Gene networks and transcription factor motifs defining the differentiation of human embryonic stem cells into hepatocyte like cells
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Date
2015
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Abstract
Das Potenzial von humanen embryonalen Stammzellen (hESCs), in Hepatozyten-ähnliche
Zellen (hepatocyte like cells, HLCs) zu differenzieren, ermöglicht Hepatozyten in
unbegrenzter Anzahl für pharmakologische und toxikologische Untersuchungen sowie für
Zelltherapien zur Behandlung von Leberversagen herzustellen. Obwohl zahlreiche
wissenschaftliche Untersuchungen über die erfolgreiche Herstellung von HLCs aus hESCs
berichten, existieren immer noch kontroverse Angaben über die Ausprägung des
Differenzierungsgrades zu homogenen Populationen von reifen Hepatozyten.
Das primäre Ziel dieser Promotionsarbeit war es, ein umfassenderes Verständnis der
Eigenschaften von stammzellabgeleiteten HLCs zu erreichen. Hierfür wurden durch die
Verwendung eines bewährten Protokolls hESCs zu HLCs differenziert. Die Charakterisierung
und Identität der stammzellabgeleiteten HLCs erfolgte durch die Ermittlung der
Genexpressionsignatur mittels GeneChip® Human Genome U133 Plus 2.0 Arrays. Die HLCs
Genexpressionsignatur wurde anschließend mit der Genexpressionsignatur von hESCs und
von frisch isolierten adulten Hepatozyten sowie von bis zu 14 Tage lang kultivierten adulten
Hepatozyten verglichen. Durch die Anwendung eines breiten Spektrums von
bioinformatischen Analyseprogrammen konnten die Gennetzwerke für erfolgreiche und
erfolglose Hepatozytendifferenzierung sowie die involvierten regulativen
Transkriptionsfaktoren identifiziert werden.
Die Analyse der regulatorischen Gennetzwerke zeigte, dass HLCs einen hybriden Zelltyp
darstellen, welcher Gensignaturen von Leber-, Darm-, Bindegewebs- und Stammzellen zeigt.
Der unerwünschte “Colon”-Phänotyp stand hierbei im Zusammenhang mit
XIV
Transkriptionsfaktoren wie KLF5 und NKX2-3 sowie dem CDX2-Transkriptionsnetzwerk.
Durch Clusteranalysen konnten stark korrelierende Gengruppen identifiziert werden, welche
sowohl mit Funktionen der reifen Leber und einer Herunterregulierung der Zellproliferation
zusammenhängen, als auch dem Expressionslevel der adulten Hepatozyten nahe kamen.
Allerdings erreichten drei weiteren Gencluster nicht das Expressionslevel adulter
Hepatozyten. Zwei dieser Cluster beinhalteten die Schlüsseltranskriptionsfaktoren SOX11,
FOXQ1 und YBX3. Die dritte nicht erfolgreich exprimierte Clustergruppe, welche u.a. durch
die Transkriptionsfaktoren HNF1A, CAR, FXR und PXR kontrolliert wird, zeigte signifikante
Überschneidungen mit Genen, die in kultivierten Hepatozyten eher unterdrückt waren als in
frisch isolierten Hepatozyten. Dies deutet darauf hin, dass in den gegenwärtig verwendeten invitro
Kulturbedingungen essentielle Stimuli für den Erhalt der Genexpression von
Hepatozyten fehlen. Demzufolge könnten hierdurch auch die vergleichbaren Defizite von
HLCs erklärt werden.
Zusammenfassend kann festgestellt werden, dass durch den in dieser Arbeit verwendeten
Ansatz zur Untersuchung von Genregulationsnetzwerken wichtige Transkriptionsfaktoren
identifiziert werden konnten, welche sich als Interventionsziele zur Verbesserung der
Differenzierung von hESCs zur reiferen HLCs anbieten.
In the past decade, it has been recognized that human embryonic stem cells (hESCs) differentiation into hepatocyte like cells (HLCs) could offer an unlimited supply of hepatocytes for pharmacology, toxicology, and cell therapy for liver failure. Many research efforts claimed to have successfully engineered HLCs from hESCs. However, the degree of differentiation and identity of HLCs remains controversial. The primary goal of this thesis work was to obtain a comprehensive understanding of HLCs identity. Thus, HLCs were differentiated from hESCs using a well-established protocol. Genome-wide gene expression programs of hESCs and HLCs were analyzed using GeneChip® Human Genome U133 Plus 2.0 arrays. The resulting gene expression profiles of HLCs and hESCs were compared to freshly isolated adult hepatocytes and adult hepatocytes cultivated for up to 14 days. Application of a broad range of bioinformatic tools and data mining approaches led to elucidation of gene networks and transcription factors (TFs) involved in the regulation of gene expression suggesting successful and failed hepatocyte differentiation. Gene regulatory network analysis revealed that HLCs represent a hybrid cell type with features of the liver, intestine, fibroblast, and stem cells. The undesirable “colon” phenotype was associated with TFs such as KLF5, NKX2-3, as well as CDX2 transcriptional networks. Cluster analysis identified highly correlated groups of genes associated with mature liver functions and downregulated proliferation-associated genes, which approach levels of adult hepatocytes. However, three further clusters failed to reach the gene expression levels of adult hepatocytes. Key TFs of two of these clusters include SOX11, FOXQ1, and YBX3. The third XVI cluster group, controlled by TFs such as HNF1A, CAR, FXR, and PXR, significantly overlaps with genes that are repressed in cultured adult hepatocytes relative to freshly isolated adult hepatocytes, suggesting that the current in vitro conditions lack stimuli essential for maintaining gene expression in hepatocytes, which consequently explains the corresponding functional deficiency of HLCs. In conclusion, the present gene regulatory network approach identified critical transcription factors for interventions to improve differentiation of hESCs to functional maturated hepatocytes.
In the past decade, it has been recognized that human embryonic stem cells (hESCs) differentiation into hepatocyte like cells (HLCs) could offer an unlimited supply of hepatocytes for pharmacology, toxicology, and cell therapy for liver failure. Many research efforts claimed to have successfully engineered HLCs from hESCs. However, the degree of differentiation and identity of HLCs remains controversial. The primary goal of this thesis work was to obtain a comprehensive understanding of HLCs identity. Thus, HLCs were differentiated from hESCs using a well-established protocol. Genome-wide gene expression programs of hESCs and HLCs were analyzed using GeneChip® Human Genome U133 Plus 2.0 arrays. The resulting gene expression profiles of HLCs and hESCs were compared to freshly isolated adult hepatocytes and adult hepatocytes cultivated for up to 14 days. Application of a broad range of bioinformatic tools and data mining approaches led to elucidation of gene networks and transcription factors (TFs) involved in the regulation of gene expression suggesting successful and failed hepatocyte differentiation. Gene regulatory network analysis revealed that HLCs represent a hybrid cell type with features of the liver, intestine, fibroblast, and stem cells. The undesirable “colon” phenotype was associated with TFs such as KLF5, NKX2-3, as well as CDX2 transcriptional networks. Cluster analysis identified highly correlated groups of genes associated with mature liver functions and downregulated proliferation-associated genes, which approach levels of adult hepatocytes. However, three further clusters failed to reach the gene expression levels of adult hepatocytes. Key TFs of two of these clusters include SOX11, FOXQ1, and YBX3. The third XVI cluster group, controlled by TFs such as HNF1A, CAR, FXR, and PXR, significantly overlaps with genes that are repressed in cultured adult hepatocytes relative to freshly isolated adult hepatocytes, suggesting that the current in vitro conditions lack stimuli essential for maintaining gene expression in hepatocytes, which consequently explains the corresponding functional deficiency of HLCs. In conclusion, the present gene regulatory network approach identified critical transcription factors for interventions to improve differentiation of hESCs to functional maturated hepatocytes.
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Keywords
Stem cells, Hepatocyte like cells, Transcription factors, Freshly isolated human hepatocytes