Homozygous shiverer mice (The Jackson Laboratory, Bar Harbor, ME) were crossed with homozygous rag2-null immunodeficient mice (Shinkai et al., 1992) on the C3h background (Taconic, Germantown, NY, USA) to generate shi/shi × rag2−/− myelin-deficient, immunodeficient mice (Windrem et al., 2008). In addition, rag1−/− normally-myelinated immunodeficient mice (B6.129S7-Rag1tm1Mom/J), were obtained from the Jackson Laboratory and bred in our lab. Suspensions of single-cells or small clusters of hiPSC-derived GPCs were spun down to 100,000 cells/μl. Neonates were anesthetized by cooling, and transplanted bilaterally in the corpus callosum with a total of 200,000 cells, as described (Windrem et al., 2004); see Method Details section for transplant procedure. At 3 months of age (shi/shi × rag2−/−) or after completion of behavioral testing at 6–9 months (rag1−/− only), transplanted mice were anesthetized with pentobarbital, then perfusion fixed with cold HBSS+/+ followed by 4% paraformaldehyde (PF) with a 2 hour post-fixation in cold PF. All animal procedures were approved by the University of Rochester’s Committee on Animal Resources.
