Lineage tracing of direct astrocyte-to-neuron conversion in the mouse cortex

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.

Central nervous system and peripheral cell labeling by vascular endothelial cadherin-driven lineage tracing in adult mice

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.

Genetic lineage tracing identifies adaptive mechanisms of pancreatic islet β cells in various mouse models of diabetes with distinct age of initiation

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.

Genetic lineage tracing identifies cardiac mesenchymal-to-adipose transition in an arrhythmogenic cardiomyopathy model

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.

Dual recombinases-based genetic lineage tracing for stem cell research with enhanced precision

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.

Connecting past and present:single-cell lineage tracing

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.

Embryonic lineage tracing with Procr-CreER marks balanced hematopoietic stem cell fate during entire mouse lifespan

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.

MEMOIR: A Novel System for Neural Lineage Tracing

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

The Limbal Niche and Its Role in Maintaining Corneal Regeneration

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. .

Unexpected BrdU inhibition on astrocyte-to-neuron conversion

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.

New insight into dental epithelial stem cells:Identification,regulation,and function in tooth homeostasis and repair

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.

Resolving the lineage relationship between malignant cells and vascular cells in glioblastomas

Glioblastoma multiforme(GBM),a highly malignant and heterogeneous brain tumor,contains various types of tumor and non-tumor cells.Whether GBM cells can trans-differentiate into non-neural cell types,including mural cells or endothelial cells(ECs),to support tumor growth and invasion remains controversial.Here we generated two genetic GBM models de novo in immunocompetent mouse brains,mimicking essential pathological and molecular features of human GBMs.Lineage-tracing and transplantation studies demonstrated that,although blood vessels in GBM brains underwent drastic remodeling,evidence of trans-differentiation of GBM cells into vascular cells was barely detected.Intriguingly,GBM cells could promiscuously express markers for mural cells during gliomagenesis.Furthermore,single-cell RNA sequencing showed that patterns of copy number variations(CNVs)of mural cells and ECs were distinct from those of GBM cells,indicating discrete origins of GBM cells and vascular components.Importantly,single-cell CNV analysis of human GBM specimens also suggested that GBM cells and vascular cells are likely separate lineages.Rather than expansion owing to trans-differentiation,vascular cell expanded by proliferation during tumorigenesis.Therefore,cross-lineage trans-differentiation of GBM cells is very unlikely to occur during gliomagenesis.Our findings advance understanding of cell lineage dynamics during gliomagenesis,and have implications for targeted treatment of GBMs.

Cellular Mechanism of Mouse Atrial Development

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.

Temporal-spatial Generation of Astrocytes in the Developing Diencephalon

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.

scRNA-seq profiling of neonatal and adult thymus-derived CD4+ T cells by a T cell origin-time tracing model

It is well documented that the neonatal thymus-derived (neonatal-TD) regulatory T cells (Treg) are essential to prevent lethal autoimmune diseases and allergies, and neonatal and adult thymus possesses distinct output potentials for naïve T cells, including Treg. However, the molecular features and detailed functional differences between neonatal-TD and adult thymus-derived (adult-TD) T cells in terms of their ability to maintain immune homeostasis during long-term environmental influences are still largely unknown, partially due to the lack of appropriate animal models to precisely trace these cells at specific time points. In this study, neonatal-TD and adult-TD CD4+ T cells from the spleen and Peyer's patches were traced for 9 weeks by a T cell origin-time tracing mouse model and analysed by single-cell RNA sequencing. More Treg but fewer naïve T cells were found in neonatal-TD CD4+ T cells from both tissues than those from adult-TD counterparts. Interestingly, the neonatal-TD Treg in both the spleen and Peyer's patches exhibited augmented expression of Foxp3, Gata3, Ctla4, Icos, Il2ra, Tgfb1, and Nrp1, as well as enriched Gene Ontology terms like T cell activation and tolerance induction, indicating an enhanced immunosuppressive function. These results were further confirmed by flow cytometry analysis and in vitro immune suppression assays. Flow cytometry also revealed a significantly higher proportion of neonatal-TD Treg in total Treg than that of adult-TD counterparts, suggesting the longer lifespan of neonatal-TD Treg. To investigate the intrinsic features of neonatal-TD and adult-TD CD4+ T cells, a shortened tracing time was performed. Surprisingly, the neonatal-TD and adult-TD CD4+ T cells had similar proportions of Treg and did not exhibit significant differences in Foxp3, Gata3, Ctla4, Icos, Il2ra, and Tgfb1 expression levels after tracing for 12 days. On the other hand, neonatal-TD Treg present an increased Nrp1 expression level compared with adult-TD counterparts, indicating the enhanced stability. Together, our work reveals that the neonatal-TD Treg are more immunosuppressive, which is likely shaped primarily by environmental factors.

Recent advances in single-cell sequencing technologies

Single-cell omics sequencingwas first achieved for the transcriptome in 2009,whichwas followed by fast development of technologies for profiling the genome,DNA methylome,3D genome architecture,chromatin accessibility,histone modifications,etc.,in an individual cell.In this review we mainly focus on the recent progress in four topics in the single-cell omics field:single-cell epigenome sequencing,single-cell genome sequencing for lineage tracing,spatially resolved single-cell transcriptomics and third-generation sequencing platform-based single-cell omics sequencing.We also discuss the potential applications and future directions of these single-cell omics sequencing technologies for different biomedical systems,especially for the human stem cell field.

Intestinal stem cells and intestinal organoids

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.

Transgenic mouse models for studying adult neurogenesis

The mammalian hippocampus shows a remarkable capacity for continued neurogenesis throughout life. Newborn neurons, generated by the radial neural stem cells （NSCs）, are important for learning and memory as well as mood control. During aging, the number and responses of NSCs to neurogenic stimuli diminish, leading to decreased neurogenesis and age-associatedcognitive decline and psychiatric disorders. Thus, adult hippocampal neurogenesis has garnered significant interest because targeting it could be a novel potential therapeutic strategy for these disorders. However, if we are to use nenrogenesis to halt or reverse hippocampal-related pathology, we need to understand better the core molecular machinery that governs NSC and their progeny. In this review, we summarize a wide variety of mouse models used in adult neurogenesis field, present their advantages and disadvantages based on specificity and efficiency of labeling of different cell types, and review their contribution to our understanding of the biology and the heterogeneity of different cell types found in adult neurogenic niches.

Tbr2-expressing intermediate progenitor cells in the adult mouse hippocampus are unipotent neuronal precursors with limited amplification capacity under homeostasis

Neurogenesis persists in two locations of the adult mammalian brain, the subventricular zone of the lateral ventricles and the subgranular zone of the dentate gyrus in the hippocampus. In the adult subgranular zone, radial glial- like cells （RGLs） are multipotent stem cells that can give rise to both astrocytes and neurons. In the process of generating neurons, RGLs divide asymmetrically to give rise to one RGL and one intermediate progenitor cell （IPC）. IPCs are considered to be a population of transit amplifying cells that proliferate and eventually give rise to mature granule neurons. The properties of individual IPCs at the clonai level are not well understood. Furthermore, it is not clear whether IPCs can generate astrocytes or revert back to RGLs, besides generating neurons. Here we developed a genetic marking strategy for clonal analysis and lineage-tracing of individual Tbr2-expressing IPCs in the adult hippocampus in vivo using Tbr2-CreERT2 mice. Using this technique we identified Tbr2-CreERT2 labeled IPCs as unipotent neuronal precursors that do not generate astrocytes or RGLs under homeostasis. Additionally, we showed that these labeled IPCs rapidly generate immature neurons in a synchronous manner and do not undergo a significant amount of amplification under homeostasis, in animals subjected to an enriched environment/running, or in animals with different age. In summary, our study suggests that Tbr2-expressing IPCs in the adult mouse hippocampus are unipotent precursors and rapidly give rise to immature neurons without major amplification.

Decoding the annulus fibrosus cell atlas by scRNA-seq to develop an inducible composite hydrogel:A novel strategy for disc reconstruction

Low back pain is one of the most serious public health problems worldwide and the major clinical manifestation of intervertebral disc degeneration(IVDD).The key pathological change during IVDD is dysfunction of the annulus fibrosus(AF).However,due to the lack of an in-depth understanding of AF biology,the methods to reconstruct the AF are very limited.In this study,the mice AF cell atlas were decoded by single-cell RNA sequencing to provide a guide for AF reconstruction.The results first identify a new population of AF cells,fibrochondrocyte-like AF cells,which synthesize both collagen Ⅰ and collagen Ⅱ and are potential functional cells for AF reconstruction.According to the dual features of the AF extracellular matrix,a composite hydrogel based on the acylation of methacrylated silk fibroin with methacrylated hyaluronic acid was produced.To obtain the ability to stimulate differentiation,the composite hydrogels were combined with a fibrochondrocyte-inducing supplement.Finally,reconstruction of the AF defects,by the novel AF stem cell-loaded composite hydrogel,could be observed,its amount of chondroid matrices recovered to 31.7% of AF aera which is significantly higher than that in other control groups.In summary,this study decodes the AF cell atlas,based on which a novel strategy for AF reconstruction is proposed.