Figure 1.
Characteristic magnitudes and time domains of mechanical signals applied in studies of multipotent cell differentiation.
Each data point represents one study. The shape of the data point portrays the lineage to which the multipotent cell committed. Blue data points depict deviatoric (shape changing) stresses, i.e. shear stress magnitudes [dyn/cm2], and duration of signal over the time course of the study [days]. Red data points depict dilatational stresses (volume changing stresses, i.e. hydrostatic compression and tension, [log10Pa]). The yellow plane (opaque to transparent reflecting respective likelihood of the stress ranges) overlay represents dilatational and deviatoric stress ranges predicted during cell fate determination in utero [5], [13], [15]–[43].
Table 1.
Magnitude and time of mechanical stresses applied in stem cell differentiation studies.
Figure 2.
In situ mapping of stem cell stresses and strains.
4D (x,y,z,t) image of microsphere displacements (A, C, E) and microbead displacements (B, D, F) shows flow fields and surface strains, respectively, in live stem cells subjected to fluid flow at low (A,B), high (C,D), and very high (E,F) densities. Calcein Green stains live cells. Red and white arrows indicate velocity of flow (microsphere displacement and direction: A, C, E) and strain (microbead displacements: B, D, F), respectively. Red, green, and white dots (B, D, F) show respective microbead positions at 0, 30, and 60 minutes after flow. Stresses imparted by flow at cell surfaces are calculated from the experimentally determined velocity gradient (slope of Fig. S1, Equation 3). Cell surface strains (deformations) are calculated using experimentally measured microbead displacements on cell surfaces.
Figure 3.
Computational predictions (CFD-FEM) of strains on cell surfaces for flow rates imparting 0.2 (A,B) and 1 dyn/cm2 (C,D).
Each computational prediction includes velocity magnitude and displacement in XY plane, assuming an elastic modulus of 0.9 Pa (A and C), and in the Z direction, assuming an elastic modulus of 90 Pa (B and D) at 16,500 cells/cm2 (1); computational predictions were carried out for 35,000 cells/cm2 (2), and 86,500 cells/cm2 (3) cell seeding densities. Topological color map indicates displacements on surface of seeded cells. The geometry of the seeded cells is imported from a 3D reconstruction of confocal images. Color arrows show flow velocity (flow field) in the near the vicinity of cells.
Figure 4.
Mechanical testing (stress - strain analysis) of live stem cells using novel microbead tracking method.
Cell surface deformation (strain) is mapped in situ concomitant to delivery of controlled mechanical cues via fluid flow. (A) Two dimensional (2D) images of microsphere displacements on cell surfaces. Calcein green stain marks live stem cells. Red, green, and blue dots indicate respective positions of microbeads at 0, 30, and 60 minutes after flow application. Control shows positions of microbeads in absence of flow. (B) Schematic depiction of flow and non-flow orientation/direction with respect to initial position of microbead (t0, red). (C) Comparison of microbead displacements on cell surfaces from control cohorts and cohorts exposed to flow. Error bars show standard errors (n = 3), * indicates statistical significance (p<0.05).
Figure 5.
Experimental and computational analysis of flow regimes around live stem cells.
(A) 2D micrographs of μ-PIV experiments show flow regimes in the apical and basal regions of the live stem cell (target shear stress imparted by flow: 0.2 dyn/cm2). (B,C) Validated computational fluid dynamics (CFD) predictions of velocity gradients (which drive shear gradients and hence target shear stresses) in the apical and basal regions of cells, at flow rates including 0.2 and 1 dyn/cm2, respectively. * indicates statistical significance, defined by p<0.05. Error bars show standard errors (n = 3).
Figure 6.
Parametric estimation of the stem cell's mechanical properties.
Displacements in the XY plane (A) and Z direction (B) were predicted computationally, using values varied parametrically (by orders of magnitude) about a known measured value for osteoblasts (900 Pa). By comparing experimentally measured displacements with displacements predicted for model cells with estimated moduli, a best estimate for the elastic and shear moduli of the model stem cell line could be made. The vertical axes are depicted in a logarithmic scale. C. Through linear interpolation, parametric estimates were made for elastic and shear modulus of the model stem cell, for flow generating a target shear of 1 dyn/cm2 and for three seeding conditions (different densities). Error bars show standard errors (n = 3).
Figure 7.
Stress - strain - fate plots to probe the live stem cell's mechanome, in a case study in live stem cells exposed to 1 dyn/cm2 shear stress via fluid flow.
(A) After mesenchymal condensation (red dotted box, E11.5 in the mouse), the first step in skeletogenesis, stem cells follow lineage commitment paths toward chondrogenic (orange), osteogenic (blue), and adipogenic (green) fates. Transcription levels for specific proteins (red text) give a “chronological fingerprint” for each stage of development over time [1]. (B) For live stem cells exposed to 1 dyn/cm2 shear stress over 60 minutes, rtPCR data provide fold changes in gene expression of markers above or below baseline (no flow exposure) gene expression levels, based on analysis of previously reported data [31]. (C) Stress - strain - fate plot of live stem cells exposed to 1 dyn/cm2 shear stress over 60 minutes, where fate is indicated by color (osteogenic: blue, chondrogenic: orange). Linear regression of experimental data shows distinct slopes for each density and resultant fates, including osteogenic (blue: 1.04 [Pa], R2∼0.7, 16,500 cell density [cells/cm2]; navy: 0.9997 [Pa], R2∼0.78, 35,000 cell density [cells/cm2]), and chondrogenic lineage commitment (orange, 1.7124 [Pa], R2>0.8, 86,500 cell density [cells/cm2]). Red overlay shapes indicate mean strain stress at each density. Ellipses show 95% density area for each density. D. Local (apical), normal stress-strain (XY plane) relationship, depicting lineage commitment, including osteogenic (blue: 3.8513 [Pa] at 16,500 cell density [cells/cm2]; navy: 4.8513 [Pa] at 35,000 cell density [cells/cm2]) and chondrogenic lineages (orange: 3.8525 [Pa] at 86,500 cell density [cells/cm2]); all R2>0.8. Red shapes indicate mean stress and strain at each density. Ellipses indicate 95% density area for each density. E. Local (basal), normal stress-strain (XY plane) relationship, depicting lineage commitment, including osteogenic (blue: 4.9413 [Pa] at 16,500 cell density [cells/cm2]; navy: 4.8059 [Pa] at 35,000 cell density [cells/cm2]) and chondrogenic lineages (orange: 4.0653 [Pa] at 86,500 cell density [cells/cm2]); all R2>0.8. Each data point is shown from cohorts of cells in the field of view.