Embryo development undergoes critical morphological transformations post-implantation, largely driven by the complex and dynamic microenvironment of the uterus. Despite advances, current 3D culture models inadequately recapitulate the uterine environment necessary for studying embryo-uterus interactions. In this work, we engineer a hydrogel inspired by the properties of the decidua, incorporating Matrigel to support blastocyst implantation and embryo development in vitro. Our findings reveal that embryos cultured within this hydrogel system successfully progress to an early organogenesis-like stage, including the development of first and second heart fields, mimicking natural embryogenesis. Moreover, we identify that the mechanical properties, particularly stress relaxation, play a crucial role in facilitating focal adhesion (FA) formation between the trophoblast and the hydrogel. Additionally, the degradation of the hydrogel by embryo-secreted metalloproteinases (MMP2 and MMP9) creates a favorable environment for continued embryonic growth and development. These insights contribute to a deeper understanding of how the external environment regulates embryo development and offer an enhanced approach for in vitro embryo culture. The complex interactions between the uterus microenvironment and the embryo during development are not fully understood. Here, authors engineer a 3D hydrogel culture system to investigate how the physical and biochemical properties of the uterine microenvironment impact embryo development in vitro.

Single-cell assay for transposase-accessible chromatin with sequencing (scATAC-seq) provides insights into transcriptional regulation, but there remain challenges in cell identity annotation due to data sparsity and limited gene regulatory annotations, particularly in non-model organisms. To address these limitations, we present ATACompass, a sequence-based framework for cell annotation that directly encodes chromatin accessibility peak sequences using the DNA foundation model HyenaDNA and large language models. By incorporating controlled sequence mutations for data augmentation, ATACompass achieves performance competitive with or superior to existing state-of-the-art methods in intra-species tasks. Critically, ATACompass enables zero-shot cross-species cell annotation by restricting input data to conserved sequences shared between species, allowing non-model organism data to be annotated without species-specific prior knowledge. Furthermore, the integration of datasets from representative evolutionary lineages enhances predictive accuracy, enabling comprehensive annotation across species. In summary, ATACompass provides a gene annotation-independent framework for studying cell types and regulatory landscapes, especially in non-model organisms.

Pyrogens, including endotoxins and non-endotoxins, are key factors that impact the safety of parenterally administered drugs as contaminants. Currently, traditional pyrogen detection methods primarily rely on the Rabbit Pyrogen Test (RPT) and the Limulus Amebocyte Lysate (LAL) assay, both of which are derived from animal-based systems. However, both methods possess certain limitations. The RPT demonstrates lower reproducibility and a higher false-positive rate compared to the LAL assay; in contrast, the LAL test is limited to detecting endotoxins from Gram-negative organisms. The Monocyte Activation Test (MAT), which is based on monocytes, has emerged as the most promising alternative to traditional pyrogen detection methods. However, the MAT is operationally complex, time-consuming, and exhibits significant individual variability; it typically requires a substantial volume of human blood, thereby considerably limiting its practical application. To overcome these limitations, we developed a rapid pyrogen detection method based on monocytes derived from human embryonic stem cells (hESCs), integrated with a luciferase reporter system. By leveraging the stability and pluripotency of hESCs, we can obtain numerous hESC-derived monocytes (hESC-Mono) through directed in vitro differentiation. These hESC-Mono exhibit batch-to-batch consistency and closely resemble peripheral blood monocytes in functionality. We have demonstrated that hESC-Mono possess sensitive reporting capabilities for at least three types of pyrogens: lipopolysaccharide (LPS), a major component of the cell wall of Gram-negative bacteria; lipoteichoic acid (LTA), a key constituent of the cell wall of Gram-positive bacteria; and fungal Zymosan. Furthermore, we have confirmed that hESC-Mono maintain stable expression of Toll-like receptors, which are among the primary determinants of the reliable detection of pyrogens. Consequently, we have successfully developed and validated a novel pyrogen detection method based on the integration of an in vitro hPSC-directed differentiation system with a luciferase-based reporter assay, thereby offering a promising alternative to traditional pyrogen detection methods.

Background: Pancreatic cancer is notorious for its aggressive behavior and poor prognosis, largely due to delayed diagnosis and early dissemination. Identifying reliable serum biomarkers could enhance early detection and risk stratification. Long non-coding RNAs (lncRNAs) have been recognised as regulators of tumour biology; however, the clinical and biochemical relevance of lncRNA DRAIC in pancreatic cancer remains unclear. Methods: Serum samples were obtained from 100 patients diagnosed with pathologically confirmed pancreatic cancer and 100 ageand sex-matched controls. Quantitative real-time polymerase chain reaction (qPCR) was employed to quantify the circulating DRAIC expression levels. We analysed the associations between serum DRAIC levels and clinicopathological characteristics, including vascular involvement and lymph node metastasis. Receiver operating characteristic (ROC) curve analyses were performed to assess the diagnostic and predictive performance of DRAIC. Paired serum samples obtained before and after surgical resection were additionally analysed. Results: Serum DRAIC expression was significantly elevated in patients with pancreatic cancer compared with controls. ROC analysis demonstrated robust diagnostic potential (AUC = 0.943). Elevated DRAIC levels were significantly associated with lymph node metastasis and vascular involvement. DRAIC showed strong predictive performance for lymph node metastasis (AUC = 0.906), whereas its predictive value for vascular involvement was moderate. Notably, serum DRAIC levels were significantly reduced following surgical resection. Conclusion: Circulating lncRNA DRAIC is markedly dysregulated in pancreatic cancer and demonstrates potential utility as a serum-based biochemical biomarker, particularly for evaluating lymph node metastasis. These findings provide a basis for further mechanistic studies and validation in larger cohorts.

Currently, no targeted therapy exists for idiopathic pulmonary fibrosis (IPF). The hallmark pathological feature of excessive extracellular matrix (ECM) deposition severely undermines the efficacy of mesenchymal stem cell (MSC)-based treatments. While existing MSC therapeutic strategies primarily focus on modulating inflammation in early stages, they have not yet established precise interventions addressing the core pathological mechanism-ECM dysregulation. Previous studies demonstrated the therapeutic potential of human embryonic stem cell (hESCs)-derived immunity-and-matrix-regulatory cells (IMRCs) in lung injury and fibrosis models. However, the critical biomarkers and underlying mechanisms mediating IMRCs' efficacy in IPF remain poorly understood. In this study, we generated MMP1 knockout IMRCs (IMRCs-MMP1 KO) using CRISPR-based gene editing. We then characterized whether MMP1 ablation affected key properties of IMRCs, including cell morphology, proliferation, migration, marker protein expression, transcriptomic profile, and cytokine secretion. Subsequently, the ability of IMRCs-MMP1 KO to degrade collagen was tested using in vivo and in vitro pulmonary fibrosis models. MMP1 knockout was successfully achieved and did not compromise typical IMRC characteristics or impair their immunomodulatory capacity. However, MMP1 deficiency significantly attenuated the ability of IMRCs to degrade TGF-β1-induced collagen I deposition in A549 cells. Importantly, wild-type IMRCs demonstrated superior therapeutic efficacy in ameliorating bleomycin-induced lung injury and fibrosis in mice compared with IMRCs-MMP1 KO. Furthermore, IMRCs exhibited significantly greater capability to directly degrade the pericellular collagen I and modulate fibroblasts' activation progression within fibrotic lung tissues in a MMP1-dependent manner. In summary, our data establish that MMP1 plays an essential functional role in IMRC-mediated attenuation of PF. MMP1 thus represents a key therapeutic biomarker for IMRC-based treatment. This work provides a foundation for developing stem cell therapies tailored to the pathological features of IPF, potentially enabling adaptive treatment strategies.

Stem cell-derived extracellular vehicles (EVs) hold great therapeutic potential for myocardial infarction (MI). However, the efficient production of EVs with high bioactivity remains a critical bottleneck limiting their clinical translation. Here, we demonstrate that conditioned photobiomodulation (PBM) with green light is capable of activating human embryonic stem cells (hESCs) to secrete more EVs with superior cardioprotective activity. These PBM-reprogrammed hESC-EVs improve cardiac recovery in a murine MI model by promoting cardiomyocyte proliferation and angiogenesis while inhibiting apoptosis. Notably, we validate that these EVs similarly enhance the survival and proliferation of human cardiomyocytes, underscoring their translational potential. Further analysis reveals that this benefit is due to the higher miR-423-3p content in reprogrammed hESC-EVs, which enhances glycolytic metabolism and restores mitochondrial function by regulating the ZBTB7A/PKM2 axis. Moreover, we synthesize a methacryloyl hydrogel microneedle patch with superior biocompatibility, biodegradability, and mechanical strength for loading hESC-EVs, and convey the patch to the infarcted heart via a modified delivery apparatus. This system ensures the precise and sustained delivery of EVs to ischemic myocardium, offering a potent treatment for MI. Collectively, this optical and biomaterials-based approach efficiently prepares EVs with higher cardioprotective activity, providing new therapeutic strategies for heart disease.

Clearance of aberrant cerebral amyloid-β (Aβ) deposits represents a promising therapeutic strategy for Alzheimer's disease (AD), yet current anti-Aβ immunotherapy raises safety concerns due to frequent adverse effects. Extracellular targeted protein degradation (eTPD) offers an approach for safe and efficient clearance of disease-causing proteins. Here, we develop a next-generation eTPD platform, synthetic peptide-programmed lysosome-targeting chimeras (SPYTACs), using entirely synthesized bispecific peptides. Leveraging low-density lipoprotein receptor-related protein 1 (LRP1), SPYTACs effectively facilitate targeted degradation of extracellular proteins and enable transcytosis across the blood-brain barrier. In vivo administration of SPYTACs effectively reduces peripheral and cerebral Aβ burden, attenuates synapse loss, and improves cognitive function in 5×FAD mice at both prodromal and symptomatic stages. Notably, SPYTAC treatment shows fewer side effects, including intracerebral hemorrhage and inflammation, compared with conventional immunotherapies. The high modularity and genetic encodability enable SPYTACs to target customized disease-causing proteins, underscoring their therapeutic versatility and translational promise across diverse diseases driven by pathogenic proteins.

Gene therapy is evolving from gene addition to precise genome editing, enabling the direct correction of disease-causing mutations. Breakthrough technologies, such as clustered regularly interspaced short palindromic repeats-CRISPR-associated protein (CRISPR-Cas) nucleases, base editors, prime editors, and CRISPR-associated transposases are reshaping the therapeutic landscape. This review covers the progression of precision editing technologies and their clinical applications, spanning from ex vivo therapies to in vivo treatments targeting vital organs. The rise of personalized medicine, highlighted by therapies, such as carbamoyl phosphate synthetase 1 editing, underscores the shift toward N-of-1 medicine for rare diseases. Clinical trial progress, delivery and accessibility challenges, and the role of AI in optimizing editing tools and predicting outcomes are also discussed. These innovations are transforming genetic medicine, offering the promise of safer, more durable, and personalized cures.

Mammalian synthetic biology holds great promise for treating complex diseases but faces challenges such as functional leakage and imprecise control dynamics. The advent of synthetic promoters (synPs) and transcription factors (synTFs) has expanded the genetic toolkit, and their coordinated integration enables precise, intelligent, and multidimensional regulation. Advances in promoter engineering and modular synTF design, aided by artificial intelligence, have shifted the field from empirical, trial-and-error discovery to rational, predictive design. This progress has facilitated the construction of synthetic circuits that integrate multiple endogenous and exogenous inputs through logic gates, feedback loops, and tunable systems. Such innovations support dynamic, spatiotemporally precise control, enhancing therapeutic precision and reducing off-target effects. By addressing key translational requirements, including multi-input sensing, tunable expression, and high orthogonality, integrated synP-synTF systems are advancing sophisticated mammalian therapeutics. This review summarizes progress in engineered and integrated systems, highlighting dynamic regulatory strategies and their therapeutic applications.

Cryopreservation-integrated bioprinting represents a promising approach for tissue regeneration by combining cell-laden bioink freezing with direct post-thaw printing, bypassing traditional culturing steps. However, key challenges remain: ice crystallization compromises cellular viability, while hydrogel structural integrity deteriorates, impairing printability. We present a biphasic bioink platform for cryopreservation-enabled three-dimensional (3D) bioprinting-CAMP (Cryopreservation for Adhesion and Maintenance Printing), which enables direct 3D printing at 4-8°C post liquid nitrogen storage (-196°C). CAMP inhibits ice recrystallization through hydrogen bond-mediated water immobilization, achieving approximately 80% cell viability without the use of toxic cryoprotectants. Cryopreserved cells in the bioink retained focal adhesions and increased phosphorylated FAK expression, and the bioink exhibited approximately ten fold higher ice recrystallization inhibition than phosphate-buffered saline. Mechanistically, CAMP suppressed cell death via phospho-FAK signaling. In vivo evaluation using a rat femoral defect model demonstrated the therapeutic efficacy of CAMP, with cryopreserved constructs promoting complete bone regeneration within three months. CAMP overcomes the key limitations of conventional biofabrication by combining cell cryopreservation, bioprinting, and functional tissue formation into a single workflow. By bridging cryopreservation and bioprinting, CAMP represents a significant advance toward clinically viable, ready-to-implant engineered tissues.

Clearance of aberrant cerebral amyloid-D (AD) deposits represents a promising therapeutic strategy for Alzheimer's disease (AD), yet current anti-AD immunotherapy raises safety concerns due to frequent adverse effects. Extracellular targeted protein degradation (eTPD) offers an approach for safe and efficient clearance of disease-causing proteins. Here, we develop a next-generation eTPD platform, synthetic peptide-programmed lysosome-targeting chimeras (SPYTACs), using entirely synthesized bispecific peptides. Leveraging low-density lipoprotein receptor-related protein 1 (LRP1), SPYTACs effectively facilitate targeted degradation of extracellular proteins and enable transcytosis across the blood-brain barrier. In vivo administration of SPYTACs effectively reduces peripheral and cerebral AD burden, attenuates synapse loss, and improves cognitive function in 5 & times;FAD mice at both prodromal and symptomatic stages. Notably, SPYTAC treatment shows fewer side effects, including intracerebral hemorrhage and inflammation, compared with conventional immunotherapies. The high modularity and genetic encodability enable SPYTACs to target customized disease-causing proteins, underscoring their therapeutic versatility and translational promise across diverse diseases driven by pathogenic proteins.

Totipotency is the first cell fate emerged from fertilization, but remains poorly understood at the molecular level. Totipotent blastomeres are characterized by the presence of topologically associating domains (TADs) with significantly weakened structural integrity. In this study, we performed high-resolution 3D genome architecture profiling of two established mouse totipotent-like models, chemically induced totipotent stem cells and totipotent blastomere-like cells, and revealed that both largely retained TADs found in inner cell mass /embryonic stem cells. Yet, amid the apparent TAD conservation, TAD strength was indeed considerably weakened upon the acquisition of totipotency. Integrative analysis of epigenetic and Hi-C data revealed that pluripotency genes underwent coordinated epigenetic landscape remodeling and 3D contact reorganization, which collectively drove pluripotency silencing. Epigenetic remodeling was also observed at totipotency gene loci. This work represents the first systematic effort to benchmark totipotency models at the 3D genome level and provides a framework to establish totipotency through 3D genome folding.

Why eukaryotic genomes are universally divided among multiple chromosomes remains an unresolved question. Although yeast and mouse cells can tolerate chromosomal fusions without impairing viability, we show here that chromosome length in mammalian cells is constrained by a biophysical limit governed by spindle geometry. Using engineered mouse cells carrying fused chromosomes of defined sizes, we identify 308 Mb as the maximal length tolerated for faithful mitosis. Chromosomes exceeding this threshold disrupt segregation, leading to daughter cell re-coalescence and polyploidization. Aurora B kinase regulates this process by modulating spindle elongation; its inhibition induces mitotic failure even in chromosome configurations within the tolerated threshold of 308 Mb. These findings explain the structural basis for genome fragmentation in animals and reveal a general mechanism linking chromosome size, spindle dynamics, and genome stability. Engineered chromosome fusions reveal a biophysical limit set by spindle geometry to constrain chromosome length, explaining the structural basis for genome fragmentation in mammals. Engineered chromosome fusions reveal a biophysical limit set by spindle geometry to constrain chromosome length, explaining the structural basis for genome fragmentation in mammals.

The development of sensitive, accurate, and multimodal approaches for the detection of viral gene fragments and the diagnosis of infections is essential for effective pandemic management across various contexts. This study introduces a CRISPR switch integrated with strand displacement amplification (SDA) for the binary channel detection of SARS-CoV-2 gene fragments and the diagnosis of SARS-CoV-2 infections. In the conventional channel, a specific single gene fragment can directly facilitate the formation of a three-way junction, thereby initiating the SDA process and resulting in the production of a substantial amount of single-stranded DNA. In the logical channel, two gene fragments can first induce the release of a substitute, which subsequently leads to the formation of the three-way junction and the ensuing SDA process. The single-stranded SDA product acts as the target sequence that activates the CRISPR switch, which performs reporter cleavage functions, thereby generating enhanced and detectable fluorescence signals. This method achieves sensitive and selective detection of SARS-CoV-2 gene fragments, with limits of detection (LODs) of 1.0 aM for the ORF1ab gene and 0.9 aM for the N gene in the conventional channel and 3.7 aM for simultaneous detection of both ORF1ab and N in the logical channel. Furthermore, accurate detection of these gene fragments in real samples obtained from patients exhibiting upper respiratory symptoms was successfully conducted, along with the corresponding diagnosis of SARS-CoV-2 infections. Consequently, this method represents a novel binary channel approach for viral gene detection and holds significant promise for clinical diagnosis and potential future epidemic control.

Spinal cord repair demands biomaterials that replicate the aligned axonal architecture and mechanical softness of native tissue. However, most current scaffolds fail to support three-dimensional alignment and neuronal differentiation of human neural stem cells (hNSCs) in hydrated, low-stiffness environments. Here, we present NEAT (nanoengineered extrusion-aligned tract), a shear-stress-driven 3D bioprinting strategy that utilizes norbornene-functionalized collagen (NorCol) to generate highly aligned, mechanically stable hydrogels without post-processing. NEAT preserves the native triple-helical structure of collagen, supports hierarchical fibrillar organization, and enables rapid photopolymerization for long-term culture (>8 weeks). When encapsulated in NEAT constructs, human NSCs exhibited enhanced alignment and accelerated neuronal differentiation, guided by the optimized fibrillar architecture. In a rat model of complete spinal cord transection, NEAT implants promoted robust axonal reconnection, synapse formation, and significant functional locomotor recovery. This strategy bridges topographical control, cellular programming, and functional integration, providing a powerful platform for neural tissue engineering and spinal cord regeneration.

Non-viral targeted integration of large DNA cargoes into human primary T cells typically requires the induction of genomic double-strand breaks (DSBs), a process associated with cytotoxicity and potential tumorigenic chromosomal abnormalities. Here we report PRIME-In, a novel genome-editing platform that uses a prime editing-engineered donor template coupled with either single (PRIME-In 1.0) or paired (PRIME-In 2.0) genomic nicks to enable precise integration of substantial DNA payloads into human cells without reliance on DSB repair pathways. Compared with traditional DSB-dependent methods, PRIME-In demonstrates markedly enhanced editing efficiency and specificity while eliminating detectable on-target and off-target chromosomal aberrations. Subsequent refinement of reagent composition and delivery protocols enabled PRIME-In-mediated engineering of primary human T cells with minimal toxicity, achieving up to 50% integration efficiency for a 3-kb CAR construct. These advances establish PRIME-In as a transformative platform for streamlining the non-viral production of genome-edited T cells, offering substantial potential for T cell-based immunotherapies.

The functional integrity of a mammalian chromosome is shaped by its long-term, co-evolution with species-specific nuclear environment. How chromosomes co-adapt with their native environment to define 3D architecture and transcriptional activity remain poorly understood, largely due to a lack of experimental models capable of systematically dissecting this co-evolution relationship. Here, we report a cross-species chromosome substitution (CROSS) method, a robust genomic engineering method that enables the stable, scarless replacement of host chromosomes with evolutionarily divergent orthologs. By integrating microcell-mediated chromosome transfer with CRISPR/Cas9, we imported the intact 158-Mb rat X chromosome into mouse embryonic stem cells, and subsequently achieved targeted substitution of its endogenous mouse counterpart, maintaining stability and integrity. Using this model, we found that rat-specific LINE1 and RatSatRep2 repeats failed to adequately recruit host SETDB1 in mouse cells, leading to localized erosion of H3K9me3 heterochromatin. This further triggered 3D structural remodeling, characterized by the de novo formation of topologically associating domain (TAD) boundaries that aberrantly activated adjacent genes—including Rhox5 , the master regulator of the Rhox cluster—impairing cellular differentiation. Our method provides a powerful chromosome engineering platform for dissecting how genomic sequences and epigenetic mechanisms cooperate in regulating chromosome architecture and function, and for evaluating the structural and functional fidelity of large-scale synthetic or heterologous DNA across species.