Expansion microscopy (ExM) increases the effective resolving power of any microscope by expanding the sample with swellable hydrogel. Since its invention, ExM has been successfully applied to a wide range of cell, tissue, and animal samples. Still, fluorescence signal loss during polymerization and digestion limits molecular-scale imaging using ExM. Here, we report the development of label-retention ExM (LR-ExM) with a set of trifunctional anchors that not only prevent signal loss but also enable high-efficiency labeling using SNAP and CLIP tags. We have demonstrated multicolor LR-ExM for a variety of subcellular structures. Combining LR-ExM with superresolution stochastic optical reconstruction microscopy (STORM), we have achieved molecular resolution in the visualization of polyhedral lattice of clathrin-coated pits in situ.

Stem cells undergo drastic morphological alterations during differentiation. While extensive studies have been performed to examine the cytoskeletal remodeling, there is a growing interest to determine the morphological, structural and functional changes of the nucleus. The current study is therefore aimed at quantifying the extent of remodeling of the nuclear morphology of human mesenchymal stem cells during biochemically-induced adipogenic differentiation. Results show the size of nuclei decreased exponentially over time as the lipid accumulation is up-regulated. Increases in the lipid accumulation appear to lag the nuclear reorganization, suggesting the nuclear deformation is a prerequisite to adipocyte maturation. Furthermore, the lamin A/C expression was increased and redistributed to the nuclear periphery along with a subsequent increase in the nuclear aspect ratio. To further assess the role of the nucleus, a nuclear morphology with a high aspect ratio was achieved using microcontact-printed substrate. The cells with an elongated nuclear shape did not efficiently undergo adipogenesis, suggesting the cellular and nuclear processes associated with stem cell differentiation at the early stage of adipogenesis cause a change in the nuclear morphology and cannot be abrogated by the morphological cues. In addition, a novel computational biomechanical model was generated to simulate the nuclear shape change during differentiation and predict the forces acting upon the nucleus. This effort led to the development of computational scaling approach to simulate the experimentally observed adipogenic differentiation processes over 15 days in less than 1.5 hours.

Background and Objectives Photobiomodulation (PBM) describes the influence of light irradiation on biological tissues. Laser light in the near‐infrared (NIR) spectrum has been shown to mitigate pain, reduce inflammation, and promote wound healing. The cellular mechanism that mediates PBM's effects is generally accepted to be at the site of the mitochondria, leading to an increased flux through the electron transport chain and adenosine triphosphate (ATP) production. Moreover, PBM has been demonstrated to reduce oxidative stress through an increased production of reactive oxygen species (ROS)‐sequestering enzymes. The aim of the study is to determine whether these PBM‐induced effects expedite or interfere with the intended stem cell differentiation to the adipogenic lineage. Study Design/Materials and Methods To determine the effects of 1064 nm laser irradiation (fluence of 8.8–26.4 J/cm2) on human mesenchymal stem cells (hMSCs) undergoing adipogenic differentiation, the ATP and ROS levels, and adipogenic markers were quantitatively measured. Results At a low fluence (8.8 J/cm2) the ATP increase was essentially negligible, whereas a higher fluence induced a significant increase. In the laser‐stimulated cells, PBM over time decreased the ROS level compared with the non‐treated control group and significantly reduced the extent of adipogenesis. A reduction in the ROS level was correlated with a diminished lipid accumulation, reduced production of adipose‐specific genetic markers, and delayed the chemically intended adipogenesis. Conclusion We characterized the use of NIR light exposure to modulate adipogenesis. Both the ATP and ROS levels in hMSCs responded to different energy densities. The current study is expected to contribute significantly to the growing field of PBM as well as stem cell tissue engineering by demonstrating the wavelength‐dependent responses of hMSC differentiation. Lasers Surg. Med. © 2020 Wiley Periodicals LLC.

Cell mechanics has been shown to regulate stem cell differentiation. We have previously reported that altered cell stiffness of mesenchymal stem cells can delay or facilitate biochemically directed differentiation. One of the factors that can affect the cell stiffness is cholesterol. However, the effect of cholesterol on differentiation of human mesenchymal stem cells remains elusive. In this paper, we demonstrate that cholesterol is involved in the modulation of the cell stiffness and subsequent adipogenic differentiation. Rapid cytoskeletal actin reorganization was evident and correlated with the cell's Young's modulus measured using atomic force microscopy. In addition, the level of membrane-bound cholesterol was found to increase during adipogenic differentiation and inversely varied with the cell stiffness. Furthermore, cholesterol played a key role in the regulation of the cell morphology and biomechanics, suggesting its crucial involvement in mechanotransduction. To better understand the underlying mechanisms, we investigated the effect of cholesterol on the membrane–cytoskeleton linker proteins (ezrin and moesin). Cholesterol depletion was found to upregulate the ezrin expression which promoted cell spreading, increased Young's modulus, and hindered adipogenesis. In contrast, cholesterol enrichment increased the moesin expression, decreased Young's modulus, and induced cell rounding and facilitated adipogenesis. Taken together, cholesterol appears to regulate the stem cell mechanics and adipogenesis through the membrane-associated linker proteins.

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