Expression of α-Smooth Muscle Actin Regulates the Function Of Mesenchymal Stromal Cells

Mesenchymal stromal cells (MSCs) are defined by their ability to undergo clonal expansion, multi-lineage differentiation and tissue repair/regeneration. Due to these properties, MSCs have been suggested and partly utilized to therapeutically treat a wide range of fibroproliferative diseases, including fibrosis and cancer. However, whether the mechanical and biochemical microenvironments present in these disease conditions drive MSCs into a pro-fibrotic `myofibroblast' fate has not been systematically investigated. We observed that rat and human MSCs become alpha-smooth muscle actin (SMA) positive myofibroblasts under the influence of mechanical stiffness and profibrotic cytokines. When sorted on basis of SMA expression, SMA-negative MSCs had high clonogenic potential and SMA-positive MSCs displayed a bias towards osteogenic fate. Plating of SMA-positive MSCs on soft culture substrates resulted in reduced SMA levels and bias toward the adipogenic fate. Knockdown of SMA in SMA-positive MSCs resulted in elevated clonogenic capacity. We reveal that SMA expression itself is part of the mechanism driving fibrogenic activation of MSCs by increasing F-actin levels and regulating the nuclear localization/activity of the mechanosensitive transcription factor YAP. Using human sarcoma patient samples, we correlated low SMA expression levels with inactive YAP/TAZ in human adiposarcoma/liposarcoma as opposed to high SMA expression and active YAP/TAZ in human osteosarcoma. We conclude that SMA mediated contraction plays a critical role in mechanically regulating clonal expansion and multi-lineage differentiation of MSCs by controlling YAP/TAZ activation. Next, to investigate the effect of extracellular matrix stiffness on the tissue repair capacity of MSCs in vivo, we isolated and sub-cultured rat MSCs from bone marrow to establish on either soft or stiff substrates. Stiff substrate expansion primed MSCs towards fibrotic behaviour that was maintained even after exposing these cells to soft substrates. Conversely, soft substrate priming protected MSCs from myofibroblast activation upon subsequent exposure to stiff substrates, establishing a `mechanical memory' in MSCs. Using a rat wound healing model to reproduce aspects of hypertrophic scars, we demonstrated that transplantation of soft primed MSCs reduced scar contraction and increased vascularization as compared to rigid-primed cells. Transplantation of stiff primed MSCs to the scar model resulted in increased collagen fiber deposition, remodeling, and alignment. The higher levels of myofibroblasts forming in stiff primed MSC treated wounds coincided with increased scar contracture when compared to either soft-primed MSC transplants or cell-free controls. We conclude that the stiffness of culture substrates used for MSC expansion dictate the regenerative potential of MSCs in a fibro-proliferative disease setting. We propose that isolation as well as expansion of MSCs on soft substrates would provide non-fibrogenic cells for applications in regenerative medicine. Next, to investigate the effect of extracellular matrix stiffness on the tissue repair capacity of MSCs in vivo, we isolated and sub-cultured rat MSCs from bone marrow to establish on either soft or stiff substrates. Stiff substrate expansion primed MSCs towards fibrotic behaviour that was maintained even after exposing these cells to soft substrates. Conversely, soft substrate priming protected MSCs from myofibroblast activation upon subsequent exposure to stiff substrates, establishing a `mechanical memory' in MSCs. Using a rat wound healing model to reproduce aspects of hypertrophic scars, we demonstrated that transplantation of soft primed MSCs reduced scar contraction and increased vascularization as compared to rigid-primed cells. Transplantation of stiff primed MSCs to the scar model resulted in increased collagen fiber deposition, remodeling, and alignment. The higher levels of myofibroblasts forming in stiff primed MSC treated wounds coincided with increased scar contracture when compared to either soft-primed MSC transplants or cell-free controls. We conclude that the stiffness of culture substrates used for MSC expansion dictate the regenerative potential of MSCs in a fibro-proliferative disease setting. We propose that isolation as well as expansion of MSCs on soft substrates would provide non-fibrogenic cells for applications in regenerative medicine.

Keywords: Actin
fibrosis
mechanobiology
mesenchymal stromal cells
myofibroblasts
YAP
Advisor: Hinz, Boris