Fusion of the Transcription Domain to iPS Factors Radically Enhances Reprogramming

AlleleBlog:

Induced pluripotent stem cells (iPSCs) can be achieved through introduction of a small group of stem cell specific transcription factors. Ever since this was first demonstrated by Takahashi and Yamanaka, there have been relentless efforts for improving the efficiency of this generally inefficient process. There is also a general opinion that iPSCs are different from each other and from embryonic stem cells (ESCs) in various aspects, depending on the method of the induction. As a result, another focus of the reprogramming field has been to find ways for creating iPSCs that are as close to ESCs as possible. One of the parameters for defining stem cell status is their epigenetic characters; epigenetic changes have been demonstrated to occur during reprogramming of subsequent differentiation.

In fact, it seems that reprogramming can be largely described as a process composed of chromatin remodeling and specific transcription activation. Strong transcription activators are known to effectively recruit multiple chromatin remodeling complexes when exerting their functions. A good example is MyoD, a master transcription factor for skeletal myogenesis that can “single-handedly” switch (transdifferentiate) the fate of differentiated cells. Hirai et al. speculated that since MyoD is such a strong transcription factor, it may be able to increase chromatin accessibility to iPS factors if fused together. When transduced on retroviral vectors, Oct-TAD (Transcription Activation Domain) of MyoD, in combination with Sox2 and Klf4, increased the number of iPSC colonies by 40-fold. Additionally, these iPSCs appeared to quickly adopt stem cell gene expression profiles, days faster than when traditional Oct4, Sox2, c-Myc, and Klf4 were used; and sometimes the levels of pluripotency genes even exceeds those seen in ESCs. Amazingly, when using the fusion assisted method some colonies are formed without the help of feeder cells, a requirement of ESCs grown in similar medium. Does this mean that these iPSCs can even be more “stem-like” than embryonic stem cells?

Like MyoD, VP16, also widely known for its strong transcription activation domain, when fused to iPS factors, was shown to exhibit a similar stimulation effect on reprogramming. Although the details of the fusion arrangements and specificity appear to differ between MyoD and VP16, the fact that two research groups could achieve similar results using comparable strategies provides a good argument that other labs should at least consider this method when creating mouse or human iPSCs. Previously in our blog we have discussed using iPS factor mRNAs, a method originally developed by Warren et al., for substantially shortening the time required for reprogramming and making it more robust across cell types and media conditions. If the new TAD-fusion factors are used also in the mRNA format, then the protocol might be further shortened and simplified. If successful, this non-integrating approach could become a dominant method in the field, even making competitive non-integrating method such as Sendai and plasmid-based miRNA irrelevant.

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