Differentiation potential, lineage commitment and gene expression profile of human cortical neural progenitor cells derived from pluripotent stem cells

The human cerebral cortex is composed of the variety of neurons and glial cells that are organized into six different layers. During development, this complex structure originates from a simple neuroepithelium. As neurogenesis continues the neural stem and progenitor cells residing in the ventricular zone (VZ) and subventricular zone (SVZ), generate cortical projection neurons in an inside-out order. The neurons within each layer have particular functions, gene expression patterns and morphologies. These neurons are born at defined stages during development. The developmental mechanisms that regulate neural progenitor fate specification during cerebral cortex development remained elusive. Stem cell based systems allow at least partial recapitulation of the important aspects of human cortical neurogenesis in a simple and accessible cell culture manner. These systems have been successfully used to understand specific mechanisms associated with human cortical development and disorders.
Here, I studied the fate potential of human cortical neural progenitors derived from 2-dimensional (2D) in vitro corticogenesis. I also investigated how neural stem cells/progenitors generate the great diversity of neurons during in vitro cortical differentiation. I propose that fate potential of the neural progenitor (NP) pool changes during human cortical development. I employed retrovirus birth dating to investigate timing of neurogenesis. I found that not only is the early progenitor pool multipotent and generates both deep and upper layer neurons but also the late progenitors are capable to give rise to deep and upper layer neurons. It has been suggested that both intrinsic and extrinsic factors mediate fate specification of neural progenitors during development. I first tried to find the transcriptional program, which regulates the competence of NPCs during differentiation. I isolated the NPs based on CD184+, CD24+, CD271-, CD44- expression at sequential stages during in vitro corticogenesis and studied the transcriptional profile of these cells. The analysis confirmed the dynamic transcriptional program of NPs over the course of differentiation. Late sorted NPs give rise to more upper layer neurons and surprisingly to a high proportion of deep layer neurons in comparison to early progenitors. Moreover, NPs (isolated at early and late stages of differentiation) when co-cultured with the cells from different differentiation stage alter the proportions and types of generated neurons in response to environmental signals.
This thesis is organized in five chapters. The first chapter provides an introduction to early human cerebral cortex development as well as a summary of common strategies for in vitro cortical differentiation derived from human pluripotent stem cells (hPSCs). The chapter ends with the aims of the project. The second chapter contains the main results that are presented as a research manuscript. “Human pluripotent stem cell derived neural progenitors display two modes of neural fate determination”. Chapter two also includes my collaboration in a research project in our lab entitled “Neurodegeneration associated TDP-43 induces p53-mediated cell death of stem cells and neurons”
In chapter two I also present my result during collaboration in the project “Multigene delivery in primary and stem cells”. The two papers entitled “Highly efficient baculovirus-mediated multigene” and “Baculovirus-based genome editing in primary cells” are attached as an appendix. In Chapter three and four, I summarize and discussed our results and refer to the limitations of our system. Chapter five contains the detailed methods and protocols that have been used in the thesis.