Mammalian neural plate border stem cells capable of forming neural tube and neural crest cell lineages including central and peripheral neurons

Abstract

The present invention relates to a method for producing mammalian neural plate border stem cells (NPBSCs), comprising: (a) differentiation of mammalian pluripotent stem cells by (a-i) culturing mammalian pluripotent stem cells in pluripotent stem cell medium for about 24 to about 96 hours, wherein the pluripotent stem cell medium comprises: (i) an inhibitor of the activin/TGF-β signalling pathway; (ii) an inhibitor of the BMP signalling pathway; (iii) an activator of the canonical WNT signalling pathway; and (iv) an activator of the Hedgehog signalling pathway; subsequently (a-ii) culturing the cells obtained in step (a-i) for about 24 to about 96 hours in a neural medium, wherein the neural medium comprises: (i) an inhibitor of the Activin/TGF-β signalling pathway; (ii) an inhibitor of the BMP signalling pathway; (iii) an activator of the canonical WNT signalling pathway; and (iv) an activator of the Hedgehog signalling pathway; subsequently (a-iii) culturing the cells obtained in step (a-ii) for about 24 to about 96 hours in a neural medium, wherein the neural medium comprises: (i) an activator of the canonical WNT signalling pathway; (ii) an activator of the Hedgehog signalling pathway; and (iii) an inhibitor of oxidation; and (b) plating the obtained differentiated mammalian pluripotent stem cells in NPBSCs expansion medium, wherein the NPBSCs expansion medium comprises (i) an activator of the canonical WNT signalling pathway; (ii) an activator of the Hedgehog signalling pathway; and (iii) an inhibitor of oxidation; and expanding the cells in the NPBSCs expansion medium for about 24 to about 96 hours; (c) splitting the cells obtained in (b) and further expanding the cells in the NPBSCs expansion medium; and (d) repeating step (c) at least two times. The present invention further relates to neural plate border stem cells obtainable by the method of the invention and the use of the cells of the invention in medicine.

1 . A method for producing mammalian neural plate border stem cells (NPBSCs), comprising:
 (a) differentiation of mammalian pluripotent stem cells by
 (a-i) culturing mammalian pluripotent stem cells in pluripotent stem cell medium for about 24 to about 96 hours, wherein the pluripotent stem cell medium comprises:
 (i) an inhibitor of the activin/TGF-β signalling pathway; 
 (ii) an inhibitor of the BMP signalling pathway; 
 (iii) an activator of the canonical WNT signalling pathway; and 
 (iv) an activator of the Hedgehog signalling pathway; subsequently 
 
 (a-ii) culturing the cells obtained in step (a-i) for about 24 to about 96 hours in a neural medium, wherein the neural medium comprises:
 (i) an inhibitor of the activin/TGF-β signalling pathway; 
 (ii) an inhibitor of the BMP signalling pathway; 
 (iii) an activator of the canonical WNT signalling pathway; and 
 (iv) an activator of the Hedgehog signalling pathway; subsequently 
 
 (a-iii) culturing the cells obtained in step (a-ii) for about 24 to about 96 hours in a neural medium, wherein the neural medium comprises:
 (i) an activator of the canonical WNT signalling pathway; 
 (ii) an activator of the Hedgehog signalling pathway; and 
 (iii) an inhibitor of oxidation; 
 
   (b) plating the obtained differentiated mammalian pluripotent stem cells in NPBSCs expansion medium, wherein the NPBSCs expansion medium comprises
 (i) an activator of the canonical WNT signalling pathway; 
 (ii) an activator of the Hedgehog signalling pathway; and 
 (iii) an inhibitor of oxidation 
 and expanding the cells in the NPBSCs expansion medium for about 24 to about 96 hours; 
   (c) splitting the cells obtained in (b) and further expanding the cells in the NPBSCs expansion medium; and   (d) repeating step (c) at least two times.   
     
     
         2 . The method of  claim 1 , wherein the differentiated mammalian pluripotent stem cells are plated in step (b) at a density of about 1000 to 100,000 per cm 2 . 
     
     
         3 . The method of  claim 1 , wherein the NPBSCs are characterised by the expression of at least three markers selected from the group consisting of FORSE1, MSX1, PHOX2B, PAX3, PAX6, SOX1, SOX2, NESTIN, IRX3, HOXA2, HOXB2, HES5, DACH1, PLZF, LMO3, EVI1 and ASCL1. 
     
     
         4 . The method of  claim 1 , wherein the NPBSCs are characterised by a lack of expression of at least one of the markers OCT4, NANOG, AFP, T, SOX17, EOMES, GSH2, OLIG2, CK8, CK18, NKX2.2, NKX6.1, HOXB8, HOXA5, FOXA2 and VCAM-1. 
     
     
         5 . The method of  claim 1 , wherein the NPBSCs are characterised by
 (i) the expression of SOX1, MSX1 and PHOX2B and the lack of expression of NKX6.1 and VCAM-1; or   (ii) the expression of SOX2, IRX3, and MSX1 and the lack of expression of HOXB8, HOXA5 and VCAM-1.   
     
     
         6 . The method of  claim 1 , further comprising differentiating the NPBSCs obtained in step (d) into:
 (i) peripheral nervous system neurons;   (ii) central nervous system neurons;   (iii) midbrain dopaminergic neurons;   (iv) motor neurons;   (v) neural crest-derived mesenchymal cells;   (vi) astrocytes; or   (vii) neural rosettes.   
     
     
         7 . The method of  claim 6 , wherein the differentiation of NPBSCs into peripheral nervous system neurons comprises:
 (i) culturing the NPBSCs obtained in step (d) in a neural medium comprising an activator of the canonical WNT signalling pathway for about 48 to 72 hours;   (ii) adding an activator of the BMP pathway to the culture of step (i) for about 192 hours; and   (iii) culturing the cells obtained in step (ii) for about 336 hours in a neural medium containing at least two different neurotrophins and an inhibitor of oxidation;   thereby differentiating the NPBSCs into peripheral nervous system neurons.   
     
     
         8 . The method of  claim 6 , wherein the differentiation of NPBSCs into central nervous system neurons comprises:
 (i) culturing the NPBSCs obtained in step (d) in a neural medium;   thereby differentiating the NPBSCs into central nervous system neurons.   
     
     
         9 . The method of  claim 6 , wherein the differentiation of NPBSCs into midbrain dopaminergic neurons comprises:
 (i) culturing the NPBSCs obtained in step (d) in a neural medium comprising
 (a) an activator of the FGF signaling pathway, 
 (b) an activator of the hedgehog signaling pathway and 
 (c) an inhibitor of oxidation, 
 for about 168 to about 192 hours; 
   (ii) changing the medium to a neural medium comprising
 (a) at least two different neurotrophins, 
 (b) an inhibitor of oxidation; 
 and culturing the cells for about 24 to about 96 hours; and 
   (iii) further culturing the cells in a neural medium comprising
 (a) at least two different neurotrophins; and 
 (b) an inhibitor of oxidation; 
   thereby differentiating the NPBSCs into midbrain dopaminergic neurons.   
     
     
         10 . The method of  claim 6 , wherein the differentiation of NPBSCs into motor neurons comprises:
 (i) culturing the NPBSCs obtained in step (d) in a neural medium comprising an activator of the hedgehog signaling pathway for about 24 to about 48 hours;   (ii) adding retinoic acid to the culture of step (i) for about 168 to about 192 hours; and   (iii) further culturing the cells in a neural medium comprising at least two different neurotrophins,   thereby differentiating the NPBSCs into motor neurons.   
     
     
         11 . The method of  claim 6 , wherein the differentiation of NPBSCs into neural crest-derived mesenchymal cells comprises:
 (i) culturing the NPBSCs obtained in step (d) in a neural medium comprising an activator of the canonical WNT signalling pathway for about 48 to 72 hours; and   (ii) culturing the cells obtained in step (i) in a cell culture medium comprising serum,   thereby differentiating the NPBSCs into neural crest-derived mesenchymal cells.   
     
     
         12 . The method of  claim 6 , wherein the differentiation of NPBSCs into astrocytes comprises:
 (i) culturing the NPBSCs obtained in step (d) in a cell culture medium comprising an activator of FGF signalling for about 12 to 96 hours; and   (ii) culturing the cells obtained in step (i) in a cell culture medium comprising fetal calf serum, fetal bovine serum and/or CNTF for about 14 to 60 days;   thereby differentiating the NPBSCs into astrocytes.   
     
     
         13 . The method of  claim 6 , wherein the differentiation of NPBSCs into neural rosette cells comprises:
 (i) culturing the NPBSCs obtained in step (d) in a neural medium comprising an activator of FGF signalling for about 12 to 96 hours;   thereby differentiating the NPBSCs into neural rosette cells.   
     
     
         14 . The method of  claim 1 , wherein
 the cells obtained are free or substantially free of pathogens.   
     
     
         15 . Neural plate border stem cells obtained by the method of  claim 1 . 
     
     
         16 . (canceled) 
     
     
         17 . (canceled) 
     
     
         18 . A method for treatment of a disease selected from the group consisting of Parkinson's disease, amyotrophic lateral sclerosis, spinal muscular atrophy, peripheral neuropathy, Hirschsprung's disease, DiGeorge syndrome, familial dysautonomia, congenital insensitivity to pain with anhididrosis and Charcot-Marie-Tooth disease, comprising administering to a subject presenting with said disease neural plate border stem cells obtained by the method of  claim 1 .