mRNA Reprogramming of T8993G Leigh's Syndrome Fibroblast Cells to Create Induced Pluripotent Stem Cell Models for Mitochondrial Disorders

Abstract

Early molecular and developmental events impacting many incurable mitochondrial disorders are not fully understood and require generation of relevant patient-and disease-specific stem cell models. In this study, we focus on the ability of a nonviral and integration-free reprogramming method for deriving clinical-grade induced pluripotent stem cells (iPSCs) specific to Leigh's syndrome (LS), a fatal neurodegenerative mitochondrial disorder of infants. The cause of fatality could be due to the presence of high abundance of mutant mitochondrial DNA (mtDNA) or decline in respiration levels, thus affecting early molecular and developmental events in energy-intensive tissues. LS patient fibroblasts (designated LS1 in this study), carrying a high percentage of mutant T8993G mtDNA, were reprogrammed using a combined mRNA-miRNA nonviral approach to generate human iPSCs (hiPSCs). The LS1-hiPSCs were evaluated for their self-renewal, embryoid body (EB) formation, and differentiation potential, using immunocytochemistry and gene expression profiling methods. Sanger sequencing and next-generation sequencing approaches were used to detect the mutation and quantify the percentage of mutant mtDNA in the LS1-hiPSCs and differentiated derivatives. Reprogrammed LS-hiPSCs expressed pluripotent stem cell markers including transcription factors OCT4, NANOG, and SOX2 and cell surface markers SSEA4, TRA-1-60, and TRA-1-81 at the RNA and protein level. LS1-hiPSCs also demonstrated the capacity for self-renewal and multilineage differentiation into all three embryonic germ layers. EB analysis demonstrated impaired differentiation potential in cells carrying high percentage of mutant mtDNA. Nextgeneration sequencing analysis confirmed the presence of high abundance of T8993G mutant mtDNA in the patient fibroblasts and their reprogrammed and differentiated derivatives. These results represent for the first time the derivation and characterization of a stable nonviral hiPSC line reprogrammed from a LS patient fibroblast carrying a high abundance of mutant mtDNA. These outcomes are important steps toward understanding disease origins and developing personalized therapies for patients suffering from mitochondrial diseases.

Leigh Syndrome: A Tale of Two Genomes

“…With iPSCs, some of these problems can be resolved as fibroblast derived from diseased patients could be reprogrammed and potentially differentiated into any cell/tissue to better elucidate the pathological mechanism of a specific mitochondrial disorder on various cells/tissues. Many recent studies have demonstrated the potential for generating human iPSC patient-specific models from LS carrying mutations in MT-ATP6 and MT-ND5 genes ( Galera-Monge et al, 2016 ; Zurita-Diaz et al, 2016 ; Grace et al, 2019 ). Another study combined somatic cell nuclear transfer (SCNT) and iPSC technology to perform a metabolic rescue in the iPSCs generated from these patients, demonstrating the significant possibilities that iPSC models hold, as they could potentially be used to simultaneously study pathogenesis and develop treatments for patients with LS and other mitochondrial diseases ( Ma et al, 2015 ).…”

Human iPSC-derived cerebral organoids model features of Leigh Syndrome and reveal abnormal corticogenesis

“…show obvious deficits in cell growth (Grace et al, 2019). Given the fact the neuroectoderm 244 expansion phase happens during days 7-10, the degeneration of the MT-ATP6/PDH organoids 245 after embedding may recapitulate these previously reported observations.…”

“…Defective organoid formation in this cell line was significantly higher than control and the other two LS cell lines (Figure S4B). A previous report showed that when iPSCs generated from fibroblasts harboring the same T8993G mitochondrial mutation were differentiated into EBs, there was rapid regression and death after 7 days in suspension, while the monolayer culture did not show obvious deficits in cell growth (Grace et al, 2019). Given the fact the neuroectoderm expansion phase happens during days 7-10, the degeneration of the MT-ATP6/PDH organoids after embedding may recapitulate these previously reported observations.…”

“…The ability to reprogram somatic cells into induced pluripotent stem cells (iPSCs), followed by differentiation into specific lineages has become a useful tool for complex disease modeling (Kelava and Lancaster, 2016; Di Lullo and Kriegstein, 2017; Pașca, 2018; Quadrato et al, 2016). In the context of LS, iPSCs have been successfully generated from patients with mutations in mitochondrially encoded ATP Synthase Membrane Subunit 6 (MT-ATP6) (Galera-Monge et al, 2016; Grace et al, 2019; Lorenz et al, 2017; Ma et al, 2015), mitochondrially encoded NADH:Ubiquinone Oxidoreductase Core Subunit 3 (MT-ND3) subunit (Hattori et al, 2016) and the nuclear-encoded gene Surfeit locus protein 1 (SURF1) (Inak et al, 2019, 2021). These iPSC-model systems have been proposed for drug discovery (Inak et al, 2017; Lorenz et al, 2017) as well as testing platforms for potential metabolic rescue treatments (Ma et al, 2015).…”

Modelling Mitochondrial Disease in Human Pluripotent Stem Cells: What Have We Learned?

“…Heteroplasmy often appears to remain stable following directed differentiation [ 45 , 87 , 91 ]. Therefore, it is practical to check heteroplasmy levels before and after differentiation to ensure they are within the phenotypic range for patients with the same mutation [ 45 , 171 , 172 ]. Additionally, use of mitochondrial targeted editing strategies like mitoTALENs and DdCBEs could help achieve a greater level of control prior to differentiation [ 80 , 81 , 83 ].…”