'Cellfie' of the month, Guest blog

Guest blog: ‘Cellfie’ of the month. The Dopaminergic Neuron.

Shown good the bad and the ugly of embryonic stem cells

Explaimed that they need feeding every day to keep them happy but when they are unhappy their default is to turn into neurons or a type of brain cell

Ive noticed recently that the odd colony of stem cells amongst my tower of cell culture plates had started to sprout these projections. Why? Because my stem cells were unhappy and started to differentiate into neurons.

This gave me an idea. Feel like Ive shown you all there is to show about embryonic stem cells and what they look like. So as I saw these neuronal projections coming from the odd colony I thought it would be good to rope an expert in to tell you more about neurons specifically dopaminergic neurons.

'Cellfie' of the month

‘Cellfie’ of the month: February 2017

How is it February already?! I swear it was only last weekend that I was in bed all day nursing my New Year’s Day hangover! I would like to think there would be some signs that Spring is on it’s way – but that is not looking likely here as I look out of the office window and this wet, cold and miserable day here in the UK!

But it is Friday – or should I say Fri-yay! – and it’s a brand new month with a brand new Cellfie!

As tomorrow (4th February) is World Cancer Day – I thought I would share some images of the cancer stem cells I work on.

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So you might now be thinking  – I thought you worked with embryonic stem cells, why are you working on cancer stem cells?

There is a very simple answer to this question – I work on both! Embryonic stem cells are my main focus but due to the similarities between them and cancer stem cells I do dabble in some comparison work.

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So. lets start off my asking what are cancer stem cells and what do they look like?

Much like embryonic stem cells can turn into, or differentiate, into any cell type that we can find in our bodies, cancer stem cells can become any of the cell types that make up the tumour which it came from! Ideally, these are the cells that we need to make sure we eradicate with treatments like chemotherapy and radiotherapy – because even if only one of these cancer stem cells survives, it can form a completely new tumour all by itself!!! So essentially – cancer stem cells are the stem cells of a tumour. The ones I use are from testicular cancer.

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I have shown in previous ‘Cellfie’ of the month blog posts that embryonic stem cells in the lab, like to grow in colonies. However, cancer stem cells like to grow in one flat sheet across the bottom of our cell culture plates in what we call a monolayer. The dark circles are the large nuclei of the cells which is home to the cell’s DNA . And those circles are surrounded by the cytoplasm which is where the cell makes all its proteins.

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What similarities are there between embryonic stem cells and cancer stem cells?

The key similarities for my research are that they express the pluripotency markers OCT4, SOX2 and NANOG – like the embryonic stem cells do, as shown using one of my immunocytochemistry images below, and they have a similar metabolism – or way of producing their energy through a process called glycolysis. They can also continuously make identical copies of themselves, called self-renewal.

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An immunocytochemistry image of some of my cancer stem cells. The green staining indicates that they are expressing  a key stem cell protein in their nucleus called OCT4.

Because of these similarities as well as a variety of others, it is thought that cancer stem cells like the ones I use could be used as a model system to study pluripotency, or ways of keeping stem cells as stem cells – especially as they are much, much cheaper and easier to grow than embryonic stem cells!

But why should YOU be interested in cancer stem cell research?

Ultimately, we want to gain some sort of clinical benefit from studying cancer stem cells to stop cancers spreading in a process called metastasis, or in combination with other therapies to stop tumours coming back again and growing uncontrollably. So, possibly in the future, if we can identify which cells are the cancer stem cells, we could try and target them more in therapies to make sure they are all killed off.

Studying these cancer stem cells could also give us clues about cancer development and how the tumours evade your body’s immune system and keep growing and growing – which in turn could give us clues for designing new cancer therapies.

It might also help us understand the biology of cancer progression. If we can work out how the cancer cells keep reproducing copies of themselves like stem cells do, and creating whole tumours which continuously grow and grow with no control – we could design new cancer therapies to stop this happening and hopefully stop certain cancers spreading!

But all in all, you should support cancer stem cell research as it will inevitably help us as researchers to develop better and more effective and perhaps personalised cancer treatments in the future!

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However – SPOILER ALERT – my research is showing that maybe these cancer stem cells are not as similar to embryonic stem cells in certain aspects as we first thought, which could be really exciting for new cancer treatments! More news on this soon hopefully!

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If anyone wants to find out more about cancer stem cells or embryonic stem cells, please do not be afraid to get in contact with me and ask some questions.

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'Cellfie' of the month

‘Cellfie’ of the month: January 2017

Welcome to the first ‘Cellfie’ of 2017!

This post might be slightly different to previous ‘Cellfie’ of the month posts because as many of the regular readers of my blog will know – I work on embryonic stem cells and they need to be fed and looked after every day. As I am still working at a university, the building I work in shuts down over the Christmas break. That combined with the fact that all of us in the lab don’t want to come into work over Christmas – we stop growing our cells during the holidays and freeze them down before we all go away!

But what that does mean is that when we come back in January – there are no cells to do experiments on! We need to get out cells up and running from scratch!

So, two weeks into the lab life of 2017 – our cell culture incubators currently look like this:

They are basically empty! We have three incubators that we use and we need them to look more like this before we can start doing some experiments:

As a side note – I am loving the new feature on Snapchat where you can make your own stickers. It made making this image of what we want our incubators to look like inside so much easier – even if it is a little messy!

Full of plates of cells! Enough for everyone in the lab to be doing all their own different experiments on!

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But as with science – it is not as simple as getting the cells out of the freezer again and then we are away again! Oh no! For us working on embryonic stem cells, it is a much longer process for various reasons – as I will try and explain more now.

Firstly – when you thaw some cells out they grow slower than normal, so you need to give them some time to get growing at a normal pace.

Next – for our stem cells to grow, we need to prepare the feeder layers that they grow on. But what are these feeder layers? Well, we use cells called mouse embryonic fibroblasts or MEFs for short. Basically we need to plate these cells into the wells of our cell culture plates BEFORE we add our stem cells! If we put our stem cells onto plates without the feeder layer, they just wouldn’t stick down and wouldn’t grow. So, the feeder layers are basically a sticky surface that the stem cells can attach to so they can keep growing and growing. Another use of our feeder layers is that they produce different proteins called growth factors and cytokines that help our stem cells to grow too.

Here is a look down the microscope at our MEF feeder layer – my ‘cellfie’ of the month photo:

It probably doesn’t look like much, but you can probably notice there is a layer of cells at the bottom of our plate. But when we look under the microscope at a higher magnification you can see what these cells actually look like:

The third reason why it takes a bit of time for our stem cells to get up and running is that, we actually need to take the cells back OFF of the feeder layers so we can use them for experiments. Yes really!

In this case, instead of growing our stem cells on MEFs we grow them on a different layer that doesn’t involve cells. This layer does the same job as the MEFs and is a sticky surface for our stem cells to stick to, but as it is not made of cells doesn’t give our stem cells the proteins they need for growth. But we can ONLY use the cells grown on these layers  in our experiments because the MEF feeder layers contain cells and therefore DNA and proteins which would affect the results we see. The cells also need to be taken off the MEFs and grown on this new cell-free layer for about 2 weeks before we can use them.

Another reason that it takes time before we have cells to use it that we often want to use cells that grow under 5% or low oxygen conditions. When we take our cells out of the freezer and start them growing again we do this under 20% or high oxygen concentrations. So, when we put cells at the lower oxygen conditions to use, we also need to give them time to adapt – and allow the cells to start making different proteins so they can continue to grow under these lower oxygen conditions. Much like with the cell-free feeder layer, the stem cells need to be at 5% oxygen for about 2 weeks before we can use them in experiments.

The final reason it is going to take some time for me to get some cells to start doing experiments on is bulking up. To save on resources, all the above is done with as few cells as possible in as few plates as possible. Once they are on the cell-free feeder layer and have been at the right oxygen  concentration for about 2 weeks, we need to start increasing the amount of cells we have so I can get some for experiments, so all my lab mates can have cells to do experiments on AND there are still some stock plates going in case we have any problems.

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So – we are trying to reduce this time as much as possible. At the moment we have cells at both 5% and 20% oxygen concentrations and they have been there for nearly long enough now – but I only put them onto our cell-free layer this weekend, so it will be at least another week before there is cells ready for experiments, but fingers crossed it is all going to plan so far and I will have cells by the end of the month!

Hopefully that is a quick insight into starting up embryonic stem cell culture from scratch, and hasn’t confused anyone too much! If it has got a bit complicated, I am more than willing for you to ask me some questions so I can help you understand. Don’t be afraid to get in contact with me. You can comment below or contact me on Facebook, Twitter or Instagram.

S.x

'Cellfie' of the month

‘Cellfie’ of the month: December 2016

Up until now all the embryonic stem cell colonies I have shown you as part of my ‘Cellfie’ series have been beautiful, nice looking colonies with happy and pluripotent stem cells.

That is about to change!

So you’ve seen the good, now for the bad and the ugly!

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As part of my research I have to take hundreds of images and show that the drugs and chemicals I put on to my embryonic stem cells don’t kill them off or stop them growing. Or simply just to keep record of what my cells looked like in each experiment I do.

A lot of my research looks at the metabolism of embryonic stem cells – which involves looking at all the chemical reactions they use to keep themselves alive. I’m particularly interested in the particular chemical reactions in a process called glycolysis. Glycolysis is a metabolic pathway that embryonic stem cells rely heavily on to generate all the energy they need to keep growing and replicating.

So as a part of this month’s ‘Cellfie of the month’ I thought I would show you an example of what my stem cell colonies look like when they are not happy (which happens far too often for my liking!).

As I mentioned my embryonic stem cells rely heavily on the process called glycolysis! But I sometimes add a drug called a glycolytic inhibitor which as the name suggests stops the action of glycolysis in my cells so I can investigate the effects on my proteins of interest.

In this particular experiment I put a range of different concentrations of my glycolytic inhibitor on my cells to see which concentration was too high for my cells and started to kill them off!

This is another phase contrast image, like I showed in my last ‘Cellfie’ post, of my cells that had been treated with the highest concentration of my drug that I tried and if you compared to the image in my last ‘Cellfie’ – you can clearly see that this drug at this concentration is toxic to my cells and killing them off as this colony is really ‘patchy’ and you can see remnants of the colony.

Because this image has shown that this concentration of drug is killing of my cells, it means I cannot confidently say that any effect I might see on my protein of interest is true because the cells are unhappy and dying 🙁

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Thanks to everyone who has read and liked my ‘Cellfie’ posts and hope you have learnt something from behind the scenes of my PhD lab life. I also love answering all your questions about my research and blog posts – so please continue to ask away! Hopefully that will continue into 2017 with plenty more ‘Cellfie’ posts!

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S.x

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'Cellfie' of the month

‘Cellfie’ of the month: November 2016

Happy November y’all! With Halloween and Bonfire Night all gone for another year, the countdown to Christmas is on! But the penultimate month of 2016 has arrived and so comes with  it the penultimate Cellfie of the Month for 2016!

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In October’s Cellfie of the month, we took it back to basics looking at my embryonic stem cells and their raw beauty. If you missed it you can find it here. This time it’s going to get a bit more up close and personal by sharing with you some of my phase contrast images!

But firstly, I want to introduce to you phase contrast microscopy! Normally when you look down the microscope at some cells they are quite difficult to spot and there is very little detail. This is because the cells themselves, all their internal components and the surrounding media absorb similar amounts of light making each part almost invisible. But this is where phase contrast microscopy comes in! It takes advantage of those tiny, tiny differences in light absorption and turns them into brightness variations on the image making each cell and the components within each cell much easier to see, as you can see when you compare the image in my previous Cellfie of the month post to this one:

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Embryonic stem cells grow in colonies like this one and each of those colonies are made up of hundreds of cells. Normally only a well trained eye could notice all the cells making up a colony under a normal light microscope, but using phase contrast microscopy, we can reveal the typical cobblestone morphology of embryonic stem cell colonies to the world!

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Let’s take an even closer look!

Using phase contrast microscopy and looking at my cells under a higher magnification, we can see even more detail! So, embryonic stem cells have several unique characteristics – they express pluripotency markers like OCT4, SOX2 and NANOG like I’ve shown here, they grow in colonies with a cobblestone morphology and they also have a high nucleus to cytoplasm ratio!

Broadly speaking, cells are split up into two areas – the nucleus and the cytoplasm. The cytoplasm is the area where proteins are made and waste is removed, whereas the nucleus holds all your DNA and is where any changes in gene expression happen! But as far as embryonic stem cells are concerned, the majority of the cell is taken up by the nucleus with a small ring of cytoplasm around it, hence the high nuclear to cytoplasmic ratio, as you can see here:

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The red circle is showing the outline of one of the cells in this particular colony, whereas the yellow circle is showing the outline of the nucleus, so you can see that most of the cell is made up of nucleus! And you can probably see that in all the other cells around!

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Slowly, this Cellfie of the Month series has been showing you some of the characteristics that make an embryonic stem cell, an embryonic stem cell! Take a look back at my older posts but hopefully you now know that:

  1. Embryonic stem cells are pluripotent
  2. Embryonic stem cells express pluripotency markers like OCT4
  3. Embryonic stem cells express pluripotency markers like TRA-1-60 on their surface
  4. Embryonic stem cells can make any cell type in your body called differentiation
  5. Embryonic stem cells can make identical copies of themselves called self-renewal
  6. Embryonic stem cells grow in colonies with cobblestone morphology
  7. And finally, embryonic stem cells have a high nuclear to cytoplasmic ratio

And there is plenty more where they came from! If you want to know more, please get in touch I would love to chat to you about stem cells 🙂

S.x

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'Cellfie' of the month

‘Cellfie’ of the month: October 2016

How is October here already!?  But the temperatures here in the UK have already started to drop meaning my favourite season of the year has officially begun. Autumn to me is all about going back to basics and enjoying the simple things in life – leaves are falling off trees to grow new ones come spring, lab fashion for me in these months returns to the trusted classic combo of jeans, t-shirts and boots, Sunday evenings are all about drinking hot chocolate with marshmallows tucked up under cosy jumpers and blankets on the sofa watching NFL until as late as I possibly dare.

So, this got me thinking to bring October’s ‘Cellfie of the Month’ post back to basics, and keep it short, sweet and simple.

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None of my experiments would get done if I had no cells. So, the basis of my research is to keep my cells growing happily before I take any sort of image or manipulate them for a heap of different experiments. Before using any fancy techniques, a simple view down the microscope is all I need to see my cells in their beautifully raw and natural state.

Capturing what you see down the microscope lens on a normal day to day basis to share with you guys is a little tricky as microscopes are obviously built for you to use with both eyes! But using a trusty phone camera, I managed to capture a glimpse of a colony down one eye-piece.

Come look down the microscope with me!! ☺️

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In this image you can see just one of my beautiful embryonic stem cell colonies! They form compact, rounded colonies as you can see here and have what we call a cobblestone morphology. Imagine you are walking down the cobbled streets of Ancient Rome, or Coronation Street if you prefer 😜, if you had a circular cutter and cut a perfect circle out of the middle of one of those cobbled streets, this is what we want our embryonic stem cells to ideally look like.

Unfortunately, keeping my cells like this in a culture plate is VERY difficult! They like to stick together in colonies, and if you break the colonies up too much and create single cells – you are going to have a problem with the cells losing their pluripotent state and start differentiating into other cell types – which are usually neurons! As my research is focussing on keeping stem cells as stem cells, this is BAD NEWS for me!

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But, for now, I have lots of beautiful embryonic stem cell colonies growing, but as I am currently stuck at my desk writing my transfer thesis, I’m not using them 😦 Let’s hope they continue to grow like this for when I am back at the lab bench 🙂

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S.x

P.S. I would love to see pictures of any of your cells in their ‘raw beauty’ down the microscope, or if you don’t work with cells whatever the basics for your research are. Add them to the comments 🙂

'Cellfie' of the month

‘Cellfie’ of the month: September 2016

A new month has arrived, which means a new ‘Cellfie’ of the month!

So this is just my second post in this series where I want to give you a bit more of a behind the scenes look into PhD life by showing you some of the sights I see down a microscope and talk all things involved in keeping my cells happy and healthy ☺️. In my previous ‘Cellfie’ of the month blog, I introduced you to my favourite image that I had taken so far of my embryonic stem cells using the technique called immunocytochemistry, and introduced you to the pattern we see if the protein we are looking at is expressed on the surface of the cells.

In this blog, I am going to show you another one of my images, but this time I am looking at the expression of the protein OCT4, which as promised shows a different expression pattern!

OCT4 is a really important protein for my PhD research as it is one of the three core pluripotency factors – so when a cell is expressing OCT4, it is classed as pluripotent and could become any cell type in your body! So, for my research, as I am looking at ways to keep embryonic stem cells as stem cells (or pluripotent!), I look at the expression levels of OCT4 after most of the experiments I do to see how it changes. If the OCT4 expression goes down, we know that the conditions we used in that experiment are not good for stem cells. If OCT4 expression increases, those conditions are good to try and keep stem cells pluripotent!

OCT4 is, also, known as a transcription factor. This means it can bind to certain regions of DNA and ‘turn on’ a particular gene. DNA is stored in the nucleus of your cells, so OCT4 is also going to be found in the nucleus of my embryonic stem cells.

If you can remember from last time, the blue staining is the DAPI stain. This is added to our cells to make their nuclei glow blue under the microscope. Using this immunocytochemistry technique, I have also added an antibody to these cells that will stick to any OCT4 that is in the cells, which is shown here in green. Notice anything about these two images?

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In the science research world, our ultimate aim is to get our research published. In these publications, we might need to add some of these images, but they need to be presented in certain ways. So, in my last ‘Cellfie’ blog post, I introduced the scale bar – a small line in the bottom of your image that is a reference point so the reader knows how big the cells they are looking at are. Last time I also showed you to the merged image, or the overlay. The aim of the merged image is to confirm where a protein is expressed. For example, in the last post where I showed you the overlay for a surface marker – there were the blue dots that were the nuclei of my cells, and green areas around them. Here, we have blue dots which are again the nuclei of my cells, but we also have green dots when we are looking for OCT4 in the images above. When we merge the images together we get this:

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The blue DAPI staining and the green OCT4 staining overlap – which just confirms that OCT4 is in the nucleus of my cells as they completely overlap. Hopefully you can see how this is different to the staining in my last ‘Cellfie’ post.

So – my stem cells are expressing lots of OCT4 in the nucleus. So by showing these images in my thesis or any publications I have demonstrated that the cells I am using are pluripotent!

Another ‘cellfie’ post done! I hope you enjoyed. I would love to hear your feedback! I have something a little different up my sleeve for the next one so watch this space.
S.x