Induced Pluripotent Stem Cells - Review


Recently, pioneering work has revealed that terminally differentiated somatic cells can be reprogrammed to generate induced pluripotent stem (iPS) cells via overexpression of a defined set of transcription factors. These iPS cells are morphologically and phenotypically similar to embryonic stem (ES) cells and thus offer exciting possibilities in stem cell research and regenerative medicine.
Moreover, iPS cells are useful tools for studying the pathogenesis of human disease, for drug discovery and toxicity screening [1, 2].

Induction of pluripotent stem cells

The most widely used set of reprogramming factors, Oct4, Sox2, Klf4 and c-Myc, was identified initially by screening 24 pre-selected factors in mouse embryonic fibroblasts (MEFs) by Takahashi and Yamanaka [3].
This cocktail of transcription factors, OSKM, was shown to work for different types of somatic cells and for different species, including rhesus monkey [4] and human cells [5].
Direct reprogramming was also achieved by another team who used a partially overlapping combination, Oct4, Sox2, Nanog and Lin28, suggesting that Oct4 and Sox2 are indispensable whereas Nanog, Lin28, Klf4 and c-Myc are alternative supporting factors [6].
Subsequent studies have demonstrated that fewer factors are required; the c-Myc gene, an oncogene, is dispensable (although the efficiency of iPS cell formation is significantly lower) [7] and in some cases, such as neural stem cells, expression of only one factor (Oct4) is sufficient [8].

The generation of iPS cells is usually achieved by genetic transduction of the reprogramming genes using retroviral or lentiviral vectors. However, the use of integrating viral vectors represent an obstacle to the therapeutic translation of iPS cells as this technology can produce insertional mutagenic lesions that are potentially tumorigenic.
Two recent publications detail the use of polycistronic lentiviral vectors delivering the OSKM quartet to somatic cells in a single lentiviral construct reducing the number of genomic insertions [9, 10].
Alternative approaches to deliver the reprogramming factors with minimal or total absence of genetic modifications have been developed. These approaches include the use of LoxP sites and Cre-induced excision and piggyBac transposon excision of integrated reprogramming vector sequences [11, 12], and the use of an oriP/EBNA1-based episomal vector [13].
Non-integrating lentiviral vectors may also represent a promising approach [14].
One possible strategy to entirely replace gene delivery is protein transduction. Previous studies have demonstrated that various proteins can be delivered into cells by conjugating them with a short peptide that mediates cell penetration, such as poly-arginine [15]. Zhou et al. have designed and purified poly-arginine tagged Oct4, Sox2, Klf4 and c-Myc proteins that were found to readily enter cells and translocate into the nucleus [16]. After several cycles of protein supplementation, iPS cells were successfully generated from MEFs. Using a similar approach, Kim et al. obtained protein-induced pluripotent stem cells from human newborn fibroblasts after several rounds of treatment with cell extracts of HEK293 cell lines expressing poly-arginine tagged OSKM genes [17].

Recent studies have reported an emerging role for microRNAs (miRNAs) in the generation of iPS cells. It has been demonstrated that the reprogramming of iPS cells and subsequent differentiation to lineage specific cells can be observed by monitoring the differential expression of miRNAs, small non-coding RNAs that are critical regulators of gene expression [18]. Furthermore, the inhibition of tissue-specific miRNAs promotes the formation of iPS cells [19].

Direct reprogramming of somatic cells is currently a slow and inefficient process, in particular when the c-Myc oncogene is omitted in an effort to reduce tumorigenicity. Several chemicals have recently been reported to either enhance reprogramming efficiencies or substitute for specific reprogramming factors. Among the reported chemicals, some are known to affect chromatin modifications while others influence signal transduction pathways. Small molecules that modulate chromatin modifications include DNA methyltransferase inhibitors (RG108, 5-azacytidine), histone deacetylase inhibitors (valproic acid, trichostatin A) and a G9a histone methyltransferase (Bix-01294) [20,21,22]. Small molecules reported to potentiate reprogramming by targeting signaling pathways include the MEK inhibitor PD035901 and the TGF-beta receptor inhibitor SB431542 [23].

Among the various reprogramming strategies, the chemical approach combined to protein transduction may represent the most promising. However, substantial research and development is still required before iPS cells are ready for therapeutic applications.

1. Yokoo N. et al., 2009. The effects of cardiovactive drugs on cardiomyocytes derived from human induced pluripotent stem cells. Biochem Biophys Res Commun. 387:482-8.
2. Lian Q. et al., 2010. Future perspective of induced pluripotent stem cells for diagnosis, drug screening and treatment of human diseases. Thromb Haemost. 104(1):39-44.
3. Takahashi K. & Yamanaka S., 2006. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 126(4):663-76.
4. Liu H. et al., 2008. Generation of induced pluripotent stem cells from adult rhesus monkey fibroblasts. Cell Stem Cell. 3(6):587-90.
5. Takahashi K. et al., 2007. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 131(5):861-72.
6. Yu J. et al., 2007. Induced pluripotent stem cell lines derived from human somatic cells. Science. 318(5858):1917-20.
7. Nakagawa M. et al., 2008. Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nat Biotechnol. 26(1):101-6.
8. Kim JB. et al., 2009. Oct4-induced pluripotency in adult neural stem cells. Cell. 136(3):411-9. 9. Sommer CA. et al., 2009. Induced pluripotent stem cell generation using a single lentiviral stem cell cassette. Stem Cells. 27(3):543-9.
10. Chang CW. et al., 2009. Polycistronic lentiviral vector for "hit and run" reprogramming of adult skin fibroblasts to induced pluripotent stem cells. Stem Cells. 27(5):1042-9.
11. Soldner F. et al., 2009. Parkinson's disease patient-derived induced pluripotent stem cells free of viral reprogramming factors. Cell. 136(5):964-77.
12. Kaji K. et al., 2009. Virus-free induction of pluripotency and subsequent excision of reprogramming factors. Nature. 458(7239):771-5.
13. Yu J. et al., 2009. Human induced pluripotent stem cells free of vector and transgene sequences. Science. 324(5928):797-801.
14. Sarkis C. et al., 2008. Non-integrating lentiviral vectors. Curr Gene Ther. 8(6):430-7. Review.
15. Ogawa T. et al., 2007. Novel Protein Transduction Method by Using 11R: An Effective New Drug Delivery System for the Treatment of Cerebrovascular Diseases. Stroke, 38: 1354 - 1361.
16. Zhou H. et al., 2009. Generation of induced pluripotent stem cells using recombinant proteins. Cell Stem Cell. 4(5):381-4.
17. Kim D. et al., 2009. Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins. Cell Stem Cell. 4(6):472-6.
18. Kamata M. et al. 2010. Live cell monitoring of hiPSC generation and differentiation using differential expression of endogenous microRNAs. PLoS One. 5(7):e11834.
19. Mallanna SK. & Rizzino A., 2010. Emerging roles of microRNAs in the control of embryonic stem cells and the generation of induced pluripotent stem cells. Dev Biol. 344:16-25.
20. Shi Y et al., 2008b. Induction of pluripotent stem cells from mouse embryonic fibroblasts by Oct4 and Klf4 with small-molecule compounds. Cell Stem Cell. 2008 Nov 6;3(5):568-74.
21. Huangfu D. et al., 2008. Induction of pluripotent stem cells by defined factors is greatly improved by small-molecule compounds. Nat Biotechnol. 2008 Jul;26(7):795-7.
22. Durcova-Hills G. et al., 2008. Reprogramming primordial germ cells into pluripotent stem cells. PLoS One. 3(10):e3531.
23. Lin T. et al., 2009. A chemical platform for improved induction of human iPSCs. Nat Methods. 6(11):805-8.


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