Cytoskeletal organization of human mesenchymal stem cells (MSC) changes during their osteogenic differentiation

Abstract

Human MSCs have been studied to define the mechanisms involved in normal bone remodeling and the regulation of osteogenesis. During osteogenic differentiation, MSCs change from their characteristic fibroblast-like phenotype to near spherical shape. In this study, we analyzed the correlation between the organization of cytoskeleton of MSCs, changes in cell morphology, and the expression of specific markers (alkaline phosphatase activity and calcium deposition) of osteogenic differentiation. For osteoblastic differentiation, cells were cultured in a culture medium supplemented with 100 nM dexamethasone, 10 mM beta- glycerophosphate, and 50 microg/ml ascorbic acid. The organization of microfilaments and microtubules was examined by inmunofluorescence using Alexa fluor 594 phalloidin and anti alpha-tubulin monoclonal antibody. Cytochalasin D and nocodazole were used to alter reversibly the cytoskeleton dynamic. A remarkable change in cytoskeleton organization was observed in human MSCs during osteogenic differentiation. Actin cytoskeleton changed from a large number of thin, parallel microfilament bundles extending across the entire cytoplasm in undifferentiated MSCs to a few thick actin filament bundles located at the outermost periphery in differentiated cells. Under osteogenic culture conditions, a reversible reorganization of microfilaments induced by an initial treatment with cytochalasin D but not with nocodazole reduced the expression of differentiation markers, without affecting the final morphology of the cells. The results indicate that changes in the assembly and disassembly kinetics of microfilaments dynamic of actin network formation may be critical in supporting the osteogenic differentiation of human MSCs; also indicated that the organization of microtubules appears to have a regulatory role on the kinetic of this process.

Low Intensity Pulsed Ultrasound (LIPUS) Influences the Multilineage Differentiation of Mesenchymal Stem and Progenitor Cell Lines through ROCK-Cot/Tpl2-MEK-ERK Signaling Pathway

“…Conversely, it has been reported that cytoskeletal organization is a key regulator of some types of cell differentiation (22). In particular, previous studies have reported that MSCs actively change their cytoskeleton (63) and cell membrane shape when their differentiation is induced (64). We found that ROCK, a major molecule involved in cytoskeletal rearrangements, is involved in LIPUS-induced signal transduction.…”

High-throughput physical phenotyping of cell differentiation

“…Pronounced cell shape changes occur, mediated in part by extensive reorganization of actin into thick bundles at the cell periphery 30,31 . In addition, previous work has also demonstrated that MSCs undergo changes in mechanical properties such as Young's modulus during osteogenesis, although the nature of the change can be method dependent 32,33 .…”

Thymosin beta‐4 directs cell fate determination of human mesenchymal stem cells through biophysical effects

“…4 Moreover, actin cytoskeleton changed from a large number of thin, parallel microfilament bundles extending across the entire cytoplasm in undifferentiated MSCs to a few thick actin filament bundles located at the outermost periphery in MSC-differentiated osteoblasts. 5 These observations clearly demonstrated the role of actin filament in modulating biophysical stimuli at the early stage of osteogenic differentiation.…”

“…3 Such biophysical effects may result from cytoskeleton reorganization, cell-cell interaction, or cell-matrix interaction. 3 For example, during early osteogenic differentiation of human MSCs, remarkable changes of biophysical cues have been observed, including cytoskeleton reorganization 4,5 and integrin redistribution. 6 A stronger interaction between plasma membrane and F-actin, as well as a shorter membrane tether length, was found in MSC-differentiated osteoblasts compared to MSCs.…”