METHOD AND COMPOSITION FOR GENERATING BASAL FOREBRAIN CHOLINERGIC NEURONS (BFCNs)

Abstract

The invention relates to methods and compositions for developing basal forebrain cholinergic neurons (BFCNs) from stem cells, and in particular, BFCNs having repaired electrophysiological defects relating to one or more mutations in PSEN2, and to the use of such BFCNs in cell-based therapies to treat Alzheimer's disease.

What is claimed is: 
     
         1 . A method of generating basal forebrain cholinergic neurons (BFCNs) comprising: culturing pluripotent stem cells (PSCs) in a basal media comprising an inhibitor of transforming growth factor beta (TGF-β) signaling and an activator of sonic hedgehog (Shh) signaling to induce neuroectodermal differentiation, wherein the basal media lacks basic fibroblast growth factor (bFGF), TGF-β, lithium chloride (Li—Cl), GABA and pipecolic acid, thereby generating BFCNs. 
     
     
         2 . A method of treating a disease or disorder in a subject comprising administering a BFCN generated using the method of  claim 1  to the subject, thereby treating the disease or disorder. 
     
     
         3 . The method of  claim 2 , wherein the disease or disorder is an amyloidogenic disease. 
     
     
         4 . The method of  claim 2 , wherein the disease or disorder is associated with diminished neuronal excitability in BFCNs of the subject. 
     
     
         5 . The method of  claim 3 , wherein the disease or disorder is selected from the group consisting of systemic amyloidosis, Alzheimer's disease, mature onset diabetes, Parkinson's disease, Huntington's disease, fronto-temporal dementia, and prion-related transmissible spongiform encephalopathies. 
     
     
         6 . The method of  claim 2 , wherein the BFCN comprises a genome repaired PSEN2 mutation. 
     
     
         7 . The method of  claim 6 , wherein the PSEN2 mutation is PSEN2 N141I . 
     
     
         8 . A method of restoring neuronal excitability in basal forebrain cholinergic neurons (BFCNs) in a subject, comprising:
 a) isolating a BFCN from the subject, wherein the BFCN has a mutation in presenilin 2 (PSEN2) resulting in impaired neuronal excitability of the BFCN;   b) generating an induced pluripotent stem cell (iPSC) using the BFCN of (a);   c) repairing the PSEN2 mutation in the iPSC;   d) culturing the iPSC of (c) using the method of  claim 1  to generate a BFCN having the repaired mutation; and   e) administering the iPSC of (d) to the subject, thereby restoring neuronal excitability in BFCNs in the subject.   
     
     
         9 . The method of  claim 8 , wherein the mutation is PSEN2 N141I . 
     
     
         10 . The method of  claim 8 , wherein the subject has, or is at risk of having Alzheimer's disease. 
     
     
         11 . A method of identifying a compound for treatment or prevention of a disease or disorder associated with diminished neuronal excitability in basal forebrain cholinergic neurons (BFCNs), the method comprising:
 a) contacting a BFCN or neuronal embryoid body (NEB) generated by the method of  claim 1  with a candidate compound, wherein the BFCN comprises a mutation in presenilin 2 (PSEN2) resulting in impaired neuronal excitability of the BFCN; and   b) detecting neuronal excitability of the BFCN after contact with the candidate compound.   
     
     
         12 . The method of  claim 11 , wherein the mutation is PSEN2 N141I . 
     
     
         13 . The method of  claim 11 , wherein the disease or disorder is Alzheimer's disease. 
     
     
         14 . A basal forebrain cholinergic neuron (BFCN) generated using the method of  claim 1 . 
     
     
         15 . The BFCN of  claim 14 , wherein the genome has a recombinantly introduced marker. 
     
     
         16 . A kit for generating a basal forebrain cholinergic neuron (BFCN) comprising a culture media having an inhibitor of transforming growth factor beta (TGF-β) signaling and an activator of sonic hedgehog (Shh) signaling, wherein the basal media lacks basic fibroblast growth factor (bFGF), TGF-β, lithium chloride (Li—Cl), GABA and pipecolic acid, wherein the inhibitor comprises SB431542 and LDN193189, and wherein the activator comprises Smoothened Agonist (SAG) and purmorphamine. 
     
     
         17 . The kit of  claim 16 , wherein the kit further comprises reagents for generating an induced pluripotent stem cell (iPSC). 
     
     
         18 . The kit of  claim 16 , wherein the kit further comprises a gene-editing reagent. 
     
     
         19 . The kit of  claim 16 , wherein the kit further comprises a reagent for detecting a genetic mutation. 
     
     
         20 . The kit of  claim 19 , wherein the mutation is is PSEN2 N141I .