Posted by Elie Dolgin
[Entry posted at 23rd April 2009 05:00 PM GMT]
Comment on this news story
Researchers have attained the holy grail of cellular reprogramming: inducing pluripotency without using any DNA-based materials. Using only a cocktail of purified proteins and a chemical additive, investigators have generated induced pluripotent stem (iPS) cells that don't carry the potential burden of unexpected genetic modifications, according to a new study published online today (Apr. 23) in Cell Stem Cell.
via Flickr/CIRM
Mais do artigo em inglês aqui.
Copyright 2009 Elsevier Inc.. All rights reserved.
Cell Stem Cell, 23 April 2009
doi:10.1016/j.stem.2009.04.005
Generation of Induced Pluripotent Stem Cells Using Recombinant Proteins
Hongyan Zhou1,Shili Wu4,7,Jin Young Joo5,7,Saiyong Zhu1,Dong Wook Han5,Tongxiang Lin1,Sunia Trauger2,3,Geoffery Bien4,Susan Yao4,Yong Zhu4,Gary Siuzdak2,3,Hans R. Schöler5,Lingxun Duan6andSheng Ding1,,
1 Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
2 Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
3 Center for Mass Spectrometry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
4 ProteomTech, Inc., 3505 Cadillac Avenue, Suite F7, Costa Mesa, CA 92626, USA
5 Department of Cell and Developmental Biology, Max Planck Institute for Molecular Biomedicine, Röntgenstrasse 20, Münster 48149, Germany
6 LD Biopharma Inc., Sandown Way, San Diego, CA 92130, USA
Corresponding author
7 These authors contributed equally to this work
MAIN TEXT
Groundbreaking work demonstrated that ectopic expression of four transcription factors, Oct4, Klf4, Sox2, and c-Myc, could reprogram murine somatic cells to induced pluripotent stem cells (iPSCs) (Takahashi and Yamanaka, 2006), and human iPSCs were subsequently generated using similar genetic manipulation (Takahashi etal., 2007,Yu etal., 2007). To address the safety issues arose from harboring integrated exogenous sequences in the target cell genome, a number of modified genetic methods have been developed and produced iPSCs with potentially reduced risks (for discussion, see Yamanaka, 2009, and references therein). However, all of the methods developed to date still involve the use of genetic materials and thus the potential for unexpected genetic modifications by the exogenous sequences in the target cells. Here wereport generation of protein-induced pluripotent stem cells (piPSCs) from murine embryonic fibroblasts using recombinant cell-penetrating reprogramming proteins. We demonstrated that such piPSCs can long-term self-renew and are pluripotent invitro and invivo.
One possible way to avoid introducing exogenous genetic modifications to target cells would be to deliver the reprogramming proteins directly into cells, rather than relying on the transcription from delivered genes. Previous studies have demonstrated that various proteins can be delivered into cells invitro and invivo by conjugating them with a short peptide that mediates protein transduction, such as HIV tat and poly-arginine (Inoue etal., 2006,Michiue etal., 2005,Wadia and Dowdy, 2002). In addition, various solubilization and refolding techniques for processing inclusion body proteins expressed in E. coli to bioactive proteins have been developed to allow facile and large-scale production of therapeutic proteins (Lafevre-Bernt etal., 2008). To generate recombinant proteins that can penetrate across the plasma membrane of somatic cells, we designed and fused a poly-arginine (i.e., 11R) protein transduction domain to the C terminus of four reprogramming factors: Oct4, Sox2, Klf4, and c-Myc (see FigureS1 A online). These proteins were expressed in E. coli in inclusion bodies, which were then solubilized, refolded, and further purified (FigureS1 B). The protein identities were confirmed by mass spectrometry and western blot analysis (FigureS1 C). To test the cell permeability and stability of the proteins, we treated mouse embryonic fibroblast (MEF) cells with the recombinant proteins at various concentrations by adding them to the cell culture media for 672 hr and examining cell morphology and protein presence by immunocytochemistry. We found that the purified 11R-tagged recombinant transcription factors readily entered cells at concentrations of 0.58 g/ml within 6 hr and could translocate into nucleus (FigureS1 D). In addition, we found that the transduced proteins appeared to be stable inside cells for up to 48 hr (FigureS1 D).
We then employed this simple protein transduction protocol to reprogram OG2/Oct4-GFP reporter MEF cells. Because reprogramming through the iPSC mechanism/process typically requires sustained activity of reprogramming proteins for 710 days, we devised a strategy that involved treating the cells in four cycles. In each cycle the fibroblasts (initially seeded at the density of 5 104 cells/well in a six-well plate) were first treated overnight with the recombinant reprogramming proteins (i.e., Oct4-11R, Sox2-11R, Klf4-11R, and c-Myc-11R) at 8 g/ml in the mESC growth media supplemented withor without 1 mM valproic acid (VPA),a HDAC inhibitor that can significantly improve reprogramming efficiency (Huangfu etal., 2008b), followed by changing to the same media without the recombinant reprogramming proteins and VPA, and culturing for additional 36 hr before the next cycle of the treatment. After completing four repeated protein transductions of reprogramming proteins, the treated cells were transferred onto irradiated MEF feeder cells and simply kept in mESC growth media until colonies emerged around day 3035 (Figure1A). We obtained three GFP+ colonies per 5 104 cells when they were transduced with four proteins and treated with VPA, and one GFP+ colony per 5 104 cells when they were transduced with only three proteins (i.e., Oct4-11R, Sox2-11R, and Klf4-11R) and treated with VPA. However, we did not obtain stable GFP+ piPSC colonies by transducing the three or four reprogramming proteins only for the same period of time, although GFP-negative cell colonies were observed. Those GFP-negative cell colonies stained positive with ALP, an early pluripotency marker, suggesting they might be partially reprogrammed cells. The initial GFP+ colonies were subsequently passaged under conventional mESC growth conditions to yield piPSCs and were characterized further.
++++
Download gratuito do PDF
