Matrix Elasticity (2006) - created from Mind Map

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Review: Extracellular matrix: A dynamic microenvironment for stem cell niche (2014)

Stiffness Gradients Mimicking In Vivo Tissue Variation Regulate Mesenchymal Stem Cell Fate (2011) (https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0015978)

MSCs migrate to stiffer matrix and then differentiate into a more contractile myogenic phenotype

phenotype is not completely determined by the stiff hydrogel as some cells retain expression of a neural marker.

Mechanical

Hofstetter, C.P., Schwarz, E.J., Hess, D., Widenfalk, J., El Manira, A., Prockop, D.J., and Olson, L. (2002). Marrow stromal cells form guiding strands in the injured spinal cord and promote recovery. Proc. Natl. Acad. Sci. USA 99, 2199–2204.

Applicatoins

Review: Tissue Cells feel and respond

https://www-jstor-org.ezproxy.lib.ryerson.ca/stable/pdf/3843055.pdf?refreqid=excelsior%3A0efefe322ce31c1df6b3a6230458984c

Kim et al., 2005/McBeath et al., 2004/Wang et al., 2002:

→ “How might MSCs “feel” or sense matrix

elasticity and transduce that information into morphological changes and lineage specification?...One or

all of the nonmuscle myosin II isoforms (NMM IIA, B, & C) are...implicated in tensioning cortical actin

structures.

Embryonic cardiomyocytes beat best on a matrix with heart-like elasticity: scar-like rigidity inhibits beating (2008) (http://jcs.biologists.org/content/121/22/3794)

ECM properties

Biochemical

Structural

Heart-Specific Stiffening in Early Embryos Parallels Matrix and Myosin Expression to Optimize Beating (2013)

https://www.researchgate.net/publication/258852421_Heart-Specific_Stiffening_in_Early_Embryos_Parallels_Matrix_and_Myosin_Expression_to_Optimize_Beating

Tissue elasticity, Et, increases daily for heart to 1-2 kPa by embryonic day 4 (E4), and although this is ∼10-fold softer than adult heart, the beating contractions of E4 cardiomyocytes prove optimal at ∼Et,E4 both in vivo and in vitro

Sparsely cultured E4 cardiomyocytes on collagen-coated gels likewise show maximal contraction on matrices with native E4 stiffness, highlighting cell-intrinsic mechanosensitivity

Pluripotent stem cell-derived cardiomyocytes also prove to be mechanosensitive to matrix and thus generalize the main observation that myosin II organization and contractile function are optimally matched to the load contributed by matrix elasticity.

Matrix Elasticity Regulates Lamin-A,C Phosphorylation and Turnover with Feedback to Actomyosin (2014)

https://www.researchgate.net/publication/264832369_Matrix_Elasticity_Regulates_Lamin-AC_Phosphorylation_and_Turnover_with_Feedback_to_Actomyosin

The nucleoskeletal protein lamin-A,C increases with matrix stiffness, confers nuclear mechanical properties, and influences differentiation of mesenchymal stem cells (MSCs), whereas B-type lamins remain relatively constant [9].

tension-dependent stabilization of lamin-A,C and myosin-IIA can suitably couple nuclear and cell morphology downstream of matrix mechanics.

Scarring: Fractal Heterogeneity in Minimal Matrix Models of Scars Modulates Stiff-Niche Stem-Cell Responses Via Nuclear Exit of a Mechanorepressor (2016)

while allowing separate control over adhesive-ligand density.we show that copolymerization of collagen I with polyacrylamide produces minimal matrix models of scars (MMMS), in which fractal-fibre bundles of collagen segregate heterogeneously to the hydrogel subsurface. Matrix stiffens locally — as in scars — while allowing separate control over adhesive-ligand density.

MSCs thus form a ‘mechanical memory’ of rigidity by progressively suppressing NKX2.5, thereby elevating SMA in a scar-like state. control over adhesive-ligand density. The MMMS elicits scar-like phenotypes from mesenchymal stem cells (MSCs) that have become recognized recently as a major contributor to scarring in most or all tissues: cells spread and polarize quickly, increasing nucleoskeletal lamin-A but expressing more slowly the ‘scar marker’ smooth muscle actin (SMA)

Coordinated increase of nuclear tension and lamin-A with matrix stiffness out-competes Lamin-B Receptor which favors soft tissue phenotypes (2017)

https://www.researchgate.net/publication/319958400_Coordinated_increase_of_nuclear_tension_and_lamin-A_with_matrix_stiffness_out-competes_Lamin-B_Receptor_which_favors_soft_tissue_phenotypes

stiffness-associated increases in lamin-A,C are accompanied by decreases in lamin-B receptor (LBR), which contributes to lipid biosynthesis. Adipogenesis (soft) of MSCs increases LBR Matrix stiffness-driven contractility thus tenses the nucleus to favor lamin-A,C accumulation and oppose soft tissue phenotypes.

Cell Shape, Cytoskeletal Tension, and RhoA Regulate Stem Cell Lineage Commitment (2004) http://dx.doi.org/10.1016/S1534-5807(04)00075-9

Expressing dominant-negative RhoA committed hMSCs to become adipocytes, while constitutively active RhoA caused osteogenesis. However, the RhoA-mediated adipogenesis or osteogenesis was conditional on a round or spread shape, respectively, while constitutive activation of the RhoA effector, ROCK, induced osteogenesis independent of cell shape. This RhoA-ROCK commitment signal required actin-myosin-generated tension

Engler et al., 2004: Myotubes differentiate optimally on substrates with tissue-like stiffness: pathological implications for soft or stiff microenvironments (cited pelham, beningo) (FOLLOWS TIMELINE)

-Study had major implications for in vivo introduction of stem cells into diseased or damaged striated muscle of altered mechanical compositions and attempted to elucidate a role for matrix or tissue stiffness in striated muscle differentiation

Although muscle differentiation in cell culture has been described for many years the results from this paper appear the FIRST to show that membrane adhesion results increase exponentially with substrate stiffness

Absence of stiff ECM resistance, assembly and phosphorylation of adhesion proteins are inhibited → F-actin assembly reduced. Thus on soft substrates striation is absent

Studies here show that differentiation in culture depends intimately on optimal outside-in signalling of matrix elasticity

Relevant for attempts in introducing SCs in dystrophic SK or infarcted regions of myocardium → due to induced stiffening, implanted SCs are unlikely to fully differentiate into contractile muscle. Instruction of SCs for tissue repair require signaling pathways involved.

Discher et al., 2005: Tissue feel and respond to the stiffness of their substrate (it’s a REVIEW) (FOLLOWS TIME LINE) (CITES PELHAM, ENGLER 2004)

Contractile state can be strongly influenced by the stiffness of the anchoring substrate

“Cell aggregation of less differentiated cells such as some stem cells that assemble into “embryoid bodies”..yet to be studied with myosin inhibitors or related methods, but the principles may extend to stem cell differentiation” → this keys into our paper

“Emphasized here is the recent recognition that most tissue cells not only adhere to but also pull on their microenvironment and thereby respond to its stiffness in ways that relate to tissue elasticity.”

Focal Contacts as Mechanosensors (2001)

https://www.researchgate.net/publication/274955460_Focal_Contacts_as_Mechanosensors

Thus, as long as mDia1 is active, external tension force bypasses the requirement for ROCK-mediated myosin II contractility in the induction of focal contacts. Our experiments show that integrin-containing focal complexes behave as individual mechanosensors exhibiting directional assembly in response to local force.

Pittenger et al., 1999: Multilineage potential of adult human mesenchymal stem cells (JUST ABOUT MSCs?? → supposedly about MSC differentiation into myoblasts/osteoblasts? Cited in paper intro)

-Were able to isolate MSCs and induce their differentiation using drugs and growth factors

-Experiments showed that the isolated expanded hMSC culture would differentiate in a controlled manner to multiple lineages

-Concluded that basal nutrients, cell density, spatial organization, mechanical forces, growth factors, and cytokines had an influence on human mesenchymal cells’ differentiation

-Specific environmental cues to initiate the proliferation + differentiation of hMSCs in vivo not understood but had the ability to direct the differentiation of hMSCs in vitro to allow for study of commitment + differentiation

Watt & Hogan, 2000:

cell locomotion and focal adhesions (1997)

https://www.pnas.org/content/94/25/13661

Microinjection of fluorescent vinculin indicated that focal adhesions on flexible substrates were irregularly shaped and highly dynamic whereas those on firm substrates had a normal morphology and were much more stable

In this study, we have developed a thin polyacrylamide-based, collagen-coated flexible substrate. By maintaining a constant total concentration of acrylamide while varying the concentration of bis-acrylamide, we were able to obtain a series of chemically identical substrates with a wide range of flexibility. By using imaging techniques, we show that cells can respond to differences in substrate flexibility by altering both their adhesion structures and motile behavior. Moreover, the response appears to involve both tyrosine phosphorylation and forces generated by the actin-myosin cytoskeleton.

Characterization of Polyacrylamide Gels.The flexibility of polyacrylamide sheets with different acrylamide/bis-acrylamide ratios was determined both macroscopically and microscopically. Sheets of 69 mm × 30 mm × 1 mm were deformed with a force (F⊥) of 0.103 N (Fig. 1A), and Young’s modulus (19) was calculated according to the equation: Y = (F⊥/A)/(Δl/l), where l is the original length of the sheet, Δl is the change in length, and A is the cross-sectional area. Compliance or “stiffness” of thin gels used for cell culture was characterized microscopically by deforming with glass microneedles (20). The same needles were calibrated by measuring vertical movements of the tip after applying known submilligram weights. Gel compliance was calculated as N/μm deformation. Uncoated and collagen-coated substrates gave identical compliance and Young’s modulus values.

-Paper examined the idea that mechanical forces may play a role in adhesion responses, and that cells may actively probe and respond to mechanical cues in the surrounding environment -Examined the impact of elastic and rigid surfaces on epithelial cells (NRK, kidney) and 3T3 fibroblasts, and concluded that the cells altered their spreading and speed of movement depending on the type of matrix they were on -Used thin polyacrylamide-based, collagen-coated flexible substrate (maintaining constant concentration of acrylamide while varying bis-acrylamide allowed for the range of flexibility) → elasticity of substrate was measured by applying known forces to large suspended sheets to view recoil and also measured via calibrated needle to determine substrate “stiffness” → used KT5926 (potent inhibitor of myosin light chain kinase) + 2,3-butanedione monoxime (an inhibitor of myosin motors). Due to inhibition, cells showed neither enhanced motility or rapid changes in adhesion structures, which reflect the involvement of myosin-generated forces in these processes-Conclusion of study: Tests with 3T3 and NRK cells demonstrate ability of cultured cells to detect the flexibility of the surrounding environment . Increases in tyrosine phosphorylation, in response to substrate stiffness cause the formation of mature, stable focal adhesions (thus, signals elicited by a stiff substrate involve either the stimulation of a tyrosine kinase or inhibition of a tyrosine phosphatase). Results strongly indicate that communications through physical signals are as important as communications through chemical messengers

Most tissue cells are not viable upon dissociation and suspension in a fluid, even if soluble proteins are added to engage cell adhesion molecules e.g. integrin RGD peptide (1987)