Stretching human mesenchymal stromal cells on stiffness-customized collagen type I generates a smooth muscle marker profile without growth factor addition.

Miriam Rothdiener,Karen A Boehme,Bodo Kurz,Bernd Rolauffs,Tatiana Uynuk-Ool,Piruntha Papugy,Jan P Stegemann,Tanja Seeger,Miriam Hegemann,Ulrich Stoeckle,Phong Nguyen,Melanie L Hart,Gerd Klein,Valentin Claus,Brandan Walters,Wilhelm K Aicher

doi:10.1038/srep35840

Abstract

Using matrix elasticity and cyclic stretch have been investigated for inducing mesenchymal stromal cell (MSC) differentiation towards the smooth muscle cell (SMC) lineage but not in combination. We hypothesized that combining lineage-specific stiffness with cyclic stretch would result in a significantly increased expression of SMC markers, compared to non-stretched controls. First, we generated dense collagen type I sheets by mechanically compressing collagen hydrogels. Atomic force microscopy revealed a nanoscale stiffness range known to support myogenic differentiation. Further characterization revealed viscoelasticity and stable biomechanical properties under cyclic stretch with >99% viable adherent human MSC. MSCs on collagen sheets demonstrated a significantly increased mRNA but not protein expression of SMC markers, compared to on culture flasks. However, cyclic stretch of MSCs on collagen sheets significantly increased both mRNA and protein expression of α-smooth muscle actin, transgelin, and calponin versus plastic and non-stretched sheets. Thus, lineage-specific stiffness and cyclic stretch can be applied together for inducing MSC differentiation towards SMCs without the addition of recombinant growth factors or other soluble factors. This represents a novel stimulation method for modulating the phenotype of MSCs towards SMCs that could easily be incorporated into currently available methodologies to obtain a more targeted control of MSC phenotype.

Highlights

Application[11,12] because mesenchymal stromal cells (MSCs) lineage commitment can be enhanced by specific matrix elasticity
atomic force microscopy (AFM) revealed that all collagen type I sheets were in a relatively narrow stiffness range from approximately 7–11 kPa (Fig. 1)
In contrast to compressive stiffness determined with AFM, the tensile stiffness was determined under cyclic tension

Summary

Introduction

Application[11,12] because MSC lineage commitment can be enhanced by specific matrix elasticity. Previous studies have applied cyclic biomechanical stretch to generate myogenically differentiating MSCs15–17, based on the observation that an elongated MSC morphology is associated with increased expression of smooth muscle cell (SMC) markers[18]. We generated physiologically relevant substrates with stiffness in this range by mechanically compressing collagen type I hydrogels into dense sheets, termed compressed collagen[19]. This type of material has been used previously for generating artificial corneas[20,21], skin[22], bone[23], and bladder[24]. We applied defined cyclic stretch to the compressed collagen sheets and the adhering human bone marrow derived MSCs and assessed the expression of specific markers associated with the SMC phenotype. The ability to reliably differentiate MSCs into SMCs would be an important tool in understanding phenotype shifts in these cells, and would advance the fields of vascular biology and tissue engineering

Methods

Results

Conclusion