Elsevier

Biomaterials

Volume 32, Issue 16, June 2011, Pages 3921-3930
Biomaterials

The effect of matrix stiffness on the differentiation of mesenchymal stem cells in response to TGF-β

https://doi.org/10.1016/j.biomaterials.2011.02.019Get rights and content

Abstract

Bone marrow mesenchymal stem cells (MSCs) are a valuable cell source for tissue engineering and regenerative medicine. Transforming growth factor β (TGF-β) can promote MSC differentiation into either smooth muscle cells (SMCs) or chondrogenic cells. Here we showed that the stiffness of cell adhesion substrates modulated these differential effects. MSCs on soft substrates had less spreading, fewer stress fibers and lower proliferation rate than MSCs on stiff substrates. MSCs on stiff substrates had higher expression of SMC markers α-actin and calponin-1; in contrast, MSCs on soft substrates had a higher expression of chondrogenic marker collagen-II and adipogenic marker lipoprotein lipase (LPL). TGF-β increased SMC marker expression on stiff substrates. However, TGF-β increased chondrogenic marker expression and suppressed adipogenic marker expression on soft substrates, while adipogenic medium and soft substrates induced adipogenic differentiation effectively. Rho GTPase was involved in the expression of all aforementioned lineage markers, but did not account for the differential effects of substrate stiffness. In addition, soft substrates did not significantly affect Rho activity, but inhibited Rho-induced stress fiber formation and α-actin assembly. Further analysis showed that MSCs on soft substrates had weaker cell adhesion, and that the suppression of cell adhesion strength mimicked the effects of soft substrates on the lineage marker expression. These results provide insights of how substrate stiffness differentially regulates stem cell differentiation, and have significant implications for the design of biomaterials with appropriate mechanical property for tissue regeneration.

Introduction

Bone marrow mesenchymal stem cells (MSCs) are a valuable cell source for tissue engineering and regenerative medicine applications. MSCs are expandable and can differentiate into a variety of cell types, including smooth muscle cells (SMCs), chondrocytes, osteoblasts and adipocytes. The interactions of MSCs with the extracellular matrix (ECM) play an important role in MSC differentiation and function. ECM can be designed to guide MSC differentiation, deliver MSCs for therapies and construct tissues using MSCs. In addition to ECM cues, MSC differentiation is regulated by growth factors. It is noted that transforming growth factor β (TGF-β) promotes MSC specification into a smooth muscle (SM) lineage on stiff substrates [1], [2]. In contrast, TGF-β induces chondrogenic differentiation of MSCs in hydrogels [3]. This phenomenon suggests that the ECM could play an important role in the divergence of cell fate. We postulated that ECM stiffness modulated MSC differentiation into SMC and chondrogenic lineages.

Cells are not only sensitive to dynamic mechanical loading in ECM [4], [5], [6], [7], but are also sensitive to the mechanical properties of the ECM such as the stiffness [8], [9], [10], [11]. The range of ECM stiffness in the body is enormous, from soft, pliable brain tissue with an elastic modulus of tenths of a kilopascal (kPa) to hard, calcified bone with a modulus of hundreds of megapascals (MPa). With many orders of magnitude separating the softest and stiffest tissue in the body, these tissues contain cells that are tuned to the specific mechanical environments in which they reside. Engler et al. have demonstrated that ECM guides MSC differentiation into osteoblastic, skeletal muscular and neural lineages in a manner dependent on ECM stiffness [11]. However, whether and how ECM stiffness regulates MSC differentiation into other cell types such as SMCs and chondrogenic cells are not clear. In this study, we investigated how ECM stiffness modulates MSC differentiation into SMC and chondrogenic lineages in response to TGF-β, and explored the underlying mechanisms.

Section snippets

Materials and methods

The commercial sources of reagents are listed in Supplementary Table 1.

Matrix stiffness and cytoskeleton organization

Expanded MSCs were characterized by the expression of surface markers and the capability of differentiating into osteogenic, chondrogenic and Schwann cells (Fig. S1).

To minimize the compounding effects of soluble factors, the studies of substrate stiffness and MSC differentiation were performed in the low-serum medium unless otherwise specified. We first determined the effects of substrate stiffness on MSCs by using collagen gels. MSCs on stiff substrates exhibited extensive stress fibers (

Discussion

Our study has shown that MSC differentiation is responsive to two important stimuli in the cellular microenvironment: matrix stiffness and growth factors. Matrix stiffness alone can promote a certain lineage over another, i.e. SMC on stiff substrates, and chondrogenic and adipogenic cells on soft substrates. These results are largely correlated with the mechanical properties of the native tissue in which these cells reside. For example, native arteries have MPa stiffness, while fat stiffness is

Conclusions

In conclusion, stiff matrix promotes MSC differentiation into SMC lineage, while soft matrix (∼1 kPa) promotes MSC differentiation into chondrogenic and adipogenic lineage. Although matrix stiffness is an important determinant of stem cell differentiation, its effect may not be specific for only one lineage, and biochemical factors such as TGF-β are required, together with matrix stiffness, to define a unique differentiation pathway. Furthermore, the weak cell adhesion strength on soft matrix

Acknowledgement

This work was supported in part by grants (HL078534 and HL083900 to S.L.) and Predoctoral Fellowships (to A.D.T. and 1F31HL087728 to R. D.) from National Institute of Health"National Institute of Health. A.D.T. and R.D. are Siebel Scholars. A.W. is a postdoctoral CIRM Scholar of California Institute for Regenerative Medicine"California Institute for Regenerative Medicine training grant TG2-01164.

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