Genetically modified pigs:Emerging animal models for hereditary hearing loss

Hereditary hearing loss(HHL),a genetic disorder that impairs auditory function,significantly affects quality of life and incurs substantial economic losses for society.To investigate the underlying causes of HHL and evaluate therapeutic outcomes,appropriate animal models are necessary.Pigs have been extensively used as valuable large animal models in biomedical research.In this review,we highlight the advantages of pig models in terms of ear anatomy,inner ear morphology,and electrophysiological characteristics,as well as recent advancements in the development of distinct genetically modified porcine models of hearing loss.Additionally,we discuss the prospects,challenges,and recommendations regarding the use pig models in HHL research.Overall,this review provides insights and perspectives for future studies on HHL using porcine models.

Stem Cell-Based Hair Cell Regeneration and Therapy in the Inner Ear

Hearing loss has become increasingly prevalent and causes considerable disability,thus gravely burdening the global economy.Irreversible loss of hair cells is a main cause of sensorineural hearing loss,and currently,the only relatively effective clinical treatments are limited to digital hearing equipment like cochlear implants and hearing aids,but these are of limited benefit in patients.It is therefore urgent to understand the mechanisms of damage repair in order to develop new neuroprotective strategies.At present,how to promote the regeneration of functional hair cells is a key scientific question in the field of hearing research.Multi-ple signaling pathways and transcriptional factors trigger the activation of hair cell progenitors and ensure the maturation of newborn hair cells,and in this article,we first review the principal mechanisms underlying hair cell reproduction.We then further discuss therapeutic strategies involving the co-regulation of multiple signaling pathways in order to induce effective functional hair cell regeneration after degeneration,and we summarize current achievements in hair cell regeneration.Lastly,we discuss potential future approaches,such as small molecule drugs and gene therapy,which might be applied for regenerating functional hair cells in the clinic.

Derivation and applications of human hepatocyte-like cells

Human hepatocyte-like cells (HLCs) derived from human pluripotent stem cells (hPSCs) promise a valuable source of cells with human genetic background, physiologically relevant liver functions, and unlimited supply. With over 10 years’ efforts in this field, great achievements have been made. HLCs have been successfully derived and applied in disease modeling, toxicity testing and drug discovery. Large cohorts of induced pluripotent stem cells-derived HLCs have been recently applied in studying population genetics and functional outputs of common genetic variants in vitro. This has offered a new paradigm for genomewide association studies and possibly in vitro pharmacogenomics in the nearly future. However, HLCs have not yet been successfully applied in bioartificial liver devices and have only displayed limited success in cell transplantation. HLCs still have an immature hepatocyte phenotype and exist as a population with great heterogeneity, and HLCs derived from different hPSC lines display variable differentiation efficiency. Therefore, continuous improvement to the quality of HLCs, deeper investigation of relevant biological processes, and proper adaptation of recent advances in cell culture platforms, genome editing technology, and bioengineering systems are required before HLCs can fulfill the needs in basic and translational research. In this review, we summarize the discoveries, achievements, and challenges in the derivation and applications of HLCs.

Immunity-and-matrix-regulatory cells enhance cartilage regeneration for meniscus injuries: a phase I dose-escalation trial

Immunity-and-matrix-regulatory cells(IMRCs)derived from human embryonic stem cells have unique abilities in modulating immunity and regulating the extracellular matrix,which could be mass-produced with stable biological properties.Despite resemblance to mesenchymal stem cells(MSCs)in terms of self-renew and tri-lineage differentiation,the ability of IMRCs to repair the meniscus and the underlying mechanism remains undetermined.Here,we showed that IMRCs demonstrated stronger immunomodulatory and pro-regenerative potential than umbilical cord MSCs when stimulated by synovial fluid from patients with meniscus injury.Following injection into the knees of rabbits with meniscal injury,IMRCs enhanced endogenous fibrocartilage regeneration.In the dose-escalating phase I clinical trial(NCT03839238)with eighteen patients recruited,we found that intra-articular IMRCs injection in patients was safe over 12 months post-grafting.Furthermore,the effective results of magnetic resonance imaging(MRI)of meniscus repair and knee functional scores suggested that 5×107 cells are optimal for meniscus injury treatment.In summary,we present the first report of a phase I clinical trial using IMRCs to treat meniscus injury.Our results demonstrated that intra-articular injection of IMRCs is a safe and effective therapy by providing a permissive niche for cartilage regeneration.

Conductive PS inverse opals for regulating proliferation and differentiation of neural stem cells

The development of neural tissue engineering has brought new hope to the treatment of spinal cord injury(SCI).Up to date,various scaffolds have been developed to induce the oriented growth and arrangement of nerves to facilitate the repair after injury.In this work,a conductive and anisotropic inverse opal substrate was presented by modifying polystyrene(PS)inverse opal films with carbon nanotubes and then stretching them to varying degrees.The film had good biocompatibility,and neural stem cells(NSCs)grown on the film displayed good orientation along the stretching direction.In addition,benefiting from the conductivity and anisotropy of the film,NSCs differentiated into neurons significantly.These results suggest that the conductive and anisotropic PS inverse opal substrates possess value in nerve tissue engineering regeneration.

Genome-edited rabbits:Unleashing the potential of a promising experimental animal model across diverse diseases

Animal models are extensively used in all aspects of biomedical research,with substantial contributions to our understanding of diseases,the development of pharmaceuticals,and the exploration of gene functions.The field of genome modification in rabbits has progressed slowly.However,recent advancements,particularly in CRISPR/Cas9-related technologies,have catalyzed the successful development of various genome-edited rabbit models to mimic diverse diseases,including cardiovascular disorders,immunodeficiencies,agingrelated ailments,neurological diseases,and ophthalmic pathologies.These models hold great promise in advancing biomedical research due to their closer physiological and biochemical resemblance to humans compared to mice.This review aims to summarize the novel gene-editing approaches currently available for rabbits and present the applications and prospects of such models in biomedicine,underscoring their impact and future potential in translational medicine.

Human ESC-derived vascular cells promote vascular regeneration in a HIF-1α dependent manner

Hypoxia-inducible factor(HIF-1α),a core transcription factor responding to changes in cellular oxygen levels,is closely associated with a wide range of physiological and pathological conditions.However,its differential impacts on vascular cell types and molecular programs modulating human vascular homeostasis and regeneration remain largely elusive.Here,we applied CRISPR/Cas9-mediated gene editing of human embryonic stem cells and directed differentiation to generate HIF-ia-deficient human vascular cells including vascular endothelial cells,vascular smooth muscle cells,and mesenchymal stem cells(MsCs),as a platform for discovering cell type-specific hypox-ia-induced response mechanisms.Through comparative molecular profiling across cell types under normoxic and hypoxic conditions,we provide insight into the indispensable role of HIF-1αin the promotion of ischemic vascular regeneration.We found human MSCs to be the vascular cell type most susceptible to HIF-1a deficiency,and that transcriptional inactivation of ANKZF1,an effector of HIF-1a,impaired pro-angiogenic processes.Altogether,our findings deepen the understanding of HIF-ia in human angiogenesis and support further explorations of novel therapeutic strategies of vascular regeneration against ischemic damage.

Dock4 is required for the maintenance of cochlear hair cells and hearing function

Auditory hair cells(HCs)are the mechanosensory receptors of the cochlea,and HC loss or malfunction can result from genetic defects.Dock4,a member of the Dock180-related protein superfamily,is a guanine nucleotide exchange factor for Rac1,and previous reports have shown that Dock4 mutations are associated with autism spectrum disorder,myelodysplastic syndromes,and tumorigenesis.Here,we found that Dock4 is highly expressed in the cochlear HCs of mice.However,the role of Dock4 in the inner ear has not yet been investigated.Taking advantage of the piggyBac transposon system,Dock4 knockdown(KD)mice were established to explore the role of Dock4 in the cochlea.Compared to wild-type controls,Dock4 KD mice showed significant hearing impairment from postnatal day 60.Dock4 KD mice showed hair bundle deficits and increased oxidative stress,which eventually led to HC apoptosis,late-onset HC loss,and progressive hearing loss.Furthermore,molecular mechanism studies showed that Rac1/β-catenin signaling was significantly downregulated in Dock4 KD cochleae and that this was the cause for the disorganized stereocilia and increased oxidative stress in HCs.Overall,our work demonstrates that the Dock4/Rac1/β-catenin signaling pathway plays a critical role in the maintenance of auditory HCs and hearing function.

An optimized prime editing system for efficient modification of the pig genome

Prime editing(PE)is a recent gene editing technology that can mediate insertions or deletions and all twelve types of base-tobase conversions.However,its low efficiency hampers the application in creating novel breeds and biomedical models,especially in pigs and other important farm animals.Here,we demonstrate that the pig genome is editable using the PE system,but the editing efficiency was quite low as expected.Therefore,we aimed to enhance PE efficiency by modulating both exogenous PE tools and endogenous pathways in porcine embryonic fibroblasts(PEFs).First,we modified the peg RNA by extending the duplex length and mutating the fourth thymine in a continuous sequence of thymine bases to cytosine,which significantly enhanced PE efficiency by improving the expression of peg RNA and targeted cleavage.Then,we targeted SAMHD1,a deoxynucleoside triphosphate triphosphohydrolase(d NTPase)that impedes the reverse transcription process in retroviruses,and found that treatment with its inhibitor,cephalosporin C zinc salt(CPC),increased PE efficiency up to 29-fold(4-fold on average),presumably by improving the reverse transcription process of Moloney murine leukemia virus reverse transcriptase(M-MLV RT)in the PE system.Moreover,PE efficiency was obviously improved by treatment with a panel of histone deacetylase inhibitors(HDACis).Among the four HDACis tested,panobinostat was the most efficient,with an efficiency up to 122-fold(7-fold on average),partly due to the considerable HDACi-mediated increase in transgene expression.In addition,the synergistic use of the three strategies further enhanced PE efficiency in PEFs.Our study provides novel approaches for optimization of the PE system and broadens the application scope of PE in agriculture and biomedicine.

Dual human iPSC-derived cardiac lineage cell-seeding extracellular matrix patches promote regeneration and long-term repair of infarcted hearts

Human pluripotent stem cell-derived cardiovascular progenitor cells (hCVPCs) and cardiomyocytes (hCMs) possess therapeutic potential for infarcted hearts;however, their efficacy needs to be enhanced. Here we tested the hypotheses that the combination of decellularized porcine small intestinal submucosal extracellular matrix (SIS-ECM) with hCVPCs, hCMs, or dual of them (Mix, 1:1) could provide better therapeutic effects than the SIS alone, and dual hCVPCs with hCMs would exert synergic effects in cardiac repair. The data showed that the SIS patch well supported the growth of hCVPCs and hCMs. Epicardially implanted SIS-hCVPC, SIS-hCM, or SIS-Mix patches at 7-day post-myocardial infarction significantly ameliorated functional worsening, ventricular dilation and scar formation at 28- and 90-day post-implantation in C57/B6 mice, whereas the SIS only mildly improved function at 90-day post-implantation. Moreover, the SIS and SIS-cell patches improved vascularization and suppressed MI-induced cardiomyocyte hypertrophy and expression of Col1 and Col3, but only the SIS-hCM and the SIS-Mix patches increased the ratio of collagen III/I fibers in the infarcted hearts. Further, the SIS-cell patches stimulated cardiomyocyte proliferation via paracrine action. Notably, the SIS-Mix had better improvements in cardiac function and structure, engraftments, and cardiomyocyte proliferation. Proteomic analysis showed distinct biological functions of exclusive proteins secreted from hCVPCs and hCMs, and more exclusive proteins secreted from co-cultivated hCVPCs and hCMs than mono-cells involving in various functional processes essential for infarct repair. These findings are the first to demonstrate the efficacy and mechanisms of mono- and dual-hCVPC- and hCM-seeding SIS-ECM for repair of infarcted hearts based on the side-by-side comparison.

肝胆肿瘤中肿瘤特异性CircRNA衍生抗原肽的鉴定

基于肿瘤抗原的免疫治疗的应用受到验证免疫原性肽稀缺性的阻碍。本研究旨在研究环状RNA(circRNA)在肝胆肿瘤类器官中作为肿瘤抗原肽新来源的潜力。使用RNA测序(RNA-seq)和基于算法的评分工具,预测3950个翻译的肿瘤特异性环状RNA在27个类器官中产生18971个抗原肽。从抗原格局来看,11个氨基酸长度(mer)肽和人白细胞抗原(HLA)-A结合肽具有最高的免疫原性相关评分。在分析的3/5类器官中,有13个预测抗原肽通过质谱(MS)免疫肽组学被直接确认为HLA-A、HLA-B和HLA-C(HLA-ABC)结合肽。在流式细胞术和酶联免疫吸附试验(ELISA)中,由HLA-ABC分子呈递的circRNA衍生的肿瘤特异性肽刺激CD8(CD8)T细胞,显示CD107a干扰素γ(IFNγ)共表达和IFNγ分泌增加。免疫原性环状RNA衍生肽诱导的靶向类器官的细胞毒性T细胞活性在杀伤实验中得到验证。值得注意的是,来自circTBC1D15的抗原肽YGFNEILKK不仅被认为是类器官的HLA-ABC呈递肽,而且还显著降低了肿瘤类器官的存活率。本研究的发现强调了产生肿瘤抗原的一个关键亚群,这对靶向肿瘤特异性circRNA具有重要意义。

A narrative review of organoids for investigating organ aging:opportunities and challenges

Aging research has shifted from studying phenotypes to studying in-depth mechanisms in recent decades.However,extrapolating cellular and molecular bases of aging from studying traditional model systems to humans has been challenging.The advent of organoids holds promise for overcoming the limitations of monolayer cell culture and bridging the gap between animal models and humans.Here,we mainly discuss recent paradigms for using organoid models in studying organ aging.Pluripotent stem cells-derived organoid provides a promising platform for simulating the pathophysiology of several aging-related diseases,especially neurodegenerative diseases,and adult stem cells organoids derived from different age groups have been applied to detect aging-related functional changes.We also assess the value of organoid model systems in understanding human aging and aging-related diseases,and identify challenges to be addressed in the future,such as the immaturity of organoids,and effective methods of inducing aging.

Exosomes from antler stem cells alleviate mesenchymal stem cell senescence and osteoarthritis

Dear Editor,Stem cell therapy holds enormous and revolutionary promise to treat various age-related diseases,such as diabetes,heart failure,and Parkinson’s disease.However,low retention and survival rate of delivered stem cells,partially due to immunological rejection,constitute major hurdles for the clinical implementation of stem cell therapy(Lei et al.,2021a).Since mounting evidence showed that several types of stem cells mainly exert their therapeutic effects through the secretion of paracrine effects,exosomes,which are released by stem cells and execute most paracrine functions,have begun to draw attention in the field(Tran and Damaser,2015).Exosomes are membrane-enclosed vesicles with an average diameter of∼100 nanometers secreted by the cells,containing cytokines.

The overexpression of Rps14 in Lgr5+progenitor cells promotes hair cell regeneration in the postnatal mouse cochlea

Sensory hair cells(HCs)in the cochlea cannot regenerate spontaneously in adult mammals after being damaged by external or genetic factors.However,several genes and signaling pathways are reported to induce cochlear HC regeneration at the early neonatal stage.Rps14 encodes a ribosomal protein that is involved in the regulation of cell differentiation and proliferation in mammals.However,its roles in the cochlea have not been reported in vivo.Here,we specifically overexpressed Rps14 in Lgr5+progenitor cells in the newborn mouse cochlea and found that Rps14 conditional overexpression(cOE)mice had significantly increased the ectopic HCs,including inner and outer HCs.We further explored the source of these ectopic HCs and found no EdU+supporting cells observed in the Rps14 cOE mice.The lineage tracing results,on the other hand,revealed that Rps14 cOE mice had significantly more tdTomato+HCs in their cochleae than control mice.These results indicated that regenerated HCs by cOE of Rps14 are most likely derived from inducing the direct trans-differentiation of Lgr5+progenitor cells into HCs.Moreover,real-time qPCR results suggested that the transcription factor genes Atoh1 and Gfi1,which are important in regulating HC differentiation,were upregulated in the cochlear basilar membrane of Rps14 cOE mice.In summary,this study provides in vivo evidence that,in the postnatal mouse cochlea,Rps14 is a potential gene that can promote the spontaneous trans-differentiation of Lgr5+progenitor cells into HCs.This gene may one day be exploited as a therapeutic target for treating hearing loss.

Large-scale chemical screen identifies Gallic acid as a geroprotector for human stem cells

Dear Editor,The interventions that slow aging or promote healthy aging may provide preventative measures for age-related diseases(Zhang et al.,2015).Therefore,it is crucial to identify drugs that target aging-related pathologies and improve health-span in geroscience research.Using model organisms such as C.elegans and rodents,several small molecules capable of alleviating the onset or progression of aging,including rapamycin,nicotinamide mononucleotide,and metformin,have been discovered(Partridge et al.,2020).However,the safety and efficacy of these chemicals still need in-depth evaluation before clinical applications(Partridge et al.,2020).As a result,it is necessary to identify additional compounds with geroprotective effects for human cells to counteract the general trend of populational aging.However,transforming a promising compound into an approved drug requires enormous resources.Alternatively,repurposing previously approved drugs for new clinical applications offers a more efficient and less costly path toward drug develop-ment.Therefore,testing U.S.Food and Drug Administration(FDA)-approved drugs for geroprotective effects may dis-covernew therapeutics that have already been stringently tested in humans for safety.

The evolving views of hematopoiesis:from embryo to adulthood and from in vivo to in vitro

The hematopoietic system composed of hematopoietic stem and progenitor cells(HSPCs)and their differentiated lineages serves as an ideal model to uncover generic principles of cell fate transitions.From gastrulation onwards,there successively emerge primitive hematopoiesis(that produces specialized he-matopoietic cells),pro-definitive hematopoiesis(that produces lineage-restricted progenitor cells),and definitive hematopoiesis(that produces multipotent HSPCs).These nascent lineages develop in several transient hematopoietic sites and finally colonize into lifelong hematopoietic sites.The development and maintenance of hematopoietic lineages are orchestrated by cell-intrinsic gene regulatory networks and cell-extrinsic microenvironmental cues.Owing to the progressive methodology(e.g.,high-throughput lineage tracing and single-cell functional and omics analyses),our understanding of the developmental origin of hematopoietic lineages and functional properties of certain hematopoietic organs has been updated;meanwhile,new paradigms to characterize rare cell types,cell heterogeneity and its causes,and comprehensive regulatory landscapes have been provided.Here,we review the evolving views of HSPC biology during developmental and postnatal hematopoiesis.Moreover,we discuss recent advances in the in vitro induction and expansion of HSPCs,with a focus on the implications for clinical applications.

Development of Chinese herbal medicine for sensorineural hearing loss

According to the World Health Organization’s world report on hearing,nearly 2.5 billion people worldwide will suffer from hearing loss by 2050,which may contribute to a severe impact on individual life quality and national economies.Sensorineural hearing loss(SNHL)occurs commonly as a result of noise exposure,aging,and ototoxic drugs,and is pathologically characterized by the impairment of mechanosensory hair cells of the inner ear,which is mainly triggered by reactive oxygen species accumulation,inflammation,and mitochondrial dysfunction.Though recent advances have been made in understanding the ability of cochlear repair and regeneration,there are still no effective therapeutic drugs for SNHL.Chinese herbal medicine which is widely distributed and easily accessible in China has demonstrated a unique curative effect against SNHL with higher safety and lower cost compared with Western medicine.Herein we present trends in research for Chinese herbal medicine for the treatment of SNHL,and elucidate their molecular mechanisms of action,to pave the way for further research and development of novel effective drugs in this field.

Programmable synthetic receptors:the next-generation of cell and gene therapies

Cell and gene therapies hold tremendous promise for treating a range of difficult-to-treat diseases.However,concerns over the safety and efficacy require to be further addressed in order to realize their full potential.Synthetic receptors,a synthetic biology tool that can precisely control the function of therapeutic cells and genetic modules,have been rapidly developed and applied as a powerful solution.Delicately designed and engineered,they can be applied to finetune the therapeutic activities,i.e.,to regulate production of dosed,bioactive payloads by sensing and processing user-defined signals or biomarkers.This review provides an overview of diverse synthetic receptor systems being used to reprogram therapeutic cells and their wide applications in biomedical research.With a special focus on four synthetic receptor systems at the forefront,including chimeric antigen receptors(CARs)and synthetic Notch(synNotch)receptors,we address the generalized strategies to design,construct and improve synthetic receptors.Meanwhile,we also highlight the expanding landscape of therapeutic applications of the synthetic receptor systems as well as current challenges in their clinical translation.

CRL2^(APPBP2)-mediated TSPYL2 degradation counteracts human mesenchymal stem cell senescence

Cullin-RING E3 ubiquitin ligases(CRLs),the largest family of multi-subunit E3 ubiquitin ligases in eukaryotic cells,represent core cellular machinery for executing protein degradation and maintaining proteostasis.Here,we asked what roles Cullin proteins play in human mesenchymal stem cell(hMSC)homeostasis and senescence.To this end,we conducted a comparative aging phenotype analysis by individually knocking down Cullin members in three senescence models:replicative senescent hMSCs,Hutchinson-Gilford Progeria Syndrome hMSCs,and Werner syndrome hMSCs.Among all family members,we found that CUL2 deficiency rendered hMSCs the most susceptible to senescence.To investigate CUL2-specific underlying mechanisms,we then applied CRISPR/Cas9-mediated gene editing technology to generate CUL2-deficient human embryonic stem cells(hESCs).When we differentiated these into h MSCs,we found that CUL2 deletion markedly accelerates hMSC senescence.Importantly,we identified that CUL2 targets and promotes ubiquitin proteasome-mediated degradation of TSPYL2(a known negative regulator of proliferation)through the substrate receptor protein APPBP2,which in turn downregulates one of the canonical aging marker-P21^(waf1/cip1),and thereby delays senescence.Our work provides important insights into how CRL2^(APPBP2)-mediated TSPYL2 degradation counteracts hMSC senescence,providing a molecular basis for directing intervention strategies against aging and aging-related diseases.

Multimodal Omics Approaches to Aging and Age‑Related Diseases

Aging is associated with a progressive decline in physiological capacities and an increased risk of aging-associated disorders.An increasing body of experimental evidence shows that aging is a complex biological process coordinately regulated by multiple factors at diferent molecular layers.Thus,it is difcult to delineate the overall systematic aging changes based on single-layer data.Instead,multimodal omics approaches,in which data are acquired and analyzed using complementary omics technologies,such as genomics,transcriptomics,and epigenomics,are needed for gaining insights into the precise molecular regulatory mechanisms that trigger aging.In recent years,multimodal omics sequencing technologies that can reveal complex regulatory networks and specifc phenotypic changes have been developed and widely applied to decode aging and age-related diseases.This review summarizes the classifcation and progress of multimodal omics approaches,as well as the rapidly growing number of articles reporting on their application in the feld of aging research,and outlines new developments in the clinical treatment of age-related diseases based on omics technologies.