Future of iPSC

If you look at my medium profile right now, you can see multiple articles on how we can use several variations of stem cell therapies to cure different types of degenerative diseases. But I was wrong!

No, not wrong about the huge potential that stem cell therapies have, but I was wrong in thinking that implementing these therapies right now is our next step. When going through my research, I had a realization:

There are way too many problems with creating induced pluripotent stem cells (iPSC), that would make stem cell therapies inefficient and would take forever.

Let’s talk about the status quo before we excite ourselves with alternative reprogramming methods:

Just a reminder, this is an in-depth article that doesn’t cover most of the basic principles of stem cells and their applications, don’t worry if you don’t understand some of the things I talk about. Just visit my other articles here and come back :)

Yamanaka Factors

Back in 2006, Shinya Yamanaka discovered that you could turn normal somatic cells into pluripotent stem cells with the overexposure of 4 growth genes: OCT4, Sox2, c-MYC, KLF4. These four growth genes were found in embryonic stem cells and proved to show pluripotency in regular cells with over-expression. These four transcription factors are called Yamanaka Factors, and they are our current validated method of creating induced pluripotent stem cells (iPSC). But there are problems that have arisen from this type of reprogramming:

1) Oncogenic Transformation

There have been many studies that show tumor formation after transcription factors were implemented because of abnormalities that were found later in time after the transcription factors were introduced to suitable animal hosts. Why does this make using these transcription factors dangerous?

Let’s first breakdown the usage of each individual gene that is used in this process:

• Oct-4 is an octamer binding transcription factor that is expressed in undifferentiated cells, that deals with the self-renewal property of stem cells.
• Sox2 is a sex-determining region Y box 2, which means that it is a transcription factor that also deals with self-renewal but also pluripotency.
• KLF4 is a kruppel-like factor, which is a transcription factor that deals with proliferation and differentiation traits in stem cells.
• C-Myc is part of a family of regulatory genes, this transcription factor is also expressed in cancer for its proliferation ability..

We can see that all these traits would support the idea of pluripotent stem cells, and we can see why they were used for inducing somatic cells, but when we look at what the genes do, it is quite similar to how cancer seems to work. Cancer is a gene mutation that proliferates at high rates, and when a gene mutation happens in a stem cell population, then it would be horrible.

2) Efficiency

Not only do these four transcription factors show a high probability for tumor growth, but they also take a long time to actually create.

iPSC derivation from human cells takes around 3–4 weeks with efficiencies of 0.01 to 0.1%

That’s crazy… that means we need more and more samples just to create a functional population of stem cells that would be an effective stem cell therapy.

Welcome to what I’ve been researching ↓

What is Deterministic Reprogramming?

Multiple studies have shown alternative reprogramming protocols where rapid and up to 100% reprogramming efficiency can be obtained within a relatively short period. These types of methods are called deterministic reprogramming while using transcription factors and such are called stochastic processes.

There are a variety of deterministic reprogramming methods are being researched right now, but I want to talk about just one today:

Neutralizing Gatad2a-Chd4-Mbd3/NuRD complex to facilitate ES-like pluripotency

Before we put it all together, let’s breakdown the different components of this solution:

• Gatad2a is a gene that facilitates the deterministic reprogramming methods like the disruption of the Mbd3 gene that allows the ESC to remain in its pluripotency state
• Mbd3/NuRD complex is the gene that turns off the pluripotency state in ESC, in order for them to differentiate into different types of somatic cells
• OKSM is the Yamanaka factors that are used to create the pluripotency in these somatic cells

From the results, we can see that the efficiency at which iPSC is created is now improved and we are reaching the full potential of the translation. But how we do achieve these types of results?

Let’s look at the differentiation cycle when it comes to embryonic stem cells, and then break down where these different genes come in to prevent it from turning into a somatic cell.

We can see that there comes a stage before they differentiate into different types of cells like a muscle, blood, neurons, intestinal, pancreatic islet cells, liver cells. This is the stage where the Mbd3 gene is introduced, and the whole purpose of the gene would be to turn off the pluripotency trait so that it can turn into these different types of cells and stay that way.

Now imagine what if the cell wasn’t told to become anything?

Then it would remain pluripotent. That’s what the disruption of the Mbd3 trait does.

Let’s look at another example of a deterministic reprogramming method, where we can achieve iPS cells.

Retrovirus with infected β cells

Breaking this process down as we did with the previous method, we can clearly see different isolated aspects throughout the process.

Where we start with the reprogrammable mouse in a lab, we find the gene CD19 which acts as a biomarker for beta cells in the mouse, which helps us find the beta cells that we need.

Then the retrovirus is added to infect the beta-cell population, the retrovirus construct consists of C/EBPa-ER and hCD4. C/EBPa-ER acts as a biological monitor for the OKSM activation genes to make sure that they don’t overexpress themselves in the population because as we know from before overexpression of these genes causes oncogenicity. hCDH4 just acts as a biomarker and we use this biomarker for separate beta cells for iPSC lineage. Last not but not least, IRES is a key component when it comes to protein synthesis in the virus and after when the virus is introduced to the beta-cell population.

After that, we can see the different times that the genes in the virus are activated and how they lead to an alpha population of iPSC. As a TL;DR, the system is meant to act as a regulation system for OKSM expression.

Implications

Deterministic reprogramming methods are in the testing stage, where the are running clinical trials or have run trials very few times. But the implications for newer and more efficient methods of reprogramming stem cells are huge.

I talk about this in a previous article, where we’ve learned how to disguise stem cell populations within lipid-based nanoparticles (you can find it here), but the possibilities don’t just end there.

With newer populations that don’t face the same problems that we have today, now there can be smaller populations that don’t take as long to create so that we can now reply to demands for cures, and we can reply fast. In terms of in vivo treatment, we can see how now stem cells can remain in patient’s systems for longer and longer with a reduced chance of tumorigenicity, and provide repair faster and faster. The resupply for a specific patient or just in general wouldn’t take long either because deterministic reprogramming methods show results of fully reprogrammed iPSC cultures in a matter of 8 days.

But if everything is so great about it, why isn’t it used today?

From the ethical issues to the actual technology, there are a variety of problems that arise when people choose to experiment with embryonic stem cells.

The technological problems can always be overcome with time, but something that remains to exist for longer periods of time is the stigma around ES cells.

There are 23 countries around the world, that are authorized for embryonic stem cell research but prohibited to bring that to clinics. They are only allowed to conduct their research under the guidelines that they must keep it for the purpose of research only.

I’m not saying that this type of movement is bad, it’s actually good that scientists are using donated or extra embryos to learn about stem cell behavior, but the clinical application for the genetically neutral cells (ES cells)are crazy and very underrated.

Overall, I think that potential stem cell therapies show is huge, but are we making the most out of it? no. That’s because we’re stuck at stem cells, we must find a way to implement a super-efficient method of creating iPSC (like what I explained in this article)and then only can we look at the different therapies we can use it in.