The Role of Wnt Signaling in an Embryoid Body Generation Protocol for Hematopoiesis Studies: Investigating the Emergence of Endothelial and Hematopoietic Stem and Progenitor Cells in hiPSC-Derived Embryoid Bodies through RT-qPCR and Flow Cytometry Analysis
Typ
Examensarbete för masterexamen
Master's Thesis
Master's Thesis
Program
Biotechnology (MPBIO), MSc
Publicerad
2024
Författare
Hilpold Berntsson, Elin
Modellbyggare
Tidskriftstitel
ISSN
Volymtitel
Utgivare
Sammanfattning
Research indicates that childhood leukemia originates in utero during hematopoietic development,
emphasizing the importance of understanding embryonic hematopoiesis to identify disruptions
leading to diseases such as leukemia. However, studying human embryonic hematopoiesis
in vivo faces significant ethical and technical challenges. As an alternative, embryoid bodies
(EBs) derived from human induced pluripotent stem cells (hiPSCs) offer a promising in vitro
model for studying human developmental processes and hematological diseases. This thesis
evaluates an established EB generation protocol for its efficacy in differentiating hiPSCs into
hematopoietic stem and progenitor cells (HSPCs). Through the application of real-time quantitative
PCR, this study investigates the activation of the Wnt signaling pathway, marked by
RSPO3 gene expression, assessing its impact on cell fate decisions within the EBs. Key findings
reveal that activation of the Wnt pathway not only induces the emergence of endothelial
cells but also facilitates the specification of HSPCs. The robust upregulation of genes such as
SOX17, CDH5, RUNX1, GATA2, and HOXA9 over time validates the progressive development
of endothelial and hematopoietic lineages. Flow cytometry analysis results also confirm
the emergence of endothelial and blood cell populations. However, the response of differentiating
hiPSCs to Wnt signaling varies between independent experiments, suggesting that further
optimization of the EB generation protocol can be implemented. Overall, the results underscore
the role of Wnt signaling in lineage specification, offering valuable insights into hematopoietic
differentiation and development in a controlled environment.
Beskrivning
Ämne/nyckelord
hiPSC, hemogenic endothelium, HSPC, embryoid bodies, Wnt signaling, RSPO3