The Brain Must Grow On

Cecelia Ky-Lan Do
Jun 15
13 min read

by Andrew Nguyen

art by Hailey Kopp

Remember that time that you farted in class during quiet time in elementary school and EVERYONE looked at you and stared? What about the time your mom abandoned you in the checkout line at the grocery store, and you were scared out of your mind that you’d have to pay by yourself? Or how about the tears streaming down your face as you tried to learn long division? Well, maybe you don’t remember that, but at least you remember learning about it, right? Or maybe you remember the blinding spotlight and the echoing applause as you bowed in your middle school play? What do all of these memories have in common? Your neurons processed all of these experiences—embarrassment, fear, learning, happiness, and remembering—forming memories we hope to remember (or forget) through neurons' intricate firing and connection [1]. What if I told you all of these experiences and all your other senses are possible because of one type of cell—the stem cell? These remarkable cells give rise to every neuron and are responsible for everything you think, feel, and remember.

Stem Cell Introduction

Stem cells are nature’s best actors; they are award-winning cells that can differentiate, specializing themselves for any role the body demands. And to introduce the Meryl Streep of stem cells…drum roll please…*pause for dramatic effect*....we have THE EMBRYONIC STEM CELL! Because they can specialize into any cell in the body, embryonic stem cells can become a heart cell, intensely beating at the peak of a thriller, or a tear duct, perfectly positioning tears during a tragedy [2]. They can do any part with enough work, embodying each character and fully committing to the role. However, unlike your typical actor, they don’t just pretend—they become.

Stem Cells to Scaffolding

Before the performance comes the stringent process of casting. Not just anyone can land a role—stem cells develop at different times and stages depending on the type of cell they will become [2]. Who directs the process and keeps everything on schedule? Regulatory genes and hormones take charge, guiding growth and determining what type of neuron a stem cell will become. Every hormone is secreted into the brain at a specific time to ensure proper stepwise development. And every play needs a set, a precise scaffolding that lays the foundation for complex brain circuits [3]. This scaffold provides not just a physical framework, but also biochemical cues that help neurons develop correctly and in the right places.

Neurogenesis

Choosing the neurons requires a relatively early phase to keep a strict schedule; this happens during fetal and early postnatal brain development [4]. Around 40 days after conception, the stage is set. The ventricular zone lining the fluid-filled vesicles is like the neuronal audition space. During peak audition season, approximately 250,000 cells are welcomed to the production every minute! Still untrained and wondering what they will become, these pluripotent cells are ready to migrate to rehearsals to become full-fledged actors [5]. Much like the capacity of actors to perform any role, pluripotency is the ability to become any cell in the body. The suspense and energy are electric. Now comes the next step: figuring out where each actor should be.

DSCAM

In comes the Down Syndrome cell adhesion molecule (DSCAM), our Academy Award winner for “BEST DIRECTOR.” DSCAM ensures neurons don’t miss their cues. Sure, you could have the best cast, but without someone to organize it all, there would never be a film. DSCAM helps neurons detach from their starting positions and guide them to their correct layer to prevent them from forming inappropriate connections outside their fated regions [6]. It even prevents neurons from overlapping with another neuron’s branch-like arms (dendrites) and their projections that send electrical impulses to other neurons (axons) [7]. This process is known as self-avoidance, keeping the choreography of brain development clear [8]. These structures are how our neural actors communicate with each other.

And now, a very honorable mention for directing, coming in from humble beginnings —the thyroid gland—is Triiodothyronine (T3)! This hormone plays a crucial behind-the-scenes role in supporting neurogenesis, differentiation, migration, synaptogenesis, and myelination [9]. Neurogenesis can be thought of as a casting call to find new talent for production. T3 helps orchestrate the rigorous recruitment process to find new neurons ready to step into the spotlight. Even with the cast selected, there must be chemistry among all the members to synchronize the production. Synaptogenesis is much like a team bonding activity; neurons across this period connect to form an efficient circuit. Myelination, the process of adding fats and proteins to the axon to accelerate electrical signaling, is like a breathable, flexible, sweat-wicking costume that allows actors to move fluidly across the stage and communicate effectively. That said, each scaffold is not always in the right location or completely developed immediately. As one step culminates, another hormone or transcription factor is released into the area to provide the next instructions [10].

Migration

Stage logistics are crucial for ensuring a smooth production. Glial cells, non-neuronal support cells, are like production assistants that provide support and structure. They help neurons determine where to be transported and become fully differentiated. Some neurons migrate to their final destination using these glial cells, while others have a chemical signal that guides them to where they need to go [11]. This way, they can integrate into neuronal circuits and create the connections necessary for proper brain function. Alas, the plot thickens. Only about a third of the neurons make it to their final destination alive—the remaining either die or end up in the wrong place. The final destinations of these neurons is crucial, where erroneous migration could lead to disorders like childhood epilepsy and dyslexia [12].

Growing Neurons

Now that these star actors have their assignments and are in the right location, they can start memorizing their lines and commit to their roles. Neuronal differentiation is a highly orchestrated and complex process to create specific neurons [13]. A neuron will assume its assigned role through DNA methylation. This process is where a methyl group (CH3) is added to a cytosine nucleotide—one of the building blocks of DNA—followed by a guanine nucleotide. Through this permanent modification, the neuron signs a lifelong contract. DNA methyltransferase then blocks the DNA from transcribing those genes (Schübeler, 2015). Much like Timothée Chalamet's five-year training to become Bob Dylan, once a neuron commits, there’s no turning back—it embodies its identity fully. When methylation occurs, the DNA structure tightens the structure and makes the sequences less accessible, so the genes cannot be used to create proteins [14].

The kinds of proteins a cell creates determine the fate of a stem cell. Whether added by a scientist in a petri dish or released by the body, growth factors play a crucial role in signaling pathways. Essentially, growth factors bind to specific receptors on the cell’s surface and start signaling cascades that activate the next steps of neuronal differentiation, whether releasing other growth factors or mediating subsequent steps [15]. Together, these mechanisms ensure that each neuron fully commits to its role, following the script laid out by genetic and molecular cues. Once differentiation begins and the actors are casted, there’s no improvisation—only a carefully directed performance that shapes the intricate neural circuits of the brain.

Stem Cells

Actors often need to forget their previous role, so they can play someone new. Similarly, fully differentiated cells can be reprogrammed to a stem cell state. Research labs have developed protocols and methods to create these induced pluripotent stem cells (iPSCs) [16]. Growing neurons in vitro (outside of an organism) has many practical functions: it is more ethical, efficient, and provides a biologically similar sample. Some experiments may involve changing genetic sequences using CRISPR-Cas9, a gene editing tool, to add or knock out gene sequences that encode proteins This cannot ethically be done to living humans, as the range of effects is unknown and may be harmful. Further, iPSCs also excel in the temporal aspect; in vitro neurons require no more than eight weeks to grow, while waiting for a human brain donation takes significantly longer [17]. Reprogrammed cells provide an advantage when it comes to studying diseases. A skin biopsy can be taken from an individual with a certain disease, and the cells can be reprogrammed into a disease-specific induced pluripotent stem cell (iPSC) [18]. These stem cells can be differentiated into neurons that provide a functional cellular model of an individual with that disease.

Yamanaka Factors

Reprogramming is a meticulous process that requires much care and attention. Differentiated cells, once thought to be set in their roles, can have another chance at fame. Cells can be treated with a quartet of transcription factors, which are proteins that bind to specific regions of DNA near a gene to activate or repress gene expression. Essentially, they orchestrate what genes are turned on or off in a cell. In creating human iPSCs, Yamanaka factors—c-Myc, Kruppel-like factor 4 (Klf4), octamer-binding transcription factor 3/4 (Oct3/4), and sex-determining region Y-box 2 (Sox2)—are introduced to induce cellular reprogramming [16].

C-myc is the opening act of this multipart reprogramming show. It loosens up the crowd, relaxing the DNA so that following actors, Oct3/4 and Sox2, can take the stage and bind to the DNA. It successfully sets the mood for the night, allowing exogenous (coming from outside the cell) Oct3/4 and Sox2 to take their places and bind to their respective spots in the genome. Once the scene is set, Klf4—a versatile and skilled actor—steps in to play two roles [19]. It shuts down the cell’s old identity, erasing the cell’s former skin cell persona. Embracing its hybrid role, it also activates pluripotency genes like NANOG, an important transcription factor in reprogramming cells. NANOG works with other proteins to control gene expression to keep cells in an undifferentiated state [20].

To fully stabilize the cell’s pluripotency network, the other Yamanaka factors need to be thoroughly expressed. Oct3/4 ensures that the reprogramming protocol is efficient and activates genes to maintain the pluripotent state. Sox2 also expresses genes that are associated with pluripotency, specifically endogenous (made by the cell’s own DNA) Sox2 and NANOG [21]. By the final curtain call, the cell has forgotten its old identity and embraced its new pluripotent fate. The actors are now ready to take on any role the script of differentiation demands.

Methods of Differentiation

Now that the cells have forgotten their other roles, they can be assigned new ones. In the lab, a popular rehearsal space is in 2D culture. This is a petri dish where stem cells learn their lines and develop into their roles to become neurons. The cells can be plated on various substances to mimic the brain’s intricate environment. Although these actors can perform and practice well under these conditions, this rehearsal space isn’t a perfect replica of opening night with a bustling crowd—these 2D cultures do not perfectly simulate the environment as neurons grow in vivo [22]. The set is a bit too simple, there’s no spotlight, and the interactions feel a little… staged.

To create a more immersive environment, scientists have looked towards organoids, self-deterministic 3D tissues originating from stem cells that provide a cellular environment similar to that of a brain [23]. Organoids allow neurons to interact in ways that more closely mirror their natural performance, providing a much more complex, in vivo-like environment. Compared to growing neurons in petri dishes, organoids are much better at mimicking cellular interactions [24].

Alas, organoids are still a rehearsal space that cannot fully replicate a packed theater on opening night. To see how these neurons actually perform, these 2D and 3D neuron cultures can be placed in a live production. The differentiated neurons can be transplanted into mice brains to integrate into an in vivo network. This method provides a highly accurate way for scientists to study how neurons interact with the surrounding environment [16]. Instead of simulating a brain-like environment, the neurons would be growing in an actual developing brain. Stem cells can be specialized either by deriving them from a patient or altering them using CRISPR-Cas9 [25]. Once modified and grown, these neurons can be injected into a baby mouse’s brain to grow in a real, functional neural network. Though this in vivo transplantation puts the neurons in front of a live audience, there are always complications.

When human neurons are performing in living mice brains, the audience might boo them off stage because of their unfamiliarity. The mouse’s immune system recognizes the neurons as foreign and induces cell death via p53-mediated apoptosis. P53 is a protein that detects abnormal cells and marks them for apoptosis (programmed cell death). But the show must go on! Scientists have discovered a way of keeping our little actors on stage. Adding drugs such as Adalimumab, an anti-inflammatory agent, can help protect transplanted neurons from the mouse’s immune system. By blocking tumor necrosis factor-alpha (TNF-α), an important part of immune responses, Adalimumab acts as a security guard, quelling any violent reactions and preventing the audience from throwing our little actors out. This allows the neurons to integrate more effectively into their new stage and gives them a chance to perform the roles they worked so hard on learning [26].

Frontiers of Stem Cells and Neurons

Innovations and development in neuron differentiation protocol and technique have been nothing short of groundbreaking. Throughout our journey of the differentiation process, cells have been reprogrammed to an induced pluripotent state, learned their parts, and were put into a live performance [27]. But like any great show, there’s always another performance that will continue to undergo countless revisions. Gene expression is the director's notes—empirical evidence that helps review how well neurons in vitro resemble a human’s neurons in vivo. This is done using single-cell sequencing, a process that identifies all of the gene expression within a cell/neuron [13]. This data is then cross-analyzed with real human neurons to see how well the training process was for our little actors. Currently, only some neurons can be accurately grown in vitro, meaning that while the cast is promising, there is much polishing and rehearsing to do.

An incredible amount of progress has been made from reprogramming cells to rigorous training to differentiate specific types of neurons; the understanding of neuronal development is still growing. Just like any great thriller, we are left in suspense to see where the plot will lead us next. The stage is set, the lights are shining, and the actors are ready—the pursuit to create an in vivo-like neuron continues.

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The Brain Must Grow On

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