Generation of Human-Induced Pluripotent Stem Cells from Gut Mesentery-Derived Cells by Ectopic Expression of OCT4/SOX2/NANOG

Abstract

Induced pluripotent stem (iPS) cells have been generated from human somatic cells by ectopic expression of defined transcription factors. Application of this approach in human cells may have enormous potential to generate patient-specific pluripotent stem cells. However, traditional methods of reprogramming in human somatic cells involve the use of oncogenes c-MYC and KLF4, which are not applicable to clinical translation. In the present study, we investigated whether human fetal gut mesentery-derived cells (hGMDCs) could be successfully reprogrammed into induced pluripotent stem (iPS) cells by OCT4, SOX2, and NANOG alone. We used lentiviruses to express OCT4, SOX2, NANOG, in hGMDCs, then generated iPS cells that were identified by morphology, presence of pluripotency markers, global gene expression profile, DNA methylation status, capacity to form embryoid bodies (EBs), and terotoma formation. iPS cells resulting from hGMDCs were similar to human embryonic stem (ES) cells in morphology, proliferation, surface markers, gene expression, and epigenetic status of pluripotent cell-specific genes. Furthermore, these cells were able to differentiate into cell types of all three germ layers both in vitro and in vivo, as shown by EB and teratoma formation assays. DNA fingerprinting showed that the human iPS cells were derived from the donor cells, and are not a result of contamination. Our results provide proof that hGMDCs can be reprogrammed into pluripotent cells by ectopic expression of three factors (OCT4, SOX2, and NANOG) without the use of oncogenes c-MYC and KLF4.

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References

Aasen

, Raya

, Barrero

M.J.

et al. 2008. Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes. Nat. Biotechnol., 26:1276–1284.

Aoi

, Yae

, Nakagawa

et al. 2008. Generation of pluripotent stem cells from adult mouse liver and stomach cells. Science, 321:699–702.

Avilion

A.A.

, Nicolis

S.K.

, Pevny

L.H.

et al. 2003. Multipotent cell lineages in early mouse development depend on SOX2 function. Genes Dev, 17:126–140.

Boyer

L.A.

, Lee

T.I.

, Cole

M.F.

et al. 2005. Core transcriptional regulatory circuitry in human embryonic stem cells. Cell, 122:947–956.

Brambrink

, Foreman

, Welstead

G.G.

et al. 2008. Sequential expression of pluripotency markers during direct reprogramming of mouse somatic cells. Cell Stem Cell, 2:151–159.

Carey

B.W.

, Markoulaki

, Hanna

et al. 2009. Reprogramming of murine and human somatic cells using a single polycistronic vector. Proc. Natl. Acad. Sci. USA, 106:157–162.

Chagraoui

, Lepage-Noll

, Anjo

et al. 2003. Fetal liver stroma consists of cells in epithelial-to-mesenchymal transition. Blood, 101:2973–2982.

Chambers

, Colby

, Robertson

et al. 2003. Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell, 113:643–655.

Charbord

, Oostendorp

, Pang

et al. 2002. Comparative study of stromal cell lines derived from embryonic, fetal, and postnatal mouse blood-forming tissues. Exp. Hematol., 30:1202–1210.

10.

Darr

, Mayshar

, Benvenisty

2006. Overexpression of NANOG in human ES cells enables feeder-free growth while inducing primitive ectoderm features. Development, 133:1193–1201.

11.

Giorgetti

, Montserrat

, Aasen

et al. 2009. Generation of induced pluripotent stem cells from human cord blood using OCT4 and SOX2. Cell Stem Cell, 5:353–357.

12.

Haase

, Olmer

, Schwanke

et al. 2009. Generation of induced pluripotent stem cells from human cord blood. Cell Stem Cell., 5:434–441.

13.

Hanna

, Markoulaki

, Schorderet

et al. 2008. Direct reprogramming of terminally differentiated mature B lymphocytes to pluripotency. Cell, 133:250–264.

14.

Jaenisch

2004. Human cloning—the science and ethics of nuclear transplantation. N. Engl. J. Med., 351:2787–2791.

15.

Kaji

, Norrby

, Paca

et al. 2009. Virus-free induction of pluripotency and subsequent excision of reprogramming factors. Nature, 458:771–775.

16.

Kim

J.B.

, Zaehres

, Wu

et al. 2008. Pluripotent stem cells induced from adult neural stem cells by reprogramming with two factors. Nature, 454:646–650.

17.

Kim

, Kim

C.H.

, Moon

J.I.

et al. 2009a. Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins. Cell Stem Cell, 4:472–476.

18.

Kim

J.B.

, Greber

, Arauzo-Bravo

M.J.

et al. 2009b. Direct reprogramming of human neural stem cells by OCT4. Nature, 461:649–643.

19.

Kim

J.B.

, Sebastiano

, Wu

et al. 2009c. Oct4-induced pluripotency in adult neural stem cells. Cell, 136:411–419.

20.

Loh

Y.H.

, Wu

, Chew

J.L.

et al. 2006. The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells. Nat. Genet., 38:431–440.

21.

Loh

Y.H.

, Agarwal

, Park

I.H.

et al. 2009. Generation of induced pluripotent stem cells from human blood. Blood, 113:5476–5479.

22.

Lowry

W.E.

, Richter

, Yachechko

et al. 2008. Generation of human induced pluripotent stem cells from dermal fibroblasts. Proc. Natl. Acad. Sci. USA., 105:2883–2888.

23.

Maherali

, Sridharan

, Xie

et al. 2007. Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution. Cell Stem Cell, 1:55–70.

24.

Meissner

, Wernig

, Jaenisch

2007. Direct reprogramming of genetically unmodified fibroblasts into pluripotent stem cells. Nat. Biotechnol., 25:1177–1181.

25.

Mikkelsen

T.S.

, Hanna

, Zhang

et al. 2008. Dissecting direct reprogramming through integrative genomic analysis. Nature, 454:49–55.

26.

Mitsui

, Tokuzawa

, Itoh

et al. 2003. The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell, 113:631–642.

27.

Nakagawa

, Koyanagi

, Tanabe

et al. 2008. Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nat. Biotechnol., 26:101–106.

28.

Nichols

, Zevnik

, Anastassiadis

et al. 1998. Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell, 95:379–391.

29.

Okita

, Ichisaka

, Yamanaka

2007. Generation of germline-competent induced pluripotent stem cells. Nature, 448:313–317.

30.

Okita

, Nakagawa

, Hyenjong

et al. 2008. Generation of mouse induced pluripotent stem cells without viral vectors. Science, 322:949–953.

31.

Park

I.H.

, Zhao

, West

J.A.

et al. 2008. Reprogramming of human somatic cells to pluripotency with defined factors. Nature, 451:141–146.

32.

Silva

, Chambers

, Pollard

et al. 2006. Nanog promotes transfer of pluripotency after cell fusion. Nature, 441:997–1001.

33.

Silva

, Barrandon

, Nichols

et al. 2008. Promotion of reprogramming to ground state pluripotency by signal inhibition. PLoS Biol., 6:e253.

34.

Stadtfeld

, Brennand

, Hochedlinger

2008a. Reprogramming of pancreatic beta cells into induced pluripotent stem cells. Curr. Biol., 18:890–894.

35.

Stadtfeld

, Maherali

, Breault

D.T.

et al. 2008b. Defining molecular cornerstones during fibroblast to iPS cell reprogramming in mouse. Cell Stem Cell, 2:230–240.

36.

Stadtfeld

, Nagaya

, Utikal

et al. 2008c. Induced pluripotent stem cells generated without viral integration. Science, 322:945–949.

37.

Sun

, Panetta

N.J.

, Gupta

D.M.

et al. 2009. Feeder-free derivation of induced pluripotent stem cells from adult human adipose stem cells. Proc. Natl. Acad. Sci. USA, 106:15720–15725.

38.

Takahashi

, Tanabe

, Ohnuki

et al. 2007. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell, 131:861–872.

39.

Takahashi

, Yamanaka

2006. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 126:663–676.

40.

Thomson

J.A.

, Itskovitz-Eldor

, Shapiro

S.S.

et al. 1998. Embryonic stem cell lines derived from human blastocysts. Science, 282:1145–1147.

41.

Wernig

, Meissner

, Foreman

et al. 2007. In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature, 448:318–324.

42.

Woltjen

, Michael

I.P.

, Mohseni

et al. 2009. piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells. Nature, 458:766–770.

43.

Yamanaka

2009. Elite and stochastic models for induced pluripotent stem cell generation. Nature, 460:49–52.

44.

Yantiss

R.K.

, Spiro

I.J.

, Compton

C.C.

et al. 2000. Gastrointestinal stromal tumor versus intra-abdominal fibromatosis of the bowel wall: a clinically important differential diagnosis. Am. J. Surg. Pathol., 24:947–957.

45.

, Vodyanik

M.A.

, Smuga-Otto

et al. 2007. Induced pluripotent stem cell lines derived from human somatic cells. Science, 318:1917–1920.

46.

, Hu

, Smuga-Otto

et al. 2009. Human induced pluripotent stem cells free of vector and transgene sequences. Science, 324:797–801.

47.

Zhou

, Wu

, Joo

J.Y.

et al. 2009. Generation of induced pluripotent stem cells using recombinant proteins. Cell Stem Cell, 4:381–384.

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