Last update: Oct 2020
Creating Induced Pluripotent Stem Cells
Researchers Yamanaka and Takahashi were somewhat inspired by the MYOD1 studies outlined in the previous section. They knew there was probably a set of reprogramming factors that erased cell's epigenetic memories, and that the transcription factor responsible for the direct conversion of fibroblasts to skeletal muscle cells has already been found. Thus, they reasoned it would also be possible to identify the transcription factors inducing pluripotency in somatic cells. If those factors were activated, it would no longer be required to transplant the somatic cell nucleus into an egg or fuse it with ES cell to rejuvenate it to a pluripotent state.
Yamanaka decided to focus on 24 candidate transcription factors that switch on genes typically active in ES cells and narrow them down to the factors essential for rejuvenation. He asked his student Takahashi to conduct the experiments. Takahashi introduced the 24 factors into mouse fibroblasts by retroviral transduction.
To distinguish the fibroblasts in which the pluripotency was successfully induced from other fibroblast cells, he used the βgeo gene, which gives rise to G418 antibiotic resistance. Takahashi inserted the βgeo gene into the Fbx15 locus by homologous recombination (knockin). Since the Fbx15 promoter is active in pluripotent stem cells, the cells which were successfully rejuvenated were expressing βgeo, and therefore were resistant to high concentrations of G418. While the knockin of βgeo damaged the Fbx15 gene itself, Yamanaka showed that this gene is dispensable for the maintenance of pluripotency, so it was not an issue.
Thanks to this experimental design, fibroblasts with 24 active transcription factors that were converted into pluripotent cells successfully were also resistant to G418, which made the screening easy. The researchers obtained 22, then another 29 colonies using this method, which meant they were on the right track to finding the key transcription factors.
The team then needed to narrow down the search, trying to identify the factors that are necessary for reprogramming. They repeated the experiments, introducing 23 out of the original 24 factors, one different factor missing per experiment. If the missing factor was a key factor, no stem cell colonies would form. In the end, they identified 4 factors crucial in inducing pluripotency: Oct3/4, Sox2, c-Myc, and Klf4, which were later called Yamanaka factors.
To confirm that the cells rejuvenated by the expression of Yamanaka factors were indeed pluripotent stem cells, the researchers ran 2 tests. First, they injected the cells under the skin of immunodeficient mice, which caused a tumor to form. To be considered pluripotent, a cell has to be able to form all 3 germ layers (endoderm, mesoderm, and ectoderm). When the scientist studied the tumor tissue, they confirmed that it was indeed a teratoma, which is a tumor containing 3 germ layers. In the second test, the scientists assessed whether the cells they made could give rise to an entire organism. They transplanted their cells with GFP-tag into blastocysts (which are early embryos), which indeed resulted in fetus formation (Fig1.e,f).
Thus, in 2006, Takahashi and Yamanaka showed that they could induce a pluripotent state in differentiated somatic cells, namely mouse embryonic fibroblasts and adult mouse tail-tip fibroblasts. Cells formed this way were called induced pluripotent stem cells, or iPS cells. When the researchers compared the global gene-expression profiles of ES cells and iPS cells, they found several differences, which shows that ES and iPS cells are similar, but not identical. iPS cells can be theoretically used to create any cell type, including ES cells.
In 2007, the team also found iPS cells with germline transmission. When transplanted into blastocysts, mouse iPS cells can give rise to adult chimeras (Fig.1.f) capable of germline transmission. When a chimera is crossed with a normal mouse, offspring carrying the genetic content of an iPS cell can be formed (Fig.1.g).
The researchers also managed to produce human iPS cells from fibroblasts using the same four reprogramming factors as in mice, demonstrating the conservation of transcription factor networks between species.
Several groups of scientists have also produced iPS cells from other cell types, for example, from neuronal progenitor cells, keratinocytes, hepatocytes, B cells, kidneys, muscles, and adrenal glands. They obtained those cells using the Yamanaka factors, which showed that those factors could carry out cellular reprogramming regardless of the cell's epigenetic status.
Figure 1. Creating iPS cells from fibroblasts
Epigenetic Status and Reprogramming Efficiency
The efficiency of reprogramming somatic cells into iPS cells is usually only around 1%. This hints that to successfully reprogram a cell, on top of activating the reprogramming factors, one has to take the stochastic (random) events that maintain reprogramming into consideration.
We know that there are no major genomic differences between somatic cells and reprogrammed iPS cells. Thus, we could expect the epigenetic status, for example, DNA methylation, to be the crucial factor in successful reprogramming. When we use small-molecule compounds that inhibit histone deacetylase, increasing chromatin acetylation levels, and thus making the chromatin structure more relaxed, the reprogramming efficiency increases. This observation supports the theory outlining the importance of epigenetics in cellular reprogramming. Moreover, throughout reprogramming, somatic cell genes are silenced, and pluripotent stem cell genes are activated, again hinting towards an underlying epigenetic status change. That said, the exact mechanisms and causes of the phenomena mentioned above are not clear yet.
The efficiency of cellular reprogramming differs depending on cell type. For example, human keratinocytes can be reprogrammed into iPS cells faster and with way higher frequency than fibroblasts. In this case, it is probably caused by the fact that keratinocytes express higher levels of c-Myc and Klf4 (which are 2 of the Yamanaka factors) than fibroblasts. Thus, the conversion of keratinocytes into iPS cells is facilitated.
Applications and Limitations of iPS Cells
As you have probably worked out by now, creating iPS cells provides a good model and tool for elucidating the mechanisms of epigenetic changes induced by transcription factors. iPS cells are also used to obtain stem cells for therapeutic use (cell replacement therapy) and to study various pathological processes (disease mechanisms, toxicology).
In Regenerative Medicine
The therapeutic use of iPS cells is significantly stalled by several factors. While the public remains excited about the great potential of creating patient-specific organs for transplant using iPS cells, especially since the creation of a functional human liver in mouse host in 2013, several problems surrounding the transcription factors slow down the progress of regenerative medicine.
First and foremost, Yamanaka and Takahashi and their followers used retroviral and lentiviral vectors when introducing the 4 transcription factor genes into somatic cells that were to be converted into iPS cells. These viral vectors integrate into active genes rather than inactive ones, potentially leading to the activation of the neighboring genes, which is an undesirable effect. Worse still, most of the 4 transcription factors are oncogenic, and their prolonged overexpression may lead to cancer. When you think about it, it makes much sense. Both iPS cells and cancer cells exhibit high proliferation potential, so it is not unlikely that both of them express the same or similar proliferation-associated transcription factors. Therefore, the iPS cells that, once successfully converted, do not silence the factors properly, could potentially prompt tumorigenesis, as shown in mice.
One possible solution to this problem is replacing the retroviral and lentiviral vectors with episomal vectors, nonintegrating viral vectors, transient DNA transfection, transposons, or protein transduction. Instead of the viral vectors permanently integrating into genes, this approach facilitates the transient expression of the Yamanaka factors at levels sufficient for reprogramming initiation. Using this approach is safer, but reduces reprogramming efficiency.
Another method is to use small molecules instead of transcription factors to reprogram cells. Those molecules could be a substitute for some of the factors, but so far, nobody managed to generate iPS cells using small molecules only.
In Studying Diseases
Given numerous problems in developing viable therapeutic strategies, the focus of scientists shifted from using iPS cells in cell replacement therapy to establishing models for studying disease mechanisms.
Why are iPS cells so useful when investigating pathological processes? A big challenge in studying human diseases is obtaining in vitro disease models. Cells affected by a disease are often difficult to grow in culture, or their phenotype changes when cultured in a Petri dish. If we could obtain many cells affected by a particular disease, we could investigate the cellular physiology and molecular mechanisms of that disease better. Moreover, we could subject the cells to numerous drugs in search of therapeutic agents. Fortunately, iPS cells are an abundant source of disease-specific pluripotent cell lines. We only need to collect somatic cell samples from people affected by a disease and reprogram those cells to iPS cells to obtain our disease model.
For example, we can reprogram fibroblasts from patients with Mendelian and complex genetic disorders such as amyotrophic lateral sclerosis, type 1 diabetes, Parkinson's disease, and Duchenne muscular dystrophy, and thus obtain the disease-specific iPS cell lines. Before we can study those cell lines, we have to determine if they make a good disease model, that is if they show the disease phenotype. So far, iPS- derived cells showed partial disease phenotypes in spinal muscular atrophy and familial dysautonomia-derived cells. This shows that iPS cells might be effectively used in modeling early-onset human diseases, such as the 2 conditions mentioned above.
Unfortunately, studying late-onset diseases, for example, Parkinson's disease and amyotrophic lateral sclerosis, is way more complicated. So far, iPS cells derived from patients with those diseases did not exhibit the desired phenotype. A possible solution could be subjecting the cells to various stress conditions, such as increased levels of reactive oxygen species.
Another limitation of the iPS approach is the fact that some diseases are difficult to model using one cell only, as those conditions involve interactions among different cell types. In that case, more work is needed to recreate the disease phenotypes using complex iPS cell-based models. The disease phenotype might also be obtained if the iPS-derived cells were transplanted into immunodeficient mice.
Figure 2. iPS cells can be used to create disease models
Davis, Weintraub, Lassar, Takahashi, Yamanaka, and their many predecessors and successors have contributed significantly to our current knowledge of cellular reprogramming. After MYOD1 and Yamanaka factors were determined, the field got very popular, and the hunt for the many undiscovered transcription factors involved in different types of direct conversions between differentiated cell types began. Defined transcription factors were used to convert the lymphoid cells to myeloid cells and glial cells to neurons. Direct conversion to more distantly related differentiated cell types also became possible. The researchers even induced conversions transcending the germ layer origin (endoderm, mesoderm, or ectoderm), such as conversions of fibroblasts into neurons or hematopoietic cells.
This is to show that, contrary to the claims of Waddington, Weissman, and their contemporaries, cell fate can be very flexible and is in no way final. Differentiation is in no sense one-way traffic, and cellular identities are, in fact, determined by the cell's epigenetic status.
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Figure 1. Rossant, J. (2007). Stem cells: The magic brew. Nature 448, 260–262, Nature Publishing Group https://www.nature.com/scitable/topicpage/turning-somatic-cells-into-pluripotent-stem-cells-14431451/ [Accessed 5 May 2020].
Figure 2. Unternaehrer, J. J., & Daley, G. Q.. (2011). Induced pluripotent stem cells for modelling human diseases. Philosophical transactions of the Royal Society of London. Series B, Biological sciences, 366(1575), 2274–2285. https://doi.org/10.1098/rstb.2011.0017