Pluripotent stem cells (PSCs) offer unprecedented biomedical potential not only in relation to humans but also companion animals, particularly the horse. Despite this, attempts to generate bona fide equine embryonic stem cells have been unsuccessful. A very limited number of induced PSC lines have so far been generated from equine fibroblasts but their potential for directed differentiation into clinically relevant tissues has not been explored. In this study, we used retroviral vectors to generate induced pluripotent stem cells (iPSCs) with comparatively high efficiency from equine keratinocytes. Expression of endogenous PSC markers (OCT4, SOX2, LIN28, NANOG, DNMT3B, and REX1) was effectively restored in these cells, which could also form in vivo several tissue derivatives of the three germ layers, including functional neurons, keratinized epithelium, cartilage, bone, muscle, and respiratory and gastric epithelia. Comparative analysis of different reprogrammed cell lines revealed an association between the ability of iPSCs to form well-differentiated teratomas and the distinct endogenous expression of OCT4 and REX1 and reduced expression of viral transgenes. Importantly, unlike in previous studies, equine iPSCs were successfully expanded using simplified feeder-free culture conditions, constituting significant progress toward future biomedical applications. Further, under appropriate conditions equine iPSCs generated cells with features of cholinergic motor neurons including the ability to generate action potentials, providing the first report of functional cells derived from equine iPSCs. The ability to derive electrically active neurons in vitro from a large animal reveals highly conserved pathways of differentiation across species and opens the way for new and exciting applications in veterinary regenerative medicine.
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References
1.
CyranoskiD. (2013). Stem cells boom in vet clinics. Nature, 496:148–149.
2.
SchnabelLV, FortierLA, McIlwraithCW and NobertKM. (2013). Therapeutic use of stem cells in horses: which type, how, and when?. Vet J, 197:570–577.
3.
RadtkeCL, Nino-FongR, Esparza GonzalezBP, StryhnH and McDuffeeLA. (2013). Characterization and osteogenic potential of equine muscle tissue- and periosteal tissue-derived mesenchymal stem cells in comparison with bone marrow- and adipose tissue-derived mesenchymal stem cells. Am J Vet Res, 74:790–800.
4.
De SchauwerC, Van de WalleGR, Van SoomA and MeyerE. (2013). Mesenchymal stem cell therapy in horses: useful beyond orthopedic injuries?. Vet Q, 33:234–241.
5.
CarvalhoAM, YamadaAL, GolimMA, AlvarezLE, HussniCA and AlvesAL. (2013). Evaluation of mesenchymal stem cell migration after equine tendonitis therapy. Equine Vet J [Epub ahead of print]; DOI: 10.1111/evj.12173.
6.
FortierLA. (2012). Making progress in the what, when and where of regenerative medicine for our equine patients. Equine Vet J, 44:511–512.
7.
KisidayJD, GoodrichLR, McIlwraithCW and FrisbieDD. (2013). Effects of equine bone marrow aspirate volume on isolation, proliferation, and differentiation potential of mesenchymal stem cells. Am J Vet Res, 74:801–807.
8.
López-GonzálezR and VelascoI. (2012). Therapeutic potential of motor neurons differentiated from embryonic stem cells and induced pluripotent stem cells. Arch Med Res, 43:1–10.
9.
GuestDJ and AllenWR. (2007). Expression of cell-surface antigens and embryonic stem cell pluripotency genes in equine blastocysts. Stem Cells Dev, 16:789–796.
10.
OkitaK and YamanakaS. (2011). Induced pluripotent stem cells: opportunities and challenges. Philos Trans R Soc Lond B Biol Sci, 366:2198–2207.
11.
BretonA, SharmaR, DiazAC, ParhamAG, GrahamA, NeilC, WhitelawCB, MilneE and DonadeuFX. (2013). Derivation and characterization of induced pluripotent stem cells from equine fibroblasts. Stem Cells Dev, 22:611–621.
KhodadadiK, SumerH, PashaiaslM, LimS, WilliamsonM and VermaPJ. (2012). Induction of pluripotency in adult equine fibroblasts without c-MYC. Stem Cells Int, 2012:429160
14.
MaheraliN, AhfeldtT, RigamontiA, UtikalJ, CowanC and HochedlingerK. (2008). A high-efficiency system for the generation and study of human induced pluripotent stem cells. Cell Stem Cell, 3:340–345.
15.
AasenT, RayaA, BarreroMJ, GarretaE, ConsiglioA, GonzalezF, VassenaR, BilićJ, PekarikV, et al. (2008). Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes. Nat Biotechnol, 26:1276–1284.
16.
OhmineS, SquillaceKA, HartjesKA, DeedsMC, ArmstrongAS, ThatavaT, SakumaT, TerzicA, KudvaY and IkedaY. (2012). Reprogrammed keratinocytes from elderly type 2 diabetes patients suppress senescence genes to acquire induced pluripotency. Aging (Albany NY), 4:60–73.
17.
AbdullahAI, PollockA and SunT. (2012). The path from skin to brain: generation of functional neurons from fibroblasts. Mol Neurobiol, 45:586–595.
18.
EgawaN, KitaokaS, TsukitaK, NaitohM, TakahashiK, YamamotoT, AdachiF, KondoT, OkitaK, et al. (2012). Drug screening for ALS using patient-specific induced pluripotent stem cells. Sci Transl Med, 4:145ra104
19.
LancasterMA, RennerM, MartinCA, WenzelD, BicknellLS, HurlesME, HomfrayT, PenningerJM, JacksonAP and KnoblichJA. (2013). Cerebral organoids model human brain development and microcephaly. Nature, 501:373–339.
20.
WylieCE and ProudmanCJ. (2009). Equine grass sickness: epidemiology, diagnosis, and global distribution. Vet Clin North Am Equine Pract, 25:381–399.
21.
MohammedHO, DiversTJ, KwakJ, OmarAH, WhiteME and de LahuntaA. (2012). Association of oxidative stress with motor neuron disease in horses. Am J Vet Res, 73:1957–1962.
22.
VolkSW and TheoretC. (2013). Translating stem cell therapies: the role of companion animals in regenerative medicine. Wound Repair Regen, 21:382–394.
23.
LoeschA, MayhewTM, TangH, LaddFV, LaddAA, de MeloMP, da SilvaAA and CoppiAA. (2010). Stereological and allometric studies on neurons and axo-dendritic synapses in the superior cervical ganglia of rats, capybaras and horses. Cell Tissue Res, 341:223–237.
24.
Cebrian-SerranoA, StoutT and DinnyesA. (2013). Veterinary applications of induced pluripotent stem cells: regenerative medicine and models for disease?. Vet J, 198:34–42.
SzkolnickaD, FarnworthSL, Lucendo-VillarinB, StorckC, ZhouW, IredaleJP, FlintO and HayDC. (2014). Accurate prediction of drug-induced liver injury using stem cell-derived populations. Stem Cells Transl Med, 3:141–148.
27.
SharmaR, BarakzaiSZ, TaylorSE and DonadeuFX. (2013). Epidermal-like architecture obtained from equine keratinocytes in three-dimensional cultures. J Tissue Eng Regen Med [Epub ahead of print]; DOI: 10.1002/term.1788.
28.
BilicanB, LiveseyMR, HaghiG, QiuJ, BurrK, SillerR, HardinghamGE, WyllieDJ and ChandranS. (2014). Physiological normoxia and absence of EGF is required for the long-term propagation of anterior neural precursors from human pluripotent cells. PLoS One, 9:e85932
29.
RoseLC, FitzsimmonsR, LeeP, KrawetzR, RancourtDE and UludağH. (2013). Effect of basic fibroblast growth factor in mouse embryonic stem cell culture and osteogenic differentiation. J Tissue Eng Regen Med, 7:371–382.
30.
SommerCA, ChristodoulouC, Gianotti-SommerA, ShenSS, SailajaBS, HezroniH, SpiraA, MeshorerE, KottonDN and MostoslavskyG. (2012). Residual expression of reprogramming factors affects the transcriptional program and epigenetic signatures of induced pluripotent stem cells. PLoS One, 7:e51711
31.
FlöttmannM, ScharpT and KlippE. (2012). A stochastic model of epigenetic dynamics in somatic cell reprogramming. Front Physiol, 3:216
32.
DehmeltL and HalpainS. (2005). The MAP2/Tau family of microtubule-associated proteins. Genome Biol, 6:204
33.
ShupliakovO, HauckeV and PechsteinA. (2011). How synapsin I may cluster synaptic vesicles. Semin Cell Dev Biol, 22:393–399.
34.
LeeS, CuvillierJM, LeeB, ShenR, LeeJW and LeeSK. (2012). Fusion protein Isl1-Lhx3 specifies motor neuron fate by inducing motor neuron genes and concomitantly suppressing the interneuron programs. Proc Natl Acad Sci U S A, 109:3383–3388.
35.
LiangX, SongMR, XuZ, LanuzaGM, LiuY, ZhuangT, ChenY, PfaffSL, EvansSM and SunY. (2011). Isl1 is required for multiple aspects of motor neuron development. Mol Cell Neurosci, 47:215–222.
36.
DasenJS and JessellTM. (2009). Hox networks and the origins of motor neuron diversity. Curr Top Dev Biol, 88:169–200.
37.
MauckschC, JonesKS and ConnorB. (2013). Concise review: the involvement of SOX2 in direct reprogramming of induced neural stem/precursor cells. Stem Cells Transl Med, 2:579–583.
38.
ShiY, KirwanP and LiveseyFJ. (2012). Directed differentiation of human pluripotent stem cells to cerebral cortex neurons and neural networks. Nat Protoc, 7:1836–1846.
39.
LiuH and ZhangSC. (2011). Specification of neuronal and glial subtypes from human pluripotent stem cells. Cell Mol Life Sci, 68:3995–4008.
40.
SmuklerSR, RuncimanSB, XuS and van der KooyD. (2006). Embryonic stem cells assume a primitive neural stem cell fate in the absence of extrinsic influences. J Cell Biol, 172:79–90.
41.
RowlandJW, LeeJJ, SalewskiRP, EftekharpourE, van der KooyD and FehlingsMG. (2011). Generation of neural stem cells from embryonic stem cells using the default mechanism: in vitro and in vivo characterization. Stem Cells Dev, 20:1829–1845.
42.
TantinD. (2013). Oct transcription factors in development and stem cells: insights and mechanisms. Development, 140:2857–2866.
43.
RadzisheuskayaA, GeB, ChiaRL, dos SantosTW, TheunissenLF, CastroLF, NicholsJ and SilvaJC. (2013). A defined Oct4 level governs cell state transitions of pluripotency entry and differentiation into all embryonic lineages. Nat Cell Biol, 15:579–590.
44.
NicholsJ and SmithA. (2009). Naive and primed pluripotent states. Cell Stem Cell, 4:487–492.
45.
SonMY, ChoiH, HanYM and ChoYS. (2013). Unveiling the critical role of REX1 in the regulation of human stem cell pluripotency. Stem Cells, 31:2374–2387.
46.
SartoriC, DiDomenicoAI, ThomsonAJ, MilneE, LillicoSG, BurdonTG and WhitelawCB. (2012). Ovine-induced pluripotent stem cells can contribute to chimeric lambs. Cell Reprogram, 14:8–19.
47.
EzashiT, TeluguBP and RobertsRM. (2012). Induced pluripotent stem cells from pigs and other ungulate species: an alternative to embryonic stem cells?. Reprod Domest Anim, 47Suppl 4:92–97.
48.
SumerH, LiuJ, Malaver OrtegaLF, LimML, KhodadadiK and VermaPJ. (2011). NANOG is a key factor for induction of pluripotency in bovine adult fibroblasts. J Anim Sci, 89:2708–2716.
49.
ChanEM, RatanasirintrawootS, ParkIH, ManosPD, LohYH, HuoH, MillerJD, HartungO, RhoJ, et al. (2009). Live cell imaging distinguishes bona fide human iPS cells from partially reprogrammed cells. Nat Biotechnol, 27:1033–1037.
50.
BilicJ and Izpisua BelmonteJC. (2012). Concise review: Induced pluripotent stem cells versus embryonic stem cells: close enough or yet too far apart?. Stem Cells, 30:33–41.
51.
MontserratN, de OñateL, GarretaE, GonzálezF, AdamoA, EguizábalC, HäfnerS, VassenaR and Izpisua BelmonteJC. (2012). Generation of feeder-free pig induced pluripotent stem cells without Pou5f1. Cell Transplant, 21:815–825.
52.
RodríguezA, AllegrucciC and AlberioR. (2012). Modulation of pluripotency in the porcine embryo and iPS cells. PLoS One, 7:e49079
EsmailpourT and HuangT. (2012). TBX3 promotes human embryonic stem cell proliferation and neuroepithelial differentiation in a differentiation stage-dependent manner. Stem Cells, 30:2152–2163.
55.
TsangWH, WangB, WongWK, ShiS, ChenX, HeX, GuS, HuJ, WangC, et al. (2013). LIF-dependent primitive neural stem cells derived from mouse ES cells represent a reversible stage of neural commitment. Stem Cell Res, 11:1091–1102.
56.
DimosJT, RodolfaKT, NiakanKK, WeisenthalLM, MitsumotoH, ChungW, CroftGF, SaphierG, LeibelR, et al. (2008). Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons. Science, 321:1218–1221.
57.
KarumbayaramS, NovitchBG, PattersonM, UmbachJA, RichterL, LindgrenA, ConwayAE, ClarkAT, GoldmanSA, et al. (2009). Directed differentiation of human-induced pluripotent stem cells generates active motor neurons. Stem Cells, 27:806–811.
58.
WadeCM, GiulottoE, SigurdssonS, ZoliM, GnerreS, ImslandF, LearTL, AdelsonDL, BaileyE, et al. (2009). Genome sequence, comparative analysis, and population genetics of the domestic horse. Science, 326:865–867.
59.
CibelliJ, EmborgME, ProckopDJ, RobertsM, SchattenG, RaoM, HardingJ and MirochnitchenkoO. (2013). Strategies for improving animal models for regenerative medicine. Cell Stem Cell, 12:271–274.
60.
HardingJ, RobertsRM and MirochnitchenkoO. (2013). Large animal models for stem cell therapy. Stem Cell Res Ther, 4:23
61.
RiceCM, KempK, WilkinsA and ScoldingNJ. (2013). Cell therapy for multiple sclerosis: an evolving concept with implications for other neurodegenerative diseases. Lancet, 382:1204–1213.
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