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

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Dhiraj Indana
Dhiraj Indana
Dhiraj Indana
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Pranay Agarwal
Pranay Agarwal
Pranay Agarwal
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Nidhi Bhutani at Stanford Medicine
Nidhi Bhutani
Ovijit Chaudhuri
Ovijit Chaudhuri
Ovijit Chaudhuri
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Abstract and Figures

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.
Viscoelasticity and ligand density of alginate hydrogels regulate growth and morphogenesis of human induced pluripotent stem cells (hiPSCs). a) Schematic depicting encapsulation of single hiPSCs in 3D alginate hydrogels (cross‐sectional view shown) and methodology for tuning alginate stress‐relaxation rate independent of initial elastic modulus. Single hiPSCs proliferate and undergo morphogenetic changes to give rise to lumen‐containing cell clusters, depending on hydrogel properties. b–e) Mechanical characterization of 20 kPa alginate hydrogels using compression testing and shear rheometry. The bars and curves indicate means and standard deviation for biological replicates (n ≥ 3). b,c) Initial elastic modulus from unconfined compression tests (b) (p = 0.2807, Welch's analysis of variance (ANOVA); ns: not significant), and loss tangent from shear rheometry (c) (*p < 0.05, ***p < 0.001, ****p < 0.0001, one‐way ANOVA; ##p < 0.01, two‐tailed Spearman's rank correlation). d) Representative stress‐relaxation profiles of different 20 kPa alginate formulations at 10% compressive strain. Inset indicates applied strain profile. e) Timescale of stress relaxation τ1/2, defined here as the time when the normalized stress reaches 0.5 (*p < 0.05, ***p < 0.001, ****p < 0.0001, Welch's ANOVA; ####p < 0.0001, two‐tailed Spearman's rank correlation). f) Representative bright‐field images of hiPSC clusters on day 7 of culture in different alginate formulations with an initial elastic modulus of 20 kPa. The scale bar is 100 µm. g) Representative bright‐field images of hiPSC clusters in fast relaxing 1500 × 10⁻⁶ m RGD gels on different days of culture. The scale bar is 50 µm.
Viscoelasticity and ligand density of alginate hydrogels regulate growth and morphogenesis of human induced pluripotent stem cells (hiPSCs). a) Schematic depicting encapsulation of single hiPSCs in 3D alginate hydrogels (cross‐sectional view shown) and methodology for tuning alginate stress‐relaxation rate independent of initial elastic modulus. Single hiPSCs proliferate and undergo morphogenetic changes to give rise to lumen‐containing cell clusters, depending on hydrogel properties. b–e) Mechanical characterization of 20 kPa alginate hydrogels using compression testing and shear rheometry. The bars and curves indicate means and standard deviation for biological replicates (n ≥ 3). b,c) Initial elastic modulus from unconfined compression tests (b) (p = 0.2807, Welch's analysis of variance (ANOVA); ns: not significant), and loss tangent from shear rheometry (c) (*p < 0.05, ***p < 0.001, ****p < 0.0001, one‐way ANOVA; ##p < 0.01, two‐tailed Spearman's rank correlation). d) Representative stress‐relaxation profiles of different 20 kPa alginate formulations at 10% compressive strain. Inset indicates applied strain profile. e) Timescale of stress relaxation τ1/2, defined here as the time when the normalized stress reaches 0.5 (*p < 0.05, ***p < 0.001, ****p < 0.0001, Welch's ANOVA; ####p < 0.0001, two‐tailed Spearman's rank correlation). f) Representative bright‐field images of hiPSC clusters on day 7 of culture in different alginate formulations with an initial elastic modulus of 20 kPa. The scale bar is 100 µm. g) Representative bright‐field images of hiPSC clusters in fast relaxing 1500 × 10⁻⁶ m RGD gels on different days of culture. The scale bar is 50 µm.
… 
Hydrogel stress relaxation and ligand density regulate hiPSC viability, apoptosis, proliferation while sustaining pluripotency. a) Representative maximum intensity projection images and quantification of % live cells from live/dead assays performed on day 1 of culture. The bars indicate means and standard error of the mean (S.E.M.) (**p < 0.01, ****p < 0.0001, ns: not significant (p > 0.05), one‐way ANOVA; n ≥ 5 replicates). The scale bar is 100 µm. b,c) TUNEL assay on day 1 in 150 × 10⁻⁶ m RGD gels. Slow stress relaxation triggers apoptosis in hiPSCs. b) Representative immunohistochemical stains of nucleus (4′,6‐diamidino‐2‐phenylindole (DAPI)) and fragmented DNA (TUNEL) in 150 × 10⁻⁶ m RGD gels of different stress‐relaxation rates. The scale bar is 5 µm. c) Colocalization between DAPI and TUNEL channels is quantified to identify apoptotic cells in 150 × 10⁻⁶ m RGD gels of different stress‐relaxation rates. Pearson's R value of 1 indicates perfect correlation, 0 indicates no correlation, and −1 indicates perfect anti‐correlation. The bars indicate means and S.E.M. (****p < 0.0001, ns: not significant (p > 0.05), one‐way ANOVA; number of cells > 12 per gel). d) PicoGreen assay on days 1, 5, 7, and 9 for different alginate formulations. The curves indicate means and S.E.M. (#p < 0.05, ##p < 0.01; Pearson's correlation). Some of the error bars are too small to be seen. e) Representative immunohistochemical stains of pluripotency markers OCT4, SOX2, and NANOG on day 7 of culture in 150 × 10⁻⁶ m RGD gels. The scale bar is 25 µm. f–h) Quantification of % cells per cluster expressing SOX2 (f), OCT4 (g), or NANOG (h) on day 7 and day 14 of culture in different alginate formulations. Pluripotency markers are expressed in all alginate formulations. The bars indicate means and S.E.M. (**p < 0.01, ****p < 0.0001, ns: not significant (p > 0.05), one‐way ANOVA). All of the gels had an initial elastic modulus of 20 kPa.
Hydrogel stress relaxation and ligand density regulate hiPSC viability, apoptosis, proliferation while sustaining pluripotency. a) Representative maximum intensity projection images and quantification of % live cells from live/dead assays performed on day 1 of culture. The bars indicate means and standard error of the mean (S.E.M.) (**p < 0.01, ****p < 0.0001, ns: not significant (p > 0.05), one‐way ANOVA; n ≥ 5 replicates). The scale bar is 100 µm. b,c) TUNEL assay on day 1 in 150 × 10⁻⁶ m RGD gels. Slow stress relaxation triggers apoptosis in hiPSCs. b) Representative immunohistochemical stains of nucleus (4′,6‐diamidino‐2‐phenylindole (DAPI)) and fragmented DNA (TUNEL) in 150 × 10⁻⁶ m RGD gels of different stress‐relaxation rates. The scale bar is 5 µm. c) Colocalization between DAPI and TUNEL channels is quantified to identify apoptotic cells in 150 × 10⁻⁶ m RGD gels of different stress‐relaxation rates. Pearson's R value of 1 indicates perfect correlation, 0 indicates no correlation, and −1 indicates perfect anti‐correlation. The bars indicate means and S.E.M. (****p < 0.0001, ns: not significant (p > 0.05), one‐way ANOVA; number of cells > 12 per gel). d) PicoGreen assay on days 1, 5, 7, and 9 for different alginate formulations. The curves indicate means and S.E.M. (#p < 0.05, ##p < 0.01; Pearson's correlation). Some of the error bars are too small to be seen. e) Representative immunohistochemical stains of pluripotency markers OCT4, SOX2, and NANOG on day 7 of culture in 150 × 10⁻⁶ m RGD gels. The scale bar is 25 µm. f–h) Quantification of % cells per cluster expressing SOX2 (f), OCT4 (g), or NANOG (h) on day 7 and day 14 of culture in different alginate formulations. Pluripotency markers are expressed in all alginate formulations. The bars indicate means and S.E.M. (**p < 0.01, ****p < 0.0001, ns: not significant (p > 0.05), one‐way ANOVA). All of the gels had an initial elastic modulus of 20 kPa.
… 
hiPSC lumen formation and cluster morphology impacted by RGD density and hydrogel stress relaxation. a) Representative membrane stained (R18) images of hiPSC clusters on day 7 of culture for different hydrogel formulations. The scale bar is 100 µm. b) % Clusters with or without lumen on day 7 of culture for different alginate formulations. High RGD density and faster hydrogel stress relaxation promote lumen formation. The bars indicate means pooled from 2 biological replicates. n ≥ 20 clusters per condition (****p < 0.0001, ns: not significant (p > 0.05), Fisher's exact; ####p < 0.0001, χ² test for trend). c) Quantification of lumen volume on day 7 of culture for different hydrogel formulations. The bars indicate means and S.E.M. (****p < 0.0001, one‐way ANOVA). d) Quantification of lumen volume and total volume of cells per cluster, on different days of culture for various alginate formulations. For total cell volume per cluster quantification, both clusters with and without lumens were included. The curves indicate means and S.E.M. Some of the error bars are too small to be seen. e) Representative membrane stained (R18) images of hiPSC clusters in fast‐relaxing 1500 × 10⁻⁶ m RGD gels on different days of culture. The scale bar is 100 µm. f) Immunohistochemical stains of nucleus (DAPI) and F‐actin (Phalloidin) for representative clusters in 150 × 10⁻⁶ and 1500 × 10⁻⁶ m RGD gels on day 7 of culture. Quantification of average thickness of cell layer per cluster and cell length for different cells in a cluster. The bars indicate means and S.E.M. (****p < 0.0001, Mann–Whitney). The scale bar is 25 µm. For 150 × 10⁻⁶ m, n = 50 clusters and for 1500 × 10⁻⁶ m, n = 50 clusters. For single cell length, n = 500 cells, measured from 100 clusters. All gels had an initial elastic modulus of 20 kPa.
hiPSC lumen formation and cluster morphology impacted by RGD density and hydrogel stress relaxation. a) Representative membrane stained (R18) images of hiPSC clusters on day 7 of culture for different hydrogel formulations. The scale bar is 100 µm. b) % Clusters with or without lumen on day 7 of culture for different alginate formulations. High RGD density and faster hydrogel stress relaxation promote lumen formation. The bars indicate means pooled from 2 biological replicates. n ≥ 20 clusters per condition (****p < 0.0001, ns: not significant (p > 0.05), Fisher's exact; ####p < 0.0001, χ² test for trend). c) Quantification of lumen volume on day 7 of culture for different hydrogel formulations. The bars indicate means and S.E.M. (****p < 0.0001, one‐way ANOVA). d) Quantification of lumen volume and total volume of cells per cluster, on different days of culture for various alginate formulations. For total cell volume per cluster quantification, both clusters with and without lumens were included. The curves indicate means and S.E.M. Some of the error bars are too small to be seen. e) Representative membrane stained (R18) images of hiPSC clusters in fast‐relaxing 1500 × 10⁻⁶ m RGD gels on different days of culture. The scale bar is 100 µm. f) Immunohistochemical stains of nucleus (DAPI) and F‐actin (Phalloidin) for representative clusters in 150 × 10⁻⁶ and 1500 × 10⁻⁶ m RGD gels on day 7 of culture. Quantification of average thickness of cell layer per cluster and cell length for different cells in a cluster. The bars indicate means and S.E.M. (****p < 0.0001, Mann–Whitney). The scale bar is 25 µm. For 150 × 10⁻⁶ m, n = 50 clusters and for 1500 × 10⁻⁶ m, n = 50 clusters. For single cell length, n = 500 cells, measured from 100 clusters. All gels had an initial elastic modulus of 20 kPa.
… 
Varying hydrogel initial elastic modulus, or stiffness, from 3 to 20 kPa, has no effect on hiPSC viability, apoptosis, proliferation, pluripotency, and lumen formation. a) Representative live/dead staining images in fast relaxing 150 × 10⁻⁶ m RGD gels and quantification of % live cells on day 7 of culture. The scale bar is 500 µm. The bars indicate means and S.E.M. (p > 0.9, ns: not significant, one‐way ANOVA). b) Cluster sizes on days 1, 3, 5, and 7 of culture. Cluster size is not dependent on matrix elastic modulus. The curves indicate means and S.E.M. c) PicoGreen assay on days 1, 3, and 7 for different alginate formulations. The curves indicate means and S.E.M. (b, c) Some error bars are too small to be seen. d) TUNEL assay on day 1. Representative immunohistochemical stains of nucleus (DAPI) and fragmented DNA (TUNEL) in 0 m RGD gels of different elastic moduli. Scale bar is 5 µm. Colocalization between DAPI and TUNEL channels is quantified to identify apoptotic cells in 0 and 1500 × 10⁻⁶ m RGD gels of different elastic moduli. Pearson's R value of 1 indicates perfect colocalization, 0 indicates no correlation, and −1 indicates perfect exclusion. The bars indicate means and S.E.M. (p > 0.7, ns: not significant, one‐way ANOVA; number of cells > 8 per gel). e) Quantification of immunohistochemical stains of pluripotency markers OCT4, SOX2, and NANOG on day 7 of culture in fast relaxing gels. Plots measure % cells per cluster expressing the corresponding protein. The bars indicate means and S.E.M. (p > 0.4, ns: not significant, one‐way ANOVA). f) Quantification of % clusters with lumen on day 7 of culture in different alginate formulations. The bars indicate means and S.E.M. (ns: not significant, one‐way ANOVA).
Varying hydrogel initial elastic modulus, or stiffness, from 3 to 20 kPa, has no effect on hiPSC viability, apoptosis, proliferation, pluripotency, and lumen formation. a) Representative live/dead staining images in fast relaxing 150 × 10⁻⁶ m RGD gels and quantification of % live cells on day 7 of culture. The scale bar is 500 µm. The bars indicate means and S.E.M. (p > 0.9, ns: not significant, one‐way ANOVA). b) Cluster sizes on days 1, 3, 5, and 7 of culture. Cluster size is not dependent on matrix elastic modulus. The curves indicate means and S.E.M. c) PicoGreen assay on days 1, 3, and 7 for different alginate formulations. The curves indicate means and S.E.M. (b, c) Some error bars are too small to be seen. d) TUNEL assay on day 1. Representative immunohistochemical stains of nucleus (DAPI) and fragmented DNA (TUNEL) in 0 m RGD gels of different elastic moduli. Scale bar is 5 µm. Colocalization between DAPI and TUNEL channels is quantified to identify apoptotic cells in 0 and 1500 × 10⁻⁶ m RGD gels of different elastic moduli. Pearson's R value of 1 indicates perfect colocalization, 0 indicates no correlation, and −1 indicates perfect exclusion. The bars indicate means and S.E.M. (p > 0.7, ns: not significant, one‐way ANOVA; number of cells > 8 per gel). e) Quantification of immunohistochemical stains of pluripotency markers OCT4, SOX2, and NANOG on day 7 of culture in fast relaxing gels. Plots measure % cells per cluster expressing the corresponding protein. The bars indicate means and S.E.M. (p > 0.4, ns: not significant, one‐way ANOVA). f) Quantification of % clusters with lumen on day 7 of culture in different alginate formulations. The bars indicate means and S.E.M. (ns: not significant, one‐way ANOVA).
… 
Lumen‐containing hiPSC clusters exhibit apicobasal polarization and do not form a continuous basement membrane at the cluster boundary. a–d) Representative immunostaining images and quantifications of PAR6 (a,b) and EZRIN (c,d) localization, two different apical markers, on day 7 in fast relaxing 1500 × 10⁻⁶ m RGD gels. For quantification, position is normalized by taking the center of the lumen to be at 0 and apical surfaces at −1 and +1. The curves in the quantification plots show means and S.E.M. For PAR6 staining, n = 12 clusters and for EZRIN staining, n = 14 clusters. e–h) Representative immunostaining images and quantification (h) of 2 basement membrane proteins laminin‐111 (e), collagen IV (f), and β1 integrin (g) on day 7 in fast relaxing 1500 × 10⁻⁶ m RGD gels. For quantification, % of nonzero intensity pixels along the cluster boundary is measured in thresholded images of laminin‐111 or collagen IV or β1 integrin channel. The bars in the quantification plot show means and 95% confidence intervals (****p < 0.0001; ns: not significant (p > 0.05), Kruskal–Wallis). For laminin‐111, n = 53 clusters; for collagen IV, n = 49 clusters; and for β1 integrin, n = 45 clusters. All scale bars are 25 µm (a–g).
+2
Lumen‐containing hiPSC clusters exhibit apicobasal polarization and do not form a continuous basement membrane at the cluster boundary. a–d) Representative immunostaining images and quantifications of PAR6 (a,b) and EZRIN (c,d) localization, two different apical markers, on day 7 in fast relaxing 1500 × 10⁻⁶ m RGD gels. For quantification, position is normalized by taking the center of the lumen to be at 0 and apical surfaces at −1 and +1. The curves in the quantification plots show means and S.E.M. For PAR6 staining, n = 12 clusters and for EZRIN staining, n = 14 clusters. e–h) Representative immunostaining images and quantification (h) of 2 basement membrane proteins laminin‐111 (e), collagen IV (f), and β1 integrin (g) on day 7 in fast relaxing 1500 × 10⁻⁶ m RGD gels. For quantification, % of nonzero intensity pixels along the cluster boundary is measured in thresholded images of laminin‐111 or collagen IV or β1 integrin channel. The bars in the quantification plot show means and 95% confidence intervals (****p < 0.0001; ns: not significant (p > 0.05), Kruskal–Wallis). For laminin‐111, n = 53 clusters; for collagen IV, n = 49 clusters; and for β1 integrin, n = 45 clusters. All scale bars are 25 µm (a–g).
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ReseaRch aRticle
Viscoelasticity and Adhesion Signaling in Biomaterials
Control Human Pluripotent Stem Cell Morphogenesis in
3D Culture
Dhiraj Indana, Pranay Agarwal, Nidhi Bhutani,* and Ovijit Chaudhuri*
D. Indana, O. Chaudhuri
Department of Mechanical Engineering
Stanford University
Stanford, CA , USA
E-mail: chaudhuri@stanford.edu
P. Agarwal, N. Bhutani
Department of Orthopaedic Surgery
Stanford University School of Medicine
Stanford, CA , USA
E-mail: nbhutani@stanford.edu
The ORCID identification number(s) for the author(s) of this article
can be found under https://doi.org/./adma..
DOI: 10.1002/adma.202101966
play a key role in several morphogenetic
processes, especially during embryonic
development.[,] For example, in preim-
plantation mouse embryos, dierences in
cell contractility regulate dierentiation to
trophectoderm and inner cell mass fates
by controlling Yes-associated protein (YAP)
nuclear localization,[] and leads to the for-
mation of microlumens which coalesce to
form the blastocyst cavity.[] However, mor-
phogenetic processes during the earliest
stages of human embryogenesis are much
less understood. Human embryogenesis
involves a series of complex morphoge-
netic events which cannot be studied in
vivo or via long-term in vitro culture of
human embryos due to ethical concerns.[]
Recently, there has been a tremendous
eort toward developing human-pluripo-
tent-stem-cell-based models of embryonic
development.[–] Lumen formation is the
first morphogenetic event that pluripotent
stem cells undergo in vivo during post-
implantation human embryogenesis at the
epiblast stage.[–] Interestingly, human
pluripotent stem cells self-organize to
form lumens when cultured in recon-
stituted basement membrane (rBM)-based matrices.[–]
However, these matrices suer from loss of human induced
pluripotent stem cell (hiPSC) pluripotency over longer time-
scales[] and are poorly defined and heterogeneous with limited
tunability of matrix properties. Thus, the role of the matrix, in
regulating lumen formation by human pluripotent stem cells
remains unknown.
Engineered biomaterials are often used for D culture of
cells to model morphogenetic processes in vitro and to eluci-
date the role of dierent matrix properties in mediating mor-
phogenesis.[,–] Matrix stiness, degradability, cell–matrix
adhesion ligand type, and ligand density have been shown to
impact intestinal stem cell organoid formation and budding
morphogenesis,[,] neural tube formation,[] liver orga-
noid formation,[] and Madin–Darby canine kidney (MDCK)
cell lumenogenesis.[] More recently, scaold geometry was
engineered using hydrogel coated microchips to obtain intes-
tinal organoids with in vivo like morphology.[] On the other
hand, the impact of viscoelasticity on morphogenetic pro-
cesses is much less explored. Viscoelastic materials dissipate
mechanical energy, like viscous liquids, while exhibiting some
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 oer 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), stiness, 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.
1. Introduction
Morphogenesis is a complex but tightly regulated multicel-
lular process where cells self-organize into tissues with special-
ized macroscale form and function via dynamic integration of
cues from the mechanical microenvironment,[] chemical mor-
phogens,[] and local cell states.[] Forces and mechanical cues
Adv. Mater. 2021, 33, 
... For example, differences in relaxation response within hydrogels have been demonstrated to influence mesenchymal stem cell fate [20,21]. Similarly, these properties have been shown to influence the organization of epithelial tissues and organization of pluripotent stem cell clusters in hydrogels [22,23]. It is evident that increasing our understanding of time Poroelasticity affects solute movement at multiple scales, thus influencing nutrient transport to and from cells, as well as local signaling between cells. ...
Isolating Poroelastic and Viscoelastic Mechanisms of Soft Tissues and Hydrogels Through Sequential Microscale Indentation Testing: New Applications of Indentation Theory for Microscale Characterization
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... Our analysis of stimulated gels in the absence of cells did not reveal any biased deformation pattern, negating gel deformation as a mechanism ( Supplementary Fig. 17). More telling was when we tested an alternate tissue model-human induced pluripotent stem cells (hiPSC) hollow spheroids 88,89 . These spheroids were similar in morphology to MDCK cysts but, crucially, exhibited asymmetry in the opposite polarity of MDCK cysts-that is they thinned on the right (cathode-facing) side (Supplementary Movie 9). ...
Bioelectric stimulation controls tissue shape and size
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  • Apr 2024
Epithelial tissues sheath organs and electro-mechanically regulate ion and water transport to regulate development, homeostasis, and hydrostatic organ pressure. Here, we demonstrate how external electrical stimulation allows us to control these processes in living tissues. Specifically, we electrically stimulate hollow, 3D kidneyoids and gut organoids and find that physiological-strength electrical stimulation of ∼ 5 - 10 V/cm powerfully inflates hollow tissues; a process we call electro-inflation. Electro-inflation is mediated by increased ion flux through ion channels/transporters and triggers subsequent osmotic water flow into the lumen, generating hydrostatic pressure that competes against cytoskeletal tension. Our computational studies suggest that electro-inflation is strongly driven by field-induced ion crowding on the outer surface of the tissue. Electrically stimulated tissues also break symmetry in 3D resulting from electrotaxis and affecting tissue shape. The ability of electrical cues to regulate tissue size and shape emphasizes the role and importance of the electrical micro-environment for living tissues.
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... In the era of modern synthetic polymers like acrylates and silanes, the world turned back towards sodium alginate for its biocompatibility and tuneable viscoelastic properties. Recent reports include attempts of organoids growth and differentiation employed tuneable visco-elastic property of alginate gels to obtain desirable responses [1,2]. Besides, alginate has been a favourite polymer for industries like -food [3,4], pharmaceutical [5,6], environmental bioremediation [7,8], biomedical device manufacturing [9], tissue engineering and 3D bioprinting [10], advanced physical and molecular therapeutic techniques [11]. ...
Emergence of Anionic Counterparts of Divalent Metal Salts as the Fine-Tuners of Alginate Hydrogel Properties for Tissue Engineering and Drug Delivery Applications
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The role of anionic counter ions of divalent metal salts in alginate gelation and hydrogel properties was thoroughly investigated. Three anions were selected from the Hofmeister series viz. sulphates, acetates and chlorides paired in all permutations and combinations with calcium, zinc and copper divalent metals. Spectroscopic analysis revealed the presence of anions and their interaction with the metal atoms post-gelation. Data showed gelation time and other hydrogel properties were mostly governed by the cations. However, subtle yet significant variations in viscoelastic, water-uptake, drug-release and cytocompatibility properties were anion dependent in a cationic group. Computational modelling study showed metal-anion-alginate configurations were energetically more stable than metal-alginate models. The in vitro and in silico studies conclude that acetate anions precede the chlorides in the drug-delivery, swelling, and cytocompatibility fronts, followed by sulphate anions in each cationic group. Overall, the data provided affirmation that anions are integral part of the metal-alginate complex. Furthermore, anions offer a novel option to further fine-tune the properties of alginate hydrogels for tissue engineering and drug delivery applications. Moreover, extensive exploration of this novel avenue would enhance the usability of alginate polymers in pharma, environmental, biomedical and food industries.
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The Influence of Crosslinker Architecture on Dynamic Covalent Hydrogel Viscoelasticity
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Full-text available
  • May 2024
Dynamic covalent crosslinked (DCC) hydrogels represent a significant advance in biomaterials for regenerative medicine and mechanobiology. These gels typically offer viscoelasticity and self-healing properties that more closely mimic in vivo tissue mechanics than traditional, predominantly elastic, covalent crosslinked hydrogels. Despite their promise, the effects of varying crosslinker architecture – side chain versus telechelic crosslinks – on the viscoelastic properties of DCC hydrogels have not been thoroughly investigated. This study introduces hydrazone-based alginate hydrogels and examines how side-chain and telechelic crosslinker architectures impact hydrogel viscoelasticity and stiffness. In side-chain crosslinked gels, higher polymer concentrations, enhances stiffness and decelerates stress relaxation, while an off-stoichiometric hydrazine-to-aldehyde ratio leads to reduced stiffness and shorter relaxation time. In telechelic crosslinked gels, maximal stiffness and stress relaxation occurs at intermediate crosslinker concentrations for both linear and star crosslinkers, with higher crosslinker valency further increasing stiffness and decreasing relaxation rates. Our result suggested different ranges of stiffness and stress relaxation are accessible with the different crosslinker architectures, with side-chain crosslinking leading to gels with slower stress relaxation times relative to the other architectures, and star crosslinked gels providing increased stiffness and slower stress relaxation relative to linear crosslinked gels. The mechanical properties of hydrogels with star crosslinking are more robust to changes induced by competing chemical reactions compared to linear crosslinking. Our research underscores the pivotal role of crosslinker architecture in defining hydrogel stiffness and viscoelasticity, providing crucial insights for the design of DCC hydrogels with tailored mechanical properties for specific biomedical applications.
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Looking both ways: Electroactive biomaterials with bidirectional implications for dynamic cell–material crosstalk
Article
  • May 2024
Cells exist in natural, dynamic microenvironmental niches that facilitate biological responses to external physicochemical cues such as mechanical and electrical stimuli. For excitable cells, exogenous electrical cues are of interest due to their ability to stimulate or regulate cellular behavior via cascade signaling involving ion channels, gap junctions, and integrin receptors across the membrane. In recent years, conductive biomaterials have been demonstrated to influence or record these electrosensitive biological processes whereby the primary design criterion is to achieve seamless cell–material integration. As such, currently available bioelectronic materials are predominantly engineered toward achieving high-performing devices while maintaining the ability to recapitulate the local excitable cell/tissue microenvironment. However, such reports rarely address the dynamic signal coupling or exchange that occurs at the biotic–abiotic interface, as well as the distinction between the ionic transport involved in natural biological process and the electronic (or mixed ionic/electronic) conduction commonly responsible for bioelectronic systems. In this review, we highlight current literature reports that offer platforms capable of bidirectional signal exchange at the biotic–abiotic interface with excitable cell types, along with the design criteria for such biomaterials. Furthermore, insights on current materials not yet explored for biointerfacing or bioelectronics that have potential for bidirectional applications are also provided. Finally, we offer perspectives aimed at bringing attention to the coupling of the signals delivered by synthetic material to natural biological conduction mechanisms, areas of improvement regarding characterizing biotic–abiotic crosstalk, as well as the dynamic nature of this exchange, to be taken into consideration for material/device design consideration for next-generation bioelectronic systems.
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Mechanotransduction in Stem Cells
Article
Full-text available
  • May 2024
  • EUR J CELL BIOL
Nowadays, it is an established concept that the capability to reach a specialised cell identity via differentiation, as in the case of multi- and pluripotent stem cells, is not only determined by biochemical factors, but that also physical aspects of the microenvironment play a key role; interpreted by the cell through a force-based signalling pathway called mechanotransduction. However, the intricate ties between the elements involved in mechanotransduction, such as the extracellular matrix, the glycocalyx, the cell membrane, integrin adhesion complexes, Cadherin-mediated cell/cell adhesion, the cytoskeleton, and the nucleus, are still far from being understood in detail. Here we report what is currently known about these elements in general and their specific interplay in the context of multi- and pluripotent stem cells. We furthermore merge this overview to a more comprehensive picture, that aims to cover the whole mechanotransductive pathway from the cell/microenvironment interface to the regulation of the chromatin structure in the nucleus. Ultimately, with this review we outline the current picture of the interplay between mechanotransductive cues and epigenetic regulation and how these processes might contribute to stem cell dynamics and fate.
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Homeostatic mini-intestines through scaffold-guided organoid morphogenesis
Article
Full-text available
  • Sep 2020
  • NATURE
Epithelial organoids, such as those derived from stem cells of the intestine, have great potential for modelling tissue and disease biology1–4. However, the approaches that are used at present to derive these organoids in three-dimensional matrices5,6 result in stochastically developing tissues with a closed, cystic architecture that restricts lifespan and size, limits experimental manipulation and prohibits homeostasis. Here, by using tissue engineering and the intrinsic self-organization properties of cells, we induce intestinal stem cells to form tube-shaped epithelia with an accessible lumen and a similar spatial arrangement of crypt- and villus-like domains to that in vivo. When connected to an external pumping system, the mini-gut tubes are perfusable; this allows the continuous removal of dead cells to prolong tissue lifespan by several weeks, and also enables the tubes to be colonized with microorganisms for modelling host–microorganism interactions. The mini-intestines include rare, specialized cell types that are seldom found in conventional organoids. They retain key physiological hallmarks of the intestine and have a notable capacity to regenerate. Our concept for extrinsically guiding the self-organization of stem cells into functional organoids-on-a-chip is broadly applicable and will enable the attainment of more physiologically relevant organoid shapes, sizes and functions.
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Mechanisms of human embryo development: From cell fate to tissue shape and back
Article
Full-text available
  • Jul 2020
Gene regulatory networks and tissue morphogenetic events drive the emergence of shape and function: the pillars of embryo development. Although model systems offer a window into the molecular biology of cell fate and tissue shape, mechanistic studies of our own development have so far been technically and ethically challenging. However, recent technical developments provide the tools to describe, manipulate and mimic human embryos in a dish, thus opening a new avenue to exploring human development. Here, I discuss the evidence that supports a role for the crosstalk between cell fate and tissue shape during early human embryogenesis. This is a critical developmental period, when the body plan is laid out and many pregnancies fail. Dissecting the basic mechanisms that coordinate cell fate and tissue shape will generate an integrated understanding of early embryogenesis and new strategies for therapeutic intervention in early pregnancy loss.
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Mechano-modulatory synthetic niches for liver organoid derivation
Article
Full-text available
  • Jul 2020
The recent demonstration that primary cells from the liver can be expanded in vitro as organoids holds enormous promise for regenerative medicine and disease modelling. The use of three-dimensional (3D) cultures based on ill-defined and potentially immunogenic matrices, however, hampers the translation of liver organoid technology into real-life applications. We here use chemically defined hydrogels for the efficient derivation of both mouse and human hepatic organoids. Organoid growth is found to be highly stiffness-sensitive, a mechanism independent of acto-myosin contractility and requiring instead activation of the Src family of kinases (SFKs) and yes-associated protein 1 (YAP). Aberrant matrix stiffness, on the other hand, results in compromised proliferative capacity. Finally, we demonstrate the establishment of biopsy-derived human liver organoids without the use of animal components at any step of the process. Our approach thus opens up exciting perspectives for the establishment of protocols for liver organoid-based regenerative medicine.
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An in vitro model of early anteroposterior organization during human development
Article
Full-text available
  • Jun 2020
  • NATURE
The body plan of the mammalian embryo is shaped through the process of gastrulation, an early developmental event that transforms an isotropic group of cells into an ensemble of tissues that is ordered with reference to three orthogonal axes1. Although model organisms have provided much insight into this process, we know very little about gastrulation in humans, owing to the difficulty of obtaining embryos at such early stages of development and the ethical and technical restrictions that limit the feasibility of observing gastrulation ex vivo2. Here we show that human embryonic stem cells can be used to generate gastruloids—three-dimensional multicellular aggregates that differentiate to form derivatives of the three germ layers organized spatiotemporally, without additional extra-embryonic tissues. Human gastruloids undergo elongation along an anteroposterior axis, and we use spatial transcriptomics to show that they exhibit patterned gene expression. This includes a signature of somitogenesis that suggests that 72-h human gastruloids show some features of Carnegie-stage-9 embryos3. Our study represents an experimentally tractable model system to reveal and examine human-specific regulatory processes that occur during axial organization in early development. Human gastruloids—three-dimensional aggregates derived from human embryonic stem cells—show features of human embryos at around 19–21 days, and provide a model for the study of early human development.
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Mouse embryonic stem cells self-organize into trunk-like structures with neural tube and somites
Article
  • Dec 2020
Trunk formation in a dish Building mammalian embryos from self-organizing stem cells in culture would accelerate the investigation of morphogenetic and differentiation processes that shape the body plan. Veenvliet et al. report a method for generating embryonic trunk-like structures (TLSs) with a neural tube, somites, and gut by embedding mouse embryonic stem cell aggregates in an extracellular matrix surrogate. Live imaging and comparative single-cell transcriptomics indicate that TLS formation is analogous to mouse development. TLSs therefore provide a scalable, tractable, and accessible high-throughput platform for decoding mammalian embryogenesis at a high level of resolution. Science , this issue p. eaba4937
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Extracellular matrix plasticity as a driver of cell spreading
Article
  • Oct 2020
  • P NATL ACAD SCI USA
Significance In many biomaterial systems, plastic deformation of extracellular matrix, degradation, or relaxation of stress are coupled and therefore their effects on cell phenotype are difficult to isolate. Using a polymer plasticizing architecture that specifically decouples irreversible creep from stress relaxation and modulus, network plasticity is shown to have a biphasic relationship with cell spreading.
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Pluripotent Stem Cell-Based Cell Therapy-Promise and Challenges
Article
  • Oct 2020
Human pluripotent stem cells such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) provide unprecedented opportunities for cell therapies against intractable diseases and injuries. Both ESCs and iPSCs are already being used in clinical trials. However, we continue to encounter practical issues that limit their use, including their inherent properties of tumorigenicity, immunogenicity, and heterogeneity. Here, I review two decades of research aimed at overcoming these three difficulties.
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Effects of extracellular matrix viscoelasticity on cellular behaviour
Article
  • Aug 2020
Substantial research over the past two decades has established that extracellular matrix (ECM) elasticity, or stiffness, affects fundamental cellular processes, including spreading, growth, proliferation, migration, differentiation and organoid formation. Linearly elastic polyacrylamide hydrogels and polydimethylsiloxane (PDMS) elastomers coated with ECM proteins are widely used to assess the role of stiffness, and results from such experiments are often assumed to reproduce the effect of the mechanical environment experienced by cells in vivo. However, tissues and ECMs are not linearly elastic materials—they exhibit far more complex mechanical behaviours, including viscoelasticity (a time-dependent response to loading or deformation), as well as mechanical plasticity and nonlinear elasticity. Here we review the complex mechanical behaviours of tissues and ECMs, discuss the effect of ECM viscoelasticity on cells, and describe the potential use of viscoelastic biomaterials in regenerative medicine. Recent work has revealed that matrix viscoelasticity regulates these same fundamental cell processes, and can promote behaviours that are not observed with elastic hydrogels in both two- and three-dimensional culture microenvironments. These findings have provided insights into cell–matrix interactions and how these interactions differentially modulate mechano-sensitive molecular pathways in cells. Moreover, these results suggest design guidelines for the next generation of biomaterials, with the goal of matching tissue and ECM mechanics for in vitro tissue models and applications in regenerative medicine. This Review explores the role of viscoelasticity of tissues and extracellular matrices in cell–matrix interactions and mechanotransduction and the potential utility of viscoelastic biomaterials in regenerative medicine.
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Bioengineered pluripotent stem cell models: new approaches to explore early human embryo development
Article
  • Dec 2020
  • CURR OPIN BIOTECH
Human development is a complex process in which environmental signals and factors encoded by the genome interact to engender cell fate changes and self-organization that drive the progressive formation of the human body. Herein, we discuss engineered biomimetic platforms with controllable environments that are being used to develop human pluripotent stem cell (hPSC)-based embryo models (or embryoids) that recapitulate a wide range of early human embryonic developmental events. Coupled with genome editing tools, single-cell analysis, and computational models, they can be used to parse the spatiotemporal dynamics that lead to differentiation, patterning, and growth in early human development. Furthermore, we discuss ongoing efforts in human extraembryonic lineage derivation and what can be learned from mouse embryoid models that have used both embryonic and extraembryonic stem cells. Finally, we discuss promising bioengineering tools for the generation of more controllable systems and the need for validation of findings from hPSC-based embryoid models.
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Pluripotent stem cell models of early mammalian development
Article
  • Oct 2020
  • CURR OPIN CELL BIOL
Pluripotent stem cells derived from the early mammalian embryo offer a convenient model system for studying cell fate decisions in embryogenesis. The last 10 years have seen a boom in the popularity of two-dimensional micropatterns and three-dimensional stem cell culture systems as a way to recreate the architecture and interactions of particular cell populations during development. These methods enable the controlled exploration of cellular organization and patterning during development, using cell lines instead of embryos. They have established a new class of in vitro model system for pre-implantation and peri-implantation embryogenesis, ranging from models of the blastocyst stage, through gastrulation and toward early organogenesis. This review aims to set these systems in context and to highlight the strengths and suitability of each approach in modelling early mammalian development.
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