Discovering Yamanaka Factors — An Exploration of Yamanaka’s Nobel Prize-winning experiment
Exploring and visualising the scientific process behind one of the most significant discovery in human longevity of this century
Aging and Yamanaka’s experiment
Aging is often wrongly equated with the passage of time, unstoppable and irreversible. In 2006, scientist Shinya Yamanaka and his team proved that by activating four transcriptional factors, mammalian cells are capable of demethylating its epigenome and undoing all the effects of aging it had experienced since conception. Up-regulating these four genetic sequences — now named after its discoverer, Yamanaka — induces cellular pluripotency in adult somatic cells, making them functionally identical to embryonic stem cells (ES). Pluripotent cells are capable of differentiating into all human cell types and tissues, making them integral in many present and future therapies, some with the potential to treat currently untreatable diseases. In light of this groundbreaking discovery made 15 years ago, I decided to recreate and explain the process of Yamanaka’s Nobel Prize winning experiment. Please follow me through the whole method Yamanaka and his team used, I will attempt to help you visualise each steps along the way.
Where to begin?
The rationale behind this experiment is simple: insert a gene sequence into mouse embryonic fibroblasts (MEFs: cells that synthesize extracellular matrix and produce structural framework for animal tissues), then observe if the cell property reflects that of a pluripotent cell. The gene sequence inserted targets four factor candidates suspected of inducing pluripotency in cells, namely Sox2, Oct3/4, Klf4 and c-Myc. This means if any pluripotent property is observed in cells with the inserted gene sequence, we can verify that these four factors do in fact have links to cellular pluripotency.
Yamanaka Factors Overview
So first, what are the four Yamanaka factors?
So, what are the Yamanaka factors? Firstly, it’s important to know that all four of the Yamanaka factors (Sox2, Oct3/4, Klf4 and c-Myc; or OSKM) are transcription factors. This means that they are protein expressed by one or several particular genes (gene: sequence and region of DNA) that regulates which of the other genes are turned on (genes that generate proteins are considered “turned on”) and which are turned off. In simple terms, think of your genome as a chain of lights. The transcriptional factors are essentially individual switches, in charge of turning on and off each of the lights in the chain.
Each of the four factors are regulated by one or several genes in the human genome. To activate the Yamanaka factors in a cell requires a gene editing procedure that inserts the regulating genes into the cell’s genome.
In his experiment, Yamanaka inserted each of these four factors regulating genes through a method called retroviral transduction. Retrovirus is a type of virus that integrates its RNA into the genome of the host cell it infects, causing a permanent alteration of the cell genome (HIV-1 and HIV-2 are some famous examples of human retrovirus). Retroviral transduction is a gene editing technique that utilises this property of retrovirus, making it the vectors (vector: the vehicle carrying foreign genetic material into a cell) to deliver the gene insertion activating the four Yamanaka factors.
To be able to deliver these targeted gene sequence into cell samples, several steps are required:
1.) Creating the desired sequence to be inserted into the cells genome, plasmid construction.
2.) Packaging the constructed plasmid into the viral vector and replicating it, or transduction and packaging.
3.) Delivering the gene sequence into the cell via the created viral vector, or transfection.
This is organised roughly into the chronological order to how it is completed in a lab. Below, I will explain each of these three steps one by one in that order.
Plasmid Construction
The process of synthesising the correct genes, in DNA form, to be loaded onto the retroviral vector via Plat-E cells. I will start with the science, it might be a little difficult to follow, but please stick with me. The more convoluted concepts I will explain with analogy in the “To Visualise” section.
The Science
The first phase to plasmid construction is to create a plasmid base that’s capable of being loaded into the viral vector. This is done with the Gateway cassette rfA. Gateway cassette is a technology invented and commercialised by Invitrogen to facilitate cloning (Cloning: Making many identical copies of a piece of DNA) and inserting targeted gene sequence into a viral vector. The Gateway cassette is introduced into EcoRI/XhoI sites of a pMXs plasmid. pMXs plasmid is retroviral loading vector with multiple splicing sites(splicing sites: joining sites between exons) and acceptor sites (acceptor sites: location of where ribosome binds to during protein synthesis). It is commonly used in the process of generating retrovirus with specific payload (payload: sequence of RNA that scientists want to insert into a cell).
On the basis of pMXs plasmid previously generated, the means to induce the expression of the four desired factors have to be generated. This can be distinguished into 1.) inducing gene mutation in the parent plasmid, and 2.) amplification of targeted sequence.
For mutations, Yamanaka’s team induced mutations in the β-catenin, Stat3 and c-Myc expression by a method called PCR-based site-directed mutagenesis. β-catenin is a functional protein regulating gene transcription in human. It is found that presence of β-catenin enhances the expression of Klf4, Oct4 and Sox2, thus tying it to cellular pluripotency. Stat3 is another transcriptional factor whose activation is found to be a requirement of self-renewal in embryonic stem cell. This ties activation of Stat3 to the maintenance of pluripotency, which means if the experimental sample of induced pluripotent cells want to be sustain for an extended period of time, Stat3 activation is necessary. PCR, or polymerase chain reaction, is a common technique used to quickly replicate a sequence of genes. PCR-based site-directed mutagenesis is a technique using PCR to induce a specific change in a targeted sequence of genes.
For amplification, the coding region of candidate genes (genes associated with activation of Sox2, Oct3/4, Klf4 and c-Myc in this experiment) are amplified by a method called RT-PCR. RT-PCR, or Reverse Transcription PCR, is another variant of polymerase chain reaction used to quickly replicate DNA from a RNA base template. Reverse transcription refers to a process of generating DNA from RNA. The result generated is moved onto a Gateway donor vector (Gateway donor vector: essentially a vehicle to help integrate the gene into the plasmid), and then recombined with the pMXs base vector previously generated.
To Visualize
Now imagine a lego kit. In the kit there is three components. A base board that you can build your stuff on, a already built structure that you don’t like, and a tiny piece on the very edge that is too small to be notices. The pMXs plasmid bases is essentially like that base board, where you build the gene sequence — the structures — on. The induced gene mutation through PCR-based site-directed mutagenesis in the plasmid aims to deal with the built but undesirable structure, changing it (AKA mutating it) into something desirable. The amplification with RT-PCR is dealing with the tiny little piece that is too small, making it bigger by replicating itself. Altogether, in plasmid construction, you built a structure that you want on the baseboard by changing the structure that you don’t like and making some of the out-of-proportionally small things bigger.
Now the plasmid is generated, we will move on to the next step; loading the constructed plasmid into the viral vectors.
Retroviral Transduction and Packaging
Loading the previously generated genetic material onto the retroviral vector through Plat-E packaging cell.
The Science
Transduction of constructed plasmid onto the retroviral vector via Plat-E cells are done in vitro condition on a cell culture dish. Around 12 hours prior to transduction (Yamanaka mentioned the night before, so I assumed to be 12 hours), Plat-E cells were seeded at 8*10⁶ cells per 100mm dish. Plat-E cells is a retrovirus packaging cell line derived from 293T cell line. It is capable of producing extra stable expression of viral structural protein in the retroviral vectors produced. The culture is left for the night.
On the next day, pMXs-based retroviral vectors is introduced into Plat-E cells using Fugene 6 transfection reagent. pMXs-based retroviral vector is based on Moloney murine leukemia virus, or MMLV, a 5.9kb vector (5.9kb: the vector have a total RNA length of 5900 base)for delivering heritable genes into a cells genome. Introducing viral vector into the packaging cell line requires transfection reagent. Transfection reagent induces the viral vectors to release nucleic acids into the cytoplasm of the Plat-E cells, facilitating the vector entering the cell.
Immediately after the previous process, 27 µl of fugene 6 transfection reagent is diluted in 300µl DMEM and incubated for 5 mins. DMEM, or Dulbecco’s Modified Eagle Medium, is a common medium supporting growth of many mammalian cells. After incubation, 9µg of the plasmid DNA previously generated in plasmid construction is added into the mixture before another incubation for another 15 mins period. Mixing plasmid with transfection reagent facilitates the plasmid’s entry into Plat-E cells, hence why plasmid is not added alone to the cell samples. Following the incubation period, the mixture is added into the Plat-E cell culture. The culture is left incubated at 37 degree celsius overnight with 5% Carbon Dioxide. This incubation period allows the plasmid to enter the cell and replicate. To this point, the transduction process is complete.
To Visualize
The lego structure you built previously needed to be packaged. You send your legos into a packaging facility where many workers labours to package legos. Plat-E cells are like the workers in a packaging facility, packaging plasmid into retroviral vectors. However your not satisfied with just having the one lego structure you built packaged, you want it to be mass produced. The packaging worker tells you no problem, they’re on the job. Plat-E cells doesn’t only package the plasmid into the retroviral vector, they also replicate the packaged viral vectors. Altogether, you now have a bunch of packaged retroviral vector with the genetic information you want.
The retroviral vectors are finally packaged and replicated. You are now ready to move to the final step, delivering the gene into the cell samples.
Retroviral Transfection
Delivering the retroviral vectors to the cell samples and allowing the infection and genetic material integration into the cell’s genome.
The Science
The transfection process will also be completed in a in vitro condition on a cell culture dish. 24 hours after the transduction is completed, targeted cell sample of mouse embryonic fibroblast (MEF) is seeded at 3*10⁵ per 100mm dish. Before seeding of the cell sample, the dish must first be conditioned to allow any potential induced pluripotency to occur. For this reason, the dish is first layered with mitomycin C treated STO Feeder cells. Mitomycin C is a class of anti-cancer chemotherapy associated drugs, this prevents any carcinogenic cell in the culture to replicate. STO feeder cells are integral in the process of establishment and maintenance of pluripotent cell culture, promoting cell proliferation.
24 hours after the cells are seeded, supernatants containing the viral vectors are then extracted from the Plat-E culture. The fluid is then filtered through a 0.45mm cellulose acetate filter before supplemented with 4mg/ml polybrene. Cellulose acetate membrane filters have hydrophilic properties and very low protein binding capacity; this meaning they are the ideal filter for any experiment which requires most of the protein to be recovered. The supplemented 4mg/ml polybrene is used for increased efficiency of retroviral transfection in cell cultures. In this step, the packaged viral vector are extracted from Plat-E cells and refined; preparing it for the actual transfection process.
The solution after extraction and filtering is then added to the dish seeded with targeted MEF, and left to incubate for anywhere between 4–12 hours (Yamanaka again wrote 4 hours to overnight, which I assumed to be 12 hours). After the incubation period, the cells are replated in 10ml of fresh medium. Another 3 days afterward a G418 solution with 0.3mg/ml concentration is added. G418 is essentially a filtering method used to filter out the pluripotent cell from the non-pluripotent cells in the culture. I will explain how this work later on in the article. The experimental observation is then conducted 2–3 weeks afterward.
To Visualize
Now, you got the many packaged lego you ordered to be made back. It's time to deliver it to your friends and families and you're many little cousins. Instead of going to them one by one at their home, you invited them all over to present them the gift. The in-vitro environment is somewhat like this gathering, where instead of delivering the vectors into a living mouse, you extract the cells and grow them on a culture dish. Your friends and families and you’re many little cousins now arrived, so you present them with the present. The adult are quick to understand and to enjoy the lego pieces, but the kids are going to take a while. This is similar to a cell sample. How quickly the vectors integrate into the genome varies, and some cells takes a while before they read and display the change. This is the why for the may extensive incubation periods.
The vectors are delivered to your cells, and we wait to see the result. But what kind of result indicate the cell have became pluripotent? Lets move on to the method Yamanaka and his team used to test the pluripotency.
Pluripotency Test
How do we tell if a cell is pluripotent? What are the method to test that? Here I will explain how Yamanaka and his team used G418 resistance as hallmark of pluripotency.
The Science
It’s difficult to distinguish physically which cell is pluripotent and which aren’t. In trying to evaluate the pluripotent properties of the cells, Yamanaka and his team devised a system basing on G418 resistance. G418 is a amino-glycoside antibiotic, capable of inhibiting protein synthesis. Normal adult cells have very low tolerance to the exposure of G418 (sensitive to any concentration above 0.3mg/ml). To differentiate pluripotent cells from the normal level, Yamanaka and his team inserted a βgeo cassette (a fusion of the b-galactosidase and neomycin resistance genes, both similar types of antibiotic) into the mouse Fbx15 gene by homologous recombination prior to the experiment. Fbx15 genes are exclusively expressed in mouse embryonic stem cells, although it does not part take in maintaining its pluripotency. The edited mouse is then backcrossed with the parent line for 5 generation before conducting the experiment.
The inserted βgeo cassette essentially allows any expression of Fbx15 gene to provide a level of G418 resistance to the cell. This means that any expression of pluripotent properties which activate Fbx15 genes expression in the MEFs will result in a higher then normal level of G418 resistance, yielding a quantitative reading on the pluripotency of the cell. The experimental result indicates that pluripotent cells activating this modified Fbx15 gene are resistant to G418 concentration of up to 12mg/ml, more than 30 times the normal value.
To Visualize
As a science nerd myself (very proudly), pH test paper (essentially a buffer solution) is a common sight. The G418 and the cells samples essentially interact like pH test paper and acidic solution. When the concentration of G418 is high, the cell behaves in a certain way (namely dying), and when the concentration is low, the cell behaves otherwise. This change in behaviour then yields a reading on the pluripotency property of the cells.
A Final Word…
Now, lets review the few key steps Yamanaka and his team under took in their method inducing the four candidate factors associated with cellular pluripotency.
Technical TL;DR
- Firstly, the desired gene sequence is constructed on a pMXs plasmid with PCR-based site-directed mutagenesis and RT-PCR. The main method used in this process is mutation and amplification of the genes regulating the four Yamanaka factors.
- Secondly, the previously generated plasmid is packaged into pMXs-based retroviral vectors by Plat-E cells before it is replicated. This step generates retroviral vectors with the desired gene sequence attached.
- Thirdly, the retroviral vector is delivered to the cell samples in a in vitro environment. A long incubation (2–3 weeks) period is allowed prior to observation, with addition of G418 solution 72 hours into the incubation.
- Finally, observation on the pluripotency of the cell samples are conducted according to the G418 resistance hallmark.
The process Yamanaka and his team constructed is very interesting in the sense of the intricate science behind. However, the truly ground breaking aspect of this experiment comes from the result Yamanaka derives. In the part 2 of this article, I will explain the result of Yamanaka’s experiment, his interpretation to the data and researches further conducted in the following years. As humanity, there have always been a craving for rejuvenation of our body. Until the publishing of Yamanaka’s study in 2006, that remains a fantasy. The significance of this discovery can hardly be overstated, as it is one of the only keys humanity holds to halting and potentially reversing aging. Result of his experiment — as you will see next time — feeds hope into that one dream we humans had for millennials; longevity.
Continue here: part 2 coming out soon
Additional resources
Thanks for reading! I’m Henry, a 17 year old tech and longevity enthusiast on a mission to help extend human health span.
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