Phenotypic Correction of Oligodendrocytes by Concurrent Direct Reprogramming and Gene Replacement of Fibroblasts

Transcription factor-mediated direct reprogramming technologies that can convert one differentiated somatic cell to another, without an intermediate pluripotent stem cell stage, provide a new mechanism to access somatic cell types for disease modeling and possible therapeutic application. Previous efforts have focused on wild-type cells harboring transgenic reporters to signal successful reprogramming events. Outstanding questions remain of whether cells from disease backgrounds without transgenic reporters are capable of direct reprogramming and whether they phenocopy disease pathology. Here we demonstrate that fibroblasts from the shiverer mouse model of myelin disease can be directly reprogrammed to induced oligodendrocyte progenitor cells (iOPCs) by the expression of Sox10, Olig2, and Nkx6.2 without the aid of transgenic selection reporters. Shiverer mice harbor a homozygous ~20-kilobase deletion in the Myelin Basic Protein (Mbp) gene and this substantial deletion leads to abnormal oligodendrocytes resulting in hypomyelination and decreased lifespan. Shiverer iOPCs efficiently differentiated to induced oligodendrocytes (iOLs) which recapitulated the known disease phenotype of impaired axonal ensheathment and myelination capacity. Combining direct reprogramming and genetic supplementation of a minigene carrying regulatory elements and an open reading frame of MBP, restored MBP expression in differentiated iOLs and their ability to track along and ensheath microfibers in an in vitro assay of myelination capacity. Collectively these results show that iOPCs provide a rapid and accessible system to accurately model myelin disease pathology and provide promise for future therapeutic advancement for genetic myelin disorders. RNA was collected from whole cells by lysing in 1 ml TRIzol (Invitrogen) and stored at -80C until processing. Phase-lock gel tubes (5Prime) were used to enhance chloroform extraction. The aqueous phase was collected, and RNA isolation completed using the miRNeasy Kit (Qiagen) according to manufacturer’s protocol. One microgram was then subject to ribosome depletion, fragmented, and libraries prepared using the TruSeq Stranded Total RNA Kit with Ribo Zero Gold (Illumina) according to the manufacturer’s protocol and indexed using TruSeq adapters. One hundred base pair paired-end reads were generated on the Illumina HISeq 2500 (Case Western Reserve University Sequencing Core; Cleveland, OH). Samples: EpiSC5-OPC= OPCs derived from mouse epiblast stem cells Shiv-MEF= MEFs derived from shiverer mice and depleted using A2B5 microbeads Shiv-iOPC-MBP= Day 21 shiverer iOPCs without MBP transgene Shiv-iOPC-Rd1-Dox= Day 21 shiverer MEFs which were infected with the reprogramming factors but were not induced Shiv-iOPC-Rd1= Day 21 shiverer iOPCs with the MBP transgene (replicate 1) Shiv-iOPC-Rd2-Dox= Day 21 shiverer MEFs which were infected with the reprogramming factors but were not induced Shiv-iOPC-Rd2= Day 21 shiverer iOPCs with the MBP transgene (replicate 2) Shiv-iOPC-Rd3= Day 21 shiverer iOPCs with the MBP transgene (replicate 3)