My project aimed to develop a refined protocol for generating cerebral organoids from 3D aggregates of pluripotent stem cells. Different protocols to generate organoids differ in the use and timing of growth factors and other media supplements. The protocol used by the Pasca group generally results in larger organoids with many progenitor zones, and uses growth factors EGF and FGF starting at day 6, and NT-3 and BDNF at day 25. The protocol used by the Ming group generally results in organoids with rudimentary cortical layers, and does not include any growth factors after day 14. By combining these two protocols, this project attempted to combine the strengths of each protocol to ultimately produce an organoid most suitable for transplantation into cortex in vivo. Because of the organoid’s architecture and size, transplantation into large brain injuries could be a more functional avenue of repair.
By introducing Pasca media at different time points (differentiation day 15 and differentiation day 25), I found significant differences between the organoids generated with the hybrid protocol and the protocols used by the Pasca and Qian groups. Specifically, organoids that began with the Qian protocol at differentiation day 0 and started the Pasca protocol at differentiation day 15, were larger and had more progenitor zones, while still maintaining a forebrain identity. There was also an increased population of Satb2+ cells, an upper-layer cortical marker. Stains for extracellular matrix demonstrates that hybrid organoids have increased amounts of laminin. These results indicate that these organoids may have be suitable for further in vivo transplantation studies.
Having worked in the Chen Lab since my freshman year, this research experience has been incredibly rewarding. As a recipient of the Spring 2018 Pincus- Magaziner Family Undergraduate Research and Travel Fund from the College Alumni Society, I was able to expand on some of the technical skills I had learned previously in the lab such as cell culture, brain tissue sectioning, and immunohistochemistry. This grant specifically gave me the opportunity to learn how to plan and troubleshoot experiments, as well as analyze results.