The present disclosure relates to differentiating cells based on mechanical properties of the environment of the cell, in particular, elasticity of the surrounding matrix.
Normal tissue cells are generally not viable when suspended in a fluid. Thus, they are “anchorage-dependent” because to grow, such cells must adhere to a solid matrix, varying in stiffness from rigid glass to soft agar, topography, and thickness (e.g., basement membrane). Anchorage-dependent cells, therefore, are no longer viable if dissociated from the solid matrix and suspended in the culture media, even if soluble proteins are added to engage cell adhesion molecules, e.g., integrin-binding RGD peptide.
Fluids are clearly mechanically distinct from solids, which flow when stressed, whereas solids have the ability to resist sustained deformation. In most soft tissues—skin, muscle, brain, etc.—adherent cells together with an extracellular matrix constitute a relatively elastic microenvironment. Macroscopically, elasticity (measured as ‘Pascal’ or newtons/square meters) is evident in the ability of a solid tissue to recover its shape within seconds after mild poking and pinching, or even after sustained compression. At the cellular scale, normal tissue cells probe elasticity as they adhere and pull on their surroundings. Such processes are dependent in part on myosin-based contractility and transcellular adhesions—centered on integrins, cadherins, and perhaps other adhesion molecules—to transmit forces to substrates. Consequently, adhesion complexes and the actomyosin cytoskeleton, whose contractile forces are transmitted through transcellular structures, play key roles in molecular pathways.
Microenvironments and niches appear important in stem cell lineage specification and differentiation as cells can ‘feel’ tissue softness via contractile forces, generated by cross-bridging interactions of actin and myosin filaments. These forces (referred to as traction forces) are transmitted to the substrate, causing wrinkles or strains in thin films or soft gels (Harris et al., Science 208:177 (1980); Oliver et al., J. Cell Biol. 145:589 (1999); Marganski et al., Methods Enzymol. 361:197 (2003); Balaban et al., Nat. Cell Biol. 3:466 (2001); Tan et al., Proc. Natl. Acad. Sci. USA 100:1484 (2003)). The cell, in turn, responds to the resistance of the substrate by adjusting its adhesions, cytoskeleton, and overall state, e.g.—differentiation. Although considerable attention has been directed at the responsiveness of individual differentiated cells to external forces (outside-in) such as stretching and local twisting (Alenghat et al., Sci. STKE 119:pe6 (2002)), there is little understanding of how cell-exerted forces in response to the surrounding microenvironment contribute to signaling pathways effecting contractile mechanisms and ultimately cell state.
For example, adult stem cells, as part of normal regenerative processes, are believed to migrate or circulate and engraft to sites of injury, and will differentiate within these various in vivo microenvironments, ranging from compliant tissue substrates, such as brain or muscle, to rigid tissue substrates, such as bone. Mesenchymal stem cells (MSCs) are pluripotent, anchorage-dependent, and bone marrow-derived cells differentiating into various types of anchorage-dependent cells, including neurons, myoblasts, osteoblasts, and more (Gang et al., Stem Cells 22:617-624 (2004); Gilbert et al., J. Biol. Chem. 277, 2695-2701 (2002); McBeath et al., Developmental Cell 6: 483-495 (2004); Pittenger et al., Science 284:143-147 (1999); Salim et al., J. Biol. Chem. 279:40007-40016 (2004); Tanaka et al., J. Cell Biochem. 93, 454-462 (2004)) via different signaling paths. Soluble factors and cell density clearly influence these differentiation pathways chemically, but variations can also be physical (Gregory et al., Science STKE PE37 (2005); Salasznyk et al., J. Biomed. Biotechnol. 24-34 (2004)). For instance, stem cells adhere and differentiate in soft brain tissue or near rigid bone, and in vitro on soft gels or hard plastic culture dishes. However, compounding MSC-based therapies which consider physical matrix effects are normal wound healing responses, where the formation of fibrotic scar tissue will stiffen the microenvironment, and genetic disorders, such as muscular dystrophy, which increase fibrosis in affected tissues (Engler et al., 2004c, supra).
This wide range in substrate stiffness, exacerbated by disease, has been observed in vivo in many differentiated cell types to strongly influence focal adhesions and cytoskeleton (Beningo et al., J. Cell Biol. 153:881-888 (2001); Bershadshy et al., Annu. Rev. Cell Dev. Biol. 19:677-695 (2003); Discher et al., Science, 310:1139-1143 (Nov. 18, 2005); Engler et al., Biophys. J. 86:617-628 (2004a); Engler et al., J. Cell Biol. 166: 877-887 (2004c); Georges et al., J. Appl. Physiol. 98:1547-1553 (2005); Pelham et al., Proc. Natl. Acad. Sci. USA 94:13661-13665 (1997); Yeung et al., Cell Motil. Cytoskeleton 60:24-34 (2005)) and to be modulated by Ras superfamily proteins and their effectors (Gregory et al., 2005, supra; Paszek et al., Cancer Cell 8:241-254 (2005); Peyton et al., J. Cell Physiol. 204:198-209 (2005)). Rho subfamily members especially are broadly known to regulate the cytoskeleton, cell growth, and transcription, and recent studies of stem cell differentiation are also beginning to implicate cytoskeletal reorganization in vitro (Rodrigues et al., J. Cell Biochem. 93:721-731 (2004)) and Ras superfamily signaling in vivo (Benitah et al., Science 309:933-935 (2005)).
In fibroblasts, it is well established that Rho-stimulated contractility drives stress fiber and focal adhesion formation and that smooth muscle actin up-regulation correlates with contractility on rigid substrates (Chrzanowska-Wodnicka et al., J. Cell Biol. 133:1403 (1996); Hinz et al., Mol. Biol. Cell 12:2730 (2001)). Rac1, another Rho family protein, in activated macrophages promotes engulfment of soft beads, which otherwise are not engulfed (Beningo et al., 2001, supra). RhoA, in contrast, has no observable effect in these measurements. Current views of signaling pathways, especially various physical signals, clearly implicate Rac in cell motility (versus contractility) -indeed, myosin inhibition activates Rac (Katsumi et al., J. Cell Biol. 158:153 (2002)).
In addition to cell differentiation, the mechanical resistance or elasticity of a tissue cell's surrounding microenvironment adjusts spread morphology and contractile forces (Cukierman et al., Science 294:1708-1712 (2001); Engler et al., 2004a, supra; Flanagan et al., Neuroreport 13:2411-2415 (2002); Tolic-Norrelykke et al., Am. J Physiol. Cell Physiol. 283:C1254-1266 (2002)), as well as motility and viability (Engler et al., 2004c, supra; Lo et al., Biophys. J. 79:144-152 (2000); Peyton et al., 2005, supra; Wang et al., Am. J. Physiol. Cell Physiol. 279:C1345-1350 (2000); Wong et al., Langmuir 19:1908-1913 (2003)), and protein expression and signaling (Beningo et al., 2001, supra; Pelham et al., 1997, supra). The involvement of contractile-effector proteins in sensing implies that cell crawling, and thus MSC's ability migrate or circulate and engraft to sites of injury is also likely to be sensitive to substrate stiffness, as demonstrated in studies of the “cell on gel” effect with epithelial cells (Pelham et al., 1997, supra), fibroblasts (Lo et al., 2000, supra), and smooth muscle cells (Peyton et al., 2005, supra; Zaari et al., Adv. Mater. 16:2133 (2004)). With the latter cell type, crawling speed appears maximal at an intermediate stiffness and is reminiscent of crawling speed versus adhesive ligand concentration (Goodman et al., J. Cell Biol. 109:799 (1989))—mathematically modeled as a shift in the balance between ligand-mediated traction and ligand-mediated anchorage (Zaman et al., Biophys. J. 89:1389 (2005)). Additionally, smooth muscle cells on gels are slowed by inhibition of Rho kinase, suggesting that RhoA activity contributes to the tensions needed to detach any established adhesions at the rear of a motile cell (a process not needed in engulfinent) (Jay et al., J. Cell Sci. 108:387 (1995)). The dependence of cell crawling speed and direction on substrate stiffness, particularly gradients in stiffness, is referred to as “durotaxis” (Lo et al., 2000, supra).
Nevertheless, while cells have been shown to respond to externally applied forces (see, e.g., Riveline et al., J. Cell Biol. 153:1175-1186 (2001)), until the present invention there was no suggestion of a relationship between pluripotent cell differentiation and matrix elasticity and how various disease states can complicate the physical remodeling required to decrease elasticity to proper, tissue-relevant levels prior to the use of stem-cell based therapies. Thus a need remained in the art to provide a method for regulating the differentiation of mesenchymal stem cells (“MSCs”) into anchorage-dependent cell types. Moreover, similar sensitivity, growth and remodeling principles seem to apply to most anchored cells, and by regulating differentiation via contractile mechanisms, light may be shed on other matrix-altering pathologies.
A normal tissue cell not only applies forces, but also, as demonstrated in the following disclosure, responds through cytoskeleton organization (and other cellular processes) to the resistance that the cell senses, regardless of whether the resistance derives from a normal tissue matrix, synthetic substrate, or even an adjacent cell. Thus, the present invention meets the foregoing identified needs and other purposes by providing methods for regulating differentiation and cell shape of an anchorage-dependent cell.
It is, therefore, an object of the present invention to provide a method for regulating differentiation and cell shape of an anchorage-dependent cell, comprising: selecting, designing, or engineering a substrate or tissue microenvironment having an elasticity defined by elastic constant E; introducing the anchorage-dependent cell onto a substrate or into a microenvironment; and developing the anchorage-dependent cell into a differentiated cell type, wherein shape and lineage commitment (in terms of gene or protein expression, or both) are regulated by the elasticity of the underlying substrate. Depending on the controlled elasticity of the substrate, there is implemented a differentiation of the anchorage-dependent cells into at least one neurogenic, myogenic or osteogenic-type cell.
It is an additional object of the invention to provide a method wherein the subject anchorage-dependent cell is exposed to an inhibiting agent to inhibit expression of a lineage-specific regulator.
It is a further object of the invention to provide a method for regulating differentiation and cell shape of an anchorage-dependent cell, comprising: selecting, designing, or engineering a substrate or tissue microenvironment having an elasticity defined by elastic constant E; and introducing the anchorage-dependent cell onto a substrate or into a microenvironment, as above; and balancing chemo-mechanical energetics localized to cell adhesions against contractile energetics, σ, of the cell that balances cell traction stresses, τ, exerted by the cell on its underlying substrate, thereby controlling cell shape and lineage commitment. Because the cell adhesions provide necessary attachments permitting the cell to feel its microenvironment, and adhesion area increases linearly with E, larger deformation within the cell occurs on stiffer matrices and larger deformation in the substrate occurs on softer matrices.
It is yet another object of the invention to provide a method wherein the cell shape and differentiation of the subject anchorage-dependent cell is further regulated by controlling cell strain, such that there is an inverse relationship between intracellular and extracellular strains so that on stiff matrices, cell strains are large, while matrix strains are small, and on soft matrices, cell strains are small, while matrix strains are large.
Additional objects, advantages and novel features of the invention will be set forth in part in the description, examples and figures which follow, all of which are intended to be for illustrative purposes only, and not intended in any way to limit the invention, and in part will become apparent to those skilled in the art on examination of the following, or may be learned by practice of the invention.
The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings in which like numerals designate like elements. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown.
The present invention provides a method for regulating the differentiation of mesenchymal stem cells (“MSCs”) in response to tissue elasticity, with coupled regulation of non-muscle myosin II (“NMM II”) activity, as well as cytoskeletal organization. In some aspects of the invention, cell morphology shows that lineage commitment is influenced by matrix stiffness (elasticity), and that it is dependent on NMM II (particularly isoforms IIA, IIB, and IIC).
Regardless of geometry, the intrinsic resistance of a solid to a stress is measured by the solid's elastic (or Young's) modulus E, which is most simply obtained by applying a force—such as hanging a weight—to a section of tissue or other material and then measuring the relative change in length or strain. Another common method to obtain E involves controlled macro- or micro-indentation, including atomic force microscopy (AFM). The elastic modulus E is discussed, e.g., by Sugawara et al., Hearing Research 192:57-64 (2004); Taylor et al. J. Biomech. 37:1263-1269 (2004); Engler et al., 2004c, supra. Many tissues and biomaterials exhibit a relatively linear stress versus strain relation up to small strains of about 10 to 20%. The slope E of stress versus strain is relatively constant at the small strains exerted by cells (Lo et al., 2000, supra), although stiffening (increased E) at higher strains is the norm (Storm et al., Nature 435:191 (2005); Fung, A First Course in Continuum Mechanics: For Physical and Biological Engineers and Scientists (Prentice Hall, Englewood Cliffs, N.J., ed. 3, 1994).
Nonetheless, microscopic views of both natural and synthetic matrices (e.g., collagen fibrils and polymer-based mimetics (Stevens et al., Science 310:1135 (2005)), suggest that there are many subtleties to tissue mechanics, particularly concerning the length and time scales of greatest relevance to cell sensing. The elastic resistance that a cell ‘feels’ when it attaches to a substrate is governed by the elastic constant E of the substrate or tissue microenvironment. Sample preparation is also critical; for example, macroscopic elastic moduli measurements of whole brain can vary 2-fold or more, depending on sample preparation, perfusion, etc. (Gefen et al., J. Biomech. 37:1339 (2004)). In addition, many single or multi-cell probing methods involve high-frequency stressing (Hu et al., Am. J. Physioi. Cell Physiol. 287:C1184 (2004)), whereas relevant time scales for cell-exerted strains seem likely to range from seconds to hours, motivating long time studies of cell rheology (Bao et al., Nat. Mater. 2:715 (2003); Wottawah etal., Phys. Rev. Lett. 94:098103 (2005)). Regardless, comparisons of E (in units of Pascal; “Pa”) of three diverse tissues that contain a number of different cell types show that brain tissue is softer than muscle (skeletal muscle) (Engler et al., 2004c, supra; Yoshikawa et al., Biochem. Biophys. Res. Commun. 256:13 (1999)), and muscle is softer than collagenous bone (Engler et al., 2004c, supra; Taylor et al., J. Biomech. 37:1263-1269 (2004)). Although mapping soft tissue micro-elasticities at a resolution typical in histology is important, the implication here is that there are distinct elastic microenvironments for epithelial cells and fibroblasts in skin, for myotubes in fiber bundles, for neurons in brain, etc.
Correlations have long been made between increased cell adhesion and increased cell contractility (e.g., Leader et al., J. Cell Sci. 64:1 (1983)), but it now seems clear that tactile sensing of substrate stiffness feeds back on adhesion and cytoskeleton, as well as on net contractile forces, for many cell types. Seminal studies on epithelial cells and fibroblasts exploited inert polyacrylamide gels with a thin coating of covalently attached collagen (Pelham et al., 1997, supra). This adhesive ligand allows the cells to attach and, by controlling the extent of polymer cross-linking in the gels, E can be adjusted over several orders of magnitude, from extremely soft to stiff.
Because tissue elasticity is a factor in MSC differentiation, matrix elasticity is mimicked in vitro in the present invention with relatively inert, collagen I-coated polyacrylamide gels in which the concentration of bis-acrylamide cross-linking sets the elasticity (Pelham et al., 1997, supra). Solid phase gels for 2-dimensional electrophoresis generally are made of a porous polymer, such as polyacrylamide, and are constructed using known methods.
To minimize variability, it is beneficial if the materials and methods for making the gels are reproducible (see, e.g., the Examples that follow), and perhaps, produced by an automated means to reduce introduced variability. Gel monomers are mixed with agents that induce polymerization and then are poured into a mold that dictates the size and shape of the polymerized gel. For example, the catalyzed liquid gel monomer can be poured between glass plates separated uniformly over the entire surfaces thereof to produce a square or rectangular slab gel. The glass plates, separated by about a millimeter or a fraction thereof, are held in place until the gel is formed. The concentrations of polyacrylamide gels used in electrophoresis are generally stated in terms of % T (the total percentage of acrylamide in the gel by weight) and % C (the proportion of the total acrylamide that is accounted for by the cross-linker used). N,N′-methylenebisacrylamide (“bis”) is typically used as a cross-linker.
Using these tunable gel systems and sparse cultures,
In an alternative embodiment of the present invention, MSC differentiation is blocked or inhibited with an inhibitor of NMM II, blebbistatin. Nevertheless, as demonstrated herein in the Examples that follow, soluble inductive factors tend to be less selective than matrix stiffness in stimulating differentiation. Moreover, by controlling gel thickness h, the distance between a MSC and its substrate (that influences differentiation) was determined, and physically defined the microenvironment surrounding the MSCs.
As graphically shown in
Effect of Cell Morphology: Lineage Commitment Influenced by Matrix Stiffness and Dependent on Non-Muscle Myosin II
In the in vitro gel system, on soft, collagen-coated gels that mimic the elasticity of brain tissue (Ebrain≈0.1-1 kPa), the vast majority of MSCs exhibit a branched morphology (schematically shown in
In contrast, on 10-fold stiffer substrates that mimic the elasticity of striated muscle (Emuscle˜8-17 kPa), MSCs develop myoblast-like, spindle shapes as shown on the graph inset in
For cells on any substrate, blebbistatin was found to block branching, elongation, and any significant spreading of MSCs (
RNA Profiles: Lineage Commitment on Matrices of Tissue-like Stiffness
In an embodiment of the invention, RNA profiles indicated lineage commitment on matrices of tissue-like stiffness. Transcriptional profiles of early neurogenic, myogenic, and osteogenic markers were consistent with lineage identifications based above on morphology. With reference to early passage MSCs, cells on the softest gels (Ebrain˜0.1-1 kPa) showed the greatest expression of early neurogenic genes (see, for example,
Neuron-specific cytoskeletal markers such as β3-tubulin and neurofilament light chain (“NFL”), as well as adhesion proteins, such as NCAM, all contributed to an average 4-fold up-regulation of the neurogenic transcripts on the softest gels relative to expression on the other gel substrates. In contrast, MSCs grown on Emuscle-substrates (11 kPa) expressed 8-fold more myogenic message, with clear up-regulation of relevant transcriptional proteins, such as the Pax activators and myogenic factors (e.g., MyoD). On the stiffest gels (34 kPa), MSCs expressed 3-fold greater osteogenic message, up-regulating osteocalcin and the transcriptional factor CBFα1 (
Late differentiation genes, such as lineage-specific integrins (α3, α7, β1D) and morphogenetic proteins, are not yet up-regulated (as seen in
RNA levels were obtained for initially isolated MSCs (passage 4), as well as MSCs expanded in culture (up to passage 12). MSCs from these groups were plated onto 0.1, 1, 11, and 34kPa matrices, grown for 7 days, and also profiled. Data was normalized to total actin levels and scaled from 0 (no expression) to 1 (maximal expression). The names of genes in bold, italicized type in Table 1 indicate markers that are not generally expressed in the native stem cell population, i.e., normalized expression in initially isolated MSCs<0.15. Notably, there was not a dramatic RNA change between initially isolated and expanded MSCs.
Cytoskeletal Markers and Transcription Factors: Lineage Commitment
In another embodiment of the invention, cytoskeletal markers and transcription factors can also indicate lineage commitment.
Fluorescence intensity analyses and western immunoblots quantify the elasticity-specific, up-regulation of NFH (neurofilament heavy chain) and P-NFH, as well as β3-tubulin (
Although chemical agonists (Woodbury et al., J. Neurosci. Res. 69:908-917 (2002)) reportedly induce reversible branching in MSCs, ‘branched’ fibroblasts can also be induced chemically (Neuhuber et al., J. Neurosci. Res. 77:192-204 (2004)), which suggests a pan-matrix mechanism with soluble factors. In contrast, primary fibroblasts (FC7) did not branch on the soft elastic substrates (not shown), which implies that matrix stiffness-driven neurogenesis of MSCs is specific to these pluripotent cells, as well as to committed neurons.
On substrates with stiffness optimal for myogenic differentiation (Emuscle≈8-17 kPa), MSCs up-regulate the transcription factor MyoD1, localizing it to the nucleus (large arrow,
All MSC cultures use MSC GM for all experiments, except when noted.
Table 2 shows oligonucleotide array profiles for MSCs cultured on 0.1, 1, 11, and 34 kPa matrices that were normalized to actin levels, and were differentially compared to gene up-regulation (+1) to maximum down-regulation (−1). mRNA expression for MSCs cultured on 11 and 34 kPa matrices were also expressed as a fraction of mRNA expression in C2C 12 myoblasts or hFOB osteoblasts for the genes as indicated (scale: 0 to 1).
All MSC cultures use MSC GM for all experiments, except when noted.
In Table 3, oligonucleotide array profiles for MSCs cultured on 0.1, 1, 11, and 34 kPa matrices were normalized to actin levels, differentially compared to gene expression of low (4) passage MSCs, and scaled from maximum up-regulation (+1) to maximum down-regulation (−1). mRNA expression for MSCs cultured on 11 and 34 kPa matrices were also expressed as a fraction of mRNA expression in C2C12 myoblasts or hFOB osteoblasts for those indicated genes (scale: 0 to 1).
For osteogenesis, expression of the transcription factor CBFα1 is a first step since its inhibition limits osteogenesis (Gilbert et al., 2002, supra); Salim et al., 2004, supra). On osteogenic gel substrates (Eosteo≈30-40 kPa) and with standard growth media, MSCs expressed CBFα1 (see
MSCs Couple NMM II Expression to Matrix Stiffness
In yet another embodiment of the invention, MSCs couple NMM II expression to matrix stiffness. Although, cellular contractility and related signals have been postulated by others (Bick et al., Cell Adhes. Commun. 6:301-310 (1998); Engler et al., 2004c, supra; McBeath et al., 2004, supra; Muller et al., Biochem. Biophys. Res. Commun. 229:198-204 (1996); Puceat et al., Mol. Biol. Cell 14:2781-2792 (2003)) to influence differentiation, the prior art includes no report or evidence of any kind that has suggested the effect of strong, tissue-directed feedback of matrix elasticity on lineage commitment of previously pluripotent stem cells. Chronic treatment of MSCs on various gels with blebbistatin blocks expression of neurogenic, myogenic, and osteogenic markers (
Consistent with myosin regulation by substrate elasticity, a number of myosin RNAs are up-regulated on stiffer gels (11, 34 kPa) when compared to the softer matrices (
Select myosin genes appear more matrix sensitive than others, based on clustering of microarray data by variation with E (
On soft, Ebrain-gels, non-muscle myosin staining was diffuse; while on stiff Emuscle-gels, myosin striations emerged. Spacing between these nascent striations is the same for MSCs and age-matched C2C12 myocytes (1.0±0.3 μm) and is consistent with non-muscle myosin organization (Verhovsky et al., J. Cell Biol. 131:989-1002 (1995)), as well as periodic β3-integrin clusters (Giannone et al., Cell 116:431-443 (2004)) in embryonic fibroblasts. However, the striation period was smaller than the spacing set by myogenic molecular “rulers,” such as titin (TTN) (see
Chronic blebbistatin inhibition of non-muscle myosin II activity also decreased NMM IIB expression about 10-fold, to levels comparable to MSCs on soft gels (see
Rho GTP-ases Switch Signals for Lineage Specification
In an embodiment of the invention, Rho GTP-ases switched signals for lineage specification. For example, representative activator/effector and cell adhesion transcripts reveal a range of myosin-based sensitivities to substrate elasticity. Although myosin showed on average a peak expression in stem cells on Emuscle-gels (
Variance (Var) versus substrate-E spans an order of magnitude with RhoA and the mDiaphanous (mDia) homolog, DIAPH2 (Watanabe et al., Embo J. 16:3044-3056 (1992)), showing the largest variance. Since Ras and Rho family activators and their effectors generally regulate cytoskeleton, contractility, adhesion, and transcription (Kaibuchi et al., Annu. Rev. Biochem. 68:459-486 (1999)), relatively low RhoA expression on soft, neurogenic substrates appears consistent with low myosin activity (
To better identify lineage-specific activators of key transcription factors, a crosscorrelation function, Φ, was developed to compare the elasticity-dependent expression of each activator/effector gene with key transcription factor genes. These are respectively denoted as gene x(E) and gene y(E) in the function:
A value of Φ=1 indicates that genes x and y have identical elasticity-dependence, i.e., expression between genes is similar regardless of microenvironment, whereas a value of Φ=−1 indicates an inverse correlation, i.e., gene expression trends are highly variable across different matrices.
Pathway divergence is apparent in
Cell adhesions often rely on the RhoGTP-ases, effectors ROCK and mDia, to promote cytoskeletal assembly. ROCK is typically present in large, tension-generating processes associated with long, narrow focal adhesions (Riveline et al., 2001, supra) and cell shape maintenance, as well as osteogenesis (McBeath et al., 2004, supra). Myoblasts, on the other hand, can differentiate when ROCK is inhibited, but require RhoA activity via serum response factor (“SRF”) (Dhawan et al., J. Cell Sci. 117:3735-3748 (2004)), and other effectors. STARS (striated muscle activator of Rho signaling), and the formin homology protein, mDia, respectively bind actin and promote its assembly, activating SRF, but dependent on Rho (Copeland et al., Mol. Biol. Cell 13:4088-4099 (2002); Kuwahara et al., 2005, supra).
The sensitivity of adhesions to force depends on mDia (not ROCK), which seems likely to foster both the labile and punctate focal contacts of myogenesis seen in
While Rac1 is described as a pleiotropic regulator of cell adhesion and the cytoskeleton (Benitah et al., 2005, supra), both Rac1 and Cdc42 showed high activity in actin-driven motile processes, as compared to myosin-dominated contractility (Bershadsky et al., 2003, supra). The former processes predominate in growth cone migration (Sakumura et al., Biophys. J. 89:812-822 (2005)). In the present invention, RAC1 and, to a lesser extent, CDC42 showed a positive correlation with neurogenesis (
Stem Cell Differentiation: Adhesion and Contractility Balance, but Increase with Matrix Stiffness
Like the matrix-directed variation of activators, select focal adhesion transcripts, such as non-muscle α-actinin, filamin, and talin also appear to be particularly stiffness-sensitive and driven in expression by the stiff substrates (
Consistent with the transcription profiles for activators/effectors and adhesions, as well as the earliest reports of substrate-stiffness responses (Engler et al., 2004a, supra; Gaudet et al., Biophys. J. 85:3329-3335 (2003), stiff substrates were found to promote paxillin integration in the growth and elongation of focal adhesions (
Actomyosin is the contractile element of the myotubule. On very soft gels that are micropattemed with collagen strips so as to generate well-separated myotubes, actomyosin appeared diffuse after weeks in culture. However, on very stiff gels, as well as on glass micropatterns, stress fibers and strong focal adhesions predominate, suggesting a state of isometric contraction. Notably, on gels with an elasticity that approximates that of relaxed muscle bundles (E˜10 kPa), a large fraction of myotubes in culture exhibited definitive actomyosin striations. Actomyosin striation is even more prominent when cells are cultured on top of a first layer of muscle cells (as shown in Discher et al., 2005, supra). The lower myotubes attached strongly to glass and formed abundant stress fibers: whereas the upper myotubes differentiated to the more physiological, striated state. Although cell-cell contact may provide additional signals, the elasticity E of the myotubes, as measured by atomic force microscopy, was in the same range as that of gels that are optimal for differentiation, and importantly, was in the same range as that of normal muscle tissue.
Cell-cell contact appears to induce similar cell-on-gel effects for systems other than muscle. Astrocytes growing on glass, for example, appeared to provide a soft cell “stroma” adequate for neuronal branching that is similar to gels having brainlike E. Cell-cell contact may have a similar effect when cells are grown at a high density. When endothelial cells are confluent, the cells have indistinguishable morphologies on soft versus stiff substrates, whereas cells attached only to an underlying stiff surface differ in their spreading and cytoskeletal organization. Related results are also emerging from the present invention using epithelial cells and fibroblasts, as well as cardiomyocytes, showing a tendency to aggregate and form cell-cell contacts in preference to contact with soft gels.
Inhibition of actomyosin contractions largely eliminated prominent focal adhesions, whereas stimulation of contractility drives integrin aggregation into adhesions (Chrzanowska-Wodnicka et al., 1996, supra). Additionally, although microtubules have been proposed to act as “struts” in cells, and thus limit wrinkling of thin films by cells (Pletjushkina et al., Cell Motil. Cytoskeleton 48:235 (2001)), quantification of their contributions to cells on gels shows that they provide only a minor fraction of the resistance (14%) to contractile tensions. Most of a cell's tension is thus resisted by matrix (Wang et al., Proc. Natl. Acad. Sci. USA 98:7765 (2001)). On the stiffest, osteogenic gels (34 kPa), represented by thin gels of h≈0.5 μm, the adhesions were long and thin and slightly more peripheral than they appear on glass (
Materials ranging from fibrin gels and microfabricated pillars to layer-by-layer polymer assemblies (Georges et al., Appl. Physiol. 98:1547 (2005); Raeber et al., Biophys. J. 89:1374 (2005); Saez et al., in press; Wang et al, in press; Engler et al., 2004b, supra)), all suggest a similar trend of more organized cytoskeleton and larger, more stable adhesions with increasing E as outlined in the present invention, despite likely differences in adhesive ligand density and long-time elasticity. However, the responses appear to be specific to anchorage-dependent and/or relatively contractile cells.
To assess adhesions and to begin addressing length scales of “micro” environments, e.g., the volume of the environment that interacts with the cell, which is on the scale of nanometers to microns, thin polyacrylamide (PA) gels were cast with spacer beads to a thickness of h˜500 nm. This length scale allowed for low-intensity total internal reflectance fluorescence (TIRF) microscopy (Axelrod et al., J. Microsc. 129(Pt 1):19-28 (1983)). MSCs plated on thin, but stiff, matrices spread more with many more large focal adhesions (see
Adhesions provide MSCs the necessary attachments to “feel” their microenvironment through acto-myosin contractions. Mechanically, contractility equates to a cellular pre-stress, σ, that balances the traction stresses, τ, exerted on the gel by the cell (Wang et al., Am. J Physiol. Cell Physiol. 282:C606-616 (2002b)). Traction stresses (τ, force per area) were determined from bead displacements in the gel (see
Blebbistatin, which was shown above to inhibit myosin contraction and expression, prevented any of the cells from developing either a pre-stress σ (Griffin et al., 2004, supra) or a significant cortical stiffness, κ, on any matrix (see
As functions of matrix stiffness, the two differentiated cell types, the C2C12-myoblasts and hFOB-osteoblasts exhibit similar (κ/E) slopes—though distinct intercepts. By using the pre-stress results, these two differentiated cell types also show the same slope for (κ/σ) (≈0.2) as highly contractile, smooth muscle cells assayed by different techniques (Wang et al., 2002b, supra). On the other hand, MSCs appear more mechano-sensitive, with twice the slope for (κ/E) and (κ/σ). This increased mechano-sensitivity leads to a self-consistent crossover. On myogenic gels (11 kPa), MSCs and C2C12s have similar κ, whereas on osteogenic gels (34 kPa), MSCs and hFOBs have similar κ. Despite this difference, the inside-outside relationship between intracellular strains, εin (=σ/κ), and the extracellular strain field, εout (=τ/E), fits a universal power law for all cell types (see
One can think of such a strain comparison as similar to comparing intracellular and extracellular ion concentrations (Na+, K+, Ca++, etc.). In the present invention, however, the inverse relationship between intracellular and extracellular strains reveals that, on stiff matrices, cell strains are large, while matrix strains are small. Whereas, by comparison, on soft matrices, cell strains are small, while matrix strains are large. The strain thus transfers from outside to in with increasing matrix stiffness, presumably activating different pathways at different strains. However, the common power law indicates a common mechanism, consistent with the central role of myosin II.
Effective Energetics for Lineage Specification
In an embodiment of the present invention, the effective energetics for lineage specification can be determined. For example, NMM II inhibition blocks lineage commitment (see
These findings are formalized in a simple model, wherein chemo-mechanical energetics localized to adhesions are balanced against the contractile energetics of the cell. Contractility or pre-stress, σ, is assumed to act throughout the cell volume V as a global regulator of differentiation. Coupled to this, an increase in free concentration of the local, transducing activator/effector links cooperatively to collagen (with Hill coefficient m and affinity K) and to substrate elasticity (E). The net result (see Example 2) is a lineage commitment probability given by:
The effective thermal energy kbTeff in the exponential factor should relate more to cytoskeletal stochastics than to temperature. In the limit of rigid substrates where tensions (σ) are high, such as on glass, isometric pulling on adhesions will limit differentiation of MSC, as seen, e.g., in
Equation 2 fits the three differentiation peaks of
The present invention is further described in the following examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. The various scenarios are relevant for many practical situations, and are intended to be merely exemplary to those skilled in the art. These examples are not to be construed as limiting the scope of the appended claims. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident in light of the teaching provided herein.
The following Materials and Methods were utilized in the tunable systems used to provide exemplary proofs of the principles provided in the present invention.
Materials and Methods
Cell Culture: Human Mesenchymal Stem Cells (MSCS; Osiris Therapeutics; Baltimore, Md.), human osteoblasts (hFOBs; ATCC, Manassas, Va.), primary human skin fibroblasts (FC7) (Engler et al., 2004c, supra), and murine myoblasts (C2C12s; ATCC) were cultured in normal growth media listed in Table 1, supra. To chemically induce MSC differentiation, cells were placed in the appropriate induction media also listed in Table 1, supra. All cells were used at low passage number, and were subconfluently cultured. Cells were plated for experiments at ˜103 cells/cm3 and cultured for 7 days, unless otherwise noted. All chemicals were purchased from Sigma (St. Louis, Mo.) unless otherwise noted.
To inhibit proliferation, cells were exposed mitomycin C (10 μg/ml) for 2 hr and washed three times with media prior to plating. Blebbistatin (50 μM; EMD Biosciences, Inc., San Diego, Calif.), a NMM II inhibitor, was applied with every media change and was stable in culture media for up to 48 hours, as determined by thin layer chromatography.
Substrate Preparation: Cells were plated on variably compliant polyacrylamide gels according to a previously established protocol (Engler et al., 2004a, supra; Pelham et al., 1997, supra) herein incorporated by reference. Briefly, gel cross-linker N,N′methylene-bis-acrylamide and acrylamide monomer were varied in distilled water to achieve a polymerized solution with a tunable elastic modulus (Engler et al., 2004b, supra). Approximately 25 μl of the mixed solution was polymerized on a coverslip using 1/200 volume of 10% ammonium persulfate and 1/2000 volume of N,N,N′,N′-tetramethylethylenediamine. The polymerizing gel was covered with a dichlorodimethylsilane-pretreated coverslip to ensure easy detachment and a uniform polymerized gel surface. Final gels were 70-100-μm thick, as measured by microscopy.
To produce ultra-thin gels, however, 10 μl of a 1% polystyrene bead solution (d=250 nm; Polysciences, Inc., Warrington, Pa.) was added to polymerizing solutions, and a weight was added to the top coverslip to ensure that the gel thickness was defined by the spacer bead diameter. Type 1 collagen (0.25-1 μg/cm2; BD Biosciences, Rockville, Md.) was chemically cross-linked using a photoactivating cross-linker, sulfo-SANPAH (Pierce Biotechnology, Inc., Rockford, Ill.) and attachment was confirmed by fluorescence. Cells grown on glass coverslips (GL) alone were always coated non-specifically with collagen prior to cell seeding.
Differentiation Assays:
1) Morphological Changes and Immunofluorescence: Changes in cell shape (<4 days), especially the development of neurite-like branches (Engler et al., 2004c, supra) or spindle-like morphologies (Flanagan et al., 2002, supra), were quantified either by the number of membrane branches per mm of cell or by a “spindle factor,” referring to the major cell axis/minor cell axis, respectively. Cells also were stained with lineage-specific antibodies: myogenesis with Myogenesis Differentiation Protein 1 (MyoD1; Chemicon® International, Temecula, Calif.), osteogenesis with Core Binding Factor α1 (CBFα1 ; Alpha Diagnostic International, San Antonio, Tex.), and neurogenesis with phosphorylated and dephosphorylated Neurofilament Heavy chain (NFH; Stemberger Monoclonal, Berkeley, Calif.) along with paxillin (Chemicon), skeletal muscle myosin heavy chain (Zymed Laboratories, S. San Francisco, Calif.), and non-muscle myosin IIA and B (Sigma) or rhodamine-labeled phalloidin. Cells were fixed with formaldehyde, incubated in a 5% albumin blocking solution for 1 hour at 37° C., permeabilized with 0.5% Triton-X-100 and incubated overnight at 4° C. in 1:100 dilution of antibodies in PBS. Cells were then incubated for 1 hour at 37° C. in 1:500 FITC-conjugated secondary and 60 μg/mL TRITC-phalloidin. Finally, cells were incubated for 10 min. in 1:100 Hoechst 33342 (Molecular Probes Europe, Leiden, Netherlands) to label DNA. Cell morphology and fluorescently labeled cells were examined on a TE300 inverted epi-fluorescent Nikon or Olympus (TIRF) microscope, imaged on a cascade CCD camera (PhotoMetrics, Huntington, Beach, Calif.), and quantified with Scion Image (Scion Corp., Frederick, Md.).
Western blotting: Cells (with or without blebbistatin treatment, 50 μM) were also plated on 45×50 mm coverslips to obtain enough cells for western blotting. Cells were permeablized with lysis buffer (10% SDS, 25 mM NaCl, 10 nM pepstatin, and 10 nM leupeptin in distilled water), boiled for 10 minutes, placed in a reducing SDS-PAGE gel (Invitrogen, Carlsbad, Calif.) with MOPS buffer, and run against a colormetric molecular weight marker. Proteins were transferred onto nitrocellulose and blocked in a solution of 1% albumin, 50 mM Tris Buffered Saline (TBS). Membranes were rinsed 2X in 1 % Tween 20-TBS (TTBS), and then a 1:500 solution of primary antibodies were added for 2 hours. Membranes were rinsed 2X with TTBS again and 1:1000 of the secondary HRP-conjugated antibodies were added. Color development was achieved with a HRP development kit (Bio-Rad Laboratories, Hercules, Calif.). All westerns were run in duplicate, along with an addition blot for actin and Commassie blue-staining to ensure constant protein load among samples. Quantification of western blots was done by Scion Image software.
3) Oligonucleotide Array Assays: Total RNA (3-5 μg) was obtained from MSCs (with or without blebbistatin treatment) cultured on gel substrates of varying stiffness, as well as C2C12 myoblasts on 11 kPa gels and hFOB osteoblasts on 34 kPa gels, using an ethanol-spin column extraction. The samples were labeled with an Ampolabeling Linear Polymerase Reaction kit (SuperArray Bioscience, Frederick, Md.) and hybridized to custom oligonucleotide arrays. Membranes for control (C2C12, hFOB, and MSCs from flasks), experimental (MSCs on gels), and duplicate sample RNAs were processed in parallel to reduce technical variability. Chemiluminescent signals were detected on Biomax Film (Kodak) and analyzed with Scion Image software. Background-corrected signals were normalized to a control gene, β-actin.
Creep-test Micropipette Aspiration: Micropipettes were forged using a deFonbrune-type microforge (Vibratome, St. Louis, Mo.) to a radius of 2-3 μm with an approximately 25° pipette curvature so when mounted in micromanipulators (Nirishige; Japan) at an angle similar to the micropipette's curvature. The end of the pipette was flush with the cell edge (see
Images of the projection length were taken every 2 seconds in brightfield on a TE300 inverted Nikon microscope and cascade CCD camera (Photometrics), which allowed accurate fitting of the parameters ε, μ′, and τ to the data to determine the elastic and viscous moduli of each membrane.
Traction Force Measurements: Adhesive stresses, imposed on the matrix surface by an adherent cell, generate a displacement field from embedded beads within a soft substratum, which can be mapped on the cell if the gel can be approximated as a semi-infinite solid. Briefly, the well characterized traction force method (Butler et al., 2002, supra; Dembo et al., 1999, supra; Wang et al., 2002b, supra) uses bead displacements between images with, and without, the adherent cell to assemble a displacement field and determine Green's strain function given known material properties of the substratum (elastic modulus, poisson's ratio, etc). The traction field was used to obtain the cell pre-stress, i.e., the net tensile force over the cell-matrix interface carried by the actin cytoskeleton across a cross-sectional area of the cell that balances cell traction forces (Wang et al., 2002b, supra).
Accordingly, while stem cell differentiation by soluble stimuli is known, ihe strong influence of an immobilized microenvironment on cell differentiation is provided by the present invention. Myosins are known to have key roles in the relevant cell biology. NMM II, for example, is needed for neurite out-growth, as well as for myofibril assembly. RhoA associated shifts between mDia and ROCK have roles in cytoskeleton and mechano-sensing mechanisms via the serum response factor. Myosin signaling pathways in lineage commitment are summarized in
Although the following model may be far too simplistic to capture the complexities of stiffness-controlled gene regulation, the model is inspired by the atomistic complexity of cooperative release of oxygen under hydrostatic tension (Carey et al., J. Biol. Chem. 252:4102-4107 (1977)). The goal of the following minimal model is thus to fit the differentiation results presented in
ξ=1+[(K/E) coll]m Equation 4
In terms of energetics, K˜exp(−ΔG/kbT) and the matrix modulus E˜A exp(εx2/kbT). Additionally, ε is the relevant stiffness of matrix/membrane/adhesions, and x is a strain. If εx2 is small, E˜A[1+(εx2/kbT)] which implies that K is linear in E, as shown in
It was assumed that the fraction of unbound Xi matters most for lineage specificity: θ′=1−[δIn(ξ)/In(coll)]=1/ξ. This is the free and diffusible fraction of Xi (not associated with collagen) that has the strongest effect. With N as the total number of species Xi, the total unbound portion of this species is Θ′=N θ′=N/ξ, which gives a chemo-mechanical potential for N=constant as:
Gchem=−kbT In(N/ξ)=constant−kbT In [1+[(K/E) coll]m]−1 Equation 5
The Total Free Energy depends additionally on the global pre-stress (σ), acting on the cell volume, V, Gtot=Gchem+σV, which gives the lineage commitment probability (Equation5) by taking the exponential of Gtot.
Mechano-biology is a broad field encompassing the recognition in the present invention that most tissue cells not only adhere to, but also pull on, their microenvironment, and as a result anchorage-dependent cells respond to the stiffness of the underlying matrix in ways that relate to tissue elasticity. In some aspects, microenvironments that are too soft or too stiff have implications in disease, as well as development, and highlight the need to understand the important role provided herein, for matrix physical properties and how cells feel the cellular matrix. For the cell biologist, the present invention offers methods that will lead to a better understanding of mechano-biological and/or mechano-chemical pathways and offer an understanding of more biologically relevant elastic substrates, as compared with rigid coverslips and polystyrene, for in vitro studies. For the applied biologist or bioengineer, the present invention will likely lead to modified strategies for tissue repair and cell scaffolding, such as the development of fibrous scaffolds for cell seeding, where careful attention can be given to fiber flexibility. Consequently, in addition to the regulation of differentiation of anchorage-dependent mesenchymal stem cells, many applications will result from the recognition that tissue cells feel and respond to the mechanics (elasticity) of their substrate.
All patents, patent applications and publications referred to in the present specification are also fully incorporated by reference.
While the foregoing specification has been described with regard to certain preferred embodiments, and many details have been set forth for the purpose of illustration, it will be apparent to those skilled in the art that the invention may be subject to various modifications and additional embodiments, and that certain of the details described herein can be varied considerably without departing from the basic principles of the invention. Such modifications and additional embodiments are also intended to fall within the scope of the appended claims.
This invention was supported in part by funds obtained from the National Institutes of Health: “Myocytes Sense Substrate Stiffness—New materials,” grant number 11R21EB004489-01 and “Bioengineering Research Partnership—Muscular Dystrophy,” grant number 5R01AR047292-05. The U.S. government may therefore have certain rights in the invention.