matrix elasticity directs stem cell lineage specification adam j. engler 1,2, shamik sen 1,2, h. lee...
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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