Derivation of Disease-Free Induced Pluripotent Stem Cells From Patients with Pearson Marrow Pancreas Syndrome

Abstract 3

Mutations in mitochondrial DNA (mtDNA) cause several incurable diseases. In congenital mtDNA disorders, a mixture of normal and mutated mtDNA termed heteroplasmy exists at varying levels in different tissues, which determines the severity and phenotypic expression of the disease. Pearson marrow pancreas syndrome (PS) is a congenital bone marrow failure disorder caused by heteroplasmic deletions in mtDNA. The clinical hallmarks of PS include sideroblastic anemia and other cytopenias, pancreatic insufficiency, metabolic acidosis, and other systemic organ dysfunction. The cause of the hematopoietic failure in PS is unknown, and adequate cellular and animal models are lacking. Somatic cells can be directly reprogrammed using defined genetic and chemical factors to yield “induced pluripotent stem” (iPS) cells, which have the capacity to differentiate into any tissue. iPS cells are particularly amenable for modeling mtDNA disorders, as cytoplasmic genetic material is retained during reprogramming. We sought to generate iPS cells from patients with PS and related mtDNA disorders to investigate the effects of mitochondrial dysfunction on stem cells and hematopoiesis. From a patient with PS, we generated bone marrow-derived fibroblasts carrying a high heteroplasmic burden of mutant mtDNA, and reprogrammed them into iPS cells. Although reprogramming efficiency was very low and kinetics of iPS colony emergence delayed, PS-iPS cells carrying the pathogenic mutation could be generated and displayed all hallmarks of pluripotency. We observed that PS-iPS cells initially demonstrated slow growth and a propensity for differentiation, but with ongoing passage in tissue culture, these characteristics improved. Unexpectedly, we found that the proportion of mutant mtDNA decreased rapidly in the PS-iPS lines as a function of passage. By subcloning, we were able to generate iPS cell lines with virtually undetectable amounts of mutant mtDNA, but which retained a viral integration pattern confirming their nuclear genetic identity to the original, highly heteroplasmic iPS clone. From “purged” PS-iPS cells, we generated hematopoietic progenitors free of detectable mutant mtDNA, thus yielding genetically identical, disease-free iPS cells and blood cells from a patient with Pearson syndrome. Disease-free iPS cells were readily obtained from the skin-derived fibroblasts of two other patients that carried a lower burden of mutant mtDNA. Our results suggest that mitochondrial dysfunction drives the segregation or elimination of mutant mtDNA in iPS cells as a function of passage, implying that maintenance of self-renewal and pluripotency are highly dependent on intact mitochondrial function. Importantly, a similar depletion of mutant mtDNA can be observed over time in vivo in certain tissues, such as hematopoietic cells, of patients with PS and other mtDNA disorders. This work provides a unique set of in vitro cellular models carrying varying degrees of mtDNA heteroplasmy to interrogate the effects of mitochondrial dysfunction on hematopoiesis. PS-iPS cells also provide a valuable opportunity to determine the factors driving changes in mtDNA heteroplasmy in stem cells, which holds important therapeutic implications for PS and a variety of congenital and acquired mtDNA disorders.