title: Generation of human induced pluripotent stem cell reporter lines to monitor patterning during early brain development
Creators: name: Frei, Jana Simone
Publisher: name: UNSPECIFIED
Year: 2023

description: Human embryonic development is extremely difficult to study – for technical and foremost ethical reasons. The establishment and ease of generating human induced pluripotent stem cells (hiPSCs) through reprogramming of readily accessible somatic cells, e.g., fibroblasts, has opened up entirely new possibilities to study human development in 2D and 3D cell culture systems. Reporter hiPSC lines which indicate the expression of marker genes via the production of fluorescent reporter proteins allow the visualization of developmental processes within hiPSC-derived cell culture systems. With such lines, developmental processes, as for example the appearance of specific cell types and the cellular organization of tissue areas, can be monitored without the necessity to fix cells and to detect gene expression products by immunostainings or RNA in situ hybridization. In this thesis, five different - three single and two double - reporter hiPSC lines (OTX2-EGFP, OTX2-mKate2, OTX2-EGFP/GBX2-mKate2, OTX2-mKate2/GBX2-EGFP, and DLX2-mKate2) were generated to study and visualize early processes of human brain development in 3D cell aggregates, namely the spatial and temporal gene expression patterns of OTX2, GBX2, and DLX2. The products of these genes represent markers for region-specific neural precursor cell types. Fluorescence reporter expression cassettes (EGFP or mKate2) that report the expression of OTX2, GBX2, and DLX2 were introduced into the respective endogenous loci using the CRISPR/Cas9 system. The integration and number of targeted alleles were analyzed by PCR, Southern blot analysis, and DNA sequencing. Immunostainings were performed to confirm overlapping patterns of cytosolic fluorescent reporter proteins and the endogenous nuclear proteins of interest. The single and double reporter hiPSC lines were applied to investigate the spatial and temporal reporter expression patterns in neurally induced or neurally induced and regionalized embryoid bodies (EBs). Regionalization of the EBs was achieved by treating the aggregates with molecules which activate or inhibit specific morphogen pathways. Thereby, the development of cells within the EBs was controlled and tissue developed comprising predominantly neural precursor cells of a brain region of interest. The detected expression patterns of the reporter cassettes in neurally induced or neurally induced and regionalized EBs are consistent with published expression data on the respective endogenous genes in mammalian embryos and in differentiated mammalian pluripotent stem cells (PSCs). High OTX2 expression levels were reported in distinct patterns in EBs which were neurally induced via dual SMAD inhibition and the cells within the EBs were thereby directed towards a dorsal forebrain fate. To strongly activate GBX2 reporter expression within entire EBs and to simultaneously strongly reduce OTX2 reporter expression, it was necessary to neurally induce and additionally treat the EBs with high concentrations (3 µM) of the WNT pathway activator CHIR99021/CT99021 (CT) for approximately one week. In this way, neural precursor cells within the EBs were directed towards a hindbrain fate. The earlier the CT treatment was started, the faster a caudalization of cells in neurally induced EBs was achieved. A treatment of neurally induced EBs with CT concentrations ≤ 1 µM directed cells within neurally induced EBs towards precursor cells of the forebrain and midbrain. An intended and extremely strong induction of DLX2 expression was reported in EBs which were treated with Smoothened Agonist (SAG), a SHH signaling pathway activator. To cause strong induction of DLX2 reporter expression and, thus, ventralize the entire tissue it was sufficient to treat neurally induced EBs with 0.5 µM SAG for one day or 0.25 µM SAG for seven days. Although final experimental steps are necessary to fully characterize some of the generated lines, this thesis has contributed very valuable cellular tools that should greatly facilitate the controlled regionalization and the study of human brain tissue development in cell culture. The generated reporter stem cell lines can thus be used to investigate the pattern formation of embryonic brain structures and the spatial and temporal gene expression of OTX2, GBX2, and DLX2 in hiPSC-derived cell culture systems.