Update 4-26-2009, F1000 review
John Mullins with Linda Mullins
University of Edinburgh, United KingdomFollowing the complete removal of reprogramming factors, human-induced pluripotent stem cells (hiPSCs) become much more closely related to human embryonic stem cells (hESCs) as measured by global gene expression profile in this study. Thus, hiPSCs derived from patients may provide cells suitable for modelling human disease and ultimately lead to iPSC-based therapies.
In the fast-moving field of reprogramming somatic cells to pluripotency when four transcription factors, OCT4, KLF4, SOX2 and c-MYC, are introduced {1,2}, the presence of viral sequences severely limit the applications of iPSC technology due to the possibility of malignant transformation or altered differentiation potential. The authors used Cre-recombinase to remove transgene-expressing viruses and found that the resulting hiPSCs had 80% fewer genes differentially expressed in comparison to hESCs than hiPSCs with transducing factors intact. They also found that limited DOX-inducible exposure of fibroblasts to just three factors (OCT4, KLF4 and SOX2) for 12 days was sufficient to reprogram the cells, was more practical and led to reduced numbers of proviral integrations. Factor-free hiPSCs derived from Parkinson's disease patients maintained a pluripotent ESC-like state and could be differentiated into dopaminergic neurons using standard protocols. The next challenge will be to identify the conditions necessary to induce Parkinson's disease-related pathology in the cells. Cre-recombinase leaves an LTR footprint in genomic DNA, which risks gene disruption at the integration sites. Recent papers using transposons and transposase to deliver and seamlessly remove transducing factors {3,4} should overcome this problem.
References: {1} Takahashi and Yamanaka, Cell 2006, 126:663-76 [PMID: 16904174]. {2} Takahashi et al. Cell 2007, 131:861-72 [PMID: 18035408]. {3} Woltjen et al. Nature 2009, 458:766-70 [PMID: 19252478]. {4} Kaji et al. Nature 2009, 458:771-5 [PMID: 19252477].
Now I know why weeks ago when Dr. Jaenisch was presenting the Parkinson's disease data I was so excited. The data presented at that time was not limited to the PNAS paper, also this new paper.
Cell. 2009 Mar 6;136(5):964-77.
Parkinson's disease patient-derived induced pluripotent stem cells free of viral reprogramming factors.Soldner F, Hockemeyer D, Beard C, Gao Q, Bell GW, Cook EG, Hargus G, Blak A, Cooper O, Mitalipova M, Isacson O, Jaenisch R.
The Whitehead Institute, 9 Cambridge Center, Cambridge, MA 02142, USA.
Induced pluripotent stem cells (iPSCs) derived from somatic cells of patients represent a powerful tool for biomedical research and may provide a source for replacement therapies. However, the use of viruses encoding the reprogramming factors represents a major limitation of the current technology since even low vector expression may alter the differentiation potential of the iPSCs or induce malignant transformation. Here, we show that fibroblasts from five patients with idiopathic Parkinson's disease can be efficiently reprogrammed and subsequently differentiated into dopaminergic neurons. Moreover, we derived hiPSCs free of reprogramming factors using Cre-recombinase excisable viruses. Factor-free hiPSCs maintain a pluripotent state and show a global gene expression profile, more closely related to hESCs than to hiPSCs carrying the transgenes. Our results indicate that residual transgene expression in virus-carrying hiPSCs can affect their molecular characteristics and that factor-free hiPSCs therefore represent a more suitable source of cells for modeling of human disease.
However, I prefer the piggyBac + multicistron method to produce "safe" iPS. Although you can Cre out the viral sequence, you will left one copy of LoxP site there. The authors were not using multicistron expression, which is going to leave four single LoxP sites in the genome. It is dangerous. There are high chance of genome swapping - it might explain why the viral-free iPS percentage is low (2-7%).
The authors know this problem, and their solution is:
"The remaining risk of gene disruption could in the future be eliminated by targeting of the reprogramming factors as a polycistronic single expression vector flanked by loxP sites into a genomic safe-harbor locus (Carey et al., 2009)."
Proc Natl Acad Sci U S A. 2009 Jan 6;106(1):157-62.
Reprogramming of murine and human somatic cells using a single polycistronic vector.
Carey BW, Markoulaki S, Hanna J, Saha K, Gao Q, Mitalipova M, Jaenisch R.Whitehead Institute for Biomedical Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA.
Directed reprogramming of somatic cells by defined factors provides a novel method for the generation of patient-specific stem cells with the potential to bypass both the practical and ethical concerns associated with somatic cell nuclear transfer (SCNT) and human embryonic stem (hES) cells. Although the generation of induced pluripotent stem (iPS) cells has proven a robust technology in mouse and human, a major impediment to the use of iPS cells for therapeutic purposes has been the viral-based delivery of the reprogramming factors because multiple proviral integrations pose the danger of insertional mutagenesis. Here we report a novel approach to reduce the number of viruses necessary to reprogram somatic cells by delivering reprogramming factors in a single virus using 2A "self-cleaving" peptides, which support efficient polycistronic expression from a single promoter. We find that up to four reprogramming factors (Oct4, Sox2, Klf4, and c-Myc) can be expressed from a single virus to generate iPS cells in both embryonic and adult somatic mouse cells and we show that a single proviral copy is sufficient to generate iPS cells from mouse embryonic fibroblasts. In addition we have generated human induced pluripotent stem (hiPS) cell lines from human keratinocytes, demonstrating that a single polycistronic virus can reprogram human somatic cells.
[...] two methods: 1. Cre out the viral sequence, which was used by Dr. Jaenisch’s group as well (ref); 2. piggyBac system … [...]