Study of mitochondrial respiratory defects on reprogramming to human induced pluripotent stem cells

Abstract

Reprogramming of somatic cells into a pluripotent state is known to be accompanied by extensive restructuring of mitochondria and switch in metabolic requirements. Here we utilized Leber's hereditary optic neuropathy (LHON) as a mitochondrial disease model to study the effects of homoplasmic mtDNA mutations and subsequent oxidative phosphorylation (OXPHOS) defects in reprogramming. We obtained fibroblasts from a total of 6 LHON patients and control subjects, and showed a significant defect in complex I respiration in LHON fibroblasts by high-resolution respiratory analysis. Using episomal vector reprogramming, our results indicated that human induced pluripotent stem cell (hiPSC) generation is feasible in LHON fibroblasts. In particular, LHON-specific OXPHOS defects in fibroblasts only caused a mild reduction and did not significantly affect reprogramming efficiency, suggesting that hiPSC reprogramming can tolerate a certain degree of OXPHOS defects. Our results highlighted the induction of genes involved in mitochondrial biogenesis (TFAM, NRF1), mitochondrial fusion (MFN1, MFN2) and glycine production (GCAT) during reprogramming. However, LHON-associated OXPHOS defects did not alter the kinetics or expression levels of these genes during reprogramming. Together, our study provides new insights into the effects of mtDNA mutation and OXPHOS defects in reprogramming and genes associated with various aspects of mitochondrial biology.

Keywords: Leber's hereditary optic neuropathy; cellular reprogramming; induced pluripotent stem cells; mitochondria; oxidative phosphorylation.

Figure 1. Genotyping of mtDNA mutation in patient fibroblasts

Genotyping of mtDNA m.11778, m.4160 and m.14484 in patient-derived fibroblasts. Red arrows indicate the position of LHON mutation.

Figure 2. OXPHOS analysis of control and LHON fibroblasts

(A) Oxygen consumption of control and LHON fibroblasts, showing the endogenous respiration rate (Endog), ADP-stimulated cell respiration with glutamate + malate (CxI-ADP) or with succinate (CxI+II-ADP), the uncoupled maximal respiration by addition of CCCP (CxI+II-UC) and the complex II respiration with uncoupled feedback by addition of rotenone (CxII-U). n = 3 for each patient fibroblast. Error bars represent SEM. (B) Complex I respiration of individual fibroblast samples and pooled data, normalized to uncoupled maximal respiration (CxI-ADP/CxI+II-UC). (C) Uncoupled complex II respiration (CxII-U/CxI+II-UC) in control (CERA007, MRU11780, BJ) and LHON fibroblasts (LHON V31-1, LHON T1-20, LHON Q1-4). **** = p<0.0001, *** = p<0.001, ** = p<0.01, * = p<0.05, ns = not significant.

Figure 3. Reprogramming of control and LHON fibroblasts using feeder-free system

Representative pictures of (A) TRA-1-60 positive colony and (B) TRA-1-60 negative colony. (C) Quantification of hiPSC colonies generated from control and LHON fibroblasts. Blue bar indicates bona fide hiPSC colonies, as defined by TRA-1-60 expression and hESC-like morphology, per 50,000 reprogrammed cells. Red bar indicates partially reprogrammed colonies as defined by absence of TRA-1-60 expression and/or resemble non-hESC-like morphology, per 50,000 reprogrammed cells. n=3, error bars represent SEM. (D) Pooled results of hiPSC colony number generated from control and LHON fibroblasts. ns = not significant, as indicated by unpaired t-test.

Figure 4. Characterization of the derived hiPSCs

(A) Cell morphology and immunocytochemistry analysis of pluripotent markers OCT4 and TRA-1-60 in a representative control hiPSC line (MRU11780) and LHON hiPSC line (LHON Q1-4). Scale bar = 100μm. (B) In vitro embryoid body differentiation of hiPSC into cell representative of the endoderm (AFP), mesoderm (SMA) and ectoderm (NESTIN). Scale bar = 100μm. (C) hiPSCs form teratoma containing endodermal cells (e, gut-like epithelium), mesodermal cells (c, cartilaginous structure; a, adipose tissue) and ectodermal cells (nr, neural rosette). Scale bar = 50μm.

Figure 5. Expression of mitochondrial regulatory genes during reprogramming

qPCR analysis of the expression of (A) TFAM, (B) NRF1, (C) MRN1, (D) MFN2 and (E) GCAT at various time points during reprogramming to hiPSCs (day 0, 7, 13, 21, 28). Error bars represent SEM for pooled data of control (CERA007, MRU11780, BJ) or LHON (LHON Q1-4, LHON V31-1, LHON T1-20).