Matrix elasticity in vitro controls muscle stem cell fate in vivo

Almost every laboratory that grows mammalian cells today grows their cells on tissue culture plastic, which was introduced to cell culture decades ago based on properties such as inertness, transparency, and so forth. However, plastic is rigid and unlike the many soft tissues in the body. Polymer gel systems that mimic the softness of various tissues have been developed over the past decade to test and understand the effects of rigidity on cells such as muscle cells. One recent study even shows that muscle stem cells expand much better in vitro on muscle-mimetic gels and that such cells prove optimal for engraftment in muscle.

Solid tissue repair or regeneration with stem cells is a major challenge that is now motivating studies of many cell types with many methods. Th erapies are typically expected to require expansion of autologous or allogeneic cells before transplantation into damaged or diseased tissue, but an ability to control self-renewal, diff er entiation, and expansion of isolated stem cells and their progenitors is probably crucial for any successful translation to humans. Soluble factors have been the usual approach in eff orts to control stem cells, but Gilbert and colleagues show that even transplantation into mice will benefi t from more attention to insoluble factors [1].
Tissue cells are adherent, which engages many signaling pathways. Indeed, adhesion extends into a cell beyond its membrane, with active engagement of the cytoskeleton: a cell constantly probes its micro environment by physically pulling on extracellular matrix and adjacent cells. Such forces cause matrix deformations in proportion to matrix elasticity; that is, the tendency of your tissue to spring back after pinching or pulling it. At the cell level, the mechanics feed back and remodel a cell's cytoskeleton, impacting signaling pathways and cell fate. A number of recent papers have highlighted the importance of myosin contractility in the viability and fate of pluripotent embryonic stem cells [2][3][4]. Th e coupling of stem-cellgenerated forces to matrix elasticity also aff ects diff erentiation, with initial results for mesen chymal stem cells indicating that matrix elasticity directs lineage specifi cation [5]. Hematopoietic stem cells cultured on soft, elastic substrates expand twofold to threefold whereas crosslink rigidifi cation of the substrate abolishes this eff ect [6]. Matrix stiff ness indeed varies between tissues, and ranges from extremely soft bone marrow and brain tissue to rigid calcifi ed bone. Muscle is not too soft and not too stiff , as muscle needs to be suffi ciently compliant to change length in contraction. Moreover, when there are defects in compliant proteins, such as dystrophin [7], muscular dystrophies arise -which motivates therapies such as stem cell trans plantation.
At the microscale relevant to cells, the lateral elasticity of normal, fl accid skeletal muscle has been measured to have an elastic modulus of ~12 kPa [8], while rat cardiac tissue [9] and mouse cardiac tissue [10] are perhaps stiff er by up to twofold. Such elasticity is typical of many polyacrylamide gels widely used in protein separations. Gilbert and colleagues reproduced past measurements of the tibialis anterior skeletal muscle and then determined whether this level of stiff ness aff ects the behavior of muscle stem cells (MuSCs) [1]. Hydrogels were engineered to have the same elasticity as muscle tissue, or else were softer or stiff er, with laminin basement membrane protein integrated into the gels. MuSCs were thus cultured on gels of elasticities 2 kPa, 12 kPa, and 42 kPa, and also on extremely thin gels on plastic so that the cells could feel the rigid plastic beneath. Tissue culture plastic is about 100,000-fold more rigid than any soft tissue.
Surprisingly, MuSCs cultured on 12 kPa gels expanded over time and maintained a primitive phenotype, whereas MuSCs on rigid substrates did not expand and also tended to diff erentiate. Time-lapse imaging showed that while the cells divided at similar rates on both soft and rigid substrates, cells on rigid substrates died much more frequently. Two transcription factors were assayed to stage the cells: Pax7 for MuSCs, and Myogenin for

Abstract
Almost every laboratory that grows mammalian cells today grows their cells on tissue culture plastic, which was introduced to cell culture decades ago based on properties such as inertness, transparency, and so forth. However, plastic is rigid and unlike the many soft tissues in the body. Polymer gel systems that mimic the softness of various tissues have been developed over the past decade to test and understand the eff ects of rigidity on cells such as muscle cells. One recent study even shows that muscle stem cells expand much better in vitro on muscle-mimetic gels and that such cells prove optimal for engraftment in muscle. diff er entiation. Pax7 was seen in about one-third of dividing cell doublets on the 12 kPa matrices, indicating sym metric division and self-renewal; this was about fi vefold higher than doublets on rigid substrate. Myogenin immunofl uorescence also suggested a threefold lower expression on the 12 kPa matrices, consistent with a less diff erentiated state. Past studies of mesen chymal stem cells on ~12 kPa showed that both Pax7 and Myogenin were induced but only to levels well below myoblasts [5]. Earlier work with myoblasts further demonstrated that myoblasts would fuse and generate the most robustly striated myotubes on 12 kPa matrices compared with even 50% softer or 50% stiff er substrates [8]. Multiple in vitro studies have thus demonstrated that 12 kPa matrices are best for multiple stages of myogenesis.
Transplantation of matrix-controlled cells was therefore the next logical step for the fi eld. Gilbert and colleagues expanded MuSCs on 12 kPa gels for 7 days and then injected them into damaged muscle (Figure 1). While freshly isolated MuSCs that are immediately transplanted into injured muscle tissue engraft best, even one cycle of division on rigid plastic prevents any signifi cant MuSC engraftment. In comparison, just 10 MuSCs that had undergone one round of division on 12 kPa gels proved suffi cient for 10% above threshold engraftment upon transplantation. Growth of MuSCs in vitro for even a short time on muscle-mimetic matrix can thus promote self-renewal and prolong regenerative potential. Whether this material approach to stem cell expansion will apply to other progenitor cells needs to be tested. Also in need of testing for more lineages than muscle is whether matrices can be used to fi rst direct in vitro diff erentiation of more pluripotent or multipotent stem cells and then expand the early progenitors as a method to fully prime for implantation. It is nonetheless becoming clear that stem cells feel matrix elasticity as a potent insoluble factor in proliferation and diff erentiation.

Abbreviations
MuSC, muscle stem cell.