Thesis

Growing brains to study development: optimization of cerebral organoid culture from embryonic stem cells

The outermost portion of the brain, the cortex, is a six-layered structure of neurons that is responsible for higher cognitive functions such as thinking and speech. This region is the most evolutionarily new part of the brain and is disproportionately large in humans than predicted by body size. What has caused the cortex to expand across evolutionary time? Little is known about this process due to the inaccessibility to human tissue. Much of what is currently known stems from static images of human fetal tissue or extrapolation from model organisms. Mice, and other model organisms, fail to recapitulate hallmark features of human brain development. There is a need for a better system to study brain development and its disorders. One alternative is to use stem cells. Stem cells have the ability to form any cell type in the body, and in the lab can be directed to form any cell type, or tissue, of interest. Recently, Kadoshima et al. (2013) and Lancaster et al. (2013) published protocols to create organ-like structures, called organoids, that model some aspects of brain development in vitro using stem cells. Stem cells are exposed to factors normally present during development and spontaneously form cortical tissue. This system has not been well characterized and it remains to be determined how reproducible these studies are with other cell lines. This study determined cortical organoids derived from H9 human embryonic stem cells and MM13 mouse embryonic stem cells can model some aspects of in vivo cortex development with some modifications of previously published protocols. Cortical organoids derived from human cells efficiently and reproducibly formed cortical tissue that exhibited some established features of cortex development. Human cortical organoids produced a thick layer of FOXG1+ cortical progenitors, initially showed the correct apical-basal (inside-out) polarity, and formed cortical neurons with signs of laminar organization. Mouse cortical organoids also efficiently formed FOXG1+ progenitors and produced cortical neurons, but with less laminar organization than in the human organoids. This study is the first to thoroughly characterize how well cortical organoids model in vivo development and in which areas the methods could be improved.

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