Term Paper

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Abstract

Pluripotent cells hold great promise in the world of therapeutic and regenerative medicine due to

their ability to differentiate into almost any type of cell. Embryonic stem cells have been heavily

researched as potential tools in therapeutic and regenerative medicine but have not been widely

accepted due to unique ethical and clinical challenges. Kazutoshi Takahashi and Shinya

Yamanaka hypothesized that the same factors that contribute to characteristic embryonic stem

cell traits would also contribute to pluripotency of cells and could possibly be used to reprogram

differentiated cells into embryonic-like stem cell states. Utilizing retroviral transduction, they

began testing with a pool of 24 potential factors responsible for pluripotency, then narrowed that

list to 10, and eventually isolated 4 key factors (now called “Yamanaka factors”) responsible for

the pluripotency of cells. Various genetic and histological testing was performed in order to

confirm that pluripotency was induced in the mouse embryonic fibroblast cells. While these

induced pluripotent stem cells (iPSCs) were not identical to embryonic stem cells, they greatly

resemble them in morphology, proliferation, and pluripotency. While the differentiation

mechanisms of iPSCs are not yet totally understood, they present exciting possibilities in the

realm of future cellular therapies by bypassing the moral concerns of using viable embryos and

safety concerns of tissue rejection.

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Background

As embryonic development progresses, non-specialized cells called embryonic stem cells

differentiate by means of gene expression. Once cell fate is determined, undifferentiated cells

will activate and express particular genes that correlate with their specified function. Activation

causes transcription factors to initiate the transcription of genes into messenger RNA molecules

which undergo post-translational modifications and are eventually translated by ribosomes into

various proteins that help the cell perform explicit functions. Undifferentiated cells have been

explored as tools in potential medical treatments involving tissue damage, organ failure, etcetera

due to their ability to differentiate into many other types of cells. Embryonic stem cells, the most

widely studied undifferentiated cells, have great potential to be useful but present two key

problems. First, embryonic stem cells must be harvested from human blastocysts and eliminates

the embryos’ ability to continue in embryonic development, which stimulated debates regarding

the moral concerns of utilizing embryos in regenerative medicine techniques. The second

problem included the risks of tissue rejection as the compatibility between harvested embryonic

cells and the patient receiving those cells is an intricate problem to navigate.

One technique that actively sidesteps both of these key problems involves taking somatic

cells from a patient and reprogramming them to look and behave similar to embryonic stem cells

(Takahashi). It was hypothesized that the ability to control the differentiation of cells lies in the

presence of certain genetic regulatory factors. If one has the ability to isolate transcription factors

responsible for an embryonic stem cell phenotype, one could theoretically induce a differentiated

cell back to an undifferentiated state. To explore this idea Kazutoshi Takahashi and Shinya

Yamanaka selected 24 genes based on their recorded potential roles in the pluripotency,

embryonic cell phenotype, and rapid proliferation of embryonic cells and designed experiments

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to further isolate which of the 24 factors were necessary to establish and maintain the identity of

embryonic stem cells (Takahashi & Yamanaka, 2006).

Experimental Approaches

Takahashi and Yamanaka “hypothesized that the factors that play important roles in the

maintenance of embryonic stem cell identity also play pivotal roles in the induction of

pluripotency in somatic cells” (Takahashi & Yamanaka, 2006). They designed an assay in which

the activation of the Fbx15 locus (and therefore resistance to G418) indicated that a particular

transcription factor was successful in the induction of pluripotency in somatic cells. If the cells

did not gain pluripotency, the Fbx15 locus would not be activated, the cell would not exhibit

resistance to G418, and the cell would not be able to grow on the G418-containing growth

medium. First, they used viruses to individually introduce each of the 24 factors into mouse

embryonic fibroblasts by a technique called retroviral transduction (Takahashi & Yamanaka,

2006). When all 24 factors were simultaneously introduced into mouse embryonic fibroblasts,

G418 resistant colonies were observed and named “iPS-MEF24 for ‘pluripotent stem cell

induced from MEFs by 24 factors’” (Takahashi & Yamanaka, 2006). These cells were further

observed to exhibit similar morphologies and proliferation characteristics to embryonic stem

cells. However, when each factor was introduced individually, not one of them activated G418

resistance. This indicated to researchers that a specific combination of factors is necessary to

induce pluripotency in the cells. In order to narrow down from the 24 candidate genes, scientists

eliminated single factors at a time and observed the effects on cells accordingly.

There were 10 factors isolated based on the lack of G418 resistance in their absence

which were used to create iPS-MEF10 cells that exhibited further pluripotency than the

previously created iPS-MEF24 cells (Takahashi & Yamanaka, 2006). The previously described

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methodology was repeated in order to further narrow the list of 10 factors to 4 key factors

(Oct3/4, Klf4, Sox2, and c-Myc) and created iPS-MEF4 cells (Takahashi & Yamanaka, 2006). In

order to confirm the role of the 4 isolated key factors in pluripotency, scientists utilized various

genomic tests including RT-PCR, DNA microassays, western blot analyses, and southern blot

analyses which all suggested that although the iPS-MEF4 cells were not identical to embryonic

stem cells they had pluripotent abilities. In-vivo testing was utilized to further confirm

pluripotency by means of histological examination of teratomas, immunostaining of teratomas,

and fluorescent microscopy of embryos which all confirmed to presence of three distinct germ

layers and therefore pluripotency (Takahashi & Yamanaka, 2006).

Conclusion

The results collected by Takahashi and Yamanaka in 2006 were exciting, thorough, and

highly supported their hypothesis that the key factors responsible for the fundamental features of

embryonic stem cells are also responsible for pluripotency. They clearly showed that a specific

combination of 4 key factors (now referred to as “Yamanaka factors”) is capable of

reprogramming an adult fibroblast cell back to an undifferentiated, pluripotent state (Wolff &

Purvis, 2019). The numerous and meticulous avenues of confirming pluripotency in the iPS cells

(both in-vitro and in-vivo) by genetic and histological testing was a huge strength of this study.

An area of concern in this study is that very little was discussed about the safety of utilizing both

retroviral transduction (viral vectors) and oncogenes (c-Myc and Klf4) in iPS cells that will

potentially be used in various therapeutic applications.

Future Direction

Takahashi and Yamanaka’s results from their 2006 study were certainly groundbreaking

in the field of cellular biology and have generated many further studies regarding the

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mechanisms behind iPSCs and their possible therapeutic uses. Since the publication of their

findings, it has been confirmed that their experimental premise can also be applied to human

fibroblasts (Wolff & Purvis, 2019). Various studies have investigated the reprogramming

potential of cells to find that in-vitro all cell types can reprogram equitably, but in-vivo this was

not the case (Wolff & Purvis, 2019). From a medicinal standpoint I would personally like to see

a study involving the growth of iPSCs under the extrinsic factors resembling human tissue under

stress of specific diseases. Different pathologies create different molecular environments for cells

in the body (such as inflammation) therefore the clinical efficiency of iPSCs under these

conditions would be highly dependent on how they operate in the presence of specific molecules

or under certain stressors.

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References

Takahashi, Kazutoshi, and Shinya Yamanaka. “Induction of Pluripotent Stem Cells from Mouse

Embryonic and Adult Fibroblast Cultures by Defined Factors.” Cell, vol. 126, no. 4, 2006,

pp. 663–676., doi:10.1016/j.cell.2006.07.024.

Wolff, Samuel C., and Jeremy E. Purvis. “Reprogramming Favors the Elite.” Science, vol. 364,

no. 6438, 2019, pp. 330–331., doi:10.1126/science.aax1681.