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

Abstract

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.