Lead supervisor: W. Wagner, Co-supervisor: L. De Laporte Helmholtz-Institute for Biomedical Engineering, Division of Stem Cell Biology
Hypothesis: Cell-cell interaction and mechanical stimuli influence self-organization and cell-fate decisions in 3D embryoid bodies.
Self-organization of embryoid bodies. A) Transcriptome and epigenome reveal continuous changes during differentiation of induced pluripotent stem cells (iPSCs) towards embryoid bodies (EBs). B) The EBs often develop cavitated structures. C) Exemplary analysis of an EB at day 7 with areas of PAX6 (ectoderm) and GATA6 (endoderm) expressing cells.
Background: Aggregates of induced pluripotent stem cells (iPSCs) form embryoid bodies (EBs) that differentiate toward the three germ layers (endoderm, mesoderm, and ectoderm), and recapitulate molecular changes of early embryogenesis. We anticipate that this self-organization is directly governed by cell-cell interaction and external mechanical stimuli (REF-A, REF-B).
Aims: To understand how self-organization and cell-fate decisions are influenced by cell-cell interaction and mechanical stimuli in EBs, we will investigate (i) expression of adhesion proteins and cytoskeletal organization, (ii) determine effects of mechanical stimuli on lineage-specific differentiation, (iii) estimate mechanical stability of EBs and (iv) determine the functional relevance of specific adhesion proteins and signal cascades in early EB formation and differentiation. Approach: Embryoid bodies of defined size are generated with our recently described method based on micro-contact printing of vitronectin (REF). Distribution of cell adhesion proteins and germ layer specific markers are analyzed by confocal, two-photon and light-sheet microscopy. The heterogeneity of EBs is further analyzed with single-cell RNA sequencing. As a continuation of our previous work 13,19we now investigate the impact of the matrix elasticity of hydrogels on growth and differentiation of EBs. The impact of various hydrogels will be systematically evaluated. As readout, we will investigate DNA methylation changes, which are specifically acquired by directed differentiation toward specific germ layers. Finally, the relevance of specific cell adhesion proteins for early cell-fate decisions and EB stability are elucidated with knockout iPSC-lines generated with CRISPR-Cas9 or CRISPR-Off Technology.