Generation of micropatterned circular colonies of Pluripotent Stem Cells and spatial characterization of specific markers of the three germ layers in response to BMP4

JOAQUIN SMUCLER; ALAN MIKEAS MOBBS; GUSTAVO SEVLEVER; SANTIAGO MIRIUKA; CARLOS LUZZANI; ALEJANDRO LA GRECA; ARIEL WAISMAN

Mar del Plata

Congreso; Reunion anual de sociedades de biociencias; 2022

Human Pluripotent Stem Cells (hPSC) have the capacity to self-renew and differentiate in vitro into specialized cells. While the spatial distribution of cell populations is a key variable in regulating cell fate, common cell culture practices generate colonies of different geometries in an aleatory manner. Control over colony spatiality affects cell-cell interaction and specific signaling pathways that can influence pluripotency and differentiation. Circular colonies recapitulate part of the biophysical cues present in the embryonic disc before gastrulation. Furthermore, when hPSCs are cultured in these conditions, activation of BMP4 signaling induces a geometric-specific differentiation that resembles early gastrulation.In this work, we developed a device capable of generating functionalized surfaces of extracellular matrix (ECM) proteins in various shapes and sizes for hPSCs culture, called micropatterns. A Laminin-coated surface was treated with UV-C light through circular opaque patterns of 1000 um diameter. Light denatures the ECM protein in specific regions, in a stencil-like way, leaving only small delimited portions of functional laminin where cells can adhere.Our laboratory studies mesodermal lineage differentiation using BMP4. We thus analyzed BMP4 effects on micropatterned cell colonies as a validation of the device generated. Cells were cultured in circular colonies of 1000 µm for 24 hours and then treated with BMP4 for 42 hours. Immunofluorescence was performed against SOX2 (ectoderm), SOX17 (endoderm) and TBXT (mesoderm). Using quantitative bioinformatic analysis, we found a positive center for SOX2, a middle ring for TBXT, and an outer ring for SOX17. We observed the spatial distribution of key factors within the colony consistent with the distribution on the germinal disc. The device generates cellular micropatterns that have features of the embryonic disc in vitro, resulting in a novel tool to create gastruloids in a fast and reproducible way.