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Generation of human induced pluripotent stem cell reporter lines to monitor patterning during early brain development

Frei, Jana Simone (2023)
Generation of human induced pluripotent stem cell reporter lines to monitor patterning during early brain development.
Technische Universität Darmstadt
doi: 10.26083/tuprints-00019713
Dissertation, Erstveröffentlichung, Verlagsversion

Kurzbeschreibung (Abstract)

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.

Typ des Eintrags: Dissertation
Erschienen: 2023
Autor(en): Frei, Jana Simone
Art des Eintrags: Erstveröffentlichung
Titel: Generation of human induced pluripotent stem cell reporter lines to monitor patterning during early brain development
Sprache: Englisch
Referenten: Nuber, Prof. Dr. Ulrike A. ; Cardoso, Prof. Dr. M. Cristina
Publikationsjahr: 2023
Ort: Darmstadt
Kollation: XIII, 120, XXIX Seiten
Datum der mündlichen Prüfung: 22 November 2022
DOI: 10.26083/tuprints-00019713
URL / URN: https://tuprints.ulb.tu-darmstadt.de/19713
Kurzbeschreibung (Abstract):

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.

Alternatives oder übersetztes Abstract:
Alternatives AbstractSprache

Die menschliche Embryonalentwicklung ist aus technischen und vor allem aus ethischen Gründen sehr schwer zu untersuchen. Die Etablierung und einfache Erzeugung menschlicher induzierter pluripotenter Stammzellen (hiPSCs) durch Reprogrammierung leicht zugänglicher somatischer Zellen, z. B. Fibroblasten, hat ganz neue Möglichkeiten zur Untersuchung der menschlichen Entwicklung in 2D- und 3D-Zellkultursystemen eröffnet. Reporter-hiPSC-Linien, die die Expression von Markergenen über die Produktion fluoreszierender Reporterproteine anzeigen, ermöglichen die Visualisierung von Entwicklungsprozessen innerhalb hiPSC-abgeleiteter Zellkultursysteme. Beispielsweise können das Auftreten bestimmter Zelltypen und Änderungen der zellulären Organisation von Gewebebereichen mit solchen Linien beobachtet werden, ohne dass die Zellen fixiert und Genprodukte beispielsweise mittels Antikörperfärbungen oder RNA-in-situ-Hybridisierung nachgewiesen werden müssen.

In dieser Doktorarbeit wurden fünf verschiedene Reporterstammzelllinien – drei mit einem und zwei mit zwei integrierten Reporterkonstrukten – hergestellt (OTX2-EGFP, OTX2-mKate2, OTX2-EGFP/GBX2-mKate2, OTX2-mKate2/GBX2-EGFP und DLX2-mKate2), um frühe Prozesse der humanen Gehirnentwicklung in dreidimensionalen Zellaggregaten zu untersuchen und zu visualisieren. Die Expressionsprodukte der Gene OTX2, GBX2 und DLX2 stellen Marker für regionsspezifische neurale Vorläuferzelltypen dar. Expressionskassetten für fluoreszierende Reporterproteine (EGFP oder mKate2), welche die Expression von OTX2, GBX2 und DLX2 anzeigen, wurden in die entsprechenden endogenen Loci mithilfe des CRISPR/Cas9-Systems eingebracht.

Die Integration und Anzahl der editierten Allele wurden mittels PCR, Southern-Blot-Analyse und DNA-Sequenzierungen untersucht. Immunfärbungen wurden durchgeführt, um zu bestätigen, dass die Muster der zytosolischen Reporterproteine mit denen der endogenen nukleären Proteine übereinstimmen. Die Einzel- und Doppelreporterstammzelllinien wurden verwendet, um räumliche und zeitliche Reporterexpressionsmuster in neural-induzierten oder neural-induzierten und regionalisierten Embryoid Bodies (EBs) zu untersuchen. Die Regionalisierung der EBs wurde durch Behandlung der Aggregate mit Molekülen erreicht, welche bestimmte Morphogensignalwege aktivieren oder inhibieren. Auf diese Weise wird die Entwicklung der Zellen in EBs gesteuert und es entstehen Zellverbände, die vorwiegend aus neuralen Vorläuferzellen einer bestimmten Gehirnregion bestehen. Die beobachteten Expressionsmuster der Reporterkassetten in neural-induzierten oder neural-induzierten und regionalisierten EBs stimmen mit veröffentlichten Expressionsdaten der entsprechenden endogenen Gene in Säugetierembryonen und in differenzierten Säugetier-PSCs überein.

Hohe OTX2-Expressionsniveaus wurden in EBs detektiert, welche mithilfe einer zweifachen SMAD-Hemmung neural-induziert und dadurch hauptsächlich zu Gewebe des dorsalen Vorderhirns entwickelt wurden. Um eine starke Aktivierung der GBX2-Reporterexpression in gesamten EBs und gleichzeitig eine starke Reduktion der OTX2-Reporterexpression zu erreichen, war es notwendig die Zellaggregate neural zu induzieren und zusätzlich für circa eine Woche mit hohen Konzentrationen (3 µM) des WNT-Signalweg-Aktivators CHIR99021/CT99021 (CT) zu behandeln. Auf diese Weise wurde die Entwicklung der neural-induzierten Zellen zu Vorläuferzellen mit Hinterhirn-Identität in den EBs gesteuert. Je früher die CT-Behandlung begonnen wurde, desto schneller wurde eine solche Kaudalisierung der Zellen in neural-induzierten EBs erreicht. Wurden die neural-induzierten EBs mit CT-Konzentrationen ≤ 1 µM behandelt, entstanden eher Vorläuferzellen des Vorderhirns bzw. Mittelhirns. Eine sehr starke DLX2-Expression konnte in EBs detektiert werden, die mit Smoothened Agonist (SAG), einem Aktivator des SHH-Signalwegs, behandelt wurden. Um eine hohe DLX2-Expression zu erreichen und eine Ventralisierung des gesamten Zellverbands zu erzielen, war es ausreichend, neural-induzierte EBs mit 0.5 µM SAG für einen Tag oder mit 0.25 µM SAG für die Dauer von sieben Tagen zu behandeln.

Obgleich noch letzte experimentelle Schritte notwendig sind, um drei der fünf erzeugten Linien vollständig zu charakterisieren, wurden in dieser Arbeit sehr wertvolle zelluläre Werkzeuge hergestellt und validiert, die die kontrollierte Regionalisierung und die Untersuchung der Entwicklung von menschlichem Hirngewebe in Zellkultur erheblich erleichtern.

Mit den erstellten Reporterstammzelllinien können die Musterbildung embryonaler Gehirnstrukturen und die räumliche und zeitliche Expression der Gene OTX2, GBX2 und DLX2 in hiPSC-abgeleiteten Zellkultursystemen untersucht werden.

Deutsch
Status: Verlagsversion
URN: urn:nbn:de:tuda-tuprints-197139
Sachgruppe der Dewey Dezimalklassifikatin (DDC): 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie
Fachbereich(e)/-gebiet(e): 10 Fachbereich Biologie
10 Fachbereich Biologie > Stammzell- und Entwicklungsbiologie
Hinterlegungsdatum: 18 Apr 2023 11:09
Letzte Änderung: 19 Apr 2023 04:59
PPN:
Referenten: Nuber, Prof. Dr. Ulrike A. ; Cardoso, Prof. Dr. M. Cristina
Datum der mündlichen Prüfung / Verteidigung / mdl. Prüfung: 22 November 2022
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