Bead Interaction Potential from Physical confinement signals regulate the organization of stem cells in three dimensions

a) The piece-wise interaction potential (in units of the effective thermal energy) is shown as a function of separation (in units of the bead diameter). For short distances, a constant force is used which yields a softer repulsion than the full Lennard-Jones interaction (shown as a dashed line). For intermediate distances, the Lennard-Jones potential is used which contains both a repulsive and an attractive component. At large distances the potential is truncated such that the beads have a finite attraction range. Simulations were carried out to understand the influence of cell-substrate (ɛcs) and cell-cell (ɛcc) interactions on embryonic stem cells growing within different levels of confinement. A simulation rendered with a χ (ɛcs/ɛcc) value of 0.1 (a) in 100 µm channels and (b) on a flat surface, revealing that with a low cell-substrate adhesion, cells are capable of aggregating in a spheroidal manner through purely diffusive means. When generated with a χ of 4, under (c) 100 µm and (d) flat conditions, cells demonstrate typical flat island morphology irrespective of the confinement1,-3

ESPResSo input parameters from Physical confinement signals regulate the organization of stem cells in three dimensions

During embryogenesis, the spherical inner cell mass (ICM) proliferates in the confined environment of a blastocyst. Embryonic stem cells (ESCs) are derived from the ICM, and mimicking embryogenesis <i>in vitro</i>, mouse ESCs (mESCs) are often cultured in hanging droplets. This promotes the formation of a spheroid as the cells sediment and aggregate owing to increased physical confinement and cell-cell interactions. In contrast, mESCs form two-dimensional monolayers on flat substrates and it remains unclear if the difference in organization is owing to a lack of physical confinement or increased cell-substrate versus cell-cell interactions. Employing microfabricated substrates, we demonstrate that a single geometric degree of physical confinement on a surface can also initiate spherogenesis. Experiment and computation reveal that a balance between cell-cell and cell-substrate interactions finely controls the morphology and organization of mESC aggregates. Physical confinement is thus an important regulatory cue in the three-dimensional organization and morphogenesis of developing cells

The effects of inhibiting actomyosin dynamics and E-cadherin function on mESC organization on flat substrates from Physical confinement signals regulate the organization of stem cells in three dimensions

Embryonic stem cells were seeded onto a flat PDMS surface with inhibitory drugs (a) blebbistatin (2µM)(n=24), (b) Y27632 (10µM)(n=20) and (c) SMIFH2 (10µM)(n=28) for a 48hr incubation period (Scale bar = 50 µm and applies to all). Contrary to cells grown in a confined microenvironment, inhibition of actin contractility, mechanotransduction signaling and actin polimerization respectively had no effect on aggregate morphology whilst on a two dimensional surface. (d) No significant difference was found with regards to the planar Isotropy (Ip, black) or globular isotropy (Ig, red) when we used any of the inhibitory conditions

Supplementary Figure 5 from Physical confinement signals regulate the organization of stem cells in three dimensions

During embryogenesis, the spherical inner cell mass (ICM) proliferates in the confined environment of a blastocyst. Embryonic stem cells (ESCs) are derived from the ICM, and mimicking embryogenesis <i>in vitro</i>, mouse ESCs (mESCs) are often cultured in hanging droplets. This promotes the formation of a spheroid as the cells sediment and aggregate owing to increased physical confinement and cell-cell interactions. In contrast, mESCs form two-dimensional monolayers on flat substrates and it remains unclear if the difference in organization is owing to a lack of physical confinement or increased cell-substrate versus cell-cell interactions. Employing microfabricated substrates, we demonstrate that a single geometric degree of physical confinement on a surface can also initiate spherogenesis. Experiment and computation reveal that a balance between cell-cell and cell-substrate interactions finely controls the morphology and organization of mESC aggregates. Physical confinement is thus an important regulatory cue in the three-dimensional organization and morphogenesis of developing cells

Manipulating cell-substrate interactions through matrix protein deposition from Physical confinement signals regulate the organization of stem cells in three dimensions

To confirm that collagen is responsible for the cell-substrate adhesion on PDMS channels, cells were seeded without collagen and incubated for 48hrs. Whether on the (a) 100 µm channel or the (b) flat substrate, no cells had successfully adhered (Scale bar = 100 µm and applies to (b) as well). (c) In an attempt to reverse the spheroidal geometry observed in the channels, we increased cell-substrate adhesion by depositing fibronectin in addition to collagen. Increased cell substrate adhesion appeared to have no effect of embryonic stem cells growth, as the aggregate still formed a highly three dimensional geometry. Interestingly, the increased substrate interaction promoted cell adhesion at the top of the channels; however, these cells formed typical two dimensional flat aggregates.To ascertain whether the observed changes in geometry are the result of a higher cell density, cells were seeded to match the density of 100 µm channels (~450 cells/mm2). Shown are cells at (d) 12 hrs and (e) 48 hours after seeding, displaying a typical island shaped morphology. Scale bar = 100 µm

Supplementary Figure 4 from Physical confinement signals regulate the organization of stem cells in three dimensions

During embryogenesis, the spherical inner cell mass (ICM) proliferates in the confined environment of a blastocyst. Embryonic stem cells (ESCs) are derived from the ICM, and mimicking embryogenesis <i>in vitro</i>, mouse ESCs (mESCs) are often cultured in hanging droplets. This promotes the formation of a spheroid as the cells sediment and aggregate owing to increased physical confinement and cell-cell interactions. In contrast, mESCs form two-dimensional monolayers on flat substrates and it remains unclear if the difference in organization is owing to a lack of physical confinement or increased cell-substrate versus cell-cell interactions. Employing microfabricated substrates, we demonstrate that a single geometric degree of physical confinement on a surface can also initiate spherogenesis. Experiment and computation reveal that a balance between cell-cell and cell-substrate interactions finely controls the morphology and organization of mESC aggregates. Physical confinement is thus an important regulatory cue in the three-dimensional organization and morphogenesis of developing cells

Free Energy of a Polymer in Slit-like Confinement from the Odijk Regime to the Bulk

We directly measure the free energy of confinement for semiflexible polymers from the nanoscale to bulk regimes in slit-like confinement. We use convex lens-induced confinement (CLiC) microscopy of DNA to directly count molecules at equilibrium in a single chamber of smoothly increasing height. Our data, acquired across a continuum of confinement regimes, provide a bridge with which to connect scaling theories established for qualitatively different regimes. We present new experimental data and simulations that connect the Odijk theory describing sub-persistence-length confinement, the interpolation model by Chen and Sullivan extending Odijk to moderate confinement, and the Casassa theory describing the transition from moderate confinement to bulk. Further, this work establishes a robust, quantitative platform for understanding and manipulating biopolymers at the nanoscale, with key applications and insights toward emerging genomic analysis tools