Importantly expression of SMA is not simply a marker for

Importantly, expression of α-SMA is not simply a marker for MF activation, but also the driver of cell function and fate. Increased expression of α-SMA directly reduces the clonal potential of hMSCs and guides their differentiation toward osteoblasts. Hence, α-SMA not only identifies osteoprogenitors in hMSC populations as shown by others before (Grcevic et al., 2012; Kalajzic et al., 2008), but may be part of the mechanism driving differentiation. Analysis of bone marrow-derived hMSCs treated with TGF-β1 and/or exposed to fibrosis-stiff culture substrates has previously revealed a fibrogenic MF activation program (Park et al., 2011; Wang et al., 2004). Our results additionally demonstrate that neo-expression of α-SMA associates with reduced clonogenicity and lineage differentiation potential of hMSCs and that this program is reversible. SMA(+) hMSC can be deactivated to lose fibrotic MF features by reducing substrate stiffness. Originally considered to be a terminal differentiation state of various precursor cells, deactivation of the MF has been shown in recent experimental models of kidney and liver fibrosis (Hecker et al., 2011; Kisseleva and Brenner, 2013). In vitro, the depletion of MF features in fibroblasts and MSCs is achieved by culture on soft silicone substrates (Achterberg et al., 2014; Balestrini et al., 2012; Goffin et al., 2006; Park et al., 2011) or by treatment with anti-fibrotic growth factors (Desai et al., 2014). Our results add to these findings that SMA(+) hMSC populations, deactivated to lose MF features, regain adipogenic lineage differentiation potential rather than turning into α-SMA-negative fibroblasts. Hence, α-SMA-positive tlr signaling are likely derived from previously α-SMA-negative hMSCs and expression of α-SMA reversibly reduced their clonogenicity and lineage potential. Our findings have important implications for hMSC therapy in fibroproliferative diseases, including tumor formation and development of fibrosis. Our in vivo data show that the percentage of α-SMA-expressing MFs in sarcomas correlates with the degree of YAP/TAZ activation. A wide variety of different tumors have been shown to accumulate stromal cells that are positive for α-SMA and perform MF functions, including stiffening of the stroma and promoting tumor progression (Hinz et al., 2012; Öhlund et al., 2014). Consistently, YAP expressed in cancer-associated fibroblasts was recently shown to play an important role in controlling of cytoskeleton-regulating genes, as well as tumor cell invasion and ECM stiffening (Calvo et al., 2013). We propose a feedforward loop of MSC-to-MF activation in the tumor microenvironment, leading to higher contractile cells and stiffer ECM, which both lead to increased YAP/TAZ activity and conversion of regenerative MSCs into fibrotic MFs. Interrupting this feedforward loop will have important consequences for hMSC potential in clinical applications. First, specific inhibitors of α-SMA such as the SMA-FP or shRNA strategies may be co-delivered with hMSCs to exert fibrosis-inhibitory effects on the resident fibrotic cell population in the lesion. Second, suppressing MSC-to-MF activation during cell culture expansion will enhance the fraction of valuable stem cells and reduce the risk of fibrosis upon implantation. We have shown that explantation and continued culture on soft culture substrates renders populations of lung fibroblasts resistant to subsequent mechanical activation on stiff substrates over several consecutive passages (Balestrini et al., 2012). This concept of cells acquiring a “mechanical memory” has recently been confirmed for MSC lineage programs on a shorter timescale with YAP/TAZ being involved (Yang et al., 2014). It is yet unclear why hMSCs and other mesenchymal cells that have been cultured on conventional stiff culture dishes are not similarly primed and can at least acutely (up to 8 days in our experiments) lose fibrogenic character upon short-term exposure (5 days) to soft substrates. In our own studies, we found it to be essential to use substrates with a pathophysiological stiffness range (1–100 kPa) and to never expose cells to tissue culture plastic for mechanical priming to occur (Balestrini et al., 2012).