Molecular maps of synovial cells in inflammatory arthritis using an optimized synovial tissue dissociation protocol.

In this study, we optimized the dissociation of synovial tissue biopsies for single-cell omics studies and created a single-cell atlas of human synovium in inflammatory arthritis. The optimized protocol allowed consistent isolation of highly viable cells from tiny fresh synovial biopsies, minimizing the synovial biopsy drop-out rate. The synovium scRNA-seq atlas contained over 100,000 unsorted synovial cells from 25 synovial tissues affected by inflammatory arthritis, including 16 structural, 11 lymphoid, and 15 myeloid cell clusters. This synovial cell map expanded the diversity of synovial cell types/states, detected synovial neutrophils, and broadened synovial endothelial cell classification. We revealed tissue-resident macrophage subsets with proposed matrix-sensing (FOLR2+COLEC12high) and iron-recycling (LYVE1+SLC40A1+) activities and identified fibroblast subsets with proposed functions in cartilage breakdown (SOD2highSAA1+SAA2+SDC4+) and extracellular matrix remodeling (SERPINE1+COL5A3+LOXL2+). Our study offers an efficient synovium dissociation method and a reference scRNA-seq resource, that advances the current understanding of synovial cell heterogeneity in inflammatory arthritis.

Single-cell Bayesian deconvolution.

Individual cells exhibit substantial heterogeneity in protein abundance and activity, which is frequently reflected in broad distributions of fluorescently labeled reporters. Since all cellular components are intrinsically fluorescent to some extent, the observed distributions contain background noise that masks the natural heterogeneity of cellular populations. This limits our ability to characterize cell-fate decision processes that are key for development, immune response, tissue homeostasis, and many other biological functions. It is therefore important to separate the contributions from signal and noise in single-cell measurements. Addressing this issue rigorously requires deconvolving the noise distribution from the signal, but approaches in that direction are still limited. Here, we present a non-parametric Bayesian formalism that performs such a deconvolution efficiently on multidimensional measurements, providing unbiased estimates of the resulting confidence intervals. We use this approach to study the expression of the mesodermal transcription factor Brachyury in mouse embryonic stem cells undergoing differentiation.

Isolation of circulating tumor cells.

Circulating tumor cells (CTCs) enter the vasculature from solid tumors and disseminate widely to initiate metastases. Mining the metastatic-enriched molecular signatures of CTCs before, during, and after treatment holds unique potential in personalized oncology. Their extreme rarity, however, requires isolation from large blood volumes at high yield and purity, yet they overlap leukocytes in size and other biophysical properties. Additionally, many CTCs lack EpCAM that underlies much of affinity-based capture, complicating their separation from blood. Here, we provide a comprehensive introduction of CTC isolation technology, by analyzing key separation modes and integrated isolation strategies. Attention is focused on recent progress in microfluidics, where an accelerating evolution is occurring in high-throughput sorting of cells along multiple dimensions.

Label-free cell tracking enables collective motion phenotyping in epithelial monolayers.

Collective cell migration is an umbrella term for a rich variety of cell behaviors, whose distinct character is important for biological function, notably for cancer metastasis. One essential feature of collective behavior is the motion of cells relative to their immediate neighbors. We introduce an AI-based pipeline to segment and track cell nuclei from phase-contrast images. Nuclei segmentation is based on a U-Net convolutional neural network trained on images with nucleus staining. Tracking, based on the Crocker-Grier algorithm, quantifies nuclei movement and allows for robust downstream analysis of collective motion. Because the AI algorithm required no new training data, our approach promises to be applicable to and yield new insights for vast libraries of existing collective motion images. In a systematic analysis of a cell line panel with oncogenic mutations, we find that the collective rearrangement metric, D2 min, which reflects non-affine motion, shows promise as an indicator of metastatic potential.

Automated measurement of cell mechanical properties using an integrated dielectrophoretic microfluidic device.

Cell mechanics is closely related to and interacts with cellular functions, which has the potential to be an effective biomarker to indicate disease onset and progression. Although several techniques have been developed for measuring cell mechanical properties, the issues of limited measurement data and biological significance because of complex and labor-intensive manipulation remain to be addressed, especially for the dielectrophoresis-based approach that is difficult to utilize flow measurement techniques. In this work, a dielectrophoresis-based solution is proposed to automatically obtain mass cellular mechanical data by combining a designed microfluidic device integrated the functions of cell capture, dielectrophoretic stretching, and cell release and an automatic control scheme. Experiments using human umbilical vein endothelial cells and breast cells revealed the automation capability of this device. The proposed method provides an effective way to address the low-throughput problem of dielectrophoresis-based cell mechanical property measurements, which enhance the biostatistical significance for cellular mechanism studies.

Methods to separate nuclear soluble fractions reflecting localizations in living cells.

To understand various intranuclear functions, it is important to know when, what, and how proteins enter the nucleus. Although many methods and commercial kits for nuclear fractionation have been developed, there are still no methods for obtaining a complete nuclear proteome. Soluble nuclear proteins are often lost during fractionation. We developed remarkably improved methods to obtain nuclear soluble fractions by optimizing the conditions of selective permeabilization of the plasma membrane. As a result, 10 million cells could be separated into the cytoplasmic and nuclear soluble fractions more precisely in a 1.5-mL test tube. Moreover, the addition of an inhibitor to prevent leakage from the nucleus retained small proteins in the nucleus. Because of the simple protocols and easy application for multiple samples, our methods are expected to be applied to various studies on spatiotemporal changes of dynamic nuclear proteins, such as signal transduction.

Use of dual-electron probes reveals the role of ferritin as an iron depot in ex vivo erythropoiesis.

In the finely regulated process of mammalian erythropoiesis, the path of the labile iron pool into mitochondria for heme production is not well understood. Existing models for erythropoiesis do not include a central role for the ubiquitous iron storage protein ferritin; one model proposes that incoming endosomal Fe3+ bound to transferrin enters the cytoplasm through an ion transporter after reduction to Fe2+ and is taken up into mitochondria through mitoferrin-1 transporter. Here, we apply a dual three-dimensional imaging and spectroscopic technique, based on scanned electron probes, to measure Fe3+ in ex vivo human hematopoietic stem cells. After seven days in culture, we observe cells displaying a highly specialized architecture with anchored clustering of mitochondria and massive accumulation of nanoparticles containing high iron concentrations localized to lysosomal storage depots, identified as ferritin. We hypothesize that lysosomal ferritin iron depots enable continued heme production after expulsion of most of the cellular machinery.

In situ fiducial markers for 3D correlative cryo-fluorescence and FIB-SEM imaging.

Imaging of cells and tissues has improved significantly over the last decade. Dual-beam instruments with a focused ion beam mounted on a scanning electron microscope (FIB-SEM), offering high-resolution 3D imaging of large volumes and fields-of-view are becoming widely used in the life sciences. FIB-SEM has most recently been implemented on fully hydrated, cryo-immobilized, biological samples. Correlative light and electron microscopy workflows combining fluorescence microscopy (FM) with FIB-SEM imaging exist, whereas workflows combining cryo-FM and cryo-FIB-SEM imaging are not yet commonly available. Here, we demonstrate that fluorescently labeled lipid droplets can serve as in situ fiducial markers for correlating cryo-FM and FIB-SEM datasets and that this approach can be used to target the acquisition of large FIB-SEM stacks spanning tens of microns under cryogenic conditions. We also show that cryo-FIB-SEM imaging is particularly informative for questions related to organelle structure and inter-organellar contacts, nuclear organization, and mineral deposits in cells.

Combinatorial expression of cell cycle regulators is more suitable for immortalization than oncogenic methods in dermal papilla cells.

The immortalized cell is an essential research tool that uses robust growth properties for the functional investigation of gene products. Immortalized mammalian cells have mainly been established using three methods: expression of simian vacuolating virus 40 T antigen (the SV40 method); human papilloma virus-derived oncoprotein E6/E7 (the E6/E7 method); or combinatorial expression of R24C mutant cyclin-dependent kinase 4, cyclin D1, and telomerase reverse transcriptase (the K4DT method). However, it is unclear as to which method is optimal for an in vitro model. Here, we compared the biological characteristics and genome-wide expression profiles of immortalized human dermal papilla cells generated by the SV40, E6/E7, or K4DT method. To our knowledge, this is the first study to comprehensively compare expression profiles to determine the optimal immortalization method for maintaining the original nature of the wild-type cells. These data would be valuable to scientists aiming to establish new immortalized cell lines.

Degradation of Mutant Protein Aggregates within the Endoplasmic Reticulum of Vasopressin Neurons.

Misfolded or unfolded proteins in the ER are said to be degraded only after translocation or isolation from the ER. Here, we describe a mechanism by which mutant proteins are degraded within the ER. Aggregates of mutant arginine vasopressin (AVP) precursor were confined to ER-associated compartments (ERACs) connected to the ER in AVP neurons of a mouse model of familial neurohypophysial diabetes insipidus. The ERACs were enclosed by membranes, an ER chaperone and marker protein of phagophores and autophagosomes were expressed around the aggregates, and lysosomes fused with the ERACs. Moreover, lysosome-related molecules were present within the ERACs, and aggregate degradation within the ERACs was dependent on autophagic-lysosomal activity. Thus, we demonstrate that protein aggregates can be degraded by autophagic-lysosomal machinery within specialized compartments of the ER.

Bright Ferritin-a Reporter Gene Platform for On-Demand, Longitudinal Cell Tracking on MRI.

A major unresolved challenge in cell-based regenerative medicine is the absence of non-invasive technologies for tracking cell fate in deep tissue and with high spatial resolution over an extended interval. MRI is highly suited for this task, but current methods fail to provide longitudinal monitoring or high sensitivity, or both. In this study, we fill this technological gap with the first discovery and demonstration of in vivo cellular production of endogenous bright contrast via an MRI genetic reporter system that forms manganese-ferritin nanoparticles. We demonstrate this technology in human embryonic kidney cells genetically modified to stably overexpress ferritin and show that, in the presence of manganese, these cells produce far greater contrast than conventional ferritin overexpression with iron or manganese-permeable cells. In living mice, diffusely implanted bright-ferritin cells produce the highest and most sustained contrast in skeletal muscle. The bright-ferritin platform has potential for on-demand, longitudinal, and sensitive cell tracking in vivo.

A DNA-Based FLIM Reporter for Simultaneous Quantification of Lysosomal pH and Ca2+ during Autophagy Regulation.

pH and Ca2+ play important roles in regulating lysosomal activity and lysosome-mediated physiological and pathological processes. However, effective methods for simultaneous determination of pH and Ca2+ is the bottleneck. Herein, a single DNA-based FLIM reporter was developed for real-time imaging and simultaneous quantification of pH and Ca2+ in lysosomes with high affinity, in which a specific probe for recognition of Ca2+ was assembled onto a DNA nanostructure together with pH-responsive and lysosome-targeted molecules. The developed DNA reporter showed excellent biocompatibility and long-term stability up to ∼56 h in lysosomes. Using this powerful tool, it was discovered that pH was closely related to Ca2+ concentration in lysosome, whereas autophagy can be regulated by lysosomal pH and Ca2+. Furthermore, Aß-induced neuronal death resulted from autophagy abnormal through lysosomal pH and Ca2+ changes. In addition, lysosomal pH and Ca2+ were found to regulate the transformation of NSCs, resulting in Rapamycin-induced antiaging.

PhotoGal4: A Versatile Light-Dependent Switch for Spatiotemporal Control of Gene Expression in Drosophila Explants.

We present here PhotoGal4, a phytochrome B-based optogenetic switch for fine-tuned spatiotemporal control of gene expression in Drosophila explants. This switch integrates the light-dependent interaction between phytochrome B and PIF6 from plants with regulatory elements from the yeast Gal4/UAS system. We found that PhotoGal4 efficiently activates and deactivates gene expression upon red- or far-red-light irradiation, respectively. In addition, this optogenetic tool reacts to different illumination conditions, allowing for fine modulation of the light-dependent response. Importantly, by simply focusing a laser beam, PhotoGal4 induces intricate patterns of expression in a customized manner. For instance, we successfully sketched personalized patterns of GFP fluorescence such as emoji-like shapes or letterform logos in Drosophila explants, which illustrates the exquisite precision and versatility of this tool. Hence, we anticipate that PhotoGal4 will expand the powerful Drosophila toolbox and will provide a new avenue to investigate intricate and complex problems in biomedical research.

Off-Peak 594-nm Light Surpasses On-Peak 532-nm Light in Silencing Distant ArchT-Expressing Neurons In Vivo.

For large brain volume manipulations using optogenetics, both effective opsin excitation and efficient light delivery with minimal light absorption are required to minimize the illuminating light intensity and concomitant off-target effects. ArchT, a widely used potent inhibitory opsin, is commonly activated by 532-nm light, which lies on its in vitro excitation peak. However, 532-nm light also lies on a peak range of the hemoglobin absorption spectrum. Therefore, we predicted that 594-nm light is superior in suppressing distant ArchT-expressing neurons, which is slightly off the ArchT-excitation-plateau and largely off the peak of the hemoglobin absorption spectrum. We quantitatively tested this prediction by the electrophysiological recording of the rat cortex in vivo. At illumination distances greater than 500 µm, 594-nm light was more effective than 532-nm light. Its superiority increased with distance. These results validate our prediction and highlight the significance of excitation-absorption trade-off in selecting illumination wavelength for optogenetics in vivo.

Voltage Imaging of Cardiac Cells and Tissue Using the Genetically Encoded Voltage Sensor Archon1.

Precise measurement of action potentials (APs) is needed to observe electrical activity and cellular communication within cardiac tissue. Voltage-sensitive dyes (VSDs) are traditionally used to measure cardiac APs; however, they require acute chemical addition that prevents chronic imaging. Genetically encoded voltage indicators (GEVIs) enable long-term studies of APs without the need of chemical additions, but current GEVIs used in cardiac tissue exhibit poor kinetics and/or low signal to noise (SNR). Here, we demonstrate the use of Archon1, a recently developed GEVI, in hiPSC-derived cardiomyocytes (CMs). When expressed in CMs, Archon1 demonstrated fast kinetics comparable with patch-clamp electrophysiology and high SNR significantly greater than the VSD Di-8-ANEPPS. Additionally, Archon1 enabled monitoring of APs across multiple cells simultaneously in 3D cardiac tissues. These results highlight Archon1's capability to investigate the electrical activity of CMs in a variety of applications and its potential to probe functionally complex in vitro models, as well as in vivo systems.

A Conditional Dependency on MELK for the Proliferation of Triple-Negative Breast Cancer Cells.

The role of maternal and embryonic leucine zipper kinase (MELK) in cancer cell proliferation has been contentious, with recent studies arriving at disparate conclusions. We investigated the in vitro dependency of cancer cells on MELK under a range of assay conditions. Abrogation of MELK expression has little effect under common culture conditions, in which cells are seeded at high densities and reach confluence in 3-5 days. However, MELK dependency becomes clearly apparent in clonogenic growth assays using either RNAi or CRISPR technologies to modulate MELK expression. This dependency is in sharp contrast to that of essential genes, such as those encoding classic mitotic kinases, but is similar to that of other oncogenes including MYC and KRAS. Our study provides an example demonstrating some of the challenges encountered in cancer target validation, and reveals how subtle, but important, technical variations can ultimately lead to divergent outcomes and conclusions.