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.

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.

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.

CRISPR/Cas系统在谱系追踪中的应用

CRISPR/Cas系统是细菌和古生菌的自适应免疫系统,其对基因组高效精准的编辑,极大地推动了发育生物学、表观遗传学、药物开发、疾病治疗等多个学科和研究领域的发展.CRISPR/Cas9系统诱导基因组DNA产生双链断裂,以非同源末端连接(non-homologous end joining,NHEJ)的方式进行修复,因此会在剪切位置随机地引入短的插入和删除(insertions and deletions,indels).这些引入的indels作为区分细胞的标签,被称为条形码.细胞条形码技术已经被用于谱系追踪、基因组功能筛选等.而测序技术的飞速发展和成本的大幅度降低以及单细胞转录组测序技术的出现,可以在时间和空间层面同时对数百万个单细胞进行谱系追踪,记录细胞活动.本综述讨论了CRISPR/Cas系统的工作原理、细胞条形码技术和单细胞测序技术(scRNA-seq),以及两者结合产生的单细胞谱系追踪技术.

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.

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.