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Optimization of Cerebral Organoid Culture from Embr CALIFORNIA STATE UNIVERSITY, NORTHRIDGE Growing Brains to Study Development: Optimization of Cerebral Organoid Culture from Embryonic Stem Cells A thesis submitted in partial fulfillment of the requirements For the degree of Master of Science in Biology By Jessie Erin Buth December 2015 The thesis of Jessie Erin Buth is approved: __________________________________ ____________________________________ Dr. Aida Metzenberg Date __________________________________ ____________________________________ Dr. Randy Cohen Date __________________________________ ____________________________________ Dr. Cindy Malone, Chair Date California State University, Northridge ii Table of Contents Signature Page ii List of Figures iii Abstract vi Chapter 1: Introduction 1 Chapter 2: Methods 15 Chapter 3: Results 23 Chapter 4: Discussion 50 References 55 Appendix: Supplemental Methods 59 List of Figures Figure 1: Human organoid protocol (Cell line H9) and morphology at various time points. 23 Figure 2: Review of cortex development. 24 Figure 3: V-bottomed 96-well plates improve aggregate formation and generates a thick neuroepithelial layer on day 18 compared to U-bottomed 96-well plates. 25 Figure 4: Plating at 9,000 cells per well on day 0 generates thickest continuous neuroepithelial layer compared to other cell numbers. 26 Figure 5: Addition of the BMP inhibitor (LDN193189) does not improve the efficiency of producing FOXG1+ cortical progenitors and inhibits formation of continuous N-cadherin+ apical membrane. 27 Figure 6: Fold change in gene expression of cortical markers increases as stem cell markers decrease in human cortical organoids over time by qPCR. 28 Figure 7: Human embryonic stem cells efficiently form cortical progenitors with 81.7% of total live cells per organoid positive for cortical marker FOXG1 at day 18. 29 Figure 8: Initial apical-basal polarity of human cortical organoids. 31 Figure 9: Organoids form cortical neurons with signs of laminar organization. 32 Figure 10: Basal-radial glial-like cells form in the subventricular zone of human cortical organoids and increase in number over time. 34 iii Figure 11: Basal radial glia and intermediate progenitor cells dividing in the subventricular zone increase in number over time. 35 Figure 12: Summary of current human organoid culture. 36 Figure 13: Optimized mouse organoid protocol (Cell line MM13). 37 Figure 14: Plating mouse embryonic stem cells at 5,000 cells per well on day 0 generates most consistent aggregates compared to other cell numbers and transfer to high oxygen produces more rosettes. 38 Figure 15: 3µM IWR1e (Wnt inhibitor) induced highest fold change in Foxg1 expression at day 7 relative to undifferentiated mESCs by qPCR. 39 Figure 16: qPCR data shows mouse cortical organoids upregulate cortical genes by day 5 (Line MM13 + 3µM IWR1e). 40 Figure 17: Mouse embryonic stem cells efficiently form cortical progenitors with 92.9% of total live cells per organoid positive for cortical marker Foxg1 at day 5. 41 Figure 18: 1:1 mixture of GMEM/KSR based media and N2 based media followed by N2 media supplemented with CDLC on day 7 “D” produces largest rosette structure positive for cortical markers. 42 Figure 19: 1:1 mixture of GMEM/KSR based media and N2 based media followed by N2 media supplemented with CDLC on day 7 “D” produces largest rosette structure with intermediate progenitors and cortical markers. 44 Figure 20: Summary of current mouse organoid culture. 45 iv Figure 21: Foxp1 expression overlaps with apical and basal radial glial cells and Foxp4 expression overlaps with intermediate progenitors in human cortical organoids over time. 46 Figure 22: Foxp1/Foxp4 expression partially overlaps and Foxp4 expression overlaps with apical radial glia in mouse cortical organoids over time. 47 v Abstract Growing Brains to Study Development: Optimization of Cerebral Organoid Culture from Embryonic Stem Cells By Jessie Erin Buth Master of Science in Biology 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 vi 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. vii CHAPTER 1: INTRODUCTION Humans have one of the largest brains of all animals relative to body mass (Hofman 2014). The outermost region, the cortex, is a six-layered structure of neurons that is involved with higher cognitive functions such as thinking and fine motor control. The cortex is the most evolutionarily new part of the brain and is disproportionately large in humans compared to other primates. How the human cortex expanded across evolutionary time remains difficult to study due to the inaccessibility to tissue. In vitro models of cortex development are a promising way to study human-specific features of cortical development that could not be recapitulated in model organisms. This chapter will review what is known about cortex development, and the following chapters will report on optimized protocols to model cortex development in 3D organ-like structures derived from human and mouse embryonic stem cells and discuss two genes hypothesized to be involved with cortical expansion over evolutionary time. Early in development of the brain, regional cues alter gene expression in a concentration-dependent manner in order to persuade cells to adopt different fates (Shimogori et al. 2004). These cues are called morphogens; the major classes of morphogens that set up early patterning in the brain include wingless-wnt (Wnt), bone- morphogenic proteins (BMP), fibroblast growth factor (FGF), and sonic hedgehog (Shh). These proteins vary in concentration across the rostral-caudal and dorsal-ventral axes (Shimogori et al. 2004). The cortex arises from the telencephalon in an area with a low concentration of these signals and begins as a population of multipotent neural stem cells (Shimogori et al. 2004). 1 The cortex extends from a layer of neural stem cells that line the lateral ventricles. First, neural stem cells expand in cell number by proliferative divisions, and then later differentiate into the six-layered structure of neurons seen in adults (Rakic 1995; Jessell and Sanes 2000). In 1995, Rakic first proposed the radial-unit hypothesis, which has become the basis for our understanding of cortex expansion. The radial unit hypothesis postulates that changes in regulatory genes that control how long the proliferative and differentiative phases are control the surface area and thickness of the cortex, with a larger surface area having more convolutions and folds as in the human cortex (Rakic 1995). The radial unit is defined as a group of neurons that arise from one founder cell, to form a vertical column of neurons (Rakic 1995). The number of founder cells determines the surface area of the cortex, and the number of neurons within a column determines the thickness (Rakic 1995). Rakic provides a simplified example to represent how timing could influence cortex size, by comparing the timing of the proliferative and differentiative phases of human and macaque. It is argued that during the proliferative phase, there is an exponential increase in cells, because each round of cell division doubles the pool of founder cells; thus, even a few days increase in the proliferative phase could allow the human cortex to have 23 or 24 more founder cells than the macaque (Rakic 1995). During the second phase, neurons are formed in a linear fashion because each founder cell can add one neuron
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