Restricted accessResearch articleFirst published online 2014-11
Conversion of Partially Reprogrammed Cells to Fully Pluripotent Stem Cells Is Associated with Further Activation of Stem Cell Maintenance- and Gamete Generation-Related Genes
Somatic cells are reprogrammed to induced pluripotent stem cells (iPSCs) by overexpression of a combination of defined transcription factors. We generated iPSCs from mouse embryonic fibroblasts (with Oct4-GFP reporter) by transfection of pCX-OSK-2A (Oct4, Sox2, and Klf4) and pCX-cMyc vectors. We could generate partially reprogrammed cells (XiPS-7), which maintained more than 20 passages in a partially reprogrammed state; the cells expressed Nanog but were Oct4-GFP negative. When the cells were transferred to serum-free medium (with serum replacement and basic fibroblast growth factor), the XiPS-7 cells converted to Oct4-GFP-positive iPSCs (XiPS-7c, fully reprogrammed cells) with ESC-like properties. During the conversion of XiPS-7 to XiPS-7c, we found several clusters of slowly reprogrammed genes, which were activated at later stages of reprogramming. Our results suggest that partial reprogrammed cells can be induced to full reprogramming status by serum-free medium, in which stem cell maintenance- and gamete generation-related genes were upregulated. These long-term expandable partially reprogrammed cells can be used to verify the mechanism of reprogramming.
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References
1.
TakahashiK and YamanakaS. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 126:663–676.
2.
OkitaK, IchisakaT and YamanakaS. (2007). Generation of germline-competent induced pluripotent stem cells. Nature, 448:313–317.
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.
6.
EbertAD, YuJ, RoseFFJr., MattisVB, LorsonCL, ThomsonJA and SvendsenCN. (2009). Induced pluripotent stem cells from a spinal muscular atrophy patient. Nature, 457:277–280.
7.
TakahashiK, TanabeK, OhnukiM, NaritaM, IchisakaT, TomodaK and YamanakaS. (2007). Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell, 131:861–872.
8.
ArakiR, JinchoY, HokiY, NakamuraM, TamuraC, AndoS, KasamaY and AbeM. (2010). Conversion of ancestral fibroblasts to induced pluripotent stem cells. Stem Cells, 28:213–220.
9.
GolipourA, DavidL, LiuY, JayakumaranG, HirschCL, TrckaD and WranaJL. (2012). A late transition in somatic cell reprogramming requires regulators distinct from the pluripotency network. Cell Stem Cell, 11:769–782.
10.
HanssonJ, RafieeMR, ReilandS, PoloJM, GehringJ, OkawaS, HuberW, HochedlingerK and KrijgsveldJ. (2012). Highly coordinated proteome dynamics during reprogramming of somatic cells to pluripotency. Cell Rep, 2:1579–1592.
11.
LiR, LiangJ, NiS, ZhouT, QingX, LiH, HeW, ChenJ, LiF, et al. (2010). A mesenchymal-to-epithelial transition initiates and is required for the nuclear reprogramming of mouse fibroblasts. Cell Stem Cell, 7:51–63.
12.
MikkelsenTS, HannaJ, ZhangX, KuM, WernigM, SchorderetP, BernsteinBE, JaenischR, LanderES and MeissnerA. (2008). Dissecting direct reprogramming through integrative genomic analysis. Nature, 454:49–55.
13.
PoloJM, AnderssenE, WalshRM, SchwarzBA, NefzgerCM, LimSM, BorkentM, ApostolouE, AlaeiS, et al. (2012). A molecular roadmap of reprogramming somatic cells into iPS cells. Cell, 151:1617–1632.
14.
BuganimY, FaddahDA, ChengAW, ItskovichE, MarkoulakiS, GanzK, KlemmSL, van OudenaardenA and JaenischR. (2012). Single-cell expression analyses during cellular reprogramming reveal an early stochastic and a late hierarchic phase. Cell, 150:1209–1222.
15.
Sancho-MartinezI and Izpisua BelmonteJC. (2013). Stem cells: Surf the waves of reprogramming. Nature, 493:310–311.
16.
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.
17.
SilvaJ, BarrandonO, NicholsJ, KawaguchiJ, TheunissenTW and SmithA. (2008). Promotion of reprogramming to ground state pluripotency by signal inhibition. PLoS Biol, 6:e253
18.
SridharanR, TchieuJ, MasonMJ, YachechkoR, KuoyE, HorvathS, ZhouQ and PlathK. (2009). Role of the murine reprogramming factors in the induction of pluripotency. Cell, 136:364–377.
19.
SilvaJ, NicholsJ, TheunissenTW, GuoG, van OostenAL, BarrandonO, WrayJ, YamanakaS, ChambersI and SmithA. (2009). Nanog is the gateway to the pluripotent ground state. Cell, 138:722–737.
20.
TheunissenTW, van OostenAL, Castelo-BrancoG, HallJ, SmithA and SilvaJC. (2011). Nanog overcomes reprogramming barriers and induces pluripotency in minimal conditions. Curr Biol, 21:65–71.
21.
YuJ, VodyanikMA, Smuga-OttoK, Antosiewicz-BourgetJ, FraneJL, TianS, NieJ, JonsdottirGA, RuottiV, et al. (2007). Induced pluripotent stem cell lines derived from human somatic cells. Science, 318:1917–1920.
22.
SzaboPE, HubnerK, ScholerH and MannJR. (2002). Allele-specific expression of imprinted genes in mouse migratory primordial germ cells. Mech Dev, 115:157–160.
23.
BlellochR, VenereM, YenJ and Ramalho-SantosM. (2007). Generation of induced pluripotent stem cells in the absence of drug selection. Cell Stem Cell, 1:245–247.
24.
OkitaK, HongH, TakahashiK and YamanakaS. (2010). Generation of mouse-induced pluripotent stem cells with plasmid vectors. Nat Protoc, 5:418–428.
25.
KimMJ, ChoiHW, JangHJ, ChungHM, Arauzo-BravoMJ, ScholerHR and DoJT. (2013). Conversion of genomic imprinting by reprogramming and redifferentiation. J Cell Sci, 126:2516–2524.
26.
OkitaK, NakagawaM, HyenjongH, IchisakaT and YamanakaS. (2008). Generation of mouse induced pluripotent stem cells without viral vectors. Science, 322:949–953.
27.
WernigM, MeissnerA, ForemanR, BrambrinkT, KuM, HochedlingerK, BernsteinBE and JaenischR. (2007). In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature, 448:318–324.
28.
MaheraliN and HochedlingerK. (2008). Guidelines and techniques for the generation of induced pluripotent stem cells. Cell Stem Cell, 3:595–605.
29.
GreberB, WuG, BernemannC, JooJY, HanDW, KoK, TapiaN, SabourD, SterneckertJ, TesarP and ScholerHR. (2010). Conserved and divergent roles of FGF signaling in mouse epiblast stem cells and human embryonic stem cells. Cell Stem Cell, 6:215–226.
30.
NiwaH, OgawaK, ShimosatoD and AdachiK. (2009). A parallel circuit of LIF signalling pathways maintains pluripotency of mouse ES cells. Nature, 460:118–122.
31.
YingQL, NicholsJ, ChambersI and SmithA. (2003). BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell, 115:281–292.
32.
FiczG, BrancoMR, SeisenbergerS, SantosF, KruegerF, HoreTA, MarquesCJ, AndrewsS and ReikW. (2011). Dynamic regulation of 5-hydroxymethylcytosine in mouse ES cells and during differentiation. Nature, 473:398–402.
33.
FiczG, HoreTA, SantosF, LeeHJ, DeanW, ArandJ, KruegerF, OxleyD, PaulYL, et al. (2013). FGF signaling inhibition in ESCs drives rapid genome-wide demethylation to the epigenetic ground state of pluripotency. Cell Stem Cell, 13:351–359.
34.
ChenJ, LiuJ, HanQ, QinD, XuJ, ChenY, YangJ, SongH, YangD, et al. (2010). Towards an optimized culture medium for the generation of mouse induced pluripotent stem cells. J Biol Chem, 285:31066–31072.
35.
Di StefanoB, BueckerC, UngaroF, PrigioneA, ChenHH, WellingM, EijpeM, MostoslavskyG, TesarP, et al. (2010). An ES-like pluripotent state in FGF-dependent murine iPS cells. PLoS One, 5:e16092
36.
WuG, HanD, GongY, SebastianoV, GentileL, SinghalN, AdachiK, FischedickG, OrtmeierC, et al. (2013). Establishment of totipotency does not depend on Oct4A. Nat Cell Biol, 15:1089–1097.
37.
BronsIG, SmithersLE, TrotterMW, Rugg-GunnP, SunB, Chuva de Sousa LopesSM, HowlettSK, ClarksonA, Ahrlund-RichterL, PedersenRA and VallierL. (2007). Derivation of pluripotent epiblast stem cells from mammalian embryos. Nature, 448:191–195.
38.
KoivistoH, HyvarinenM, StrombergAM, InzunzaJ, MatilainenE, MikkolaM, HovattaO and TeerijokiH. (2004). Cultures of human embryonic stem cells: serum replacement medium or serum-containing media and the effect of basic fibroblast growth factor. Reprod Biomed Online, 9:330–337.
39.
JiaoJ, DangY, YangY, GaoR, ZhangY, KouZ, SunXF and GaoS. (2012). Promoting reprogramming by FGF2 reveals that the extracellular matrix is a barrier for reprogramming fibroblasts to pluripotency. Stem Cells, 31:729–740.
40.
EstebanMA, WangT, QinB, YangJ, QinD, CaiJ, LiW, WengZ, ChenJ, et al. (2010). Vitamin C enhances the generation of mouse and human induced pluripotent stem cells. Cell Stem Cell, 6:71–79.
41.
StadtfeldM, ApostolouE, FerrariF, ChoiJ, WalshRM, ChenT, OoiSS, KimSY, BestorTH, et al. (2012). Ascorbic acid prevents loss of Dlk1-Dio3 imprinting and facilitates generation of all-iPS cell mice from terminally differentiated B cells. Nat Genet, 44:398–405, S1–S2.
42.
GafniO, WeinbergerL, MansourAA, ManorYS, ChomskyE, Ben-YosefD, KalmaY, ViukovS, MazaI, et al. (2013). Derivation of novel human ground state naive pluripotent stem cells. Nature, 504:282–286.
43.
KurodaT, TadaM, KubotaH, KimuraH, HatanoSY, SuemoriH, NakatsujiN and TadaT. (2005). Octamer and Sox elements are required for transcriptional cis regulation of Nanog gene expression. Mol Cell Biol, 25:2475–2485.
44.
RoddaDJ, ChewJL, LimLH, LohYH, WangB, NgHH and RobsonP. (2005). Transcriptional regulation of nanog by OCT4 and SOX2. J Biol Chem, 280:24731–24737.
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