Toward developing human organs via embryo models and chimeras.

Developing functional organs from stem cells remains a challenging goal in regenerative medicine. Existing methodologies, such as tissue engineering, bioprinting, and organoids, only offer partial solutions. This perspective focuses on two promising approaches emerging for engineering human organs from stem cells: stem cell-based embryo models and interspecies organogenesis. Both approaches exploit the premise of guiding stem cells to mimic natural development. We begin by summarizing what is known about early human development as a blueprint for recapitulating organogenesis in both embryo models and interspecies chimeras. The latest advances in both fields are discussed before highlighting the technological and knowledge gaps to be addressed before the goal of developing human organs could be achieved using the two approaches. We conclude by discussing challenges facing embryo modeling and interspecies organogenesis and outlining future prospects for advancing both fields toward the generation of human tissues and organs for basic research and translational applications.

A patterned human neural tube model using microfluidic gradients.

The human nervous system is a highly complex but organized organ. The foundation of its complexity and organization is laid down during regional patterning of the neural tube, the embryonic precursor to the human nervous system. Historically, studies of neural tube patterning have relied on animal models to uncover underlying principles. Recently, models of neurodevelopment based on human pluripotent stem cells, including neural organoids1-5 and bioengineered neural tube development models6-10, have emerged. However, such models fail to recapitulate neural patterning along both rostral-caudal and dorsal-ventral axes in a three-dimensional tubular geometry, a hallmark of neural tube development. Here we report a human pluripotent stem cell-based, microfluidic neural tube-like structure, the development of which recapitulates several crucial aspects of neural patterning in brain and spinal cord regions and along rostral-caudal and dorsal-ventral axes. This structure was utilized for studying neuronal lineage development, which revealed pre-patterning of axial identities of neural crest progenitors and functional roles of neuromesodermal progenitors and the caudal gene CDX2 in spinal cord and trunk neural crest development. We further developed dorsal-ventral patterned microfluidic forebrain-like structures with spatially segregated dorsal and ventral regions and layered apicobasal cellular organizations that mimic development of the human forebrain pallium and subpallium, respectively. Together, these microfluidics-based neurodevelopment models provide three-dimensional lumenal tissue architectures with in vivo-like spatiotemporal cell differentiation and organization, which will facilitate the study of human neurodevelopment and disease.

Stem cell-based models of early mammalian development.

The complex process by which a single-celled zygote develops into a viable embryo is nothing short of a miraculous wonder of the natural world. Elucidating how this process is orchestrated in humans has long eluded the grasp of scientists due to ethical and practical limitations. Thankfully, pluripotent stem cells that resemble early developmental cell types possess the ability to mimic specific embryonic events. As such, murine and human stem cells have been leveraged by scientists to create in vitro models that aim to recapitulate different stages of early mammalian development. Here, we examine the wide variety of stem cell-based embryo models that have been developed to recapitulate and study embryonic events, from pre-implantation development through to early organogenesis. We discuss the applications of these models, key considerations regarding their importance within the field, and how such models are expected to grow and evolve to achieve exciting new milestones in the future.

Controlled modelling of human epiblast and amnion development using stem cells.

Early human embryonic development involves extensive lineage diversification, cell-fate specification and tissue patterning1. Despite its basic and clinical importance, early human embryonic development remains relatively unexplained owing to interspecies divergence2,3 and limited accessibility to human embryo samples. Here we report that human pluripotent stem cells (hPSCs) in a microfluidic device recapitulate, in a highly controllable and scalable fashion, landmarks of the development of the epiblast and amniotic ectoderm parts of the conceptus, including lumenogenesis of the epiblast and the resultant pro-amniotic cavity, formation of a bipolar embryonic sac, and specification of primordial germ cells and primitive streak cells. We further show that amniotic ectoderm-like cells function as a signalling centre to trigger the onset of gastrulation-like events in hPSCs. Given its controllability and scalability, the microfluidic model provides a powerful experimental system to advance knowledge of human embryology and reproduction. This model could assist in the rational design of differentiation protocols of hPSCs for disease modelling and cell therapy, and in high-throughput drug and toxicity screens to prevent pregnancy failure and birth defects.

Metabolic-Glycoengineering-Enabled Molecularly Specific Acoustic Tweezing Cytometry for Targeted Mechanical Stimulation of Cell Surface Sialoglycans.

In this study, we developed a novel type of dibenzocyclooctyne (DBCO)-functionalized microbubbles (MBs) and validated their attachment to azide-labelled sialoglycans on human pluripotent stem cells (hPSCs) generated by metabolic glycoengineering (MGE). This enabled the application of mechanical forces to sialoglycans on hPSCs through molecularly specific acoustic tweezing cytometry (mATC), that is, displacing sialoglycan-anchored MBs using ultrasound (US). It was shown that subjected to the acoustic radiation forces of US pulses, sialoglycan-anchored MBs exhibited significantly larger displacements and faster, more complete recovery after each pulse than integrin-anchored MBs, indicating that sialoglycans are more stretchable and elastic than integrins on hPSCs in response to mechanical force. Furthermore, stimulating sialoglycans on hPSCs using mATC reduced stage-specific embryonic antigen-3 (SSEA-3) and GD3 expression but not OCT4 and SOX2 nuclear localization. Conversely, stimulating integrins decreased OCT4 nuclear localization but not SSEA-3 and GD3 expression, suggesting that mechanically stimulating sialoglycans and integrins initiated distinctive mechanoresponses during the early stages of hPSC differentiation. Taken together, these results demonstrated that MGE-enabled mATC uncovered not only different mechanical properties of sialoglycans on hPSCs and integrins but also their different mechanoregulatory impacts on hPSC differentiation, validating MGE-based mATC as a new, powerful tool for investigating the roles of glycans and other cell surface biomolecules in mechanotransduction.

Molecular identification and functional analysis of X-linked inhibitor of apoptosis -associated factor-1 (XAF1) in grass carp, Ctenopharyngodon idella.

X-linked inhibitor of apoptosis protein (XIAP) -associated factor 1 (XAF1) is an interferon-stimulated gene which exhibits pro-apoptosis effect. In this study, XAF1 was characterized from grass carp Ctenopharyngodon idella and its expression pattern and function were analyzed. The open reading frame (orf) of XAF1 is 789 nucleotides (nt) encoding 262 amino acids. SMART online search results showed that a C2H2-type and six C2HC-type zinc-fingers were found in XAF1, however, the XAF1 of grass carp showed high sequence identity to zebrafish (71%), low sequence identity to tetrapods (21-22%). Rt-qPCR results showed that XAF1 was constitutively expressed in all tested organs/tissues with highest expression in blood. An inductive expression of XAF1 at mRNA level was observed in peripheral blood leucocytes (PBLs) and C. idellus kidney cells (CIKs) after treatment with C. idellus recombinant interferon-γ (rIFNg). Overexpressing XAF1 in CIKs exhibited resistance against grass carp reovirus (GCRV) and more sensitivity to cisplatin. These results implied a functional homologue of XAF1 in evolution, however the mechanism may require further investigation.

Disruption of intestinal structure, tight junction complex, immune response and microbiota after chronic exposure to copper in swamp eel (Monopterus albus).

As an essential micronutrient, copper is crucial in aquatic organisms' growth and development. Numerous studies have consistently reported that excessive intake of copper can have harmful effects on organisms. However, there are limited studies on the impact of copper on the intestine of the swamp eel (Monopterus albus). This study aimed to investigate the changes of intestinal histopathology, tight junction complex, immune response, and microbiota in swamp eel treated with 0 mg/L Cu2+, 0.05 mg/L Cu2+, and 0.10 mg/L Cu2+ for 56 d. Intestinal histopathology showed major changes such as the increased number of erythrocytes and goblet cells in the lamina propria, and separation of the lamina propria. The expression of genes involved in tight junction complex (ZO-1, Claudin-3, Claudin-12 and Claudin-15) was significantly changed. In addition, copper exposure significantly increased the mRNA levels of TLR3, TLR7, TLR8, NF-κB, I-κB, TNF-α and IL-8, especially in 0.10 mg/L Cu2+ group. In contrast, the relative expression level of anti-inflammatory cytokine TGF-ß was significantly decreased after exposure to copper. Analysis of the intestinal microbiome showed the intestinal microbiota of swamp eels in the control and copper exposure groups were dominated by Firmicutes and Proteobacteria at the phylum level. Notably, copper exposure changed the diversity of the intestinal microbiota and decreased the relative abundance of Firmicutes and Proteobacteria in the intestine of swamp eel. Collectively, this study demonstrates that chronic copper exposure induces intestinal pathologic changes and inflammatory response, disrupts the intestinal microbial diversity and microbiota composition, and decreases intestinal barrier function in swamp eel, which enhances our understanding of copper-induced intestinal toxicity in fish.

Effects of haloxyfop-p-methyl on the developmental toxicity, neurotoxicity, and immunotoxicity in zebrafish.

Pesticides are extensively used in agricultural production, and their residues in soil, water, and agricultural products have become a threat to aquatic ecosystem. In this study, the toxicity of haloxyfop-p-methyl, an aryloxyphenoxypropionate herbicide was studied using the model animal zebrafish. The development of zebrafish larvae was affected by haloxyfop-p-methyl including spinal deformities, decreased body length, slow heart rate, and large yolk sac area. Behavior analysis revealed that behavior activity of larvae was weakened significantly including shortened displacement distance, reduced swimming speed, increased angular speed winding degrees, in accordance with higher AChE activity. Besides, exposure to haloxyfop-p-methyl could induce oxidative stress companied by the increased intents of ROS, MDA and increased activities of CAT and SOD. In immunotoxicity, haloxyfop-p-methyl not only reduced the innate immune cells such as neutrophils and macrophages, but also affected T cells mature in thymus. Furthermore, haloxyfop-p-methyl could induce neutrophils apoptosis, accompanied with the upregulation of the expression of proapoptotic protein such as Bax and P53 and the downregulation of the expression of antiapoptotic protein Bcl-2. In addition, haloxyfop-p-methyl could induce the expression of Jak, STAT and proinflammatory cytokine genes (IFN-γ, TNF-α, and IL-8). These results indicate that haloxyfop-p-methyl induces developmental toxicity, neurotoxicity, and immunotoxicity in zebrafish, providing a perspective on the toxicological mechanism of haloxyfop-p-methyl in teleosts.

The Mosquito Larvicidal Activity of Lignans from Branches of Cinnamomum camphora chvar. Borneol.

The chemical investigation of branches of Cinnamomum camphora chvar. Borneol guided by mosquito larvicidal activity led to the isolation of fourteen known lignans (1-14). Their structures were elucidated unambiguously based on comprehensive spectroscopic analysis and comparison with the literature data. This is the first report of these compounds being isolated from branches of Cinnamomum camphora chvar. Borneol. Compounds 3-5 and 8-14 were isolated from this plant for the first time. All compounds isolated were subjected to anti-inflammatory, mosquito larvicidal activity and cytotoxic activity evaluation. Compounds (1-14) showed significant mosquito larvicidal activity against Culex pipiens quinquefasciatus with lethal mortality in 50% (LC50), with values ranging from 0.009 to 0.24 µg/mL. Among them, furofuran lignans(1-8) exhibited potent mosquito larvicidal activity against Cx. p. quinquefasciatus, with LC50 values of 0.009-0.021 µg/mL. From the perspective of a structure-activity relationship, compounds with a dioxolane group showed high mosquito larvicidal activity and have potential to be developed into a mosquitocide.

Stem-cell-based embryo models for fundamental research and translation.

Despite its importance, understanding the early phases of human development has been limited by availability of human samples. The recent emergence of stem-cell-derived embryo models, a new field aiming to use stem cells to construct in vitro models to recapitulate snapshots of the development of the mammalian conceptus, opens up exciting opportunities to promote fundamental understanding of human development and advance reproductive and regenerative medicine. This Review provides a summary of the current knowledge of early mammalian development, using mouse and human conceptuses as models, and emphasizes their similarities and critical differences. We then highlight existing embryo models that mimic different aspects of mouse and human development. We further discuss bioengineering tools used for controlling multicellular interactions and self-organization critical for the development of these models. We conclude with a discussion of the important next steps and exciting future opportunities of stem-cell-derived embryo models for fundamental discovery and translation.

A base-mediated aerobic oxidative synthesis of cyclopent-2-enol derivatives from doubly activated cyclopropanes and substituted acetonitriles.

We report here that polysubstituted cyclopent-2-enols can be constructed by the one-pot reaction of doubly activated cyclopropanes and α-EWG substituted acetonitriles under mild basic conditions via a domino-ring-opening-cyclization/deacylation/oxidation sequence. Moreover, the synthetic applications of these cyclopent-2-enols have been demonstrated in the late-stage derivatization into functionalized cyclopentapyrimidin-4-ones and 2-hydroxy cyclopentanones with good yields.

Effects of chlorobromoisocyanuric acid on embryonic development and immunotoxicity of zebrafish.

Although chlorobromoisocyanuric acid has been widely used in agriculture, its deleterious toxicity on aquatic organisms remains rare. In this study, zebrafish were exposed to chlorobromoisocyanuric acid (0, 30, 40, and 50 mg/L) from 10 to 96 h post-fertilization (hpf). We found a significant reduction in immune cell numbers (neutrophils and macrophages) and the area of thymus at 96 hpf. The expression of immune-related genes and pro-inflammatory cytokines genes were upregulated. Besides, chlorobromoisocyanuric acid triggered neutrophils cell apoptosis. The mRNA and protein levels of pro-apoptotic p53 pathway and the Bax/Bcl-2 ratio further indicated the underlying mechanism. Furthermore, the oxidative stress was observed that the accumulation of reactive oxygen species and malondialdehyde significantly increased. Subsequently, the antioxidant agent astaxanthin significantly attenuated the level of oxidative stress and the dysregulation of inflammatory response. In summary, our results showed that chlorobromoisocyanuric acid induced developmental defects and immunotoxicity of zebrafish, partly owing to oxidative stress and cell apoptosis.

Novel synthesis of fused spiro piperidone-cyclopropanes from cyclopropyl amides and electron-deficient alkenes.

We report here that a series of bridged O,O-ketal fused spiro piperidone-cyclopropane derivatives 3 can be constructed with excellent yields and good diastereoselectivity by the one-pot reaction of 1-acylcyclopropanecarboxamides 1 with electron-deficient alkene 2a (EWG = CHO) via the domino process involving [4 + 2] annulation/intermolecular electrophilic addition/intramolecular cyclization. Furthermore, reactions of 1 with 2b/2c (EWG = CN, COOMe), leading to spiro piperidone-cyclopropane derivatives 4 or 5 by base catalyst selection, were also presented.

A novel and efficient method to induce allospecific CD8+ memory T lymphocytes.

The aim of the current study was to establish a simple method for effectively inducing memory T lymphocytes by the intraperitoneal injection of spleen lymphocytes into mice. In total, 75 mice were divided into the following groups: an injection group administered three doses of spleen lymphocytes (1 × 106 , 5 × 106 , and 1 × 107 cells), a transplantation group in which a 0.25-cm2 skin section from C57BL/6 mice was transplanted onto the back of the recipient, and a control group in which an equal volume of phosphate-buffered saline was injected. At 1, 2, or 3 months following transplantation, the following parameters were evaluated: quantity of T lymphocytes, percentage of cluster of differentiation 8+ (CD8+ ) memory T cells, and proliferation index of purified CD8+ memory T cells. No significant differences among groups were detected at 1 month (p > .05). However, the injection group administered 1 × 106 cells exhibited the highest proportion of CD8+ memory T cells among all groups at 2 months, and the proportions of CD8+ T cells were higher in the three injection groups than in the skin transplantation and control groups at 3 months. The proportions of memory T cells were higher in the injection groups administered 5 × 106 or 1 × 107 cells than in the skin transplantation and control groups at 3 months. The newly established method effectively induces memory T lymphocytes via the intraperitoneal injection of spleen lymphocytes in vivo and has potential applications in the field of immunotherapy.

Multiplexed Luminescence Oxygen Channeling Immunoassay Based on Dual-Functional Barcodes with a Host-Guest Structure: A Facile and Robust Suspension Array Platform.

The development of a powerful immunoassay platform with capacities of both simplicity and high multiplexing is promising for disease diagnosis. To meet this urgent need, for the first time, a multiplexed luminescent oxygen channeling immunoassay (multi-LOCI) platform by implementation of LOCI with suspension array technology is reported. As the microcarrier of the platform, a unique dual-functional barcode with a host-guest structure composed of a quantum dot host bead (QDH) and LOCI acceptor beads (ABs) is designed, in which QDH provides function of high coding capacity while ABs facilitate the LOCI function. The analytes bridge QDH@ABs and LOCI donor beads (DBs) into a close proximity, forming a QDH@ABs-DBs "host-guest-satellite" superstructure that generates both barcode signal from QDH and LOCI signal induced by singlet oxygen channeling between ABs and DBs. Through imaging-based decoding, different barcodes are automatically distinguished and colocalized with LOCI signals. Importantly, the assay achieves simultaneous detection of multiple analytes within one reaction, simply by following a "mix-and-measure" protocol without the need for tedious washing steps. Furthermore, the multi-LOCI platform is validated for real sample measurements. With the advantages of robustness, simplicity, and high multiplexing, the platform holds great potential for the development of point-of-care diagnostics.

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.

Mechanics-guided embryonic patterning of neuroectoderm tissue from human pluripotent stem cells.

Classic embryological studies have successfully applied genetics and cell biology principles to understand embryonic development. However, it remains unresolved how mechanics, as an integral driver of development, is involved in controlling tissue-scale cell fate patterning. Here we report a micropatterned human pluripotent stem (hPS)-cell-based neuroectoderm developmental model, in which pre-patterned geometrical confinement induces emergent patterning of neuroepithelial and neural plate border cells, mimicking neuroectoderm regionalization during early neurulation in vivo. In this hPS-cell-based neuroectoderm patterning model, two tissue-scale morphogenetic signals-cell shape and cytoskeletal contractile force-instruct neuroepithelial/neural plate border patterning via BMP-SMAD signalling. We further show that ectopic mechanical activation and exogenous BMP signalling modulation are sufficient to perturb neuroepithelial/neural plate border patterning. This study provides a useful microengineered, hPS-cell-based model with which to understand the biomechanical principles that guide neuroectoderm patterning and hence to study neural development and disease.

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.

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.