Embryonic stem cells and neural progenitor cells derived therefrom

Abstract

The present invention provides undifferentiated human embryonic stem cells, methods of cultivation and propagation and production of differentiated cells. In particular it relates to the production of human ES cells capable of yielding somatic differentiated cells in vitro, and committed progenitor cells such as neural progenitor cells capable of giving rise to mature somatic cells including neural cells and/or glial cells and uses thereof. The invention also provides methods that generate in vitro and in vivo models of controlled differentiation of ES cells towards the neural lineage. The model and the cells generated along the pathway of neural differentiation may be used for the study of the cellular and molecular biology of human neural development, for the discovery of genes, growth factors, and differentiation factors that play a role in neural differentiation and regeneration, for drug discovery and for the development of screening assays for teratogenic, toxic and neuroprotective effects.

1 - 7 . (canceled)  
     
     
         8 . A differentiated committed human progenitor cell line capable of differentiation and propagation into mature neurons or glial cells said cell line derived from undifferentiated human embryonic stem cells.  
     
     
         9 . The differentiated committed human progenitor cell line according to  claim 8  capable of establishing a graft in a recipient brain.  
     
     
         10 . The differentiated committed human progenitor cell line according to  claim 9  capable of differentiating in vivo into other cell lineages including neurons and glial cells such as astrocytes and oligodendrocytes.  
     
     
         11 . A neural progenitor cell differentiated in vitro from an undifferentiated human embryonic stem cell.  
     
     
         12 . The neural progenitor cell according to  claim 11  wherein said cell is capable of proliferation.  
     
     
         13 . The neural progenitor cell according to  claim 11  wherein said cell is capable of differentiating to a mature neuron cell or glial cell.  
     
     
         14 . The neural progenitor cell according to  claim 11  wherein said cell is capable of transdifferentiation into other cell lineages to generate stem cells and differentiated cells of non-neural phenotype.  
     
     
         15 . The differentiated neural progenitor cell according to  claim 8  or  11  characterised by expressed markers including markers of the neuroectodermal lineage; markers of neural progenitor cells; neuro-filament proteins; monoclonal antibodies including MAP 2  ab; glutamate; synaptophysin; glutamic acid decarboxylase; tyrosine hydroxylase; β-tubulin; β-tubulin III; GABA Aα 2  receptor, glial fibrillary acidic protein (GFAP), galactocerebroside (gal C), 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase), pip, DM-20 and O4.  
     
     
         16 . The neural progenitor cell according to  claim 15  which expresses markers of neuroectoderm and neural progenitor cells selected from the group including NCAM, nestin, vimentin and the transcriptional factor Pax-6, and do not express Oct-4.  
     
     
         17 . The neural progenitor cell according to  claim 16  wherein said cell is capable of establishing a graft in a recipient brain.  
     
     
         18 . The neural progenitor cell according to  claim 17  wherein said cell can incorporate extensively into a recipient brain.  
     
     
         19 . The neural progenitor cell according to  claim 18  wherein said cell is capable of migrating along host brain pathways.  
     
     
         20 . The neural progenitor cell according to  claim 19  wherein said cell is responsive to host environmental signals.  
     
     
         21 . The neural progenitor cell according to  claim 20  wherein said cell differentiates in response to local host environmental signals.  
     
     
         22 . The neural progenitor cell according  claim 20  wherein said cell is capable of differentiation to other cell lineages in a recipient brain.  
     
     
         23 . The enriched preparation of neural progenitor cells including an enriched population of cells according to  claim 15 .  
     
     
         24 . The enriched preparation of neural progenitor cells according to  claim 23  wherein said cells are capable of prolonged undifferentiated proliferation.  
     
     
         25 . The enriched preparation of neural progenitor cells according to  claim 23  wherein said cells are capable of differentiation into neurons, mature neurons and glial cells.  
     
     
         26 . The enriched preparation of neural progenitor cells according to  claim 25  wherein said cells are capable of establishing a graft in a recipient brain in the absence of tumors.  
     
     
         27 . The enriched preparation of neural progenitor cells according to  claim 26  wherein said cells may be recovered from cryopreservation.  
     
     
         28 - 38 . (canceled)  
     
     
         39 . A method of inducing somatic differentiation of stem cells in vitro into progenitor cells said method comprising: 
 obtaining undifferentiated embryonic stem cells; and    providing a differentiating signal under conditions which are non-permissive for stem cell renewal, do not kill cells and/or induces unidirectional differentiation toward extraembryonic lineages.    
     
     
         40 - 45 . (canceled)  
     
     
         46 . The method according to  claim 39  wherein the conditions for inducing somatic differentiation of stem cells are selected from any one of the following including: 
 culturing the undifferentiated stem cells for prolonged periods and at high density on a fibroblast feeder cell layer to induce differentiation;    culturing the undifferentiated stem cells in serum free media;    culturing the undifferentiated stem cells on a differentiation inducing fibroblast feeder layer and wherein said fibroblast feeder layer does not induce extra embryonic differentiation and cell death;    culturing to a high density in monolayer or on semi-permeable membranes so as to create structures mimicing the postimplantation phase of human development; or    culturing in the presence of a chemical differentiation factor selected from the group including bone morphogenic protein- 2  or antagonists thereof.    
     
     
         47 . The differentiated progenitor cell prepared by the method according to  claim 39 .  
     
     
         48 . The differentiated progenitor cell according to  claim 47  selected from the group including a neural progenitor cell or mesodermal progenitor cell including hemangioblast or hematopoietic stem cells.  
     
     
         49 . The differentiated progenitor cell according to  claim 48  which is a neural progenitor cell capable of differentiating into a neuron cell or a glial cell.  
     
     
         50 . A method of inducing somatic cells from embryonic stem cell derived somatic progenitors, said method comprising: 
 obtaining a source of embryonic stem cell derived somatic progenitors;    culturing the progenitor cells on an adhesive substrate; and    inducing the cells to differentiate to somatic cells under conditions which favour somatic differentiation.    
     
     
         51 . (canceled)  
     
     
         52 . The method according to  claim 50  wherein the embryonic stem cell-derived progenitor cell is prepared according to the method of  claim 39 .  
     
     
         53 . The method according to  claim 50  wherein the embryonic stem cell-derived progenitor cell is selected from the group consisting of a neural progenitor cell or mesodermal progenitor cell including hemangioblast or hematopoietic stem cells.  
     
     
         54 . The method according to  claim 50  wherein the embryonic stem cell-derived  
     
     
         55 . The method according to  claim 54  wherein the progenitor cells are cultured on an adhesive substrate selected from poly-D-lysine and laminin or poly-D-lysine and fibronectin.  
     
     
         56 - 63 . (canceled)  
     
     
         64 . A method of producing an enriched preparation of human ES derived neural progenitor cells, said method comprising: 
 obtaining an undifferentiated human embryonic stem cell comprising obtaining an in vitro fertilised human embryo and growing the embryo to a blastocyst stage of development;    removing inner cells mass (ICM) cells from the embryo;    culturing ICM cells under conditions which do not induce extraembryonic differentiation and cell death, and promote proliferation of undifferentiated stem cells;    recovering stem cells;    inducing somatic differentiation of the embryonic stem cell to a neural progenitor cell comprising obtaining undifferentiated embryonic stem cells; providing a differentiating signal under conditions which are non-permissive for stem cell renewal, do not kill cells or induces unidirectional differentiation toward extraembryonic lineages;    identifying a neural progenitor cell by expressed markers of primitive neuroectoderm and neural stem cells such as polysialyated N-CAM, intermediate filament proteins such as nestin and vimentin and the transcription factor Pax- 6 ; and    culturing the neural progenitor cells to promote proliferation and propagation.    
     
     
         65 - 85 . (canceled)