Forcing Stem Cells to Behave: A Biophysical Perspective of the Cellular Microenvironment

When combinatorial antibody libraries are rendered infectious for eukaryotic cells, the integrated antibody genotype and cellular phenotype become permanently linked and each cell becomes a selection system unto itself. These systems should be ideal for the identification of proteins and pathways that regulate differentiation so long as selection systems can be devised. Here we use a selection system based on the ability of secreted antibodies to alter the morphology of colonies expressing them when grown in soft agar. Importantly, this approach is different from all previous studies in that it used a pure discovery format where unbiased libraries that were not preselected against any known protein were used as probes. As such, the strategy is analogous to classical forward genetic approaches except that it operates directly at the protein level. This approach led to the identification of integrinbinding agonist antibodies that efficiently converted human stem cells to dendritic cells. intracellular combinatorial antibodies | phenotypic selections

Section: Discussionsupporting

confidence: 78%

Converting stem cells to dendritic cells by agonist antibodies from unbiased morphogenic selections

Yea

Zhang

Xie

et al. 2013

“…We propose that the mechanically gated ion channel Piezo1 is an important determinant of mechanosensitive lineage choice in neural stem cells and may play similar roles in other multipotent stem cells. (1,2). Mechanical effects on fate are particularly relevant for stem cell transplant therapy, because stem cells encounter diverse mechanical signals upon engraftment (3,4).…”

mentioning

confidence: 99%

Stretch-activated ion channel Piezo1 directs lineage choice in human neural stem cells

Pathak¹,

Nourse

Tran³

et al. 2014

452

455

Neural stem cells are multipotent cells with the ability to differentiate into neurons, astrocytes, and oligodendrocytes. Lineage specification is strongly sensitive to the mechanical properties of the cellular environment. However, molecular pathways transducing matrix mechanical cues to intracellular signaling pathways linked to lineage specification remain unclear. We found that the mechanically gated ion channel Piezo1 is expressed by brainderived human neural stem/progenitor cells and is responsible for a mechanically induced ionic current. Piezo1 activity triggered by traction forces elicited influx of Ca 2+ , a known modulator of differentiation, in a substrate-stiffness-dependent manner. Inhibition of channel activity by the pharmacological inhibitor GsMTx-4 or by siRNA-mediated Piezo1 knockdown suppressed neurogenesis and enhanced astrogenesis. Piezo1 knockdown also reduced the nuclear localization of the mechanoreactive transcriptional coactivator Yes-associated protein. We propose that the mechanically gated ion channel Piezo1 is an important determinant of mechanosensitive lineage choice in neural stem cells and may play similar roles in other multipotent stem cells. (5) and that the mechanical properties of the culturing environment before transplantation can influence the outcome of in vivo stem cell transplants (6). Hence, a molecular and mechanistic understanding of how stem cells process mechanical cues and how this processing results in downstream signaling events and ultimately in fate decisions is needed for greater control over the fate of transplanted cells.Studies in mesenchymal and neural stem cells have revealed the involvement of focal adhesion zones and cytoskeletal proteins, such as integrins, nonmuscle myosin II (7), Rho GTPases (8-10), and vinculin (11), that participate in the generation of cellular traction forces. Recent work also has identified the nucleoskeletal protein lamin-A (12) and the transcriptional coactivators Yap (Yesassociated protein) and Taz (transcriptional coactivator with PDZbinding motif) (13) in mechanotransduction in mesenchymal stem cells. However, the mechanisms by which mechanical cues detected by cellular traction forces are transduced to downstream intracellular pathways of differentiation remain unclear.Ion channels are involved, directly or indirectly, in the transduction of all forms of physical stimuli-including sound, light, temperature, mechanical force, and even gravity-into intracellular signaling pathways. Hence, we wondered whether ion channels could be involved in transducing matrix mechanical cues to intracellular signaling pathways linked to lineage specification. In particular, we focused here on cationic stretch-activated channels (SACs) because they are known to detect mechanical forces with high sensitivity and broad dynamic range and because they are permeable to Ca 2+ , an important second messenger implicated in cell fate (14,15). We examined the role of SACs in neural stem cells, for which mechanical cues influence specification alo...

“…The ECM confers mechanical integrity to tissues and provides chemical, anatomical, and mechanical signals to cells, influencing differentiation, development, and pathogenesis (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11). The extent to which mechanical cues are generated and received by individual cells has been studied both experimentally and theoretically (12)(13)(14). Progress has also been made in understanding the signaling pathways that cells use to sense external mechanics.…”

mentioning

confidence: 99%

Rapid disorganization of mechanically interacting systems of mammary acini

Shi

Ghosh

Engelke

et al. 2013

133

175

Cells and multicellular structures can mechanically align and concentrate fibers in their ECM environment and can sense and respond to mechanical cues by differentiating, branching, or disorganizing. Here we show that mammary acini with compromised structural integrity can interconnect by forming long collagen lines. These collagen lines then coordinate and accelerate transition to an invasive phenotype. Interacting acini begin to disorganize within 12.5 ± 4.7 h in a spatially coordinated manner, whereas acini that do not interact mechanically with other acini disorganize more slowly (in 21.8 ± 4.1 h) and to a lesser extent (P < 0.0001). When the directed mechanical connections between acini were cut with a laser, the acini reverted to a slowly disorganizing phenotype. When acini were fully mechanically isolated from other acini and also from the bulk gel by box-cuts with a side length <900 μm, transition to an invasive phenotype was blocked in 20 of 20 experiments, regardless of waiting time. Thus, pairs or groups of mammary acini can interact mechanically over long distances through the collagen matrix, and these directed mechanical interactions facilitate transition to an invasive phenotype.mechanobiology | cancer E pithelial cells are physically coupled to their neighbors and are also in contact with the ECM. The ECM confers mechanical integrity to tissues and provides chemical, anatomical, and mechanical signals to cells, influencing differentiation, development, and pathogenesis (1-11). The extent to which mechanical cues are generated and received by individual cells has been studied both experimentally and theoretically (12)(13)(14). Progress has also been made in understanding the signaling pathways that cells use to sense external mechanics. Cell-ECM interactions are mediated in part by transmembrane proteins typified by the integrins, which link the cell's internal actin cytoskeleton with extracellular collagen fibers (15). Cells use multiple approaches for integrating mechanical cues with biochemical cues provided by soluble factors; for example, there is extensive multilayered cross-talk between integrin and TGF-β signaling (16,17). It is now also clear that key developmental regulators, such as Notch, can be directly activated by mechanical force (18,19).Less is known about the interactions of organized multicellular structures with the ECM and how those interactions affect tissue architecture, composition, and stability, as well as the molecular pathways by which these effects are mediated. In certain situations, contractile multicellular structures are able to mechanically reorganize biopolymer networks over long distances and in a highly directional manner, generating what are variously referred to as fibers, tracts, cables, straps, or lines. Regions of highly directional collagen alignment and concentration have been seen in systems ranging from single cells and tumor explants to human clinical samples (6,8,10,(20)(21)(22). Vader et al. demonstrated that the formation of long collagen lines is ...