title: A high-efficiency system for the generation and study of human induced pluripotent stem cells.
abstract: Direct reprogramming of human fibroblasts to a pluripotent state has been achieved through ectopic expression of the transcription factors OCT4, SOX2, and either cMYC and KLF4 or NANOG and LIN28. Little is known, however, about the mechanisms by which reprogramming occurs, which is in part limited by the low efficiency of conversion. To this end, we sought to create a doxycycline-inducible lentiviral system to convert primary human fibroblasts and keratinocytes into human induced pluripotent stem cells (hiPSCs). hiPSCs generated with this system were molecularly and functionally similar to human embryonic stem cells (hESCs), demonstrated by gene expression profiles, DNA methylation status, and differentiation potential. While expression of the viral transgenes was required for several weeks in fibroblasts, we found that 10 days was sufficient for the reprogramming of keratinocytes. Using our inducible system, we developed a strategy to induce hiPSC formation at high frequency. Upon addition of doxycycline to hiPSC-derived differentiated cells, we obtained "secondary" hiPSCs at a frequency at least 100-fold greater than the initial conversion. The ability to reprogram cells at high efficiency provides a unique platform to dissect the underlying molecular and biochemical processes that accompany nuclear reprogramming.
authors.affiliation: Department of Stem Cell and Regenerative Biology, Harvard Stem Cell Institute, Massachusetts General Hospital Center for Regenerative Medicine, Massachusetts General Hospital Cancer Center, Boston, MA 02114, USA.
authors.forename: Nimet
authors.initials: N
authors.lastname: Maherali
authors.forename: Tim
authors.initials: T
authors.lastname: Ahfeldt
authors.forename: Alessandra
authors.initials: A
authors.lastname: Rigamonti
authors.forename: Jochen
authors.initials: J
authors.lastname: Utikal
authors.forename: Chad
authors.initials: C
authors.lastname: Cowan
authors.forename: Konrad
authors.initials: K
authors.lastname: Hochedlinger
mesh_terms: D054337:Cell Dedifferentiation; D065150:Cellular Reprogramming; D003584:Cytological Techniques / Q000379:methods*; D005347:Fibroblasts / Q000378:metabolism; D005821:Genetic Techniques; D005822:Genetic Vectors / Q000378:metabolism; D006801:Humans; D000090062:Kruppel-Like Factor 4; D016086:Lentivirus / Q000235:genetics; D039904:Pluripotent Stem Cells / Q000378:metabolism*
references.citation: Nature. 2007 Jul 19;448(7151):313-7
references.pmid: 17554338
references.citation: Nat Biotechnol. 2008 Aug;26(8):916-24
references.pmid: 18594521
references.citation: Curr Biol. 2008 Jun 24;18(12):890-4
references.pmid: 18501604
references.citation: Cell Stem Cell. 2008 Mar 6;2(3):230-40
references.pmid: 18371448
references.citation: Bioinformatics. 2006 May 1;22(9):1111-21
references.pmid: 16522673
references.citation: Proc Natl Acad Sci U S A. 2008 Feb 26;105(8):2883-8
references.pmid: 18287077
references.citation: Dev Cell. 2008 Jun;14(6):818-29
references.pmid: 18539112
references.citation: Science. 2008 Aug 1;321(5889):699-702
references.pmid: 18276851
references.citation: Nat Biotechnol. 2008 Jul;26(7):795-7
references.pmid: 18568017
references.citation: Science. 2007 Dec 21;318(5858):1917-20
references.pmid: 18029452
references.citation: Nature. 2008 Jan 10;451(7175):141-6
references.pmid: 18157115
references.citation: Cell. 2006 Aug 25;126(4):663-76
references.pmid: 16904174
references.citation: Cell Stem Cell. 2007 Jun 7;1(1):55-70
references.pmid: 18371336
references.citation: Nature. 2007 Jul 19;448(7151):318-24
references.pmid: 17554336
references.citation: Cell. 2008 Apr 18;133(2):250-64
references.pmid: 18423197
references.citation: Nat Genet. 2000 Apr;24(4):372-6
references.pmid: 10742100
references.citation: Cell. 2007 Nov 30;131(5):861-72
references.pmid: 18035408
references.citation: Nature. 2008 Jul 3;454(7200):49-55
references.pmid: 18509334
references.citation: Stem Cells. 2008 Apr;26(4):903-11
references.pmid: 18238855