“Once development was ended, the fonts of growth and regeneration of axons and dendrites dried up irrevocably. In the adult centers, the neural paths are something fixed and immutable: everything may die, nothing may be regenerated”
This is one of the most famous quotes of Santiago Ramon y Cajal, a man whose accomplishments in the field have earned him the title ‘The Father of Neuroscience’. He also won the Nobel Prize for Medicine.
However, if there’s one thing we know about science, it’s that it is always progressing. Over the years, we’ve learned that Santiago’s beliefs about the inability of the central nervous system to regenerate were incorrect.
This is thanks to the discovery of neurogenesis. And it’s a concept that changes everything we think we know about our brains.
A brief introduction to neural cells
Arguably one of the most mysterious parts of the human body, the brain has been the subject of research for longer than documented history. Much of this research has been more intangible, focusing on how the brain controls our desires and fears. Some would call this philosophy.
That being said, the topic of today’s conversation is not on philosophy but the physical structure of what our brains are made of: neurons and glial cells.
Neurons are the powerhouse of our brain, they’re the cells that do what we commonly associate with ‘thinking’. By forming connections called synapses, they transfer messages throughout the nervous system telling our bodies what, when, and how to do something. Every movement you’ve made in your life, from opening a door to scrolling through this article is thanks to these guys.
Then we have glial cells, think of them as the assistant. Their job is to facilitate neurons through metabolic and mechanical support. For example, oligodendrocytes are a specialized type of glial cell that forms the myelin sheath. This is a layer of fatty substances that insulates neurons and allows them to send the signals that make you, well .. you.
What are neural stem cells?
If you’ve opened a news channel on TV, you’ve probably heard of stem cells. Many people have heard this term but don’t quite understand what it means due to the media circus either depicting them as miracle cures or an evil and unnatural science.
Here’s the truth: stem cells are any cell in the human body that is unspecialized and can self-renew. This means they can perform two main functions:
- dividing indefinitely
- giving rise to cells that go through the process of cellular differentiation
Cellular differentiation is when cells change from one type to another. This is usually when cells become more specialized by developing features that allow them to perform a specific function. This is regulated by which genes, the code that tells our cells what do, are activated and inactivated in a cell.
To get a more in-depth understanding, you can learn about the basic terms and definitions regarding stem cells in my introductory article here.
Neural stem cells are simply a sub-group of stem cells that can only differentiate into neural cells (neurons and glial cells).
Where are neural stem cells found?
Currently, neural stem cells have been located in two parts of the brain: the sub-ventricular zone (SVZ) and the dentate gyrus (DG) of the hippocampus.
Don’t be intimidated by the big words, here are some lovely diagrams that can help you visualize.
Why is neurogenesis so important?
As we learned through Ramon’s quote, the process of neurogenesis was thought to be the equivalent of alchemy in many scientific circles.
Simply put, neurogenesis is the process through which new neurons are formed in our brain.
For the longest time, scientists believed that we are born and die with the same number of neurons. This all changed thanks to Joseph Altman, who proved adult neurogenesis was proven to be possible in 1962 through the discovery of neural stem cells.
Before him, there was no hope for people who had suffered injuries to their central nervous system.
“Oh, you got a spinal cord injury that damages your central nervous system’s cells? Aw, that sucks. Nothing we can do about it, though :)”
Now, things have changed. Research that would take decades in normal circumstances can now be done exponentially faster by using neural stem cells that divide indefinitely, thereby giving an infinite source of neurons to be used in studies. It also means finding cures for neurodegenerative diseases such as Alzheimer's might not be impossible as once thought.
With all the benefits neural stem cell research could bring, the question becomes: how do we study neural stem cells in the first place?
To do that, we would need a process that allows us to study them outside of the brain.. Luckily for us, this very process exists! It’s called a neurosphere assay and it’s what this simulation is based of.
What is a neurosphere assay?
In biology, studying cells in a live subject is difficult. Instead of trying to study the cells in their natural environment, we often take samples of them to study in a lab. This process of studying cells outside their normal environment is called an in-vitro study.
Instead of poking around in someone’s brain to study neural stem cells, we make neurosphere assays. These are an in-vitro technique of isolating small clusters of undifferentiated neural stem cells to study their biology.
What can we study with the neurosphere assay?
Neurospheres can be used to study the intrinsic characteristics of all types of neural cells, including neural stem cells.
Here are a few examples of things scientists can study using neurosphere assays:
- How neural stem cell multipotency (ability to differentiate into different cell types) is affected by age
- Modeling how neurodegenerative diseases develop
- Screening neuroactive compounds — chemical agent made by a neuron that affects other neurons
- Testing the effects of pharmaceuticals on neural stem cells and differentiated neural cells
- Studying what factors affect how neural stem cells differentiate into the three main neural cell lineages: neurons, astrocytes, and oligodendrocytes
These are just a few examples, but the bottom line is this: neurosphere assays are a critical tool that allows researchers to learn more about the cellular and molecular mechanism that dictate how our neural cells develop and work.
A simplified example of the protocol
To give context for the simulation, it’s important to understand the method through which neurospheres are created in the first place!
Without further ado, here’s a step-by-step explanation of the protocol for a neurosphere assay:
1. Prepping the materials 🧴
The procedure begins by warming solutions to the right temperature and preparing surgical tools
This includes the cell culture media, trypsin, and trypsin-inhibitor.
Surgical tools include instruments such as a scalpel and forceps.
2. Tissue dissection 🔪
Scientists locate the lateral ventricle of a mouse’s brain and trim away tissue using a scalpel.
The tissue is then broken up using the scalpel into smaller chunks.
3. Preparing the neurosphere 🧠
The process to prepare the cultures has many steps. For clarity, I broke it down into three main sub-steps
a) Spin the sample in a centrifuge 🔄
A centrifuge is a machine that spins around samples. This process separates the solid and non-solid parts of a sample called the pellet and supernatant.
The supernatant is discarded through a process called aspiration while the pellet is cultured into the neurospheres.
Trypsin-inhibitor and more neurosphere media is added to the pellet. This is necessary as active trypsin would prevent the neurospheres from forming.
b) Add the necessary solutions 🍹
The three main solutions used in this process are cell culture media, trypsin, and trypsin inhibitor.
- cell culture media — gel or liquid that provides the necessary amino acids, nutrients, and growth factors that regulate the cell cycle
- trypsin — enzyme that breaks down the proteins that allow cells to stick together
- trypsin-inhibitor — protein that neutralizes trypsin
Cell culture media is added throughout the process.
The trypsin is added before centrifuging to ensure cells aren’t clumped and trypsin-inhibitor is added after centrifuging since the cells need to stick together to form neurospheres.
c) Triturate the cell suspension 💉
Trituration is the process of repeatedly passing the cell suspension through a pipette to break up chunks of solid matter in the sample.
4. Count cells 🔢
A device called a hemocytometer is used to count the number of cells in the suspension
5. Add cells to a plate 🍽️
250 microlitres of the cell culture is put into each well of a plate. The neurospheres are incubated for several days after at 37° (body temperature) to allow them to grow.
Several steps of this procedure such as the adding of the solutions and spinning the samples in the centrifuge are done several times throughout the protocol.
I haven’t described the repeated steps for time’s sake.
6. Examining the neurospheres 🔎
After the incubation period, clusters of the neural cells (neurospheres) will appear.
Only some of the cells in each cluster will be true neural stem cells, while the rest are in different stages of differentiation.
Now that we’ve gone over the basics of what a neurosphere assay is and how it’s made, it’s time to dive into the simulator.
The Neurosphere Simulator
The Neurosphere Simulator is a freely accessible, interactive simulation made by the Laboratory of Neurobiology at Northeastern University that models tissue growth caused by neural stem cells.
What is the objective of the simulator?
Its purpose it to allow users to see how changes in the parameters can impact neurospheres, giving you a better understanding of neurobiology and neural stem cells.
How does the Neurosphere Simulator work?
The Neurosphere Simulator runs on a discrete modeling framework, meaning that it operates in microscopic dimensions.
Specifically, it runs on cellular automata (CA) modeling. This type of simulation models the way a biological organism can replicate itself and was first proposed by John von Neumann in the 1940s.
Agents are the subject of CA modelling. In this case, they would be the neural cells in the neurosphere.
They are placed on a certain number of squares on a two-dimensional lattice.
The agents interact with each other in ways that depend on the pre-chosen rules of the simulation that the user can manipulate. An example of a rule could be how each cell division is executed at set time intervals (iteration t).
Here are some basic cellular processes that the Neurosphere simulator takes into account:
Asymmetric Cell Divison
This is a process that’s quite common among stem cells. After undergoing mitosis, only one cell remains a stem cell while the other is more specialized. The more specialized version of a stem cell is called a progenitor cell.
Progenitors are the middle ground between a stem cell and a specialized cell. They’re not fully specialized but they’re not considered stem cells because they lost the ability to divide indefinitely. They usually go through a certain number of mitosis rounds until the result is a specialized cell.
The simulator categorizes cells based on their proliferative potential (how many different cell types a cell can divide into).
- Stem cells — indefinite proliferative potential
- Progenitor cells — limited proliferative potential
- Specialized cells — no proliferative potential
When some of the neighbouring squares are occupied, mitosis is less likely.
When all of the neighbouring squares are occupied, mitosis cannot occur.
Daughter position selection
The mother cell will always remain in the same square after mitosis: this is called a deterministic process
The daughter cell has a 25% chance to be placed in each of the four squares surrounding the mother cell: this is called a stochastic process
After mitosis, one of the two daughter cells often undergoes programmed cell death known as apoptosis.
This is due to the asymmetric division of proteins that control whether a cell will undergo apoptosis.
It is also affected by local cell density, meaning cells in the simulation are more likely to undergo apoptosis if there are too many cells around them. This is to lower the rates of contact inhibition.
Here is an example of what would happen if a symmetrically dividing progenitor cell underwent mitosis and three neighbouring squares were occupied:
The dead bodies of cells that underwent apoptosis cause debris. The removal of this debris by microglia and macrophages is called phagocytosis. This clears space for new cells, reducing the rates of contact inhibition.
There is a 25% chance of the daughter cell to be placed in each of the four squares surrounding the mother cell.
The first thing I did was find a baseline for the simulation by running its default settings. I used this as the control group that I compared the trials where I manipulated the independent variable to.
There are many hypotheses you can test using the Neurosphere Simulator since there are so many combinations of variables you can adjust to see how the simulation plays out.
For time’s sake, here are two of the experiments I ran using the simulation:
How does proliferative potential impact stem cell differentiation?
The first thing I was interested in observing was how proliferative potential (number of divisions after which a progenitor cell differentiates) impacted my results. To do this, I reduced the value of proliferative potential from 7 to 1.
Here’s a comparison of the results from the two trials:
From the simulation, we can tell that decreasing proliferative potential decreases everything from the number of differentiated and progenitor cells to the number of dead cells.
How does phagocytosis impact stem cell differentiation?
The second thing I wanted to investigate was how increasing clearance time, how long after the dead cell dies is it cleared from its spot on the lattice, impacted the results.
Here’s a comparison of the results from trial 1 and trial 3.
As can be seen from the graph, the number of differentiated cells did decrease but the more noticeable difference was in the ratio of progenitor cells to dead cells. In trial 1, there were consistently more progenitor cells than dead ones while the opposite was true for trial 3.
This indicates that the more efficient phagocytosis (clearing of dead cells), the more progenitor cells that remain to differentiate.
As someone actively researching the stem cell field, the Neurosphere Simulator was a great resource for many reasons.
- Data-driven — the simulation is based on data derived from scientific research done at the Laboratory of Neurobiology at Northeastern University
- Virtual — due to the fact that it’s online, even people who don’t have access to laboratories where neurospheres are made can use it
- Free — no financial barrier, unlike other resources
- Interactive — there are several variables that the user can adjust, allowing them to see how the adjustments are connecting to stem cell differentiation
- Intuitive design — the site itself is well-designed and easy to navigate
- Built-in tutorial — the tutorial offered on the website concisely explains different concepts that the simulation models such as phagocytosis and contact inhibition, allowing you to have a clear idea of how to use the simulator
- Visual component — unlike certain resources that solely represent data in dense sheets, the Neurosphere Simulator provides a graph and lattice-simulation side by side so that the user can easily see how a neurosphere would grow given the conditions they set
Stem cell research is a growing field investigating how we can harness the power of undifferentiated cells to further the medical and technological field.
To study neural stem cells, scientists grow them outside the body using a process called the neurosphere assay which produces clusters of cells with different types of neural cells as well as stem cells in them.
The Neurosphere Simulator is an online tool that simulates the growth of neurospheres and allows you to see the changes in the neurosphere growth when you adjust certain variables, giving you a better understanding of how stem cells work.