Creating ground-state human iPSCs

Murine pluripotent stem cells can exist in two distinct states, blastocyst-derived LIF-dependent embryonic stem cells (ESCs) and epiblast-derived bFGF-dependent stem cells (EpiSCs). Murine ESCs and similar iPSC lines are more of the “ground-state” in terms of developmental status, as reflected by the lack of X chromosome inactivation in female cells and their abilities to pass as single cells. Human iPSCs, like human ES cells, are more similar to mouse EpiSCs. Unfortunately these human pluripotent stem cells are difficult to genetically manipulate, e.g. knockin or knockout. They also grow slowly, with doubling time averaging 36 hours. In order to create ground-state human iPSCs, several approaches have been tested, including reprogramming iPSC-derived fibroblasts, continuously expressing 5 iPS factors (Oct4, Sox2, Nanog, c-Myc, and Klf4), or using chemicals to inhibit chromatin modifying enzyme HDAC. While these approaches succeeded to certain degrees, the resulting cell lines seem to have some limitations, such as limited passage numbers.

Retinoic acid (RA) signaling is involved in many aspects of embryonic development. RA receptor (RAR), together with one of its heterodimerization partners, steroid hormone receptor Lrh-1, was recently found to be able to synergize with the 4 common iPS factors (Oct4, Sox2, Klf4, and c-Myc) to induce mouse and human fibroblasts into ground-state iPSCs. The pluripotent cells created by the so-called F6 factor combination show no X chromosome inactivation if from female origin, can fully activate the endogenous Oct4 promoter, express Rex1 (which is specific to mouse ESCs, not EpiSCs), and grow with a 16 hour doubling time. All these mouse ESC-like features were achieved without detectable expression of the exogenous factors once iPSC colonies formed, indicating transient F6 expression is capable of effectively initiating endogenous stem cell factors. Remarkably, these stem cells can maintain their undifferentiated status in mouse ESC medium for 50 passages or more. This work, published this month in Proceedings of National Academy of Science USA [1], provided the stem cell research and application field with a very desirable choice of human stem cells.

As opposed to ~16 days with F4, it appears that the time required to induce adult fibroblasts into pluripotent stem cells is as short as 4 days if F6 factors are introduced on a murine stem cell virus (MSCV) vector with an integrated piggyback transposon. As the authors noted in their discussion, the speed-up benefit should be particularly advantageous for transient transfection approaches such as mRNA reprogramming. The bottom line from this paper and the engineered factor papers (see the previous AlleleBlog article under “iPS and other Stem Cells”) is that iPSC reprogramming is only going to get faster, which means that hopefully in the near future creating iPSCs will become a routine experiment as easy as a simple transfection.

Wang, W., J. Yang, et al. (2011). “Rapid and efficient reprogramming of somatic cells to induced pluripotent stem cells by retinoic acid receptor gamma and liver receptor homolog 1.” Proc Natl Acad Sci U S A.

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Tags: EpiSCs, feeder cells, ground state stem cells, HDAC, iPSC, Lrh-1, Lrh1, RAR, RAR heterodimer, stem cell medium, X chromosome inactivatin

Solving the world’s problems with new biotechnology

The ability to isolate, create, synthesize, or artificially evolve living organisms towards desirable phenotypes may be increasingly important for solving many of the problems the world is facing. Such problems may include creating renewable energy using biowaste, finding biocontrol products that kill food-spoiling fungi “organically”, or assaying pathogens in the field using synthetic biological detection systems. With the arrival of synthetic biology, “it is possible to design and assemble chromosomes, genes and gene pathways, and even whole genomes”, according to the J. Craig Venter Institute. That is, if you know which genes or gene pathways you would need to put into the synthetic genome that would lead to the desired traits. So far, most published synthetic biology work involves bringing in transcription factors from a non-host source to set up an artificial network like circadian oscillators, showing that it can be done and it is interesting.

Through the process of evolution biological systems aptly self-engineer favorable traits in order to survive, but these changes require millions of years to manifest. However, there are quicker adaptations to environmental cues, such as developing antibiotic resistance, which can be achieved through a small number of mutations in hundreds or even dozens of generations. The question is how to harness this kind of adaptation for new strains that can be used as products with defined purposes? As a first requirement, you must have an assay for identifying the wanted mutants or method for augmenting their subpopulation, which is not necessarily easy and normally takes some clever designs to establish. Since evolutionary success in nature results from continuous “rounds” of gene mutagenesis, expression and selection, an evolution in the lab should ideally proceed with continuity. Previously, each round of mutation and selection takes a few days to complete. Recently, Esvelt et al. in David Liu’s lab at Harvard demonstrated one way of doing in vitro continuous evolution, by creating a lagoon of mixed E. coli and phages. By continuous dilution of the phage population through outflow, those phages that remain in the pool with properties that help them propagate in the host bacteria will have a better chance to regenerate and accumulate mutations towards the design of the assay [1].

Another aspect of natural evolution is that it occurs in a heterogeneous environment separated into niches of subpopulations with uneven stress levels. Although most evolutions with human intervention were conducted in a homologous population under the same stress and selection, a spatially complex environment may speed up evolution. This may not be easy to imagine, but if a mutant acquires some level of resistance to its environmental stress level and has a chance to move to join a population under higher stress, its relative fitness will likely increase. In addition, in a smaller population in the niche under higher stress, the mutant with marginally beneficial properties acquired under lower pressure can take over more quickly. This was demonstrated by Zhang et al. who showed that with a gradient of antibiotics applied to an array of microwells interconnected through tiny channels, new resistant strains can evolve in less than a day. Without the gradient, or separate the interconnected niches into discrete wells, no resistant populations could be obtained [2].

With more understandings like these and equipped with large scale gene synthesis, chromosome assembly, and deep sequencing technologies, we should see increasing numbers of human-made organisms serving special needs for food, health, energy, and the environment. Synthetic biology or artificial evolution won’t solve all the world’s problems, but if applied effectively and diligently, they can certainly help with many critical aspects as the technology “coevolves” with the environment.

[1] Kevin M. Esvelt, Jacob C. Carlson, & David R. Liu. “A system for the continuous directed evolution of biomolecules” Nature 499, 2011.
Qiucen Zhang, Guillaume Lambert, David Liao, Hyunsung Kim, Kristelle Robin, Chih-kuan Tung, Nader Pourmand, Robert H. Austin. “Acceleration of Emergence of Bacterial Antibiotic Resistance in Connected Microenvironments” Science 333, 2011.

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Tags: biocontrol, bioenergy, biofuel, biowaste, chromosome assembly, deep sequencing, food fungus, green energy, in vitro evolution, microenvironment, next generation sequencing, synthetic biology

Fusion of the Transcription Domain to iPS Factors Radically Enhances Reprogramming

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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Tags: chromatin remodeling, epegenetic markers, footprint free, iPS cells, iPSCs, mRNA iPSCs, MyoD-Oct, reprogramming, TAD-Oct, VP16-Oct