Lab-Grown Human Brains?

A fascinating technology that has emerged within the past decade is the ability to grow miniature versions of brain-like organs called “cerebral organoids” in the lab. This article takes you through an original research paper that was key in the development of this process.

First, some background on how the human brain develops…

The brain starts out with some cells that form a tube. Cells at the inner wall of the tube turn into another type of cell. Many of these cells will go on to ultimately become neurons. As they develop into more neuron-like cells, they move outward through the wall of the tube, eventually ending with the most mature neurons in the outermost layer, which will become the cerebral cortex. This tube forms some layers or “zones” that contain different types of cells at different stages of maturity. These zones all have long and fancy sounding names like “subventricular zone” or “inner fiber layer”. But instead of getting caught up in remembering all the names, it’s more advantageous to picture the general process. There are a couple of key players that will come up again though. The cells that form the tube are called neuroectoderm, and the cells at the inside of the tube are called radial glia. In humans, there is a unique small layer of cells called “outer radial glia”. Mice, for example, don’t have this, and the structure of the rodent neural tube overall just isn’t as elaborate as in humans. This leads to the striking difference in brain size and function in humans versus rodents. Still, mice and other rodents are often used to study human disease. There is an increasing need for better alternatives. At the time this paper was written, some neural tissue had previously been grown and successfully maintained in the lab, but it didn’t have all of the organized layers and types of cells described above.

In this paper, the authors developed an improved type of lab-grown brain tissue to use in the study of disease. They did this by using human pluripotent stem cells, which are cells that are capable of becoming almost any type of cell in the body. They started by making clusters of these stem cells. Once clustered together, the process of differentiation begins. Differentiation means stem cells are starting to turn into other types of cells, becoming different from each other. A combination of chemicals, growth factors, and physical stimuli all contribute to determining what type of cell a stem cell will ultimately turn into. In this case, these clusters of stem cells were triggered to develop into neuroectoderm – the type of cells that form the neural tube when brain development begins. This makes neuroectoderm a logical place to start if we’re trying to form a brain-like structure in the lab. These clusters of neuroectoderm cells were then embedded in small drops of a jelly-like substance called Matrigel. Matrigel contains lots of proteins that form a structure for cells to grow into, providing physical stimuli that helps the cells develop into more complex brain-like tissues. This step was a key discovery in this paper that enabled the authors to generate more complex brain-like organoids than previous studies. The process of further cell differentiation into neural cells takes 8-10 days and 20-30 days for more defined brain-like regions to form. 

The authors reported that these cerebral organoids could grow up to 4mm in diameter and survive over 10 months. However, they were missing a circulatory system that could bring nutrients and oxygen to cells at their dense center. Consequently, cells in the center of the tissues would begin to die off, limiting their size. Despite these limitations, the authors noted many regions that were similar to brain structure, at least in shape and form, including cerebral cortex, networks of fluid-filled spaces, retina (light sensitive cells that aid in vision) and meninges (membranes protecting parts of the brain). But notably, all of these regions were not present in every organoid produced. For example, the retinal cell regions were only observed in a few organoids. Still, all organoids had regions similar to dorsal cortex (an area at the back of the brain involved in cognitive functions). This is an important observation because this region differed the most between humans and rodents. The researchers observed that these dorsal cortex neurons had characteristic long, branching structures resembling axons, the long, slender tail-like structures that help transmit information between neurons. The scientists also saw spontaneous increases in calcium, suggesting some level of neural activity. 

The authors next wanted to try out applying this method of making cerebral organoids to study the disease microcephaly. Microcephaly is a condition where brain size is smaller. This is thought to be due to changes in the genes that control cellular growth. At the time, microcephaly couldn’t be studied optimally using available methods, like mouse experiments, due to the differences in human and rodent brain development. In this study, the researchers identified an actual patient with microcephaly. They were even able to determine the gene mutation which had caused the patient to have the disease. Genes are regions in the DNA that are like an instruction template for making proteins. Proteins can be thought of as molecular machines that control the cell. Changes in genes, called mutations, can alter their corresponding proteins. Similarly as if you were to rip a page out of an instruction manual, shortening or truncating a gene can result in a protein that doesn’t work correctly anymore, or maybe isn’t even produced at all. That’s just what happened in this patient with microcephaly, who had loss of this protein with the very long name: CDK5RAP2. 

The authors took a type of cell from the patient’s skin, and used them to make cerebral organoids. Just as stem cells can become lots of different types of cells, some cells found in skin can be made to turn back into stem cells under the right conditions. So, the authors took a sample of the patient’s skin cells, converted them in the lab back to stem cells, and then used those stem cells to make cerebral organoids. Now, the authors had cerebral organoids with the patient's mutation. 

This patient’s cerebral organoids were smaller overall, as would be expected since they had a mutation that causes microcephaly. Also, they had disorganized placement and orientation of some cell types, and imbalanced populations of neurons and radial glia cells. Of course, this paper didn’t provide any cures yet, but understanding what causes neurological diseases is the first step. Here, the researchers found that too many of the radial glia cells were turning into neurons as the patient’s organoids developed. This was part of why microcephaly couldn’t really be studied well in mice, because mice don’t have a large amount of radial glia, and they don’t appear to be critical for proper mouse brain development. Culturing patient-derived cerebral organoids was a major breakthrough towards developing alternative experimental models to study brain diseases and disorders.

However, it should be remembered that although the researchers saw many similarities to human cerebral cortex in their experiments, these organoids were still just not anywhere near as complex in structure as actual human brains. But as technology progresses, could there eventually be some ethical concerns or barriers with these types of studies?

Reference for the paper discussed in this article:
Lancaster, Madeline A., et al. "Cerebral organoids model human brain development and microcephaly." Nature 501.7467 (2013): 373-379.