Essential environmental cues from the satellite cell niche : optimizing proliferation and differentiation
Citation for published version (APA):
Boonen, K. J. M., Rosaria - Chak, K. Y., Baaijens, F. P. T., Schaft, van der, D. W. J., & Post, M. J. (2008). Essential environmental cues from the satellite cell niche : optimizing proliferation and differentiation. Poster session presented at Mate Poster Award 2008 : 13th Annual Poster Contest.
Document status and date: Published: 01/01/2008 Document Version:
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Proliferation
Conclusions
Elasticity of the substrate influenced proliferation and maturation: Cells grown on coverslips and substrates of 21 kPa (close to physiological elasticity of skeletal muscle), proliferated most and for the longest duration. In addition, maturation (evaluated by cross-striations and spontaneous contractions), was best on coverslips, followed by 21 and 80 kPa gels and lastly 3 kPa gels.
The extracellular matrix proteins used for coating of the substrate only influenced differentiation of the cells: more and thinner myotubes were found on poly-lysine and laminin, compared to Matrigel, ECL-gel and Collagen IV.
References
[1] Collins, C. A., I. Olsen, et al. (2005). Cell 122(2): 289-301.
[2] Boonen, K.J.M., and Post, M.J (2008). Tissue Eng Part B Rev [3] Engler, A. J., S. Sen, et al. (2006). Cell 126(4): 677-89
Essential environmental cues
from the satellite cell niche
Optimizing proliferation and differentiation
K.J.M. Boonen, K.Y. Rosaria-Chak, F.P.T. Baaijens, D.W.J. van der Schaft, M.J. Post
/ Department of Biomedical Engineering/ Biomechanics and Tissue Engineering
Introduction
Satellite cells (SCs) are very efficient in regenerating muscle defects in vivo. However, their functionality in vitro has been disappointing.1 We hypothesized this is due to loss of the natural niche2 and anticipated that this niche
needs to be mimicked in culture conditions.2 Therefore, we explored the influence of substrate elasticity3 and protein
coating on differentiation and proliferation capacity of SCs.
Material and Methods
Cell isolation and culture: Single fibers were isolated from
muscles of C57BL/6 mice, and SCs were liberated with a 19G needle. Passage 0 cells were plated (1000 cells/cm2)
on coated coverslips or polyacrylamide (PA) gels (figure 1).
Differentiation: MyoD/Myosin/DAPI immunocytochemical
stainings were performed to evaluate differentiation and maturation.
Proliferation: SCs were exposed to BrdU for 16 hours, after
which BrdU incorporation was detected with a microscope.
Results
Differentiation & Maturation
Figure 4: Proliferation of SCs on poly-lysine-coated
substrates. Proliferation was significantly higher and for a longer duration of SCs cultured on glass, followed by cells cultured on 21 kPa gels, then cells cultured on 80 kPa gels and lastly cells cultured on 3 kPa gels. No differences were found in proliferation between protein coatings (data not shown).
Figure 3: Cross-striated myotubes (arrows) grown on different elasticities.
Myosin cross-striations and spontaneous contractions (both signs of maturation) were observed most in myotubes grown on glass, followed by myotubes on 21 and 80 kPa gels, and were not observed in myotubes cultured on 3 kPa gels.
Figure 2: Differentiation on coated coverslips after 11 days.
Timing of differentiation did not depend on elasticity or protein coating. However, more and thinner myotubes were formed on laminin and poly-lysine, compared to Matrigel (not shown), ECL-gel and Collagen IV.
Figure 1: Preparation of polyacrylamide (PA) gels with
elastic moduli ranging from 3 to 80 kPa. Coverslips and PA gels were coated with MatrigelTM, ECL-gel, collagen IV, poly-D-lysine and laminin.
Poly-lysine 0 0.25 0.5 0.75 1 4 6 8 11 15 Time (days) ra ti o B rdU 3 kPa 21 kPa 80 kPa coverslip Poly. (3 kPa) Poly. (21 kPa) Poly. (80 kPa) Poly. (coverslip)
