FAK, vinculin, and talin control mechanosensitive YAP nuclear localization.

Focal adhesions (FAs) are nanoscale complexes containing clustered integrin receptors and intracellular structural and signaling proteins that function as principal sites of mechanotransduction in part via promoting the nuclear translocation and activation of the transcriptional coactivator yes-associated protein (YAP). Knockdown of FA proteins such as focal adhesion kinase (FAK), talin, and vinculin can prevent YAP nuclear localization. However, the mechanism(s) of action remain poorly understood. Herein, we investigated the role of different functional domains in vinculin, talin, and FAK in regulating YAP nuclear localization. Using genetic or pharmacological inhibition of fibroblasts and human mesenchymal stem cells (hMSCs) adhering to deformable substrates, we find that disruption of vinculin-talin binding versus talin-FAK binding reduces YAP nuclear localization and transcriptional activity via different mechanisms. Disruption of vinculin-talin binding or knockdown of talin-1 reduces nuclear size, traction forces, and YAP nuclear localization. In contrast, disruption of the talin binding site on FAK or elimination of FAK catalytic activity did not alter nuclear size yet still prevented YAP nuclear localization and activity. These data support both nuclear tension-dependent and independent models for matrix stiffness-regulated YAP nuclear localization. Our results highlight the importance of vinculin-talin-FAK interactions at FAs of adherent cells, controlling YAP nuclear localization and activity.

Combined transcriptomics and metabolomics analysis reveals lipid metabolic disruption in swamp eel (Monopterus albus) under chronic waterborne copper exposure.

Excessive copper can induce many adverse effects although it's an essential trace element in organisms. The effects of copper on the lipid metabolism have aroused increasing attention. This study investigated the liver lipid metabolism in swamp eel (Monopterus albus, M. albus) chronically exposed to 0, 10, 50, and 100 µg/L Cu2+ for 56 days. The results showed that copper increased the contents of triglyceride (TG), total cholesterol (T-CHO), non-esterified fatty acids (NEFA), and lipid droplets. Transcriptomic analysis found 1901 differentially expressed genes (DEGs) and 140 differential alternative splicing (DAS) genes in the 50 µg/L Cu2+ group, and 1787 DEGs and 184 DAS genes in the 100 µg/L Cu2+ group, respectively, which were enriched in peroxisome proliferator-activated receptor (PPAR), adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK), and other signaling pathways. The expression levels of key genes related to PPAR and AMPK signaling pathways were significantly down-regulated after chronic exposure to Cu2+. Meanwhile, metabolomics analysis showed that 52 and 110 differentially expressed metabolites (DEMs) were identified, which were mainly enriched in glycerophospholipids metabolism and steroid synthesis. Moreover, combined analysis of transcriptome and metabolome showed that glycerophospholipid metabolism co-enriched 19 down-regulated DEGs and 4 down-regulated DEMs. Taken together, our results suggested that chronic waterborne copper exposure promoted lipid synthesis, disrupted the metabolic homeostasis of glycerophospholipid, and led to excessive hepatic lipid deposition in M. albus. The combined omics approach enhanced our understanding of copper pollution to lipid metabolism.

Microcystin-LR-induced autophagy regulates oxidative stress, inflammation, and apoptosis in grass carp ovary cells in vitro.

MC-LR is one of the cyanotoxins produced by fresh water cyanobacteria. Previous studies showed that autophagy played an important role in MC-LR-induced reproduction toxicity. However, information on the toxicological mechanism is limited. In this study, MC-LR could induce autophagy and apoptosis in GCO cells in vitro. In GCO cells that had been exposed to MC-LR, the inhibitor of 3-MA effectively decreased cell viability and damaged cell ultrastructure. Oxidative stress was significantly increased in the 3-MA + MC-LR group, accompanied by significantly increased MDA content and decreased CAT activity and GST, SOD1, GPx, and GR expression levels (P < 0.05). Inflammation was more serious in the 3-MA + MC-LR group than that of MC-LR group, which was evidenced by increasing expression levels of TNFα, IL11, MyD88, TNFR1, TRAF2, JNK, CCL4, and CCL20 (P < 0.05). Interestingly, the significant decrease of Caspase-9, Caspase-7, and Bax expression and significant increase of Bcl-2 and Bcl-2/Bax ratio in 3-MA + MC-LR group compared to MC-LR group, suggesting that extent of apoptosis were reduced. Taken together, these results indicated that MC-LR induced autophagy and apoptosis in GCO cells, however, the inhibition of autophagy decreased the extent of apoptosis, induced more serious oxidative stress and inflammation, which eventually induced cell death. Our findings provided some information for exploring the toxicity of MC-LR, however, the role of autophagy require further study in vivo.

[Pollution Characteristics and Emission Factors of PCDD/Fs from Iron and Steel Industry].

The pollution level, emission characteristics, and emission factors of PCDD/Fs from a number of steel plants were investigated in a particular province of China. The results showed that the concentration of PCDD/Fs was at a low level and decreased by 1-2 orders of magnitude compared with that in 2005-2019. In detail, the concentrations of PCDD/Fs ranged from 0.003-0.557 ng·m-3(I-TEQ), and the mean value was 0.165 ng·m-3 for the sintering process. Moreover, the concentrations of PCDD/Fs ranged from 0.006 to 0.057 ng·m-3, and the mean value was 0.025 ng·m-3 for the electric furnace process. In addition, the concentration of PCDD/Fs in the iron and steel industry from 2005 to 2020 increased first and then decreased, especially after the implementation of the new emission standard and the ultra-low emission control of conventional pollutants such as smoke, showing a significant decline. The results of fingerprint analysis showed that 2,3,7,8-TCDF was the largest congener contributing to the mass concentration, and lower chlorinated PCDFs were increased. This result differed from those of previous studies in which highly chlorinated PCDFs and PCDDs dominated, indicating that the generation source of PCDD/Fs had changed. The congener and isomer profiles of PCDD/Fs in flue gas from the sintering process were similar to those in the flue gas from the electric furnace process. Additionally, showing the characteristics of the typical high-temperature thermal process, the de novo synthesis may be the dominant mechanism of formation of PCDD/Fs in the sintering process and electric furnace process. The emission factor was 0.003-0.5 µg·t-1 (I-TEQ), and the average emission factor was (0.18±0.22) µg·t-1 for the sintering process. The emission factor was 0.04-0.5 µg·t-1, and the average emission factor was (0.27±0.23) µg·t-1 for the electric furnace process. These values were far lower than those of the standard toolkit for identification and quantification of dioxin and furan emissions released by UNEP in 2013 and the emission factors in the dioxin emission inventory of China in 2004. It is suggested that the emission factors of PCDD/Fs in the iron and steel industry of China should be studied and updated.

Machine learning-assisted imaging analysis of a human epiblast model.

The human embryo is a complex structure that emerges and develops as a result of cell-level decisions guided by both intrinsic genetic programs and cell-cell interactions. Given limited accessibility and associated ethical constraints of human embryonic tissue samples, researchers have turned to the use of human stem cells to generate embryo models to study specific embryogenic developmental steps. However, to study complex self-organizing developmental events using embryo models, there is a need for computational and imaging tools for detailed characterization of cell-level dynamics at the single cell level. In this work, we obtained live cell imaging data from a human pluripotent stem cell (hPSC)-based epiblast model that can recapitulate the lumenal epiblast cyst formation soon after implantation of the human blastocyst. By processing imaging data with a Python pipeline that incorporates both cell tracking and event recognition with the use of a CNN-LSTM machine learning model, we obtained detailed temporal information of changes in cell state and neighborhood during the dynamic growth and morphogenesis of lumenal hPSC cysts. The use of this tool combined with reporter lines for cell types of interest will drive future mechanistic studies of hPSC fate specification in embryo models and will advance our understanding of how cell-level decisions lead to global organization and emergent phenomena. Insight, innovation, integration: Human pluripotent stem cells (hPSCs) have been successfully used to model and understand cellular events that take place during human embryogenesis. Understanding how cell-cell and cell-environment interactions guide cell actions within a hPSC-based embryo model is a key step in elucidating the mechanisms driving system-level embryonic patterning and growth. In this work, we present a robust video analysis pipeline that incorporates the use of machine learning methods to fully characterize the process of hPSC self-organization into lumenal cysts to mimic the lumenal epiblast cyst formation soon after implantation of the human blastocyst. This pipeline will be a useful tool for understanding cellular mechanisms underlying key embryogenic events in embryo models.

Spatially resolved cell polarity proteomics of a human epiblast model.

Critical early steps in human embryonic development include polarization of the inner cell mass, followed by formation of an expanded lumen that will become the epiblast cavity. Recently described three-dimensional (3D) human pluripotent stem cell-derived cyst (hPSC-cyst) structures can replicate these processes. To gain mechanistic insights into the poorly understood machinery involved in epiblast cavity formation, we interrogated the proteomes of apical and basolateral membrane territories in 3D human hPSC-cysts. APEX2-based proximity bioinylation, followed by quantitative mass spectrometry, revealed a variety of proteins without previous annotation to specific membrane subdomains. Functional experiments validated the requirement for several apically enriched proteins in cyst morphogenesis. In particular, we found a key role for the AP-1 clathrin adaptor complex in expanding the apical membrane domains during lumen establishment. These findings highlight the robust power of this proximity labeling approach for discovering novel regulators of epithelial morphogenesis in 3D stem cell-based models.

Effect of Massage on the TLR4 Signalling Pathway in Rats with Neuropathic Pain.

This study set out to investigate the effect of massage on the Toll-like receptor 4 (TLR4) signalling pathway in the dorsal root ganglia of rats that had undergone spinal nerve ligation (SNL), with the hypothesis that massage could be used as an analgesic. Forty female SD rats were randomly divided into 5 groups: the control group, sham-operated group, model group, sham massage group, and massage group. There were 8 rats in each group. SNL rat models were established in the model group, sham massage group, and massage group. Rats in the sham-operated group underwent surgery to expose the vertebral nerves, but no further procedures were performed. The control group consisted of intact animals. The rats in the massage group underwent massage using a massage simulation machine once a day for 14 d in succession; the hind limbs of the rats in the sham massage group were gently touched with a cloth bag once a day for 14 continuous days. The rats in the control group, the sham-operated group, and the model group did not receive any intervention and were observed for 14 d. Paw withdrawal thermal latency (PWTL) and paw withdrawal mechanical threshold (PWMT) of rats in each group were detected 1 d before modelling and at 1, 3, 7, and 14 d after modelling. Fourteen days after modelling, the expression levels of TLR4, IRAK1, TRAF6, TNF-α, and IL-6 were detected in all rats. The PWTL and PWMT of SNL rats were decreased, while these parameters were elevated after massage. SNL rats showed higher levels of TLR4, IRAK1, TRAF6, IL-6, and TNF-α, and massage effectively lowered the expression levels of these molecules. Inhibiting activation of the TLR4 signalling pathway, which can reduce the release of inflammatory factors, may be one mechanism by which massage treats neuropathic pain.

Microengineered human amniotic ectoderm tissue array for high-content developmental phenotyping.

During early post-implantation human embryogenesis, the epiblast (EPI) within the blastocyst polarizes to generate a cyst with a central lumen. Cells at the uterine pole of the EPI cyst then undergo differentiation to form the amniotic ectoderm (AM), a tissue essential for further embryonic development. While the causes of early pregnancy failure are complex, improper lumenogenesis or amniogenesis of the EPI represent possible contributing factors. Here we report a novel AM microtissue array platform that allows quantitative phenotyping of lumenogenesis and amniogenesis of the EPI and demonstrate its potential application for embryonic toxicity profiling. Specifically, a human pluripotent stem cell (hPSC)-based amniogenic differentiation protocol was developed using a two-step micropatterning technique to generate a regular AM microtissue array with defined tissue sizes. A computer-assisted analysis pipeline was developed to automatically process imaging data and quantify morphological and biological features of AM microtissues. Analysis of the effects of cell density, cyst size and culture conditions revealed a clear connection between cyst size and amniogenesis of hPSC. Using this platform, we demonstrated that pharmacological inhibition of ROCK signaling, an essential mechanotransductive pathway, suppressed lumenogenesis but did not perturb amniogenic differentiation of hPSC, suggesting uncoupled regulatory mechanisms for AM morphogenesis vs. cytodifferentiation. The AM microtissue array was further applied to screen a panel of clinically relevant drugs, which successfully detected their differential teratogenecity. This work provides a technological platform for toxicological screening of clinically relevant drugs for their effects on lumenogenesis and amniogenesis during early human peri-implantation development, processes that have been previously inaccessible to study.

Molecular characterization of caspase-8-like and its expression induced by microcystin-LR in grass carp (Ctenopharygodon idella).

Caspase-8, an initiator caspase, plays a vital role in apoptosis. In this study, caspase-8-like (named as Cicaspase-8-like), a homologue of caspase-8, was identified in grass carp (Ctenopharygodon idella). The full-length cDNA sequence of CiCaspase-8-like was 1409 bp and contained a 162 bp 5'-UTR, a 239 bp 3'-UTR and a 1008 bp coding sequence. The putative amino acids sequence was 335 residues long, including a large subunit (P20) and a small subunit (P10), but lacking conserved death effector domains. A histidine active site DHSQMDAFVCCVLSHG and a cysteine active-site motif KPKLFFIQACQG were found in P20. Phylogenetic analysis showed that Cicaspase-8-like clustered with the caspase-8 and caspase-8-like of other fish and grouped closely with Carassius auratus caspase-8-like. Quantitative real-time PCR revealed that the Cicaspase-8-like mRNA were expressed constitutively in all tested tissues from healthy grass carp, with high expression level in the blood, spleen, liver and gill, indicating its role in immune reaction. The expression of Cicaspase-8-like mRNA was decreased significantly in the liver because of the stress caused by microcystin-LR (MC-LR) (75 and 100 µg MC-LR/kg BW) at 24 h and 96 h post injection (P < 0.05), but it was increased significantly in grass carp treated with 25 µg MC-LR/kg BW at 24 h (P < 0.05) post injection. Cleaved fragments of Cicaspase-8-like were observed using western blot analysis, and the expression of Cicaspase-8-like protein was increased after MC-LR treatments. Moreover, the expression of both caspase-9 and caspase-3 mRNA increased significantly after treatment with the three doses of MC-LR. TUNEL assay results showed remarkable changes in apoptosis after the MC-LR treatment. These results suggest that Cicaspase-8-like is an important caspase and plays an essential role in MC-LR-induced apoptosis.

Intein-mediated expression and purification of common carp IFN-γ and its protective effect against spring viremia of carp virus.

IFN-γ is a pleiotropic cytokine with significant roles in antiviral, antitumor and immune regulation. It could be used as an immuno-enhancer to improve fish protectiveness against pathogens. In this study, the prokaryotic expression plasmid pTwin1-N-IFN-γ was constructed to express Cyprinus carpio (common carp) IFN-γ fused with a chitin binding domain (CBD) and a self-cleavable intein-tag, Synechocystis sp DnaB. The recombinant protein CBD-DnaB-IFN-γ with the molecular weight of 44.25 kD was successfully expressed in soluble form, and the rIFN-γ (approximate 18.61 kD) was further cleaved and eluted under pH = 7.0 at 25 °C. rIFN-γ could be recognized by western blotting with rabbit anti-grass carp IFN-γ polyclonal antibody. Cytotoxicity studies on EPC cells showed that only 500 ng/ml rIFN-γ had a subtle effect on cells growth and its proliferation rate was reduced to 76.2%. EPC cells incubated with 100 ng/ml rIFN-γ showed significantly higher resistance against SVCV, reducing the TCID50/ml by more than 800-fold. In vivo studies suggested that intraperitoneal injection of rIFN-γ significantly improved the survival rate of common carps compared with SVCV challenge alone. These results implied that rIFN-γ would act as an immuno-enhancer in carp aquaculture.

Biophysical Phenotyping and Modulation of ALDH+ Inflammatory Breast Cancer Stem-Like Cells.

Cancer stem-like cells (CSCs) have been shown to initiate tumorigenesis and cancer metastasis in many cancer types. Although identification of CSCs through specific marker expression helps define the CSC compartment, it does not directly provide information on how or why this cancer cell subpopulation is more metastatic or tumorigenic. In this study, the functional and biophysical characteristics of aggressive and lethal inflammatory breast cancer (IBC) CSCs at the single-cell level are comprehensively profiled using multiple microengineered tools. Distinct functional (cell migration, growth, adhesion, invasion and self-renewal) and biophysical (cell deformability, adhesion strength and contractility) properties of ALDH+ SUM149 IBC CSCs are found as compared to their ALDH- non-CSC counterpart, providing biophysical insights into why CSCs has an enhanced propensity to metastasize. It is further shown that the cellular biophysical phenotype can predict and determine IBC cells' tumorigenic ability. SUM149 and SUM159 IBC cells selected and modulated through biophysical attributes-adhesion and stiffness-show characteristics of CSCs in vitro and enhance tumorigenicity in in vivo murine models of primary tumor growth. Overall, the multiparametric cellular biophysical phenotyping and modulation of IBC CSCs yields a new understanding of IBC's metastatic properties and how they might develop and be targeted for therapeutic interventions.

Identification and characterization of the interferon-γ-inducible lysosomal thiol reductase gene in Chinese soft-shelled turtle, Pelodiscus sinensis.

The reduction of disulfide bonds of exogenous antigens is crucial to the MHC-II class antigen processing and presenting pathway and is catalysed by interferon-γ-inducible lysosomal thiol reductase (GILT). In this study, a reptile GILT gene from Chinese soft-shelled turtle, Pelodiscus sinensis (PsGILT), was identified. The full-length cDNA of PsGILT is 1631 nucleotides (nt), including a 5'-untranslated region (UTR) of 3 nt, a 3'-UTR of 860 nt and an open reading frame (ORF) of 768 nt encoding 255 amino acids (aa). The conserved features in known GILTs, such as signal peptide, CXXC motif, GILT signature sequence, N-glycosylation site and conserved cysteines, were all found in the putative PsGILT protein. Genomic analysis revealed that PsGILT kept the "7 exons and 6 introns" structure of vertebrate GILT genes. PsGILT was expressed in all examined organs/tissues and was mainly expressed in spleen and blood. Increased mRNA expression levels of PsIFN-γ and PsGILT in PBLs were observed after induction with LPS, PolyI:C and recombinant IFN-γ (rIFN-γ). We also tested the reductase activity of rGILT in vitro and found that it could reduce intact human IgG into H chains and L chains. These above results implied that PsGILT may play an important role in resisting bacterial and viral infections, like other vertebrate GILTs.

Acoustic Actuation of Integrin-Bound Microbubbles for Mechanical Phenotyping during Differentiation and Morphogenesis of Human Embryonic Stem Cells.

Early human embryogenesis is a dynamic developmental process, involving continuous and concomitant changes in gene expression, structural reorganization, and cellular mechanics. However, the lack of investigation methods has limited the understanding of how cellular mechanical properties change during early human embryogenesis. In this study, ultrasound actuation of functionalized microbubbles targeted to integrin (acoustic tweezing cytometry, ATC) is employed for in situ measurement of cell stiffness during human embryonic stem cell (hESC) differentiation and morphogenesis. Cell stiffness, which is regulated by cytoskeleton structure, remains unchanged in undifferentiated hESCs, but significantly increases during neural differentiation. Further, using the recently established in vitro 3D embryogenesis models, ATC measurements reveal that cells continue to stiffen while maintaining pluripotency during epiblast cyst formation. In contrast, during amniotic cyst formation, cells first become stiffer during luminal cavity formation, but softens significantly when cells differentiate to form amniotic cysts. These results suggest that cell stiffness changes not only due to 3D spatial organization, but also with cell fate change. ATC therefore provides a versatile platform for in situ measurement of cellular mechanical property, and cell stiffness may be used as a mechanical biomarker for cell lineage diversification and cell fate specification during embryogenesis.

Magnetothermal heating facilitates the cryogenic recovery of stem cell-laden alginate-Fe3O4 nanocomposite hydrogels.

Constructs of magnetic nanocomposite hydrogels microencapsulated with stem cells are of great interest as smart materials for tissue engineering and regenerative medicine. Due to the short shelf life of such biocomposites at an ambient temperature, their long-term storage and banking at cryogenic temperatures are essential for the "off-the-shelf" availability of such biocomposites for widespread clinical applications. However, high-quality cryogenic recovery of stem cell-nanocomposite hydrogel constructs has not yet been achieved due to the damage to cells and/or microstructures of hydrogel constructs caused by ice formation, particularly during warming from cryogenic temperatures. Herein, stem cell-magnetic nanocomposite hydrogel constructs, which have an inherent magnetothermal property provided by embedded magnetic nanoparticles, are explored to achieve ultra-rapid cryogenic warming. The binding of water molecules by the hydrogel combined with the magnetothermal heating greatly suppressed ice formation during both cryogenic cooling and warming. Thus, the cryogenic recovery of nanocomposite hydrogel constructs with intact microstructures and fully functional stem cells from ultra-low temperatures was successfully achieved. We further demonstrated that magnetic nanocomposite hydrogels microencapsulated with stem cells could be conveniently manipulated for a self-assembled 3D culture. Together, we have developed a highly efficient and easy-to-perform approach for the cryogenic recovery of stem cell-encapsulated magnetic nanocomposite hydrogel constructs. Our results will facilitate the applications of such stem cell-magnetic nanocomposite hydrogels in regenerative medicine and tissue engineering.

[Discussion of Emissions and Health Risk of Polycyclic Aromatic Hydrocarbons (PAHs) from the Retreading Process of Waste Tires].

The emissions characteristics of 16 kinds of polycyclic aromatic hydrocarbons (PAHs) in ambient air during the waste tire retreading process (open-air storage, mixing, vulcanization, and grinding processes) and in workers' dormitory were analyzed. In addition, the occupational health risk of the workers was evaluated. Results showed that PAHs were detected in all retreading processes and in the workers' dormitory. The highest concentration site was the mixing process, followed by open-air storage and vulcanization process. The lowest concentration point was in the grinding process. The average concentration of PAHs in the workers' dormitory was 11.1 ng·m-3. The PAHs at all sampling points were largely phenanthrene (Phe), fluoranthene (Flu), anthracene (Ant), and pyrene (Pry), which also had a stronger linear correlation with the total PAH concentration. An analysis of the benzene rings showed that three ring and four ring were the majority, while two ring, five ring, and six ring components accounted for less than 10%. Results of the possible influencing factors of the PAHs revealed that the open-air storage and dormitory might be affected by a combustion source, but the mixing, vulcanization, and grinding processes might be affected by rubber oil. The principal component analysis (PCA) and cluster analysis showed that the spatial location of all sites would significantly influence the distribution of PAHs during the tire retreading process. The health risk assessment showed that occupational workers had a lower risk of lifelong cancer, and there was little influence on life expectancy.

Modulation of Micro RNA Expression and Osteoblast Differentiation by Nanotopography.

PURPOSE: This study evaluated the expression pattern of micro RNAs (miRNAs) on a surface with nanotopography compared with a smooth surface (control). MATERIALS AND METHODS: Human mesenchymal stem cells (hMSCs) were plated on different surfaces and compared at 3, 7, and 14 days for alkaline phosphatase (ALP) activity, expression of genes (osterix [OSX], runt-related transcription factor 2 [RUNX2], bone morphogenetic protein 2 [BMP2], and ALP), and expression of miRNAs. Western blot was also used to detect osteogenic proteins (BMP2, OSX, and osteocalcin [OCN]). Scanning electron microscopy of cells plated onto the surfaces was obtained. RESULTS: ALP activity on different surfaces was significantly greater in the nanotopography surface. At day 14, there was a 3.5-fold and a 9-fold increase for the RUNX2 and OSX genes, respectively. BMP2 and ALP also increased by fourfold and sevenfold compared with the control. Protein levels for OSX and BMP2 were also upregulated compared with the control group. Using RNA sequencing technology (RNA-Seq), a total of 117 miRNAs were found to be differentially expressed comparing the control (day 7) with the nanosurface (day 14). Forty-five miRNAs were upregulated, and 72 were downregulated. Several of the miRNAs that were differently expressed regulate osteogenic genes. For example, hsa-miR-135b-5p targets OCN, BSP, RUNX2, COL15A1, and OSX; hsa-miR-122-5p targets OPN; hsa-miR-196a-5p targets BMP4; hsa-miR-26b-5p targets BMP2; and hsa-miR-148b-3p targets OPN. CONCLUSION: Surfaces with nanotopography have the potential to improve the osseointegration response in order to reduce the osseointegration time and also increase bone formation around the implants, improving areas with low bone quality. Within the limitation of this study, nanotopography surfaces affected MSC differentiation to osteoblasts. Several miRNAs were differentially regulated by surface topography. These miRNAs could be related to the differentiation response to help control the osseointegration process.

An apicosome initiates self-organizing morphogenesis of human pluripotent stem cells.

Human pluripotent stem cells (hPSCs) self-organize into apicobasally polarized cysts, reminiscent of the lumenal epiblast stage, providing a model to explore key morphogenic processes in early human embryos. Here, we show that apical polarization begins on the interior of single hPSCs through the dynamic formation of a highly organized perinuclear apicosome structure. The membrane surrounding the apicosome is enriched in apical markers and displays microvilli and a primary cilium; its lumenal space is rich in Ca2+ Time-lapse imaging of isolated hPSCs reveals that the apicosome forms de novo in interphase, retains its structure during mitosis, is asymmetrically inherited after mitosis, and relocates to the recently formed cytokinetic plane, where it establishes a fully polarized lumen. In a multicellular aggregate of hPSCs, intracellular apicosomes from multiple cells are trafficked to generate a common lumenal cavity. Thus, the apicosome is a unique preassembled apical structure that can be rapidly used in single or clustered hPSCs to initiate self-organized apical polarization and lumenogenesis.

Tracking the tumor invasion front using long-term fluidic tumoroid culture.

The analysis of invading leader cells at the tumor invasion front is of significant interest as these cells may possess a coordinated functional and molecular phenotype which can be targeted for therapy. However, such analyses are currently limited by available technologies. Here, we report a fluidic device for long-term three-dimensional tumoroid culture which recapitulated the tumor invasion front, allowing for both quantification of invasive potential and molecular characterization of invasive leader cells. Preliminary analysis of the invasion front indicated an association with cell proliferation and higher expression of growth differentiation factor 15 (GDF15). This device makes real-time tracking of invading leader cell phenotypes possible and has potential for use with patient material for clinical risk stratification and personalized medicine.

A pluripotent stem cell-based model for post-implantation human amniotic sac development.

Development of the asymmetric amniotic sac-with the embryonic disc and amniotic ectoderm occupying opposite poles-is a vital milestone during human embryo implantation. Although essential to embryogenesis and pregnancy, amniotic sac development in humans remains poorly understood. Here, we report a human pluripotent stem cell (hPSC)-based model, termed the post-implantation amniotic sac embryoid (PASE), that recapitulates multiple post-implantation embryogenic events centered around amniotic sac development. Without maternal or extraembryonic tissues, the PASE self-organizes into an epithelial cyst with an asymmetric amniotic ectoderm-epiblast pattern that resembles the human amniotic sac. Upon further development, the PASE initiates a process that resembles posterior primitive streak development in a SNAI1-dependent manner. Furthermore, we observe asymmetric BMP-SMAD signaling concurrent with PASE development, and establish that BMP-SMAD activation/inhibition modulates stable PASE development. This study reveals a previously unrecognized fate potential of human pluripotent stem cells and provides a platform for advancing human embryology.Early in human embryonic development, it is unclear how amniotic sac formation is regulated. Here, the authors use a human pluripotent stem cell-based model, termed the post-implantation amniotic sac embryoid, to recapitulate early embryogenic events of human amniotic sac development.

Acoustic tweezing cytometry enhances osteogenesis of human mesenchymal stem cells through cytoskeletal contractility and YAP activation.

Human mesenchymal stem cells (hMSCs) have great potential for cell-based therapies for treating degenerative bone diseases. It is known that mechanical cues in the cell microenvironment play an important role in regulating osteogenic (bone) differentiation of hMSCs. However, mechanoregulation of lineage commitment of hMSCs in conventional two-dimensional (2D) monocultures or bioengineered three-dimensional (3D) tissue constructs remains suboptimal due to complex biomaterial design criteria for hMSC culture. In this study, we demonstrate the feasibility of a novel cell mechanics and mechanobiology tool, acoustic tweezing cytometry (ATC), for mechanical stimulation of hMSCs. ATC utilizes ultrasound (US) pulses to actuate functionalized lipid microbubbles (MBs) which are covalently attached to hMSCs via integrin binding to exert forces to the cells. ATC stimulation increases cytoskeletal contractility of hMSCs regardless of the cell area. Furthermore, ATC application rescues osteogenic differentiation of hMSCs in culture conditions that are intrinsically repressive for hMSC osteogenesis (e.g., soft cell culture surfaces). ATC application activates transcriptional regulator YAP to enhance hMSC osteogenesis. Our data further show that F-actin, myosin II, and RhoA/ROCK signaling functions upstream of YAP activity in mediating ATC-stimulated hMSC osteogenesis. With the capability of applying controlled dynamic mechanical stimuli to cells, ATC provides a powerful tool for mechanoregulation of stem cell behaviors in tissue engineering and regenerative medicine applications.