoma culture producing monoclonal antibody specific to mouse LNGFR. Supernatants were screened using live Schwann cells isolated from mouse sciatic nerve.
  • Panel A shows that most cells are stained with anti-LNGFR antibody (red staining; open arrows).
  • Panel B shows Schwann cell nuclei counter stained with DAPI. Comparison with Panel A reveals a few cells not labeled by anti-LNGFR antibody (blue staining; open arrows).
  • Figures 17 A and B depict the identification of smooth muscle cells in neural crest cultures.
  • Cultures of neural crest stem cells were fixed and double- labeled with antibodies to p75-LNGFR (Panel B, orange staining), and SMA (Panel B, green staining).
  • the cultures were also labeled with DAPI, a nuclear dye (Panel B, blue ovals).
  • DAPI a nuclear dye
  • a phase contrast image of the microscopic field is shown in Figure 17A. Note that the p75 + cells (Panel B, solid arrow) do not express SMA ⁇ whereas the SMA+ cells (Panel B, open arrows) do not express p75.
  • Figures 18 A and B demonstrate that individual neural cres cells can generate neurons, glia and smooth muscle cells.
  • the figures illustrate three views of a clone derived from a single p75+ neural crest founder cell, grown for two weeks in standard medium.
  • a neuron is identifiable in the clone by virtue of peripherin expression (Panel B, arrowhead) and long neurites (Panel A).
  • Glia are identifiable by GFAP expression (Panel C, orange staining, open arrows), and a smooth muscle cell is identified by staining with anti-SMA (Panel C, green staining, closed arrow). Nuclei of all cells have been labeled blue with DAPI (Panel C).
  • Figures 19 A, B and C demonstrate that smooth muscle cell differentiation is promoted by fetal bovine serum. Shown are three views of a colony of neural crest cells grown in 5% fetal bovine serum. These cells do not express p75-LNGFR under these conditions. Cells visible by phase-contrast (Panel A) express both SMA (Panel B, red staining) and also desmin (Panel C, green staining).
  • Figures 20 A and B demonstrate that neural crest-derived smooth muscle cells express calponin.
  • the culture is similar to that in Fig. 19, except the cells were doubly-labeled with anti-SMA (Panel B, red staining) and calponin (Panel B, green staining). Cells that co-express both markers stain orange due to blending of the two colors (Panel B).
  • Figure 21 demonstrates that bone marrow phogenic protein 2 (“BMP2”) induces autonomic neuronal differentiation of NCSCs.
  • NCSCs were cultured in rBMP2 (Panels A-D) or control medium (Panels E and F), fixed and immunostained with antibody to peripherin (Panels B and F) or monoclonal antibody B2 (Panel D) followed by phycoerythrin conjugated second antibody and DAPI counterstain.
  • Phase contrast views presented by way of comparison indicate neuronal phase-bright colonies in Panel A, which are peripherin + (Panel B)in contrast to the colonies in Panels E and F which are peripherin- resemble undifferentiated neurons.
  • Figure 22 demonstrates that recombinant BMP2 or TGF ⁇ induce differentiation of NCSCs to smooth muscle.
  • NCSCs were cultured in rBMP2(Panels A-C) or rTGF ⁇ (Panels D-F), fixed and triply-labeled with peripherin (second antibody bound to HRP), anti-SMA (green staining) and calponin (red staining). Cells that co-express both SMA ans calponin stain counterstain. Phase contrast views are presented for purposes of comparison in Panels A and D. 25% of colonies in rBMP-2 contained SM and neurons (Panels A-C) while 99% of colonies in contained SM-like cells (Panels D-F).
  • Figure 23 demonstrates that recombinant BMP2 and TGF- ⁇ induce distinct responses over a wide range of doses.
  • Cells cultured in rBMP2(Panels A and B) or rTGF- ⁇ (Panel C) were fixed 4 days after the addition of factors, stained for the markers indicated, and the proportion of different colony types determined.
  • rBMP2 both neuron-only and SM-only colonies are obtained at essentially all doses.
  • Figure 24 demonstrates instructive effects of BMP2 and TGF ⁇ l based on serial observation of identified clones. Individual founder cells were identified and photographed at day 0 (DO) in control medium, and then rBMPS (Panel B) or rTGF ⁇ 1 (Panel C) was added to some plates while others were maintained as controls (Panel A) . The same clones were photographed every 24 hr for the next 4 days (Dl, D2, etc.) In rBMP2
  • Figure 25 demonstrate that BMP2 induces MASH1 expression in NCSCs based on labeling with an anti-MASHl monoclonal antibody.
  • NCSCs were grown at clonal density in rBMP2 (Panels A and B) or in control medium (Panels C and D) for 12 hr, then fixed and labeled with anti-MASHl monoclonal antibody.
  • rBMP2 Panels A and B
  • control medium Panels C and D
  • the invention is directed, in part, to the isolation and clonal propagation of non-transformed mammalian neural crest stem cells and to multipotent neural stem cells from other embryonic and adult tissue.
  • the invention also includes the production of neural crest stem cell and multipotent neural stem cell derivatives including progenitor and more differentiated cells of the neuronal and smooth muscle lineages.
  • the invention is illustrated using neural crest stem cells isolated from the rat.
  • the invention encompasses all mammalian neural crest stem cells and multipotent neural stem cells and their derivatives and is not limited to neural crest stem cells from the rat.
  • Mammalian neural crest stem cells and multipotent neural stem cells and their progeny can be isolated from tissues from human and non- human primates, equines, canines, felines, bovines, porcines, lagomorphs, etc.
  • the invention encompasses several important methodological innovations: 1) the use of monoclonal antibodies to the low-affinity Nerve Growth Factor Receptor (LNGFR) as a cell surface marker to isolate and identify neural crest stem cells, a method extensible to other neural stem cell populations as well; 2) the development of cell culture substrates and medium compositions which permit the clonal expansion of undifferentiated neural crest cells; 3) the development of culture substrates and medium compositions which permit the differentiation of mammalian neural crest cells into their differentiated derivatives (including but not restricted to peripheral neurons and smooth muscle cells) in clonal culture.
  • LNGFR Low-affinity Nerve Growth Factor Receptor
  • the invention also provides neural crest stem cells and other multipotent neural stem cells. It is important to understand that such cells could not be identified as stem cells without the development of the isolation and cell culture methodologies summarized above.
  • the identification of a neural stem cell requires that several criteria be met: 1) that the cell be an undifferentiated cell capable of generating one or more kinds of differentiated derivatives; 2) that the cell have extensive proliferative capacity; 3) that the cell be capable of self-renewal or self-maintenance (Hall et al. (1989) Development 106:619; Potten et al. (1990) Crypt. Development 110: 1001).
  • a stem cell as obligatorily capable of "unlimited” self-renewal is applicable only to regenerating tissues such as skin or intestine. In the case of a developing embryo stem cells may have limited self-renewal capacity but be stem cells nevertheless. Potten et al. (1990) Crypt. Development 110: 1001.
  • the development of clonal culture methods permitted the demonstration of criteria 1 and 2 herein.
  • the development of sub-clonal culture methods i.e. , the ability to clone single neural stem cells, and then re-clone progeny cells derived from the original founder cell further permitted the demonstration herein of criterion 3.
  • neural crest cell is a progenitor cell but not a stem cell, because it does not have self-renewal capacity. If this were the case, then upon sub-cloning of neural crest cell clones, the resulting "secondary" clones could contain either neurons or smooth muscle cells, but not both. This is not observed.
  • non-transformed cells means cells which are able to grow in vitro without the need to immortalize the cells by introduction of a virus or portions of a viral genome containing an oncogene(s) which confers altered growth properties upon cells by virtue of the expression of viral genes within the transformed cells. These viral genes typically have been introduced into cells by means of viral infection or by means of transfection with DNA vectors containing isolated viral genes.
  • the term "genetically-engineered cell” refers to a cell into which a foreign (i.e. , non-naturally occurring) nucleic acid, e.g. , DNA, has been introduced.
  • the foreign nucleic acid may be introduced by a variety of techniques, including, but not limited to, calcium-phosphate-mediated transfection, DEAE-mediated transfection, microinjection, retroviral transformation, protoplast fusion and lipofection.
  • the genetically-engineered cell may express the foreign nucleic acid in either a transient or long-term manner. In general, transient expression occurs when foreign DNA does not stably integrate into the chromosomal DNA of the transfected cell. In contrast, long-term expression of foreign DNA occurs when the foreign DNA has been stably integrated into the chromosomal DNA of the transfected cell.
  • an “immortalized cell” means a cell which is capable of growing indefinitely in culture due to the introduction of an "immortalizing gene(s)" which confers altered growth properties upon the cell by virtue of expression of the immortalizing gene(s) within the genetically engineered cell.
  • Immortalizing genes can be introduced into cells by means of viral infection or by means of transfection with vectors containing isolated viral nucleic acid encoding one or more oncogenes. Viruses or viral oncogenes are selected which allow for the immortalization but preferably not the transformation of cells.
  • Immortalized cells preferably grow indefinitely in culture but do not cause tumors when introduced into animals.
  • transformed cell refers to a cell having the properties of 1) the ability to grow indefinitely in culture and 2) causing tumors upon introduction into animals. "Transformation” refers to the generation of a transformed cell.
  • the term "feeder-cell independent culture” or grammatical equivalents means the growth of cells in vitro in the absence of a layer of different cells which generally are first plated upon a culture dish to which cells from the tissue of interest are added.
  • the "feeder” cells provide a substratum for the attachment of the cells from the tissue of interest and additionally serve as a source of mitogens and survival factors.
  • the feeder- cell independent cultures herein utilize a chemically defined substratum, for example fibronectin (FN) or poly-D-lysine (PDL) and mitogens or survival factors are provided by supplementation of the liquid culture medium with either purified factors or crude extracts from other cells or tissues.
  • FN fibronectin
  • PDL poly-D-lysine
  • the cells in the culture dish are primarily cells derived from the tissue of interest and do not contain other cell types required to support the growth of the cells derived from the tissue of interest.
  • the term "clonal density” means a density sufficiently low enough to result in the isolation of single, non-impinging cells when plated in a culture dish, generally about 225 cells/100 mm culture dish.
  • the term "neural crest stem cell” means a cell derived from the neural crest which is characterized by having the properties (1) of self- renewal and (2) asymmetrical division; that is, one cell divides to produce two different daughter cells with one being self (renewal) and the other being a cell having a more restricted developmental potential, as compared to the parental neural crest stem cell.
  • each cell division of a neural crest stem cell gives rise to an asymmetrical division. It is possible that a division of a neural crest stem cell can result only in self-renewal, in the production of more developmentally restricted progeny only, or in the production of a self- renewed stem cell and a cell having restricted developmental potential.
  • multipotent neural stem cell refers to a cell having properties similar to that of a neural crest stem cell but which is not necessarily derived from the neural crest. Rather, as described hereinafter, such multipotent neural stem cells can be derived from various other tissues including neural epithelial tissue from the brain and/or spinal cord of the adult or embryonic central nervous system or neural epithelial tissue which may be present in tissues comprising the peripheral nervous system. In addition, such multipotent neural stem cells may be derived from other tissues such as lung, bone and the like utilizing the methods disclosed herein.
  • a neural crest stem cell is a multipotent neural stem cell derived from a specific tissue, i.e. , the embryonic neural tube.
  • neural crest stem cells are further characterized by a neural cell-specific surface marker.
  • Such surface markers in addition to being found on neural crest stem cells may also be found on other multipotent neural stems derived therefrom, e.g. , glial and neuronal progenitor cells of the peripheral nervous system (PNS) and central nervous system (CNS).
  • PNS peripheral nervous system
  • CNS central nervous system
  • An example is the cell surface expression of a nerve growth factor receptor on neural crest stem cells. In rat, humans and monkeys this nerve growth factor receptor is the low-affinity nerve growth factor receptor (LNGFR).
  • Such stem cells may also be characterized by the expression of nestin, an intracellular intermediate filament protein.
  • Neural crest stem cells may be further characterized by the absence of markers associated with mature PNS neuronal or glial cells.
  • this nerve growth factor receptor is the low-affinity nerve growth factor receptor (LNGFR) .
  • LNGFR low-affinity nerve growth factor receptor
  • Such stem cells may also be characterized by the expression of nestin, an intracellular intermediate filament protein.
  • Neural crest stem cells may be further characterized by the absence of markers associated with mature PNS neuronal or, glial cells. In the rat, such markers include sulfatide, glial fibrillary acidic protein (GFAP) and myelin protein P 0 in PNS glial cells, and peripherin and neuro filament in PNS neuronal cells.
  • markers include sulfatide, glial fibrillary acidic protein (GFAP) and myelin protein P 0 in PNS glial cells, and peripherin and neuro filament in PNS neuronal cells.
  • LNGFR is a receptor for nerve growth factor, a neurotrophic factor shown to be responsible for neuronal survival in vivo.
  • LNGFR is found on several mammalian cell types including neural crest cells and Schwann cells (glial cells of the PNS) as well as on the surface of cells in the ventricular zone throughout the embryonic central nervous systems.
  • neural crest cells and Schwann cells glial cells of the PNS
  • Schwann cells glial cells of the PNS
  • Neuron 5:283- 296 which studied such cells in the rat and chick systems, respectively.
  • Antibodies specific for LNGFR have been identified for LNGFR from rat monoclonal antibodies 217c (Peng, W.W. et al.
  • monoclonal antibodies specific for LNGFR from any desired mammalian species are generated by first isolating the nucleic acid encoding the LNGFR protein.
  • One protocol for obtaining such nucleic acid sequences uses one or more nucleic acid sequences from a region of the LNGFR gene which is highly conserved between mammalian species, e.g. , rat and human, as a hybridization probe to screen a genomic library or a cDNA library derived from mammalian tissue from the desired species (Sambrook, J. et al. (1989) Cold Spring Harbor Laboratory Press. Molecular Cloning: A Laboratory Manual. 2nd Ed. , pp. 8.3-8.80, 9.47-9.58 and 11.45-11.55).
  • the cloned LNGFR sequences are then used to express the LNGFR protein or its extracellular (ligand binding) domain in an expression host from which the LNGFR protein is purified. Purification is performed using standard techniques such as chromatography on gel filtration, ion exchange or affinity resins.
  • the purified LNGFR is then used to immunize an appropriate animal (e.g. , mouse, rat, rabbit, hamster) to produce polyclonal antisera and to provide spleen cells for the generation of hybridoma cell lines secreting monoclonal antibodies specific for LNGFR of the desired species (Harlow, E. et al. (1988) Cold Spring Harbor Laboratory Press, Antibodies: A Laboratory Manual, pp. 139-242).
  • a novel screening method can be used to detect the production of antibody against LNGFR or any other surface marker which characterizes a multipotent neural stem cell or progeny thereof.
  • the method can be practiced to detect animals producing polyclonal antibodies against a particular antigen or to identify and select hybridomas producing monoclonal antibodies against such antigens.
  • serum from an immunized animal or supernatent from a hybridoma culture is contacted with a live neural cell which displays a surface marker characteristic of a particular neural cell line.
  • a particularly preferred method is to use labeled antibody which is specific for the immunoglobulins produced by the species which is immunized with the particular antigen and which is a source for polyclonal serum and spleen cells for hybridoma formation.
  • the live neural cell used in the foregoing antibody assay is dependent upon the particular surface marker for which an antibody is desired.
  • a monoclonal antibody for mouse LNGFR was identified using a dissociated primary culture of Schwann cells.
  • mouse fibroblasts acted as a negative control.
  • primary cultures of other cell lines can be used to detect monoclonal antibodies to LNGFR.
  • forebrain cholinergic neurons or sensory neurons can be used.
  • a primary culture of epithelial cells can be used as a negative control.
  • PDGFR Platelet Derived Growth Factor Receptor
  • FGF Fibroblast Growth Factor
  • SCFR Stem Cell Factor Receptor
  • Monoclonal antibodies against an antigenic determinant from one species may react against that antigen from more than one species.
  • the antibody directed against the human LNGFR molecule also recognizes LNGFR on monkey cells.
  • Nestin a second marker in the neural crest stem cell, is an intermediate filament protein primarily located intracellular ly, which has been shown to be present in CNS neuroepithelial cells and Schwann cells in the peripheral nervous system of rats (Friedman et al. (1990) J. Comp. Neurol. 295:43-51). Monoclonal antibodies specific for rat nestin have been isolated: Rat 401, (Hockfield, S. et al. (1985) J. Neurosci. 5(72):3310-3328V A polyclonal rabbit anti-nestin antisera has been reported which recognizes mouse nestin. Reynolds, D.A. et al. (1992) Science 255: 1707-1710).
  • the DNA sequences encoding the rat nestin gene have been cloned. Lendahl, U. et al. (1990) Cell 60:585-595). These DNA sequences are used to isolate nestin clones from other mammalian species. These DNA sequences are then used to express the nestin protein and monoclonal antibodies directed against various mammalian nestins are generated as described above for LNGFR.
  • Glial fibrillary acidic protein is an intermediate filament protein specifically expressed by astrocytes and glial cells of the CNS and by Schwann cells, the glial cells of the PNS (Jessen, K.R. et al. (1984) . Neurocvtologv 13:923-934 and Fields, K.L. et al. (1989) J. Neuroimmuno. 8:311-330). Monoclonal antibodies specific for GFAP have been reported (Debus et al. (1983) Differentiation 25: 193-203). Mouse and human GFAP genes have been cloned (Cowan, N.J. et al. (1985) N.Y. Acad. Sci.
  • neuronal progenitor cell refers to a cell which is intermediate between the fully differentiated neuronal cell and a precursor multipotent neural stem cell from which the fully differentiated neuronal cell develops.
  • neuronal progenitor cells are derived according to the methods described herein for isolating such cells from various tissues including adult and embryonic CNS and PNS tissue as well as other tissues which may potentially contain such progenitors.
  • PNS neuronal progenitor cell means a cell which has differentiated from a mammalian neural crest stem cell which is committed to one or more PNS neuronal lineages and is a dividing cell but does not yet express surface or intracellular markers found on more differentiated, non-dividing PNS neuronal cells.
  • Such progenitor cells are preferably obtained from neural crest stem cells isolated from the embryonic neural crest which have undergone further differentiation. However, equivalent cells may be derived from other tissue.
  • PNS neuronal progenitor cells When PNS neuronal progenitor cells are placed in appropriate culture conditions they differentiate into mature PNS neurons expressing the appropriate differentiation markers, for example, peripherin, neurofilament and high-polysialic acid neural cell adhesion molecule (high PSA-NCAM).
  • peripherin a 57 kDa intermediate filament protein, is expressed in adult rodents primarily in peripheral neurons. More limited expression of peripherin is found in some motoneurons of the spinal cord and brain stem and a limited group of CNS neurons. Peripherin is expressed in rat embryos primarily in neurons of peripheral ganglia and in a subset of ventral and lateral motoneurons in the spinal cord. Gorham, J.D. et al. (1990) Dev.
  • Neurofilaments are neuron-specific intermediate filament proteins.
  • Three neurofilament (NF) proteins have been reported: NF68, a 68 kD protein also called NF-L (Light); NF160, a 160 kD protein also called NF-M (Medium); NF200, a 200 kD protein also called NF-H (Heavy).
  • NF68 neurofilament
  • NF-L Light
  • NF160 a 160 kD protein also called NF-M (Medium)
  • NF200 a 200 kD protein also called NF-H (Heavy).
  • the DNA sequences encoding the rat NF200 and NF160 proteins have been cloned (Dautigny, A. et al. (1988) Biochem. Biophvs. Res. Commun. 154: 1099- 1106 and Napolitano, E.W. et al. (1987) J.
  • mice and humans All three NF protein genes have been cloned in mice and humans.
  • Mouse NF68 nucleic acid sequences were reported in Lewis, S.A. et al. (1985) J. Cell Biol. 700:843-850.
  • Mouse NF160 nucleic acid sequences were reported in Levy, E. et al. (1987) Eur. J. Biochem. 166:71- 77.
  • Mouse NF200 nucleic acid sequences were reported in Shneidman, P.S. et al. (1988) Mol. Brain Res. 4:217-231. In humans, nucleic acid sequences were reported for: NF68, Julien, J.-P. et al.
  • NF + means expression of one or more of the three NF proteins.
  • factors permissive for PNS neuronal cell differentiation means compounds, such as, but not limited to, protein or steroid molecules or substrates such as FN or PDL, which permit at least a neural crest stem cell to become restricted to the PNS neuronal lineage.
  • Such lineage-restricted progeny of neural crest stem cells include PNS neuronal progenitor cells, which are at least bipotential, in that they can divide to give rise to self, as well as, more mature, non-dividing PNS neurons.
  • growth factors from the TGF- ⁇ superfamily means growth factors related to transforming growth factor beta-1 ("TGF ⁇ - 1 "). Such TGF- ⁇ superfamily growth factors may or may not exert a similar biological effect to TGF ⁇ -1, the prototypic member of the TGF- ⁇ superfamily.
  • TGF ⁇ - 1 transforming growth factor beta-1
  • rTGF- ⁇ l transforming growth factor beta-1
  • TGF ⁇ 2 and TGF ⁇ 3 yielded similar results as TGF ⁇ l .
  • members of the TGF- ⁇ superfamily of growth factors include but are not limited to naturally occurring analogues (e.g. TGF ⁇ -2, ⁇ -3, ⁇ 4), and any known synthetic or natural analogues of TGF ⁇ -1 in addition to related growth factors exemplified by bone marrow phogenic proteins 2 and 4
  • BMP-2 and "BMP-4". These compounds can be purified from natural sources or may be produced by recombinant DNA techniques and may or may not be substantially pure. Variants and fragments retaining the property of causing differentiation are included in the definition of the members of this superfamily.
  • bone marrow phogenic protein refers to a group of growth factors which are members of the TGF- ⁇ superfamily. Under comparable culture conditions BMP2 and BMP4 produced neurons and some SM cells, while TGF ⁇ l produced only SM cells. Shah et al.(1996) Cell 85:331-343. As indicated in the examples, when neural stem cells are contacted with certain factors permissive for neuronal and glial cell differentiation, such cells differentiated into neurons, glia and a subpopulation referred to as "O" cells. As disclosed in Example 10, these O cells are, in fact, smooth muscle cells.
  • the factors which are permissive for differentiation to neuronal and/or glial cells are also permissive for the differentiation of neural stem cells to smooth muscle cells.
  • the growth factors described herein can be administered individually or in combination with each other.
  • the term "instructive factor” or grammatical equivalents refers to one or more factors which are capable of causing the differentiation of neural stem cells primarily to a single lineage, e.g. , glial, neuronal or smooth muscle cell.
  • a factor which is instructive for smooth muscle cell differentiation is one which causes differentiation of neural stem cells to smooth muscle cells at the expense of the differentiation of such stem cells into other lineages such as glial or neuronal cells.
  • mammalian serum contains one or more factors which are instructive factors for the production of smooth muscle cells.
  • mammalian serum contains one or more instructive factors for smooth muscle cell differentiation
  • instructive factors can be identified by fractionating mammalian serum and adding back one or more such fractions to a neural stem cell culture to identify one or more fractions containing instructive factors for smooth muscle cell differentiation. Positive fractions can then be further fractionated and reassayed until the one or more components required for instructive differentiation to smooth muscle cells are identified.
  • Mammalian neural crest stem cell compositions which serve as a source for neural crest cell derivatives such as neuronal and glial progenitors of the PNS which in turn are a source of PNS neurons and glia.
  • Methods are provided for the isolation and clonal culture of neural crest stem cells, in the absence of feeder cells. In the examples provided, these methods utilize a chemically defined medium which is supplemented with chick embryo extract as a source of mitogens and survival factors. Factors present in the extract of chicken embryos allow the growth and self renewal of rat neural crest stem cells.
  • media used to isolate and propagate rat neural crest stem cells can be used to isolate and propagate neural crest stem cells from other mammalian species, such as human and non-human primates, equines, felines, canines, bovines, porcines, lagomo ⁇ hs, etc.
  • Culture conditions provided herein allow the isolation self-renewal and differentiation of mammalian neural crest stem cells and their progeny. These culture conditions may be modified to provide a means of detecting and evaluating growth factors relevant to mammalian neural crest stem cell self-renewal and the differentiation of the stem cell and its progeny. These modifications include, but are not limited to, changes in the composition of the culture medium and/or the substrate and in the specific markers used to identify either the neural crest stem cell or their differentiated derivatives. Culture conditions are provided which allow the differentiation of mammalian neural crest stem cells into the PNS neuronal, glial and smooth muscle lineages in the absence of feeder cell layers.
  • these culture conditions utilize a substratum comprising fibronectin alone or in combination with poly-D-lysine.
  • human fibronectin is utilized for the culturing of rat neural crest stem cells and their progeny.
  • Human fibronectin can be used for the culturing of neural crest stem cells isolated from avian species as well as from any mammal, as the function of the fibronectin protein is highly conserved among different species. Cells of many species have fibronectin receptors which recognize and bind to human fibronectin.
  • neural crest stem cells In order to isolate the subject neural crest stem cells, it is necessary to separate the stem cell from other cells in the embryo. Initially, neural crest cells are obtained from mammalian embryos.
  • the region containing the caudal-most 10 somites are dissected from early embryos (equivalent to gestational day 10.5 day in the rat). These trunk sections are transferred in a balanced salt solution to chilled depression slides, typically at 4°C, and treated with collagenase in an appropriate buffer solution such as Howard's Ringer's solution. After the neural tubes are free of somites and notochords, they are plated onto fibronectin (FN)-coated culture dishes to allow the neural crest cells to migrate from the neural tube.
  • FN fibronectin
  • the crest cells are removed from the FN-coated plate by treatment with a Trypsin solution, typically at 0.05% .
  • the suspension of detached cells is then collected by centrifugation and plated at an appropriate density, generally 225 cells/ 100mm dish in an appropriate chemically defined medium.
  • This medium is preferentially free of serum and contains components which permit the growth and self-renewal of neural crest stem cells.
  • the culture dishes are coated with an appropriate substratum, typically a combination of FN and poly-D-lysine (PDL).
  • Procedures for the identification of neural crest stem cells include incubating cultures of crest cells for a short period of time, generally 20 minutes, at room temperature, generally about 25 °C, with saturating levels of antibodies specific for a particular marker, e.g. , LNGFR. Excess antibody is removed by rinsing the plate with an appropriate medium, typically L15 medium (Gibco) supplemented with fresh vitamin mix and bovine serum albumin (L- 15 Air). The cultures are then incubated at room temperature with a fluorochrome labeled secondary antibody, typically Phycoerythrin R- conjugated secondary antibody (TAGO) at an appropriate dilution for about 20 minutes. Excess secondary antibodies are then removed using an appropriate medium, such as L-15 Air.
  • an appropriate medium typically L15 medium (Gibco) supplemented with fresh vitamin mix and bovine serum albumin (L- 15 Air).
  • TAGO Phycoerythrin R- conjugated secondary antibody
  • the plates are then covered with the chemically defined growth medium and examined with a fluorescence microscope.
  • Individual LNGFR + clones are isolated by fluorescence activated cell sorting (FACS) or, more typically, by marking the plate under the identified clone. The markings are typically made to a diameter of 3-4 mm, which generally allows for the unambiguous identification of the progeny of the founder cell at any time during an experiment. If desired, individual LNGFR + clones are removed from the original plate by trypsinization with the use of cloning cylinders.
  • Procedures for permitting the differentiation of stem cells include the culmring of isolated stem cells in a medium permissive for differentiation to a desired lineage, such as Schwann cell differentiation (SCD) medium. Other procedures include growth of isolated stem cells on substrates capable of permitting differentiation, such as FN or FN and PDL. Procedures for the serial subcloning of stem cells and their derivatives include the trypsinization of individual clones, as described above, followed by replating the clone on a desired substrate and culturing in a desired medium, such as a chemically defined medium suitable for maintenance of stem cells or SCD medium permissive for the differentiation of said neural crest stem cells. Crest cells may be identified following serial subcloning by live-cell labeling with an antibody directed against LNGFR, as described above.
  • neurogulin/GGF can instructively influence multipotent, self-renewing rodent neural crest stem cells(NCSCs) (Stemple and Anderson (1992) Cell 71 :973-985) to differentiate to glia in vitro (Shah et al. (1994) Cell 77:349-360; while this study demonstrated that the fate could be promoted by an environmental signal, it left open the question of how alternative fates might be chosen.
  • the neuronal fate of NCSCs might be promoted by other extrinsic cues.
  • neural crest cells might be predisposed to select a neuronal fate in the absence of extrinsic influence.
  • EPO erythropoietin
  • the methods described herein provide the basis of functional assays which allow for the identification and production of cellular compositions of mammalian cells which have properties characteristic of neural crest stem cells, glial, neuronal, smooth muscle progenitor cells or multipotent stem cell precursor of such progenitor cells.
  • tissue other than embryonic neural tubes it is necessary to separate the progenitor and/or multipotent stem cells from other cells in the tissue.
  • the methods presented in the examples for the isolation of neural crest stem cells from neural tubes can be readily adapted for other tissues by one skilled in the art.
  • a single cell suspension is made from the tissue; the method used to make this suspension will vary depending on the tissue utilized. For example, some tissues require mechanical disruption of the tissue while other tissues require digestion with proteolytic enzymes alone or in combination with mechanical disruption in order to create the single cell suspension.
  • Tissues such as blood already exists as a single cell suspension and no further treatment is required to generate a suspension, although hypotonic lysis of red blood cells may be desirable.
  • the single cell suspension Once the single cell suspension is generated it may be enriched for cells expressing LNGFR or other neural cell-specific markers on their surface.
  • One protocol for the enrichment for LNGFR + cells is by incubating the cell suspension with antibodies specific for LNGFR and isolating the LNGFR + cells. Enrichment for cells expressing a neural cell-specific surface marker is particularly desirable when these cells represent a small percentage (less than 5%) of the starting population.
  • the isolation of cells which have complexed with an antibody for a neural cell-specific surface marker such as is carried out using any physical method for isolating antibody-labeled cells.
  • Such methods include fluorescent-activated cell sorting in which case the cells, in general, are further labeled with a fluorescent secondary antibody that binds the anti- LNGFR antibody, e.g. , mouse anti-LNGFR and fluorescein label goat anti- mouse IgG; panning in which case the antibody-labeled cells are incubated on a tissue-culture plate coated with a secondary antibody; Avidin-sepharose chromatography in which the anti-LNGFR antibody is biotinylated prior to incubation with the cell suspension so that the complexed cells can be recovered on an affinity matrix containing avidin (i.e.
  • the cells are plated at clonal density, generally 225 cells/lOO mm dish, in an appropriate chemically defined medium on a suitable substrate as described in the examples for isolation of rat neural crest stem cells.
  • neural crest-like stem cells e.g. , a multipotent neural stem cell
  • Other types of multipotent neural stem cells are identified by differentiation to other cell types such as CNS neural or glial cells or their progenitors.
  • multipotent neural stem cells may not be obtained. Rather, further differentiated cell types such as glial, neuronal or smooth muscle progenitor cells may be obtained.
  • SM cells are one of the normal derivatives of the neural crest, although in avians they derive from an anterior region of the neural crest (the cardiac neural crest; Kirby (1987) Pediatr. Res. 21:219-224), rather than from the trunk region (which corresponds to the region from which our NCSCs are obtained).
  • the trunk crest has the capacity to give rise to SM if transplanted to anterior regions (Nakamura and Ayer-Le Lievre (1982) J. Embryol. Exp. Morphol. 70: 1-19.
  • the ability to elicit SM differentiation from rodent trunk NCSCs may reflect a developmental potential that is available to these cells in vivo.
  • the available fate mapping data (Serbedzija et al.(1990) Development 108:605-612) do not exclude a contribution of trunk neural crest to SM in mammals.
  • SM cells Although the development of SM cells is of considerable relevance for human disease (Kirby and Waldo (1990) Circulation 82:332-340, their development from precursor cells in mammals is poorly understood (see Schwartz et al. (1990; Owens, (1995) Phvsiol. Rev. 75:487-517 and references therein). While SM cell differentiation has been obtained from cell lines such as ES- like cells (Edwards et al.(1983) Mol. Cell. Biol. 3:2280-2286. the present invention represents the first case in which de novo differentiation of these cells from a naturally occurring precursor has been elicited in vitro. Such a system should open the way to further studies aimed at understanding the factors that control the differentiation and maturation of this important cell type. Chamley-Campbell et al. (1979) Phvsiol. Rev. 59: 1-61.
  • TGF ⁇ l super family members may be utilized to instructively influence cell fate decisions, rather than selectively support survival of lineage-committed progenitors.
  • Members of the TGF- ⁇ superfamily of growth factors are expressed at sites where autonomic neurons differentiate.
  • bone morphogenic protein 2 BMP2
  • BMP2 bone morphogenic protein 2
  • Some SM cell differentiation is also observed in BMP2 in contrast to TGF ⁇ l , the prototypic member of the TGF ⁇ superfamily, which drives virtually all NCSCs to a SM fate.
  • BMP2 bone morphogenic protein 2
  • a clonal culture system has been developed(Stemple and Anderson (1992) Cell 71 :973-985) which has permitted detailed investigation of the action of growth factors on rodent neural crest cells. Initially, the promotion of neuregulin/GGF on glial as opposed to neuronal differentiation was demonstrated. Shah et al. (1994) Cell 77:349-360. More recently, SM differentiation has been added to the NCSC repertoire and as being promoted by TGF ⁇ l . In contrast, a related factor, BMP2/4, promotes primarily autonomic neuronal differentiation although some SM differentiation is observed. Clonal analysis and serial observations of living clones strongly indicates that both TGF ⁇ l and BMP2 act instructively rather than selectively.
  • Transplantation assay systems described herein provide the basis of functional assays which allow for the identification of mammalian cells which have properties characteristic of neural crest stem cells, multipotent neural stem cells and/or neuronal, glial or smooth muscle progenitor cells.
  • Cells of interest identified by either the in vivo or in vitro assays described above, are transplanted into mammalian hosts using standard surgical procedures. Using standard techniques, it is possible to deliver neural crest cells to a developing mammalian or avian embryo or to any tissue or compartment of the adult animal (e.g. , brain, peritoneal cavity, etc.).
  • the transplanted cells and their progeny are distinguished from the host cells by the presence of species specific antigens or by the expression of an introduced marker gene.
  • transplanted cells and their progeny are also stained for markers of mature neurons and glia in order to examine the developmental potential of the transplanted cells.
  • This transplantation assay provides a means to identify neural crest stem cells by their functional properties in addition to the in vitro culture assays described above.
  • the transplantation of cells having characteristics of multipotent neural stem cells, neural crest stem cells or progenitors of neuronal, glial or smooth muscle cells provides a means to investigate the therapeutic potential of these cells for neurological disorders of the PNS and CNS in animal models.
  • PNS disorders in mice include the trembler and shiver er strains.
  • the trembler mutation involves a defect in the structural gene for peripheral myelin protein 22 (PMP22). This mutation results in the defective myelination of axons in the PNS.
  • An analogous disorder is seen in humans, Charcot-Marie-Tooth syndrome, which results in progressive neuropathic muscular atrophy.
  • mice results in a severe myelin deficiency throughout the CNS and a moderate hypo-myelination in the PNS. Severe shivering episodes are seen 12 days after birth.
  • An analogous disorder is seen in humans, Guillaum-Barre' disease, which is characterized by an acute febrile polyneuritis.
  • Cells having characteristics of multipotent neural stem cells, neural crest stem cells or neuronal, glial or smooth muscle progenitors of the PNS or CNS are introduced into a mammal exhibiting a neurological disorder to examine the therapeutic potential of these cells.
  • These cells are preferably isolated from a mammal having similar MHC genotypes or the host mammal is immunosuppressed using drugs such as cyclosporin A.
  • the cells are injected into an area containing various peripheral nerves known to be effected in a particular mammal or into the spinal cord or brain for mammals which show involvement of the CNS.
  • the cells are injected at a range of concentrations to determine the optimal concentration into the desired site.
  • the cells are introduced in a plasma clot or collagen gel to prevent rapid dispersal of cells from the site of injection.
  • Desired therapeutic effects in the above mutant mice include the reduction or cessation of seizures or improved movement of lower motor extremities.
  • multipotent neural stem cells such as the neural crest stem cell and defining culture conditions which allow the clonal propagation and differentiation of said stem cells.
  • Having possession of a multipotent neural stem cell or a neural crest stem cell allows for identification of growth factors associated with self regeneration.
  • growth factors associated with self regeneration there may be as yet undiscovered growth factors associated with (1) with the early steps of restriction of the stem cell to a particular lineage; (2) the prevention of such restriction; and (3) the negative control of the proliferation of the stem cell or its derivatives.
  • the multipotent neural stem cell, neural crest stem cell, progeny thereof or immortalized cell lines derived therefrom are useful to: (1) detect and evaluate growth factors relevant to stem cell regeneration; (2) detect and isolate ligands, such as growth factors or drugs, which bind to receptors expressed on the surface of such cells or their differentiated progeny (e.g.
  • GGF Glial Growth Factor
  • NDF Neu Differentiation Factor
  • the stem cells may be subsequently preferentially differentiated into neurons or smooth muscle cells using media containing growth factors.
  • growth factors from the TGF- ⁇ superfamily is illustrated in Example 11 , as exemplified by TGF- ⁇ and BMP2.
  • the appropriate dose of TGF- ⁇ superfamily growth factors may be determined in a number of ways which will depend on the TGF- ⁇ superfamily growth factor used. As shown for TGF- ⁇ and BMP2 in Figure 24, an initial range of doses may be tested and an appropriate range set. For example, 0.0001 to about lOOnM may be used with a preferred range from about 0.001 to about lOnM. The most preferred dose range may vary depending upon the TGF- ⁇ superfamily growth factor used.
  • the TGF- ⁇ superfamily growth factor is substantially pure. In other embodiments, the TGF- ⁇ superfamily growth factor is in culture media and may be one of many ingredients in the culture media.
  • PNS peripheral nervous system
  • TGF- ⁇ superfamily growth factors to differentiate stem cells into neurons and smooth muscle cells is not limited to PNS-derived cells. Rather, the method can be used on stem cells derived from the central nervous system (CNS). Putative CNS stem cells have been reported (see WO 93/01275 and Reynold et al., Science 255: 1707 (1992)). The effective dosages for CNS stem cells can be determined in the same way as for PNS-derived stem cells.
  • neural crest stem cells have been passaged for at least six-ten generations in culture. Although it may be unnecessary to immortalize those or other multipotent neural stem cell lines or progenitor cell lines obtained by the methods described herein, once a cell line has been obtained it may be immortalized to yield a continuously growing cell line useful for screening trophic or differentiation factors or for developing experimental transplantation therapies in animals. Such immortalization can be obtained in multipotent neural stem cells or progenitors of glial, neuronal and smooth muscle cells by genetic modification of such cells to introduce an immortalizing gene.
  • immortalizing genes include: (1) nuclear oncogenes such as v- myc, N-myc, T antigen and Ewing's sarcoma oncogene (Fredericksen et al. (1988) Neuron 1:439-448; Bartlett, P. et al. (1988) Proc. Natl. Acad. Sci. USA 85:3255-3259, and Snyder, E.Y. et al. (1992) Cell 68:33-51), (2) cytoplasmic oncogenes such as bcr-abl and neurofibromin (Solomon, E. et al.
  • oncogenes include v-myc and the SV40 T antigen.
  • Foreign (heterologous) nucleic acid may be introduced or transfected into multipotent neural stem cells or their progeny.
  • a multipotent neural stem cell or its progeny which harbors foreign DNA is said to be a genetically- engineered cell.
  • the foreign DNA may be introduced using a variety of techniques.
  • foreign DNA is introduced into multipotent neural stem cells using the technique of retroviral transfection.
  • Recombinant retroviruses harboring the gene(s) of interest are used to introduce marker genes, such as the E. coli ⁇ -galactosidase (lacZ) gene, or oncogenes.
  • the recombinant retroviruses are produced in packaging cell lines to produce culture supernatants having a high titer of virus particles (generally 10 5 to 10° pfu/ml).
  • the recombinant viral particles are used to infect cultures of the neural stem cells or their progeny by incubating the cell cultures with medium containing the viral particles and 8 ⁇ g/ml polybrene for three hours .
  • the cells are rinsed and cultured in standard medium.
  • the infected cells are then analyzed for the uptake and expression of the foreign DNA.
  • the cells may be subjected to selective conditions which select for cells that have taken up and expressed a selectable marker gene.
  • the foreign DNA is introduced using the technique of calcium-phosphate-mediated transfection.
  • a calcium-phosphate precipitate containing DNA encoding the gene(s) of interest is prepared using the technique of Wigler et al. (1979) Proc. Natl. Acad. Sci. USA 76: 1373- 1376.
  • Cultures of the neural stem cells or their progeny are established in tissue culture dishes. Twenty four hours after plating the cells, the calcium phosphate precipitate containing approximately 20 ⁇ g/ml of the foreign DNA is added. The cells are incubated at room temperature for 20 minutes. Tissue culture medium containing 30 ⁇ M chloroquine is added and the cells are incubated overnight at 37°C. Following transfection, the cells are analyzed for the uptake and expression of the foreign DNA. The cells may be subjected to selection conditions which select for cells that have taken up and expressed a selectable marker gene.
  • HBSS Hank's Balanced Salt Solution
  • Trunk sections were treated with collagenase (152 units/mg) (Worthington Biochemical, Freehold, New Jersey) made to a concentration of 0.75 mg/ml in Howard's Ringer's solution (per 1 liter of dH 2 0: NaCl 7.2g; CaCl 2 0.17g; KC1 0.37g) and sterilized, by passage through a 0.22 ⁇ m filter prior to use.
  • the collagenase solution was exchanged at least 3 times and with each exchange the trunk sections were vigorously triturated by passage through a pasteur pipet. After incubation at 37 °C for 20 minutes in humidified CO 2 atmosphere, the trunk sections were triturated very gently until most of the neural tubes were free and clean of somites and notochords.
  • the collagenase solution was quenched by repeated exchanges with cold complete medium (described below).
  • the neural tubes were plated onto fibronectin-coated (substrate preparation is described below) 60mm tissue culture dishes (Corning, Corning, New York) that had been rinsed with complete medium. After a 30 minute incubation to allow the neural tubes to attach, dishes were flooded with 5 ml of medium. After a 24 hour culture period, using an L-shaped electrolytically sharpened tungsten needle and an inverted phase contrast microscope equipped with a 4X objective lens, each neural tube was carefully scraped away from the neural crest cells that had migrated onto the substrate. Crest cells were removed by a 2 minute 37oC treatment with 0.05 % Trypsin solution (Gibco). The cells were centrifuged for 4 minutes at 2000 r.p.m. and the pellet was resuspended into 1 ml of fresh complete medium. Typically the cells were plated at a density of 225 cells/100 mm dish.
  • Tissue culture dishes were coated with human plasma fibronectin (New York Blood Center, New York, New York) in the following way. Lyophilized fibronectin was resuspended in sterile distilled water (dH 2 O) to a concentration of 10 mg/ml and stored at -80 °C until used. The fibronectin stock was diluted to a concentration of 250 mg/ml in Dulbecco's phosphate buffered saline (D-PBS) (Gibco). The fibronectin solution was then applied to tissue culture dishes and immediately withdrawn.
  • D-PBS Dulbecco's phosphate buffered saline
  • PDL Polv-D-Lvsine
  • PDL FN Substrate Sterile poly-D-Lysine
  • the PDL solution was applied to tissue culture plates and immediately withdrawn. The plates were allowed to dry at room temperature, rinsed with 5 ml of dH 2 O and allowed to dry again. Fibronectin was then applied, as described above, over the PDL.
  • a serum-free, chemically defined basal medium was developed based on the formulations of several existing defined media.
  • This basal medium consists of L15-CO 2 formulated as described by Hawrot, E. et al. (1979) Methods in Enzymology 58: 574-583 supplemented with additives described by Bottenstein, J.E. et al. (1979) Proc. Natl. Acad. Sci. USA 76:514-517 and further supplemented with the additives described by Sieber-Blum, M. et al. (1985) Exp. Cell Res. 755:267-272.
  • the final recipe is given here: to LI 5- CO 2 add, 100 ⁇ g/ml transferrin (Calbiochem, San Diego, California), 5 ⁇ g/ml insulin (Sigma, St. Louis, MO), 16 ⁇ g/ml putrescine (Sigma), 20 nM progesterone (Sigma), 30 nM selenious acid (Sigma), 1 mg/ml bovine serum albumin, crystallized (Gibco), 39 pg/ml dexamethasone (Sigma), 35 ng/ml retinoic acid (Sigma), 5 ⁇ g/ml -d, 1-tocopherol (Sigma), 63 ⁇ g/ml ⁇ - hydroxybuyrate (Sigma), 25 ng/ml cobalt chloride (Sigma), 1 ⁇ g/ml biotin (Sigma), 10 ng/ml oleic acid (Sigma), 3.6 mg/ml glycerol, 100 ng/ml - melanocyte stimulating hormone (S
  • CEE chick embryo extract
  • CEE is prepared as follows: chicken eggs were incubated for 11 days at
  • FCS fetal calf serum
  • CEE CEE is preferred as a supplement, as in the presence of FCS, most of the cells derived from the neural crest exhibit a flattened, fibroblastic morphology and expression of LNGFR is extinguished. In the absence of both FCS and CEE, clone formation from neural crest cells was greatly attenuated.
  • E10.5 neural tubes were explanted onto a fibronectin (FN) substratum, many of the neural crest cells that emigrated from the neural tubes over the next 24 hours expressed the low-affinity NGF receptor (LNGFR), recognized by monoclonal antibodies 192-Ig and 217c.
  • LNGFR low-affinity NGF receptor
  • Neural crest cells were labeled with antibodies as follows: For cell surface antigens, such as LNGFR, it was possible to label the living cells in culture. The cultures were incubated with primary antibody solution for 20 minutes at room temperature. The cultures were washed twice with L15 medium (Gibco) supplemented with 1: 1 :2, fresh vitamin mix (FVM) (Hawrot, E. et al. (1979), ibid), and 1 mg/ml bovine serum albumin (LI 5 Air). The cultures were then incubated for 20 minutes at room temperature with Phycoerythrin R conjugated secondary antibody (TAGO) at a dilution of 1 :200 in L-15 Air.
  • L15 medium Gibco
  • FVM fresh vitamin mix
  • TAGO Phycoerythrin R conjugated secondary antibody
  • cytoplasmic antigens For the staining of cytoplasmic antigens, fixed cells were first treated with a blocking solution comprising D-PBS, 0.1 % Tween-20 (Bio-Rad Laboratories, Richmond, California) and 10% heat inactivated normal goat serum (NGS) for 15 minutes at room temperature. Primary antibodies were diluted with a solution of D-PBS, 0.1 % Tween-20 and 5 % NGS. The fixed cells were incubated overnight at 4°C in primary antibody solution then rinsed twice with DPBS, 0.05% Tween-20. Fluorescent secondary antibodies were diluted with D-PBS, 1 % NGS and applied to cells for 1 hour at room temperature. The cells were rinsed twice with D-PBS, 0.05% Tween-20. To prevent photobleaching, a solution of 8 mg/ml N-propyl gallate in glycerol was placed over the stained cells prior to fluorescence microscopy.
  • D-PBS 0.1 % Tween-20
  • NGS
  • Mouse monoclonal anti-GFAP G-A-5 (Debus et al. (1983) Differentiation 25: 193-203) was purchased from Sigma and used at a 1 : 100 dilution.
  • Mouse monoclonal anti-NF200, SMI39 was purchased from Sternberger Monoclonals Inc. , Baltimore, Maryland and used at a 1: 100 dilution. SMI39 reactivity is equivalent to the 06-53 monoclonal antibody described by
  • FIG. 1 An individual neural crest cell co-expressing both nestin and LNGFR is shown in Figure 2, panels A-C.
  • Panel A shows the individual neural crest cell in phase contrast.
  • Panels B and C show this cell following staining with both anti-LNGFR (panel B) and anti-nestin (panel C).
  • Panel D-F show that the clonal progeny of this nestin + , LNGFR + neural crest cell also co-express nestin and LNGFR.
  • FIG 3 provides a flow chart depicting the following cell cloning experiments.
  • plating medium refers to the complete medium, described above and differentiation medium refers to SCD medium, described below.
  • differentiation medium refers to SCD medium, described below.
  • FCS-free, CEE-containing medium complete or plating medium
  • single neural crest cells Figure 4, panel A, phase contrast and panel B, LNGFR staining
  • Figure 4 panel C phase contrast
  • neuronal cells contained a mixture of neuronal and non-neuronal cells (see below) .
  • These neuronal cells could be labeled by antibodies to pan- neuronal markers such as neurofilament ( Figure 4, panel E, anti-NF160 staining) and high-poly sialyic acid (PSA) NCAM (Figure 4, panel D, anti- NCAM staining), as well as by an antibody to peripherin, an intermediate filament protein that is preferentially expressed by peripheral nervous system (PNS) neurons ( Figure 4, panel F).
  • PNS peripheral nervous system
  • the neuron-containing clones also contained non-neuronal cells. These cells continued to express LNGFR and nestin, in contrast to the neurons, and displayed an elongated morphology characteristic of Schwann cells. While immature Schwann cells are known to express both LNGFR and nestin, these markers are insufficient to identify Schwann cells in this system since they are expressed by the neural crest precursor cell as well. Expression of more definitive Schwann cell markers was elicited by transferring the cells into a medium known to enhance Schwann cell differentiation. This medium, called Schwann cell differentiation (SCD) medium, contained both 10% FCS and 5 ⁇ M forskolin, an activator of adenylate cyclase.
  • SCD Schwann cell differentiation
  • Figure 5 shows the expression of a Schwann cell phenotype by neural crest- derived glia.
  • Clones plated initially on FN were allowed to grow for a week in complete medium, then transferred into SCD medium and allowed to grow for another 1-2 weeks prior to fixation and immunocytochemistry.
  • Cells of two morphologies, one elongated and the other flattened can be seen in phase contrast (Panels A and D).
  • GFAP glial fibrillary acidic protein
  • O 4 glial fibrillary acidic protein
  • Triple-labeling of such "mature" clones with polyclonal anti-peripherin and monoclonal 0 4 and anti-GFAP antibodies revealed that sulfatide and GFAP were not expressed by the peripherin-positive neurons and that these two glial markers were coincident in the non-neuronal cell population ( Figure 6).
  • Figure 6 shows a clone from a single founder cell in phase contrast (Panel A) which expresses LNGFR (Panel B).
  • This clone was allowed to proliferate and differentiate in complete medium (containing CEE and lacking serum) and then transferred into SCD medium (containing serum and forskol in). After approximately 10 days, the culture was fixed and triple-labeled with rabbit anti-peripherin (Panels C and D, in green/yellow), anti-GFAP (IgG) (Panel C, in red) and O 4 (IgM) (Panel D, blue). Panels C and D are two separate fields from the same clone.
  • GFAP is expressed by astrocytes and sulfatide is expressed by oligodendrocytes in the CNS, the co-expression of these two markers in the same cell is unique to peripheral glial cells (Jessen, K.R. et al. (1990) Devel. 709:91-103 and Mirsky, R. et al. (1990) Devel. 709: 105-116).
  • FIG. 7 provides a flow chart summarizing these serial subcloning experiments.
  • plaque medium refers to complete medium containing CEE and lacking FCS
  • differentiation medium refers to SCD medium containing FCS and forskolin.
  • clones were harvested and replated as follows. The primary clones were examined microscopically to ensure that there were no impinging colonies and that the whole clone fits within the inscribed circle. Using sterile technique throughout the procedure, glass cloning cylinders (3mm id.) were coated on one end with silicone grease (Dow Corning) and placed about the primary clone so that the grease formed a seal through which medium could not pass. The cells were removed from the cylinder by first treating them with 100 ml of 0.05 % Trypsin solution (Gibco) for 3 minutes at 37° C in a humidified 5 % CO 2 incubator.
  • 0.05 % Trypsin solution Gibco
  • the apparent labeling of neurons in panel C is an artifact due to bleed-through into the fluorescein channel of the Texas Red fluorochrome used on the goat anti-rabbit secondary antibody in panel B.
  • Substrate Composition Influences the Developmental Fate of Multipotent Neural Crest Cells
  • FIG. 13 provides a flow chart summarizing these experiments. These experiments were performed to demonstrate that differences in attachment and/or survival do not account for differences in eventual clone composition. Subsequently, one group of cells was exposed to PDL as an overlay in liquid media (0.05 mg/ml) after 48 hrs, while a sister culture was retained on FN alone as a control ( Figure 13). Clones expressing LNGFR were identified by live cell surface labeling at the time of the PDL overlay and the development of only LNGFR + clones was further monitored. After two weeks, the cultures were transferred to SCD medium for an additional 10 days of culture, and their phenotypes then scored as previously described.
  • Neural crest stem cells are identified by two general criteria: by their antigenic phenotype, and by their functional properties. These functional properties may be assessed in culture (in vitro), as described above, or they may be assessed in an animal (in vivo). The above examples described how the self-renewal and differentiation of neural crest stem cells can be assayed in vitro, using clonal cell cultures. However, these properties may also be determined by transplanting neural crest cells into a suitable animal host.
  • Such an assay requires a means of delivering the cells and of identifying the transplanted cells and their progeny so as to distinguish them from cells of the host animal.
  • a means of delivering the cells and of identifying the transplanted cells and their progeny so as to distinguish them from cells of the host animal.
  • neural crest cell cultures are prepared as described earlier. After a suitable period in primary or secondary culture, neural crest cells are identified by live cell-labeling with antibodies to LNGFR, and removed from the plate using trypsin and a cloning cylinder, as described in previous examples. The cells are diluted into serum-containing medium to inhibit the trypsin, centrifuged and resuspended to a concentration of 10 6 - 10 7 cells per milliliter. The cells are maintained in a viable state prior to injection by applying them in small drops (ca. 10 ⁇ l each) to a 35 mm petri dish, and evaporation is prevented by overlaying the droplets with light mineral oil. The cells are kept cold by keeping the petri dishes on ice.
  • Neural crest cells are drawn into a sharpened glass micropipette (with a sealed tip and hole in the side to prevent clogging during penetration of tissues) by gentle suction.
  • the pipette is inserted into the lower third of the deciduum and a volume of approximately 0.5 ⁇ l is expelled containing approximately 1000 cells.
  • the micropipette is withdrawn and the incision is sutured shut.
  • the mother is sacrificed, and individual embryos are removed, fixed and analyzed for the presence and phenotype of cells derived from the injected neural crest cells.
  • rat neural crest cells are injected into a mouse embryo (following suitable immunosuppression of the mother or using a genetically immunodeficient strain such as the SCID strain of mice), the injected cells are identified by endogenous markers such as Thyl or major histocompatibility complex (MHC) antigens using monoclonal antibodies specific for the rat Thyl or MHC antigens.
  • endogenous markers such as Thyl or major histocompatibility complex (MHC) antigens using monoclonal antibodies specific for the rat Thyl or MHC antigens.
  • an exogenous genetic marker is introduced into the cells prior to their transplantation as a means of providing a marker on or in the injected cells.
  • neural crest cells in culture are incubated with a suspension of replication- defective, helper-free retrovirus particles harboring the lacZ gene, at a titer of 10 5 - 10 6 pfu/ml in the presence of 8 ⁇ l/ml polybrene for four hours.
  • the cells are then washed several times with fresh medium and prepared for injection as described above.
  • the harvested embryos are then assayed for expression of ⁇ -galactosidase by whole mount staining according to standard procedures.
  • the blue cells (indicating expression of the lacZ gene) will correspond to the progeny of the injected neural crest cells. This procedure can be applied to any tissue or any stage of development in any animal suitable for transplantation studies.
  • NCSCs Neural Crest Stem Cells
  • NCSCs are infected with a replication-incompetent, recombinant retrovirus harboring the foreign gene of interest.
  • This foreign gene is under the control of the long terminal repeats (LTRs) of the retrovirus, in this case a Moloney Murine Leukemia Virus (MoMuLv) (Cepko et al. (1984) CeU 57: 1053-1062).
  • LTRs long terminal repeats
  • MoMuLv Moloney Murine Leukemia Virus
  • the foreign gene is under the control of a distinct promoter-enhancer contained within the recombinant portion of the virus (i.e. , CMV or RSV LTR).
  • CMV or RSV LTR a distinct promoter-enhancer contained within the recombinant portion of the virus
  • the E. coli ⁇ -galactosidase gene was used, because it provides a blue histochemical reaction product that can easily be used to identify the genetically-engineered cells, and
  • Rat NCSC cultures were established as described above. Twenty-four hours after replating, the cells were exposed to a suspension of ⁇ -galactosidase- containing retrovirus (Turner et al. (1987) Nature 525: 131-136) with a titer of approximately 10 5 -10° pfu/ml in the presence of 8 ⁇ g/ml polybrene. Following a 3 hr exposure to the viral suspension, the cultures were rinsed and transferred into standard medium. After three days of growth in this medium, the transformed cells were visualized using the X-gal histochemical reaction (Sanes et al. (1986) EMBO J. 5:3133-3142) Fig.
  • Panel A shows the NCSC culture three days after infection with the lacZ containing retrovirus, after fixation and staining using the X-gal reaction, ⁇ - galactosidase-expressing cells are indicated by the solid arrows. Non- expressing cells in the same microscopic field are visualized by phase contrast microscopy (B), and are indicated by open arrows. The blue, ⁇ - galactosidase "1" cells represented approximately 5-10% of the total cells in the culture as visualized by phase-contrast microscopy (Fig. 15, Panel B).
  • NCSCs are transfected with an expression plasmid using the calcium phosphate method (Wigler et al. (1979) Proc. Natl. Acad. Sci. USA 76: 1373-1376).
  • the ⁇ -galactosidase gene was used to facilitate visualization of the transfected cells.
  • the vector pRSVlacZ was used, in which the ⁇ -galactosidase gene (lacZ) is under the control of the Rous Sarcoma Virus (RSV) LTR, and the SV40 intron and poly A-addition site are provided at the 3' end of the gene (Johnson et al. (1992) Proc. Natl. Acad. Sci. USA 59:3596-3600).
  • lacZ ⁇ -galactosidase gene
  • RSV40 intron and poly A-addition site are provided at the 3' end of the gene
  • NCSCs were established in 35 mm tissue culture dishes. 24 hr after plating, a calcium phosphate precipitate containing approximately 20 ⁇ g/ml of pRSVlacZ was prepared. 123 ⁇ l of this precipitate was added to each dish, and incubated at room temperature for 20 minutes . Two ml of standard medium containing 30 ⁇ M chloroquine was then added to each dish and incubation was continued overnight at 37 °C. The next day, the medium was replaced and incubation continued for a further two days. The cultures were then fixed and assayed for ⁇ -galactosidase expression by the standard X-gal reaction. Approximately 10% of the NCSCs expressed the lacZ reaction product.
  • NCSC cultures are established as described above.
  • the cultures are exposed, in the presence of 8 ⁇ g/ml polybrene, to a suspension of retrovirus harboring an oncogene preferably selected from the immortalizing oncogenes identified herein.
  • retroviruses contain, in addition to the oncogene sequences, a gene encoding a selectable marker, such as hisD, driven by the SV40 early promoter-enhancer (Stockschlaeder, M.A.R. et al. (1991) Human Gene Therapy 2:33). Cells which have taken up the hisD gene are selected for by growth in the presence of L-histidinol at a concentration of 4 mM.
  • selection can be based upon growth in the presence of neomycin (500 ⁇ g/ml).
  • NCSCs are infected with the above retroviruses which are concentrated to a titer of greater than 10 6 pfu/ml by centrifugation. The virus is applied to the cells in two sequential incubations of 4-8 hours each in the presence of 8 ⁇ g/ml polybrene.
  • the cells are grown in the presence of 4 mM L-histinol or 500 ⁇ g/ml neomycin (G418) for 5-10 days.
  • Cells which survive the selection process are screened for expression of LNGFR by live-cell labeling using the monoclonal antibody 192 Ig as described above.
  • Colonies containing a homogeneous population of LNGFR + cells are cloned using a cloning cylinder and mild trypsinization, and transferred into duplicate FN/pDL-coated 96-well plates. After a short period of growth, one of the plates is directly frozen (Ramirez-Solis, R. et al. (1992) Meth. Enzymol.. in press).
  • the cells in the other plate are replated onto several replicate 96-well plates, one of which is maintained for carrying the lines.
  • the cells on the other plates are fixed and analyzed for the expression of antigenic markers.
  • Successful immortalization is indicated by (1) the cells homogeneously maintain an antigenic phenotype characterized by LNGFR + , nestin + , lin- (where "lin” refers to lineage markers characteristic of differentiated neuronal or glial crest derivatives, including neurofilament, peripherin, hi PSA-NCAM, GFAP, 04 and P 0 ); and (2) the cell population is phenotypically stable over several weeks of passage (as defined by lack of differentiation to morphologically- and antigenically-recognizable neurons and/or glia).
  • the ability of the lines to differentiate is tested by transferring them to conditions that promote differentiation (omission of CEE in the case of neurons and addition of serum and 5 ⁇ M forskolin for Schwann cells). Maintenance of the ability to differentiate is a desirable, although not necessary, property of the constitutively-immortalized cells.
  • mice The isolation of such cells from mice is particularly desirable, as that species is the experimental organism of choice for genetic and immunological studies or human disease.
  • a genomic DNA fragment encoding the extracellular domain (ligand binding domain) of that protein was expressed in E. coil, as a fusion protein with glutathione-S- transferase (Lassar et al. (1989) CeU 55:823-831).
  • a probe for the extracellular domain based on either of the known DNA sequences for rat and human LNGFR is used to screen a mouse genomic library.
  • a cloned insert from a positively hybridizing clone is excised and recombined with DNA encoding glutathione with appropriate expression regulation sequences and transfected into E. coli.
  • the fusion protein was affinity -purified on a glutathione-Sepharose column, and injected into rats. Sera obtained from tail bleeds of the rats were screened by surface-labeling of live Schwann cells isolated from mouse sciatic nerve by standard procedures (Brockea et al. (1979) In Vitro 75:773-778. Surface labeling was with labelled goat anti-rat antibody Following a boost, fusions were carried out between the rat spleen cells and mouse myeloma cells.
  • O Cells are Smooth Muscle Cells
  • SM smooth muscle
  • SMA smooth muscle actin
  • a marker of smooth muscle cells Skalli et al (1966) J. Cell Biol. 103:2787-2796.
  • the cultures were counter- stained with anti-p75 to identify the neural crest stem cells.
  • the anti-SMA antibody labeled a significant number of cells (Fig. 17B, open arrows), and these cells did not express p75 on their surface and were clearly distinct from the p75 -expressing neural crest stem cells (Fig. 17B, closed arrow).
  • a clonal analysis was performed. Individual p75 + neural crest stem cells were identified and allowed to develop for two weeks in culture. The resultant clones were then fixed and triply-labeled with antibody to peripherin (to detect neurons), GFAP (to detect glia) and SMA (to detect smooth muscle cells). As shown in Fig. 18, within the same clone it was possible to identify neurons (Figs. 18A, 18B, arrowhead), glia (Figs. 18C, open arrows) and smooth muscle cells (Fig. 18C, closed arrow), confirming that the neural crest stem cell is able to generate all three lineages in our culture system.
  • neural crest stem cells are progenitors of smooth muscle, as well as of neurons and glia, and indicate that they can be induced to differentiate to smooth muscle in culture using fetal bovine serum. Such differentiation occurs at the expense of neuronal and glial differentiation, which does not occur in the present of fetal bovine serum (Stemple et al. (1992), Cell 71 : 973-985.
  • neural crest stem cells should be useful for identifying smooth muscle differentiation factors present in fetal bovine serum, as well as for identifying other growth, survival or differentiation factors for smooth muscle present in other sources.
  • Neural Crest Stem Cells Preferentially Differentiate to Neurons or Smooth Muscle Cells
  • NCSCs grown at clonal density in standard culture medium undergo symmetrical, self-renewing divisions for at least 5-6 days in vitro.
  • Stemple and Anderson (1992) Cell 71:973-985 Neurons do not begin to differentiate in such cultures until 10-15 days of incubation. Moreover, clones containing only neurons are never observed; rather the neurons differentiate together with nonneuronal cells such as glia.
  • neural crest stem cells respond to growth factors from the TGF- ⁇ superfamily neural crest stem cells were grown in 1.6 nM recombinant bone marrow phogenic protein 2 ("rBMP2").
  • rBMP2 bone marrow phogenic protein 2
  • Many neuron-only colonies identified by their neurite-bearing morphology and expression of peripherin developed within 3-4 days ( Figures 21A and 21B).
  • At this dose — 50% of the colonies contained only neurons; 20% -25 % contained neurons (about as many per colony as in the neuron-only colonies) as well as large flat cells; the remainder consisted only of such flat cells.
  • 75 % of colonies grown in rBMP2 contained neurons after 4 days.
  • rBMP2 promotes the differentiation of autonomic neurons, which are either nonsympathetic or which require additional signals (Groves et al.(1995) Development 121:887-901) to express markers characteristic of the sympathetic sublineage (for review, see Patterson and Nawa (1993) Cell 72/Neuron 10(SuppL): 123-137).
  • a subset of the colonies in rBMP2 also contained large, flat cells that suggested they could be a mesectodermal derivative of the neural crest, such as smooth muscle (Chamley-Campbell et al.(1979) Physiol. Rev. 59: 1-61: Ito and Shah et al.(1996) Cell 85:331-343. Many of the flat cells expressed ⁇ SMA, a well-characterized SM marker (Owens (1995) Physiol. Rev.
  • TGF ⁇ l recombinant TGF ⁇ l
  • Figure 22D recombinant TGF ⁇ l
  • Figure 22F 12% has at least one ⁇ SMA + or calponin + cell together with SM-like, marker- negative cells, while 5.6% + 1.8% of the colonies contained only marker- negative but SM-like cells.
  • TGF ⁇ l No neurons or glial cells were observed to develop under these conditions.
  • 95% of the colonies consisted primarily of undifferentiated NCSCs, although some SM cells were present.
  • TGF ⁇ 2 and TGF ⁇ 3 yielded similar results as TGF ⁇ l (data not shown).
  • a clonal analysis was performed in order to distinguish whether BMP2 and TGF ⁇ l act to influence differentiation by multipotent neural crest stem cells, or rather to support survival of subpopulations of pre-committed neuronal or SM precursors, respectively.
  • Individual NCSCs were identified shortly after plating, growth factors were added to some, and their subsequent survival and differentiation assessed after 4 days. Selective survival of subsets of clones was not observed. Daily observation of the cultures indicated that none of them contained neurons prior to death; in fact, many contained cells with a SM-like morphology.

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Abstract

Procédé de production d'une population de neurones mammaliens et/ou de cellules musculaires lisses, qui consiste à mettre au moins une cellule neurale souche en contact avec un milieu de culture contenant un ou plusieurs facteurs de croissance de la superfamille de TGF-β et à détecter la différenciation de la cellule souche par rapport à une population de neurones ou de cellules musculaires lisses.
PCT/US1998/008364 1997-04-24 1998-04-23 Procedes de differenciation de cellules neurales souches WO1998048001A1 (fr)

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US4479797P 1997-04-24 1997-04-24
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US08/846,028 US6001654A (en) 1994-01-28 1997-04-25 Methods for differentiating neural stem cells to neurons or smooth muscle cells using TGT-β super family growth factors
US08/846,028 1997-04-25

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EP1897937A1 (fr) * 2006-09-07 2008-03-12 NeuroProgen GmbH Leipzig Méthode de culture de cellules neuronales progénitrices
US7544511B2 (en) 1996-09-25 2009-06-09 Neuralstem Biopharmaceuticals Ltd. Stable neural stem cell line methods
US7560553B1 (en) 2003-08-08 2009-07-14 Neuralstem, Inc. Use of fuse nicotinamides to promote neurogenesis
US7691629B2 (en) 2004-11-17 2010-04-06 Neuralstem, Inc. Transplantation of human neural cells for treatment of neurodegenerative conditions
US8293488B2 (en) 2002-12-09 2012-10-23 Neuralstem, Inc. Method for screening neurogenic agents
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US9334478B2 (en) 2001-08-24 2016-05-10 Advanced Cell Technology, Inc. Differentiating ES cells using a tenascin
US9540611B2 (en) 2010-07-28 2017-01-10 Neuralstem, Inc. Methods for treating and/or reversing neurodegenerative diseases and/or disorders
US9750769B2 (en) 2014-10-20 2017-09-05 Neuralstem, Inc. Stable neural stem cells comprising an exogenous polynucleotide coding for a growth factor and methods of use thereof
EP3305889A4 (fr) * 2015-06-02 2018-12-19 National Institute of Advanced Industrial Science and Technology Procédé permettant d'induire une différenciation des cellules de crête neuronale en cellules du système nerveux autonome
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US9465025B2 (en) 1996-05-23 2016-10-11 Neuralstem, Inc. Stable neural stem cell lines
US7544511B2 (en) 1996-09-25 2009-06-09 Neuralstem Biopharmaceuticals Ltd. Stable neural stem cell line methods
WO2000052143A3 (fr) * 1999-03-05 2001-02-15 California Inst Of Techn Isolation et enrichissement de cellules neurales souches provenant d'un tissu non cultive sur la base de l'expression de marqueurs de surface cellulaire
AU774289B2 (en) * 1999-03-05 2004-06-24 California Institute Of Technology The isolation and enrichment of neural stem cells from uncultured tissue based on cell-surface marker expression
WO2000052143A2 (fr) * 1999-03-05 2000-09-08 California Institute Of Technology Isolation et enrichissement de cellules neurales souches provenant d'un tissu non cultive sur la base de l'expression de marqueurs de surface cellulaire
AU2012205203B2 (en) * 2001-08-24 2015-07-30 Ocata Therapeutics, Inc. Screening assays for identifying differentiation-inducing agents and production of differentiated cells for cell therapy
AU2017265022B2 (en) * 2001-08-24 2019-12-05 Ocata Therapeutics, Inc. Screening assays for identifying differentiation-inducing agents and production of differentiated cells for cell therapy
US9334478B2 (en) 2001-08-24 2016-05-10 Advanced Cell Technology, Inc. Differentiating ES cells using a tenascin
US8293488B2 (en) 2002-12-09 2012-10-23 Neuralstem, Inc. Method for screening neurogenic agents
US7560553B1 (en) 2003-08-08 2009-07-14 Neuralstem, Inc. Use of fuse nicotinamides to promote neurogenesis
US8058434B2 (en) 2003-08-08 2011-11-15 Neuralstem, Inc. Compositions to effect neuronal growth
US8030492B2 (en) 2003-08-08 2011-10-04 Neuralstem, Inc. Compositions to effect neuronal growth
US7858628B2 (en) 2003-08-08 2010-12-28 Neuralstem, Inc. Use of fused nicotinamides to promote neurogenesis
US8362262B2 (en) 2003-08-08 2013-01-29 Neuralstem, Inc. Compositions to effect neuronal growth
US8674098B2 (en) 2003-08-08 2014-03-18 Neuralstem, Inc. Compositions to effect neuronal growth
US8460651B2 (en) 2004-11-17 2013-06-11 Neuralstem, Inc. Methods of treating amyotrophic lateral sclerosis (ALS)
US8236299B2 (en) 2004-11-17 2012-08-07 Neuralstem, Inc. Transplantation of human neural cells for treatment of neurodegenerative conditions
US9220730B2 (en) 2004-11-17 2015-12-29 Neuralstem, Inc. Methods of treating ischemic spasticity
US7691629B2 (en) 2004-11-17 2010-04-06 Neuralstem, Inc. Transplantation of human neural cells for treatment of neurodegenerative conditions
US9744194B2 (en) 2004-11-17 2017-08-29 Neuralstem, Inc. Methods of treating ischemic spasticity
US10286010B2 (en) 2004-11-17 2019-05-14 Neuralstem, Inc. Methods of treating neurodegenerative conditions
WO2008028531A1 (fr) * 2006-09-07 2008-03-13 Neuroprogen Gmbh Leipzig procédé de culture de cellules précurseurs neurales
EP1897937A1 (fr) * 2006-09-07 2008-03-12 NeuroProgen GmbH Leipzig Méthode de culture de cellules neuronales progénitrices
US9540611B2 (en) 2010-07-28 2017-01-10 Neuralstem, Inc. Methods for treating and/or reversing neurodegenerative diseases and/or disorders
US9750769B2 (en) 2014-10-20 2017-09-05 Neuralstem, Inc. Stable neural stem cells comprising an exogenous polynucleotide coding for a growth factor and methods of use thereof
US10702555B2 (en) 2014-10-20 2020-07-07 Neuralstem, Inc. Stable neural stem cells comprising an exogenous polynucleotide coding for a growth factor and methods of use thereof
EP3305889A4 (fr) * 2015-06-02 2018-12-19 National Institute of Advanced Industrial Science and Technology Procédé permettant d'induire une différenciation des cellules de crête neuronale en cellules du système nerveux autonome
US11186821B2 (en) 2015-06-02 2021-11-30 National Institute Of Advanced Industrial Science And Technology Method for inducing differentiation of neural crest cells into neurons of the autonomic nervous system

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WO1998048001A9 (fr) 1999-03-25

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