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Embryoid bodies (EBs) resemble self-organizing aggregates of pluripotent stem cells that recapitulate some aspects of early embryogenesis. Within few days, the cells undergo a transition from rather homogeneous epithelial-like pluripotent stem cell colonies into a three-dimensional organization of various cell types with multifaceted cell–cell interactions and lumen formation—a process associated with repetitive epithelial-mesenchymal transitions. In the last few years, culture methods have further evolved to better control EB size, growth, cellular composition, and organization—e.g., by the addition of morphogens or different extracellular matrix molecules. There is a growing perception that the mechanical properties, cell mechanics, and cell signaling during EB development are also influenced by physical cues to better guide lineage specification; substrate elasticity and topography are relevant, as well as shear stress and mechanical strain. Epithelial structures outside and inside EBs support the integrity of the cell aggregates and counteract mechanical stress. Furthermore, hydrogels can be used to better control the organization and lineage-specific differentiation of EBs. In this review, we summarize how EB formation is accompanied by a variety of biomechanical parameters that need to be considered for the directed and reproducible self-organization of early cell fate decisions.
Species | Cell Type | Mechanical Stimulus | Parameters Tested | Duration | Readout | Ref. | |
---|---|---|---|---|---|---|---|
Substrate Elasticity | Human | ESC | Rigidity of PDMS micropost arrays | 1.92–1218.4 kPa | 24 hours | Traction force measurements; pluripotency and cytoskeletal changes (IF) | [93] |
Human | iPSC | Elastomer pillars of different heights on a rigid substrate | 3–168 kPa | Up to 8 days | Morphology; viability; proliferation; pluripotency (IF); cardiac differentiation (IF, FACS, Ca2+ assays, electrical stimulation) | [85] | |
Substrate Topography | Human | iPSC | Polyimide substrate with submicrometer groove-ridge structure | 340 nm, 650 nm, 1400 nm periodicity, 200 nm depth; compared to unstructured | 3 days | Morphology; pluripotency (IF, qRT-PCR); YAP/TAZ expression (WB), gene expression profiles | [94] |
Colony Size | Human | ESC, EB | Size of initial 2D colony and EB size via micropatterning | 200, 400, 800 µm | 4–22 days | Morphology; pluripotency (FACS) and germ layer differentiation (qRT-PCR, IF) | [70] |
Mouse | ESC, EB | Size of initial 2D colony and cell density via laser direct-write cell printing | Colony size: 200–3000 µm; cell density: <25,000 cells, 25,000–125,000 cells, >125,000 cells | 3–8 days | Morphology | [71] | |
Mouse, human | ESC, EB | Size of EBs; substrate hydrophobicity | Colony size: <100 µm, 100–300 µm, >300 µm; surface chemical properties: agarose, PEG, pHEMA, PDMS, TCP, LAC | 4–20 days | Morphology; viability; proliferation; (germ layer) differentiation potential (qRT-PCR, FACS) | [72] | |
Mouse | ESC, EB | Size of EBs via adhesive stencils with different diameter | 100–500 µm diameter | 20 days | Germ layer differentiation (IF, qRT-PCR) | [73] | |
Shear Stress | Mouse | ESC | Laminar shear stress | 1.5–10 dyn/cm2 | 3 days | Cell density; cell cycle (FACS, ELISA); endothelial differentiation (IF, WB, qRT-PCR) | [75] |
Mouse | ESC, EB | Shear stress in rotary suspension culture | Comparison of static and rotary suspension culture (25–55 rpm) | 12 hours to 7 days | Morphology; viability; proliferation; cyst formation; germ layer formation (qRT-PCR) | [76] | |
Mouse | ESC, EB | Y-channel microfluidic system with two different media | Laminar flow 50–200 µL/min | 5 days | Differentiation potential (WB, IF) | [78] | |
Mouse | EB | Microfluidic chip system with continuous laminar flow and shear stress | Comparison of static culture and laminar flow 2 µL/min | 21 days | Viability; proliferation; steroidogenic differentiation (hormone release, IF, ELISA) | [77] | |
Mouse | ECS, EB | Comparison of static conditions and shear stress as pre-condition | 0–5 dyn/cm2 | 48 hours before EB formation; up to 10 days of EB culture | Morphology; pluripotency and endothelial differentiation (IF, qRT-PCR, FACS) cellular organization | [79] | |
Mouse | ESC, EB | Shear stress in rotary orbital suspension culture | 0.7–2.5 dyn/cm2; 20–60 rpm | EB culture for 7 days; up to day 12 of differentiation | Morphology (IF); pluripotency and germ layer differentiation (qRT-PCR, FACS); global gene expression (PCR array analysis) | [80] | |
Mechanical Strain | Mouse | ESC, EB | Mechanical strain by short-term magnetization via incorporated RGD-conjugated paramagnetic beads | Stimulation pulses using short-term magnetization; 0.128–0.4 Tesla | 1 hour stimulation for up to 7 days | Morphology; viability; protein expression and cardiomyogenesis (β1 integrin inhibition, FACS, WB, IF) | [82] |
Mouse | ESC, EB | Mechanical strain by (cyclic) stretching and compression between two microtips with a magnetic tissue stretcher | Stretching amplitude of 50% of original size (+ cyclic stretching 1 Hz, 10% amplitude, twice daily for 2 hours) | 3 days; with three additional days of cyclic stretching | Morphology; viability; proliferation; pluripotency and germ layer differentiation (qRT-PCR, IF) | [39] | |
Mouse | ESC, EB | Mechanical strain by stretching device (Flexercell Strain Unit) | 10% elongation of undifferentiated EBs | 2 hours stretching on day 4 of EB generation | Monitoring of intracellular [Ca2+]i; Expression of angiogenesis guidance molecules; Expression of pro-angiogenic growth factors; ROS generation | [83] | |
Mouse | ESC, EB | Mechanical strain by stretching device (Flexercell Strain Unit) | 5%, 10%, or 20% elongation of undifferentiated EBs | 2 hours stretching on day 4 of EB generation | Staining of capillary-like structures; counting of beating bodies; staining of sarcomeric α-actinin; upregulation of NADPH oxidase subunits; ROS generation; inhibition of mechanical-strain stimulated MAPKs | [84] | |
3D Culture in Hydrogel | Human | ESC, EB | Agarose 3D culture system | Comparison of agarose 3D culture and suspension and hanging droplet culture | 7 days up to 8 weeks | In vivo teratoma assay; morphology; germ layer differentiation (IF) | [95] |
Human | ESC | Hydrogel based material for switching between alginate and collagen via ionic de-crosslinking | Change hydrogel composition and in matrix elasticity from 21.37 ± 5.37 kPa (alginate) to 4.87 ± 1.64 kPa (collagen) | 21 days | Viability; proliferation; pluripotency and germ layer differentiation (qRT-PCR) | [96] | |
Human | ESC, EB | Dextran-acrylate and PEG hydrogel +/- RGD and VEGF | Comparison of dextran-acrylate and PEG hydrogel +/- RGD and VEGF | 10 days | Viability; vascular differentiation (FACS, IF, qRT-PCR) | [88] | |
Human | ESC, EB | Biodegradable polymer scaffolds (50:50 PLGA:PLLA) | Medium supplemented with different growth factors | 14 days in vitro; 14 days in vivo | Proliferation; germ layer differentiation (qRT-PCR); transplantation into SCID mice (IF) | [89] | |
Human | iPSC, EB | Concave PEG hydrogel microstructures via 3D projection printing; initial cell number | Comparison between concave and flat gels; low (250,000/mL) and high (750,000/mL) cell density | Up to 10 days | Morphology; culture duration; pluripotency and germ layer differentiation (IF); cyst formation | [90] | |
Mouse | ESC, EB | Hybrid hydrogels (GelMA, PEG) with varying matrix elasticity | Analysis of a hybrid GelMA (3 wt%)/PEG (10 wt%) hydrogel | Up to 7 days | Vasculogenic and cardiogenic differentiation (qRT-PCR, IF) | [91] | |
Mouse | ESC, EB | PEG hydrogel with and without RGD | Comparison of different PEG gels; 150 µm EBs and 450 µm EBs | Up to 15 days | Morphology; endothelial and cardiac differentiation (contraction behavior, IF, qRT-PCR) | [92] |