Machine learning based approach to pH imaging and classification of single cancer cells.

The ability to identify different cell populations in a noninvasive manner and without the use of fluorescence labeling remains an important goal in biomedical research. Various techniques have been developed over the last decade, which mainly rely on fluorescent probes or nanoparticles. On the other hand, their applications to single-cell studies have been limited by the lengthy preparation and labeling protocols, as well as issues relating to reproducibility and sensitivity. Furthermore, some of these techniques require the cells to be fixed. Interestingly, it has been shown that different cell types exhibit a unique intracellular environment characterized by specific acidity conditions as a consequence of their distinct functions and metabolism. Here, we leverage a recently developed pH imaging modality and machine learning-based single-cell segmentation and classification to identify different cancer cell lines based on their characteristic intracellular pH. This simple method opens up the potential to perform rapid noninvasive identification of living cancer cells for early cancer diagnosis and further downstream analyses.

Localized mechanical stimulation of single cells with engineered spatio-temporal profile.

In vivo, cells are frequently exposed to multiple mechanical stimuli arising from the extracellular microenvironment, with a deep impact on many biological functions. On the other hand, current methods for mechanobiology do not allow one to easily replicate in vitro the complex spatio-temporal profile of such mechanical signals. Here we introduce a new platform for studying the mechanical coupling between single cells and a dynamic extracellular environment, based on active substrates for cell culture made of Fe-coated polymeric micropillars. Under the action of quasi-static external magnetic fields, each group of pillars produces synchronous mechanical stimuli at different points of the cell membrane, thanks to the highly controllable pillars' deflection. This method allows one to apply complex stress fields, resulting in the parallel application of localized forces with tunable intensity and temporal profile. The platform has been validated by studying the cellular response to periodic stimuli in NIH3T3 fibroblasts. We find that low-frequency mechanical stimulation affects the actin cytoskeleton, nuclear morphology, and H2B core-histone dynamics and induces MKL transcription-cofactor translocation from nucleus to cytoplasm. The unique capability of the proposed platform to apply stimuli with a tunable temporal profile and high parallelism on a cell culture holds great potential for the investigation of mechanotransduction mechanisms in cells and tissues.

Nuclear deformability and telomere dynamics are regulated by cell geometric constraints.

Forces generated by the cytoskeleton can be transmitted to the nucleus and chromatin via physical links on the nuclear envelope and the lamin meshwork. Although the role of these active forces in modulating prestressed nuclear morphology has been well studied, the effect on nuclear and chromatin dynamics remains to be explored. To understand the regulation of nuclear deformability by these active forces, we created different cytoskeletal states in mouse fibroblasts using micropatterned substrates. We observed that constrained and isotropic cells, which lack long actin stress fibers, have more deformable nuclei than elongated and polarized cells. This nuclear deformability altered in response to actin, myosin, formin perturbations, or a transcriptional down-regulation of lamin A/C levels in the constrained and isotropic geometry. Furthermore, to probe the effect of active cytoskeletal forces on chromatin dynamics, we tracked the spatiotemporal dynamics of heterochromatin foci and telomeres. We observed increased dynamics and decreased correlation of the heterochromatin foci and telomere trajectories in constrained and isotropic cell geometry. The observed enhanced dynamics upon treatment with actin depolymerizing reagents in elongated and polarized geometry were regained once the reagent was washed off, suggesting an inherent structural memory in chromatin organization. We conclude that active forces from the cytoskeleton and rigidity from lamin A/C nucleoskeleton can together regulate nuclear and chromatin dynamics. Because chromatin remodeling is a necessary step in transcription control and its memory, genome integrity, and cellular deformability during migration, our results highlight the importance of cell geometric constraints as critical regulators in cell behavior.

Human embryonic stem cell differentiation into odontoblastic lineage: an in vitro study.

AIM: The aim of this study was to differentiate human embryonic stem cells (hESCs) into odontoblastic lineage in an optimized culture milieu. METHODOLOGY: In Phase 1, hESCs were differentiated into mesenchymal stem cells (H9-MSCs). In Phase 2, H9-MSCs were then differentiated into odontoblast-like cells (H9-Odont) under the stimulation of FGF-8 and BMP-4. Alternatively, H9-MSCs were differentiated into osteogenic lineage (H9-Osteo). In Phase 3, H9-Odont were seeded on 17% EDTA-treated dentine substrates in the presence of FGF-8 and BMP-4 for further differentiation. All experiments were performed in triplicate (n = 3). One-way anova was used to test hESC differentiation into different cell types. Post hoc Tukey's test was used to compare between groups. P < 0.05 was considered statistically significant. RESULTS: H9-Odont expressed the odontoblastic marker DSPP gene 125.47 ± 0.1 (SD)-folds higher compared with H9-MSCs at mRNA level (real-time RT-PCR). Additionally, the flow cytometry results revealed 53.1 ± 3.4 (SD) % of DSP (+) cells in H9-Odont. Alternatively, H9-Osteo expressed 5.9 ± 2.2 (SD) % of DSP (+) cells. Moreover, the SEM results demonstrated that H9-Odont were found to undergo morphological changes from a fibroblast-like shape into more rounded shapes with cytoplasmic extensions into the dentinal tubules when seeded on 17% EDTA-treated dentine substrate in the presence of FGF-8 and BMP-4. However, H9-Osteo and H9-MSCs did not show similar morphological changes under similar culture milieu. CONCLUSION: This study supports the potential of hESCs as a stable, consistent, unlimited and 'off-the-shelf' cell source to obtain odontoblastic cells for future clinical and research applications.