Clinical-Grade Stem-Cell Based Model Systems for Mitochondrial Diseases

Mutations in the mitochondrial DNA (mtDNA) lead to many human mitochondrial disorders with impaired bioenergetics. The clinical severity of the disease may also depend on the load of mutant mtDNA and bioenergetic dysfunction. The development of FDA –approved therapies for primary mitochondrial disorders has been hindered by genetic and clinical heterogeneity, limited knowledge of natural history of disease progression, and lack of clear clinical endpoints. Two recent studies have reported the derivation of human induced pluripotent stem cells (hiPSCs) using viral methods from two human diseased fibroblast cell lines carrying different mtDNA mutations. These studies demonstrated that reprogramming led to the generation of isogenic hiPSC clones; each containing different mutant mtDNA levels despite identical nuclear genomes. An important finding was that cell fate decisions in the hiPSCs were directly linked to the extent of mutant mtDNA burden. To further advance the search for improved cell models and therapies, we have used a highly efficient mRNA nuclear reprogramming technology, considered as the fastest and safest approach for generating integration-free, virus-free, clinical grade human induced pluripotent stem cells (hiPSCs). We are focusing our efforts on Leigh’s Syndrome (LS), as there is no current cure for LS nor an adequate model for understanding the rapid fatality associated with the disease. Our results indicate that (a) successful reprogramming of a LS fibroblast (carrying 8993T>G) was achieved (b) LS-hiPSC exhibited multiple transcription factors and cell surface markers representative of a pluripotent stem cell; (c) LS-hiPSC exhibited ability to differentiate into all three embryonic germ layers (d) LS-hiPSCs and differentiated derivatives continue to exhibit the 8993T>G mutation. Our results demonstrate the generation and characterization of a stable clinical-grade LS-hiPSC cell based model that now provide us the opportunity to investigate the effects of pathogenic mtDNA burdens on altered bioenergetics; and to monitor and quantitate mtDNA dynamics during differentiation into specialized cell types.