Effect of geometrical constraints on human pluripotent stem cell nuclei in pluripotency and differentiation

Abstract

Mechanical stimuli and geometrical constraints transmitted across the cytoskeleton to the nucleus affect the nuclear morphology and cell function. Human pluripotent stem cells (hPSCs) represent an effective tool for evaluating transitions in nuclear deformability from the pluripotent to differentiated stage, and for deciphering the underlying mechanisms. We report the first study that investigates the nuclear deformability induced by geometrical constraints of hPSCs both in the pluripotent stage and during early germ layer specification. We specifically developed micro-structured surfaces coupled with high-content imaging analysis algorithms to quantitatively characterize nuclear deformability. Our results show that hPSCs possess high nuclear deformability, which does not alter pluripotency. We observed nuclear deformability transition along early germ layer specification: during early ectoderm differentiation nuclear deformability is strongly reduced, during early endoderm differentiation nuclei keep a deformed shape and during early mesoderm specification they show an intermediate behaviour. Different mRNA expressions between hPSCs differentiated on flat and micro-structured surfaces have been observed along early mesoderm and early endoderm specification. In order to better understand the mechanisms of the nuclear deformability transition observed during early ectoderm differentiation, we also employed cytoskeletal and nuclear protein inhibitors to evaluate their role in determining the nuclear shape. Actin and nesprin are essential for maintaining deformed nuclei, while lamin A/C and intermediate filaments confer rigidity to the nucleus. This study suggests that nuclear deformability is highly regulated during differentiation.

Dystrophin Cardiomyopathies: Clinical Management, Molecular Pathogenesis and Evolution towards Precision Medicine

“…Mechanosensing pathways respond not only to substrate rigidity but also to geometric stimuli. In vitro cardiac and muscle tissues are characterized by cell alignment and anisotropy, and the effect of this geometrical organization on the maturation of iPSC-derived cells has been demonstrated in vitro by using micro- and nano-structured substrates, shown to support the formation of cell-to-cell junctions, building foundations for the generation of functional grafts, showing a contractile phenotype, increasing parameters such as beating rate and tissue-specific protein arrangements (e.g., sarcomeric α-actinin, connexin 43 and troponins) [ 209 , 225 , 226 ].…”

A Biophysical Model for Curvature-Guided Cell Migration

“…As such, mechanobiology tells us that adhesion and migration, if controlled and understood, can provide a way to manipulate cell fate (4,5) and avoid severe pathologies (6). Recent findings in cell mechanobiology have shown the correlation between the mechanical state perceived by the cell and, how it differentiates, proliferates, and dies (7)(8)(9)(10). Designing efficient technologies to manipulate cells fate relies partly on understanding the mechanisms triggered by the complex combination of mechanical cues induced by their surrounding environment (11,12).…”

PnBA/PDMAA‐Based Iron‐Loaded Micropillars Allow for Discrete Cell Adhesion and Analysis of Actuation‐Related Molecular Responses

“…PDMS microwells were generated by replica molding from a micropillared Si wafer (1 cm × 1 cm square) that was fabricated by a standard photolithography technique. The detailed process can be found elsewhere 43. The micropillared Si wafer was subsequently silanized by (tridecafluoro‐1,1,2,2‐tetrahydrooctyl)1‐trichlorosilane vapor under vacuum for 1 h. PDMS (base:curing agent at a ratio of 10:1) was poured over the micropillared Si master, degassed under vacuum for 1 h and cured at 60 °C for 6 h. The PDMS microwell‐stamp was peeled off from the Si wafer and used as a template for the subsequent fabrication of P(nBA‐ co ‐5%MABP) micropillars.…”