You are hereSeptember 20, 2010 | Pluripotent Stem Cells
Yamanaka Factors, Version 2.0
About Minh: Minh was born in Minnesota and grew up primarily in the Twin Cities of Minneapolis and Saint Paul. Studying for his undergraduate degree at the University of Minnesota, he received his bachelor’s degree in Chemistry in 1998. In 2004 Minh graduated with a PhD in Chemical Biology which studied RNA-protein interactions that govern HIV replication with Dr. Karin Musier-Forsyth. He then did a Postdoc at the Department of Chemistry & Chemical Biology at Harvard University, studying single molecule fluorescent spectroscopy with Dr. Xiaowei Zhuang. Wanting to move to more translational research, Minh took a position with Dr. Mathew Warman at the Children’s Hospital in Boston, where he studied normal and malignant bone and cartilage development in a mouse model system. Returning home in 2008, Minh moved to Dr. Dan Kaufman’s laboratory at the University of Minnesota, Stem Cell Institute, where his current research focuses on generating, characterizing, and understanding human iPSC reprogramming.
Yamanaka Factors, Version 2.0
Minh K. Hong and Dan S. Kaufman
Advances in pluripotent stem cell biology have been fast and furious ever since the production of mouse induced pluripotent stem cells (iPSCs) were first described by ectopic expression of Oct3/4, Sox2, Klf4, and c-Myc, commonly referred to as the “Yamanaka Factors” (Takahashi and Yamanaka 2006). These factors have also been shown effective at reprogramming human (Takahashi et al. 2007), rat (Liao et al. 2009), monkey (Liu et al. 2008), and pig (Esteban et al. 2009; Ezashi et al. 2009; Wu et al. 2009) somatic cells to iPSCs. Notably, another set of partially-overlapping genes Oct3/4, Sox2, Nanog, and Lin28, considered the “Thomson Factors,” can also produce iPSCs (Yu et al. 2007). Therefore, multiple strategies are available for this amazing ability to generate novel pluripotent cells. Understanding the mechanisms that control reprogramming of cells into pluripotency, and then differentiating these cells, is critical for their use in regenerative medicine (Muller et al. 2009). However, one significant lingering concern, is the tumourigenic potential of iPSCs. Initial studies demonstrated that over 15% of mice derived from Yamanaka factors iPSCs developed tumors (Okita et al. 2007). Some tumorigenic potential is often attributed to the reprogramming factor c-Myc, which is notoriously known to be oncogenic (Dang et al. 2006). However, iPSCs can be formed without the need of exogenous c-Myc (at a greatly reduced efficiency) and interestingly, mice derived from these cells do not demonstrate enhanced tumorigenesis (Nakagawa et al. 2008).
The proto-oncogenic Myc family members, c-Myc (MYC), N-Myc (MYCN), and L-Myc (MYCL1), are vital for many cellular processes including proliferation, apoptosis, and differentiation. Through exhaustive studies in normal and cancer cells, Myc has been demonstrated to dimerize with Myc-associated factor-X (Max) and has DNA binding activity allowing for transcriptional activation and DNA modulation activities (Shafa et al. 2010) and can directly interact and inhibit activity of the transcription factor Miz-1 (ZBTB17) resulting in suppression of gene expression (Eilers and Eisenman 2008). From studies investigating the biological role of Myc proteins, it would be tempting to simply rationalize that Myc functions to enhance iPSC reprogramming by inhibiting differentiation and promoting proliferation. However, the role of Myc in iPSC reprogramming is far from straightforward and a recent report by Yamanaka and coworkers has compared how the various Myc proteins can affect iPSC reprogramming (Nakagawa et al. 2010). The researchers determined that the quality of the iPSC colonies generated were inversely proportional to the transformation activity of the Myc variant and their findings may have broad implications in generating future iPSCs for biologically relevant uses.
In this study, adult human dermal fibroblasts (AHDF) were transduced with OCT4, SOX2, and KLF4 in the absence or presence of Myc family members. Colony formation was scored three weeks post transduction based on ESC-like morphology. L-Myc increased the efficiency of iPSC generation more so than c-Myc or N-Myc (efficiency using L-Myc was approximately 5 fold that of c-Myc). Furthermore, L-Myc produced more putative colonies with a greater percentage of colonies determined to be fully reprogrammed. This observation is striking given the fact that among the Myc family members, L-Myc has the lowest propensity to transform cells in culture. To assess the molecular differences between Myc members during iPSC reprogramming, the authors utilized various c-Myc and L-Myc mutants, which were deficient in either their transformation activity or ability to bind to Miz-1 and Max. The researchers observed that wild type L-Myc and c-Myc mutations that reduced or eliminated transformation activity showed the greatest ability to generate human iPSCs. The Miz-1 binding mutant did not enhance iPSC reprogramming compared to using wild type c-Myc. Since Myc is capable of inducing apoptosis through Miz-1 interactions (Patel and McMahon 2006), this data suggests that Myc-mediated apoptosis does not play a role in iPSC reprogramming. Interestingly, the Myc mutants deficient in Max binding did not enhance reprogramming validating the requirement of Myc transcriptional regulation for efficient reprogramming. To further investigate the differences in transcriptional modulation using c-Myc versus L-Myc, microarray analysis was performed on fibroblasts transduced with c-Myc, L-Myc, or the mutant Myc variants. Analysis showed that ectopic expression of c-Myc increased expression of many pluripotent genes and in agreement with mouse studies by Plath and coworkers (Sridharan et al. 2009), down regulated human fibroblast-specific genes. In addition, L-Myc or the transformation-deficient c-Myc further suppressed fibroblast-specific genes.
Comparison of mouse and human iPSC derivation shows some similarities, but also key differences for the role of L-Myc. Unlike in the human model system, c-Myc produced more putative mouse iPSC colonies than L-Myc. However, using L-Myc did result in a higher percentage of fully reprogrammed colonies. Whereas chimeras and progeny mice derived from c-Myc iPSCs are known to have increased tumor formation and mortality rates (Okita et al. 2007), the L-Myc iPSC-derived mice did not demonstrate enhanced tumorigenicity compared to wild type mice. This observation is consistent with the reduced transformation activity of L-Myc and its reduced oncogenic potential compared to c-Myc. Taken together, L-Myc produces more fully reprogrammed and potentially less tumorigenic iPSC colonies, though the colony formation efficiency is greater in human fibroblasts. However, it still remains unclear if using L-Myc to reprogram human fibroblasts would generate less tumorigenic iPSCs.
Going forward, it is unlikely that iPSCs produced through the use of stably integrated genes will be used directly for therapies. However, this study helps to elucidate the necessary transcriptional regulation that occurs in iPSC reprogramming and adds to the growing understanding of Myc in the stem cell and cancer biology fields. In addition, modifying current protocols to substitute c-Myc for L-Myc, may be beneficial for producing more biologically relevant models for studying disease as well as pharmaceutical drug screening. Our group (and many others) are keenly interested in generating patient-specific human iPSC lines utilizing transient reprogramming strategies. Since adult donor cells and terminally differentiated cells have reduced reprogramming abilities, we have been using various types of patient samples to generate iPSCs, including dermal fibroblasts, blood progenitors, mesenchymal stem/stromal cells, and gingival fibroblasts. Many of the transient strategies to produce iPSCs utilize c-Myc including: non-integrating virus (Fusaki et al. 2009; Zhou and Freed 2009); plasmid and vector-based methods (Okita et al. 2008; Yu et al. 2009); and protein transduction (Zhou et al. 2009). With L-Myc’s robust ability to enhance iPSC reprogramming along with its low tumorigenic potential, studies utilizing L-Myc transiently to produce potentially therapeutic iPSCs is now clearly warranted.
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*Department of Medicine and Stem Cell Institute, University of Minnesota, 420 Delaware St. SE MMC 480, Minneapolis, MN 55455, USA