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'Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors'

A summary of the results of Takahashi, et al. (2008) Cell 126(2008): 663-676. Web. Feb. 2014.

Hannah H., Kirsty W., Brittany P.


What is a Stem Cell?

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Stem cells are cells typically found in the very early stages of development that have not yet been differentiated. At this stage, they are said to be pluripotent, meaning have the potential to give rise to all somatic cell types in the body (Murnaghan). Stem cells retain the ability to replicate and divide almost limitlessly, thus maintaining a population of stem cells. Thus stem cells are defined by two key properties: 1) ability to self renew (i.e. to divide and to give rise to some daughter cells that retain their pluripotency); as well as 2) the ability to differentiate (i.e. to divide and give rise to cells that can differentiate into a variety of specialized adult cell types)

There are two major types of stem cells: embryonic stem cells, and adult stem cells. Embryonic stem cells originate in eggs that have been fertilized in vitro, while adult stem cells are undifferentiated cells found among differentiated somatic cells in a tissue of the body. The key difference in their competence is that embryonic stem cells can give rise to virtually any somatic cell type in that organism, adult stem cells are only able to produce differentiated cells of that specific tissue type in the body (NIH website).

In their 2006 article in Cell, Kazutoshi Takahashi and Shinya Yamanaka were able to genetically reprogram differentiated cells such that they behaved similarly to embryonic stem cells. They called these cells induced pluripotent stem cells or iPS cells.

What Determines Pluripotency?

Previous work on stem cells has established that somatic cells can be reprogrammed to become stem cells through two methods: either by transferring material from the nucleus of the differentiated adult cell into oocytes (egg cells), or by combination (fusion) of somatic and embryonic stem (ES) cells into one multinuclear cell (Takahashi & Yamanaka, 2008). This shows that the oocyte or stem cell can in someway reprogram the differentiated somatic cell nucleus and induce pluripotency. Kazutoshi Takahashi and Shinya Yamanaka were able in this work to define the specific genes that are responsible for causing this reprogramming.

Transcription factors Oct3/4, Nanog, and Sox2 all function in embryonic stem cells and early embryos to keep the cells pluripotent (Takahashi & Yamanaka, 2008). In tumors, the genes Stat3, E-Ras, Klf4, c-myc, and beta-catenin are more highly expressed, which lead to these genes being candidates for the genes that might have the ability to reprogram a differentiated cell. In this paper, these factors were studied to see if they would help confer pluripotency on somatic cells. Twenty-four different factors were evaluated to see if they could induce pluripotency in somatic cells.

To measure if the candidate genes were important in pluripotency, the cells were subjected to high concentrations of G418, an antibiotic that blocks polypeptide synthesis. The beta-geo locus was inserted into Fbx15 gene to select for those cells that were pluripotent. Those that were resistant to G418 were determined to be important in the induction of pluripotency, because Fbx15 is only expressed in cells that are pluripotent. Thus only if a pluripotent state was induced in the adult cells would the beta-geo (G418 resistance gene) be activated. Therefore they could select for cells that were in a pluripotent state by plating the cells on the G418 selective media.

Yamanaka and Takahashi: Results

24 candidate genes were identified to test for pluripotency, which was determined by resistance to G418 (Takahashi & Yamanaka, 2008). All 24 of these candidate genes were introduced into mouse embryonic fibroblasts (MEFs) using retroviral transduction. Resulting in "clones exhibiting morphology similar to embryonic stem cells (ES), including a round shape, large nucleoli, and scant cytoplasm," and the ability to proliferate like embryonic stem cells.

Yamanaka and Takahashi identified 10 factors from these 24 without which there was no colony formation 10 days after transduction. When each of these 10 factors were withdrawn individually, Oct3/4, Klf4, Sox2, and c-Myc were identified as key factors in the generation of induced pluripotent stem (iPS) cells from MEFs. Finally, they showed that the introduction of these four transcription factors is sufficent for the induction of iPS cells from differentiated cell types.

Significance and Ethical Issues of Embryonic vs iP Stem Cells

Stem cells are important in that they are capable of dividing for a long time and can develop into specialized cells from an unspecialized state (NIH website). They can also reproduce themselves over and over again. There are two types of naturally occurring stem cells: embryonic and adult. Embryonic stem cells are especially important because they eventually give rise to every cell, tissue, and organ in the fetus’ body. These are found within the fetus and the umbilical cord. Adult stem cells can be found in already developed tissues of infants, children, and adults (Bethesda). The problem with adult stem cells is that they can only give rise to cells within their resident organs. For example, an adult stem cell extracted from a kidney can only give rise to other cells that makeup a kidney. Adult stem cells do not provide the developmental flexibility that embryonic stem cells provide, making them less valuable at the clinical level. It’s been shown that embryonic stem cells can be used to treat diseases such as Parkinson’s, diabetes, and spinal cord injuries (NIH website). However, there are many ethical issues surrounding the methods used to obtain embryonic stem cells, preventing this revolutionary discovery from taking full flight.

To try to avoid the ethical issues while still producing cells as flexible as embryonic stem cells, scientists have developed induced pluripotent stem cells, or iPS cells. These are adult stem cells that have been experimentally reprogrammed into a stem cell-like state. This allows scientists to bypass the need for embryos and each individual can have their own line of pluripotent stem cells. Ethical problems are still present though. It has been found that viruses are an effective way to reprogram the adult cells but this has the potential to trigger the expression oncogenes, potentially causing cancer in the patient (Brind'Amour). There also is hesitancy to alter the human genome in a way that may have larger or more permanent consequences than desired.

Shinya Yamanaka's discovery that mature cells can be reprogrammed to become pluripotent was so ground-breaking that it won the Nobel Prize in Physiology or Medicine in 2012.

References

NIH website - "What are induced pluripotent stem cells?" National Institutes of Health, U.S. Department of Health and Human Services, 2009. <http://stemcells.nih.gov/info/basics/pages/basics10.aspx>

Brind'Amour, K. "Ethics and Induced Pluripotent Stem Cells". Embryo Project Encyclopedia. 10 June 2009. http://embryo.asu.edu/handle/10776/1986.

Image source: http://www.csa.com/discoveryguides/stemcell/overview.php

Murnaghan, Ian. "Pluripotent Stem Cells." Explore Stem Cells. N.p., 30 Oct 2013. Web. 26 Feb 2014. <http://www.explorestemcells.co.uk/pluripotentstemcells.html>.

Takahashi, Kazutoshi, and Shinya Yamanaka. "Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors." Cell 126(2008): 663-676. Web. Feb. 2014.


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