The recipe for human serotonin neurons

If you want to study human neurons directly, you may be out of luck. Scientific ethics, rightfully so, doesn't let us just open up any human head and experiment on their brains. Luckily, talented scientists have found ways around studying live neurons in humans. Using cultured human pluripotent stem cells (hPSCs), we can make neurons by using specific signaling proteins that tell the stem cells what to turn into. In this way, we can grow human neurons in a dish that we can experiment on directly. However, there is one small caveat: there are hundreds of different types of neurons, and this may not even include genetic diversity within specific groups of neurons. The first neurons created from hPSCs were a bit indistinct. Some of them were excitatory, some were inhibitory, and some couldn't be classified. Inducing stem cells to become neurons was an amazing first step, but given the diversity of neurons, if we truly want to understand each sub-type, we would want to create and experiment on each group. As an example, there is a small population of neurons that produce serotonin that are major players in human psychological disorders. If we could make serotonergic neurons it would help us study how drugs affect them specifically.

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Serotonergic neurons are located in the Raphe Nuclei of the brain and represent a rather small portion of neurons in humans - about 300,000 of the near 100 billion neurons. Although small in number, they have a diverse repertoire of tasks in the brain. Serotonin producing neurons help regulate mood, sleep, and cognition, to name just a few jobs. These neurons are also the major target of drugs that treat mood disorders like depression and bipolar disorder. Most drugs used to treat mood disorders affect serotonergic neurons directly by preventing either the degradation of serotonin or its removal from the neuronal synapse. Both of these effects lead to higher levels of serotonin at the synapse and an improvement of symptoms. To better study new drugs and their effects on human serotonergic neurons we need to understand what they are doing to the neurons themselves. As I was saying before, this would be best done if we could culture human serotonergic neurons.

Recently, researchers from the University of Wisconsin set out to create serotonergic neurons from hPSCs. In order to do this, they had to study their development textbooks. Every cell in the body can trace its history back to a stem cell. In a developing animal, stem cells in the blastocyst slowly transform into all of the different cell types in the body. Depending on the position within the early embryo, they will receive varying amounts of different signaling proteins, which will determine the cell they will turn into. Stem cells will slowly get more mature, meaning the amount of different types of cells it can turn into slowly decreases. As an example, an embryonic stem cell can turn into any cell in the human body, whereas a blood stem cell can only turn into any type of blood cell. One would consider a blood stem cell more mature than an embryonic stem cell. A fully differentiated cell, that is, a cell that has a distinct identity, like a skin cell or muscle cell, is completely mature and can't turn into any other cell type. To create serotoninergic neurons, fully mature cells, the researchers studied the signaling proteins needed to create these neurons from the first stem cells. Luckily, there was a vast amount of work that went into studying neuronal development that helped these scientists determine how to make serotonergic neurons. Years of data can be used to figure out which proteins are necessary to turn stem cells into serotonergic neurons. Using this information, the researchers developed a three step process (outlined in the first figure above). First, they use hPSCs to create rostral hindbrain neural stem cells (NSCs). This protocol to do this is established, more or less, but they did have to play with the concentration of the proteins to induce the specific type of NSCs they wanted. They did this by using proteins that include TGF-β inhibitors and BMP inhibitors. TGF-β and BMP are well studied proteins that we know inhibit stem cells from becoming neurons. Inhibiting them allows NSCs to develop. Next, they took their NSCs and differentiated them further, into ventral rostral NSCs. They did this using another well characterized protein, Sonic hedgehog (Shh). Shh is well established to promote a ventral fate in NSCs (ventral meaning these NSCs would be located on the ventral portion of a growing embryos neural tube). Following this, their last step, they added FGF4, a signaling protein that is known to work with Shh to promote serotoninergic neuron development; it was only a matter of doing it at the right time. After this whole process, they started to see neurons develop. The only question was whether or not they were serotonergic neurons.

To see whether these were actual serotonergic neurons, they did several tests. They stained for markers in the neurons that would be specific to serotonergic neurons - for example, serotonin (shown in the figure above in red). They also stuck small glass pipets into them to study their electrical properties. This is the gold-standard way of studying neurons. They found that the neurons fire spontaneously at the same rate serotonergic neurons in the body usually fire (shown in the left half of the figure to the left), and have similar action potentials. Finally, the authors showed that these neurons release serotonin, and respond to known drugs that target serotonergic neurons by increasing their production of serotonin (shown in the right half of the figure above. The drugs shown are tramadol and escitalopram oxalate). From this data, it appears as though the researchers were able to successfully differentiate serotonergic neurons from stem cells in about one month. Most importantly, and shown in the right half of the figure above, is how they can directly study these neurons' responses to drugs. This platform gives us the ability to test how and where different drugs affect human serotonin neurons. Not only that, but because we can develop serotonin neurons from induced stem cells, we could test drugs in serotonergic neurons derived from the cells of the person who will be using the drug. This protocol is also more efficient than differentiating serotonergic neurons from human fibroblasts (skin cells), which had been done before. Because this technique follows a more ‘normal' pathway of development, and because this protocol doesn't use viruses to deliver the necessary signaling proteins, it may be healthier for the cells. They also show more definitively that these neurons are serotonergic. Overall, this research represents a major type of neuron that we can put into the ‘toolbox' of neurons we can now grow and test directly. Hopefully, this will allow for better drug screening and development, leading to better treatments for mood disorders. Maybe it's only a matter of time until we can create recipes for every cell in the body.

Resources:

Lu, J., et al. Generation of serotonin neurons from human pluripotent stem cells. Nature Biotechnology, 34, 89-94 (2016).

Gaspar, P., Nedelec, S. Serotonin neurons in a dish. Nature Biotechnology, 34, 41-42 (2016).

Image credits:

All images are augments from the Lu et al. paper referenced above.