A cellular dissection and reconstruction of the human cortex [electronic resource]

Deciphering the development and function of a complex organ like the brain requires an assortment of approaches. The work presented here follows two generally complementary strategies, first dissecting and characterizing the basic cellular components of the mouse and human brain, and secondly using a human stem cell-based approach to model cortical development in vitro. This work further highlights the function and development of one particular central nervous system (CNS) cell type--the astrocyte. Contrary to previous dogma, astrocytes are not merely passive bystanders residing within the CNS. Rather, these cells choreograph neural development and actively contribute to the formation and maintenance of neural circuits by controlling synapse formation, function, and elimination. Understanding human astrocyte evolution, function, and maturation is a common theme that permeates the following studies. The work presented here begins with the systematic transcriptional dissection of the mouse cortex to its primary cell type components. These data enabled the construction of a transcriptome database that provides a resource for understanding the functional and molecular partitioning that occurs within the various CNS cell types. Additionally, the use of RNA-sequencing revealed thousands of examples of cell-type specific splicing, which results in far more transcriptomic diversity within the brain than previously appreciated. To supplement this work, we tailored these techniques to acutely purify cell types from the human cortex, using specimens from neurosurgical resections. Our particular focus centered on understanding the molecular and functional evolution of human astrocytes, which differ significantly from those found in rodents. Additionally, we discovered a profound distinction between two types of human astrocytes--astrocyte progenitor cells found throughout fetal development, and mature cells found from adolescence onward. In parallel with our efforts to purify cell types from the rodent and human CNS, we pursued a stem cell-based approach to model normal human cortical development. We used induced pluripotent stem cells (iPSCs) to generate a reproducible 3D culture system that generates a laminated cerebral cortex-like structure, named human cortical spheroids (hCS). hCS contain neurons from both deep and superficial cortical layers and map transcriptionally to in vivo fetal development. The neurons residing in hCS are electrophysiologically mature, display spontaneous activity, are surrounded by nonreactive astrocytes and form functional synapses. We then used hCS as a system for studying human astrocytes in vitro by adapting our techniques for purifying human astrocytes from neurosurgical samples to purifying iPSC-derived astrocytes from hCS. Not only do iPSC-derived astrocytes demonstrate a high transcriptional fidelity to primary human astrocytes, but by maintaining hCS in culture for over 1.5 years, we were able to observe the in vitro maturation of human astrocytes across various time points. Together, the work presented here has revealed new features of CNS cell type diversity and evolution, and has established platforms for the future investigation of nervous system pathologies.

Advisor: Barres Ben
Gitler Aaron D. primary advisor. Thesis advisor
Monje-Deisseroth Michelle
Reimer Richard J. advisor. Thesis advisor
Theriot Julie advisor. Thesis advisor