Cell Stemness can be achieved by various reprogramming techniques namely, somatic cell nuclear transfer, cell fusion, cell extracts, and introduction of transcription factors from which induced pluripotent stem cells (iPSCs) are obtained. iPSCs are valuable cell sources for drug screening and human disease modeling. Alternatives to virus-based introduction of transcription factors include application of DNA-free methods and introduction of chemically defined culturing conditions. However, the possibility of tumor development is still a hurdle. By taking advantage of NTERA-2 cells, a human embryonal carcinoma cell line, we obtained partially differentiated cells and examined the dedifferentiation capacity of regenerative tissue from rabbit ears. Results indicated that treatment of partially differentiated NTERA-2 cells with the regenerating tissue-conditioned medium (CM) induced expression of key pluripotency markers as examined by real-time polymerase chain reaction, flow cytometry, and immunocytochemistry techniques. In this study, it is reported for the first time that the CM obtained from rabbit regenerating tissue contains dedifferentiation factors, taking cells back to the pluripotency. This system could be a simple and efficient way to reprogram the differentiated cells and generate iPSCs for clinical applications as this system is not accompanied by any viral vector, and reprograms the cells within 10 days of treatment. The results may convince the genomic experts to study the unknown signaling pathways involved in the dedifferentiation by regenerating tissue-CM to authenticate the reprogramming model.
Get full access to this article
View all access options for this article.
References
1.
AasenT., RayaA., BarreroM.J., GarretaE., ConsiglioA., GonzalezF., VassenaR., BilicJ., PekarikV., TiscorniaG., EdelM., BouéS., and Izpisúa BelmonteJ.C. (2008). Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes. Nat. Biotechnol., 26, 1276–1284.
2.
AndrewsP.W. (1984). Retinoic acid induces neuronal differentiation of a cloned human embryonal carcinoma cell line in vitro. Dev. Biol., 103, 285–293.
3.
AndrewsP.W., CasperJ., DamjanovI., Duggan-KeenM., GiwercmanA., HataJ., von KeitzA., LooijengaL.H., MillanJ.L., OosterhuisJ.W., PeraM., SawadaM., SchmollH.J., SkakkebaekN.E., van PuttenW., and SternP. (1996). Comparative analysis of cell surface antigens expressed by cell lines derived from human germ cell tumours. Int. J. Cancer., 66, 806–816.
4.
AndrewsP.W., DamjanovI., SimonD., BantingG.S., CarlinC., DracopoliN.C., and FoghJ. (1984). Pluripotent embryonal carcinoma clones derived from the human teratocarcinoma cell line Tera-2. Differentiation in vivo and in vitro. Lab. Invest., 50, 147–162.
5.
AndrewsP.W., MatinM.M., BahramiA.R., DamjanovI., GokhaleP., and DraperJ.S. (2005). Embryonic stem (ES) cells and embryonal carcinoma (EC) cells: opposite sides of the same coin. Biochem. Soc. Trans., 33, 1526–1530.
6.
AoiT., YaeK., NakagawaM., IchisakaT., OkitaK., TakahashiK., ChibaT., and YamanakaS. (2008). Generation of pluripotent stem cells from adult mouse liver and stomach cells. Science, 321, 699–702.
7.
BahramiA.R., MatinM.M., and AndrewsP.W. (2005). The CDK inhibitor p27 enhances neural differentiation in pluripotent NTERA2 human EC cells, but does not permit differentiation of 2102Ep nullipotent human EC cells. Mech. Dev., 122, 1034–1042.
8.
BosP.K., van OschG.J., FrenzD.A., VerhaarJ.A., and Verwoerd-VerhoefH.L. (2001). Growth factor expression in cartilage wound healing: temporal and spatial immunolocalization in a rabbit auricular cartilage wound model. Osteoarthritis Cartilage, 9, 382–389.
9.
CarlsonB.M. (2005). Some principles of regeneration in mammalian systems. Anat Rec B New Anat. 287, 4–13.
10.
ChenS., DoJ.T., ZhangQ., YaoS., YanF., PetersE.C., ScholerH.R., SchultzP.G., and DingS. (2006). Self-renewal of embryonic stem cells by a small molecule. Proc. Natl. Acad. Sci. U S A., 103, 17266–17271.
11.
CowanC.A., AtienzaJ., MeltonD.A., and EgganK. (2005). Nuclear reprogramming of somatic cells after fusion with human embryonic stem cells. Science, 309, 1369–1373.
12.
DoJ.T., and ScholerH.R. (2004). Nuclei of embryonic stem cells reprogram somatic cells. Stem Cells, 22, 941–949.
13.
DraperJ.S., PigottC., ThomsonJ.A., and AndrewsP.W. (2002). Surface antigens of human embryonic stem cells: changes upon differentiation in culture. J. Anat., 200, 249–258.
14.
FrebergC.T., DahlJ.A., TimoskainenS., and CollasP. (2007). Epigenetic reprogramming of OCT4 and NANOG regulatory regions by embryonal carcinoma cell extract. Mol. Biol. Cell., 18, 1543–1553.
15.
FusakiN., BanH., NishiyamaA., SaekiK., and HasegawaM. (2009). Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome. Proc. Jpn. Acad. Ser. B Phys. Biol. Sci., 85, 348–362.
16.
GossR.J., and GrimesL.N. (1972). Tissue interactions in the regeneration of rabbit ear holes. Amer. Zool., 12, 151–157.
17.
GossR.J., and GrimesL.N. (1975). Epidermal downgrowths in regenerating rabbit ear holes. J. Morphol., 146, 533–542.
18.
GrimesL.N., and GossR.J. (1970). Regeneration of holes in rabbit ears. Amer. Zool., 10, 537.
19.
GrinnellK.L., and BickenbachJ.R. (2007). Skin keratinocytes pre-treated with embryonic stem cell-conditioned medium or BMP4 can be directed to an alternative cell lineage. Cell Prolif. 40, 685–705.
20.
HansisC., BarretoG., MaltryN., and NiehrsC. (2004). Nuclear reprogramming of human somatic cells by xenopus egg extract requires BRG1. Curr. Biol., 14, 1475–1480.
21.
HuangfuD., MaehrR., GuoW., EijkelenboomA., SnitowM., ChenA.E., and MeltonD.A. (2008). Induction of pluripotent stem cells by defined factors is greatly improved by small-molecule compounds. Nat. Biotechnol., 26, 795–797.
22.
IchidaJ.K., BlanchardJ., LamK., SonE.Y., ChungJ.E., EgliD., LohK.M., CarterA.C., Di GiorgioF.P., KoszkaK., HuangfuD., AkutsuH., LiuD.R., RubinL.L., and EgganK. (2009). A small-molecule inhibitor of tgf-Beta signaling replaces sox2 in reprogramming by inducing nanog. Cell Stem Cell, 5, 491–503.
23.
KimD., KimC., MoonJ., ChungY., ChangM.Y., HanB., KoS., YangE., Yul ChaK., LanzaL., and KimK. (2009). Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins. Cell Stem Cell, 4, 472–476.
24.
LiY., ZhangQ., YinX., YangW., DuY., HouP., GeJ., LiuC., ZhangW., ZhangX., WuY., LiH., LiuK., WuC., SongZ., ZhaoY., ShiY., and DengH. (2011). Generation of iPSCs from mouse fibroblasts with a single gene, Oct4, and small molecules. Cell Res. 21, 196–204.
25.
LinT., AmbasudhanR., YuanX., LiW., HilcoveS., AbujarourR., LinX., HahmH.S., HaoE., HayekA., and DingS. (2009). A chemical platform for improved induction of human iPSCs. Nat. Methods., 6, 805–808.
26.
MaheraliN., and HochedlingerK. (2009). Tgfbeta signal inhibition cooperates in the induction of iPSCs and replaces Sox2 and cMyc. Curr. Biol., 19, 1718–1723.
27.
MaheraliN., SridharanR., XieW., UtikalJ., EminliS., ArnoldK., StadtfeldM., YachechkoR., TchieuJ., JaenischR., PlathK., and HochedlingerK. (2007). Directly reprogrammed fibroblasts show global epigenetic remodelling and widespread tissue contribution. Cell Stem Cell, 1, 55–70.
28.
MahmoudiZ., MatinM.M., SaeinasabM., Nakhaei-RadS., MirahmadiM., Mahdavi-ShahriN., MahmoudiM., and BahramiA.R. (2011). Blastema cells derived from rabbit ear show stem cell characteristics. JCMR, 3, 25–31.
29.
MarsonA., ForemanR., ChevalierB., BilodeauS., KahnM., YoungR.A., and JaenischR. (2008). Wnt signaling promotes reprogramming of somatic cells to pluripotency. Cell Stem Cell, 3, 132–135.
30.
MatinM.M., WalshJ.R., GokhaleP.J., DraperJ.S., BahramiA.R., MortonI., MooreH.D., and AndrewsP.W. (2004). Specific knockdown of Oct4 and beta2-microglobulin expression by RNA interference in human embryonic stem cells and embryonal carcinoma cells. Stem Cells, 22, 659–668.
31.
McGannC.J., OdelbergS.J., and KeatingM.T. (2001). Mammalian myotube dedifferentiation induced by newt regeneration extract. Proc. Natl. Acad. Sci. U S A., 98, 13699–13704.
32.
MiyoshiN., IshiiH., NaganoH., HaraguchiN., DewiD.L., KanoY., NishikawaS., TanemuraM., MimoriK., TanakaF., SaitoT., NishimuraJ., TakemasaI., MizushimaT., IkedaM., YamamotoH., SekimotoM., DokiY., and MoriM. (2011). Reprogramming of mouse and human cells to pluripotency using mature microRNAs. Cell Stem Cell, 8, 633–638.
33.
Nakhaei-RadS., BahramiA.R., MirahmadiM., and MatinM.M. (2012). New windows to enhance direct reprogramming of somatic cells towards induced pluripotent stem cells. Biochem Cell Biol. 90, 115–123.
34.
NiwaH., MiyazakiJ., and SmithA.G. (2000). Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat. Genet., 24, 372–376.
35.
OkitaK., IchisakaT., and YamanakaS. (2007). Generation of germline competent induced pluripotent stem cells. Nature, 448, 313–317.
36.
OkitaK., MatsumuraY., SatoY., OkadaA., MorizaneA., OkamotoS., HongH., NakagawaM., TanabeK., TezukaK., ShibataT., KunisadaT., TakahashiM., TakahashiJ., SajiH., and YamanakaS. (2011). A more efficient method to generate integration-free human iPS cells. Nat. Methods., 8, 409–412.
37.
OkitaK., NakagawaM., HyenjongH., IchisakaT., and YamanakaS. (2008). Generation of mouse induced pluripotent stem cells without viral vectors. Science, 322, 949–953.
38.
OkitaK., YamakawaT., MatsumuraY., SatoY., AmanoN., WatanabeA., GoshimaN., and YamanakaS. (2013). An efficient nonviral method to generate integration-free human-induced pluripotent stem cells from cord blood and peripheral blood cells. Stem Cells, 31, 458–466.
39.
ParkI.H., ZhaoR., WestJ.A., YabuuchiA., HuoH., InceT.A., LerouP.H., LenschM.W., and DaleyG.Q. (2008). Reprogramming of human somatic cells to pluripotency with defined factors. Nature, 451, 141–146.
40.
ShiY., Tae DoJ., DespontsC., Sik HahmH., ScholerH.R., and DingS. (2008). A combined chemical and genetic approach for the generation of induced pluripotent stem cells. Cell Stem Cell, 2, 525–528.
41.
SilvaJ., BarrandonO., NicholsJ., KawaguchiJ., TheunissenT.W., and SmithA. (2008). Promotion of reprogramming to ground state pluripotency by signal inhibition. PLoS Biol., 6, e253.
42.
SinghB.N., DoyleM.J., WeaverC.V., Koyano-NakagawaN., and GarryD.J. (2012). Hedgehog and Wnt coordinate signaling in myogenic progenitors and regulate limb regeneration. Dev. Biol., 371, 23–34.
43.
SomersA., JeanJ.C., SommerC.A., OmariA., FordC.C., MillsJ.A., YingL., SommerA.G., JeanJ.M., SmithB.W., LafyatisR., DemierreM.F., WeissD.J., FrenchD.L., GadueP., MurphyG.J., MostoslavskyG., and KottonD.N. (2010). Generation of transgene-free lung disease-specific human induced pluripotent stem cells using a single excisable lentiviral stem cell cassette. Stem Cells, 28, 1728–1740.
44.
SpergerJ.M., ChenX., DraperJ.S., AntosiewiczJ.E., ChonC.H., JonesS.B., BrooksJ.D., AndrewsP.W., BrownP.O., and ThomsonJ.A. (2003). Gene expression patterns in human embryonic stem cells and human pluripotent germ cell tumors. Proc. Natl. Acad. Sci. U S A., 100, 13350–13355.
45.
StadtfeldM., NagayaM., UtikalJ., WeirG., and HochedlingerK. (2008). Induced pluripotent stem cells generated without viral integration. Science, 322, 945–949.
46.
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.
47.
TakahashiK., and YamanakaS. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 126, 663–676.
48.
TarangerC.K., NoerA., SorensenA.L., HakelienA.M., BoquestA.C., and CollasP. (2005). Induction of dedifferentiation, genomewide transcriptional programming, and epigenetic reprogramming by extracts of carcinoma and embryonic stem cells. Mol Biol Cell. 16, 5719–5735.
49.
ThomsonJ.A., Itskovitz-EldorJ., ShapiroS.S., WaknitzM.A., SwiergielJ.J., MarshallV.S., and JonesJ.M. (1998). Embryonic stem cell lines derived from human blastocysts. Science, 282, 1145–1147.
50.
ThorntonC.S. (1957). The effect of apical cap removal on limb regeneration in Amblystoma larvae. J. Exp. Zool., 134, 357–381.
WallaceH. (1981). Vertebrate Limb Regeneration. (John Wiley and Sons Inc., New York).
53.
WarrenL., ManosP.D., AhfeldtT., LohY.H., LiH., LauF., EbinaW., MandalP.K., SmithZ.D., MeissnerA., DaleyG.Q., BrackA.S., CollinsJ.J., CowanC., SchlaegerT.M., and RossiD.J. (2010). Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell, 7, 618–630.
54.
WernigM., MeissnerA., ForemanR., BrambrinkT., KuM., HochedlingerK., BernsteinB.E., and JaenischR. (2007). In vitro reprogramming of fibroblasts into a pluripotent ES cell-like state. Nature, 448, 318–324.
55.
WilmutI., SchniekeA.E., McWhirJ., KindA.J., and CampbellK.H. (1997). Viable offspring derived from fetal and adult mammalian cells. Nature, 385, 810–813.
XieJ., QiS., XuY., TangJ., LiT., LiuX., ShuB., LiangH., and HuangB. (2008). Effects of basic fibroblast growth factors on hypertrophic scarring in a rabbit ear model. J. Cutan. Med. Surg., 12, 155–162.
58.
YuJ., VodyanikM.A., Smuga-OttoK., Antosiewicz-BourgetJ., FraneJ.L., TianS., NieJ., JonsdottirG.A., RuottiV., StewartR., SlukvinI.I., and ThomsonJ.A. (2007). Induced pluripotent stem cell lines derived from human somatic cells. Science, 318, 1917–1920.
59.
ZaravinosA., SouflaG., BizakisJ., and SpandidosD.A. (2008). Expression analysis of VEGFA, FGF2, TGFbeta1, EGF and IGF1 in human nasal polyposis. Oncol. Rep., 19, 385–391.
60.
ZhouH., WuS., Young JooJ., ZhuS., Wook HanD., LinT., TraugerS., BienG., YaoS., ZhuY., SiuzdakG., SchölerH, DuanL., and DingS. (2009). Generation of induced pluripotent stem cells using recombinant proteins. Cell Stem Cell, 4, 381–384.
61.
ZhouW., and FreedC.R. (2009). Adenoviral gene delivery can reprogram human fibroblasts to induced pluripotent stem cells. Stem Cells, 27, 2667–2674.
62.
ZhuS., LiW., ZhouH., WeiW., AmbasudhanR., LinT., KimJ., ZhangK., and DingS. (2010). Reprogramming of human primary somatic cells by OCT4 and chemical compounds. Cell Stem Cell, 7, 651–655.
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
