Cell lineage tracing is a key technology for describing the developmental history of individual progenitor cells and assembling them to form a lineage development tree.However,traditional methods have limitations of p...Cell lineage tracing is a key technology for describing the developmental history of individual progenitor cells and assembling them to form a lineage development tree.However,traditional methods have limitations of poor stability and insufficient reso-lution.As an efficient and flexible gene editing tool,CRISPR-Cas9 system has been widely used in biological research.Furthermore,CRISPR-Cas9 gene editing-based tracing methods can introduce fluorescent proteins,reporter genes,or DNA barcodes for high-throughput sequencing,enabling precise lineage analysis,significantly im-proving precision and resolution,and expanding its application range.In this review,we summarize applications of CRISPR-Cas9 system in cell lineage tracing,with special emphasis on its successful applications in traditional model animals(e.g.,zebrafish and mice),large animal models(pigs),and human cells or organoids.We also discussed its potential prospects and challenges in xenotransplantation and regenerative medicine.展开更多
Unraveling the lineage relationships of all descendants from a zygote is fundamental to advancing our understanding of developmental and stem cell biology.However,existing cell barcoding technologies in zebrafish lack...Unraveling the lineage relationships of all descendants from a zygote is fundamental to advancing our understanding of developmental and stem cell biology.However,existing cell barcoding technologies in zebrafish lack the resolution to capture the majority of cell divisions during embryogenesis.A recently developed method,a substitution mutation-aided lineage-tracing system(SMALT),successfully reconstructed high-resolution cell phylogenetic trees for Drosophila melanogaster.Here,we implement the SMALT system in zebrafish,recording a median of 14 substitution mutations on a one-kilobase-pair barcoding sequence for one-day post-fertilization embryos.Leveraging this system,we reconstruct four cell lineage trees for zebrafish fin cells,encompassing both original and regenerated fin.Each tree consists of hundreds of internal nodes with a median bootstrap support of 99%.Analysis of the obtained cell lineage trees reveals that regenerated fin cells mainly originate from cells in the same part of the fins.Through multiple times sampling germ cells from the same individual,we show the stability of the germ cell pool and the early separation of germ cell and somatic cell progenitors.Our system offers the potential for reconstructing high-quality cell phylogenies across diverse tissues,providing valuable insights into development and disease in zebrafish.展开更多
Genetic lineage tracing has been widely employed to investigate cell lineages and fate.However,conventional reporting systems often label the entire cytoplasm,making it challenging to discern cell boundaries.Additiona...Genetic lineage tracing has been widely employed to investigate cell lineages and fate.However,conventional reporting systems often label the entire cytoplasm,making it challenging to discern cell boundaries.Additionally,single Cre-lox P recombination systems have limitations in tracing specific cell populations.This study proposes three reporting systems utilizing Cre,Dre,and Dre+Cre mediated recombination.These systems incorporate td Tomato expression on the cell membrane and Phi YFP expression within the nucleus,allowing for clear observation of the nucleus and membrane.The efficacy of these systems is successfully demonstrated by labeling cardiomyocytes and hepatocytes.The potential for dynamic visualization of the cell membrane is showcased using intravital imaging microscopy or threedimensional imaging.Furthermore,by combining this dual recombinase system with the Pro Tracer system,hepatocyte proliferation is traced with enhanced precision.This reporting system holds significant importance for advancing the understanding of cell fate studies in development,homeostasis,and diseases.展开更多
Regenerating functional new neurons in the adult mammalian central nervous system has been proven to be very challenging due to the inability of neurons to divide and repopulate themselves after neuronal loss.Glial ce...Regenerating functional new neurons in the adult mammalian central nervous system has been proven to be very challenging due to the inability of neurons to divide and repopulate themselves after neuronal loss.Glial cells,on the other hand,can divide and repopulate themselves under injury or diseased conditions.We have previously reported that ectopic expression of NeuroD1 in dividing glial cells can directly convert them into neurons.Here,using astrocytic lineage-tracing reporter mice(Aldh1l1-CreERT2 mice crossing with Ai14 mice),we demonstrate that lineage-traced astrocytes can be successfully converted into NeuNpositive neurons after expressing NeuroD1 through adeno-associated viruses.Retroviral expression of NeuroD1 further confirms that dividing glial cells can be converted into neurons.Importantly,we demonstrate that for in vivo cell conversion study,using a safe level of adeno-associated virus dosage(10^10–10^12 gc/mL,1μL)in the rodent brain is critical to avoid artifacts caused by toxic dosage,such as that used in a recent bioRxiv study(2×10^13 gc/mL,1μL,mouse cortex).For therapeutic purpose under injury or diseased conditions,or for non-human primate studies,adeno-associated virus dosage needs to be optimized through a series of dose-finding experiments.Moreover,for future in vivo gliato-neuron conversion studies,we recommend that the adeno-associated virus results are further verified with retroviruses that mainly express transgenes in dividing glial cells in order to draw solid conclusions.The study was approved by the Laboratory Animal Ethics Committee of Jinan University,China(approval No.IACUC-20180330-06)on March 30,2018.展开更多
The functional heterogeneity of hematopoietic stem cells(HSCs) has been comprehensively investigated by single-cell transplantation assay.However,the heterogeneity regarding their physiological contribution remains an...The functional heterogeneity of hematopoietic stem cells(HSCs) has been comprehensively investigated by single-cell transplantation assay.However,the heterogeneity regarding their physiological contribution remains an open question,especially for those with life-long hematopoietic fate of rigorous selfrenewing and balanced differentiation capacities.In this study,we revealed that Procr expression was detected principally in phenotypical vascular endothelium co-expressing DII4 and CD44 in the midgestation mouse embryos,and could enrich all the HSCs of the embryonic day 11.5(E11.5) aortagonad-mesonephros(AGM) region.We then used a temporally restricted genetic tracing strategy to irreversibly label the Procr-exp res sing cells at E9.5.Interestingly,most labeled mature HSCs in multiple sites(such as AGM) around E11.5 were functionally categorized as lymphomyeloid-balanced HSCs assessed by direct transplantation.Furthermore,the labeled cells contributed to an average of 7.8% of immunophenotypically defined HSCs in E14.5 fetal liver(FL) and 6.9% of leukocytes in peripheral blood(PB) during one-year follow-up.Surprisingly,in aged mice of 24 months,the embryonically tagged cells displayed constant contribution to leukocytes with no bias to myeloid or lymphoid lineages.Altogether,we demonstrated,for the first time,the existence of a subtype of physiologically long-lived balanced HSCs as hypothesized,whose precise embryonic origin and molecular identity await further characterization.展开更多
Memory by Engineered Mutagenesis with Optical In situ Readout（MEMOIR）is a novel strategy for lineage tracing that combines Cas9/g RNA and sequential multiplexed single-molecule RNA fluorescence hybridization（seqFIS...Memory by Engineered Mutagenesis with Optical In situ Readout（MEMOIR）is a novel strategy for lineage tracing that combines Cas9/g RNA and sequential multiplexed single-molecule RNA fluorescence hybridization（seqFISH）[1],which was created by Cai Long et al.at the California Institute of Technology[2].In MEMOIR,dynamic cellular event histories are recorded,then read out in single cells using seq FISH.Here,we introduce the展开更多
Understanding the contribution of endothelial cells to the progenitor pools of adult tissues has the potential to inform therapies for human disease.To address whether endothelial cells transdifferentiate into non-vas...Understanding the contribution of endothelial cells to the progenitor pools of adult tissues has the potential to inform therapies for human disease.To address whether endothelial cells transdifferentiate into non-vascular cell types,we performed cell lineage tracing analysis using transgenic mice engineered to express a fluorescent marker following activation by tamoxifen in vascular endothelial cadherin promoter-expressing cells(VEcad-CreERT2;B6 Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze).Activation of target-cell labeling following 1.5 months of ad libitum feeding with tamoxifen-laden chow in 4–5 month-old mice resulted in the tracing of central nervous system and peripheral cells that include:cerebellar granule neurons,ependymal cells,skeletal myocytes,pancreatic beta cells,pancreatic acinar cells,tubular cells in the renal cortex,duodenal crypt cells,ileal crypt cells,and hair follicle stem cells.As Nestin expression has been reported in a subset of endothelial cells,Nes-CreERT2 mice were also utilized in these conditions.The tracing of cells in adult Nes-CreERT2 mice revealed the labeling of canonical progeny cell types such as hippocampal and olfactory granule neurons as well as ependymal cells.Interestingly,Nestin tracing also labeled skeletal myocytes,ileal crypt cells,and sparsely marked cerebellar granule neurons.Our findings provide support for endothelial cells as active contributors to adult tissue progenitor pools.This information could be of particular significance for the intravenous delivery of therapeutics to downstream endothelial-derived cellular targets.The animal experiments were approved by the Boise State University Institute Animal Care and Use Committee(approval No.006-AC15-018)on October 31,2018.展开更多
1 Introduction Dissecting the dynamics of cell statesiscrucial for understanding various biological processes,such as tissue development and tumor drug responses.Recent advancements in single-cell lineage tracing(scLT...1 Introduction Dissecting the dynamics of cell statesiscrucial for understanding various biological processes,such as tissue development and tumor drug responses.Recent advancements in single-cell lineage tracing(scLT)technologies provide effective ways to track single-cell lineages through heritable cellular barcodes,while simultaneously detecting the molecular states of cells by sequencing[1].展开更多
During the pathogenesis of type 1 diabetes(T1D) and type 2 diabetes(T2D), pancreatic islets, especially the β cells, face significant challenges. These insulin-producing cells adopt a regeneration strategy to compens...During the pathogenesis of type 1 diabetes(T1D) and type 2 diabetes(T2D), pancreatic islets, especially the β cells, face significant challenges. These insulin-producing cells adopt a regeneration strategy to compensate for the shortage of insulin, but the exact mechanism needs to be defined. High-fat diet(HFD) and streptozotocin(STZ) treatment are well-established models to study islet damage in T2D and T1D respectively. Therefore, we applied these two diabetic mouse models, triggered at different ages, to pursue the cell fate transition of isletβ cells. Cre-LoxP systems were used to generate islet cell type-specific(α, β, or δ) green fluorescent protein(GFP)-labeled mice for genetic lineage tracing, thereinto β-cell GFP-labeled mice were tamoxifen induced. Single-cell RNA sequencing(scRNA-seq) was used to investigate the evolutionary trajectories and molecular mechanisms of the GFP-labeled β cells in STZ-treated mice. STZ-induced diabetes caused extensive dedifferentiation of β cells and some of which transdifferentiated into α or δ cells in both youth-and adulthood-initiated mice while this phenomenon was barely observed in HFD models. β cells in HFD mice were expanded via self-replication rather than via transdifferentiation from α or δ cells, in contrast, α or δ cells were induced to transdifferentiate into β cells in STZ-treated mice(both youthand adulthood-initiated). In addition to the re-dedifferentiation of β cells, it is also highly likely that these “α or δ” cells transdifferentiated from pre-existing β cells could also re-trans-differentiate into insulin-producing β cells and be beneficial to islet recovery. The analysis of ScRNA-seq revealed that several pathways including mitochondrial function, chromatin modification, and remodeling are crucial in the dynamic transition of β cells. Our findings shed light on how islet β cells overcome the deficit of insulin and the molecular mechanism of islet recovery in T1D and T2D pathogenesis.展开更多
Stem cell research has become a hot topic in biology,as the understanding of stem cell biology can provide new insights for both regenerative medicine and clinical treatment of diseases.Accurately deciphering the fate...Stem cell research has become a hot topic in biology,as the understanding of stem cell biology can provide new insights for both regenerative medicine and clinical treatment of diseases.Accurately deciphering the fate of stem cells is the basis for understanding the mechanism and function of stem cells during tissue repair and regeneration.Cre-loxP-mediated recombination has been widely applied in fate mapping of stem cells for many years.However,nonspecific labeling by conventional cell lineage tracing strategies has led to discrepancies or even controversies in multiple fields.Recently,dual recombinase-mediated lineage tracing strategies have been developed to improve both the resolution and precision of stem cell fate mapping.These new genetic strategies also expand the application of lineage tracing in studying cell origin and fate.Here,we review cell lineage tracing methods,especially dual genetic approaches,and then provide examples to describe how they are used to study stem cell fate plasticity and function in vivo.展开更多
Central to the core principle of cell theory,depicting cells'history,state and fate is a fundamental goal in modern biology.By leveraging clonal analysis and sin-gle-cell RNA-seq technologies,single-cell lineage t...Central to the core principle of cell theory,depicting cells'history,state and fate is a fundamental goal in modern biology.By leveraging clonal analysis and sin-gle-cell RNA-seq technologies,single-cell lineage trac-ing provides new opportunities to interrogate both cell states and lineage histories.During the past few years,many strategies to achieve lineage tracing at single-cell resolution have been developed,and three of them(in-tegration barcodes,polylox barcodes,and CRISPR barcodes)are noteworthy as they are amenable in experimentally tractable systems.Although the above strategies have been demonstrated in animal develop-ment and stem cell research,much care and effort are still required to implement these methods.Here we review the development of single-cell lineage tracing,major characteristics of the cell barcoding strategies,applications,as well as technical considerations and limitations,providing a guide to choose or improve the single-cell barcoding lineage tracing.展开更多
Arrhythmogenic cardiomyopathy(ACM)is one of the most common inherited cardiomyopathies,characterized by progressive fibrofatty replacement in the myocardium.However,the cellular origin of cardiac adipocytes in ACM rem...Arrhythmogenic cardiomyopathy(ACM)is one of the most common inherited cardiomyopathies,characterized by progressive fibrofatty replacement in the myocardium.However,the cellular origin of cardiac adipocytes in ACM remains largely unknown.Unraveling the cellular source of cardiac adipocytes in ACM would elucidate the underlying pathological process and provide a potential target for therapy.Herein,we generated an ACM mouse model by inactivating desmosomal gene desmoplakin in cardiomyocytes;and examined the adipogenic fates of several cell types in the disease model.The results showed that SOX9^(+),PDGFRa^(+),and PDGFRb^(+)mesenchymal cells,but not cardiomyocytes or smooth muscle cells,contribute to the intramyocardial adipocytes in the ACM model.Mechanistically,Bmp4 was highly expressed in the ACM mouse heart and functionally promoted cardiac mesenchymal-to-adipose transition in vitro.展开更多
tAstrocytes are the largest glial population in the mammalian brain.However,we have a minimal understanding of astrocyte development,especially fate specification in different regions of the brain.Through lineage trac...tAstrocytes are the largest glial population in the mammalian brain.However,we have a minimal understanding of astrocyte development,especially fate specification in different regions of the brain.Through lineage tracing of the progenitors of the third ventricle(3V)wall via in-utero electroporation in the embryonic mouse brain,we show the fate specification and migration pattern of astrocytes derived from radial glia along the 3V wall.Unexpectedly,radial glia located in different regions along the 3V wall of the diencephalon produce distinct cell types:radial glia in the upper region produce astrocytes and those in the lower region produce neurons in the diencephalon.With genetic fate mapping analysis,we reveal that the first population of astrocytes appears along the zona incerta in the diencephalon.Astrogenesis occurs at an early time point in the dorsal region relative to that in the ventral region of the developing diencephalon.With transcriptomic analysis of the region-specific 3V wall and lateral ventricle(LV)wall,we identified cohorts of differentially-expressed genes in the dorsal 3V wall compared to the ventral 3V wall and LV wall that may regulate astrogenesis in the dorsal diencephalon.Together,these results demonstrate that the generation of astrocytes shows a spatiotemporal pattern in the developing mouse diencephalon.展开更多
In recent years, stem cells have been a focal point in research designed to evaluate the efficacy of ophthalmologic therapies, specifically those for corneal conditions. The corneal epithelium is one of the few region...In recent years, stem cells have been a focal point in research designed to evaluate the efficacy of ophthalmologic therapies, specifically those for corneal conditions. The corneal epithelium is one of the few regions of the body that maintains itself using a residual stem cell population within the adjacent limbus. Stem cell movement has additionally captivated the minds of researchers due to its potential application in different body regions. The cornea is a viable model for varying methods to track stem cell migratory patterns, such as lineage tracing and live imaging from the limbus. These developments have the potential to pave the way for future therapies designed to ensure the continuous regeneration of the corneal epithelium following injury via the limbal stem cell niche. This literature review aims to analyze the various methods of imaging used to understand the limbal stem cell niche and possible future directions that might be useful to consider for the better treatment and prevention of disorders of the cornea and corneal epithelium. .展开更多
The intestinal epithelium is one of the most rapidly renewing tissues,which is fueled by stem cells at the base of the crypts.Strategies of genetic lineage tracing and organoids,which capture major features of origina...The intestinal epithelium is one of the most rapidly renewing tissues,which is fueled by stem cells at the base of the crypts.Strategies of genetic lineage tracing and organoids,which capture major features of original tissues,are powerful avenues for exploring the biology of intestinal stem cells in vivo and in vitro,respectively.The combination of intestinal organoideculturing system and genetic modification approaches provides an attractive platform to uncover the mechanism of colorectal cancer and genetic disorders in the human minigut.Here,we will provide a comprehensive overview of studies on intestinal epithelium and intestinal stem cells.We will also review the applications of organoids and genetic markers in intestinal research studies.Furthermore,we will discuss the advantages and drawbacks of organoids as disease models compared with mice models and cell lines.展开更多
5-Bromo-2′-deoxyuridine(BrdU)is a halogenated pyrimidine that can be incorporated into newly synthesized DNA during the S phase of the cell cycle.BrdU is widely used in fate-mapping studies of embryonic and adult neu...5-Bromo-2′-deoxyuridine(BrdU)is a halogenated pyrimidine that can be incorporated into newly synthesized DNA during the S phase of the cell cycle.BrdU is widely used in fate-mapping studies of embryonic and adult neurogenesis to identify newborn neurons,however side effects on neural stem cells and their progeny have been reported.In vivo astrocyte-to-neuron(AtN)conversion is a new approach for generating newborn neurons by directly converting endogenous astrocytes into neurons.The BrdU-labeling strategy has been used to trace astrocyte-converted neurons,but whether BrdU has any effect on the AtN conversion is unknown.Here,while conducting a NeuroD1-mediated AtN conversion study using BrdU to label dividing reactive astrocytes following ischemic injury,we accidentally discovered that BrdU inhibited AtN conversion.We initially found a gradual reduction in BrdU-labeled astrocytes during NeuroD1-mediated AtN conversion in the mouse cortex.Although most NeuroD1-infected astrocytes were converted into neurons,the number of BrdU-labeled neurons was surprisingly low.To exclude the possibility that this BrdU inhibition was caused by the ischemic injury,we conducted an in vitro AtN conversion study by overexpressing NeuroD1 in cultured cortical astrocytes in the presence or absence of BrdU.Surprisingly,we also found a significantly lower conversion rate and a smaller number of converted neurons in the BrdU-treated group compared with the untreated group.These results revealed an unexpected inhibitory effect of BrdU on AtN conversion,suggesting more caution is needed when using BrdU in AtN conversion studies and in data interpretation.展开更多
Tooth enamel,a highly mineralized tissue covering the outermost area of teeth,is always damaged by dental caries or trauma.Tooth enamel rarely repairs or renews itself,due to the loss of ameloblasts and dental epithel...Tooth enamel,a highly mineralized tissue covering the outermost area of teeth,is always damaged by dental caries or trauma.Tooth enamel rarely repairs or renews itself,due to the loss of ameloblasts and dental epithelial stem cells(DESCs)once the tooth erupts.Unlike human teeth,mouse incisors grow continuously due to the presence of DESCs that generate enamel-producing ameloblasts and other supporting dental epithelial lineages.The ready accessibility of mouse DESCs and wide availability of related transgenic mouse lines make mouse incisors an excellent model to examine the identity and heterogeneity of dental epithelial stem/progenitor cells;explore the regulatory mechanisms underlying enamel formation;and help answer the open question regarding the therapeutic development of enamel engineering.In the present review,we update the current understanding about the identification of DESCs in mouse incisors and summarize the regulatory mechanisms of enamel formation driven by DESCs.The roles of DESCs during homeostasis and repair are also discussed,which should improve our knowledge regarding enamel tissue engineering.展开更多
During the development of mammalian heart, the left and right atria play an important role in cardiovascular circulation. The embryonic atrium is mainly formed by the differentiation of progenitor cells and the prolif...During the development of mammalian heart, the left and right atria play an important role in cardiovascular circulation. The embryonic atrium is mainly formed by the differentiation of progenitor cells and the proliferation of cardiomyocytes, while the postnatal atrium is primarily shaped by the increase in the volume of cardiomyocytes. Cell proliferation and differentiation of atrial development is the basis for its functions such as “blood reservoir” and “supplementary pump”. Deep understanding the cellular mechanism of atrial development is imperative to explore the causes of common congenital arrhythmia heart diseases such as atrial fibrillation. We used genetically engineered mouse reproduction knowledge, lineage tracing method based on CreloxP system, molecular biology and immunofluorescence technology to track the cardiomyocyte lineage of Nppa-GFP mouse line with stereo fluorescence microscope and ultra-high-speed confocal microscope. Besides the atrium of Nppa-CreER;Rosa26 tdTomato mouse was examined during embryonic (E10.5 - E18.5) and postnatal (P0, P3, P5, P7, P14, P28, P8w) stage. Immunofluorescence results revealed that Nppa-positive cells labeled TNNI3-positive cardiomyocytes and protruded into the atrial cavity at the beginning of E11.5 - E12.0 and during subsequent development to form Nppa-positive myocardial trabeculae. Thick comb-shaped myocardium was observed after birth, and we suspect that this was particularly important for the normal contractile activity and pumping function of the atrium. Additionally, non-single origin of Nppa-positive trabecular myocardiocytes were revealed through Tamoxifen-induced lineage tracing experiment. Our findings reveal proliferation dynamics and non-comprehensive fate decisions of cardiomyocytes that produce the distinct architecture of the atrium chamber.展开更多
Tissue-resident memory T cells(T_(RM))play a key role in defense against pathogen invading barrier sites and other non-lymphoid tissues.How T_(RM)cells are maintained in various tissues,and how they relate to antigen-...Tissue-resident memory T cells(T_(RM))play a key role in defense against pathogen invading barrier sites and other non-lymphoid tissues.How T_(RM)cells are maintained in various tissues,and how they relate to antigen-experienced memory T cells in lymphoid organs are not fully understood.By barcode-based lineage tracing and single-cell transcriptome analysis,we found a distinct population of CD69^(+)CD103^(+)virusspecific CD8^(+)T cells in draining lymph nodes(dLNs)following intranasal influenza infection.Intriguingly,these dLN T_(RM)cells and lung T_(RM)cells shared similar function-neutral barcode contents and transcriptomic features,implicating local circulation between the lung and dLNs in the maintenance of resident memory.Ablation of CXCR3 from CD8^(+)T cells,which impairs lung T_(RM)generation,severely reduced the abundance of dLN T_(RM)cells,suggesting that dLN T_(RM)cells are connected to lung T_(RM)cells by retrograde migration.Our screen for chemokines and chemokine receptors implicated the CCR5-CCL5 axis in promoting lung-to-dLN migration.Temporary CCR5 blockade by intratracheal administration of Maraviroc,a CCR5 inhibitor,reduced the abundance of dLN T_(RM)cells without affecting lung T_(RM)cells.By intratracheal cell transfer,CCR5-deficient CD8^(+)T cells were found impaired in lung-to-dLN migration.Finally,dLN T_(RM)cells actively participated in the secondary response and could reconstitute lung T_(RM)cells following influenza infection.Our results support a model in which lung-to-dLN T_(RM)retrograde migration helps maintain the CD8^(+)memory resident in the respiratory tract and optimizes the local Tcell response to reinfection.展开更多
The effective and translational strategy to regenerate knee meniscal fibrocartilage remained challenging.Herein,we first identified vascular smooth muscle cells(VSMCs)transdifferentiated into fibrochondrocytes and par...The effective and translational strategy to regenerate knee meniscal fibrocartilage remained challenging.Herein,we first identified vascular smooth muscle cells(VSMCs)transdifferentiated into fibrochondrocytes and participated in spontaneous meniscal regeneration using smooth muscle cell lineage tracing transgenic mice meniscal defect model.Then,we identified low-intensity pulsed ultrasound(LIPUS)acoustic stimulus enhanced fibrochondrogenic transdifferentiation of VSMCs in vitro and in vivo.Mechanistically,LIPUS stimulus could up-regulate mechanosensitive ion channel Piezo1 expression and then activate the transforming growth factorβ1(TGFβ1)signal,following repression of the Notch signal,consequently enhancing fibrochondrogenic transdifferentiation of VSMCs.Finally,we demonstrated that the regular LIPUS stimulus enhanced anisotropic native-like meniscal fibrocartilage tissue regeneration in a beagle canine subtotal meniscectomy model at 6 months postoperatively.The single-cell RNA sequencing analysis confirmed the role of VSMC fibrochondrogenic transdifferentiation in meniscal regeneration.展开更多
基金supported by Institute of Laboratory Animal Sciences,Chinese Academy of Medical Sciences and Comparative Medicine Center,Peking Union Medical College,Collaborative Innovation Program of the Chinese Academy of Sciences(22SH19)Nonprofit Central Research Institute Fund of Chinese Academy of Medical Sciences(2023-PT180-01).
文摘Cell lineage tracing is a key technology for describing the developmental history of individual progenitor cells and assembling them to form a lineage development tree.However,traditional methods have limitations of poor stability and insufficient reso-lution.As an efficient and flexible gene editing tool,CRISPR-Cas9 system has been widely used in biological research.Furthermore,CRISPR-Cas9 gene editing-based tracing methods can introduce fluorescent proteins,reporter genes,or DNA barcodes for high-throughput sequencing,enabling precise lineage analysis,significantly im-proving precision and resolution,and expanding its application range.In this review,we summarize applications of CRISPR-Cas9 system in cell lineage tracing,with special emphasis on its successful applications in traditional model animals(e.g.,zebrafish and mice),large animal models(pigs),and human cells or organoids.We also discussed its potential prospects and challenges in xenotransplantation and regenerative medicine.
基金supported by the National Key R&D Program of China(2021YFA1302500 and 2021YFA1302501)the National Natural Science Foundation of China(32293190,32293191,31970570,and 32200492).
文摘Unraveling the lineage relationships of all descendants from a zygote is fundamental to advancing our understanding of developmental and stem cell biology.However,existing cell barcoding technologies in zebrafish lack the resolution to capture the majority of cell divisions during embryogenesis.A recently developed method,a substitution mutation-aided lineage-tracing system(SMALT),successfully reconstructed high-resolution cell phylogenetic trees for Drosophila melanogaster.Here,we implement the SMALT system in zebrafish,recording a median of 14 substitution mutations on a one-kilobase-pair barcoding sequence for one-day post-fertilization embryos.Leveraging this system,we reconstruct four cell lineage trees for zebrafish fin cells,encompassing both original and regenerated fin.Each tree consists of hundreds of internal nodes with a median bootstrap support of 99%.Analysis of the obtained cell lineage trees reveals that regenerated fin cells mainly originate from cells in the same part of the fins.Through multiple times sampling germ cells from the same individual,we show the stability of the germ cell pool and the early separation of germ cell and somatic cell progenitors.Our system offers the potential for reconstructing high-quality cell phylogenies across diverse tissues,providing valuable insights into development and disease in zebrafish.
基金supported by the National Key Research&Development Program of China(2021YFA0805100,2023YFA1800700,2019YFA0110403,2019YFA0802000)the National Science Foundation of China(82088101,32370885,92368103,32370897)the Westlake Education Foundation,and the Benyuan Charity Fund,Research Funds of Hangzhou Institute for Advanced Study(2022ZZ01015 and B04006C01600515)。
文摘Genetic lineage tracing has been widely employed to investigate cell lineages and fate.However,conventional reporting systems often label the entire cytoplasm,making it challenging to discern cell boundaries.Additionally,single Cre-lox P recombination systems have limitations in tracing specific cell populations.This study proposes three reporting systems utilizing Cre,Dre,and Dre+Cre mediated recombination.These systems incorporate td Tomato expression on the cell membrane and Phi YFP expression within the nucleus,allowing for clear observation of the nucleus and membrane.The efficacy of these systems is successfully demonstrated by labeling cardiomyocytes and hepatocytes.The potential for dynamic visualization of the cell membrane is showcased using intravital imaging microscopy or threedimensional imaging.Furthermore,by combining this dual recombinase system with the Pro Tracer system,hepatocyte proliferation is traced with enhanced precision.This reporting system holds significant importance for advancing the understanding of cell fate studies in development,homeostasis,and diseases.
基金This study was supported by the National Natural Science Foundation of China(No.U1801681,to GC and No.31970906,to WL)Guangdong Science and Technology Department(‘Key technologies for treatment of brain disorders’,No.2018B030332001,to GC)+2 种基金the Natural Science Foundation of Guangdong Province of China(No.2020A1515011079,to WL and No.2020A1515010854,to QW)the internal funding from Jinan University(No.21616110,to GC)the Young Scientists Fund of the National Natural Science Foundation of China(No.31701291,to WL).
文摘Regenerating functional new neurons in the adult mammalian central nervous system has been proven to be very challenging due to the inability of neurons to divide and repopulate themselves after neuronal loss.Glial cells,on the other hand,can divide and repopulate themselves under injury or diseased conditions.We have previously reported that ectopic expression of NeuroD1 in dividing glial cells can directly convert them into neurons.Here,using astrocytic lineage-tracing reporter mice(Aldh1l1-CreERT2 mice crossing with Ai14 mice),we demonstrate that lineage-traced astrocytes can be successfully converted into NeuNpositive neurons after expressing NeuroD1 through adeno-associated viruses.Retroviral expression of NeuroD1 further confirms that dividing glial cells can be converted into neurons.Importantly,we demonstrate that for in vivo cell conversion study,using a safe level of adeno-associated virus dosage(10^10–10^12 gc/mL,1μL)in the rodent brain is critical to avoid artifacts caused by toxic dosage,such as that used in a recent bioRxiv study(2×10^13 gc/mL,1μL,mouse cortex).For therapeutic purpose under injury or diseased conditions,or for non-human primate studies,adeno-associated virus dosage needs to be optimized through a series of dose-finding experiments.Moreover,for future in vivo gliato-neuron conversion studies,we recommend that the adeno-associated virus results are further verified with retroviruses that mainly express transgenes in dividing glial cells in order to draw solid conclusions.The study was approved by the Laboratory Animal Ethics Committee of Jinan University,China(approval No.IACUC-20180330-06)on March 30,2018.
基金supported by grants from the National Key R&D Program of China (2017YFA0103401 and 2016YFA0100601)the National Natural Science Foundation of China(31425012,31930054,31871173 and 81890991)the Program for Guangdong Introducing Innovative and Entrepreneurial Teams (2017ZT07S347)
文摘The functional heterogeneity of hematopoietic stem cells(HSCs) has been comprehensively investigated by single-cell transplantation assay.However,the heterogeneity regarding their physiological contribution remains an open question,especially for those with life-long hematopoietic fate of rigorous selfrenewing and balanced differentiation capacities.In this study,we revealed that Procr expression was detected principally in phenotypical vascular endothelium co-expressing DII4 and CD44 in the midgestation mouse embryos,and could enrich all the HSCs of the embryonic day 11.5(E11.5) aortagonad-mesonephros(AGM) region.We then used a temporally restricted genetic tracing strategy to irreversibly label the Procr-exp res sing cells at E9.5.Interestingly,most labeled mature HSCs in multiple sites(such as AGM) around E11.5 were functionally categorized as lymphomyeloid-balanced HSCs assessed by direct transplantation.Furthermore,the labeled cells contributed to an average of 7.8% of immunophenotypically defined HSCs in E14.5 fetal liver(FL) and 6.9% of leukocytes in peripheral blood(PB) during one-year follow-up.Surprisingly,in aged mice of 24 months,the embryonically tagged cells displayed constant contribution to leukocytes with no bias to myeloid or lymphoid lineages.Altogether,we demonstrated,for the first time,the existence of a subtype of physiologically long-lived balanced HSCs as hypothesized,whose precise embryonic origin and molecular identity await further characterization.
基金supported by the grants from the National Natural Science Foundation of China (81271003)the Ministry of Science and Technology of China (2013CB967400, 2012CB966300, and ZJ2014-ZD-002)
文摘Memory by Engineered Mutagenesis with Optical In situ Readout（MEMOIR）is a novel strategy for lineage tracing that combines Cas9/g RNA and sequential multiplexed single-molecule RNA fluorescence hybridization（seqFISH）[1],which was created by Cai Long et al.at the California Institute of Technology[2].In MEMOIR,dynamic cellular event histories are recorded,then read out in single cells using seq FISH.Here,we introduce the
基金supported by the National Institutes Health(grant Nos.5P20GM109095 and P20GM103408)Boise State University(to BEM)
文摘Understanding the contribution of endothelial cells to the progenitor pools of adult tissues has the potential to inform therapies for human disease.To address whether endothelial cells transdifferentiate into non-vascular cell types,we performed cell lineage tracing analysis using transgenic mice engineered to express a fluorescent marker following activation by tamoxifen in vascular endothelial cadherin promoter-expressing cells(VEcad-CreERT2;B6 Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze).Activation of target-cell labeling following 1.5 months of ad libitum feeding with tamoxifen-laden chow in 4–5 month-old mice resulted in the tracing of central nervous system and peripheral cells that include:cerebellar granule neurons,ependymal cells,skeletal myocytes,pancreatic beta cells,pancreatic acinar cells,tubular cells in the renal cortex,duodenal crypt cells,ileal crypt cells,and hair follicle stem cells.As Nestin expression has been reported in a subset of endothelial cells,Nes-CreERT2 mice were also utilized in these conditions.The tracing of cells in adult Nes-CreERT2 mice revealed the labeling of canonical progeny cell types such as hippocampal and olfactory granule neurons as well as ependymal cells.Interestingly,Nestin tracing also labeled skeletal myocytes,ileal crypt cells,and sparsely marked cerebellar granule neurons.Our findings provide support for endothelial cells as active contributors to adult tissue progenitor pools.This information could be of particular significance for the intravenous delivery of therapeutics to downstream endothelial-derived cellular targets.The animal experiments were approved by the Boise State University Institute Animal Care and Use Committee(approval No.006-AC15-018)on October 31,2018.
基金supported by the National Key Research and Development Program of China(Nos.2020YFA0712403 and 2021YFF1200901)the National Natural Science Foundation of China(NSFC)(Grant Nos.62133006 and 92268104)+1 种基金the Tsinghua University Initiative Scientific Research Program(No.20221080076)the China Postdoctoral Science Foundation(No.2022M721839).
文摘1 Introduction Dissecting the dynamics of cell statesiscrucial for understanding various biological processes,such as tissue development and tumor drug responses.Recent advancements in single-cell lineage tracing(scLT)technologies provide effective ways to track single-cell lineages through heritable cellular barcodes,while simultaneously detecting the molecular states of cells by sequencing[1].
基金supported by the National Natural Science Foundation of China(81830023,82070803,82100838,82100837,81900708)。
文摘During the pathogenesis of type 1 diabetes(T1D) and type 2 diabetes(T2D), pancreatic islets, especially the β cells, face significant challenges. These insulin-producing cells adopt a regeneration strategy to compensate for the shortage of insulin, but the exact mechanism needs to be defined. High-fat diet(HFD) and streptozotocin(STZ) treatment are well-established models to study islet damage in T2D and T1D respectively. Therefore, we applied these two diabetic mouse models, triggered at different ages, to pursue the cell fate transition of isletβ cells. Cre-LoxP systems were used to generate islet cell type-specific(α, β, or δ) green fluorescent protein(GFP)-labeled mice for genetic lineage tracing, thereinto β-cell GFP-labeled mice were tamoxifen induced. Single-cell RNA sequencing(scRNA-seq) was used to investigate the evolutionary trajectories and molecular mechanisms of the GFP-labeled β cells in STZ-treated mice. STZ-induced diabetes caused extensive dedifferentiation of β cells and some of which transdifferentiated into α or δ cells in both youth-and adulthood-initiated mice while this phenomenon was barely observed in HFD models. β cells in HFD mice were expanded via self-replication rather than via transdifferentiation from α or δ cells, in contrast, α or δ cells were induced to transdifferentiate into β cells in STZ-treated mice(both youthand adulthood-initiated). In addition to the re-dedifferentiation of β cells, it is also highly likely that these “α or δ” cells transdifferentiated from pre-existing β cells could also re-trans-differentiate into insulin-producing β cells and be beneficial to islet recovery. The analysis of ScRNA-seq revealed that several pathways including mitochondrial function, chromatin modification, and remodeling are crucial in the dynamic transition of β cells. Our findings shed light on how islet β cells overcome the deficit of insulin and the molecular mechanism of islet recovery in T1D and T2D pathogenesis.
基金supported by the Strategic Priority Research Program of the Chinese Academy of Sciences(XDB19000000 and XDA16010507)the National Key Research and Development Program of China(2019YFA0110403 and 2019YFA0802000)National Natural Science Foundation of China(31730112,31625019,91849202,and 82008810001)。
文摘Stem cell research has become a hot topic in biology,as the understanding of stem cell biology can provide new insights for both regenerative medicine and clinical treatment of diseases.Accurately deciphering the fate of stem cells is the basis for understanding the mechanism and function of stem cells during tissue repair and regeneration.Cre-loxP-mediated recombination has been widely applied in fate mapping of stem cells for many years.However,nonspecific labeling by conventional cell lineage tracing strategies has led to discrepancies or even controversies in multiple fields.Recently,dual recombinase-mediated lineage tracing strategies have been developed to improve both the resolution and precision of stem cell fate mapping.These new genetic strategies also expand the application of lineage tracing in studying cell origin and fate.Here,we review cell lineage tracing methods,especially dual genetic approaches,and then provide examples to describe how they are used to study stem cell fate plasticity and function in vivo.
基金supported in part by National Key R&D Program of China(2018YFA0801402,2018YFA0107200)the Strategic Priority Research Program of the Chinese Academy of Sciences(XDA16020404)+3 种基金Guangdong Basic and Applied Basic Research Foundation(2019B151502054)Frontier Research Program of Bioland Laboratory(Guangzhou Regenerative Medicine and Health Guangdong Laboratory,2018GZR110105013)Jiazi Research Innovative Project of Bioland Laboratory(2019GZR110108001)Science and Technology Planning Project of Guangdong Province(2020B1212060052).
文摘Central to the core principle of cell theory,depicting cells'history,state and fate is a fundamental goal in modern biology.By leveraging clonal analysis and sin-gle-cell RNA-seq technologies,single-cell lineage trac-ing provides new opportunities to interrogate both cell states and lineage histories.During the past few years,many strategies to achieve lineage tracing at single-cell resolution have been developed,and three of them(in-tegration barcodes,polylox barcodes,and CRISPR barcodes)are noteworthy as they are amenable in experimentally tractable systems.Although the above strategies have been demonstrated in animal develop-ment and stem cell research,much care and effort are still required to implement these methods.Here we review the development of single-cell lineage tracing,major characteristics of the cell barcoding strategies,applications,as well as technical considerations and limitations,providing a guide to choose or improve the single-cell barcoding lineage tracing.
基金supported by the National key R&D Program of China(2018YFA0108100,2018YFA0108700,2017YFA0105602)the National Natural Science Foundation of China(31871474,81720108004,81974019)+7 种基金the Chinese Postdoctoral Science Foundation(2022M710144)the“Shuguang Program”supported by Shanghai Education Development Foundation and Shanghai Municipal Commission(17SG54)Shanghai Rising-Star Program(20QA1406900,22QA1409300)the Research Team Project of Natural Science Foundation of Guangdong Province of China(2017A030312007)the Science and Technology Planning Project of Guangdong Province(2022B1212010010)the Key Program of Guangzhou Science Research Plan(201904020047)the Special Project of Dengfeng Program of Guangdong Provincial People’s Hospital(DFJH201812,KJ012019119,KJ012019423)the Shanghai Tech University Start-Up Fund。
文摘Arrhythmogenic cardiomyopathy(ACM)is one of the most common inherited cardiomyopathies,characterized by progressive fibrofatty replacement in the myocardium.However,the cellular origin of cardiac adipocytes in ACM remains largely unknown.Unraveling the cellular source of cardiac adipocytes in ACM would elucidate the underlying pathological process and provide a potential target for therapy.Herein,we generated an ACM mouse model by inactivating desmosomal gene desmoplakin in cardiomyocytes;and examined the adipogenic fates of several cell types in the disease model.The results showed that SOX9^(+),PDGFRa^(+),and PDGFRb^(+)mesenchymal cells,but not cardiomyocytes or smooth muscle cells,contribute to the intramyocardial adipocytes in the ACM model.Mechanistically,Bmp4 was highly expressed in the ACM mouse heart and functionally promoted cardiac mesenchymal-to-adipose transition in vitro.
基金supported by the National Natural Science Foundation of China(31871477 and 32170971)the Natural Science Foundation of Shanghai(18ZR1403800)the National Key Basic Research Program of China(973 Program,2014CB965001).
文摘tAstrocytes are the largest glial population in the mammalian brain.However,we have a minimal understanding of astrocyte development,especially fate specification in different regions of the brain.Through lineage tracing of the progenitors of the third ventricle(3V)wall via in-utero electroporation in the embryonic mouse brain,we show the fate specification and migration pattern of astrocytes derived from radial glia along the 3V wall.Unexpectedly,radial glia located in different regions along the 3V wall of the diencephalon produce distinct cell types:radial glia in the upper region produce astrocytes and those in the lower region produce neurons in the diencephalon.With genetic fate mapping analysis,we reveal that the first population of astrocytes appears along the zona incerta in the diencephalon.Astrogenesis occurs at an early time point in the dorsal region relative to that in the ventral region of the developing diencephalon.With transcriptomic analysis of the region-specific 3V wall and lateral ventricle(LV)wall,we identified cohorts of differentially-expressed genes in the dorsal 3V wall compared to the ventral 3V wall and LV wall that may regulate astrogenesis in the dorsal diencephalon.Together,these results demonstrate that the generation of astrocytes shows a spatiotemporal pattern in the developing mouse diencephalon.
文摘In recent years, stem cells have been a focal point in research designed to evaluate the efficacy of ophthalmologic therapies, specifically those for corneal conditions. The corneal epithelium is one of the few regions of the body that maintains itself using a residual stem cell population within the adjacent limbus. Stem cell movement has additionally captivated the minds of researchers due to its potential application in different body regions. The cornea is a viable model for varying methods to track stem cell migratory patterns, such as lineage tracing and live imaging from the limbus. These developments have the potential to pave the way for future therapies designed to ensure the continuous regeneration of the corneal epithelium following injury via the limbal stem cell niche. This literature review aims to analyze the various methods of imaging used to understand the limbal stem cell niche and possible future directions that might be useful to consider for the better treatment and prevention of disorders of the cornea and corneal epithelium. .
基金supported by the National Key Research and Development Program of China(2017YFA0103603 to X.W.)supported by Beijing Natural Science Foundation(Z190016 to X.W.)。
文摘The intestinal epithelium is one of the most rapidly renewing tissues,which is fueled by stem cells at the base of the crypts.Strategies of genetic lineage tracing and organoids,which capture major features of original tissues,are powerful avenues for exploring the biology of intestinal stem cells in vivo and in vitro,respectively.The combination of intestinal organoideculturing system and genetic modification approaches provides an attractive platform to uncover the mechanism of colorectal cancer and genetic disorders in the human minigut.Here,we will provide a comprehensive overview of studies on intestinal epithelium and intestinal stem cells.We will also review the applications of organoids and genetic markers in intestinal research studies.Furthermore,we will discuss the advantages and drawbacks of organoids as disease models compared with mice models and cell lines.
基金supported by the Natural Science Foundation of Guangdong Province of China,Nos.2021A1515011237(to WL),2020A1515010854(to QSW)the National Natural Science Foundation of China,Nos.U1801681(to GC),31701291(to WL)the Guangdong Province Science and Technology Planning Project of China,No.2018B030332001(to GC)。
文摘5-Bromo-2′-deoxyuridine(BrdU)is a halogenated pyrimidine that can be incorporated into newly synthesized DNA during the S phase of the cell cycle.BrdU is widely used in fate-mapping studies of embryonic and adult neurogenesis to identify newborn neurons,however side effects on neural stem cells and their progeny have been reported.In vivo astrocyte-to-neuron(AtN)conversion is a new approach for generating newborn neurons by directly converting endogenous astrocytes into neurons.The BrdU-labeling strategy has been used to trace astrocyte-converted neurons,but whether BrdU has any effect on the AtN conversion is unknown.Here,while conducting a NeuroD1-mediated AtN conversion study using BrdU to label dividing reactive astrocytes following ischemic injury,we accidentally discovered that BrdU inhibited AtN conversion.We initially found a gradual reduction in BrdU-labeled astrocytes during NeuroD1-mediated AtN conversion in the mouse cortex.Although most NeuroD1-infected astrocytes were converted into neurons,the number of BrdU-labeled neurons was surprisingly low.To exclude the possibility that this BrdU inhibition was caused by the ischemic injury,we conducted an in vitro AtN conversion study by overexpressing NeuroD1 in cultured cortical astrocytes in the presence or absence of BrdU.Surprisingly,we also found a significantly lower conversion rate and a smaller number of converted neurons in the BrdU-treated group compared with the untreated group.These results revealed an unexpected inhibitory effect of BrdU on AtN conversion,suggesting more caution is needed when using BrdU in AtN conversion studies and in data interpretation.
文摘Tooth enamel,a highly mineralized tissue covering the outermost area of teeth,is always damaged by dental caries or trauma.Tooth enamel rarely repairs or renews itself,due to the loss of ameloblasts and dental epithelial stem cells(DESCs)once the tooth erupts.Unlike human teeth,mouse incisors grow continuously due to the presence of DESCs that generate enamel-producing ameloblasts and other supporting dental epithelial lineages.The ready accessibility of mouse DESCs and wide availability of related transgenic mouse lines make mouse incisors an excellent model to examine the identity and heterogeneity of dental epithelial stem/progenitor cells;explore the regulatory mechanisms underlying enamel formation;and help answer the open question regarding the therapeutic development of enamel engineering.In the present review,we update the current understanding about the identification of DESCs in mouse incisors and summarize the regulatory mechanisms of enamel formation driven by DESCs.The roles of DESCs during homeostasis and repair are also discussed,which should improve our knowledge regarding enamel tissue engineering.
文摘During the development of mammalian heart, the left and right atria play an important role in cardiovascular circulation. The embryonic atrium is mainly formed by the differentiation of progenitor cells and the proliferation of cardiomyocytes, while the postnatal atrium is primarily shaped by the increase in the volume of cardiomyocytes. Cell proliferation and differentiation of atrial development is the basis for its functions such as “blood reservoir” and “supplementary pump”. Deep understanding the cellular mechanism of atrial development is imperative to explore the causes of common congenital arrhythmia heart diseases such as atrial fibrillation. We used genetically engineered mouse reproduction knowledge, lineage tracing method based on CreloxP system, molecular biology and immunofluorescence technology to track the cardiomyocyte lineage of Nppa-GFP mouse line with stereo fluorescence microscope and ultra-high-speed confocal microscope. Besides the atrium of Nppa-CreER;Rosa26 tdTomato mouse was examined during embryonic (E10.5 - E18.5) and postnatal (P0, P3, P5, P7, P14, P28, P8w) stage. Immunofluorescence results revealed that Nppa-positive cells labeled TNNI3-positive cardiomyocytes and protruded into the atrial cavity at the beginning of E11.5 - E12.0 and during subsequent development to form Nppa-positive myocardial trabeculae. Thick comb-shaped myocardium was observed after birth, and we suspect that this was particularly important for the normal contractile activity and pumping function of the atrium. Additionally, non-single origin of Nppa-positive trabecular myocardiocytes were revealed through Tamoxifen-induced lineage tracing experiment. Our findings reveal proliferation dynamics and non-comprehensive fate decisions of cardiomyocytes that produce the distinct architecture of the atrium chamber.
基金supported in part by the National Natural Science Foundation of China(T2225005)the National Health Commission(2023ZD0519900,2023ZD0520300)Shanxi Medical University-Tsinghua Collaborative Innovation Center for Frontier Medicine。
文摘Tissue-resident memory T cells(T_(RM))play a key role in defense against pathogen invading barrier sites and other non-lymphoid tissues.How T_(RM)cells are maintained in various tissues,and how they relate to antigen-experienced memory T cells in lymphoid organs are not fully understood.By barcode-based lineage tracing and single-cell transcriptome analysis,we found a distinct population of CD69^(+)CD103^(+)virusspecific CD8^(+)T cells in draining lymph nodes(dLNs)following intranasal influenza infection.Intriguingly,these dLN T_(RM)cells and lung T_(RM)cells shared similar function-neutral barcode contents and transcriptomic features,implicating local circulation between the lung and dLNs in the maintenance of resident memory.Ablation of CXCR3 from CD8^(+)T cells,which impairs lung T_(RM)generation,severely reduced the abundance of dLN T_(RM)cells,suggesting that dLN T_(RM)cells are connected to lung T_(RM)cells by retrograde migration.Our screen for chemokines and chemokine receptors implicated the CCR5-CCL5 axis in promoting lung-to-dLN migration.Temporary CCR5 blockade by intratracheal administration of Maraviroc,a CCR5 inhibitor,reduced the abundance of dLN T_(RM)cells without affecting lung T_(RM)cells.By intratracheal cell transfer,CCR5-deficient CD8^(+)T cells were found impaired in lung-to-dLN migration.Finally,dLN T_(RM)cells actively participated in the secondary response and could reconstitute lung T_(RM)cells following influenza infection.Our results support a model in which lung-to-dLN T_(RM)retrograde migration helps maintain the CD8^(+)memory resident in the respiratory tract and optimizes the local Tcell response to reinfection.
基金supported by the National Natural Science Foundation of China(no.82172420)China Postdoctoral Science Foundation(2022M710260)Peking University Medicine Sailing Program for Young Scholars’Scientific&Technological Innovation(BMU2023YFJHMX008 and BMU2023YFJHPY023).
文摘The effective and translational strategy to regenerate knee meniscal fibrocartilage remained challenging.Herein,we first identified vascular smooth muscle cells(VSMCs)transdifferentiated into fibrochondrocytes and participated in spontaneous meniscal regeneration using smooth muscle cell lineage tracing transgenic mice meniscal defect model.Then,we identified low-intensity pulsed ultrasound(LIPUS)acoustic stimulus enhanced fibrochondrogenic transdifferentiation of VSMCs in vitro and in vivo.Mechanistically,LIPUS stimulus could up-regulate mechanosensitive ion channel Piezo1 expression and then activate the transforming growth factorβ1(TGFβ1)signal,following repression of the Notch signal,consequently enhancing fibrochondrogenic transdifferentiation of VSMCs.Finally,we demonstrated that the regular LIPUS stimulus enhanced anisotropic native-like meniscal fibrocartilage tissue regeneration in a beagle canine subtotal meniscectomy model at 6 months postoperatively.The single-cell RNA sequencing analysis confirmed the role of VSMC fibrochondrogenic transdifferentiation in meniscal regeneration.
