Aplnr sinyalinin mesoderm doku ve mezenkimal kök hücre farklılaşmasındaki ve migrasyonundaki düzenleyici rolü

Embryonic morphogenesis is the most complicated cell movement event that is controlled by individual or mass form of cell migration during gastrulation of the embryo. Establishment and maintenance of the cell populations during gastrulation movements in response to cell-to-cell contact and signaling molecules is not only crucial for determination of body plan and tissue patterning but also required for generation of progenitor populations for adult tissue regeneration. Embryonic stem cells (ESCs) as pluripotent stem cells are the most appropriate model to recapitulate early embryonic development, gastrulation and adult tissue morphogenesis of humans. Embryoid bodies (EBs) as ESC aggregates in suspension culture construct a regionalized tissue patterning that is displaying gastrulation and organized germ layer structure and gene expression patterning of germ layers in vitro. Wnt signaling pathway is the fundamental regulatory pathway which triggers the morphogenetic events and initiates the gastrulation movements resulting in germ layer formation. Apelin receptor (Aplnr) has been identified as a novel Wnt target gene during hindgut differentiation of ESCs. However, the molecular mechanism and the regulatory role of Aplnr in cell migration and differentiation process in development and adult remain unclear. The main objective of my thesis project is to understand regulatory role of Aplnr signaling in cell migration and differentiation process of mesoderm tissue and mesenchymal stem cells (MSCs), which are very important for gastrulation movements, tissue specification and cell fate decision in adult tissue morphogenesis and adult tissue regeneration. The study was conducted using mouse ESC (mESC) and human ESC (hESC) lines. Peptide and small molecule induced Aplnr signaling activation as well as genetic modifications were applied to activate or inactivate the Aplnr pathway. Overexpression of Aplnr signaling pathway components was provided by 3rd generation lentiviral vector system for hESCs. CRISPR/Cas9 gene editing technique was used to knockout Aplnr pathway components in mESCs and hESCs. siRNA and shRNA strategies were utilized to downregulate Aplnr signaling pathway genes. These genetically modified ESCs were used to form EBs as three dimensional gastruloid models which were used to track cellular movements under the control of Aplnr signaling and cell fate decision during three germ layers specification. Migration and differentiation of mesodermal population into MSCs were conducted by differentiation experiments. Those cell populations were subjected to gene and protein expression analyses, including antibody array, LC/MS, mRNA and miRNA sequencing, to analyze molecular regulatory pathways. Morphological changes as well as gene and protein expression data revealed that Aplnr signaling promote mesoderm commitment possibly via EGFR, FGF and TGF-beta signaling pathways and enhances migration of ESCs-derived EBs during mesoderm differentiation. Antibody array and LC/MS protein profiling data of mESC-derived mesoderm cells demonstrated that Apelin-13 treatment enhanced cell cycle, EGFR, FGF, Wnt and Integrin signaling pathway proteins. Moreover, Aplnr signaling positively regulates MSCs differentiation from ESCs and increases cell viability, proliferation, survival, MSC characteristics and differentiation capacity of MSCs by regulating various pathways, such as EGFR, TGF-beta, Wnt, PDGF and FGF signaling pathways. Osteogenic, chondrogenic, adipogenic, odontogenic, endothelial and myogenic differentiations were significantly enhanced with Aplnr signaling activity. So, this study also lays an important foundation to generate high potential MSCs from ESCs to be used in personalized cell therapy.