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. Next-generation 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.
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References
1.
IyerS, AlsayeghK, AbrahamS and RaoRR. (2009). Stem cell-based models and therapies for neurodegenerative diseases. Crit Rev Biomed Eng, 37:321–353.
2.
PicardM, WallaceDC and BurelleY. (2016). The rise of mitochondria in medicine. Mitochondrion, 30:105–116.
3.
LottMT, LeipzigJN, DerbenevaO, XieHM, ChalkiaD, SarmadyM, ProcaccioV and WallaceDC. (2018). MITOMAP: a human mitochondrial genome database. https://mitomap.org/foswiki/bin/view/MITOMAP/CitingMitomap
DimauroS. (2011). A history of mitochondrial diseases. J Inherit Metab Dis, 34:261–276.
6.
SchonEA, DiMauroS, HiranoM and GilkersonRW. (2010). Therapeutic prospects for mitochondrial disease. Trends Mol Med, 16:268–276.
7.
LeighD. (1951). Subacute necrotizing encephalomyelopathy in an infant. J Neurol Neurosurg Psychiatry, 14:216–221.
8.
LoeffenJL, SmeitinkJA, TrijbelsJM, JanssenAJ, TriepelsRH, SengersRC and van den HeuvelLP. (2000). Isolated complex I deficiency in children: clinical, biochemical and genetic aspects. Hum Mutat, 15:123–134.
9.
HoltIJ, HardingAE, PettyRK and Morgan-HughesJA. (1990). A new mitochondrial disease associated with mitochondrial DNA heteroplasmy. Am J Hum Genet, 46:428–433.
10.
ShoffnerJM, FernhoffPM, KrawieckiNS, CaplanDB, HoltPJ, KoontzDA, TakeiY, NewmanNJ, OrtizRG, et al. (1992). Subacute necrotizing encephalopathy: oxidative phosphorylation defects and the ATPase 6 point mutation. Neurology, 42:2168–2174.
11.
TatuchY, ChristodoulouJ, FeigenbaumA, ClarkeJT, WherretJ, SmithC, RuddN, Petrova-BenedictR and RobinsonBH. (1992). Heteroplasmic mtDNA mutation (T—-G) at 8993 can cause Leigh disease when the percentage of abnormal mtDNA is high. Am J Hum Genet, 50:852–858.
12.
OlivoPD, Van de WalleMJ, LaipisPJ and HauswirthWW. (1983). Nucleotide sequence evidence for rapid genotypic shifts in the bovine mitochondrial DNA D-loop. Nature, 306:400–402.
13.
PayneBA, WilsonIJ, Yu-Wai-ManP, CoxheadJ, DeehanD, HorvathR, TaylorRW, SamuelsDC, Santibanez-KorefM and ChinneryPF. (2013). Universal heteroplasmy of human mitochondrial DNA. Hum Mol Genet, 22:384–390.
14.
WilsonIJ, CarlingPJ, AlstonCL, FlorosVI, PyleA, HudsonG, SalleveltSC, LampertiC, CarelliV, et al. (2016). Mitochondrial DNA sequence characteristics modulate the size of the genetic bottleneck. Hum Mol Genet, 25:1031–1041.
15.
WaiT, TeoliD and ShoubridgeEA. (2008). The mitochondrial DNA genetic bottleneck results from replication of a subpopulation of genomes. Nat Genet, 40:1484–1488.
16.
SamuelsDC, WonnapinijP, CreeLM and ChinneryPF. (2010). Reassessing evidence for a postnatal mitochondrial genetic bottleneck. Nat Genet, 42:471–472; author reply 472–473.
17.
TachibanaM, SparmanM, SritanaudomchaiH, MaH, ClepperL, WoodwardJ, LiY, RamseyC, KolotushkinaO and MitalipovS. (2009). Mitochondrial gene replacement in primate offspring and embryonic stem cells. Nature, 461:367–372.
18.
CravenL, TuppenHA, GreggainsGD, HarbottleSJ, MurphyJL, CreeLM, MurdochAP, ChinneryPF, TaylorRW, et al. (2010). Pronuclear transfer in human embryos to prevent transmission of mitochondrial DNA disease. Nature, 465:82–85.
19.
IyerS, XiaoE, AlsayeghK, EroshenkoN, RiggsMJ, BennettJPJr. and RaoRR. (2012). Mitochondrial gene replacement in human pluripotent stem cell-derived neural progenitors. Gene Ther, 19:469–475.
20.
CherryABC, GagneKE, McLoughlinEM, BacceiA, GormanB, HartungO, MillerJD, ZhangJ, ZonRL, et al. (2013). Induced pluripotent stem cells with a mitochondrial DNA deletion. Stem Cells, 31:1287–1297.
21.
FolmesCDL, Martinez-FernandezA, Perales-ClementeE, LiX, McDonaldA, OglesbeeD, HrstkaSC, Perez-TerzicC, TerzicA and NelsonTJ. (2013). Disease-causing mitochondrial heteroplasmy segregated within induced pluripotent stem cell clones derived from a patient with MELAS. Stem Cells, 31:1298–1308.
22.
OkitaK, IchisakaT and YamanakaS. (2007). Generation of germline-competent induced pluripotent stem cells. Nature, 448:313–317.
23.
WarrenL, ManosPD, AhfeldtT, LohYH, LiH, LauF, EbinaW, MandalPK, SmithZD, et al. (2010). Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell, 7:618–630.
24.
MandalPK and RossiDJ. (2013). Reprogramming human fibroblasts to pluripotency using modified mRNA. Nat Protoc, 8:568.
25.
SheridanSD, SurampudiV and RaoRR. (2012). Analysis of embryoid bodies derived from human induced pluripotent stem cells as a means to assess pluripotency. Stem Cells Int, 2012:738910.
26.
AbrahamS, SheridanSD, MillerB and RaoRR. (2010). Stable propagation of human embryonic and induced pluripotent stem cells on decellularized human substrates. Biotechnol Prog, 26:1126–1134.
27.
WatanabeK, UenoM, KamiyaD, NishiyamaA, MatsumuraM, WatayaT, TakahashiJB, NishikawaS, NishikawaS-I, MugurumaK and SasaiY. (2007). A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat Biotechnol, 25:681–686.
28.
BockC, KiskinisE, VerstappenG, GuH, BoultingG, SmithZD, ZillerM, CroftGF, AmorosoMW, et al. (2011). Reference maps of human ES and iPS cell variation enable high-throughput characterization of pluripotent cell lines. Cell, 144:439–452.
29.
LiH. (2012). Exploring single-sample SNP and INDEL calling with whole-genome de novo assembly. Bioinformatics, 28:1838–1844.
30.
ThorvaldsdottirH, RobinsonJT and MesirovJP. (2013). Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration. Brief Bioinform, 14:178–192.
31.
HämäläinenRH, ManninenT, KoivumäkiH, KislinM, OtonkoskiT and SuomalainenA. (2013). Tissue- and cell-type-specific manifestations of heteroplasmic mtDNA 3243A>G mutation in human induced pluripotent stem cell-derived disease model. Proc Natl Acad Sci, 110:E3622–E3630.
32.
RobintonDA and DaleyGQ. (2012). The promise of induced pluripotent stem cells in research and therapy. Nature, 481:295–305.
33.
LiaoB, BaoX, LiuL, FengS, ZovoilisA, LiuW, XueY, CaiJ, GuoX, et al. (2011). MicroRNA cluster 302–367 enhances somatic cell reprogramming by accelerating a mesenchymal-to-epithelial transition. J Biol Chem, 286:17359–17364.
34.
BuganimY, FaddahDA and JaenischR. (2013). Mechanisms and models of somatic cell reprogramming. Nat Rev Genet, 14:427–439.
35.
RaoRR and SticeSL. (2004). Gene expression profiling of embryonic stem cells leads to greater understanding of pluripotency and early developmental events. Biol Reprod, 71:1772–1778.
36.
CaiJ, ChenJ, LiuY, MiuraT, LuoY, LoringJF, FreedWJ, RaoMS and ZengX. (2006). Assessing self-renewal and differentiation in human embryonic stem cell lines. Stem Cells, 24:516–530.
37.
LoringJF and RaoMS. (2006). Establishing standards for the characterization of human embryonic stem cell lines. Stem Cells, 24:145–150.
38.
HendersonJK, DraperJS, BaillieHS, FishelS, ThomsonJA, MooreH and AndrewsPW. (2002). Preimplantation human embryos and embryonic stem cells show comparable expression of stage-specific embryonic antigens. Stem Cells, 20:329–337.
39.
NatunenS, SatomaaT, PitkänenV, SaloH, MikkolaM, NatunenJ, OtonkoskiT and ValmuL. (2011). The binding specificity of the marker antibodies Tra-1-60 and Tra-1-81 reveals a novel pluripotency-associated type 1 lactosamine epitope. Glycobiology, 21:1125–1130.
40.
RaoRR, JohnsonAV and SticeSL. (2007). Cell surface markers in human embryonic stem cells. Methods Mol Biol, 407:51–61.
41.
CarrollCJ, BrilhanteV and SuomalainenA. (2014). Next-generation sequencing for mitochondrial disorders. Br J Pharmacol, 171:1837–1853.
42.
LiM, SchonbergA, SchaeferM, SchroederR, NasidzeI and StonekingM. (2010). Detecting heteroplasmy from high-throughput sequencing of complete human mitochondrial DNA genomes. Am J Hum Genet, 87:237–249.
43.
TangS and HuangT. (2010). Characterization of mitochondrial DNA heteroplasmy using a parallel sequencing system. Biotechniques, 48:287–296.
44.
WahlestedtM, AmeurA, MoraghebiR, NorddahlGL, StenG, WoodsNB and BryderD. (2014). Somatic cells with a heavy mitochondrial DNA mutational load render induced pluripotent stem cells with distinct differentiation defects. Stem Cells, 32:1173–1182.
45.
FolmesCD, NelsonTJ, Martinez-FernandezA, ArrellDK, LindorJZ, DzejaPP, IkedaY, Perez-TerzicC and TerzicA. (2011). Somatic oxidative bioenergetics transitions into pluripotency-dependent glycolysis to facilitate nuclear reprogramming. Cell Metab, 14:264–271.
46.
BukowieckiR, AdjayeJ and PrigioneA. (2014). Mitochondrial function in pluripotent stem cells and cellular reprogramming. Gerontology, 60:174–182.
47.
ZhangJ, NuebelE, DaleyGQ, KoehlerCM and TeitellMA. (2012). Metabolic regulation in pluripotent stem cells during reprogramming and self-renewal. Cell Stem Cell, 11:589–595.
48.
IyerS, BergquistK, YoungK, GnaigerE, RaoRR and BennettJPJr. (2012). Mitochondrial gene therapy improves respiration, biogenesis, and transcription in G11778A Leber's hereditary optic neuropathy and T8993G Leigh's syndrome cells. Hum Gene Ther, 23:647–657.
EbnerS, LangR, MuellerEE, EderW, OellerM, MoserA, KollerJ, PaulweberB, MayrJA, SperlW and KoflerB. (2011). Mitochondrial haplogroups, control region polymorphisms and malignant melanoma: a study in middle European Caucasians. PLoS One, 6:e27192.
51.
GovatatiS, SaradammaB, MalempatiS, DasiD, ThupuraniMK, NageshN, ShivajiS, BhanooriM, TamanamRR, NallanchakravarthulaV and PasupuletiSR. (2017). Association of mitochondrial displacement loop polymorphisms with risk of colorectal cancer in south Indian population. Mitochondrial DNA A DNA Mapp Seq Anal, 28:632–637.
52.
TipirisettiNR, GovatatiS, PullariP, MalempatiS, ThupuraniMK, PeruguS, GuruvaiahP, RaoKL, DigumartiRR, et al. (2014). Mitochondrial control region alterations and breast cancer risk: a study in South Indian population. PLoS One, 9:e85363.
53.
ZhangJ, ZhangZX, DuPC, ZhouW, WuSD, WangQL, ChenC, ShiQ, ChenC, et al. (2015). Analyses of the mitochondrial mutations in the Chinese patients with sporadic Creutzfeldt-Jakob disease. Eur J Hum Genet, 23:86–91.
54.
KimJ, AmbasudhanR and DingS. (2012). Direct lineage reprogramming to neural cells. Curr Opin Neurobiol, 22:778–784.
55.
EfeJA, HilcoveS, KimJ, ZhouH, OuyangK, WangG, ChenJ and DingS. (2011). Conversion of mouse fibroblasts into cardiomyocytes using a direct reprogramming strategy. Nat Cell Biol, 13:215–222.
56.
ShiY, KirwanP and LiveseyFJ. (2012). Directed differentiation of human pluripotent stem cells to cerebral cortex neurons and neural networks. Nat Protoc, 7:1836–1846.
57.
KirwanP, Turner-BridgerB, PeterM, MomohA, ArambepolaD, RobinsonHP and LiveseyFJ. (2015). Development and function of human cerebral cortex neural networks from pluripotent stem cells in vitro. Development, 142:3178–3187.
58.
DharaSK, HasneenK, MachacekDW, BoydNL, RaoRR and SticeSL. (2008). Human neural progenitor cells derived from embryonic stem cells in feeder-free cultures. Differentiation, 76:454–464.
59.
ShinS, MitalipovaM, NoggleS, TibbittsD, VenableA, RaoR and SticeSL. (2006). Long-term proliferation of human embryonic stem cell-derived neuroepithelial cells using defined adherent culture conditions. Stem Cells, 24:125–138.
60.
MarianiJ, SimoniniMV, PalejevD, TomasiniL, CoppolaG, SzekelyAM, HorvathTL and VaccarinoFM. (2012). Modeling human cortical development in vitro using induced pluripotent stem cells. Proc Natl Acad Sci U S A, 109:12770–12775.
61.
AldanaBI, ZhangY, LihmeMF, BakLK, NielsenJE, HolstB, HyttelP, FreudeKK and WaagepetersenHS. (2017). Characterization of energy and neurotransmitter metabolism in cortical glutamatergic neurons derived from human induced pluripotent stem cells: a novel approach to study metabolism in human neurons. Neurochem Int, 106:48–61.
62.
ZhouY, Al-SaaidiRA, Fernandez-GuerraP, FreudeKK, OlsenRK, JensenUB, GregersenN, HyttelP, BolundL, et al. (2017). Mitochondrial spare respiratory capacity is negatively correlated with nuclear reprogramming efficiency. Stem Cells Dev, 26:166–176.
63.
LorenzC and PrigioneA. (2017). Mitochondrial metabolism in early neural fate and its relevance for neuronal disease modeling. Curr Opin Cell Biol, 49:71–76.
64.
CliffTS and DaltonS. (2017). Metabolic switching and cell fate decisions: implications for pluripotency, reprogramming and development. Curr Opin Genet Dev, 46:44–49.
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