Lead supervisor: W. Wagner, Co-supervisor: L. De Laporte, Junior supervisor: R. Goetzke 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.
Organization of embyroid bodies (A) Scheme of an embryoid body cross section at day 6. (B) Confocal microscopy image of embryoid body embedded in fibrin gel showing cavity formation and differential expression of E-cadherin (green) and OCT4 (red). Nuclei are counterstained with DAPI (blue). (C) 2D iPSC colony transfected with lentiviral vectors containing red, green and blue (RGB) fluorophores. This approach allows the progeny of cells to be tracked within iPSC colonies and embryoid bodies. (B, C): unpublished data.
Background: Pluripotent stem cells grow into organoids with luminal structures33,34, and such self-organization recapitulates processes of early embryogenesis15,35. Aggregates of induced pluripotent stem cells (iPSCs) form embryoid bodies (EBs) consisting of outer cells, which differentiate toward endodermal epithelium and deposit a basement membrane, and adjacent epiblast cells, which polarize into a columnar epithelium. The remaining core cells, which are not in contact with the basement membrane, die by caspase-dependent apoptosis, creating a proamniotic-like cavity36. Recently, it has been suggested that lumenogenesis may be initiated by highly organized perinuclear apicosome structures37. We anticipate that cell-cell interaction and external mechanical stimuli govern directed differentiation in this model system.
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: EBs of defined size are generated from human iPSCs using AggreWell Plates or a large particle sorter (COPAS). Distribution of cell adhesion proteins, primary cilia, and pluripotency factors are analyzed by confocal, two-photon and light-sheet microscopy. As an example, the heterogeneity of EBs is analyzed with single-cell RNA sequencing. As a continuation of our previous work13,19 we now investigate the impact of the matrix elasticity of hydrogels on growth and differentiation of EBs. Mechanical stability of EBs is addressed by rheological measurements, osmotic pressure and magnetic micromanipulators. 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-Cas9n Technology.