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Methods of generating pluripotent cells from somatic cells

A cell and primary cell technology, applied in the direction of artificially induced pluripotent cells, biochemical equipment and methods, animal cells, etc., can solve problems such as developmental potential limitations

Inactive Publication Date: 2010-08-11
THE GENERAL HOSPITAL CORP +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

iPS cells selected by Fbx15 contribute to various tissues in mid-gestational embryos, however, these mid-gestational death embryos demonstrate that iPS cells have limited developmental potential compared with ES cells

Method used

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  • Methods of generating pluripotent cells from somatic cells
  • Methods of generating pluripotent cells from somatic cells
  • Methods of generating pluripotent cells from somatic cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0158] Example 1: Generation of iPS cells using fibroblasts selectable by Nanog

[0159] Female mouse embryonic fibroblasts (MEFs) carrying GFP-IRES-Puro cassettes in the endogenous Nanog locus are called Nanog-GFP-puro (Hatano, S.Y.; Tada, M.; Kimura, H.; Yamaguchi , S.; Kono, T.; Nakano, T.; Suemori, H.; Nakatsuji, N. and Tada, T. (2005) Mech Dev 122, 67-79), which were encoded with Oct4, Sox2, c- Retroviral infection of the Myc-T58A mutant and cDNA of Klf4, which stabilized the protein (Sears, R.; Nuckolls, F.; Haura, E.; Taya, Y. ; Tamai, K. and Nevins, J.R. (2000) Genes Dev 14, 2501-2514). In contrast to previously reported selection for Fbx15 at 3 days post-infection (Takahashi and Yamanaka, 2006), selection for Nanog expression 3 days post-infection did not result in a population of cells, suggesting that the kinetics of reactivation of Fbx15 and Nanog genes differ. Resistant cell populations reproducibly emerged when selection was performed 7 or more days after inf...

Embodiment 2

[0161] Example 2: Nanog-selectable iPS cells confer ES cell-like phenotype on somatic cells

[0162] To determine whether iPS cells that can be selected by Nanog have functional properties similar to ES cells, the ability to impose an ES-like phenotype on somatic cells in the context of cell fusion was tested. Cells were derived from the puromycin-resistant 2D4 iPS cell line and hygromycin-resistant MEFs ( Figure 2A ). Two weeks after fusion, seven double-resistant tetraploid hybrid clones with ES cell-like morphology and continuing to express Nanog-GFP were recovered ( Figure 2B and the data is not shown). A hybrid cell population was recovered when control Nanog-GFP-puro ES cells were fused with hygromycin-resistant MEFs. To test for pluripotency, the hybrid cells were injected into immunocompromised mice; after four weeks, teratomas containing cell types characteristic of all three germ layers were isolated (data not shown).

[0163] As an assay for somatic genome rep...

Embodiment 3

[0164] Example 3: Ectopic Oct4 expression is dispensable for maintenance of iPS cells

[0165] 2D4 iPS cells selected by Fbx15 showed persistent retroviral expression of Oct4 and Sox2 with negligible expression from their respective endogenous loci, indicating a constant need for exogenously supplied factors to maintain iPS cell self-renewal and multiplication. performance (Takahashi and Yamanaka, 2006). To corroborate the gene expression data indicating efficient retroviral gene silencing in iPS cells, it was determined whether sustained Oct4 expression is necessary for the maintenance of iPS cells using fibroblasts carrying a doxycycline-inducible Oct4 transgene in their genomes Genetic testing was performed (Hochedlinger, K.; Yamada, Y.; Beard, C and Jaenisch, R. (2005) Cell 121, 465-477) ( Figure 3A ).

[0166] To initially determine whether the Oct4-inducible system could be used to obtain cell populations, Oct4-inducible MEFs were retrovirally infected with Sox2, c-...

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Abstract

Disclosed herein are methods to select for the generation of mouse and human pluripotent stem cells during developmental reprogramming. The methods described herein relate to the selection of induced pluripotent stem cells, i.e., pluripotent stem cells generated or induced from differentiated cells without a requirement for genetic selection. Described herein are particular embodiments for selection of reprogrammed cells based on 1) colony morphology, or 2) X chromosome reactivation in female cells.

Description

[0001] This application claims priority under 35 U.S.C. § 119(e) (35 U.S.C. §119(e)) to U.S. Provisional Patent Application Serial No. 60 / 932,267, filed on May 30, 2007, and grants Incorporated by reference in its entirety. Background technique [0002] By nuclear transfer (Wakayama, T.; Perry, A.C.; Zuccotti, M.; Johnson, K.R. and Yanagimachi, R. (1998) Nature 394, 369-374; Wilmut, I.; Schnieke, A.E.; McWhir, J.; Kind, A.J. and Campbell, K.H. (1997) Nature 385, 810-813) and cell fusion (Cowan, C.A.; Atienza, J.; Melton, D.A. and Eggan, K. (2005) Science 309, 1369-1373; Tada, M.; Takahama, Y.; Abe, K.; Nakatsuji, N. and Tada, T. (2001) Curr Biol 11, 1553-1558) Cell reprogramming allows re-establishment of a pluripotent state in the nucleus of somatic cells (Hochedlinger , K. and Jaenisch, R. (2006) Nature 441, 1061-1067). Although the molecular mechanisms of nuclear reprogramming have not been fully elucidated, cell fusion experiments have suggested that reprogramming factor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/06C12N5/074
CPCC12N2501/603C12N5/0696C12N2501/605C12N2501/604C12N2510/00C12N2501/602C12N2501/606
Inventor 康拉德·霍希德林根尼梅特·马赫拉里
Owner THE GENERAL HOSPITAL CORP
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