BME404 Lab Module 3: BM-MSC fate depents on ECM elasticity as well as soluble biologic modifier Background Stem cell

fate is influenced by a number of factors and interactions that require robust control for safe and effective regeneration of functional tissue. Coordinated interactions with soluble factors, other cells, and extracellular matrices define a local biochemical and mechanical niche (Fig 1A) with complex and dynamic regulation that stem cells sense. Control over stem cell trafficking, survival, proliferation, and differentiation within a complex in vivo milieu is extremely challenging. Whether in vitro or in vivo, cells generate force and are often exposed to force and both can influence stem cell fates. The very first stages of cell differentiation in embryogenesis are indeed blocked after knockout of ubiquitous force-generating myosin. Stem cells may well have more than the typical ensemble of force-coupled signaling pathways as a means to sensitize themselves to microenvironments that range-physicallyfrom flowing fluids and strained tissues to solid tissues of varied elasticity (Fig 1B). Indeed, when mesenchymal stem cells (MSCs) are grown on firm gels that mimic the elasticity of muscle and that are coated with collagen I, myogenic markers are upregulated, whereas when MSCs are grown on rigid gels that mimic pre-calcified bone, the cells appear osteogenic (Engler AJ. Cell. 2006). Added induction factors can either augment or oppose this programming of MSCs by matrix. Over the last 10 years, the importance of substrate stiffness as a mechanism for modulating cell shape and phenotype has been increasing studied, Work on differentiated tissue-forming cell types suggests that each responds to a different and specific range of substrate stiffness and often exhibits the most in vivo-like morphology when the gel stiffness matches its native tissue compliance. As seen from the use of various gel matrices with well-controlled elasticity and non-limiting ligand density, most of cells are found to adhere, to spread, to assemble their cytoskeleton and to anchor more strongly to stiff substrates compared with soft substrates(Fig. 1C & 1D). Extra cell matrix can also be a more potent differentiation cue for MSCs than standard induction cocktails. Polyacrylamide offers a well-characterized system for modulating substrate stiffness while maintaining control over adhesive ligand density and composition. In module 3, we will use polyacrylamide gel (PA gel) with different stiffness to observe how the mechanics of matrix systems to influence stem cells differentiation.

Fig. 1. A) Stem cell niche. Soluble and matrix-bound factors combine with cell-cell contact, cell-matrix adhesion, and gradients to direct cell fate. B) Forces and ECM in stem cell trafficking. Soft tissue elasticity scale ranging from soft brain, fat, and striated muscle, to stiff cartilage and pre-calcified bone. C) In vitro substrates that mimic soft and stiff tissue microenvironments show that cells anchor more strongly to stiff substrates, building focal adhesions and actin-myosin stress fibers. D) Proposed force-sensing mechanism within stem cells. Changes in protein folding as forces are exerted to expose binding sites. Cells on soft matrix with weak intra-cellular forces cannot sufficiently change the conformation of a mechanically-sensitive protein of interest to expose acryptic binding site, making it non-functional. By comparison, cells on stiff matrix generate high tension, which causes the protein to unfold to such a degree that the binding site is rendered non-functional.

Overview of Procedure

Fig. 2. The fates of stem cells. In chemically induced differentiation, MSCs are cultured with different induce medium, resulting in different differentiated cell type. Also, there is a mechanically induced differentiation pathway. Module Objectives: 1. To determine how the mechanics of matrix systems to influence stem cells differentiation. 2. To compare the stiffness of matrix (mechanical factor) and chemical factors induced stem cell differentiation. Recommending Reading Materials: 1. Reilly GC, Engler AJ. Intrinsic extracellular matrix properties regulate stem cell differentiation. J Biomech. 2010 Jan 5;43(1):55-62. 2. Winer JP, Janmey PA, McCormick ME, Funaki M. Bone marrow-derived human mesenchymal stem cells become quiescent on soft substrates but remain responsive to chemical or mechanical stimuli. Tissue Eng Part A. 2009 Jan;15(1):147-54. 3. Kandow CE, Georges PC, Janmey PA, Beningo KA. Polyacrylamide hydrogels for cell mechanics: steps toward optimization and alternative uses. Methods Cell Biol. 2007;83:29-46. 4. Engler AJ, Sen S, Sweeney HL, Discher DE. Matrix elasticity directs stem cell lineage specification. Cell. 2006 Aug 25;126(4):677-89. 5. George PC, Janmey PA. Cell type-specific response to growth on soft materials. J Appl Physiol. 2005 Apr;98(4):1547-53.

6. Engler AJ, Richert L, Wong JY, Picart C, Discher DE. Surface probe measurements of the elasticity of sectioned tissue, thin gels and polyelectrolyte multilayer films: correlations between substrate stiffness and cell adhesion. Surf Sci.2004. 570; 142-154. 7. Pelham RJ, Wang YL. Cell locomotion and focal adhesions are regulated by substrate flexibility. Proc Natl Acad Sci. 1997. 94; 13661-13665.

Module 3. Experiment 5 (March 5, 2012, 8:00-11:00) Location: HSC T16-035 common lab Aim: 1. To acquaint PA gel formula and preparation process for cell culture 2. To seed human MSCs on PA gels Materials: 1. 2. 3. 4. 5. hMSC cell suspension Standard hMSC growth medium (SGM) Fibronectin-mixed PA gels MSC GM: Low glucose DMEM + 20% FBS + 1% penicillin/streptomycin MSC MIM: Low glucose DMEM + 20% FBS + 1% penicillin/streptomycin + 100 nM Dexamethasone + 50 uM Hydrocortisone Stock solution (ml) 1 2 3 4 5 6 10 % APS PBS TEMED 30% acrylamide 1% bisacrylamide 1.25 mg/ml Fibronectin Total PA gel 1 Soft gels 350:1 0.04 6.13 0.004 1.333 0.24 0.25 8 PA gel 2 Medium gels 35:1 0.04 4.17 0.004 1.333 2.4 0.25 8 PA gel 3 Hard gels 24:1 0.04 2.85 0.004 1.333 3.52 0.25 8

Note: APS: 1/200 final volume, TEMED: 1/2000 final volume, final concentration of acrylamide 5%, final concentration of bisacrylamide: 0.03%, 0.3%, 0.44% (from group 1 to 3). Question: what is the purpose of hFN in the PA gel? Procedure (under aseptic conditions) 1. Make small pieces of PA gel and put them into 12-well plates appropriate wells, rinse one time with PBS, kept in MSC-GM over week end . 2. Seed cells (density: 500 cells/ml x 2 ml) into appropriate wells of each of 12-well plates in MSC-SGM. 3. After 24 hours, transfer the MSC seeded_PA gel pieces into new wells. 4. Maintain the cell-seeded wells in a 37C, 5% CO2 incubator for 1 week, re-feed the cells every 2 days. Each group has duplicate PA gel samples for each condition.

Module 3. Experiment 6 (March 12, 2012, 8:00-11:00) Induction of hMSC differentiation to Myoblast by MSC-MIM #1. Control: Keep cells in MSC-SGM for 7 days. Fixed with 3.7% formaldehyde (see experiment 7) and keep the fixed sample at 4C until March 19, 2012 for experiment 7. #2. Control: Keep cells in MSC-SGM for 14 days #3. Induction experiment: Keep cells in MSC-SGM for 7 days, change medium to MSC-MIM for 7 days. Procedure for #2 and #3: 1. Remove SGM . 2. Add 2 ml/well of MSC-SGM to control group or 2 ml/well of MSC-MIM to Induction group. 3. Maintain the cells-seeded plates at 37C, 5% CO2 incubator for 1 week, re-feed the cells every 3 days Group 1 #1 and #2 Group 2 #1 and #3 Group 3 #2 and #3

Module 3. Experiment 7 (March 19, 2012, 8:00-11:00) Location: HSC T16-035 common lab Aim 1: To assay myoblast-like cells differentiation by immuno-staining Materials 1. 2. 3. 4. 5. 6. 7. 8. MSCs culture from Experiment 5 PBS 1% BSA-PBS 0.4% Triton X-100 Parafilm Aluminum foil 3.7% formaldehyde-PBS Primary antibody Pri-SP-M: Mouse anti-MyoD1, pre-made solution 1:100 dilution in 1% BSAPBS SP-c: Alexa Fluor 647 phalloidin, the vial contents be dissolved in 1.5 ml methanol to yield a final concentration of 200 units/ml (Stock solution). Work solution: 1:100 dilution in 1% BSA-PBS Sec-SP-M: Alexa fluor 488 Goat anti-mouse IgG (H+L), work solution: 1:100 dilution in 1% BSA-PBS DAPI: work solution: 1:200 dilution in 1% BSA-PBS Fluorescence microscope Staining target Actin MyoD Differentiation or cytoskeleton indicator Cytoskeleton Myoblast-like cell differentiation marker

Staining Protocol SP-c SP-M

Procedure (all steps handle at room temperature) 1. discard media from each well. 2. Wash 2 x 1000 ul with PBS 3. Fix cells with 1000 ul 3.7% formaldehyde-PBS for 10 min. 4. Wash 3 x with 1000 ul PBS 5. Permeabilize cell membrane with 1000 ul/well 0.4% Triton X-100, 5 min at room temperature. 6. Wash 3 x with 1000 ul PBS. 7. Add 1000 ul/well primary antibody Pri-SP_M and maintain for 1 hour 8. Rinse 3 X with 1000 ul/well PBS with 2-3 min gentle agitation per wash. 9. Add 1000 ul/well labeling mixture (SP-c/Sec-SP-M/DAPI), 30 min at room temperature. 10. Wash 3 x with 1000 ul/well PBS 11. Add 1000 ul/well PBS 12. Seal plates in parafilm and wrap in aluminum foil. 13. Store plates at 4C until imaging acquisition

14. View and take pictures of all samples by using Fluorescence Microscope at excitation of 488 nm for Myo D, 647 nm for phalloidin, or DAPI. Data analysis: Compare changes of cell number from day 7 to day 14, staining of cells under all conditions (reference peer-reviewed journals)

Module 3 Lab Report Module 3 lab report questions will be posted on Monday, March 26, 2011. The report should be completed by groups and would be presented on April 9, 2012