Ming Wai Lau Centre for Reparative Medicine

Nature Communications 7: Article number: 10774 (2016); Published: 8 March 2016; Updated: 19 July 2016. The affiliation details for Boon-Seng Soh, Lei Bu and Ronald A. Li are incorrect in this Article. The correct addresses of these authors are listed below: Boon-Seng Soh Cardiovascular Research Center, Massachusetts General Hospital, 185 Cambridge Street, Boston, Massachusetts 02114, USA; Department of Stem Cell and Regenerative Biology, Harvard University, 7 Divinity Avenue, Cambridge, Massachusetts 02138, USA; Li Dak-Sum Research Centre-HKU-Karolinska Institutet Collaboration on Regenerative Medicine, University of Hong Kong, Hong Kong, China and Department of Cell and Molecular Biology and Medicine, Karolinska Institute, Stockholm S-171 77, Sweden.

Fluorescence imaging in near-infrared IIb (NIR-IIb, 1500-1700 nm) spectrum holds a great promise for tissue imaging. While few inorganic NIR-IIb fluorescent probes have been reported, their organic counterparts are still rarely developed, possibly due to the shortage of efficient materials with long emission wavelength. Herein, we propose a molecular design philosophy to explore pure organic NIR-IIb fluorophores by manipulation of the effects of twisted intramolecular charge transfer and aggregation-induced emission at the molecular and morphological levels. An organic fluorescent dye emitting up to 1600 nm with a quantum yield of 11.5% in the NIR-II region is developed. NIR-IIb fluorescence imaging of blood vessels and deeply-located intestinal tract of live mice based on organic dyes is achieved with high clarity and enhanced signal-to-background ratio. We hope this study will inspire further development on the evolution of pure organic NIR-IIb dyes for bio-imaging.

. However, current methods capture only one layer of omics information at a time, precluding the possibility of examining the mechanistic relationship across the central dogma of molecular biology. Here, we present two technologies for spatially resolved, genome-wide, joint profiling of the epigenome and transcriptome by cosequencing chromatin accessibility and gene expression, or histone modifications (H3K27me3, H3K27ac or H3K4me3) and gene expression on the same tissue section at near-single-cell resolution. These were applied to embryonic and juvenile mouse brain, as well as adult human brain, to map how epigenetic mechanisms control transcriptional phenotype and cell dynamics in tissue. Although highly concordant tissue features were identified by either spatial epigenome or spatial transcriptome we also observed distinct patterns, suggesting their differential roles in defining cell states. Linking epigenome to transcriptome pixel by pixel allows the uncovering of new insights in spatial epigenetic priming, differentiation and gene regulation within the tissue architecture. These technologies are of great interest in life science and biomedical research.

The human ovary orchestrates sex hormone production and undergoes monthly structural changes to release mature oocytes. The outer lining of the ovary (cortex) has a key role in defining fertility in women as it harbors the ovarian reserve. It has been postulated that putative oogonial stem cells exist in the ovarian cortex and that these can be captured by DDX4 antibody isolation. Here, we report single-cell transcriptomes and cell surface antigen profiles of over 24,000 cells from high quality ovarian cortex samples from 21 patients. Our data identify transcriptional profiles of six main cell types; oocytes, granulosa cells, immune cells, endothelial cells, perivascular cells, and stromal cells. Cells captured by DDX4 antibody are perivascular cells, not oogonial stem cells. Our data do not support the existence of germline stem cells in adult human ovaries, thereby reinforcing the dogma of a limited ovarian reserve.

Abstract Cellular function in tissue is dependent on the local environment, requiring new methods for spatial mapping of biomolecules and cells in the tissue context 1 . The emergence of spatial transcriptomics has enabled genome-scale gene expression mapping 2–5 , but the ability to capture spatial epigenetic information of tissue at the cellular level and genome scale is lacking. Here we describe a method for spatially resolved chromatin accessibility profiling of tissue sections using next-generation sequencing (spatial-ATAC-seq) by combining in situ Tn5 transposition chemistry 6 and microfluidic deterministic barcoding 5 . Profiling mouse embryos using spatial-ATAC-seq delineated tissue-region-specific epigenetic landscapes and identified gene regulators involved in the development of the central nervous system. Mapping the accessible genome in the mouse and human brain revealed the intricate arealization of brain regions. Applying spatial-ATAC-seq to tonsil tissue resolved the spatially distinct organization of immune cell types and states in lymphoid follicles and extrafollicular zones. This technology progresses spatial biology by enabling spatially resolved chromatin accessibility profiling to improve our understanding of cell identity, cell state and cell fate decision in relation to epigenetic underpinnings in development and disease.

Restriction of intramolecular motion (RIM) is the widely-accpeted mechanism of aggregation-induced emission (AIE). In this concise and comprehensive perspective, four mechanistic models related to different nonradiative pathways are summarized with examples to disclose the connotation of RIM, and meaningful mechanistic topics for future researches are advised.

Fluorescence imaging in the second near-infrared window (NIR-II, 1000-1700 nm) using small-molecule dyes has high potential for clinical use. However, many NIR-II dyes suffer from the emission quenching effect and extremely low quantum yields (QYs) in the practical usage forms. The AIE strategy has been successfully utilized to develop NIR-II dyes with donor-acceptor (D-A) structures with acceptable QYs in the aggregate state, but there is still large room for QY improvement. Here, we rationally designed a NIR-II emissive dye named TPE-BBT and its derivative (TPEO-BBT) by changing the electron-donating triphenylamine unit to tetraphenylethylene (TPE). Their nanoparticles exhibited ultrahigh relative QYs of 31.5% and 23.9% in water, respectively. By using an integrating sphere, the absolute QY of TPE-BBT nanoparticles was measured to be 1.8% in water. Its crystals showed an absolute QY of 10.4%, which is the highest value among organic small molecules reported so far. The optimized D-A interaction and the higher rigidity of TPE-BBT in the aggregate state are believed to be the two key factors for its ultrahigh QY. Finally, we utilized TPE-BBT for NIR-II photoluminescence (PL) and chemiluminescence (CL) bioimaging through successive CL resonance energy transfer and Förster resonance energy transfer processes. The ultrahigh QY of TPE-BBT realized an excellent PL imaging quality in mouse blood vessels and an excellent CL imaging quality in the local arthrosis inflammation in mice with a high signal-to-background ratio of 130. Thus, the design strategy presented here brings new possibilities for the development of bright NIR-II dyes and NIR-II bioimaging technologies.

Mammalian cells autonomously activate hypoxia-inducible transcription factors (HIFs) to ensure survival in low-oxygen environments. We report here that injury-induced hypoxia is insufficient to trigger HIF1α in damaged epithelium. Instead, multimodal single-cell and spatial transcriptomics analyses and functional studies reveal that retinoic acid–related orphan receptor γt + (RORγt + ) γδ T cell–derived interleukin-17A (IL-17A) is necessary and sufficient to activate HIF1α. Protein kinase B (AKT) and extracellular signal–regulated kinase 1/2 (ERK1/2) signaling proximal of IL-17 receptor C (IL-17RC) activates mammalian target of rapamycin (mTOR) and consequently HIF1α. The IL-17A–HIF1α axis drives glycolysis in wound front epithelia. Epithelial-specific loss of IL-17RC, HIF1α, or blockade of glycolysis derails repair. Our findings underscore the coupling of inflammatory, metabolic, and migratory programs to expedite epithelial healing and illuminate the immune cell–derived inputs in cellular adaptation to hypoxic stress during repair.

Fibrotic scar tissue limits central nervous system regeneration in adult mammals. The extent of fibrotic tissue generation and distribution of stromal cells across different lesions in the brain and spinal cord has not been systematically investigated in mice and humans. Furthermore, it is unknown whether scar-forming stromal cells have the same origin throughout the central nervous system and in different types of lesions. In the current study, we compared fibrotic scarring in human pathological tissue and corresponding mouse models of penetrating and non-penetrating spinal cord injury, traumatic brain injury, ischemic stroke, multiple sclerosis and glioblastoma. We show that the extent and distribution of stromal cells are specific to the type of lesion and, in most cases, similar between mice and humans. Employing in vivo lineage tracing, we report that in all mouse models that develop fibrotic tissue, the primary source of scar-forming fibroblasts is a discrete subset of perivascular cells, termed type A pericytes. Perivascular cells with a type A pericyte marker profile also exist in the human brain and spinal cord. We uncover type A pericyte-derived fibrosis as a conserved mechanism that may be explored as a therapeutic target to improve recovery after central nervous system lesions.

Hydrogels that are self-assembled by peptides have attracted great interest for biomedical applications. However, the link between chemical structures of peptides and their corresponding hydrogel properties is still unclear. Here, we showed a combinational approach to generate a structurally diverse hydrogel library with more than 2,000 peptides and evaluated their corresponding properties. We used a quantitative structure-property relationship to calculate their chemical features reflecting the topological and physicochemical properties, and applied machine learning to predict the self-assembly behavior. We observed that the stiffness of hydrogels is correlated with the diameter and cross-linking degree of the nanofiber. Importantly, we demonstrated that the hydrogels support cell proliferation in culture, suggesting the biocompatibility of the hydrogel. The combinatorial hydrogel library and the machine learning approach we developed linked the chemical structures with their self-assembly behavior and can accelerate the design of novel peptide structures for biomedical use.

Mature oligodendrocytes (MOLs) show transcriptional heterogeneity, the functional consequences of which are unclear. MOL heterogeneity might correlate with the local environment or their interactions with different neuron types. Here, we show that distinct MOL populations have spatial preference in the mammalian central nervous system (CNS). We found that MOL type 2 (MOL2) is enriched in the spinal cord when compared to the brain, while MOL types 5 and 6 (MOL5/6) increase their contribution to the OL lineage with age in all analyzed regions. MOL2 and MOL5/6 also have distinct spatial preference in the spinal cord regions where motor and sensory tracts run. OL progenitor cells (OPCs) are not specified into distinct MOL populations during development, excluding a major contribution of OPC intrinsic mechanisms determining MOL heterogeneity. In disease, MOL2 and MOL5/6 present different susceptibility during the chronic phase following traumatic spinal cord injury. Our results demonstrate that the distinct MOL populations have different spatial preference and different responses to disease.

Abstract Near‐infrared (NIR) chemiluminescence (CL) emission is highly favorable for deep‐tissue imaging, but chemically conjugated NIR CL emitters with the aggregation‐induced emission (AIE) property for biotechnology are seldom reported. Herein, an AIE‐active NIR CL emitter, TBL, is synthesized by conjugating luminol unit with electron‐accepting benzothiadiazole and an electron‐donating triphenylamine, and subsequently TBL dots are prepared by using F127 as the surfactant. The CL emission of TBL dots can last continuously for over 60 min and can be employed for quantitative (in vitro) and qualitative (in vivo) detection of 1 O 2 . Strikingly, the NIR CL emission can penetrate through tissues with a total thickness of over 3 cm, exhibiting significantly better performance than NIR fluorescence emission and blue CL emission. Moreover, the successful differentiation of tumor and normal tissues by TBL‐based CL imaging in vivo also paves the way for CL‐guided cancer diagnosis and surgery.

Cardiotoxicity is one of the most serious side effects of cancer chemotherapy. Current approaches to monitoring of chemotherapy-induced cardiotoxicity (CIC) as well as model systems that develop in vivo or in vitro CIC platforms fail to notice early signs of CIC. Moreover, breast cancer (BC) patients with preexisting cardiac dysfunctions may lead to different incident levels of CIC. Here, a model is presented for investigating CIC where not only induced pluripotent stem cell (iPSC)-derived cardiac tissues are interacted with BC tissues on a dual-organ platform, but electrochemical immuno-aptasensors can also monitor cell-secreted multiple biomarkers. Fibrotic stages of iPSC-derived cardiac tissues are promoted with a supplement of transforming growth factor-β 1 to assess the differential functionality in healthy and fibrotic cardiac tissues after treatment with doxorubicin (DOX). The production trend of biomarkers evaluated by using the immuno-aptasensors well-matches the outcomes from conventional enzyme-linked immunosorbent assay, demonstrating the accuracy of the authors' sensing platform with much higher sensitivity and lower detection limits for early monitoring of CIC and BC progression. Furthermore, the versatility of this platform is demonstrated by applying a nanoparticle-based DOX-delivery system. The proposed platform would potentially help allow early detection and prediction of CIC in individual patients in the future.

Probing histone modifications at a single-cell level in thousands of cells has been enabled by technologies such as single-cell CUT&Tag. Here we describe nano-CUT&Tag (nano-CT), which allows simultaneous mapping of up to three epigenomic modalities at single-cell resolution using nanobody-Tn5 fusion proteins. Multimodal nano-CT is compatible with starting materials as low as 25,000-200,000 cells and has significantly higher sensitivity and number of fragments per cell than single-cell CUT&Tag. We use nano-CT to simultaneously profile chromatin accessibility, H3K27ac, and H3K27me3 in juvenile mouse brain, allowing for discrimination of more cell types and states than unimodal single-cell CUT&Tag. We also infer chromatin velocity between assay for transposase-accessible chromatin (ATAC) and H3K27ac in the oligodendrocyte lineage and deconvolute H3K27me3 repressive states, finding two sequential waves of H3K27me3 repression at distinct gene modules during oligodendrocyte lineage progression. Given its high resolution, versatility, and multimodal features, nano-CT allows unique insights in epigenetic landscapes in complex biological systems at the single-cell level.

Abstract Silver staining, which exploits the special bioaffinity and the chromogenic reduction of silver ions, is an indispensable visualization method in biology. It is a most popular method for in‐gel protein detection. However, it is limited by run‐to‐run variability, background staining, inability for protein quantification, and limited compatibility with mass spectroscopic (MS) analysis; limitations that are largely attributed to the tricky chromogenic visualization. Herein, we reported a novel water‐soluble fluorogenic Ag + probe, the sensing mechanism of which is based on an aggregation‐induced emission (AIE) process driven by tetrazolate‐Ag + interactions. The fluorogenic sensing can substitute the chromogenic reaction, leading to a new fluorescence silver staining method. This new staining method offers sensitive detection of total proteins in polyacrylamide gels with a broad linear dynamic range and robust operations that rival the silver nitrate stain and the best fluorescent stains.

Protein is one of the four most important biomacromolecules in living systems. The detection, quantification, localization, and characterization of proteins is essential for an understanding of biological fundamentals, as well as for the diagnostics and treatment of protein-related diseases. By using intrinsic and extrinsic fluorescence, different techniques have been established to study proteins, many of which are now being routinely used in research laboratories and clinics. This review summarizes the applications of aggregation-induced emission (AIE) fluorescence in protein science. In contrast to traditional fluorescent dyes, the activation of AIE dyes is mainly attributed to the restriction of intramolecular motions. This unique turn-on mechanism of AIE dyes allows researchers to develop novel fluorogenic strategies for sensitive, selective, and reliable analysis of proteins. This review focuses on introducing AIE strategies for 1) detection, localization, and quantification of proteins; 2) probing polymer conformational transitions of proteins; 3) characterization of protein-ligand interactions; and 4) evaluation of enzyme activities. Perspectives and challenges with respect to this emerging field of protein characterization are offered.

Proprioceptive neurons (PNs) are essential for the proper execution of all our movements by providing muscle sensory feedback to the central motor network. Here, using deep single cell RNAseq of adult PNs coupled with virus and genetic tracings, we molecularly identify three main types of PNs (Ia, Ib and II) and find that they segregate into eight distinct subgroups. Our data unveil a highly sophisticated organization of PNs into discrete sensory input channels with distinct spatial distribution, innervation patterns and molecular profiles. Altogether, these features contribute to finely regulate proprioception during complex motor behavior. Moreover, while Ib- and II-PN subtypes are specified around birth, Ia-PN subtypes diversify later in life along with increased motor activity. We also show Ia-PNs plasticity following exercise training, suggesting Ia-PNs are important players in adaptive proprioceptive function in adult mice.

Stem cell-based embryo models offer unprecedented experimental tools for studying early human development. The usefulness of embryo models hinges on their molecular, cellular and structural fidelities to their in vivo counterparts. To authenticate human embryo models, single-cell RNA sequencing has been utilized for unbiased transcriptional profiling. However, an organized and integrated human single-cell RNA-sequencing dataset, serving as a universal reference for benchmarking human embryo models, remains unavailable. Here we developed such a reference through the integration of six published human datasets covering development from the zygote to the gastrula. Lineage annotations are contrasted and validated with available human and nonhuman primate datasets. Using stabilized Uniform Manifold Approximation and Projection, we constructed an early embryogenesis prediction tool, where query datasets can be projected on the reference and annotated with predicted cell identities. Using this reference tool, we examined published human embryo models, highlighting the risk of misannotation when relevant references are not utilized for benchmarking and authentication.

COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2, has resulted in global social and economic disruption, putting the world economy to the largest global recession since the Great Depression. To control the spread of COVID-19, cutting off the transmission route is a critical step. In this work, the efficient inactivation of human coronavirus with photodynamic therapy (PDT) by employing photosensitizers with aggregation-induced emission characteristics (DTTPB) is reported. DTTPB is designed to bear a hydrophilic head and two hydrophobic tails, mimicking the structure of phospholipids on biological membranes. DTTPB demonstrates a broad absorption band covering the whole visible light range and high molar absorptivity, as well as excellent reactive oxygen species sensitizing ability, making it an excellent candidate for PDT. Besides, DTTPB can target membrane structure, and bind to the envelope of human coronaviruses. Upon light irradiation, DTTPB demonstrates highly effective antiviral behavior: human coronavirus treated with DTTPB and white-light irradiation can be efficiently inactivated with complete loss of infectivity, as revealed by the significant decrease of virus RNA and proteins in host cells. Thus, DTTPB sensitized PDT can efficiently prevent the infection and the spread of human coronavirus, which provides a new avenue for photodynamic combating of COVID-19.

Abstract Different types of spiral ganglion neurons (SGNs) are essential for auditory perception by transmitting complex auditory information from hair cells (HCs) to the brain. Here, we use deep, single cell transcriptomics to study the molecular mechanisms that govern their identity and organization in mice. We identify a core set of temporally patterned genes and gene regulatory networks that may contribute to the diversification of SGNs through sequential binary decisions and demonstrate a role for NEUROD1 in driving specification of a I c -SGN phenotype. We also find that each trajectory of the decision tree is defined by initial co-expression of alternative subtype molecular controls followed by gradual shifts toward cell fate resolution. Finally, analysis of both developing SGN and HC types reveals cell-cell signaling potentially playing a role in the differentiation of SGNs. Our results indicate that SGN identities are drafted prior to birth and reveal molecular principles that shape their differentiation and will facilitate studies of their development, physiology, and dysfunction.