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

JOAQUÍN SMUCLER; ALAN MIQUEAS MOBBS; GUSTAVO SEVLEVER; SANTIAGO MIRIUKA; CARLOS LUZZANI; ALEJANDRO DAMIAN LA GRECA; ARIEL WAISMAN

Mar del Plata

Congreso; Reunion anual de la Sociedad Argentina de Investigaciones Clínicas 2022; 2022

Human Pluripotent Stem Cells (hPSC) have the capacity to self-renew and differentiate invitro into specialized cells. While the spatial distribution of cell populations is a key variablein regulating cell fate, common cell culture practices generate colonies of differentgeometries in an aleatory manner. Control over colony spatiality affects cell-cell interactionand specific signaling pathways that can influence pluripotency and differentiation. Circularcolonies recapitulate part of the biophysical cues present in the embryonic disc beforegastrulation. Furthermore, when hPSCs are cultured in these conditions, activation of BMP4signaling induces a geometric-specific differentiation that resembles early gastrulation.In this work, we developed a device capable of generating functionalized surfaces ofextracellular matrix (ECM) proteins in various shapes and sizes for hPSCs culture, calledmicropatterns. A Laminin-coated surface was treated with UV-C light through circular opaquepatterns of 1000 um diameter. Light denatures the ECM protein in specific regions, in astencil-like way, leaving only small delimited portions of functional laminin where cells canadhere.Our laboratory studies mesodermal lineage differentiation using BMP4. We thus analyzedBMP4 effects on micropatterned cell colonies as a validation of the device generated. Cellswere cultured in circular colonies of 1000 μm for 24 hours and then treated with BMP4 for 42hours. Immunofluorescence was performed against SOX2 (ectoderm), SOX17 (endoderm)and TBXT (mesoderm). Using quantitative bioinformatic analysis, we found a positive centerfor SOX2, a middle ring for TBXT, and an outer ring for SOX17. We observed the spatialdistribution of key factors within the colony consistent with the distribution on the germinaldisc.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.