Matrix elasticity regulates the secretory profile of human bone marrow-derived multipotent mesenchymal stromal cells (MSCs)

doi:10.1016/j.bbrc.2009.09.051

Biochemical and Biophysical Research Communications

Volume 389, Issue 4, 27 November 2009, Pages 663-667

https://doi.org/10.1016/j.bbrc.2009.09.051 Get rights and content

Abstract

The therapeutic efficacy of multipotent mesenchymal stromal cells (MSCs) is attributed to particular MSC-derived cytokines and growth factors. As MSCs are applied locally to target organs or home there after systemic administration, they experience diverse microenvironments that are biochemically and biophysically distinct. Here we use well-defined in vitro conditions to study the impact of substrate elasticity on MSC-derived trophic factors. By varying hydrogel compliance, the elasticity of brain and muscle tissue was mimicked. We screened >90 secreted factors at the protein level, finding a diverse elasticity-dependent expression pattern. In particular, IL-8 was up-regulated as much as 90-fold in MSCs cultured for 2 days on hard substrates, whereas levels were consistently low on soft substrates. In summary, we show substrate elasticity directly affects MSC paracrine expression, a relevant finding for therapies administering MSCs in vivo.

Introduction

Bone marrow-derived multipotent mesenchymal stromal cells (MSCs) are of great interest for tissue engineering strategies [1] as they can be readily isolated from adult donors, expanded in vitro, and differentiate into osteoblasts, chondrocytes and adipocytes [2]. Due to their robust differentiation into these mesenchymal lineages, they have been used in a number of clinical trials for bone and cartilage reconstruction [3]. In addition, MSCs show promising immune modulatory effects that are being studied in Phase I–III clinical trials to treat graft-versus-host disease and autoimmune diseases [4]. MSCs also have a therapeutic benefit when administered to areas of acute ischaemic injury, e.g. myocardial and cerebral (reviewed in [5], [6]).

Although MSCs have long been recognized to produce a myriad of chemokines, cytokines and growth factors [7], only recently has the role of specific factors responsible for MSC-derived trophic effects started to be unravelled (reviewed in [4]). Importantly, matrix elasticity has been shown to regulate MSC lineage specification [8]. This is particularly relevant for therapeutic aims involving the direct application of MSCs into different tissues/organs, exposing them to a spectrum of elastic environments. Here we test the impact of substrate compliance on trophic factor expression by using model substrates mimicking the elasticity of brain and muscle tissue. We show that human MSCs differentially express trophic factors in response to matrix compliance.

Section snippets

Materials and methods

Culture substrates. Hydrogel components were purchased from Glycosan BioSystems (Salt Lake City, USA). The chemical synthesis and characteristics of the reagents have been detailed elsewhere [9], [10]. Hydrogel substrates of variable stiffness were prepared using 1% w/v or 8% w/v of the crosslinker polyethylene glycol diacrylate (M_w 3400 g/mol) added to thiol-modified hyaluronic acid (10 mg/ml) and gelatine (10 mg/ml) and allowed to polymerise for ∼90 min. Hydrogels were then washed extensively with

Results

To test the effect of substrate compliance on MSCs’ secretory profile, we used hydrogels in which substrate elasticity was tailored to 1–2 and 15–20 kPa by titrating the amount of crosslinker (Fig. 1A). The initial MSC response (day 2) to the hydrogels was qualitatively assessed by examining cell spreading/shape after staining the cell cytoskeleton with phalloidin. We found cells acquired a more stretched/elongated shape on soft substrates compared to stiff substrates (Fig 1B), indicating that

Discussion

MSC-derived cytokines and growth factors are thought to play an essential part in regenerative therapies by modulating the immune response, inhibiting fibrosis and apoptosis, inducing angiogenesis, and stimulating tissue resident stem/progenitor cells to contribute to tissue regeneration (reviewed in [5]). The myriad of trophic factors produced by MSCs have been proposed as one of the key features of their therapeutic versatility [7]. Thus understanding the impact of micro-environmental cues,

Disclosure statement

The authors have no competing financial interests.

Acknowledgments

We would like to thank Dr. Konrad Schneider and Woranan Panyanuwat for providing measurement time and performing flat punch measurements, respectively. Dr. Tilo Pompe is gratefully acknowledged for helpful discussions and Laurel Rohde for proofreading the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft, “Collaborative Research Centre: Cells into tissues- stem cell and progenitor commitment and interactions during tissue formation” (SFB 655).

References (32)

A.J. Engler et al.
Matrix elasticity directs stem cell lineage specification
Cell
(2006)
K. Ghosh et al.
Cell adaptation to a physiologically relevant ECM mimic with different viscoelastic properties
Biomaterials
(2007)
R. McBeath et al.
Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment
Dev. Cell
(2004)
D.H. Kim et al.
Gene expression profile of cytokine and growth factor during differentiation of bone marrow-derived mesenchymal stem cell
Cytokine
(2005)
C.H. Liu et al.
Cytokine interactions in mesenchymal stem cells from cord blood
Cytokine
(2005)
J.Y. Oh et al.
Cytokine secretion by human mesenchymal stem cells cocultured with damaged corneal epithelial cells
Cytokine
(2009)
D. Baksh et al.
Adult mesenchymal stem cells: characterization, differentiation, and application in cell and gene therapy
J. Cell Mol. Med.
(2004)
D.J. Prockop
Marrow stromal cells as stem cells for nonhematopoietic tissues
Science
(1997)
A. Giordano et al.
From the laboratory bench to the patient’s bedside: an update on clinical trials with mesenchymal stem cells
J. Cell. Physiol.
(2007)
A. Uccelli et al.
Mesenchymal stem cells in health and disease
Nat. Rev. Immunol.
(2008)

J.S. Burchfield et al.

Role of paracrine factors in stem and progenitor cell mediated cardiac repair and tissue fibrosis

Fibrogenesis Tissue Repair

(2008)

K.H. Schuleri et al.

Mesenchymal stem cells for cardiac regenerative therapy

Handb. Exp. Pharmacol.

(2007)

A.I. Caplan et al.

Mesenchymal stem cells as trophic mediators

J. Cell. Biochem.

(2006)

K. Ghosh et al.

Rheological characterization of in situ cross-linkable hyaluronan hydrogels

Biomacromolecules

(2005)

L. Cheng et al.

Flat-punch indentation of viscoelastic material

J. Polym. Sci. B: Polym. Phys.

(2000)

F.P. Seib et al.

Engineered extracellular matrices modulate the expression profile and feeder properties of bone marrow-derived human multipotent mesenchymal stromal cells

Tissue Eng. A

(2009)

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¹: www.crt-dresden.de.

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Matrix elasticity regulates the secretory profile of human bone marrow-derived multipotent mesenchymal stromal cells (MSCs)

Abstract

Introduction

Section snippets

Materials and methods

Results

Discussion

Disclosure statement

Acknowledgments

Cell

Biomaterials

Dev. Cell

Cytokine

Cytokine

Cytokine

Adult mesenchymal stem cells: characterization, differentiation, and application in cell and gene therapy

J. Cell Mol. Med.

Marrow stromal cells as stem cells for nonhematopoietic tissues

Science

From the laboratory bench to the patient’s bedside: an update on clinical trials with mesenchymal stem cells

J. Cell. Physiol.

Mesenchymal stem cells in health and disease

Nat. Rev. Immunol.

Role of paracrine factors in stem and progenitor cell mediated cardiac repair and tissue fibrosis

Fibrogenesis Tissue Repair

Mesenchymal stem cells for cardiac regenerative therapy

Handb. Exp. Pharmacol.

Mesenchymal stem cells as trophic mediators

J. Cell. Biochem.

Rheological characterization of in situ cross-linkable hyaluronan hydrogels

Biomacromolecules

Flat-punch indentation of viscoelastic material

J. Polym. Sci. B: Polym. Phys.

Engineered extracellular matrices modulate the expression profile and feeder properties of bone marrow-derived human multipotent mesenchymal stromal cells

Tissue Eng. A