Human mesenchymal stem cells (hMSCs), during in vitro expansion, gradually lose their distinct spindle morphology, self-renewal ability, multi-lineage differentiation potential and enter replicative senescence. This loss of cellular function is a major roadblock for clinical applications which demand cells in large numbers. Here, we demonstrate a novel role of substrate stiffness in the maintenance of hMSCs over long-term expansion. When serially passaged for 45 days from passage 3 to passage 18 on polyacrylamide gel of Young's modulus E =5 kPa, hMSCs maintained their proliferation rate and showed nine times higher population doubling in comparison to their counterparts cultured on plastic Petri-plates. They did not express markers of senescence, maintained their morphology and other mechanical properties such as cell stiffness and cellular traction, and were significantly superior in adipogenic differentiation potential. These results were demonstrated in hMSCs from two different sources, umbilical cord and bone marrow. In summary, our result shows that a soft gel is a suitable substrate to maintain the stemness of mesenchymal stem cells. As preparation of polyacrylamide gel is a well-established, and well-standardized protocol, we propose that this novel system of cell expansion will be useful in therapeutic and research applications of hMSCs.
Microcontact printing (µCP) is a commonly used technique for patterning proteins of interest on substrates. The cells take the shape of these printed patterns. This technique is used to explore the effect of cellular morphology on their various functions such as survival, differentiation, migration, etc. An essential step for µCP is to fabricate a stamp from a silicon mould, prepared using lithography. Lithography is cost intensive and needs a high level of expertise to handle the instrumentation. Also, one stamp can be used to print patterns of one size and shape. Here, to overcome these limitations, we devised a low-cost fabrication technique using readily available objects such as injection needles and polystyrene beads. We patterned the C2C12, myoblasts cells on the shapes printed using lithography-free fabricated stamps. We further exploited the surface curvature of the stamp to vary the size of the print either by changing the applied load and/or the substrate stiffness. We showed that the print dimension could be predicted well by using JKR theory of contact mechanics. Moreover, some innovative improvisations enabled us to print complex shapes, which would be otherwise difficult with conventional lithography technique. We envisage that this low cost and easy to fabricate method will allow many research laboratories with limited resources to perform exciting research which is at present out of their reach.
Single enzymatic cascade, ubiquitously found in cellular signaling networks, is a phosphorylation-dephosphorylation reaction cycle causing a transition between inactive and active states of a protein catalysed by kinase and phosphatase, respectively. Steady-state information processing ability of such a cycle (e.g., MAPK cascade) has been classified into four qualitatively different operating regimes, viz ., hyperbolic (H), signal-transducing (ST), threshold-hyperbolic (TH) and ultrasensitive (U). These four regimes represent qualitatively different dose-response curves, that is, relationship between concentrations of input kinase (e.g., pMEK) and response activated protein (e.g., pERK). Regimes were identified using a deterministic model accounting for population-averaged behavior only. Operating regimes can be strongly influenced by the inherently present cell-to-cell variability in an ensemble of cells which is captured in the form of pMEK and pERK distributions using reporter-based single-cell experimentation. In this study, we show that such experimentally acquired snapshot pMEK and pERK distribution data of a single MAPK cascade can be directly used to infer the underlying operating regime even in the absence of a dose-response curve. This deduction is possible primarily due to the presence of a monotonic relationship between experimental observables R IQR , ratio of the inter-quartile range of the pERK and pMEK distribution pairs and R M , ratio of the medians of the distribution pair. We demonstrate this relationship by systematic analysis of a quasi-steady state approximated model superimposed with an input gamma distribution constrained by the stimulus strength specific pMEK distribution measured on Jurkat-T cells stimulated with PMA. As a first, we show that introduction of cell-to-cell variability only in the upstream kinase achieved by superimposition of an appropriate input pMEK distribution on the dose-response curve can predict bimodal response pERK distribution in ST regime. Implementation of the proposed method on the input-response distribution pair obtained in stimulated Jurkat-T cells revealed that while low-dosage PMA stimulation preserves the H regime observed in resting cells, high-dosage causes H to ST regime transition.
Operating regime of a single enzymatic cascade such as ubiquitously conserved MAPK building block provides insights into the nature of the sensitivity of the steady-state dose response that maps the upstream kinase and the downstream activated substrate concentrations. Steady-state response can be viewed either at a single-cell level in an ensemble of cells or at population-average level. Four operating regimes, viz., hyperbolic, threshold-hyperbolic, signal transducing and saturated, have been identified using the population-average level dose response curve. However, cell-to-cell variability exists in enzymatic cascades. This variability is captured using reporter based experimentation at ensemble level which permits detection of snapshot(s) of ensemble-level distribution of phosphorylated proteins. As a result, often the corresponding underlying steady-state dose response curve may not be available. We consider the question if the underyling operating regime can be directly inferred from ensemble level snapshot upstream kinase (input) and downstream phosphorylated substrates (response) distributions. In order to address this question, we use mathematical model of a single enzymatic cascade based on quasi-steady state approximation superimposed with an input distribution constrained by single-cell level experimental measurements of the MAPK cascade in Jurkat E6.1 cells stimulated with Phorbol Myristate Acetate (PMA). We prove that, under steady-state conditions, a monotonic relationship between the R IQR (=ratio of the inter-quartile range of the response and input distributions) and R m (=ratio of medians of the two distributions), both of which are experimental observables, can be used to identify the underlying operating regime. We also show that the identification of the unimodal vs bimodal nature of the response distributions can further lead to identification of the potential parameter range in the planes of Michaelis-Menten constants K 1 and K 2 , the two key parameters that dictate the operating regimes. We implement the proposed method on the stimulus strength dependent steady-state single-cell 3 level pMEK (input) and pERK (response) distributions in Jurkat E6.1 cells treated with PMA.While cells stimulated using low concentrations of PMA are likely to operate in hyperbolic regime, those exposed to higher concentrations may lie in signal-transducing regime. Author's summarySingle enzymatic cascade, a ubiquitously found key building block in biological signaling networks, exhibits different steady-state behaviour at population (ensemble) level compared to population-averaged response. Detection at ensemble level typically achieved by nonplasmid based reporter permits snapshot fluorescence distribution measurement. We ask if there are signatures of fluorescence distribution of input kinase and response activated protein that can help decipher the underlying dose-response belonging to four distinct operating regimes a single enzymatic cascade exhibits. Based on simultaneously measured snapshot input (pME...
Cells self-organize to give patterns that are essential for tissue functioning. While the effects of biochemical and mechanical cues are relatively well studied, the role of stiffness inhomogeneity on cellular patterning is unexplored. Using a rigid structure embedded in soft polyacrylamide (PAA) gel, we show that such mechanical inhomogeneity leads to long-range self-organized cellular patterns. Our results reveal that this patterning depends on cellular traction and cell morphology. Depending on a suitable combination of cellular morphology and traction, the information about the presence of embedded structure gets relayed outward. In response to this relay, the cells reorient their axis and migrate towards the embedded structure leading to the observed long-range (20-35 cell length) patterning. To predict the possibility of pattern formation, we present a dimensionless number ′ ′ combining the governing parameters. We have also shown that the pattern can be tailor-made by pre-designing sub-surface structures, a potential tool for tissue engineering. This mechanism of directed migration driven long-range pattern formation in response to mechanical inhomogeneity may be involved during several pathophysiological conditions, a proposition that needs further investigation. One Sentence Summary:Substrate inhomogeneity and cooperative cellular traction together lead to cellular migration and long-range pattern formation.
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