Evaluation of the influence of growth medium composition on cell elasticity

Recently, there has been an increasing interest in using the biomechanical properties of cells as biomarkers to discriminate between normal and cancerous cells. However, few investigators have considered the influence of the growth medium composition when evaluating the biomechanical properties of the normal and diseased cells. In this study, we investigated the variation in Young's modulus of non-malignant MCF10A and malignant MDA-MB-231 breast cells seeded in five different growth media under controlled experimental conditions. The average Young's modulus of MDA-MB-231 cells was significantly lower (p<0.0001) than the mean Young's modulus of MCF10A cells when compared in identical medium compositions. However, we found that growth medium composition affected the elasticity of MCF10A and MDA-MB-231 cells. The average Young's modulus of both cell lines decreased by 10-18% when the serum was reduced from 10% to 5% and upon addition of epidermal growth factor (EGF, 20 ng/ml) to the medium. Though these elasticity changes might have some biological impact, none was statistically significant. However, the elasticity of MCF10A was significantly more responsive than MDA-MB-231 cells to the medium composition supplemented with EGF, cholera toxin (CT), insulin (INS) and hydrocortisone (HC), which are recommended for routine cultivation of MCF10A cells (M5). MCF10A cells were significantly softer (p<0.002) when grown in medium M5 compared to a standard MDA-MB-231 medium (M1). The investigation of the effects of culture medium composition on the elastic properties of cells highlights the need to take these effects into consideration when interpreting elasticity measurements in cells grown in different media.     

Estimation of cell Young's modulus of adherent cells probed by optical and magnetic tweezers: influence of cell thickness and bead immersion

A precise characterization of cell elastic properties is crucial for understanding the mechanisms by which cells sense mechanical stimuli and how these factors alter cellular functions. Optical and magnetic tweezers are micromanipulation techniques which are widely used for quantifying the stiffness of adherent cells from their response to an external force applied on a bead partially embedded within the cell cortex. However, the relationships between imposed external force and resulting bead translation or rotation obtained from these experimental techniques only characterize the apparent cell stiffness. Indeed, the value of the estimated apparent cell stiffness integrates the effect of different geometrical parameters, the most important being the bead embedding angle 2gamma, bead radius R, and cell height h. In this paper, a three-dimensional finite element analysis was used to compute the cell mechanical response to applied force in tweezer experiments and to explicit the correcting functions which have to be used in order to infer the intrinsic cell Young's modulus from the apparent elasticity modulus. Our analysis, performed for an extensive set of values of gamma, h, and R, shows that the most relevant parameters for computing the correcting functions are the embedding half angle gamma and the ratio h(u)/2R, where h(u) is the under bead cell thickness. This paper provides original analytical expressions of these correcting functions as well as the critical values of the cell thickness below which corrections of the apparent modulus are necessary to get an accurate value of cell Young's modulus. Moreover, considering these results and taking benefit of previous results obtained on the estimation of cell Young's modulus of adherent cells probed by magnetic twisting cytometry (MTC) (Ohayon, J., and Tracqui, P., 2005, Ann. Biomed. Eng., 33, pp. 131-141), we were able to clarify and to solve the still unexplained discrepancies reported between estimations of elasticity modulus performed on the same cell type and probed with MTC and optical tweezers (OT). More generally, this study may strengthen the applicability of optical and magnetic tweezers techniques by insuring a more precise estimation of the intrinsic cell Young's modulus (CYM).     

Modelling the mechanical properties of single suspension-cultured tomato cells

BACKGROUND AND AIMS: The relationship between composition and structure of plant primary cell walls, and cell mechanical properties is not fully understood, partly because intrinsic properties of walls such as Young's modulus cannot be obtained readily. The aim of this work is to show that Young's modulus of walls of single suspension-cultured tomato cells can be determined by modelling force-deformation data. METHODS: The model simulates the compression of a cell between two flat surfaces, with the cell treated as a liquid-filled sphere with thin compressible walls. The cell wall and membrane were taken to be permeable, but the compression was so fast that water loss could be neglected in the simulations. Force-deformation data were obtained by compressing the cells in micromanipulation experiments. RESULTS:Good fits were obtained between the model and low-strain experimental data, using the modulus and initial inflation of the cell as adjustable parameters. The mean Young's modulus for 2-week-old cells was found to be 2.3 +/- 0.2 GPa at pH 5. This corresponds to an instantaneous bulk modulus of elasticity of approx. 7 MPa, similar to a value found by the pressure probe method. However, Young's modulus is a better parameter, as it should depend only on the composition and structure of the cell wall, not on bulk cell behaviour. This new method has been used to show that Young's modulus of cultured tomato cell walls is at its lowest at pH 4.5, the pH optimum for expansin activity. CONCLUSIONS:The linear elastic model is very suitable for estimating wall Young's modulus from micromanipulation experiments on single tomato cells. This is a powerful method for determining cell wall material properties.     

Modulation of cellular mechanics during osteogenic differentiation of human mesenchymal stem cells

Recognition of the growing role of human mesenchymal stem cells (hMSC) in tissue engineering and regenerative medicine requires a thorough understanding of intracellular biochemical and biophysical processes that may direct the cell's commitment to a particular lineage. In this study, we characterized the distinct biomechanical properties of hMSCs, including the average Young's modulus determined by atomic force microscopy (3.2 +/- 1.4 kPa for hMSC vs. 1.7 +/- 1.0 kPa for fully differentiated osteoblasts), and the average membrane tether length measured with laser optical tweezers (10.6 +/- 1.1 microm for stem cells, and 4.0 +/- 1.1 microm for osteoblasts). These differences in cell elasticity and membrane mechanics result primarily from differential actin cytoskeleton organization in these two cell types, whereas microtubules did not appear to affect the cellular mechanics. The membrane-cytoskeleton linker proteins may contribute to a stronger interaction of the plasma membrane with F-actins and shorter membrane tether length in osteoblasts than in stem cells. Actin depolymerization or ATP depletion caused a two- to threefold increase in the membrane tether length in osteoblasts, but had essentially no effect on the stem-cell membrane tethers. Actin remodeling in the course of a 10-day osteogenic differentiation of hMSC mediates the temporally correlated dynamical changes in cell elasticity and membrane mechanics. For example, after a 10-day culture in osteogenic medium, hMSC mechanical characteristics were comparable to those of mature bone cells. Based on quantitative characterization of the actin cytoskeleton remodeling during osteodifferentiation, we postulate that the actin cytoskeleton plays a pivotal role in determining the hMSC mechanical properties and modulation of cellular mechanics at the early stage of stem-cell osteodifferentiation.     

Simple display system of mechanical properties of cells and their dispersion

The mechanical properties of cells are unique indicators of their states and functions. Though, it is difficult to recognize the degrees of mechanical properties, due to small size of the cell and broad distribution of the mechanical properties. Here, we developed a simple virtual reality system for presenting the mechanical properties of cells and their dispersion using a haptic device and a PC. This system simulates atomic force microscopy (AFM) nanoindentation experiments for floating cells in virtual environments. An operator can virtually position the AFM spherical probe over a round cell with the haptic handle on the PC monitor and feel the force interaction. The Young's modulus of mesenchymal stem cells and HEK293 cells in the floating state was measured by AFM. The distribution of the Young's modulus of these cells was broad, and the distribution complied with a log-normal pattern. To represent the mechanical properties together with the cell variance, we used log-normal distribution-dependent random number determined by the mode and variance values of the Young's modulus of these cells. The represented Young's modulus was determined for each touching event of the probe surface and the cell object, and the haptic device-generating force was calculated using a Hertz model corresponding to the indentation depth and the fixed Young's modulus value. Using this system, we can feel the mechanical properties and their dispersion in each cell type in real time. This system will help us not only recognize the degrees of mechanical properties of diverse cells but also share them with others.     

An extended modeling of the micropipette aspiration experiment for the characterization of the Young's modulus and Poisson's ratio of adherent thin biological samples: numerical and experimental studies

The micropipette aspiration (MA) experiment remains a quite widely used micromanipulation technique for quantifying the elastic modulus of cells and, less frequently, of other biological samples. However, moduli estimations derived from MA experiments are only valid if the probed sample is non-adherent to the rigid substrate. This study extends this standard formulation by taking into account the influence of the sample adhesion. Using a finite element analysis of the sample aspiration into the micropipette, we derived a new expression of the aspirated length for linear elastic materials. Our results establish that (i) below a critical value, the thickness h of the probed sample must be considered to get an accurate value of its Young's modulus (ii) this critical value depends both on the Poisson's ratio and on the sample adhesivity. Additionally, we propose a novel method which allows the computation of the intrinsic Young's modulus of the adherent probed sample from its measured apparent elasticity modulus. Thanks to the set of computational graphs we derived from our theoretical analysis, we successfully validate this method by experiments performed on polyacrylamide gels. Interestingly, the original procedure we proposed allows a simultaneous quantification of the Young's modulus and of the Poisson's ratio of the adherent gel. Thus, our revisited analysis of MA experiments extends the application domain of this technique, while contributing to decrease the dispersion of elastic modulus values obtained by this method.     

An Insight into Cell Elasticity and Load-Bearing Ability. Measurement and Theory

We have studied the elasticity and load bearing ability of plant tissue at the cellular level, using onion (Allium cepa) epidermal cells. The Young's modulus and Poisson's ratio of the cells were obtained by loading a tensile force on onion epidermal peels of different turgor pressures, and measuring the elongation and the lateral contraction of the peels. The Young's moduli and the Poisson's ratios ranged from 3.5 to 8.0 MPa and 0.18 to 0.30, respectively. To determine the effects of cell elasticity and turgor pressure on the cell's ability to bear load, we loaded a small glass ball onto a cell and measured the projected contact area between the ball and the cell. Unlike previous studies, we considered the cell as a whole entity, and utilized the Boussinesq's solution to derive the relevant equations that related the elastic parameters and cell deformation. For cells with a turgor pressure > or = 0.34 MPa, the predicted contact area agreed well with the measured area. The equations could also predict cell turgor pressure with a deviation from the measured value of 0.01 MPa. This study gives strong support to ball tonometry, a new method of measuring cell turgor pressure.     

Researching into the cellular shape, volume and elasticity of mesenchymal stem cells, osteoblasts and osteosarcoma cells by atomic force microscopy

Within the bone lie several different cell types, including osteoblasts (OBs) and mesenchymal stem cells (MSCs). The MSCs are ideal targets for regenerative medicine of bone due to their differentiation potential towards OBs. Human MSCs exhibit two distinct morphologies: rapidly self-renewing cells (RS) and flat cells (FC) with very low proliferation rates. Another cell type found in pathological bone conditions is osteosarcoma. In this study, we compared the topographic and morphometric features of RS and FC cells, human OBs and MG63 osteosarcoma cells by atomic force microscopy (AFM). The results demonstrated clear differences: FC and hOB cells showed similar ruffled topography, whereas RS and MG63 cells exhibited smoother surfaces. Furthermore, we investigated how selected substrates influence cell morphometry. We found that RS and MG63 cells were flatter on fibrous substrates such as polystyrene and collagen I, but much more rounded on glass, the smoothest surface. In contrast, cells with large area, namely FC and hOB cells, did not exhibit pronounced changes in flatness with regards to the different substrates. They were, however, remarkably flatter in comparison to RS and MG63 cells. We could explain the differences in flatness by the extent of adhesion. Indeed, FC and hOB cells showed much higher content of focal adhesions. Finally, we used the AFM to determine the cellular Young's modulus. RS, FC and hOB cells showed comparable stiffness on the three different substrates, while MG63 cells demonstrated the unique feature of increased elasticity on collagen I. In summary, our results show, for the first time, a direct comparison between the morphometric and biophysical features of different human cell types derived from normal and pathological bone. Our study manifests the opinion that along with RNA, proteomic and functional research, morphological and biomechanical characterization of cells also reveals novel cell features and interrelationships.     

Comparative study of hematopoietic differentiation between human embryonic stem cell lines

Directed differentiation of human embryonic stem cells (hESCs) into any desired cell type has been hailed as a therapeutic promise to cure many human diseases. However, substantial roadblocks still exist for in vitro differentiation of hESCs into distinct cell types, including T lymphocytes. Here we examined the hematopoietic differentiation potential of six different hESC lines. We compare their ability to develop into CD34(+) or CD34(+)CD45(+) hematopoietic precursor populations under several differentiation conditions. Comparison of lymphoid potential of hESC derived- and fetal tissue derived-hematopoietic precursors was also made. We found diverse hematopoietic potential between hESC lines depending on the culture or passage conditions. In contrast to fetal-derived hematopoietic precursors, none of the CD34(+) precursors differentiated from hESCs were able to develop further into T cells. These data underscore the difficulties in the current strategy of hESC forward differentiation and highlight distinct differences between CD34(+) hematopoietic precursors generated in vitro versus in vivo.     

Differing lectin binding profiles among human embryonic stem cells and derivatives aid in the isolation of neural progenitor cells

Human embryonic stem cells (hESCs) and their differentiated progeny allow for investigation of important changes/events during normal embryonic development. Currently most of the research is focused on proteinacous changes occurring as a result of differentiation of stem cells and little is known about changes in cell surface glycosylation patterns. Identification of cell lineage specific glycans can help in understanding their role in maintenance, proliferation and differentiation. Furthermore, these glycans can serve as markers for isolation of homogenous populations of cells. Using a panel of eight biotinylated lectins, the glycan expression of hESCs, hESCs-derived human neural progenitors (hNP) cells, and hESCs-derived mesenchymal progenitor (hMP) cells was investigated. Our goal was to identify glycans that are unique for hNP cells and use the corresponding lectins for cell isolation. Flow cytometry and immunocytochemistry were used to determine expression and localization of glycans, respectively, in each cell type. These results show that the glycan expression changes upon differentiation of hESCs and is different for neural and mesenchymal lineage. For example, binding of PHA-L lectin is low in hESCs (14±4.4%) but significantly higher in differentiated hNP cells (99±0.4%) and hMP cells (90±3%). Three lectins: VVA, DBA and LTL have low binding in hESCs and hMP cells, but significantly higher binding in hNP cells. Finally, VVA lectin binding was used to isolate hNP cells from a mixed population of hESCs, hNP cells and hMP cells. This is the first report that compares glycan expression across these human stem cell lineages and identifies significant differences. Also, this is the first study that uses VVA lectin for isolation for human neural progenitor cells.     

Spatial and temporal dependence of the cerebral endothelial cells elasticity

The reliable determination of the mechanical properties of a living cell is one of the most important challenges of the atomic force microscopic measurements. In the present study the spatial and temporal dependency of the force measurements on cerebral endothelial cells was investigated. Besides imaging the cells, two different sequences of force measurements were applied: Acquisition of force curves in short time at several points across the cell surface investigating spatial dependence of the elasticity. Acquisition of force curves for long time at a previously determined place, over the cell nucleus, which provides the temporal stability/variation of the measured forces/values. Three different stages of endothelial cell cultures of the hCMEC/D3 cells were used: sub-confluent living, confluent living, and confluent fixed cells. The Young's modulus was calculated from the force curves using the Hertz model and the results were plotted against time or location correspondingly. The rational of using the three stage of culture was to clarify whether the observed effect belongs to the individual cell, to the ensemble of cells or just to some, not living cell component. In case of sub-confluent cells the results revealed a softer nuclear region compared to the periphery, while an attenuated oscillation like fluctuation in time, with a period of about 10-30 min, was observed. Confluent living cells showed similar tendencies to the sub-confluent cells, but the changes were larger and the temporal oscillations had longer period. The spatial dependency of the elasticity on confluent cells was confirmed by force-volume measurement too. In case of fixed cells neither spatial nor temporal differences were observed between the nuclear and peripheral region, however the Young's modulus and the error of the measurement was larger, compared to the sub-confluent living cells.     

Sickle cell trait human erythrocytes are significantly stiffer than normal

Atomic force microscopy (AFM) allows for high-resolution topography studies of biological cells and measurement of their mechanical properties in physiological conditions. In this work, AFM was employed to measure the stiffness of abnormal human red blood cells from human subjects with the genotype for sickle cell trait. The determined Young's modulus was compared with that obtained from measurements of erythrocytes from healthy subjects. The results showed that Young's modulus of pathological erythrocytes was approximately three times higher than in normal cells. Observed differences indicate the effect of the polymerization of sickle hemoglobin as well as possible changes in the organization of the cell cytoskeleton associated with the sickle cell trait.     

The effect of porosity and mineral content on the Young's modulus of elasticity of compact bone

The Young's modulus of elasticity, the calcium content and the volume fraction (1-porosity) of 23 tension specimens and 80 bending specimens, taken from compact bone of 18 species of mammal, bird and reptile, were determined. There was a strong positive relationship between Young's modulus and both calcium content and volume fraction. A power law model fits the data better than a linear model. Young's modulus has a roughly cubic relationship with both calcium content and volume fraction. Over 80% of the total variation in Young's modulus in this data set is explained by these two variables.     

Graphene enhances the cardiomyogenic differentiation of human embryonic stem cells

Graphene has drawn attention as a substrate for stem cell culture and has been reported to stimulate the differentiation of multipotent adult stem cells. Here, we report that graphene enhances the cardiomyogenic differentiation of human embryonic stem cells (hESCs) at least in part, due to nanoroughness of graphene. Large-area graphene on glass coverslips was prepared via the chemical vapor deposition method. The coating of the graphene with vitronectin (VN) was required to ensure high viability of the hESCs cultured on the graphene. hESCs were cultured on either VN-coated glass (glass group) or VN-coated graphene (graphene group) for 21 days. The cells were also cultured on glass coated with Matrigel (Matrigel group), which is a substrate used in conventional, directed cardiomyogenic differentiation systems. The culture of hESCs on graphene promoted the expression of genes involved in the stepwise differentiation into mesodermal and endodermal lineage cells and subsequently cardiomyogenic differentiation compared with the culture on glass or Matrigel. In addition, the culture on graphene enhanced the gene expression of cardiac-specific extracellular matrices. Culture on graphene may provide a new platform for the development of stem cell therapies for ischemic heart diseases by enhancing the cardiomyogenic differentiation of hESCs.     

Improved lentiviral gene transfer into human embryonic stem cells grown in co-culture with murine feeder and stroma cells

Genetic modification of human embryonic stem cells (hESCs) using biophysical DNA transfection methods are hampered by the very low single cell survival rate and cloning efficiency of hESCs. Lentiviral gene transfer strategies are widely used to genetically modify hESCs but limited transduction efficiencies in the presence of feeder or stroma cells present problems, particularly if vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped viral particles are applied. Here, we investigated whether the recently described semen derived enhancer of virus infection (SEVI) and alternative viral envelope proteins derived from either Gibbon ape leukaemia virus (GALV) or feline leukaemia virus (RD114) are applicable for transducing hESCs during co-culture with feeder or stroma cells. Our first set of experiments demonstrates that SEVI has no toxic effect on murine or hESCs and that exposure to SEVI does not interfere with the pluripotency-associated phenotype. Focusing on hESCs, we were able to further demonstrate that SEVI increases the transduction efficiencies of GALV and RD114 pseudotyped lentiviral vectors. More importantly, aiming at targeted differentiation of hESCs into functional somatic cell types, GALV pseudotyped lentiviral particles could efficiently and exclusively transduce hESCs grown in co-culture with OP9-GFP stroma cells (which were often used to induce differentiation into haematopoietic derivatives).     

Production of erythriod cells from human embryonic stem cells by fetal liver cell extract treatment

Background  

We recently developed a new method to induce human stem cells (hESCs) differentiation into hematopoietic progenitors by cell extract treatment. Here, we report an efficient strategy to generate erythroid progenitors from hESCs using cell extract from human fetal liver tissue (hFLT) with cytokines. Human embryoid bodies (hEBs) obtained of human H1 hESCs were treated with cell extract from hFLT and co-cultured with human fetal liver stromal cells (hFLSCs) feeder to induce hematopoietic cells. After the 11 days of treatment, hEBs were isolated and transplanted into liquid medium with hematopoietic cytokines for erythroid differentiation. Characteristics of the erythroid cells were analyzed by flow cytometry, Wright-Giemsa staining, real-time RT-PCR and related functional assays.     

Nodal inhibits differentiation of human embryonic stem cells along the neuroectodermal default pathway

Genetic studies in fish, amphibia, and mice have shown that deficiency of Nodal signaling blocks differentiation into mesoderm and endoderm. Thus, Nodal is considered as a major inducer of mesendoderm during gastrulation. On this basis, Nodal is a candidate for controlling differentiation of pluripotent human embryonic stem cells (hESCs) into tissue lineages with potential clinical value. We have investigated the effect of Nodal, both as a recombinant protein and as a constitutively expressed transgene, on differentiation of hESCs. When control hESCs were grown in chemically defined medium, their expression of markers of pluripotency progressively decreased, while expression of neuroectoderm markers was strongly upregulated, thus revealing a neuroectodermal default mechanism for differentiation in this system. hESCs cultured in recombinant Nodal, by contrast, showed prolonged expression of pluripotency marker genes and reduced induction of neuroectoderm markers. These Nodal effects were accentuated in hESCs expressing a Nodal transgene, with striking morphogenetic consequences. Nodal-expressing hESCs developing as embryoid bodies contained an outer layer of visceral endoderm-like cells surrounding an inner layer of epiblast-like cells, each layer having distinct gene expression patterns. Markers of neuroectoderm were not upregulated during development of Nodal-expressing embryoid bodies, nor was there induction of markers for definitive mesoderm or endoderm differentiation. Moreover, the inner layer expressed markers of pluripotency, characteristic of undifferentiated hESCs and of epiblast in mouse embryos. These results could be accounted for by an inhibitory effect of Nodal-induced visceral endoderm on pluripotent cell differentiation into mesoderm and endoderm, with a concomitant inhibition of neuroectoderm differentiation by Nodal itself. There could also be a direct effect of Nodal in the maintenance of pluripotency. In summary, analysis of the Nodal-expressing phenotype suggests a function for the transforming growth factor-beta (TGF-beta) growth factor superfamily in pluripotency and in early cell fate decisions leading to primary tissue layers during in vitro development of pluripotent human stem cells. The effects of Nodal on early differentiation illustrate how hESCs can augment mouse embryos as a model for analyzing mechanisms of early mammalian development.