Matrix Elasticity Directs Stem Cell lineage SpecificationAdam J. Engler1,2, Shamik Sen1,2, H. Lee Sweeney1, and Dennis E. Discher1,2,3,4

1Pennsylvania Muscle Institute2School of Engineering and Applied Science3Cell & Molecular Biology Graduate Group4Physics Graduate GroupUniversity of Pennsylvania

Jason IpGraduate Student

Introduction• Tissue-level stiffness influences lineage

specification as evident through:▫ 1) Cell morphology▫ 2) Transcript profiles▫ 3) Marker proteins▫ 4) Stability of responses

• The ability of MSCs to sense matrix elasticity requires:▫ 1) The ability to pull against the matrix▫ 2) Transduction of mechanical forces into biological signals

• Nonmuscle myosin II (NMM II) are the suspected mediators▫ Evidence: Tension on cortical actin

Introduction• Anchorage-dependent cells studied

▫ Neurons, Myoblasts, Osteoblasts

• Degree of bis-acrylamide crosslinking determines elasticity

• Matrix elasticity measured by elastic modulus E, where Ebrain<Emuscle<Eosteoid

• Blebbistatin▫ Blocks branching, elongation, and spreading of MSCs on any

substrate▫ Inhibits actin activation of NMM II ATPase activity

Agenda

• Experimental results▫ 1) Tissue elasticity and differentiation▫ 2) Neurogenic branching and osteogenic microenvironments▫ 3) Elastically dependent proteins and transcript profiles▫ 4) Induction through media and matrix▫ 5) Matrix-dependent myosin expression▫ 6) Stiffer matrices and cell tension

Result #1: Tissue Elasticity and Differentiation

Neurogenic Myogenic Osteogenic

• Cell morphology suggests lineage specification determined by E of the substrate

• Naïve MSCs are initially small and round, but develop differently as E changes

Result #1: Tissue Elasticity and Differentiation

Neurogenic Myogenic Osteogenic

• Microarray profiling of MSC transcript markers with varying matrix stiffness

• MSC markers taken from early to mid/late development range

• Blebbistatin-treated cultures lacked specification

Result #2: Neurogenic Branching and Osteogenic Microenvironments

• Experiment driven by uncertainty of matrix-induced neurogenesis

• DMSO causes both MSCs and fibroblasts to appear branched

• Neurogenic branching on matrices of varying stiffness reveal increased branching only on softest gel in the smallest area

• Fibroblasts did not exhibit branching

Result #2: Neurogenic Branching and Osteogenic Microenvironments

• Experiment driven by uncertainty of the role of osteoid – the crosslinked collagen precursor to bone secreted by osteoblasts

• Osteoid is suspected to be the matrix that facilitates MSC to preosteoblast transition

• Measurements (by AFM) of the compliance and thickness of osteoid reveal Eosteoid to be similar to that of concentrated collagen gel

Result #3: Elastically Dependent Proteins and Transcript Profiles

• Cytoskeletal markers and transcription factors also indicated lineage specification

• Upregulation of markers shown by immunofluroescence

• Fluorescence peaks occur at E typical of each cell type

Result #4: Induction Through Media and Matrix

• Myoblast induction media (MIM) is known to promote myogenesis and the expression of myogenic proteins

• Expression of MyoD in C2C12 committed myoblasts is statistically similar to MSCs on myogenic matrix mixed with MIM

• Blebbistatin stops cell spreading but maintains baseline MyoD expression

• Fluorescence peaks occur at E typical of each cell type

Result #4: Induction Through Media and Matrix

• Key points:▫ Lack of MyoD expression by spindle-

shaped blebbistatin-treated MSCs, no spreading

▫ Induced expression through MIM of unspread cells,

▫ MIM-induced MyoD expression on ‘incorrect’ matrices, no specification, only “trans-differentiation”

• Key conclusion:▫ NMM II is in important to lineage

specification

Result #5: Matrix-Dependent Myosin Expression

• Multiple myosins are suspected to be involved in tensioning the ECM

• Myosins in MSCs couple expression to matrix stiffness and reveal key role for NMM IIs

• The kinetics of NMM IIB suggests it generates higher force than NMM IIA

• NMM IIB is upregulated on stiffer matrices, downregulated on softer matrices

Result #5: Matrix-Dependent Myosin Expression

• NMM IIB is upregulated on stiffer matrices, downregulated on softer matrices

• Matrix sensitivity is revealed by:▫ 1) Microarray data clustered by

RNA variation▫ 2) Western blotting▫ 3) Myosin organization in

striation patterns

Result #6: Stiffer Matrices and Cell Tension

• Stiffer substrates promote focal adhesion growth and elongation

• This implies a greater activity of NMM II in probing the microenvironment through actin-myosin contractions

• Contractility can be measured by cellular prestress σ, traction stress τ, and cell cortex stiffness κ

• Blebbistatin prevents cells from developing stress or stiffness with environment

• The results show that the stiffer the matrix, the stiffer the cells are

• Implications for stem cell therapies▫Regenerative therapy of tissue could be complicated

with injured or scarred tissue displaying nonspecific and heterogeneous microenvironment

▫Possible applications include cardiomyoplasty, muscular dystrophy, and neuroplasty

▫Pre-committing stem cells in vitro could optimize matrix specificity for a desired lineage specification

Conclusions

• Induction of neurogenesis▫ Claim: Increase in branching occurs only on softest matrix▫ Rebuttle: Softest matrix reading ‘increase’ not significant relative

to other readings?

• Induction media assays▫ Blebbistatin-treated assay on myogenic matrix but not for

osteogenic matrix?▫ MSCs that are allowed to first spread react to Blebbistatin

differently than early-stage MSCs – Blebbistatin suppresses MyoD later but not earlier…unresolved issue?

▫ Media change experiment: Neuro→Myo,Osteo …but what about Myo→Neuro,Osteo and Osteo→Neuro,Myo ?

Critiques

Questions?