I investigated the effect of the founder cell type on neurosphere development…here’s what I found out
You’re in the middle of a basketball game. You carefully examine whether or not there are any opponents in front of you trying to get the ball but before you know it, you are right in front of the basketball ring, throwing it and you score. At the end your team wins…but what if that ball wasn’t for basketball and full of live cells? What if that win was getting treatment for Parkinson’s disease or ALS? Well… these balls are called neurospheres.
Neuro…whaaat?
I’m pretty sure that you might not have heard of this word before and as you are reading this or would try to type it on your own, your computer would underline it, indicating that it is misspelled, but it’s not (and yes, I am well aware that what is written with only one letter a).
At first, this word may not make sense, but if you paid attention during your biology and elementary math classes at school, you will understand it once we break it down. “Neuro” means something related to nerves or the nervous system and “sphere” refers to their shape, pretty much like a 3D-sphere :).
So, now that you know what they look like, you might ask yourself…what do they have to do with the nervous system? The answer is simple, they are formed from neural stem cells (NSCs) .
Another complicated term, huh? These are undifferentiated cells, meaning that they have yet to undergo a process through which they get specialized to do a specific function known as differentiation. Consider this as a person going to university to get a degree to prove that they’re smart enough to do the job.
However, once they get differentiated (or get their university degree), they can give rise to different cells that make up our nervous systems like our famously known neurons or less popular cells like astrocytes.
Okay…so, why would I care about a sphere of some neural stem cells?
Because these spheres could help you or your loved ones get treatment from Parkinson’s or Alzheimer’s or ALS and even stroke!
How exactly you may ask? By the help of neural stem cells inside them. As I’ve mentioned before, because they contain NSCs which can form different cells of the nervous system, they can be used for various cell therapies to treat these diseases (though they contain various cell types, including differentiated and progenitor cells as they develop with time, we will cover progenitors later in the article).
What all the three diseases have in common is that in every case, neurons and other neural cells die, leading to memory loss and movement issues. That’s when the neurospheres get into play. Since they can form neural cells, cell therapies can be developed to replace the dead ones acting as the cure to these three cell assassins. Imagine how many people would be able to improve their memory and be able to walk again!
Wait…if you want to use them to treat diseases, how can you get them in the first place?
You definitely don’t go to a pharmacy to get them and that’s where labs come in.
In order to form neurospheres, you have to proliferate single neural stem or progenitor cells under serum-free conditions and stimulate their growth by using epidermal growth factor (EGF) and fibroplast growth factor (FGT-2).
Here is a breakdown of what these words mean👇:
Neural stem cell- cells that are multipotent which means that they can differentiate into many different cell types within the nervous system e.g. neurons, astrocytes and oligodendrocytes. They can also unlimitedly replicate themselves via cell division.
Progenitor cell- similar to stem cells, they have the ability to differentiate but are “lineage-restricted” and have a more restricted proliferative potential. This means that they can only differentiate into a very specific group of cells which are usually a part of the tissue they belong in and can grow or reproduce a lower number of cells. That is why they are usually used for repair in our bodies. E.g. if your skin is damaged, skin cells should be formed to repair it, not muscle cells.
It is also important to note that progenitor cells are descendants of stem cells that then differentiate to create specialized cell types. The diagram below will help you gain some idea about it:
Proliferation- the process in which cells grow and divide , increasing the number of cells in a population. So, to proliferate the stem and progenitor cells just means make them grow and divide to increase in number.
EGF and FGT-2- growth factors that promote the proliferation of neural and progenitor cells.
Serum-free culture condition- using a culture medium (substance containing nutrients in which cells are grown) that does not contain serum. Serum is a component of blood that contains different growth factors to aid cell proliferation. In the case of culturing neurospheres, EGF and FGT-2 are used instead of serum to reduce potential contamination from the serum media and fluctuations in growth or behavior of the cells.
There you have it, once you have all of these in your lab, you may be able to have your neurospheres after 7 days with a tiny diameter of 100–200 μm but depending on the experimental conditions it can even take 2 months to see substantial growth.
Speaking of their diameter or size, there’s another factor that plays a huge role…and that is the founder cell.
What is a founder cell and how does it affect the neurospheres’ development?
A founder cell is basically the starting point of developing a neurosphere. It is a single neural stem or progenitor cell from which all the other cells within the neurosphere are derived.
The concept is based on an observation that neurospheres originate from a founder cell and are composed of its progeny (AKA composed of its descendants).
Simply, this means that the founder cell is the initial cell which proliferates and gives rise to the whole neurosphere.
Okay, so, we know that single founder cell can be either active stem cell or a progenitor cell and it can have an influence on the neurospheres, particularly their size.
You may ask yourself, who cares about their size?! As long as they can form neurons and other cells of the nervous system we might need, then we’re good. Well, it a bit more complicated than that. This is because:
- The neurosphere size influences the balance between cell proliferation and differention. Smaller neurospheres promote a more rapid cell proliferation while bigger ones may have increased differentiation into specific neural cell types. So, as a researcher, you might want to manipulate their size to get the exact cell type you need-neurons, astrocytes etc.
- Nutrient and oxygen diffusion can be a problem in larger neurospheres. In larger neurospheres, it can be challenging for the innermost cells to get enough of oxygen and nutrients which can affect their health and functionality. Therefore, you have to consider the size since you would prefer to work with cells that are healthy and able to function.
- The size of neurospheres can affect the transplantation efficiency. If we want to use neurospheres for therapeutical purposes, then they need to be transplanted. Smaller neurospheres could be a better option because they can be delivered easily into the target areas and integrate with the existing tissues.
Now that we know how important the size is to make neurospheres healthy and functional to use them in research and help to cure neurodegenerative diseases, I decided to investigate how the founder cell type affects neurospheres’ size.
To be more specific, in which case would the neurosphere be larger, using active stem or progenitor cell as the founder cell?
Experiment time🧪🧫: investigating the founder cell effect on neurosphere size
Now that we are done with all the theoretical part to give you background information, it’s time to have some fun…in our simulation labs! I am going to use the neurosphere simulator for this designed by Northeastern Neurobiology.
Hypothesis we are going to test today: active stem cells form larger neurospheres than progenitor cells.
Procedure: run 10 simulations to see the development of the neurospheres in each case, with active stem and progenitor cell. We do this 10 times to get reliable results. At the end, count the number of live cells in the neurosphere each time and compare. The lattice size is large while the rest of the paramets are set as the default setting.
Here’s what the simulation looks like. It has different parameters that affect the development of neurospheres but now we will focus on the founder cell type only.
Once you select the parameter changes by moving the circles either to the left or right for each one, you click on run the model.
Afterwards, it will take some time to process it and depending on how fast your internet connection is, you will get a diagram and a line graph.
For those who are curious the dots which represent the cells are colored is because each color represents a specific cell type inside the neurosphere. Blue dots are have been differentiated cells, orange dots are progenitor cells and red dots are stem cells.
Counting “live cells” includes counting the number of differentiated, progenitor and stem cells at the end of iteration step=100 (t). Iteration step refers to a single cycle of the simulation process and in this case, the time-based trends and progression of the simulation.
Here is the table of the results showing the number of live cells with active stem and progenitor cell as founder cells:
If we do some simple math to calculate the average number of live cells with each type of founder cell (that is by adding each number and divide by 10 as there are 10 samples available) the average number of live cells with active stem cell as a founder cell is 53.9≈54 and with progenitor cell is 37.8≈38.
Observation: as we can see, the neurospheres formed from progenitor cells are smaller.
This can be explained by the fact that progenitor cells have a restricted proliferative potential meaning that they have a lower capacity to self-renew and form new cells, forming smaller neurospheres.
Also, it has been known that if a progenitor cell acts as the founder cell, the neurospheres will contain a higher proportion of progenitor cells within them which have a lower capacity of self-renewal as we know, hence, the smaller neurospheres.
Another interesting aspect that we can explore based on the results is some factors that can limit neurosphere growth:
- Cell heterogeneity-we know that neurospheres consist of different cell types like stem cells, progenitor cells and so on. However, because there are cells present which have different proliferative potentials and differentiation capacities, this can affect the neurosphere’s ability to grow and differentiation potential (ability to differentiate into different neural cells like neurons, astrocytes etc.) limiting their use in regenerative medicine.
- Optimal time for passaging neurospheres-studies have shown that after a certain period of growth (around 5–9 days), neurospheres are ready to be passaged. This means that they should be transferred into another culture dish to support further growth, maintain their health and promote further differentiation into neural cells, so that could affect the size too.
Large VS small neurospheres: which one wins🏆?
As we have gone through the factors that should be taken into consideration in terms of the neurosphere size, it may seem like smaller neurospheres are a better option because the innermost cells don’t have a problem with getting oxygen or nutrients and are easier to transplant.
But it ultimately comes down to what you are researching and the outcome you want to get.
Although neurospheres could be the key to treating neurodegenerative diseases, we still need to do a lot of research until we start to use them in large-scale cell therapies. Therefore, it is important to consider the main aim of your research to identify the perfect neurosphere size for that case.
Large neurospheres:
- These neurospheres are optimal for studying intrinsic specifications. This refers to traits of cells that determine their development and function. In neurospheres, it’s bout understanding how NSCs behave when they are removed from their normal external environment (that environment is usually found in the central nervous system).
- They are also suitable for studying quantitative statements. Quantitative statements are numerical measures that provide specific information about something. In neurospheres, it’s related to making quantitative statements about the number of precursor cells (cells that can differentiate into a single cell type) in the neurosphere using data to describe and compare the characteristics of cells.
Small neurospheres:
- They can be used as a valuable model for differentiation studies. As we know, neurospheres can develop into different neural cell types like neurons, astrocytes and oligodendrocytes. However, smaller neurospheres are more suitable for studying this as they are easier to dissociate (breaking down the 3D neurosphere structure into individual cells). Since they are used for researching differentiation and can be dissociated easily, they could be the source of cells for transplantation in regenerative medicine.
- As neurospheres contain neural stem cells (NSCs), they can be used for cell therapies as I’ve mentioned. Smaller neurospheres might be more suitable for this as they are able to produce functional progenitor cells (specifically if the founder cell itself is a progenitor as we have seen, due to higher proportion of progenitor cells inside them). Progenitor cells are used as a part of therapies because it has been known that they integrate with the rest of the cells in damaged areas quite well. Also, they are able to differentiate into specific cell types, so you get only the type that the damaged area of the nervous system needs.
Final thoughts and takeaways
As regenerative medicine embarks on the journey of developing strategies to restore the function or regenerate organs and tissues back to normal after some type of damage or disease, we shall not forget about its potential to be the key to treating neurogenerative diseases and secondary neurodegeneration caused by things like stroke, leading to loss of functioning tissues in the damaged area and people’s basic abilities such as walking and speaking.
A strategy that could help to deal with this is cell therapy with neurospheres but there is still a ton of research that needs to be done until it is implemented. However, taking baby-steps towards this goal by understanding the basics of neurosphere development, such as factors affecting their size, could help this goal to become a reality a tad bit earlier even if it may not seem that significant.
But for now, I would like to wrap up this article by summarizing the key takeaways for you to gain some understanding about neurospheres and hopefully make you interested in this field of research as you finish reading:
- Neurospheres are 3D structures that contain different types of cells, such as neural stem cells, progenitor cells etc which could differentiate into various cell types of the nervous system like neurons, astrocytes and oligodendrocytes.
- In order to form the neurospheres, you have to proliferate single stem or progenitor cell (or make them grow and increase in number). That single cell acts as the starting point of its development known as the founder cell.
- Most studies argue that when progenitor cells act as founder cells, the neurospheres are smaller than stem cells’. This is due to their restricted proliferative potential (ability to self-renew and form new cells as a result).
- Ultimately, their optimal size depends on what you want to investigate and use them for.
- In the context of regenerative medicine, neurospheres can be used to restore damaged areas of the central nervous system (CNS)and even treat neurodegenerative diseases as they are able to differentiate into different cells of the CNS that could replace the damaged ones but more research needs to be done.
References
1) https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7521883/
2) http://neurosphere.cos.northeastern.edu
3) https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4575628/
4) https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10074836/
5) https://pubmed.ncbi.nlm.nih.gov/24228937/
6) https://www.sciencedirect.com/science/article/abs/pii/S0014488603002711
7) https://stemcellres.biomedcentral.com/articles/10.1186/s13287-021-02238-4
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