Isolation of fusion-competent myoblasts and therapeutic applications thereof related to muscular dystrophy

Abstract

Described are methods of isolating high purity myoblasts that are used to create novel DMD model systems and methods using human induced pluripotent stem cells (hiPSCs). Also described are therapeutic methods based on the use of there myoblasts.

1 . A drug discovery method comprising the following steps:
 a. obtain one or more fibroblasts from a patient with muscular dystrophy;   b. reprogram the one or more fibroblast into human induced pluripotent stem cells;   c. direct the pluripotent stem cells into myoblasts in a mixture by incubating the pluripotent stem cells with an inhibitor of GSK-3β and an inhibitor of Notch signaling;   d. select myoblasts from the mixture by contacting myoblasts with NCAM(5.H1) antibody, HNK1 antibody or a combination thereof to form purified myoblasts; and   e. treat purified myoblasts with one or more agent(s) to from rescued myoblasts so that the rescued myoblasts look more like a normal myoblast under similar conditions.   
     
     
         2 . The method of  claim 1 , wherein the agent is dual SMAD inhibitor compounds. 
     
     
         3 . The method of  claim 1 , where the agent is a vector carrying the DYSTROPHIN gene. 
     
     
         4 . The method of  claim 1 , wherein the fibroblasts are patient fibroblasts from Coriell Cell Repository. A method of using myoblast to treat a patient with muscular dystrophy comprising the following steps:
 a. obtain one or more fibroblasts from a patient with muscular dystrophy;   b. reprogram the one or more fibroblast into human induced pluripotent stem cells;   c. direct the pluripotent stem cells into myoblasts in a mixture by incubating the pluripotent stem cells with an inhibitor of GSK-3β and an inhibitor of Notch signaling;   d. select myoblasts from the mixture by contacting myoblasts with NCAM(5.H1) antibody, HNK1 antibody or a combination thereof to form purified myoblasts;   e. treat purified myoblasts with one or more agent(s) to from rescued myoblasts so that the rescued myoblasts look more like a normal myoblast under similar conditions; and   f. transplant the rescued myoblasts into the patient with muscular dystrophy.   
     
     
         6 . The method of claim  5 , wherein the patient has Duchene muscular dystrophy. 
     
     
         7 . The method of claim  5 , wherein the fibroblast come from a skin biopsy. 
     
     
         8 . The method of claim  5 , wherein the agent is dual SMAD inhibitor compounds. 
     
     
         9 . The method of claim  5 , where the agent is a vector carrying the DYSTROPHIN gene. 
     
     
         10 . The method of claim  5 , wherein the myoblast in step d are selected by contacting the myoblasts with NCAM(5.H1) antibody before contacting with HNK1 antibody. 
     
     
         11 . The method of claim  5 , wherein the patient with muscular dystrophy has a muscular dystrophy lesion and the rescued myoblasts are transplanted into the muscular dystrophy lesion. 
     
     
         12 . A method of isolating myoblasts comprising the following steps:
 a. obtain one or more fibroblasts from a patient with a disease;   b. reprogram the one or more fibroblast into human induced pluripotent stem cells;   c. direct the pluripotent stem cells into myoblasts in a mixture by incubating the pluripotent stem cells with an inhibitor of GSK-3β and an inhibitor of Notch signaling;   d. select myoblasts from the mixture by contacting myoblasts with NCAM(5.H1) antibody, HNK1 antibody or a combination thereof to form purified myoblasts.   
     
     
         13 . The method of  claim 1  wherein the inhibitor of GSK-3β is CHIR99021. 
     
     
         14 . The method of  claim 1  wherein the inhibitor of Notch signaling is DAPT. 
     
     
         15 . The method of  claim 2  wherein the dual SMAD inhibitor is selected from the group consisting of LDN193189, SB431542, or a combination thereof. 
     
     
         16 . The method of  claim 1  wherein the inhibitor of GSK-3β is CHIR99021 and the inhibitor of Notch signaling is DAPT. 
     
     
         17 . The method of  claim 16 , wherein the dual SMAD inhibitor is selected from the group consisting of LDN193189, SB431542, or a combination thereof.