A history lesson: induced pluripotent stem cells

My most recent posts have tried to be as current as possible. I enjoy writing about biological technology that hasn't become ubiquitous yet, while its future is still uncertain. But it's easier to learn and understand new techniques when you can use history to put it into perspective. Biology changes and evolves so quickly that we sometimes lose sight of where groundbreaking advancements fit into the bigger picture. So, this post will be a bit of a history lesson. We'll be talking about induced pluripotent stem cells with an emphasis on a paper from 2006 by Takahashi and Yamanaka, and along the way we'll go through some of the background that led to their breakthrough.

To state simply, a pluripotent stem cell is a cell that has the capacity to divide indefinitely and create any cell found within the three germ layers of an organism. This comprises nearly any cell you could imagine, excluding only extra-embryonic cells, such as those that make up placental tissue. When a pluripotent stem cell divides, the two resulting daughter cells can either be two new stem cells, or one stem cell and one slightly mature cell which has started down a pathway to become a specialized cell. Maturing daughter cells continue to divide until they become a fully mature cell, like an intestinal epithelial cell, or a red blood cell. Once fully mature, cells are specialized and have a specific job, but can't go back to their immature state. With this knowledge, it's logical to ask what has changed inside these cells, and what restricts them from further development.

In the late 1950s and early 1960s, a young scientist named John Gurdon was thinking about this. He asked, does the nucleus of a fully differentiated cell still contain the tools necessary to be pluripotent, or are those tools lost as the cell matures? In order to test that question, he used a technique named nuclear transplantation, in which he would remove the nucleus from a fully matured cell, and transplant it into a frog egg that has had its nucleus removed or destroyed. If the cell still had the tools to be pluripotent, but they were just inactivated, injecting the nucleus of the cell into an immature cell like an egg would allow it to become a stem cell. After doing this over 700 times, 10 of the eggs with a differentiated cell's nucleus grew up to become healthy tadpoles. At its most basic, this told him that the nucleus of some fully mature cells can still promote the production of an entire organism. Nuclei from differentiated cells still had the machinery and genetic information needed to create different cell types, but it appears to turn off once the cell matures. This showed us that any cell had the ability to be any other cell at one point, but depending on how the genetic information was expressed, it committed down one particular pathway. From a basic biology perspective, it's an exciting question: how can every cell contain the same instructions and information, but make different decisions as to how to interpret that information?

That question is still asked today. In an effort to answer it, many researchers since John Gurdon have been searching for clues as to what makes a stem cell a stem cell. Throughout the process we have seen some fascinating work. Dolly the sheep was cloned in 1996 using the same technique that Gurdon used years before, only this time it was done in a mammal, and the animal survived all the way until adulthood. Research such as this showed the cellular differentiation doesn't involve a permanent modification of the genome, and that eggs or stem cells must contain factors, active genes in this case, that can maintain or induce pluripotency. Along with transplant research, there is a host of literature focused on genetic manipulations that affect the growth of stem cells and early embryo cells. For example, OCT4 and SOX2, transcription factors in the cell, both are necessary for maintaining pluripotency at different stages of early embryonic growth. All of the research led to the hypothesis that the cocktail of factors that give a stem cell its identity could also be used to turn differentiated cells immature again. By simply introducing stem cell factors into a mature cell, one could manipulate them into believing and acting like a stem cell, the same way introducing the nucleus of a differentiated cell into a stem cell worked to produce a viable organism. The hunt was on for the right genes.

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Kazutoshi Takahashi and Shinya Yamanaka published their paper, "Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined factors" in 2006. This paper was paradigm shifting. From the research that came before them, they identified 24 different genes that could play a role in maintaining pluripotency. To start, they introduced all 24 factors into the mouse fibroblasts (shown above as MEFs, mouse embryonic fibroblasts). These are fully differentiated epithelial cells. After incubation with the 24 factors, some fibroblasts transformed into what looked like stem cell colonies (shown above. labeled ES for embryonic stem cells and iPS for induced pluripotent stem cells. Scale bars = 200 um). To identify which of these factors are completely necessary, they made 24 cultures introducing all but one of the factors into each culture. If a factor was necessary, they'd recognize it by the lack of stem cell induction. The figure below shows the amount of induced stem cell colonies per plate for each cocktail missing a given factor. It's clear that factors like 14, 15, or 20 are all necessary, as without them, there are no induced stem cells. From here, they narrowed it down to 10 factors, did the same experiment with those 10, and landed on the 4 necessary factors to induce pluripotency. The 4 factors were Oct3/4, Sox2, c-Myc, and Klf4, now known as the Yamanaka Factors, and they were sufficient on their own to induce the stem cell-like colonies.

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To prove that these stem cell look alikes really were induced pluripotent cells, they did two gold standard techniques. The first involves injection of the cells just underneath the skin of immune-compromised mice. The cells then grow and form a tumor. To judge the pluripotency of the injected cells, you study the tumor tissue to see if all of the 3 germ layers are present. A pluripotent cell must be able to create the 3 initial lineages of cells to be considered pluripotent. Next, they transplanted their cells into blastocysts, very early embryos, to see if they had the capability of forming an entire organism. They found the tumors did in fact contain all three germ layers, and, just like Gurdon's tadpoles and dolly the sheep, the cells could produce a viable fetus. The mice derived from the induced stem cells are shown below (scale bars = 2mm). The induced stem cells they created contained a GFP tag so they could distinguish which fetuses were stem cell-derived. This paper showed it was possible to induce a pluripotent state from a completely mature cell.