Human stem cell-derived neural precursors for treatment of autoimmune diseases of the central nervous system

Abstract

The present invention concerns the use of a population of cells comprising: (a) neural precursor cells committed to an oligodendroglial fate; (b) uncommitted neural precursor cells (c) differentiated oligodendrocytes; or (d) a combination of any one of (a) to (c) for the treatment of CNS autoimmune diseases, or for the preparation of a pharmaceutical composition for treating CNS autoimmune diseases, the population of cells being derived from human pluripotent stem cells. The invention also provides methods for obtaining such populations of cells, namely, neural precursor cells committed to an oligodendroglial fate as well as differentiated oligodendrocytes which then can be used in the treatment of CNS autoimmune diseases. A preferred autoimmune disease in the context of the present invention is multiple sclerosis where the population of cells is administered to the CNS for local treatment of the disease.

1 . A method for preparing a population of neural precursor cells committed to an oligodendroglial fate; comprising:
 (a) incubating early multipotent uncommitted neural precursor cells derived from human pluripotent stem cells with RA and an HH agonist at a first concentration being between about 0.5 μM and about 2.0 μM to allow the cells to propagate as floating spheres enriched with oligodendroglial precursors;   (b) allowing the floating spheres to further expand in a medium comprising a second concentration of HH agonist that is not more than about 0.5 μM to obtain an expanded population of neural precursor cells committed to an oligodendroglial fate.   
     
     
         2 . The method of  claim 1 , comprising plating the obtained expanded population of neural precursor cells committed to an oligodendroglial fate on an ECM whereby differentiation of the neural precursor cells committed to an oligodendroglial fate to terminally differentiated oligodendrocytes is obtained. 
     
     
         3 . The method of  claim 1 , wherein the HH agonist is purmorphamine. 
     
     
         4 . The method of  claim 1 , wherein incubation with HH agonist is performed in the presence of at least one mitogen. 
     
     
         5 . The method of  claim 1 , wherein the first concentration of HH agonist is about 0.5 μM, and the second concentration of HH agonist is between about 0.2 μM and about 0.5 μM. 
     
     
         6 . The method of  claim 2 , wherein the plating is performed in the absence of HH agonist and in the absence of a mitogen. 
     
     
         7 . A method for treating a subject having a CNS autoimmune disease, the method comprising administering to the subject a population of cells derived from human pluripotent stem cells, the population of cells comprising: (a) neural precursor cells committed to an oligodendroglial fate; (b) uncommitted neural precursor cells (c) differentiated oligodendrocytes; or (d) any combination of two or more of (a) to (c). 
     
     
         8 . The method of  claim 7 , wherein the population of cells comprising neural precursors committed to an oligodendroglial fate are obtainable by:
 (c) incubating early multipotent uncommitted neural precursor cells derived from human pluripotent stem cells with RA and an HH agonist at a first concentration being between about 0.5 μM and about 2.0 μM to allow the cells to propagate as floating spheres enriched with oligodendroglial progenitors;   (d) allowing the floating spheres to further expand in a medium comprising a second concentration of HH agonist that is not more than about 0.5 μM to obtain an expanded population of cells comprising neural precursor cells committed to an oligodendroglial fate.   
     
     
         9 . The method of  claim 8 , further comprising plating the thus expanded population of neural precursor cells committed to an oligodendroglial fate on an extracellular matrix thereby allowing differentiation of the committed cells to terminally differentiated oligodendrocytes. 
     
     
         10 . The method of  claim 9 , wherein the HH agonist is purmorphamine. 
     
     
         11 . The method of  claim 9 , wherein incubation with HH agonist is performed in the presence at least one mitogen. 
     
     
         12 . The method of  claim 9 , wherein the first concentration of HH agonist is about 0.5 μM, and the second concentration of HH agonist is between about 0.2 μM and about 0.5 μM. 
     
     
         13 . The method of  claim 9 , wherein the extracellular matrix is fibronectin. 
     
     
         14 . The method of  claim 7 , for the treatment of a CNS autoimmune disease associated with an inflammatory reaction. 
     
     
         15 . The method of  claim 14 , wherein the autoimmune disease is an inflammatory demyelinating disease. 
     
     
         16 . The method of  claim 15 , wherein the disease is multiple sclerosis. 
     
     
         17 . The method of  claim 7 , comprising administration to a subject in need of two or more populations of cells derived from human pluripotent stem cells, the population of cells being selected from a population of neural precursor cells committed to an oligodendroglial fate; a population of uncommitted neural precursor cells, a population of differentiated oligodendrocytes; wherein the two or more populations of cells being administered together or separately, simultaneously or in sequence. 
     
     
         18 . The method of  claim 7 , comprising local administration of said population of cells to the CNS. 
     
     
         19 . The method of  claim 18 , wherein the local administration comprises transplantation of the population of cells to the lateral ventricles or intrathecally. 
     
     
         20 . A method for producing a population of differentiating neural precursor cells committed towards oligodendroglial fate, the method comprising:
 (a) incubating early multipotent uncommitted neural precursor cells derived from human pluripotent stem cells with retinoic acid (RA) and an hedgehog (HH) agonist at a first concentration between about 0.5 μM and about 2.0 μM to allow the cells to propagate as floating spheres enriched with oligodendroglial precursors; and   (b) allowing the floating spheres to further expand in a medium comprising a second concentration of HH agonist that is not more than about 0.5 μM to obtain an expanded population of neural precursor cells committed to an oligodendroglial fate.   
     
     
         21 . A method for producing a population of differentiated oligodendrocyte cells the method comprising:
 (a) incubating early multipotent uncommitted neural precursor cells derived from human pluripotent stem cells with retinoic acid (RA) and an hedgehog (HH) agonist at a first concentration between about 0.5 μM and about 2.0 μM to allow said early multipotent NPs to propagate as floating spheres being enriched with oligodendroglial precursors;   (b) allowing the floating spheres to further expand in a medium comprising a second concentration of HH agonist that is not more than about 0.5 μM to obtain an expanded population of neural precursor cells committed to an oligodendroglial fate; and   (c) plating expanded population of neural precursor cells committed to an oligodendroglial fate on an extracellular matrix thereby allowing differentiation into oligodendrocytes.   
     
     
         22 . The method of  claim 20 , wherein the HH agonist is purmorphamine. 
     
     
         23 . The method of any one of  claim 22 , wherein incubation with HH agonist is performed in the presence of at least one mitogen. 
     
     
         24 . The method of  claim 23 , wherein the first concentration of HH agonist is about 0.5 μM, and the second concentration of HH agonist is between about 0.2 μM and about 0.5 μM. 
     
     
         25 . The method of  claim 21 , wherein the extracellular matrix is fibronectin. 
     
     
         26 . The method of any one of  claim 21 , for obtaining neural precursor cells committed to an oligodendroglial fate, the cells expressing at least one of the following markers: Olig1, Olig2, NG2, PDGFRα, GD3, O4, GalC and MBP, wherein at least Olig2 is co expressed with one or more of a marker selected from NG2, PDGFRα and GD3. 
     
     
         27 . The method of  claim 21 , for obtaining differentiated oligodendrocytes expressing at least one of the following markers: Olig1, Olig2, NG2, PDGFRα, GD3, O4, GalC and MBP, where at least Olig2 is co expressed with one or more of a marker selected from 04, GalC and MBP. 
     
     
         28 . The method of  claim 21 , wherein the cells thus obtained are expandable. 
     
     
         29 . The method of  claim 21 , wherein the plating is in the absence of one or more of an HH agonist and a mitogen. 
     
     
         30 . A population of oligodendroglial committed precursor cells obtainable by incubating floating spheres of early multipotent neural precursor cells in a medium comprising a concentration of HH agonist that is not more than about 0.5 μM; the oligodendroglial committed progenitor cells expressing one or more of the markers selected from Olig1, Olig2, NG2, PDGFRα, GD3, where at least Olig2 is co expressed with one or more of a marker selected from NG2, PDGFRα, GD3. 
     
     
         31 . The oligodendroglial committed precursor cells of  claim 30 , wherein the oligodendroglial committed precursor cells are-expandable. 
     
     
         32 . A method for promoting differentiation of early multipotent neural precursor cells towards oligodendroglial fate, the method comprising propagating floating spheres comprising early multipotent neural precursors in a medium comprising purmorphamine.