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Stem cell-derived organoids are a promising tool to model native human tissues as they resemble human organs functionally and structurally compared to traditional monolayer cell-based assays. For instance, colon organoids can spontaneously develop crypt-like structures similar to those found in the native colon. While analyzing the structural development of organoids can be a valuable readout, using traditional image analysis tools makes it challenging because of the heterogeneities and the abstract nature of organoid morphologies. To address this limitation, we developed and validated a deep learning-based image analysis tool, named D-CryptO, for the classification of organoid morphology. D-CryptO can automatically assess the crypt formation and opacity of colorectal organoids from brightfield images to determine the extent of organoid structural maturity. To validate this tool, changes in organoid morphology were analyzed during organoid passaging and short-term forskolin stimulation. To further demonstrate the potential of D-CryptO for drug testing, organoid structures were analyzed following treatments with a panel of chemotherapeutic drugs. With D-CryptO, subtle variations in how colon organoids responded to the different chemotherapeutic drugs were detected, which suggest potentially distinct mechanisms of action. This tool could be expanded to other organoid types, like intestinal organoids, to facilitate 3D tissue morphological analysis., Comment: Lab on a Chip (2022)

Psoriasis is a refractory inflammatory skin disease affecting 2 %-3 % of the world population, characterized by the infiltration and hyper-proliferation of inflammatory cells and aberrant differentiation of keratinocytes. Targeting the IL-23/ Th17 axis has been well recognized as a promising therapeutic strategy, as the IL-23/ Th17 signal plays a vital role in the pathology of psoriasis. Three pentacyclic triterpene compounds isolated from loquat leaves have been reported with significant inhibitory effects on RORγt transcription activity and Th17 cell differentiation, and excellent performance in preventing lupus nephritis pathogenesis. However, the potential effects of these pentacyclic triterpene compounds on psoriasis remain unknown. In this study, we demonstrated the potent therapeutic effects of these pentacyclic triterpene compounds on psoriasis. These three pentacyclic triterpene compounds significantly alleviated skin inflammation as well as aberrant keratinocyte proliferation in an imiquimod-induced mouse psoriasis model. These compounds also inhibited the infiltration of immune cells and the level of pro-inflammatory cytokine in the dermis, as well as the cells number and changed the cytokine profiling expression of Th17 cells. These compounds could reduce the amount of CD4+ and CD8+ T cells in local lymph node, but not in spleen, which is different from hydrocortisone, the positive control treatment. These results suggest better performance of these compounds than steroids on treating psoriasis with less side effects on the integrated immune system. In summary, our findings uncover the potent therapeutic effects of pentacyclic triterpene compounds on psoriasis, providing potential candidate compounds for drug development.

Tissue building does not occur exclusively during development. Even after a whole body is built from a single cell, tissue building can occur to repair and regenerate tissues of the adult body. This confers resilience and enhanced survival to multicellular organisms. However, this resiliency comes at a cost, as the potential for misdirected tissue building creates vulnerability to organ deformation and dysfunction-the hallmarks of disease. Pathological tissue morphogenesis is associated with fibrosis and cancer, which are the leading causes of morbidity and mortality worldwide. Despite being the priority of research for decades, scientific understanding of these diseases is limited and existing therapies underdeliver the desired benefits to patient outcomes. This can largely be attributed to the use of two-dimensional cell culture and animal models that insufficiently recapitulate human disease. Through the synergistic union of biological principles and engineering technology, organ-on-a-chip systems represent a powerful new approach to modeling pathological tissue morphogenesis, one with the potential to yield better insights into disease mechanisms and improved therapies that offer better patient outcomes. This Review will discuss organ-on-a-chip systems that model pathological tissue morphogenesis associated with (1) fibrosis in the context of injury-induced tissue repair and aging and (2) cancer.

In recent years, research has begun to touch upon the associations between the natural environment and individuals' subjective wellbeing; however, whether such associations would vary among different groups of people remains under-studied. Using four waves (2010-2013) of data from the Chinese General Social Survey, we investigate the group-specific correlation between environment-specifically, greenspace exposure-and individuals' happiness in contemporary urban China. For this study, the normalized difference vegetation index at the county level is a proxy for greenspace exposure. The results from the ordered logit model indicate that county-level green vegetation is associated with individuals' happiness level, but such effects vary with a household's relative deprivation. While greenspace exposure is positively associated with happiness among individuals from less deprived families, it is negatively associated with happiness among those from deprived households. These findings contribute to the body of knowledge of the heterogeneous effect of greenspace on residents' subjective well-being.

Background Maternal stress is potentially a modifiable risk factor for spontaneous preterm birth (sPTB). However, epidemiologic findings on the maternal stress-sPTB relationship have been inconsistent. Methods To investigate whether the maternal stress-sPTB associations may be modified by genetic susceptibility, we performed genome-wide gene × stress interaction analyses in 1490 African-American women from the Boston Birth cohort who delivered term (n = 1033) or preterm (n = 457) infants. Genotyping was performed using Illumina HumanOmni 2.5 array. Replication was performed using data from the NICHD genomic and Proteomic Network (GPN) for PTB research. Results rs35331017, a T-allele insertion/deletion polymorphism in the protein-tyrosine phosphatase receptor Type D (PTPRD) gene, was the top hit that interacted significantly with maternal lifetime stress on risk of sPTB (PG × E = 4.7 × 10-8). We revealed a dose-responsive association between degree of stress and risk of sPTB in mothers carrying the insertion/insertion genotype, but an inverse association was observed in mothers carrying the heterozygous or deletion/deletion genotypes. This interaction was replicated in African-American (PG × E = 0.088) and Caucasian mothers (PG × E = 0.023) from the GPN study. Conclusion We demonstrated a significant maternal PTPRD × stress interaction on sPTB risk. This finding, if further confirmed, may provide new insight into individual susceptibility to stress-induced sPTB. Impact This was the first preterm study to demonstrate a significant genome-wide gene-stress interaction in African Americans, specifically, PTPRD gene variants can interact with maternal perceived stress to affect risk of spontaneous preterm birth.The PTPRD × maternal stress interaction was demonstrated in African Americans and replicated in both African Americans and Caucasians from the GPN study.Our findings highlight the importance of considering genetic susceptibility in assessing the role of maternal stress on spontaneous preterm birth.

Ochratoxin A (OTA) is a mycotoxin ubiquitous in feeds and foodstuffs. The water-insoluble pentacyclic triterpene bioactive compound, ursolic acid (UA), is widespread in various cuticular waxes of edible fruits, food materials, and medicinal plants. Although studies have reported that oxidative stress was involved in both the nephrotoxicity of OTA and the renoprotective function of UA, the role of stress-responsive Lon protease 1 (Lonp1) in the renoprotection of UA against OTA is still unknown. In this study, cell viability, reactive oxygen species (ROS) production, and several proteins' expressions of human embryonic kidney 293T (HEK293T) cells in response to UA, OTA, and/or Lonp1 inhibitor CDDO-me treatment were detected to reveal the protective mechanism of UA against OTA-induced renal cytotoxicity. Results indicated that a 2 h-treatment of 1 μM UA could significantly alleviate the ROS production and cell death induced by a 24 h-treatment of 8 μM OTA in HEK293T cells (P 0.05). Compared with the control, the protein expressions of Lonp1, Aco2 and Hsp75 were significantly inhibited after 8 μM OTA treating for 24 h (P 0.05), which could be notably reversed by the pre-treatment and post-treatment of 1 μM UA (P 0.05). The protein expressions of Lonp1, Aco2 and Hsp75 were inhibited by the addition of CDDO-me. The three protein expression trends were similar before and after the addition of CDDO-me. In conclusion, OTA could inhibit the expression of Lonp1, suppressing Aco2 and Hsp75 as a result, thereby activating ROS and inducing cell death in HEK293T cells, which could be alleviated by UA pre-treatment.

COVID-19 mRNA vaccines elicit strong T and B cell responses to the SARS-CoV-2 spike glycoprotein in both SARS-CoV-2 naïve and experienced patients. However, it is unknown whether the post-vaccine CD4+ T cell responses seen in patients with a history of COVID-19 are due to restimulation of T cell clonotypes that were first activated during natural infection or if they are the result of new clones activated by the vaccine.To address this question, we analyzed the SARS-CoV-2 spike glycoprotein-specific CD4+ T cell receptor repertoire before and after vaccination in 10 COVID-19 convalescent patients and 4 SARS-CoV-2 naïve healthy donor vaccine recipients. We used the viral Functional Expansion of Specific T cells (ViraFEST) assay to quantitatively identify specific SARS-CoV-2 and common cold coronavirus CD4+ T cell clonotypes post COVID-19 disease resolution and post mRNA SARS-CoV-2 vaccination.We found that while some preexisting T cell receptor clonotypes persisted, the post-vaccine repertoire consisted mainly of vaccine-induced clones and was largely distinct from the repertoire induced by natural infection. Vaccination-induced clones led to an overall maintenance of the total number of SARS-CoV-2 reactive clonotypes over time through expansion of novel clonotypes only stimulated through vaccination. Additionally, we demonstrated that the vaccine preferentially induces T cells that are only specific for SARS-CoV-2 antigens, rather than T cells that cross-recognize SARS-CoV-2/common cold coronaviruses.These data demonstrate that SARS-CoV-2 vaccination in patients with prior SARS-CoV-2 infection induces a new antigen-specific repertoire and sheds light on the differential immune responses induced by vaccination versus natural infection.Bloomberg∼Kimmel Institute for Cancer Immunotherapy, The Johns Hopkins University, The Bill and Melinda Gates Foundation, NCI U54CA260492, NIH.

The osseointegration of metallic implants is reliant on a cascade of molecular interactions and the delivery of macromolecules to the implant environment that occurs before substantial bone formation. Early blood vessel formation is a requisite first step in the healing timeline for osteoid formation, where vascular development can be accelerated as a result of controlled hypoxic conditioning. In this study, alginate-derived xerogel films containing varied concentrations of disodium succinate salt which has been shown to induce pseudohypoxia (short-term hypoxic effects while maintaining an oxygenated environment) were developed. Xerogels were characterized for their morphology, succinate release over time and cellular response with osteoblast-mimicking Saos-2 and human umbilical vein endothelial cells (HUVEC). Scanning electron microscopy revealed a multiscale topography that may favour osseointegration and alamarBlue assays indicated no cytotoxic effects during in vitro proliferation of Saos-2 cells. pH measurements of eluted succinate reach 95 % of peak value after 7 h of immersion for all gels containing 10 mM of succinate or less, and 60 % within the first 40 min. In vitro exposure of HUVECs to succinate-conditioned media increased the net concentration of total proteins measured by bicinchoninic acid (BCA) assay and maintains stable vascular endothelial growth factor (VEGF) and extracellular platelet-derived growth factor (PDGF) for vessel formation through comparison of enzyme-linked immunosorbent assays (ELISAs) of the culture media and cell lysate. Tube formation assays also showed a sustained increase in tube diameter across the first 48 h of HUVEC culture when succinate concentrations of 1 and 10 μM in the xerogel. Overall, the succinate-alginate films serve as a prospective organic coating for bone-interfacing implant materials which may induce temporary pseudohypoxic conditions favourable for early angiogenesis and bone regeneration in vivo at succinate concentrations of 1 or 10 μM.

Vasculature is a key component of many biological tissues and helps to regulate a wide range of biological processes. Modeling vascular networks or the vascular interface in organ-on-a-chip systems is an essential aspect of this technology. In many organ-on-a-chip devices, however, the engineered vasculatures are usually designed to be encapsulated inside closed microfluidic channels, making it difficult to physically access or extract the tissues for downstream applications and analysis. One unexploited benefit of tissue extraction is the potential of vascularizing, perfusing, and maturing the tissue in well-controlled, organ-on-a-chip microenvironments and then subsequently extracting that product for in vivo therapeutic implantation. Moreover, for both modeling and therapeutic applications, the scalability of the tissue production process is important. Here we demonstrate the scalable production of perfusable and extractable vascularized tissues in an "open-top" 384-well plate (referred to as IFlowPlate), showing that this system could be used to examine nanoparticle delivery to targeted tissues through the microvascular network and to model vascular angiogenesis. Furthermore, tissue spheroids, such as hepatic spheroids, can be vascularized in a scalable manner and then subsequently extracted for in vivo implantation. This simple multiple-well plate platform could not only improve the experimental throughputs of organ-on-a-chip systems but could potentially help expand the application of model systems to regenerative therapy.

T-cell receptor sequencing (TCRseq) enables tracking of T-cell clonotypes recognizing the same antigen over time and across biological compartments. TCRseq has been used to test if cross-reactive antitumor T cells are responsible for development of immune-related adverse events (irAEs) following immune checkpoint blockade. Prior studies have interpreted T-cell clones shared among the tumor and irAE as evidence supporting this, but interpretations of these findings are challenging, given the constraints of TCRseq. Here we capitalize on a rare opportunity to understand the impact of potential confounders, such as sample size, tissue compartment, and collection batch/timepoint, on the relative proportion of shared T-cell clones between an irAE and tumor specimens. TCRseq was performed on tumor-involved and -uninvolved tissues, including an irAE, that were obtained throughout disease progression and at the time of rapid autopsy from a patient with renal cell carcinoma treated with programmed death-1 (PD-1) blockade. Our analyses show significant effects of these confounders on our ability to understand T-cell receptor overlap, and we present mitigation strategies and study design recommendations to reduce these errors. Implementation of these strategies will enable more rigorous TCRseq-based studies of immune responses in human tissues, particularly as they relate to antitumor T-cell cross-reactivity in irAEs following checkpoint blockade.

Decellularization efforts must balance the preservation of the extracellular matrix (ECM) components while eliminating the nucleic acid and cellular components. Following effective removal of nucleic acid and cell components, decellularized ECM (dECM) can be solubilized in an acidic environment with the assistance of various enzymes to develop biological scaffolds in different forms, such as sheets, tubular constructs, or three-dimensional (3D) hydrogels. Each organ or tissue that undergoes decellularization requires a distinct and optimized protocol to ensure that nucleic acids are removed, and the ECM components are preserved. The objective of this study was to optimize the decellularization process for dECM isolation from human lung tissues for downstream 2D and 3D cell culture systems. Following protocol optimization and dECM isolation, we performed experiments with a wide range of dECM concentrations to form human lung dECM hydrogels that were physically stable and biologically responsive. The dECM based-hydrogels supported the growth and proliferation of primary human lung fibroblast cells in 3D cultures. The dECM is also amenable to the coating of polyester membranes in Transwell™ Inserts to improve the cell adhesion, proliferation, and barrier function of primary human bronchial epithelial cells in 2D. In conclusion, we present a robust protocol for human lung decellularization, generation of dECM substrate material, and creation of hydrogels that support primary lung cell viability in 2D and 3D culture systems

Ochratoxin A (OTA) is one of the mycotoxins, which widely pollutes food systems and seriously threatens human health. OTA's target organ is the kidney. Exosome, as one of the extracellular vesicles, could be secreted by all kinds of cells. It contains different proteins, nucleic acid, and lipid, which are decided by their donor cells and could be uptake by the recipient cells, release their contents, and affect the recipient cell's life activity. In this study, a 24 h-treatment with 5 μM OTA was found to significantly reduce the cell viability of HEK293 cells and meanwhile to provide a sufficient quantity of exosomes, thus this concentration and time were selected for subsequent experiments. In addition, exosomes extracted by ultracentrifugation had higher purity, fewer impurities, and uniform morphology than that by the ExoQuick-TC kit. Furthermore, these exosomes increased ROS levels and decreased mitochondrial membrane potential in HEK293 cells. By RNA-seq, the cytotoxicity mechanisms induced by OTA-treated HEK293 cell-derived exosomes (EXO-OTA) and OTA were mainly the metabolism of proteins and the cell cycle respectively. Also, it proved that exosomes deliver partial OTA-induced cytotoxicity.

BACKGROUND: Recent studies have reported T cell immunity to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in unexposed donors, possibly due to crossrecognition by T cells specific for common cold coronaviruses (CCCs). True T cell crossreactivity, defined as the recognition by a single TCR of more than one distinct peptide-MHC ligand, has never been shown in the context of SARS-CoV-2. METHODS: We used the viral functional expansion of specific T cells (ViraFEST) platform to identify T cell responses crossreactive for the spike (S) glycoproteins of SARS-CoV-2 and CCCs at the T cell receptor (TCR) clonotype level in convalescent COVID-19 patients (CCPs) and SARS-CoV-2–unexposed donors. Confirmation of SARS-CoV-2/CCC crossreactivity and assessments of functional avidity were performed using a TCR cloning and transfection system. RESULTS: Memory CD4(+) T cell clonotypes that crossrecognized the S proteins of SARS-CoV-2 and at least one other CCC were detected in 65% of CCPs and unexposed donors. Several of these TCRs were shared among multiple donors. Crossreactive T cells demonstrated significantly impaired SARS-CoV-2–specific proliferation in vitro relative to monospecific CD4(+) T cells, which was consistent with lower functional avidity of their TCRs for SARS-CoV-2 relative to CCC. CONCLUSIONS: Our data confirm, for what we believe is the first time, the existence of unique memory CD4(+) T cell clonotypes crossrecognizing SARS-CoV-2 and CCCs. The lower avidity of crossreactive TCRs for SARS-CoV-2 may be the result of antigenic imprinting, such that preexisting CCC-specific memory T cells have reduced expansive capacity upon SARS-CoV-2 infection. Further studies are needed to determine how these crossreactive T cell responses affect clinical outcomes in COVID-19 patients. FUNDING: NIH funding (U54CA260492, P30CA006973, P41EB028239, R01AI153349, R01AI145435-A1, R21AI149760, and U19A1088791) was provided by the National Institute of Allergy and Infectious Diseases, the National Cancer Institute, and the National Institute of Biomedical Imaging and Bioengineering. The Bloomberg~Kimmel Institute for Cancer Immunotherapy, The Johns Hopkins University Provost, and The Bill and Melinda Gates Foundation provided funding for this study.

BACKGROUND: While stress and the absence of social support during pregnancy have been linked to poor health outcomes, the underlying biological mechanisms are unclear. METHODS: We examined whether adverse experiences during pregnancy alter DNA methylation (DNAm) in maternal epigenomes. Analyses included 250 African-American mothers from the Boston Birth Cohort. Genome-wide DNAm profiling was performed in maternal blood collected after delivery, using the Infinium HumanMethylation450 Beadchip. Linear regression models, with adjustment of pertinent covariates, were applied. RESULTS: While self-reported maternal psychosocial lifetime stress and stress during pregnancy was not associated with DNAm alterations, we found that absence of support from the baby’s father was significantly associated with maternal DNAm changes in TOR3A, IQCB1, C7orf36, and MYH7B, and that lack of support from family and friends was associated with maternal DNA hypermethylation on multiple genes, including PRDM16 and BANKL. CONCLUSIONS: This study provides intriguing results suggesting biological embedding of social support during pregnancy on maternal DNAm, warranting additional investigation, and replication.

Parkinson's disease (PD) is the second most common neurodegenerative disorder. It is characterized by the degeneration of nigral dopaminergic (DA) neurons. While over 90% of cases are idiopathic, without a clear etiology, mutations in many genes have been linked to rare, familial forms of PD. It has been quite challenging to develop effective animal models of PD that capture salient features of PD. The discovery of induced pluripotent stem cells (iPSCs) makes it possible to generate patient-specific DA neurons to study PD. Here, we review the methods for the generation of iPSCs and discuss previous studies using iPSC-derived neurons from monogenic forms of PD. These investigations have revealed several converging pathways that intersect with the unique vulnerabilities of human nigral DA neurons. With the rapid development in stem cell biology, it is possible to generate patient-specific neurons that will be increasingly similar to those in the brain of the patient. Combined with the ability to edit the genome to generate isogenic iPSCs, the generation and analysis of patient-specific midbrain DA neurons will transform PD research by providing a valuable tool for mechanistic study and drug discovery.

Background Maternal smoking affects more than half a million pregnancies each year in the US and is known to result in fetal growth restriction as measured by lower birthweight and its associated long-term consequences. Maternal smoking also has been linked to altered fetal DNA methylation (DNAm). However, what remains largely unexplored is whether these DNAm alterations are merely markers of smoking exposure or if they also have implications for health outcomes. This study tested the hypothesis that fetal DNAm mediates the effect of maternal smoking on newborn birthweight. Methods This study included mother–newborn pairs from a US predominantly urban, low-income multi-ethnic birth cohort. DNAm in cord blood were determined using the Illumina Infinium MethylationEPIC BeadChip. After standard quality control and normalization procedures, an epigenome-wide association study (EWAS) of maternal smoking was performed using linear regression models, controlling for maternal age, education, race, parity, pre-pregnancy body mass index, alcohol consumption, gestational age, maternal pregestational/gestational diabetes, child sex, cord blood cell compositions and batch effects. To quantify the degree to which cord DNAm mediates the smoking-birthweight association, the VanderWeele-Vansteelandt approach for single mediator and structural equational model for multiple mediators were used, adjusting for pertinent covariates. Results The study included 954 mother–newborn pairs. Among mothers, 165 (17.3%) ever smoked before or during pregnancy. Newborns with smoking exposure had on average 258 g lower birthweight than newborns without exposure (P GFI1, AHRR, CYP1A1, and CNTNAP2; 8 of those, located in the first three genes, were Bonferroni significantly associated with newborn birthweight and mediated the smoking-birthweight association. The combined mediation effect of the three genes explained 67.8% of the smoking-birthweight association. Conclusions Our study not only lends further support that maternal smoking alters fetal DNAm in a multiethnic population, but also suggests that fetal DNAm substantially mediates the maternal smoking–birthweight association. Our findings, if further validated, indicate that DNAm modification is likely an important pathway by which maternal smoking impairs fetal growth and, perhaps, even long-term health outcomes.

PD-1 blockade unleashes CD8 T cells1, including those specific for mutation-associated neoantigens (MANA), but factors in the tumour microenvironment can inhibit these T cell responses. Single-cell transcriptomics have revealed global T cell dysfunction programs in tumour-infiltrating lymphocytes (TIL). However, the majority of TIL do not recognize tumour antigens2, and little is known about transcriptional programs of MANA-specific TIL. Here, we identify MANA-specific T cell clones using the MANA functional expansion of specific T cells assay3 in neoadjuvant anti-PD-1-treated non-small cell lung cancers (NSCLC). We use their T cell receptors as a ‘barcode’ to track and analyse their transcriptional programs in the tumour microenvironment using coupled single-cell RNA sequencing and T cell receptor sequencing. We find both MANA- and virus-specific clones in TIL, regardless of response, and MANA-, influenza- and Epstein–Barr virus-specific TIL each have unique transcriptional programs. Despite exposure to cognate antigen, MANA-specific TIL express an incompletely activated cytolytic program. MANA-specific CD8 T cells have hallmark transcriptional programs of tissue-resident memory (TRM) cells, but low levels of interleukin-7 receptor (IL-7R) and are functionally less responsive to interleukin-7 (IL-7) compared with influenza-specific TRM cells. Compared with those from responding tumours, MANA-specific clones from non-responding tumours express T cell receptors with markedly lower ligand-dependent signalling, are largely confined to HOBIThigh TRM subsets, and coordinately upregulate checkpoints, killer inhibitory receptors and inhibitors of T cell activation. These findings provide important insights for overcoming resistance to PD-1 blockade., Single-cell RNA sequencing and T cell receptor sequencing are combined to identify transcriptional programs specific to mutation-associated neoantigen-specific T cells in non-small cell lung cancers treated with anti-PD-1, providing insights into resistance to PD-1 blockade.

Naive human pluripotent stem cells (hPSCs) can be used to generate mature human cells of all three germ layers in mouse-human chimeric embryos. Here, we describe a protocol for generating mouse-human chimeric embryos by injecting naive hPSCs converted from the primed state. Primed hPSCs are treated with a mammalian target of rapamycin inhibitor (Torin1) for 3 h and dissociated to single cells, which are plated on mouse embryonic fibroblasts in 2iLI medium, a condition essentially the same for culturing mouse embryonic stem cells. After 3-4 d, bright, dome-shaped colonies with mouse embryonic stem cell morphology are passaged in 2iLI medium. Established naive hPSCs are injected into mouse blastocysts, which produce E17.5 mouse embryos containing 0.1-4.0% human cells as quantified by next-generation sequencing of 18S ribosomal DNA amplicons. The protocol is suitable for studying the development of hPSCs in mouse embryos and may facilitate the generation of human cells, tissues and organs in animals.

Degeneration of photoreceptors is a major cause of blindness. Identifying new methods for the generation of photoreceptors offers valuable options for a cell replacement therapy of blindness. Here, we show that primary adult human retinal pigmented epithelium (hRPE) cells were directly converted to postmitotic neurons with various properties of photoreceptors by the neurogenic transcription factor ASCL1 and microRNA124. At Day 8 after the induction of ASCL1 and miRNA124 expression in hRPE cells, 91% of all cells were Tuj1+, and 83% of all cells were MAP2+neurons. The cone photoreceptor marker L/M-opsin, the rod photoreceptor marker rhodopsin, and the generic photoreceptor marker recoverin were expressed in 76%, 86%, and 92% of all cells, respectively. Real-time quantitative PCR measurements showed significant and continuous increases in the expression of photoreceptor markers phosducin and recoverin, rod cell markers phosphodiesterases 6 b and arrestin S-antigen, and cone cell markers L/M-opsin and S-opsin in three independent lines of primary hRPE cells at different days of transdifferentiation. Transmission electron microscopy of converted neurons showed disc-like structures similar to those found in photoreceptors. While the converted neurons had voltage-dependent Na+, K+, and Ca2+currents, light-induced change in membrane potential was not detected. The study demonstrates the feasibility of rapid and efficient transdifferentiation of adult hPRE cells to neurons with many properties of photoreceptors. It opens up a new possibility in cell replacement therapy of blindness caused by photoreceptor degeneration.

Three-dimensional (3D) tissue models such as epithelial spheroids or organoids have become popular for pre-clinical drug studies. However, different from 2D monolayer culture, the characterization of 3D tissue models from non-invasive brightfield images is a significant challenge. To address this issue, here we report a Deep-Learning Uncovered Measurement of Epithelial Networks (Deep-LUMEN) assay. Deep-LUMEN is an object detection algorithm that has been fine-tuned to automatically uncover subtle differences in epithelial spheroid morphology from brightfield images. This algorithm can track changes in the luminal structure of tissue spheroids and distinguish between polarized and non-polarized lung epithelial spheroids. The Deep-LUMEN assay was validated by screening for changes in spheroid epithelial architecture in response to different extracellular matrices and drug treatments. Specifically, we found the dose-dependent toxicity of Cyclosporin can be underestimated if the effect of the drug on tissue morphology is not considered. Hence, Deep-LUMEN could be used to assess drug effects and capture morphological changes in 3D spheroid models in a non-invasive manner.Significance of the workDeep learning has been applied for the first time to autonomously detect subtle morphological changes in 3D multi-cellular spheroids, such as spheroid polarity, from brightfield images in a label-free manner. The technique has been validated by detecting changes in spheroid morphology in response to changes in extracellular matrices and drug treatments.

Background Preeclampsia and preterm delivery (PTD) are believed to affect women’s long-term health including cardiovascular disease (CVD), but the biological underpinnings are largely unknown. We aimed to test whether maternal postpartum metabolomic profiles, especially CVD-related metabolites, varied according to PTD subtypes with and without preeclampsia, in a US urban, low-income multi-ethnic population. Methods This study, from the Boston Birth Cohort, included 980 women with term delivery, 79 with medically indicated PTD (mPTD) and preeclampsia, 52 with mPTD only, and 219 with spontaneous PTD (sPTD). Metabolomic profiling in postpartum plasma was conducted by liquid chromatography-mass spectrometry. Linear regression models were used to assess the associations of each metabolite with mPTD with preeclampsia, mPTD only, and sPTD, respectively, adjusting for pertinent covariates. Weighted gene coexpression network analysis was applied to investigate interconnected metabolites associated with the PTD/preeclampsia subgroups. Bonferroni correction was applied to account for multiple testing. Results A total of 380 known metabolites were analyzed. Compared to term controls, women with mPTD and preeclampsia showed a significant increase in 36 metabolites, mainly representing acylcarnitines and multiple classes of lipids (diacylglycerols, triacylglycerols, phosphocholines, and lysophosphocholines), as well as a decrease in 11 metabolites including nucleotides, steroids, and cholesteryl esters (CEs) (P −4). Alterations of diacylglycerols, triacylglycerols, and CEs in women with mPTD and preeclampsia remained significant when compared to women with mPTD only. In contrast, the metabolite differences between women with mPTD only and term controls were only seen in phosphatidylethanolamine class. Women with sPTD had significantly different levels of 16 metabolites mainly in amino acid, nucleotide, and steroid classes compared to term controls, of which, anthranilic acid, bilirubin, and steroids also had shared associations in women with mPTD and preeclampsia. Conclusion In this sample of US high-risk women, PTD/preeclampsia subgroups each showed some unique and shared associations with maternal postpartum plasma metabolites, including those known to be predictors of future CVD. These findings, if validated, may provide new insight into metabolomic alterations underlying clinically observed PTD/preeclampsia subgroups and implications for women’s future cardiometabolic health.

Owing to their high spatiotemporal precision and adaptability to different host cells, organ-on-a-chip systems are showing great promise in drug discovery, developmental biology studies and disease modeling. However, many current micro-engineered biomimetic systems are limited in technological application because of culture media mixing that does not allow direct incorporation of techniques from stem cell biology, such as organoids. Here, we describe a detailed alternative method to cultivate millimeter-scale functional vascularized tissues on a biofabricated platform, termed 'integrated vasculature for assessing dynamic events', that enables facile incorporation of organoid technology. Utilizing the 3D stamping technique with a synthetic polymeric elastomer, a scaffold termed 'AngioTube' is generated with a central microchannel that has the mechanical stability to support a perfusable vascular system and the self-assembly of various parenchymal tissues. We demonstrate an increase in user familiarity and content analysis by situating the scaffold on a footprint of a 96-well plate. Uniquely, the platform can be used for facile connection of two or more tissue compartments in series through a common vasculature. Built-in micropores enable the studies of cell invasion involved in both angiogenesis and metastasis. We describe how this protocol can be applied to create both vascularized cardiac and hepatic tissues, metastatic breast cancer tissue and personalized pancreatic cancer tissue through incorporation of patient-derived organoids. Platform assembly to populating the scaffold with cells of interest into perfusable functional vascularized tissue will require 12-14 d and an additional 4 d if pre-polymer and master molds are needed.

The basic helix-loop-helix-PAS transcription factor (CLOCK, Circadian locomotor output cycles protein kaput) was discovered in 1994 as a circadian clock. Soon after its discovery, the circadian clock, Aryl hydrocarbon receptor nuclear translocator-like protein 1 (ARNTL, also call BMAL1), was shown to regulate adiposity and body weight by controlling on the brain hypothalamic suprachiasmatic nucleus (SCN). Farther, circadian clock genes were determined to exert several of lipid metabolic and diabetes effects, overall indicating that CLOCK and BMAL1 act as a central master circadian clock. A master circadian clock acts through the neurons and hormones, with expression in the intestine, liver, kidney, lung, heart, SCN of brain, and other various cell types of the organization. Among circadian clock genes, numerous metabolic syndromes are the most important in the regulation of food intake (via regulation of circadian clock genes or clock-controlled genes in peripheral tissue), which lead to a variation in plasma phospholipids and tissue phospholipids. Circadian clock genes affect the regulation of transporters and proteins included in the regulation of phospholipid metabolism. These genes have recently received increasing recognition because a pharmacological target of circadian clock genes may be of therapeutic worth to make better resistance against insulin, diabetes, obesity, metabolism syndrome, atherosclerosis, and brain diseases. In this book chapter, we focus on the regulation of circadian clock and summarize its phospholipid effect as well as discuss the chemical, physiology, and molecular value of circadian clock pathway regulation for the treatment of plasma lipids and atherosclerosis.

The study produces large amounts of mature human cells in mouse embryos, paving the way for making human organs in animals., It has not been possible to generate naïve human pluripotent stem cells (hPSCs) that substantially contribute to mouse embryos. We found that a brief inhibition of mTOR with Torin1 converted hPSCs from primed to naïve pluripotency. The naïve hPSCs were maintained in the same condition as mouse embryonic stem cells and exhibited high clonogenicity, rapid proliferation, mitochondrial respiration, X chromosome reactivation, DNA hypomethylation, and transcriptomes sharing similarities to those of human blastocysts. When transferred to mouse blastocysts, naïve hPSCs generated 0.1 to 4% human cells, of all three germ layers, including large amounts of enucleated red blood cells, suggesting a marked acceleration of hPSC development in mouse embryos. Torin1 induced nuclear translocation of TFE3; TFE3 with mutated nuclear localization signal blocked the primed-to-naïve conversion. The generation of chimera-competent naïve hPSCs unifies some common features of naïve pluripotency in mammals and may enable applications such as human organ generation in animals.

Preeclampsia, which disproportionately affects Black women, is a leading cause of preterm delivery and risk for future hypertension and chronic kidney disease (CKD). Apolipoprotein L1 (APOL1) kidney risk alleles, common among Black individuals, contribute substantially to CKD disparities. Given the strong link between preeclampsia and CKD, we investigated whether maternal and fetal APOL1 risk alleles can jointly influence preeclampsia risk, and explored potential modifiers of the association between APOL1 and preeclampsia.Nested case-control study.426 Black mother-infant pairs (275 African Americans and 151 Haitians) from the Boston Birth Cohort.Maternal and fetal APOL1 risk alleles.Preeclampsia.Logistic regression models with adjustment for demographic characteristics were applied to analyze associations between fetal and maternal APOL1 risk alleles and risk of preeclampsia and to investigate the effects of modification by maternal country of origin.Fetal APOL1 risk alleles tended to be associated with an increased risk of preeclampsia, which was not statistically significant in the total genotyped population. However, this association was modified by maternal country of origin (P0.05 for interaction tests): fetal APOL1 risk alleles were significantly associated with an increased risk of preeclampsia among African Americans under recessive (odds ratio [OR], 3.6 [95% CI, 1.3-9.7]; P=0.01) and additive (OR, 1.7 [95% CI, 1.1-2.6]; P=0.01) genetic models but not in Haitian Americans. Also, maternal-fetal genotype discordance at the APOL1 locus was associated with a 2.6-fold higher risk of preeclampsia (P0.001) in African Americans.Limited sample size in stratified analyses; self-reported maternal country of origin; pre-pregnancy estimated glomerular filtration rate (eGFR) and proteinuria data in mothers were not collected; unmeasured confounding social and/or environmental factors; no replication study.This study supports the hypothesis that fetal APOL1 kidney risk alleles are associated with increased risk for preeclampsia in a recessive mode of inheritance in African Americans and suggests that maternal-fetal genotype discordance is also associated with this risk. These conclusions underscore the need to better understand maternal-fetal interaction and their genetic and environmental factors as contributors to ethnic disparities in preeclampsia.

Tissue engineering holds promise to replace damaged tissues for repair of vital organs in the human body. In cardiac repairs specifically, approaches are developed for intramyocardial delivery of cells and the epicardial delivery of tissue-engineered cardiac patches, providing benefit of cell localization and tissue structure, respectively. However, to improve cell retention and integration, there is a need for the intramyocardial delivery of functional tissues while preserving anisotropic muscle alignment. Here, a biodegradable z-wire scaffold that supports the scalable gel-free production of an array of functional cardiac tissues in a 384-well plate format is developed. The z-wire scaffold design supports cellular alignment, provides tunable mechanical support, and allows for tissue contraction. When the scaffold is imparted with magnetic properties, individual tissues can be assembled with macroscopic alignment under magnetic guidance. When used in combination with a customized surgical delivery tool, z-wire tissues can be injected directly into the myocardial wall, with controlled tissue orientation according to the injection path. This modular tissue engineering approach, in combination with the use of smart scaffolds, can expand opportunity in functional tissue delivery.

Aflatoxin B1 (AFB1) is a known carcinogen found in contaminated food and designated by the World Health Organization as a class I carcinogenic substance. AFB1 presents with carcinogenicity, teratogenicity, and mutagenicity, and the liver is the human organ most susceptible to AFB1. Zinc (Zn), which is one of the essential nutrient elements that could protect the cells from biological toxins, heavy metals, hydrogen peroxide, metal chelators and radiation, is assessed in this study for its potential to alleviate AFB1-induced cytotoxicity. Samples were divided into three groups, namely CK, AFB1, and AFB1+Zn. Protein expressions were analyzed by two-way electrophoresis combined with flight mass spectrometry, with 41 differentially expressed proteins identified in the results, mainly related to oxidative stress, cell apoptosis, DNA damage, and energy metabolism. Zn was found to regulate the expression of peroxidases (peroxiredoxin-1, peroxiredoxin-5, peroxiredoxin-6) to relieve AFB1-induced oxidative stress. Moreover, Zn could decrease the expression of pro-apoptotic genes (cleaved-caspase-3, caspase-9, and Bax) and increase the expression of anti-apoptotic genes (Bcl-2 and Bcl-xl) to alleviate the cell apoptosis induced by AFB1. In addition, AFB1 reduced intracellular ATP levels, whereas Zn supplementation boosted ATP levels and maintained homeostasis and a steady state of cellular energy metabolism by modulating AMPK-ACC phosphorylation levels, while many zinc finger proteins changed after AFB1 treatment. These results, therefore, indicate that Zn could alleviate AFB1-induced cytotoxicity by changing the expressions of zinc finger proteins in liver hepatocellular carcinoma (HepG2 cells).

As a vital member of AAA+ (ATPase associated with diverse cellular activities) protein superfamily, Lon, a homo-hexameric ring-shaped protein complex with a serine-lysine catalytic dyad, is highly conserved throughout almost all prokaryotic and eukaryotic organisms. Lon protease (LONP) plays an important role in maintaining mitoproteostasis through selectively recognizing and degrading oxidatively modified mitoproteins within mitochondrial matrix, such as oxidized aconitase, phosphorylated mitochondrial transcription factor A, etc. Furthermore, the up-regulated LONP increased mitochondrial ROS generation to promote cell survival, cell proliferation, epithelial-mesenchymal transition, and cell migration, which was attributed to the up-regulation of NADH:ubiquinone oxidoreductase core subunit S8 via interaction with chaperone Lon under hypoxic or oxidative stress in tumorigenesis. In addition, Lon also participated in protein kinase RNA (PKR)-like endoplasmic reticulum kinase signaling pathway under endoplasmic reticulum (ER) stress. In short, Lon, as a pivotal stress-responsive protein that involved in the crosstalks among mitochondria, ER and nucleus, participated in multifarious important cellular processes crucial for cell survival, such as the mitochondrial protein quality control system, the mitochondrial unfolded protein response, the mtDNA maintenance, and the ER unfolded protein response.

Background/Aims: Brown and beige adipocytes are widely recognized as potential therapeutic targets to treat obesity and related metabolic disorders, and the recruitment of brown and beige adipocytes is an essential aspect that requires attention. Although many methods of activating brown adipocytes or generating beige adipocytes have been reported, the limited number and sources are the biggest challenges. The number of white adipocytes is much greater than the number of brown adipocytes, both in human adults and fetuses. Unfortunately, human adult white adipose tissue-derived stem cell (aWAsc) has little beige adipogenic potential. However, the characteristics and beige adipogenic potential of human embryo-derived white adipose stem cells (eWAsc) still need to be investigated. Methods: To analyze the characteristics and functionality of eWAsc, we analyzed the markers of adipose precursor cells by flow cytometry. Then, differentiation and browning/beiging were induced, and the identifying markers were analyzed by real-time PCR and immunoblot. In addition, more in-depth exploration was performed using RNA-SEQ on eWAsc and aWAsc. Results: eWAsc was isolated from human embryonic white adipose tissue, and aWAsc was isolated from adult white adipose tissue by collagenase treatment. eWAsc has extreme advantages in adipogenesis capacity and browning/beiging ability in comparison to aWAsc, indicating that eWAsc may possess some special regulatory factors to promote the generation of functional brown/beige adipocytes. Greater exploration was enabled by RNA-SEQ, revealing a large number of differences at the transcriptional levels, including 1263 differentially expressed genes, 657 down- and 605 upregulated, in eWAsc compared to aWAsc. Pathway analysis revealed enrichment in cell cycle, TGF-β signaling pathway, DNA replication, and Hippo signaling pathways. Interestingly, the expression levels of C/EBPα, FGF1 and FST gene, which are related to the maturation of adipocytes, Hippo signaling pathway and TGF-β signaling pathway, were significantly higher in eWAsc than in aWAsc. These may be potential candidates and possible regulatory targets for recruiting beige adipocytes in human adipose tissue. Conclusion: Overall, we have demonstrated the molecular characteristics and excellent beige adipogenic potential of eWAsc, providing a new reference for studying human adipocytes.

Objective Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Protein kinase D1 (PKD1) is recognized as a key regulator in the progression in several solid cancers, while its clinical role in HCC is unclear. This study aimed to evaluate the correlation of PKD1 with clinical features and prognosis in HCC patients. Methods A total of 218 HCC patients who underwent resection were retrospectively enrolled. PKD1 expression in tumor (N = 218) and adjacent (N = 110) tissues was detected by immunohistochemical staining, scored by a semi-quantitative scoring method ranging from 0 to 12, and further classified as PKD1-, PKD1+, PKD1++ and PKD1+++ for analysis. Meanwhile, patients’ clinical features and survival data were acquired from the database. Results PKD1 was elevated in tumor tissues compared with adjacent tissues. Meanwhile, higher tumor PKD1 was correlated with elevated tumor size, Barcelona Clinic Liver Cancer (BCLC) stage, carbohydrate antigen 199 (CA199) level and alpha fetoprotein (AFP) level; while no correlation was found in tumor PKD1 with patients’ basic features or liver function indexes. Moreover, higher tumor PKD1 was correlated with worse overall survival (OS) in HCC patients, then further validated as an independent predictive factor for worse OS by multivariate Cox’s regression model analysis. Additionally, in Child-Pugh stage A, Child-Pugh stage B, BCLC stage 0/A, and BCLC stage B subgroups, higher tumor PKD1 was also correlated with worse OS. Conclusion Higher PKD1 in tumor tissues correlates with elevated BCLC stage, bigger tumor size, increased CA199 level, higher AFP level and worse OS in HCC patients.

Aflatoxin B1 (AFB1) is widely distributed in nature, especially in a variety of food commodities. It is confirmed to be the most toxic of all the aflatoxins. The toxicity of AFB1 has been well investigated, and it may result in severe health problems including carcinogenesis, mutagenesis, growth retardation, and immune suppression. Epigenetic modifications including DNA methylation, histone modifications and regulation of non-coding RNA play an important role in AFB1-induced disease and carcinogenesis. To better understand the evidence for AFB1-induced epigenetic alterations and the potential mechanisms of the toxicity of AFB1, we conducted a review of published studies of AFB1-induced epigenetic alterations.

To evaluate the utility of transthoracic contrast echocardiography (cTTE) using vitamin B6 and sodium bicarbonate as contrast agents for diagnosing right-to-left shunt (RLS) caused by patent foramen ovale (PFO) compared to that of transesophageal echocardiography (TEE). We investigated 125 patients admitted to our neurology department with unexplained cerebral infarction and migraine. All patients underwent cTTE using vitamin B6 and sodium bicarbonate as contrast agents, after which they underwent transthoracic echocardiography. The Doppler signal was recorded during the Valsalva maneuver, and TEE examinations were performed. The feasibility, diagnostic sensitivity, and safety of cTTE and TEE for PFO recognition were compared. Evidence of PFO was found in 49 (39.20%) patients with cTTE, more than were detected with TEE (39, 31.20%) (χ2=5.0625, P=0.0244). cTTE had a sensitivity of 92.31% and a specificity of 84.88% for diagnosing PFO, showing high concordance with TEE for PFO recognition (κ=0.72). Further, results of a semi-quantitative evaluation of PFO-RLS by cTTE were better than those with TEE (Z=-2.011, P=0.044). No significant adverse reaction was discovered during cTTE examination. cTTE using vitamin B6 and sodium bicarbonate as contrast agents has relatively good sensitivity and specificity for diagnosing RLS caused by PFO when compared with those for TEE. Using vitamin B6 and sodium bicarbonate as contrast agents to perform cTTE is recommended for detecting and diagnosing the PFO due to its simplicity, non-invasive character, low cost, and high feasibility.

To curb the high cost of drug development, there is an urgent need to develop more predictive tissue models using human cells to determine drug efficacy and safety in advance of clinical testing. Recent insights gained through fundamental biological studies have validated the importance of dynamic cell environments and cellular communication to the expression of high fidelity organ function. Building on this knowledge, emerging organ-on-a-chip technology is poised to fill the gaps in drug screening by offering predictive human tissue models with methods of sophisticated tissue assembly. Organ-on-a-chip start-ups have begun to spawn from academic research to fill this commercial space and are attracting investment to transform the drug discovery industry. This review traces the history, examines the scientific foundation and envisages the prospect of these renowned organ-on-a-chip technologies. It serves as a guide for new members of this dynamic field to navigate the existing scientific and market space.

Microphysiological systems (MPSs) have been proposed as an improved tool to recreate the complex biological features of the native niche with the goal of improving in vitro–in vivo extrapolation. In just over a decade, MPS technologies have progressed from single-tissue chips to multitissue plates with integrated pumps for perfusion. Concurrently, techniques for biofabrication of complex 3D constructs for regenerative medicine and 3D in vitro models have evolved into a diverse toolbox for micrometer-scale deposition of cells and cell-laden bioinks. However, as the complexity of biological models increases, experimental throughput is often compromised. This review discusses the existing disparity between MPS complexity and throughput, then examines an MPS-terminated biofabrication line to identify the hurdles and potential approaches to overcoming this disparity.

Precision toxicology evaluates the toxicity of certain substances by isolating a small group of cells with a typical phenotype of interest followed by a single cell sequencing-based analysis. In this in vitro attempt, ochratoxin A (OTA), a typical mycotoxin and food contaminant, is found to induce G0/G1 phase cell cycle arrest in human renal proximal tubular HKC cells at a concentration of 20 μM after a 24h-treatment. A small number of G0/G1 phase HKC cells are evaluated in both the presence and absence of OTA. These cells are sorted with a flow cytometer and subjected to mRNA and DNA methylation sequencing using Smart-Seq2 and single-cell reduced-representation bisulfite sequencing (scRRBS) technology, respectively. Integrated analysis of the transcriptome and methylome profiles reveals that OTA causes abnormal expression of the essential genes that regulate G1/S phase transition, act as signal transductors in G1 DNA damage checkpoints, and associate with the anaphase-promoting complex/cyclosome. The alteration of their DNA methylation status is a significant underlying epigenetic mechanism. Furthermore, Notch signaling and Ras/MAPK/CREB pathways are found to be suppressed by OTA. This attempt at precision toxicology paves the way for a deeper understanding of OTA toxicity and provides an innovative strategy to researchers in the toxicology and pharmacology field.

Ochratoxin A (OTA) is a nephrotoxic mycotoxin that is widely distributed in foodstuffs and feeds, meanwhile oleanolic acid (OA) is ubiquitous in various fruit skins, food materials, and medicinal herbs. Due to that OA has a nephroprotective effect, it has the poteintial to counteract OTA-induced nephrotoxicity by nutritional intervention of OA. Furthermore, tumor necrosis factor receptor-associated protein 1 (TRAP1) acts as the core of endoplasmic reticulum (ER)-mitochondria crosstalk, becoming our focus in the mechanism investigation. In this study, the cell viability, apoptosis rate, and protein expressions of human proximal tubule epithelial-originated kidney-2 (HK-2) cells in response to OTA and/or OA were determined. Results indicated that a 24 h-treatment of 1-5 μM OTA could notably induce mitochondrial-mediated and ER stress (ERS)-excitated apoptosis via inhibiting TRAP1, thereby activating CypD, Bax, Cyt-C, Cleaved Caspase-9, Cleaved Caspase-3, GRP78, p-PERK, p-eIF2α, ATF4, and CHOP and inhibiting Bcl-2 (P < 0.05). Results of the RNA interference of TRAP1 further ascertained its anti-apoptotic function via inhibiting CypD, Bax, GRP78, and CHOP and enhancing Bcl-2 (P < 0.05). The pre-treatment of 2 μM OA for 2 h could remarkably relieve OTA-induced suppression of TRAP1 (P < 0.05). In conclusion, TRAP1 played a central role in the ameliorative effect of OA on the mitochondrial-mediated and ERS-excitated apoptosis induced by OTA.

Despite the complexity and structural sophistication that 3D organoid models provide, their lack of vascularization and perfusion limit the capability of these models to recapitulate organ physiology effectively. A microfluidic platform named IFlowPlate is engineered, which can be used to culture up to 128 independently perfused and vascularized colon organoids in vitro. Unlike traditional microfluidic devices, the vascularized organoid-on-chip device with an "open-well" design does not require any external pumping systems and allows tissue extraction for downstream analyses, such as histochemistry or even in vivo transplantation. By optimizing both the extracellular matrix (ECM) and the culture media formulation, patient-derived colon organoids are co-cultured successfully within a self-assembled vascular network, and it is found that the colon organoids grow significantly better in the platform under constant perfusion versus conventional static condition. Furthermore, a colon inflammation model with an innate immune function where circulating monocytes can be recruited from the vasculature, differentiate into macrophage, and infiltrate the colon organoids in response to tumor necrosis factor (TNF)- inflammatory cytokine stimulation is developed using the platform. With the ability to grow vascularized colon organoids under intravascular perfusion, the IFlowPlate platform could unlock new possibilities for screening potential therapeutic targets or modeling relevant diseases.

The mounting number of patients with pulmonary adenocarcinoma (ADCA) is subjected to poor prognosis and heavy mortality, which prompts us to explore new potential therapeutics for lung ADCA. Herein, we reported a novel approach for lung ADCA therapy by abolishing autophagy and asparagine. We demonstrated that deprivation of asparagine by asparaginase could induce significant cytotoxicity and apoptosis in A549 and H1975 cells. During this process, autophagy was triggered by the asparaginase treatment, characterized by the autophagic flux with three main stages including formation of autophagosomes, lysosomes fused with autophagosomes, and degradation of autophagosomes by lysosomes. Importantly, suppression of autophagy could notably enhance the cytotoxicity and accelerate the caspase 3-dependent apoptosis induced by asparaginase. Furthermore, suppression of reactive oxygen species (ROS) could attenuated both the cytotoxicity and autophagy induced by asparaginase, while inhibition of autophagy promoted the generation of ROS in A549 and H1975 cells, indicating the essential role of ROS in asparagine deprivation therapy in lung ADCA cells. Our results demonstrated that targeting cytoprotective autophagy and asparagine could potently kill the ADCA cells, which highlighted a novel approach for lung ADCA therapy in the clinics.

Due to good biocompatibility and plasma membrane similarity, the nanosized exosomes are ideal drug carriers. Near-infrared (NIR) photothermal therapy is an emerging method for cancer treatment in which photothermal agents absorb the energy of external NIR light to generate high temperatures in a targeted region to effectively kill cancer cells. Gold nanorods (AuNRs) have been found to provide a prominent photothermal performance, while aptamers can precisely target surface markers on cells with high affinity and specificity. In this study, exosomes were mildly functionalized by integrating them with aptamers and AuNRs to assemble a powerful combination Apt-Exos-AuNRs (AEARs) with good specificity and an effective photothermal killing action on cancer cells. The structure, hydrodynamic diameters, zeta potential, UV-vis absorption spectra and stability of the AEARs were further characterized. In addition, using a cell model, the cancer cell targeting ability of the AEARs and its cellular uptake were observed. Moreover, its photothermal killing effect on various human cancer cells in vitro was validated by a CCK-8 assay as well as apoptosis analysis, the results of which suggest this exosomes-based nanomaterial can serve as a novel and broad-spectrum platform for precision cancer therapy.

Due to escalating drug developmental costs and limitations of cardiotoxicity screening, there is an urgent need to develop robust in vitro 3D tissue culture platforms that can both facilitate the culture of human cardiac tissues and provide noninvasive functional readouts predictive of cardiotoxicity in clinical settings. However, such platforms commonly require complex fabrication procedures that are difficult to scale up to high-throughput testing platforms. Here, innovative multimaterial processing into a scalable and functional platform is proposed in the format of a 96-well plate. Three classes of materials are integrated into the platform. An array of soft elastic microwires is used both as anchors for tissue formation as well as sensors for recording tissue contraction. Conductive carbon electrodes are embedded into the plate to drive electrical stimulation for tissue maturation and pace tissue contraction during drug testing. The bulk of the device is made of rigid polystyrene plastic to eliminate drug-absorbing polydimethylsiloxane (PDMS). The platform has higher throughput than the current state-of-the-art devices, at a significantly reduced cost of manufacturing and tissue production.

Microengineered biomimetic systems for organ-on-a-chip or tissue engineering purposes often fail as a result of an inability to recapitulate the in vivo environment, specifically the presence of a well-defined vascular system. To address this limitation, we developed an alternative method to cultivate three-dimensional (3D) tissues by incorporating a microfabricated scaffold, termed AngioChip, with a built-in perfusable vascular network. Here, we provide a detailed protocol for fabricating the AngioChip scaffold, populating it with endothelial cells and parenchymal tissues, and applying it in organ-on-a-chip drug testing in vitro and surgical vascular anastomosis in vivo. The fabrication of the AngioChip scaffold is achieved by a 3D stamping technique, in which an intricate microchannel network can be embedded within a 3D scaffold. To develop a vascularized tissue, endothelial cells are cultured in the lumen of the AngioChip network, and parenchymal cells are encapsulated in hydrogels that are amenable to remodeling around the vascular network to form functional tissues. Together, these steps yield a functional, vascularized network in vitro over a 14-d period. Finally, we demonstrate the functionality of AngioChip-vascularized hepatic and cardiac tissues, and describe direct surgical anastomosis of the AngioChip vascular network on the hind limb of a Lewis rat model.

Significant advances in biomaterials, stem cell biology, and microscale technologies have enabled the fabrication of biologically relevant tissues and organs. Such tissues and organs, referred to as organ-on-a-chip (OOC) platforms, have emerged as a powerful tool in tissue analysis and disease modeling for biological and pharmacological applications. A variety of biomaterials are used in tissue fabrication providing multiple biological, structural, and mechanical cues in the regulation of cell behavior and tissue morphogenesis. Cells derived from humans enable the fabrication of personalized OOC platforms. Microscale technologies are specifically helpful in providing physiological microenvironments for tissues and organs. In this review, biomaterials, cells, and microscale technologies are described as essential components to construct OOC platforms. The latest developments in OOC platforms (e.g., liver, skeletal muscle, cardiac, cancer, lung, skin, bone, and brain) are then discussed as functional tools in simulating human physiology and metabolism. Future perspectives and major challenges in the development of OOC platforms toward accelerating clinical studies of drug discovery are finally highlighted.

Maternal smoking during pregnancy may affect newborn DNA methylation (DNAm). However, little is known about how these associations vary by a newborn’s sex and/or maternal nutrition. To fill in this research gap, we investigated epigenome-wide DNAm associations with maternal smoking during pregnancy in African American mother-newborn pairs. DNAm profiling in cord (n=379) and maternal blood (n=300) were performed using the Illumina HumanMethylation450 BeadChip array. We identified 12 CpG sites whose DNAm levels in cord blood were associated with maternal smoking, at a false discovery rate GFI1 gene were more pronounced in male newborns than in females (P = 0.002 for maternal smoking × sex interaction at cg18146737). We further observed that maternal smoking and folate level may interactively affect cord blood DNAm level at cg05575921 in the AHRR gene (P = 5.0×10−4 for interaction): compared to newborns unexposed to maternal smoking and with a high maternal folate level (>19.2 nmol/L), the DNAm level was about 0.03 lower (P = 3.6×10−4) in exposed newborns with a high maternal folate level, but was 0.08 lower (P = 1.2×10−8) in exposed newborns with a low maternal folate level. Our data suggest that the male fetus may be more vulnerable to the impact of maternal smoking on DNAm than the female; adequate maternal folate levels may partly counteract the impact of maternal smoking on DNAm. These findings may open new avenues of inquiry regarding sex differences in response to environmental insults and novel strategies to mitigate their intergenerational health effects through optimization of maternal nutrition.

In this work, a multilayer-modified paper-based colorimetric sensing platform with improved color uniformity and intensity was developed for the sensitive and selective determination of uric acid and glucose with smartphone as signal readout. In detail, chitosan, different kinds of chromogenic reagents, and horseradish peroxidase (HRP) combined with a specific oxidase, e.g., uricase or glucose oxidase (GOD), were immoblized onto the paper substrate to form a multilayer-modified test paper. Hydrogen peroxide produced by the oxidases (uricase or GOD) reacts with the substrates (uric acid or glucose), and could oxidize the co-immoblized chromogenic reagents to form colored products with HRP as catalyst. A simple strategy by placing the test paper on top of a light-emitting diode lamp was adopted to efficiently prevent influence from the external light. The color images were recorded by the smartphone camera, and then the gray values of the color images were calculated for quantitative analysis. The developed method provided a wide linear response from 0.01 to 1.0 mM for uric acid detection and from 0.02 to 4.0 mM for glucose detection, with a limit of detection (LOD) as low as 0.003 and 0.014 mM, respectively, which was much lower than for previously reported paper-based colorimetric assays. The proposed assays were successfully applied to uric acid and glucose detection in real serum samples. Furthermore, the enhanced analytical performance of the proposed method allowed the non-invasive detection of glucose levels in tear samples, which holds great potential for point-of-care analysis. Graphical abstract ᅟ.

Organoid technology and organ-on-a-chip engineering have emerged as two distinct approaches for stem cell-derived 3D tissue preparation. Their strategic integration can address each approach's limitations and provide a path toward a superior, synergistic strategy of constructing tissues that will truly deliver on the promise of regenerative and precision medicine.

In this review, we introduce a new concept, precision toxicology: the mode of action of chemical- or drug-induced toxicity can be sensitively and specifically investigated by isolating a small group of cells or even a single cell with typical phenotype of interest followed by a single cell sequencing-based analysis. Precision toxicology can contribute to the better detection of subtle intracellular changes in response to exogenous substrates, and thus help researchers find solutions to control or relieve the toxicological effects that are serious threats to human health. We give examples for single cell isolation and recommend laser capture microdissection for in vivo studies and flow cytometric sorting for in vitro studies. In addition, we introduce the procedures for single cell sequencing and describe the expected application of these techniques to toxicological evaluations and mechanism exploration, which we believe will become a trend in toxicology.

Adhesion molecule signaling is critical to human pluripotent stem cell (hPSC) survival, self-renewal, and differentiation. Thus, hPSCs are grown as clumps of cells on feeder cell layers or poorly defined extracellular matrices such as Matrigel. We sought to define a small molecule that would initiate adhesion-based signaling to serve as a basis for a defined substrate for hPSC culture. Soluble angiopoeitin-1 (Ang-1)-derived peptide QHREDGS added to defined serum-free media increased hPSC colony cell number and size during long- and short-term culture when grown on feeder cell layers or Matrigel, i.e. on standard substrates, without affecting hPSC morphology, growth rate or the ability to differentiate into multiple lineages both in vitro and in vivo. Importantly, QHREDGS treatment decreased hPSC apoptosis during routine passaging and single-cell dissociation. Mechanistically, the interaction of QHREDGS with β1-integrins increased expression of integrin-linked kinase (ILK), increased expression and activation of extracellular signal-regulated kinases 1/2 (ERK1/2), and decreased caspase-3/7 activity. QHREDGS immobilization to polyethylene glycol hydrogels significantly increased cell adhesion in a dose-dependent manner. We propose QHREDGS as a small molecule inhibitor of hPSC apoptosis and the basis of an affordable defined substrate for hPSC maintenance.

Nephrotoxicity is the most prominent toxicological effects of ochratoxin A (OTA). We have previously shown that autophagy might be involved in OTA-induced early renal cytotoxicity, but the mechanisms of action are unknown. Since OTA is known to induce mitochondrial damage and Nix is a selective autophagy receptor for mitochondrial clearance, the objective of this study was to investigate whether Nix mediates autophagic response to OTA-induced renal cytotoxicity. Our results showed that OTA induced autophagic and mitophagic activitits. Nix shRNA HEK 293 cells were more sensitive than scrambled shRNA cells to OTA-induced cell death, and differentially affect the mRNA expression of SDHA, AIFM1, and Bad and protein expression of AIF, VDAC, SDHA and LONP1 after OTA treatment. In particular, up-regulation of the pro-apoptotic Bad and AIF after OTA treatment was prominent only in Nix shRNA cells, which might explain the higher ratio of cell death. These results might indicate that Nix plays a critical role in the cellular protection against OTA toxicity through autophagy and mitochondria.

Summary Tissue engineering using cardiomyocytes derived from human pluripotent stem cells holds a promise to revolutionize drug discovery, but only if limitations related to cardiac chamber specification and platform versatility can be overcome. We describe here a scalable tissue-cultivation platform that is cell source agnostic and enables drug testing under electrical pacing. The plastic platform enabled on-line noninvasive recording of passive tension, active force, contractile dynamics, and Ca2+ transients, as well as endpoint assessments of action potentials and conduction velocity. By combining directed cell differentiation with electrical field conditioning, we engineered electrophysiologically distinct atrial and ventricular tissues with chamber-specific drug responses and gene expression. We report, for the first time, engineering of heteropolar cardiac tissues containing distinct atrial and ventricular ends, and we demonstrate their spatially confined responses to serotonin and ranolazine. Uniquely, electrical conditioning for up to 8 months enabled modeling of polygenic left ventricular hypertrophy starting from patient cells.

Organ-on-a-chip devices, also known as microphysiological systems, have gained significant attention in recent years. Recent advances in tissue engineering and microfabrication have enabled these devices to provide more precise control over cellular microenvironments to mimic the tissue-level or organ-level function of the human body. These more complex tissue models can provide either an improvement in the functional expression and maturation of cells or an avenue to probe biological events and function that would otherwise be difficult to visualize and mechanistically study. This high-value information, when complimented with the existing gold-standards of cell-based assays and animal models, could potentially lead to more informed decision-making in drug development. A prevalent biological component in many organ-on-a-chip devices is an engineered vascular interface that is present in almost all organs of the human body. The vasculature and the vascular interface are particularly susceptible to biomechanical forces, they function as the conduits for inter-cellular and inter-organ interactions, and regulate drug transport. In this review, we examine the various approaches taken to model the human vasculature with an emphasis on the engineering of organ-specific vasculatures, and discuss various challenges and opportunities ahead as the field advances.