WO2008105919A2 - Nanomechanical characterization of cellular activity - Google Patents

Abstract

Methods for the characterization of the nanomechanical properties of cells to determine their differentiation state and methods for influencing the differentiation states of cells by nanomechanical processes are disclosed.

Description

NANOMECHANICAL CHARACTERIZATION OF CELLULAR ACTIVITY

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority under to United States Provisional Patent Application Serial Number 60/823,334 filed August 23, 2006, which is incorporated herein in its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to using nanomechanical processes to characterize cell activity. Specifically, the present invention provides methods to characterize and induce stem cell differentiation by nanomechanical processes. Additionally the present invention provides methods to measure the effects of drugs on cells using nanomechanical processes.

BACKGROUND OF THE INVENTION

[0003] Many biological processes taking place inside the living cell rely on the nanomechanical properties of cellular substructures and the cell membrane or wall itself. The atomic force microscope (AFM) yields information on the integrity and local nanomechanical properties of mammalian and microbial cellular membranes under normal and stressed metabolic conditions.

[0004] The atomic force microscope is a scanning probe microscope that measures a local property, such as topography, mechanical properties, thermal and electrical properties, optical absorption or magnetism, with a probe or "tip" placed very close to the sample. The small probe-sample separation makes it possible to take measurements over a small area. Measurements are taken as the microscope raster-scans the probe over the sample while measuring the local property in question.

[0005] Because the AFM can image biological samples at sub-nanometer resolution in their natural aqueous environment, it has potential for characterization of living cells. Using the AFM, it is possible to observe living cells under physiologic conditions, detecting and applying small forces with high sensitivity and determine sample height the high resolution. Examples of cellular processes that can be measured with the AFM include activation of platelets, exocytosis, movement of cells, and cell division.

[0006] In particular, elasticity measurements provide information into various cellular processes such as cell division and cell migration. Two-dimensional mapping of the cellular elasticity is achieved by recording force curves while the tip is raster-scanned across the cell in a method called force mapping. From the force curve, the cell topography at different loading forces as well as the local elastic or Young's modulus can be calculated. [0007] Therefore, methods are needed for determining the differentiation state of cells in order to successfully utilize the use of these cells in a variety of therapeutic uses including tissue regeneration. Additionally methods are needed to induce specific differentiation states in stem cells to generate the large numbers of transplantable cells of appropriate differentiation state needed for cellular regenerative therapies.

SUMMARY OF THE INVENTION

[0008] The present invention provides methods for the characterization of cells by nanomechanical processes and for influencing the differentiation of cells by application of nanomechanical forces.

[0009] By analyzing the characteristics of cellular processes and determining the nanomechanical properties, cellular differentiation can be measured using the atomic force microscope (AFM) to determine specific differentiated states. Additionally, application of localized forces, through the use of localized probes, magnetic fields, nanopatterned substrates or substrates designed to apply localized forces may inhibit, enhance or direct cellular differentiation.

[0010] In one embodiment of the present invention, a method is provided for determining the differentiation state of one or more cells comprising determining at least one nanomechanical characteristic of one or more cells. In another embodiment, the determining step comprises applying a nanomechanical force to the cells

[0011] In another embodiment of the present invention, a method is provided for influencing the differentiation state of cells comprising applying a nanomechanical force to the cells.

[0012] In another embodiment, the cells are stem cells. In another embodiment, the stem cells are embryonic stem cells.

[0013] In another embodiment, the nanomechanical characteristic is selected from the group consisting of cell motion, elasticity and viscoelastic properties.

[0014] In another embodiment, the cells are disposed on a cantilever prior to application of a nanomechanical force.

[0015] In another embodiment, the nanomechanical force is applied with an atomic force, an electrical signal, a physical force or a magnetic force. In another embodiment, the nanomechanical force is applied with magnetic beads. In another embodiment, the nanomechanical force is applied with an atomic force microscope. In another embodiment, the nanomechanical force is applied by an electrical signal. In another embodiment, the magnetic force is an extremely low frequency magnetic force.

[0016] In another embodiment of the present invention, the nanomechanical force is a local lateral force or a local normal force.

[0017] In another embodiment, applying the physical force comprises growing the cells on a polymeric substrate, the polymeric substrate capable of stretching or bending and wherein stretching or bending the polymeric substrate applies a force to the cells. In another embodiment, the polymeric substrate is coated with collagen.

[0018] In one embodiment of the present invention, influencing the differentiation state of cells comprises inhibiting or enhancing the differentiation state.

[0019] In another embodiment, applying a physical force comprises coating a carbon nanotube ATF tip with a DNA binding protein; inserting the coated ATF tip into a nucleus of a cell; allowing the binding protein to being to DNA sequences in the nucleus; and applying a nanomechanical force to the DNA sequences bound to the coated ATF tip thereby influencing the differentiation state of the cell. In another embodiment, the DNA binding protein is a zinc finger binding protein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 graphically depicts the use of the atomic force microscope (AFM) and sonocytology to characterize the transition from undifferentiated embryonic stem cells to differentiated cardiac myocytes according to the teachings of the present invention.

[0021] Figure 2 depicts directed mechanotaxis induced by the AFM according to the teachings of the present invention.

[0022] Figure 3 depicts single-cell patterning using directed mechanotaxis according to the teachings of the present invention.

[0023] Figure 4 depicts AFM deflection-mode images of large social groups and individual Myxococcus xanthus cells in air according to the teachings of the present invention. Panel A depicts a 100-μm² scan of large mounds of wild-type cells. Panel B depicts a higher-resolution scan of the marked area in panel A reveals cellular ordering along the edge of the social group in domains of -10 cells. Panel C depicts a further, higher- resolution scan of the area marked in panel B reveals the presence of pili at the cell poles. Panels D-I depict individual cells of M. xanthus mutants. (Scale bar, 2 μm); Panel D, wild- type DK1622, showing polar pili; Panel E, wild-type DK1622 cells displaying slime-like substances (^*) and extruding blebs (arrow); Panel F, pilA mutant DK10407, showing the absence of pili at the cell pole; Panel G, dif mutant SW504, showing the presence of long pili that bend toward the cell body; Panel H, stk mutant DK3088, displaying an excess of extracellular substances in the form of filaments with variable diameters from 15 to 65 nm; and Panel I, the LPS O antigen mutant HK1324.

[0024] Figure 5 depicts the twisting morphology of M. xanthus cells according to the teachings of the present invention. Panel A depicts two cells twisted around each other. (Scale bar, 2.5 μm) Panel B depicts a higher-resolution image of the cells in panel A, showing pili at the same cell poles of both cells. (Scale bar, 1 μmin both panels B and C) Panel C depicts helical twists in a single cell and a height profile of the long axis of the cell body, revealing distinct 25- to 200-nm bumps separated by ~500-nm "valleys."

[0025] Figure 6 depicts force-displacement curves according to the teachings of the present invention. Panel A depicts force curves measured on living wild-type (curve i), stk (curve ii), and glutaraldehyde-fixed wild-type M. xanthus (curve iii) cells (the curves are shifted 4 nN for clarity). Force-displacement curves were recorded as "approach" (dashed line) and "retraction" (solid line) curves. Panel B depicts force curves measured on a bare portion of the substrate before (curve i) and after (curve ii) the tip was used for force- spectroscopy measurements on the living cells.

[0026] Figure 7 depicts the effects of magnetic field on the expression of cardiac lineage-promoting genes according to the teachings of the present invention. A magnetic field (MF) was applied from the time of leukemia inhibitory factor (LIF) removal. Embryoid bodies (EBs) (Panel A) or puromycin-selected cardiomyocytes (P, panel B) were collected after three or ten additional days, respectively, and processed for RT-PCR analysis of the indicated transcripts. LIF, undifferentiated cells; C, unexposed controls; S, sham-exposed (counter-wound coils); MF, MF-exposed cells. Ethidium bromide-stained agarose gels are representative of 4 separate experiments. Panel C depicts RNase protection analysis of GAT A-4 mRNA expression. GTR1 ES cells were cultured as described in the absence (-) or presence (+) of MF. ^*Significantly different from unexposed. Panel D depicts nuclear run-off analysis of GATA-4 gene transcription in isolated ES cell nuclei. Nuclei were isolated from undifferentiated cells (LIF), EBs or P collected three or ten days after LIF removal, respectively. Each group of cells was exposed in the absence (-) or presence (+) of a MF from the time of LIF withdrawal. Row a: GATA-4 gene transcription. Row b: cyclophilin gene transcription. Autoradiograms are representative of three separate experiments. Panel E depicts immunoreactive dynorphin B (ir-dyn B) in cells (gray bars) or medium (white bars), mean ± SE (n=6). Asterisks with brackets: significant difference (1-way ANOVA, Newman Keul's test). § Significantly different from values of gray bars. [0027] Figure 8 depicts MF-primed expression of cardiac specific genes and enhanced yield of ES-derived cardiomyocytes according to the teachings of the present invention. The MF was applied from the time of LIF removal. EBs or purinocycin-selected cardiomyocytes (P) were collected after three or ten additional days, respectively. Panels A and B depict RT-PCR analysis of α-myosin heavy chain (MHC) and myosin light chain-2V (MLC) in EB (Panel A) and P (Panel B). LIF, undifferentiated cells; C, unexposed controls; S, sham- exposed (counter-wound coils); MF, MF-exposed cells. MHC immunostaining was assessed by the MF20 monoclonal antibody in undifferentiated cells (Panel C) and in cardiomyocytes derived from cells cultured in the absence (Panel D) or presence (Panel E) of MF. DNA was visualized with propidium iodide.

DETAILED DESCRIPTION OF THE INVENTION

[0028] The present invention provides methods for the characterization of cells by nanomechanical means and for influencing the differentiation of cells by nanomechanical means.

[0029] By analyzing the characteristics of cellular processes and determining the nanomechanical properties, cellular differentiation can be measured using the AFM to determine specific differentiated states. Additionally, application of localized forces, through the use of localized probes, magnetic fields, nanopatterned substrates or substrates designed to apply localized forces may inhibit, enhance or direct cellular differentiation.

[0030] Nanomechanical properties, as used herein, refers to Young's modulus or elasticity, rheological properties (shear modulus, complex shear modulus, in pashe storage modulus, loss modulus), mechanical motions of the membrane in the time and frequency domain, and contractile properties.

[0031] Correlating the nanomechanical characteristics of differentiation provides measurable characteristic signals which are correlated with implantation success and efficacy. Characterizing the differentiation pathway through nanomechanical means provides a direct single-cell method for measuring the progress of differentiation and determining optimal time points for intervening in the differentiation pathway. One method for intervention is through nanomechanical means.

[0032] Pluripotent stem cells, including embryonic stem (ES) cells, are known to differentiate into many types of tissues in early embryos. These include all tissues of the mature organism however substantial interest has been expressed in the differentiation of ES into cardiac myocytes. Typically, the differentiation efficiency is low and the differentiation process is not well understood. However, differentiation can be directed and enhanced by exposing the ES cells to dimethylsulfoxide (DMSO), modified hyaluronan esters or low frequency (50 Hz) magnetic fields. Pluripotent stem cells including, but not limited to, embryonic stem cells and adult mesenchymal stem cells, have been proposed as a source of donor cardiac cells for treatment of human hearts that have suffered cell loss or are compromised by disease or injury. Implantation of undifferentiated or completely differentiated stem cells into compromised heart tissue has not been successful to date. However, there may be an unrecognized developmental window of time during differentiation which represents the optimum time for implantation.

[0033] Differentiated cardiac myocytes produce mechanical beating motions in culture. Therefore, stem cells directed to cardiac myocyte differentiation begin to beat at a point in differentiation. This beating motion requires a major reorganization of the cell cytoskeleton and in turn a significant change in cellular nanomechanical properties. In one embodiment of the present invention, measurement of cellular nanomechanical properties as a function of differentiation progress provides a measurable characteristic to monitor cellular differentiation in individual cells. The atomic force microscope (AFM) and sonocytology can measure the vertical motion of cells including, but not limited to stem cells, during differentiation. Early stem cells may exhibit random or Brownian motion, however beating motions can be measured since beating cardiac myocytes beat with amplitudes of ~1 μm at frequencies of -0.5 Hz. Characterizing the local nanomechanical beating motion provides a direct measure of the progress of differentiation and can be correlated with functional properties of the cell. Figure 1 depicts the measurement of the transition of an individual cell from undifferentiated ES cell to differentiated cardiac myocyte using the AFM.

[0034] Additionally, the vertical or lateral motion of cells can be measured throughout the differentiation process and analyzed using fast Fourier transforms and/or auto and cross correlation functions as will be known to persons skilled in the art using commercially available software.

[0035] In another embodiment of the present invention, cell differentiation can be determined by growing populations of cells on the surface of cantilevers. In a non-limiting example, as ES cells differentiate into beating cardiac myocytes, they will induce the bending of the cantilever, leading the cantilever to beat, which is the measure of the beating motion of a group of myocytes. The beating motion of groups of cells may be different than the beating motions of single cells.

[0036] The nanomechanical properties of cells are known to be related to a wide variety of important biological properties including, but not limited to, signaling pathways, metastatic potential, cell death and division. Measurement of the local Young's modulus (elasticity) or viscoelastic response of the cells with means including, but not limited to, the AFM, can also be used to characterize the differentiation state of an individual cell. In order to produce a nanomechanical beating motion, a significant cellular architecture must develop to produce such a highly coordinated motion. Therefore, in one embodiment of the present invention provides measurement of the local cell membrane Young's modulus or viscoelastic properties to determine cytoskeletal reorganization associated with cellular differentiation.

[0037] In another embodiment of the present invention, nanomechanical characterization of stem cell differentiation will provide differentiated cardiac myocytes suitable for tissue replacement therapy for damaged hearts.

[0038] In another embodiment of the present invention, the nanomechanical characterization of the beating motion of differentiating stem cells can be influenced by contact with chemicals or magnetic fields to enhance or interfere with differentiation. Non- limiting examples of chemicals useful for influencing the differentiation of stem cells include mixed esters of hyaluron (an extracellular/intracellular matrix component) with butyric acid (a histone deacetylase inhibitor that alters chromatin structure, increasing transcription factor accessibility to target cis-acting regulatory sites) and retinoic acid (an agonist for nuclear transcription factors involved in ES cell cardiogenesis). These compounds (HBR) increase the yield of cardiac differentiation in mouse embryonic stem cells. In one embodiment, cell differentiation-inducing chemicals can be delivered by functionalized AFM cantilevers. In another embodiment, cell differentiation-inducing chemicals can be delivered by patch clamp techniques.

[0039] Stem cells can sense the extracellular and intracellular matrix as a dynamic scaffold, a developmental "niche" bearing motion-tenso-elastic properties. These properties can impact the differentiation potential of stem cells. Therefore growth of stem cells onto nanopatterned substrates with defined extracellular matrices can force specific cell shape and size changes to direct differentiation. In one non-limiting example, measurement of cell traction forces on flexible collagen-coated substrates during differentiation will provide insight into the progress of differentiation. In a further embodiment, the polymer substrate can be specifically stretched or bent to provide specific forces along a given direction or directions to inhibit, direct or enhance differentiation.

[0040] In yet another embodiment of the present invention, patch-clamp techniques can be used to measure local electrical potentials as a nanoscale electrical property related to differentiation. In still another embodiment, patch-clamp techniques can also be used to deliver electric signals to inhibit, enhance or direct differentiation. Patch-clamp techniques can also be combined with cells grown on nanopatterned surface electrodes to measure or delivery electric signals.

[0041] In another embodiment of the present invention, nanomechanical forces are delivered from carbon nanotube AFM tips modified with specific DNA binding proteins directly to specific DNA sequences. The specific DNA binding proteins useful for targeting these forces include, but are not limited to, zinc-finger binding proteins. In this embodiment, the modified nanotube tip is inserted into the cell nucleus to specific DNA sequences in the nucleosome and nanomechanical forces delivered by the AFM tip specifically inhibit, direct and or enhance differentiation.

[0042] The AFM tip is also used to apply local forces normal to the cells. A normal force is a force which acts perpendicular to the plane of the cell membrane (in this case the tip is applying a downward force perpendicular to the cell membrane). In one embodiment of the present invention, the AFM tip is placed on the surface of the cell and a normal force is applied in one direction, effecting differentiation. Forces useful in this embodiment include, but are not limited to, constant, oscillating or sporadic forces. The responses of the cell (elastic, viscoelastic or beating properties) after the application of, in a non-limiting example, a lateral force, are then measured.

[0043] The AFM tip may also be used to apply local lateral forces to a cell in a directed mechanotactic manner. Placement of the tip next to the cell and then applying a lateral force in one direction may effect differentiation. Constant lateral forces may induce the cell to move away from the tip and, in turn, change its developmental fate and inhibit or enhance differentiation. Lateral forces useful in this embodiment include, but are not limited to, constant, oscillating or sporadic forces. The responses of the cell (elastic, viscoelastic or beating properties) after the application of, in a non-limiting example, a lateral force are then measured.

[0044] Magnetic fields have been shown to affect proliferation and growth factor expression in cultured cells and interfere with endorphinergic and cholinergic systems in intact organisms as wells as inducing stem cell cardiogenesis.

[0045] As a result of the characterization of stem cells by the AFM, paradignmatic motion/elastic features can be reconstructed during a specific differentiating process and those features can then be imposed on undifferentiated cells using the AFM. The cell fate can thereby be directed with the AFM and provides methods to achieve high-throughput yields of committed stem cells with a physical stimulus of extremely low frequency magnetic fields. [0046] Extremely low frequency (ELF) magnetic fields (30-200 Hz) can have effects on cell motion and other elastic properties and the effects of amplitude, frequency and wave shape of MF can direct stem cells to different fates. In another embodiment of the present invention, magnetic beads are implanted within individual cells, attached to the external cell membrane or freely suspended in liquid culture dishes and oscillated with magnetic fields to provide local point forces to inhibit, enhance and/or direct differentiation.

[0047] The AFM tip can be used as a local probe for delivery of local forces including, but not limited to, local normal and lateral forces. By placing the tip on the substrate next to a cell, local lateral forces of known magnitude can be delivered. By measuring the lateral deflection of the cell, the applied force can be detected. As the cell moves away from the applied force (directed mechanotaxis) the lateral deflecstion will return to zero. The time required for a return to zero lateral deflection represents the response time of the cell. The response time is governed by the local nanomechanical forces of the cell and the amount of applied force.

[0048] As seen in Figure 2, the AFM tip is placed on the substrate beside the living cell. The tip can be moved and pressed against the cell to produce a net lateral force and deflection. As the cell responds to the force it moves away at a given rate which can be measured by monitoring the lateral deflection of the tip as it returns to zero.

[0049] In one embodiment of the present invention, the response time of a living cell in response to a local normal or lateral force is measured and is diagnostic of cellular nanomechanical responses.

[0050] In another embodiment of the present invention, the mechanotaxis response can measure the cellular response to drugs. Any cell type may be exposed to any type of drug and the response time used as a nanomechanical signature. These signatures may be used to monitor the effectiveness of a drug or used to differentiate between cell types, genetic mutants or metastatic potential. In a non-limiting example, the response to an anti- cytoskeletal drug is determined. The cytoskeleton is used by the cell to make nanomechanical movements and the presence of anti-cytoskeletal drugs will effect the response time. Cells with differing metastatic potential will display differing response times to a lateral force. Therefore the response time can be used as a signature for the aggressiveness of a given cancer type at the single cell level. In one embodiment of the present invention, directed mechanotaxis methods provide a rapid, single cell-based technique for the determination of cancer aggressiveness.

[0051] In yet another embodiment, cells may be patterned on a surface using directed mechanotaxis without the use of functionalized surfaces or extensive nanopatterning. This allows the controlled and intentional placement of cells in a pattern for the study of cellular activities including, but not limited to, signaling pathways, metabolic responses or any type of cellular function using one or more cells.

[0052] Figure 3 depicts single cell patterning using directed mechanotaxis. In panel A, single cells are randomly arranged in a culture dish. A lateral force (arrows) is appliced to each individual cell to direct its position. In panel B, the arrangement of cells in a particular exemplary pattern after directed mechanotaxis is depicted.

EXAMPLES

Example 1 Nanoscale Characterization of Myxococcus xanthus Cells with Atomic Force Microscopy

[0053] Bacterial Strains and Growth Conditions. Myxococcus xanthus strains of the following types were used: DK1622 (wild type), DK10407 {pilA, pilus ), SW504 (AdifA, fibril^'), DK3088 {sglA1 stk, fibrir), and HK1324 (Awzt wzm wbgA (ΩKan^r), LPS O antigen^"). The strains were grown at 32°C in CYE medium (10 g/liter casitone, 5 g/liter yeast extract, and 8 mM MgSO₄ in 10 mM Mops buffer, pH 7.6) on a rotary shaker at 225 rpm.

[0054] Cell Immobilization. For imaging in air, logarithmic-phase M. xanthus cells were collected by centrifugation at 6,000 x gfor 5 min, washed in Mops buffer (10 mM Mops/4 mM MgSO₄, pH 7.6), and resuspended to 1 x 10⁷ cells per mL in the same buffer. A 12-well CeI- Line glass slide (Erie Scientific, Portsmouth, NH) was cleaned with 75% ethanol and wiped dry with lens paper. Resuspended M. xanthus cells (20 μL) were added to one well for 30 min, and excess liquid was removed with filter paper. The slide was then air-dried before imaging. To image cell groups, cells were resuspended to 1 x 10⁸ cells per mL and prepared as above; a wash step was added after the 30-min incubation to remove the unattached cells.

[0055] For imaging in fluid, an 18- x 18-mm coverslip was cleaned with 75% ethanol. One drop of 1 % polyethyleneimine (PEI) (M_r 1 ,200) dissolved in deionized water was placed on one side of the glass and allowed to adsorb for 3 h, after which the drop was decanted, and the coverslip was rinsed in water and air-dried. Logarithmic-phase M. xanthus cells were collected by centrifugation at 6,000 x g for 5 min, washed with PBS (pH 7.4), and resuspended to 10⁹ cells per mL. For fixing, the cells were stirred in 2.5% (vol/vol) glutaraldehyde for 2 h at 4°C, rinsed, and resuspended in 1 mM Tris (pH 7.5). One drop of the cell suspension (with or without fixing) was placed on a PEI-coated coverslip and placed in a CentriVap concentrator (Labconco) without spinning for 20-30 min at 35°C to evaporate excess water without drying the cells. The coverslip was then adhered to a Petri dish and submerged in deionized water for AFM imaging. After imaging, cell viability was verified by streaking the M. xanthus cells on a CYE agar plate and checking growth after 24 h.

[0056] AFM. All imaging in air and fluid was carried out with a Nanoscope IV Bioscope (Veeco Digital Instruments). Oxide-sharpened cantilevers (OTR4, Olympus) with spring constants of 0.02 N/m and a tip radius of <10 nm were used in contact mode. Fluid imaging and mechanical measurements were performed at room temperature. All force measurements were recorded at a pulling rate of 1 Hz. "Height" and "deflection" images were simultaneously recorded. Deflection images do not represent the true topography of the sample; however, they consistently revealed a higher sensitivity to small surface features and yielded images with greater detail. Images presented in this study are deflection images, but the quantitative measurements of cell structures were taken from the height data on the same sample.

[0057] M. xanthus Cell Organization in a Social Group. Actively growing wild-type M. xanthus cells (DK1622) were dotted at various concentrations on glass slides and allowed to settle for 30 min. The samples were washed several times to remove the unattached cells, air dried, and imaged under AFM in contact mode. As shown in Fig. 4, panel A, attached cells were seen as large mound-like cell packs, and aligned individual cells were found connecting different "mounds." Focusing in on the edge of a cell mound, where cells were in a single layer, clearly depicted the cell alignment and organization (Fig. 4, panel B). Individual cells measured ~5 μm long (sometimes as long as 10 μm) and -800 nm wide. When a higher-resolution image was taken on the edge of the cell group, pili were clearly seen at the cell poles (arrows, Fig. 4, panel C).

[0058] Cell-Surface Ultrastructures. Wild type and S-motility mutants were imaged in air to examine their cellular ultrastructures. A lower concentration of actively growing M. xanthus cells was dotted directly from liquid culture onto glass slides. Many S-motility mutants are defective in certain extracellular structures. The pilA mutant lacks pili, the dif mutant lacks fibril material and has been reported to be overpiliated, the stk mutant overproduces fibril material, and the LPS O antigen mutant lacks LPS O antigen (a component of the outer cell wall). To examine the effects of these genetic mutations on nanoscale surface morphology under native conditions, the cells were harvested from actively growing cultures, air-dried, and imaged directly with AFM.

[0059] Polar TFP. As shown in Figure 4, panel D, type IV pili (TFP) filaments measuring -4-6 μm in length and 5-8 nm in diameter could be clearly seen at the cell poles of wild-type strain DK1622, whereas the filaments were missing from the pilA mutant (Figure 4, panel F). The filaments extended from the cell pole and spread out from the long axis of the cell, with a slight curve in all filaments. This native pili morphology is rather different from that seen in EM micrographs, where pili appeared as randomly arranged fibers. It is interesting to note that cells adjacent to each other often have pili at the same end (Figure 4, panel D). Because little sample preparation was involved for AFM imaging, this observation suggested that cells in close proximity to each other may coordinate their pili-shooting direction.

[0060] Overpiliation has been reported in the dif mutants that lack extracellular fibril material. When a dif mutant, SW504 (ΔdifA), was examined under AFM, the cells did display longer pili at cell poles (Figure 4, panel G). Notably, the pili morphology in the dif mutant also appeared to be different from that of the wild-type cells, with the majority bending backwards toward the cell body instead of shooting forward as in the wild type (Figure 4, panel G). It has been reported that pili in dif mutants fail to retract, leading to the overpiliation phenotype and S-motility defect. The pili morphology observed here might be a result of the pili overextension and may correlate with the defective pili function in the S motility in dif mutants.

[0061] Extracellular polysaccharide matrix. The dif mutants lack extracellular fibril material. SEM observations had originally defined "fibrils" as a matrix material consisting of branching extensions -30 nm in diameter that surrounds the wild-type cells. Later studies revealed that these fibrils likely form a mesh of extracellular polymeric substances over the entire cell body. When wild-type M. xanthus cells were examined by using AFM, no filamentous structures were seen on the cell body (Figures 4, panels D and E). Nevertheless, several structural features could be noticed on the AFM images. (/^') The cell surfaces appeared rather "rough," as shown in Figure 4 panel D, and (//^') slime-like structures were often seen covering or extending from the cell body, as shown in Figure 4, panel E. The dif mutant, however, appeared much "smoother" (Figure 4, panel G). To quantify the visual differences, a roughness analysis of the cellular surfaces was carried out. Roughness (R) is defined as the standard deviation of the height values (/?) away from the mean height (h₀) of a given scan line over the cell surface. The R_rms for the wild-type and pilA cells were determined to be 4.30 ± 1.09 nm and 3.43 ± 0.91 nm, respectively. However, the dif mutant displayed much less roughness (2.54 ± 0.77 nm).

[0062] The M. xanthus stk mutants are known to have constitutively high polysaccharide production and a higher-than-normal level of fibril material. When the stk mutant (DK3088) was examined under AFM, an excessive amount of slime-like substances were seen on the cell surface, and long filamentous structures were often found extending from the cell body (Figure 4, panel H). The stk mutants were known to exhibit a variety of properties including the clumping of cells during growth in liquid culture, rapid agglutination, and the formation of colonies in which cells adhere tightly to each other and the agar surface. The striking amount of extracellular substances observed under physiological conditions provided the structural basis for these phenotypes. The surface roughness of the stk mutant was also measured and averaged 7.16 ± 2.74 nm, significantly higher than that of wild-type cells (4.30 ± 1.09 nm).

[0063] Another important type of extracellular polysaccharide in M. xanthus is LPS. M. xanthus LPS is typical of Gram-negative bacteria and consists of lipid A, which forms the outer leaflet of the outer membrane bilayer; core, which is a chain of carbohydrates attached to lipid A; and O antigen, which contains a variable number of repeating oligosaccharide units and extends outward from the core. Genetic studies showed that the wzm wzt wbgA genes in the sasA locus of M. xanthus encode LPS O antigen biogenesis proteins, and the LPS O antigen mutant (HK1324, Awzm wzt wbgA) was defective in S motility. When this mutant was imaged with AFM, it exhibited a relatively "clean" cell surface (Figure 4, panel) as compared with wild type. The cell-surface roughness of the mutant was quantified and averaged 4.05 ± 1.42 nm, comparable to that of wild-type cell surface. This finding is expected because LPS O antigen mutants exhibit a wild-type level of extracellular fibril material, which presumably masks the cell wall and contributes to a wild-type-like cell-surface roughness.

[0064] Morphology of Gliding Cells. Aside from the ultrastructure imaging, some unique cell morphology was also observed. As shown in Figure 5, panel A, M. xanthus cells were occasionally seen curling their cell bodies and twisting about each other, leading to a braid- like morphology. Bending and flexing of the cell body was often seen in M. xanthus cells and was believed to be a means for directional change during gliding. The formation observed here suggested that adjacent cells can somehow coordinate their flexing to twist around each other. Interestingly, when examined at a higher resolution, extending pili could be seen at the same cell pole of both twisted cells (Figure 5, panel B), demonstrating again that pili on adjacent cells tend to shoot from the same cell ends.

[0065] Another noteworthy observation involves individual cell morphology. Earlier studies using shock-freezing and SEM revealed motility-associated surface patterns in gliding M. xanthus. The cells appeared to be "rotated" along the long axis of the cell, and the rotation patterns were lost in sodium-azide-treated cells, suggesting that the rotation was associated with active gliding. Under AFM, similar patterns were observed (Figure 5, panel C). Individual cells were seen to be twisted along their long axis, with a helical surface fold running through the cell body. A height profile of the twisted cell body revealed -25- to 100- nm "peaks" separated by ~500-nm "valleys" on the helical patterns along the cell axis (Figure 5, panel C) (600- to 1 ,000-nm separations). When the cells were treated with 0.02% sodium azide for 10 min, no twisting was seen in the entire sample, demonstrating that these morphological changes were correlated with active gliding, rather than representing a drying artifact.

[0066] Local Elasticity of Live M. xanthus Cells in Fluid. The ability of M. xanthus to flex and twist its cell body suggests the high level of flexibility in live M. xanthus cells. Because of its force-spectroscopy capacity, AFM allows the investigation of cell local flexibility or elasticity that is unattainable with any other imaging tools. Wild-type M. xanthus cells were immobilized on a glass coverslip, submerged in fluid, and probed with AFM. However, these cells were stable enough to be isolated and used for the determination of the local cell-wall elasticity or "stiffness" (Young's modulus, E) by measuring force curves (Figure 6, panel A) on the cell. Young's modulus can be determined by converting force-displacement curves (Figure 6, panel A) into force-indentation curves obtained on stable areas of cell surfaces.

[0067] During the approach curve, the force acting on the AFM cantilever was recorded as a function of the displacement of the piezoelectric crystal, which moves the cantilever toward the sample. The force acting on the cantilever remains at zero as long as the AFM cantilever is not in contact with a surface, and the force increases monotonically after contact. Based on these curves, wild-type cells were found to have a local Young's modulus of 0.25 ± 0.18 million pascals (MPa). After glutaraldehyde fixation, wild-type cells displayed an E of 1.34 ± 0.66 MPa, demonstrating a significant change in cell-surface elasticity upon glutaraldehyde treatment.

[0068] Although measurements of bacterial turgor pressure and local spring constants have been reported, very little is known about the Young's modulus of bacterial cell walls, and the AFM measurement of £ on a living bacterium in aqueous conditions has not been previously reported. Studies using "bacteria threads" have estimated E on "wet" Bacillus subtilis at -30 MPa, 100 times higher that the values we obtained with AFM on unfixed M. xanthus in aqueous conditions. AFM has been used to measure the Young's modulus on yeast cells (averaging ~1 MPa) and mammalian cells, which have highly variable and spatially dependent E, and usually falls in the 1- to 200-kPa range. The E value we obtained on wild-type M. xanthus cells therefore reflects the relative stiffness of the M. xanthus cell wall as compared with other organisms.

[0069] In Vivo Force Spectroscopy of Extracellular Fibrils on the M. xanthus Cell Surface. Contact-mediated cell-cell interactions are an important aspect of the social behavior of M. xanthus and are facilitated by extracellular fibril material that exists on the surface of the cells. The force-spectroscopy capacity of AFM allows the investigation of the nanomechanical properties of the M. xanthus surface adhesive molecules.

[0070] Wild-type cells were immobilized in liquid as described above, and force- displacement curves were measured on the cell by lowering the tip and pressing it against the cell surface with a force of -10 nN. Upon tip retraction, a sequence of rupture events occurred at distances of 1-3 μm, presumably arising from the breakage of multiple adhesions between the AFM tip and the cell-surface substances (Figure 6, panel A). The cantilever displacement always returned to its initial zero position after the series of rupture events. Similar retraction curves have been reported for other adhesive polymers (DNA, proteins, and polysaccharides), suggesting that the adhesive substances on the M. xanthus cell surface were extracellular polymeric molecules. Wild-type cells fixed with glutaraldehyde, which cross-links the extracellular polymers, displayed retraction curves consistent with small or absent adhesion events (Figure 6, panel A, curve iii), thus confirming that it is extracellular polymer that adheres to AFM tips. Force curves were also measured on bare substrate, before and after measurement on the cell, confirming that there was no contamination on the AFM tip that may have caused nonspecific adhesion between the tip and the cell surface (Figure 6, panel B). Therefore, we ascribe the adhesion events to the stretching of extracellular polymeric substances with the AFM tip.

[0071] The average adhesion force of each rupture event is -2.5 nN, significantly greater than forces obtained on most other microbial surfaces (-20-900 pN), suggesting the high degree of surface adhesiveness in this social bacterium. The stk mutants were examined under the same conditions and exhibited a greater number of major adhesions (one to nine events) compared with the wild type (one to three events). However, both cell types exhibit a similar average adhesion force (Figure 6, panel A), indicating that the fibril material from both strains is similar in its chemical adhesion properties. More than 100 measurements were made on each cell type for the force-spectroscopy study. The adhesion events on the stk mutant occurred after the tip was retracted more than 1 μm, whereas wild-type adhesion events usually ceased after -1 μm of tip retraction (Figure 6, panel A). Although the measured retraction length is partly determined by where the tip contacts the fibril, stk cells still display a 50-80% longer retraction length than do the wild type, indicating that the fibril- material molecules on the stk mutant are longer, in general, than those of the wild-type cells. These observations are consistent with the fibril-overproducing phenotype of the stk mutant and the morphological features observed by using AFM (Figure 4, panel H).

[0072] These in vivo systems are complex and very different from the idealized systems presented in previous polysaccharide force-spectroscopy studies. In an artificial system, the concentration of polysaccharide on a surface can be precisely controlled so that only one polysaccharide filament can be pulled for each force-spectroscopy measurement. On a cell surface, however, this precision is impossible to achieve. Fibril material forms a complex mesh-like structure that has not been precisely determined, but the transient and local contact between an AFM tip and a cell surface is a reasonable model for examining the adhesive property of the M. xanthus cell surface.

Example 2 Turning on Stem Cell Cardioqenesis with Extremely Low Frequency Magnetic Fields

[0073] Magnetic fields (MF) can prime the expression of genes encoding for tissue- restricted transcription factors in pluripotent mouse embryonic stem (ES) cells and ES cell exposure to MF leads to targeted cell lineage specification.

[0074] ES cell exposure to extremely low-frequency MF triggers GATA-4 and Nkx-2.5 gene expression. A sinusoidal MF (50 Hz, 0.8 mT rms) was applied to GTR1 cells, a derivative of pluripotent mouse R1 ES cells bearing the puromycin resistance gene driven by the cardiomyocyte-specific α-myosin heavy chain promoter. Treatment was performed continuously after removal of leukemia inhibitory factor (LIF) until time of collection of embryoid bodies or ES-derived cardiomyocytes (3 or 10 days from LIF withdrawal, respectively). In both groups of cells, MF remarkably increased the expression of GATA-4 and Nkx-2.5 mRNA, encoding for a zinc finger-containing transcription factor and a homeodomain that are essential for cardiogenesis in various animal species, including humans

[0075] Figure 7 depicts the effect of magnetic field on the expression of cardiac lineage- promoting genes. Magnetic field (MF) was applied from the time of LIF removal. EBs (Figure 7, panel A) or puromycin-selected cardiomyocytes (P, Figure 7, panel B) were collected after 3 or 10 additional days, respectively, and processed for RT-PCR analysis of the indicated transcripts. Ethidium bromide-stained agarose gels (Figure 7, panels A and B) are representative of 4 separate experiments. RNase protection analysis of GATA-4 mRNA expression is depicted in Figure 7, panel F. GTR1 ES cells were cultured as described in the absence (-) or presence (+) of MF. Figure 7, panel C and D depicts nuclear run-off analysis of GATA-4 gene transcription in isolated ES cell nuclei. Nuclei were isolated from undifferentiated cells (LIF), EBs or P collected 3 or 10 days after LIF removal, respectively. Each group of cells was exposed in the absence (-) or presence (+) of MF from the time of LIF withdrawal. Row a: GATA-4 gene transcription. Autoradiograms are representative of three separate experiments. Figure 7, panel E depicts immunoreactive dynorphin B (ir-dyn B) in cells (gray bars) or medium (white bars). [0076] MF induces the activation of an endorphinergic system. MF enhanced prodynorphin mRNA expression and levels of dynorphin B, a bioactive end product of the gene acting as a natural agonist of kappa opioid receptors, in both EBs and ES-derived cardiomyocytes, as well as in their incubation media (Figure 7). Prodynorphin gene and dynorphin B expression has been found to play a major role in ES cell cardiogenesis priming GATA-4 and Nkx-2.5 transcription through the activation of protein kinase C signaling and nuclear opioid receptors.

[0077] MF acts at the transcriptional level. Nuclear run-off transcriptional analysis revealed that the transcription rate of the GATA-4 gene was greatly enhanced in nuclei that had been isolated from EBs or ES-derived cardiomyocytes obtained from MF-exposed cells compared with nuclei from unexposed cells (Figure 7).

[0078] MF increases the yield of ES-derived cardiomvocvtes. The application of a magnetic field primes the expression of cardiac specific genes and enhances the yield of ES-derived cardiomyocytes (Figure 8). MF was applied from the time of LIF removal. EBs or P were collected after 3 or 10 additional days, respectively. Figure 8, panels A and B depict RT-PCR analysis of α-myosin heavy chain (MHC) and myosin light chain-2V (MLC). MHC immunostaining was assessed by the MF20 monoclonal antibody in undifferentiated cells (Figure 8, panel C) and in cardiomyocytes derived from cells cultured in the absence (Figure 8, panel D) or presence (Figure 8, panel E) of MF. DNA was visualized with propidium iodide (1 μg/mL).

[0079] Activation of a program of cardiac lineage-restricted genes was associated with an increase in the expression of the cardiac specific transcripts α-myosin heavy chain (MHC) and myosin light chain-2V (Figure 8, panels A and B). MHC expression in cardiomyocytes from MF-exposed cells was further confirmed in immunofluorescence studies (Figure 8, panels C-E). Exposure of GTR1 ES cells to MF after LIF removal and throughout 4 days of puromycin selection consistently increased the yield of ES-derived cardiomyocytes (number of beating colonies reached 180.38 ± 33.0% of the control value estimated in cardiomyocytes selected from unexposed cells).

[0080] Effect of MF on the expression of genes promoting non-myocardial lineages. It is noteworthy that expression of MyoD, a gene involved in skeletal myogenesis, was not affected in EBs derived from MF-exposed cells. EBs from exposed cells exhibited a slight increase in expression of neurogenini , a neuronal specification gene.

[0081] Magnetic fields have been shown to elicit behavioral changes in intact organisms and cell proliferation in vitro. The data showing the ability of MF to prime a gene expression pattern of cardiogenesis may profoundly affect the understanding of the biological consequences of MF exposure at cellular level. Failure of MF to affect the transcription of a gene promoting skeletal muscle determination and the faint effect on neuronal specification seem to exclude a generalized activation of repressed genes and suggests that coupling of MF with GATA-4, Nkx-2.5 and prodynorphin gene expression may represent a mechanism pertaining to ES cell cardiogenesis.

[0082] Stem cells were proposed recently as a renewable source of donor cells for the rescue of damaged tissues. However, such a rescuing potential is limited by the fact that differentiating cells withdraw early from the cell cycle, and development of strategies affording high throughput of targeted lineages from pluripotent cells would have obvious biomedical implications. Overexpression of tissue-specific genes by vector-mediated gene transfer is a cumbersome approach that may perturb normal homeostasis in stem cells and recipient tissues and is not readily envisionable in humans. The finding that MF can elicit a remarkable increase in the yield of ES-derived cardiomyocytes provides evidence for the potential use of magnetic fields in modifying the gene program of cardiac differentiation in ES cells without the aid of gene transfer technologies.

[0083] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0084] The terms "a", "an", "the" and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. "such as") provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0085] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0086] Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on those embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0087] Furthermore, numerous references have been made to patents and printed publications throughout this specification. Each of the above cited references and printed publications are individually incorporated by reference herein in their entirety.

[0088] In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described.

Claims

We claim:

1. A method of determining the differentiation state of one or more cells comprising determining at least one nanomechanical characteristic of said one or more cells.

2. A method of influencing the differentiation state of one or more cells comprising applying a nanomechanical force to said cells.

3. The method according to claim 1 wherein said determining step comprises applying a nanomechanical force to said cells.

4. The method according to either of claims 1 or 2 wherein said cells are stem cells.

5. The method according to claim 4 wherein said stem cells are embryonic stem cells.

6. The method according to either of claim 1 wherein said nanomechanical characteristic is selected from the group consisting of cell motion, elasticity and viscoelastic properties.

7. The method according to either of claims 2 or 3 wherein said cells are disposed on a cantilever prior to application of said nanomechanical force.

8. The method according to either of claims 2 or 3 wherein said nanomechanical force is applied with an atomic force, an electrical signal, a physical force or a magnetic force.

9. The method according to claim 8 wherein said nanomechanical force is applied with magnetic beads.

10. The method according to claim 8 wherein said nanomechanical force is applied with an atomic force microscope.

11. The method according to claim 8 wherein said nanomechanical force is applied by an electrical signal.

12. The method according to claim 8 wherein said magnetic force is an extremely low frequency magnetic force.

13. The method according to either of claims 2 or 3 wherein said nanomechanical force is a local lateral force.

14. The method according to either of claims 2 or 3 wherein said nanomechanical force is a local normal force.

15. The method according to claim 8 wherein applying said physical force comprises growing said cells on a polymeric substrate, said polymeric substrate capable of stretching or bending and wherein stretching or bending said polymeric substrate applies a force to said cells.

16. The method according to claim 15 wherein said polymeric substrate is coated with collagen.

17. The method according to claim 2 wherein influencing the differentiation state of said cells comprises inhibiting or promoting differentiation of said cells.

18. The method according to claim 8 wherein application of said physical force comprises: coating a carbon nanotube ATF tip with a DNA binding protein; inserting said coated ATF tip into a nucleus of a cell; allowing said binding protein to bind to DNA sequences in said nucleus; and applying a nanomechanical force to said DNA sequences bound to said coated ATF tip thereby influencing the differentiation state of said cell.

19. The method according to claim 18 wherein said DNA binding protein is a zinc finger binding protein.