1. Jason Ip Graduate Student
Matrix Elasticity Directs Stem Cell lineage Specification Adam J. Engler1,2, Shamik Sen1,2, H. Lee Sweeney1, and Dennis E. Discher1,2,3,4 1Pennsylvania Muscle Institute 2School of Engineering and Applied Science 3Cell & Molecular Biology Graduate Group 4Physics Graduate Group University of Pennsylvania
Jason Ip
Graduate Student

2. 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

3. 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

4. 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

5. Result #1: Tissue Elasticity and Differentiation
Cell morphology suggests lineage specification determined by E of the substrate
Naïve MSCs are initially small and round, but develop differently as E changes
Neurogenic
Myogenic
Osteogenic

6. Result #1: Tissue Elasticity and Differentiation
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
Neurogenic
Myogenic
Osteogenic

7. 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
Nestin, B3tubulin, MAP2, NFL are all neural marker proteins

8. 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
The bottom figures are of osteocalcin secreted by osteoblats on a glass substrate 7 days after plating
The distribution of matrix thickness and osteo-matrix elasticity show a means at values characteristic of concentrated gel

9. 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

10. 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

11. 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

12. 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
2) Because it spends more time attached to actin
3) Demonstrates expression regulation

13. 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

14. 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

15. Conclusions 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

16. Critiques Induction of neurogenesis Induction media assays
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 ?
Should have had more samples

17. Questions?