Viscoelasticity and Adhesion Signaling in Biomaterials Control Human Pluripotent Stem Cell Morphogenesis in 3D Culture

Abstract

Organoids are lumen‐containing multicellular structures that recapitulate key features of the organs, and are increasingly used in models of disease, drug testing, and regenerative medicine. Recent work has used 3D culture models to form organoids from human induced pluripotent stem cells (hiPSCs) in reconstituted basement membrane (rBM) matrices. However, rBM matrices offer little control over the microenvironment. More generally, the role of matrix viscoelasticity in directing lumen formation remains unknown. Here, viscoelastic alginate hydrogels with independently tunable stress relaxation (viscoelasticity), stiffness, and arginine–glycine–aspartate (RGD) ligand density are used to study hiPSC morphogenesis in 3D culture. A phase diagram that shows how these properties control hiPSC morphogenesis is reported. Higher RGD density and fast stress relaxation promote hiPSC viability, proliferation, apicobasal polarization, and lumen formation, while slow stress relaxation at low RGD densities triggers hiPSC apoptosis. Notably, hiPSCs maintain pluripotency in alginate hydrogels for much longer times than is reported in rBM matrices. Lumen formation is regulated by actomyosin contractility and is accompanied by translocation of Yes‐associated protein (YAP) from the nucleus to the cytoplasm. The results reveal matrix viscoelasticity as a potent factor regulating stem cell morphogenesis and provide new insights into how engineered biomaterials may be leveraged to build organoids.

Materials and extracellular matrix rigidity highlighted in tissue damages and diseases: Implication for biomaterials design and therapeutic targets

“… Matrix properties Cells Engineered matrix Findings/comments Refs Rigidity (with adhesion ligand) Mouse and human mesenchymal stem cell Alginate hydrogel, PEG dimethacrylate gel Differentiation of MSCs is regulated by substrate rigidity in 3D environment. However, unlike in 2D, adhesion ligand rather than cell morphology seems more critical in lineage commitment Huebsch et al [ 42 ] Stress relaxation 3T3 fibroblast, D1 cell Alginate hydrogel Stress relaxation is a key mechanical parameter that influences cell spreading, differentiation, and proliferation in 3D environment Chaudhuri et al [ 46 ] Stress relaxation Bovine chondrocyte Alginate hydrogel Fast stress relaxation induces pro-chondrogenesis pathway for chondrocytes whereas slow stress relaxation leads to cartilage degradation and cell death Lee et al [ 49 ] Stress relaxation Adenocarcinoma cell line (MDA-MB-231) Alginate hydrogel Cancer cells in the fast-relaxing deform the surrounding matrix effectively to allow mitotic elongation and cell division, whereas those in slow-relaxing gels fail to complete mitosis Nam et al [ 50 ] Stress relaxation (with adhesion ligand) Human induced pluripotent stem cell Alginate hydrogel RGD density and stress relaxation are crucial factors in stem cell morphogenesis Indana et al [ 51 ] Fiber elasticity, anisotropy Fibroblast Dextran methacrylated form (DexMa) Cells dynamically interact with flexible fibers. Cell mechano-responses to 3D fibrillar anisotropic environment are different from those to isotropic gel matrix Baker et al [ 54 ] Degradability Human mesenchymal stem cell Methacrylated hyaluronic acid (MeHA) Degradation-mediated cellular t...…”

“…With regard to the cellular phenomena in non-degradable but viscoelastic 3D gels, Mooney and Chaudhuri et al have intensely explored cell behaviors using engineered alginate gels, where they focused on stress-relaxation among other viscoelastic properties. The cellular behaviors in which they have taken interest include MSC lineage specification [ 46 ], chondrocyte matrix formation [ 49 ], cancer cell mitosis [ 50 ], and pluripotent stem cell morphogenesis [ 51 ]. The alginate-based gels were tuned to have varying stress relaxation rates, independent of the initial elastic modulus, rate of degradation, and cell-adhesion-ligand density.…”

“…6 C). In a recent work, human induced pluripotent stem cells (hiPSCs) were able to go through morphogenesis in 3D viscoelastic gel setting [ 51 ]. Within fast stress relaxation gels, hiPSCs displayed promoted viability, proliferation, apicobasal polarization, and ultimately lumen formation, whereas slow stress relaxation gels triggered apoptosis of the cells ( Fig.…”

“…Other than stress relaxation, increasing RGD ligand density enhances iPSC morphogenesis. Adapted with permission from Indana et al [ 51 ] in Adv. Mater.…”

Regulating Bacterial Behavior within Hydrogels of Tunable Viscoelasticity

“…[ 10 , 20 , 21 ] The current understanding, mainly from studies of bacteria or yeast embedded in hydrogels, indicate that the microbes grow inside the hydrogel network to form dense clusters at slower rates than in suspension. [ 20 , 22 , 23 ] Cell's response to the mechanical properties of their microenvironment is well known from 3D cultures of mammalian cells, whose proliferation, migration, or differentiation programs depend on the viscoelasticity [ 24 ] and the degradation kinetics of the hydrogel network. [ 25 , 26 ] Studies in engineered hydrogels with viscoelastic properties that can be modulated by the type of network crosslinks (reversible/dynamic vs permanent) and by the nature of the degradable sequences have helped to understand and quantify eukaryotic organism's mechanosensitivity range and response.…”