Lineage Tracing Methods Research Articles

Developmental mosaicism underlying EGFR-mutant lung cancer presenting with multiple primary tumors.

Although the development of multiple primary tumors in smokers with lung cancer can be attributed to carcinogen-induced field cancerization, the occurrence of multiple tumors at presentation in individuals with EGFR-mutant lung cancer who lack known environmental exposures remains unexplained. In the present study, we identified ten patients with early stage, resectable, non-small cell lung cancer who presented with multiple, anatomically distinct, EGFR-mutant tumors. We analyzed the phylogenetic relationships among multiple tumors from each patient using whole-exome sequencing (WES) and hypermutable poly(guanine) (poly(G)) repeat genotyping as orthogonal methods for lineage tracing. In four patients, developmental mosaicism, assessed by WES and poly(G) lineage tracing, indicates a common non-germline cell of origin. In two other patients, we identified germline EGFR variants, which confer moderately enhanced signaling when modeled in vitro. Thus, in addition to germline variants, developmental mosaicism defines a distinct mechanism of genetic predisposition to multiple EGFR-mutant primary tumors, with implications for their etiology and clinical management.

Genetic Tools for Cell Lineage Tracing and Profiling Developmental Trajectories in the Skin

The epidermis is the body's first line of protection against dehydration and pathogens, continually regenerating the outermost protective skin layers throughout life. During both embryonic development and wound healing, epidermal stem and progenitor cells must respond to external stimuli and insults to build, maintain, and repair the cutaneous barrier. Recent advances in CRISPR-based methods for cell lineage tracing have remarkably expanded the potential for experiments that track stem and progenitor cell proliferation and differentiation over the course of tissue and even organismal development. Additional tools for DNA-based recording of cellular signaling cues promise to deepen our understanding of the mechanisms driving normal skin morphogenesis and response to stressors as well as the dysregulation of cell proliferation and differentiation in skin diseases and cancer. In this review, we highlight cutting-edge methods for cell lineage tracing, including in organoids and model organisms, and explore how cutaneous biology researchers might leverage these techniques to elucidate the developmental programs that support the regenerative capacity and plasticity of the skin.

Tracing back primed resistance in cancer via sister cells

Exploring non-genetic evolution of cell states during cancer treatments has become attainable by recent advances in lineage-tracing methods. However, transcriptional changes that drive cells into resistant fates may be subtle, necessitating high resolution analysis. Here, we present ReSisTrace that uses shared transcriptomic features of sister cells to predict the states priming treatment resistance. Applying ReSisTrace in ovarian cancer cells perturbed with olaparib, carboplatin or natural killer (NK) cells reveals pre-resistant phenotypes defined by proteostatic and mRNA surveillance features, reflecting traits enriched in the upcoming subclonal selection. Furthermore, we show that DNA repair deficiency renders cells susceptible to both DNA damaging agents and NK killing in a context-dependent manner. Finally, we leverage the obtained pre-resistance profiles to predict and validate small molecules driving cells to sensitive states prior to treatment. In summary, ReSisTrace resolves pre-existing transcriptional features of treatment vulnerability, facilitating both molecular patient stratification and discovery of synergistic pre-sensitizing therapies.

Abstract B037: ClonMapper Duo: An scRNAseq compatible lineage tracing method to track cell-cell fusion at single-cell resolution

Abstract Spontaneous cell-cell fusion in cancer can generate genetic heterogeneity and often leads to more pathological phenotypes. While the consequences of cell-cell fusion in cancer continue to be revealed, few tools exist to determine the mechanisms and cell states which promote cell-cell fusion. To meet this need, we engineered ClonMapper Duo, a two-color, two-index expressed barcoding system which allows tracking of cell-cell fusion using live-cell imaging and lineage tracing of fusion cells in scRNAseq. The ClonMapper Duo system was created by modifying the ClonMapper DNA barcoding system to express indexed barcodes on either H2B-GFP or H2B-mCherry. Briefly, 8 unique 5-nucleotide sequences were designed by maximizing Hamming distance and 4 sequences were assigned to GFP and mCherry each. The index sequences were placed adjacent to a 15N random nucleotide in the expressed barcode region of the construct. Low-diversity GFP and mCherry barcoded cell libraries were created through lentiviral transduction of HCC1806, MDA-MB-231, and MCF-7 cells. Diversity and evenness of each library was confirmed via targeted barcode sequencing. For each cell line, homotypic GFP and mCherry barcoded cells were cocultured together for 1 week. A parallel culture was monitored using live-cell imaging to measure the rate of spontaneous cell-cell fusion and the probability of different fates (e.g. death, hybrid formation, senescence) for each cell line. Spontaneously fused cells identified by expression of both GFP and mCherry were enriched by FACS. Enriched fusion cells were expanded in parallel with the parental cocultures until ~1e5 fusion cells or 1 week was reached. Fusion and parental populations for each cell line were multiplexed for scRNAseq using the 10X Genomics 3’ CellPlex Kit. FACS enriched multibarcoded fusion cells were further bioinformatically distinguished from cell multiplets and the barcodes of spontaneously fused cells were traced to their single-barcoded cell lineage of origin. From this data set we ask: 1. Are certain cell lineages more primed for fusion than others, i.e. does a more fusogenic cell phenotype exist or is fusion random? 2. Is there a convergent state of post-fusion cells or does fusion tend to diversify phenotypes? Here, we will present the first results from this two-color, two-index live-cell imaging and scRNAseq lineage tracing system toward understanding cancer cell-cell fusion at a single cell resolution. Combining lineage traced transcriptomic data from scRNAseq with fusion rates and outcomes from live-cell imaging using ClonMapper Duo provides an unprecedented look into the causes and consequences of spontaneous cell-cell fusion in cancer. Citation Format: Andrea L. Gardner, Lan Zheng, Daylin Morgan, Kennedy Howland, Amy Brock. ClonMapper Duo: An scRNAseq compatible lineage tracing method to track cell-cell fusion at single-cell resolution [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr B037.

Genetic Lineage Traces the Differentiation Fate of Epicardial Cells during Heart Development

Objective: The genetic lineage tracing method was used to examine the Epithelial-Mesenchymal Transition (EMT) process and the contribution of epicardial cells to mesenchymal cells at various stages of fetal heart development. Methods: In Wt1-CreER; R26-tdTomato transgenic mice, tamoxifen was utilized to promote the tagging of epicardial cells with tdTomato fluorescence at E10. At E11.5, E12.5, and E16.5, embryonic hearts were harvested and photographed using confocal fluorescence microscopy and stereomicroscopy. Results: According to the findings, the tdTomato+ cells at E11.5 were still in the epicardium and had not yet moved into the myocardium. Epicardial cells began to separate from the epicardium and give rise to epicardial-derived cells at embryonic day 12.5 (E12.5). On the valve primordium, fibroblasts generated from epicardium have been found. By E16.5, many epicardial cells had moved into the myocardium and formed fibroblasts, mesenchymal cells, vascular smooth muscle cells, as well as migrated into the ventricular septum and valves, contributing to their growth and creation. Conclusions: The contribution of epicardial cells to mesenchymal cells during development is shown by genetic lineage tracing, opening up possibilities and offering references for creating relevant treatment approaches based on epicardial cells.

Single-cell lineage capture across genomic modalities with CellTag-multi reveals fate-specific gene regulatory changes.

Complex gene regulatory mechanisms underlie differentiation and reprogramming. Contemporary single-cell lineage-tracing (scLT) methods use expressed, heritable DNA barcodes to combine cell lineage readout with single-cell transcriptomics. However, reliance on transcriptional profiling limits adaptation to other single-cell assays. With CellTag-multi, we present an approach that enables direct capture of heritable random barcodes expressed as polyadenylated transcripts, in both single-cell RNA sequencing and single-cell Assay for Transposase Accessible Chromatin using sequencing assays, allowing for independent clonal tracking of transcriptional and epigenomic cell states. We validate CellTag-multi to characterize progenitor cell lineage priming during mouse hematopoiesis. Additionally, in direct reprogramming of fibroblasts to endoderm progenitors, we identify core regulatory programs underlying on-target and off-target fates. Furthermore, we reveal the transcription factor Zfp281 as a regulator of reprogramming outcome, biasing cells toward an off-target mesenchymal fate. Our results establish CellTag-multi as a lineage-tracing method compatible with multiple single-cell modalities and demonstrate its utility in revealing fate-specifying gene regulatory changes across diverse paradigms of differentiation and reprogramming.

The origin, progress, and application of cell-based cardiac regeneration therapy.

Cardiovascular disease (CVD) has become a severe threat to human health, with morbidity and mortality increasing yearly and gradually becoming younger. When the disease progresses to the middle and late stages, the loss of a large number of cardiomyocytes is irreparable to the body itself, and clinical drug therapy and mechanical support therapy cannot reverse the development of the disease. To explore the source of regenerated myocardium in model animals with the ability of heart regeneration through lineage tracing and other methods, and develop a new alternative therapy for CVDs, namely cell therapy. It directly compensates for cardiomyocyte proliferation through adult stem cell differentiation or cell reprogramming, which indirectly promotes cardiomyocyte proliferation through non-cardiomyocyte paracrine, to play a role in heart repair and regeneration. This review comprehensively summarizes the origin of newly generated cardiomyocytes, the research progress of cardiac regeneration based on cell therapy, the opportunity and development of cardiac regeneration in the context of bioengineering, and the clinical application of cell therapy in ischemic diseases.

Regionally distinct progenitor cells in the lower airway give rise to neuroendocrine and multiciliated cells in the developing human lung

Using scRNA-seq and microscopy, we describe a cell that is enriched in the lower airways of the developing human lung and identified by the unique coexpression of SCGB3A2/SFTPB/CFTR. To functionally interrogate these cells, we apply a single-cell barcode-based lineage tracing method, called CellTagging, to track the fate of SCGB3A2/SFTPB/CFTR cells during airway organoid differentiation in vitro. Lineage tracing reveals that these cells have a distinct differentiation potential from basal cells, giving rise predominantly to pulmonary neuroendocrine cells and a subset of multiciliated cells distinguished by high C6 and low MUC16 expression. Lineage tracing results are supported by studies using organoids and isolated cells from the lower noncartilaginous airway. We conclude that SCGB3A2/SFTPB/CFTR cells are enriched in the lower airways of the developing human lung and contribute to the epithelial diversity and heterogeneity in this region.

Abstract 21: A novel bladder cancer syngeneic mouse model using gene-edited organoids derived from bladder urothelium

Abstract In recent years, immuno-checkpoint inhibitors have expanded the range of treatment options in advanced bladder cancer, but there are still many issues to be overcome, such as drug resistance, effect of combination therapy and prediction of adverse events. To address these issues, there is an urgent need for useful animal models with normal immune system. About half of the human muscle invasive bladder cancers (MIBCs) show p53 mutations in which over 60% are missense mutations. Additionally, the downregulation of tumor suppressor gene PTEN is known in human MIBC. We also investigated cell of origin of chemical-induced mouse bladder cancer using lineage tracing method and found that Krt5 expressing cells with Trp53 alteration efficiently give a rise of high-grade MIBC with squamous differentiation. Based on these results, we have been working on establishing MIBC mouse model using genetically engineered mouse (GEM) in which Cas9 and target gene mutation express specifically in Krt5 expressing cells by Cre-LoxP system. We have also used CRISPR/Cas9 gene editing technique and 3D organoid culture system. We generated organoids from the GEM bladder urothelium with Krt5CreERT2/+; LSL-Cas9; LSL-Trp53R172H/+. For further gene editing, we infected adeno-associated virus with sgRNA to the organoids. The organoid with Trp53R172H/+ and Pten knock-out showed tumorigenicity in immunodeficient mice. The organoid-derived tumors are histologically similar with the basal-squamous subtype of human bladder cancer. Moreover, these tumors exhibited wild type (WT) Trp53 loss of heterozygosity in genomic level. Similarly, we confirmed tumorigenicity in immunodeficient mice with organoids from Trp53flox/flox; Ptenflox/flox and Trp53R172H/flox; Ptenflox/flox mice, but not from Trp53R172H/flox;Pten+/+ mice or Trp53R172H/+; Ptenflox/flox mice, suggesting that both loss of WT Trp53 and Pten loss are required for tumor formation in this setting. RNA sequencing showed Pten loss is associated with up-regulation of metabolism related gene sets and loss of WT Trp53 is associated with up-regulation of proliferation related gene sets. Furthermore, Trp53R172H/flox; Ptenflox/flox organoids showed higher engraftment rate in immunocompetent mice with less infiltration of CD8+ cells compared with Trp53flox/flox; Ptenflox/flox organoids, suggesting that gain of function of mutant Trp53 may affect immune microenvironment. Our data illustrates the usefulness of clinically relevant syngeneic bladder cancer mouse model from GEM-derived organoids for the research of tumor immunity. Citation Format: Akihiro Hamada, Yuki Kita, Takeshi Sano, Ryosuke Ikeuchi, Hideaki Takada, Kenji Nakamura, Toru Sakatani, Takayuki Goto, Atsuro Sawada, Shusuke Akamatsu, Takashi Kobayashi. A novel bladder cancer syngeneic mouse model using gene-edited organoids derived from bladder urothelium [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 21.

Origins of pathological myofibroblasts in lung fibrosis: insights from lineage tracing mouse models in the single-cell RNA sequencing era.

Idiopathic pulmonary fibrosis (IPF) is a rare interstitial lung disease with a poor prognosis. Chronic microinjuries, mainly caused by environmental factors to an aging alveolar epithelium, would lead to the aberrant differentiation and accumulation of aberrant mesenchymal cells with a contractile phenotype, known as fibrosis-associated myofibroblasts, which trigger abnormal extracellular matrix accumulation and fibrosis. The origin of those pathological myofibroblasts in pulmonary fibrosis is not fully understood to date. Lineage tracing methods using mouse models have opened new avenues for studying cell fate in a pathological context. This review aims to present a nonexhaustive list of different potential sources of those harmful myofibroblasts during lung fibrosis, based on these in vivo approaches, and considering the normal and fibrotic lung cellular atlas recently established by single-cell RNA sequencing.

New Tools for Lineage Tracing in Cancer In Vivo

During tumor evolution, cancer cells can acquire the ability to proliferate, invade neighboring tissues, evade the immune system, and spread systemically. Tracking this process remains challenging, as many key events occur stochastically and over long times, which could be addressed by studying the phylogenetic relationships among cancer cells. Several lineage tracing approaches have been developed and employed in many tumor models and contexts, providing critical insights into tumor evolution. Recent advances in single-cell lineage tracing have greatly expanded the resolution, scale, and readout of lineage tracing toolkits. In this review, we provide an overview of static lineage tracing methods, and then focus on evolving lineage tracing technologies that enable reconstruction of tumor phylogenies at unprecedented resolution. We also discuss in vivo applications of these technologies to profile subclonal dynamics, quantify tumor plasticity, and track metastasis. Finally, we highlight outstanding questions and emerging technologies for building comprehensive cancer evolution roadmaps.

Gastric stem cell research and gastric organoids

The stomach is a complex organ lined with ordered epithelium consisting of different adult stem cell (ASC) pools. In the previous decade, research into gastric epithelial stem cells has been performed using lineage tracing methods, and several putative ASC markers in the gastric gland have been identified, although their roles in homeostasis maintenance and the origin of cancer remain to be clarified. With advances in gastric stem cell research, 3-dimensional (3D) organoid culture has been developed on the basis of in-depth insights into the control of stem cell self-renewal, proliferation, and differentiation. Since the initial report that single intestinal stem cells have the ability to generate long-lived 3D structures that exhibit budding forms and self-renewal, tissue-specific adaptations of this method have been established in various organs, such as the small intestine, colon, liver, and stomach. In the murine stomach, putative ASCs isolated from the corpus and antrum generate gastric organoids that can simulate organ-specific cells to some extent. In addition, a few trials have been conducted to generate long-lived 3D organoids using human-derived ASCs and pluripotent stem cells. We hope that this review will provide comprehensive knowledge on gastric stem cell research and gastric organoids.

CellDestiny: A RShiny application for the visualization and analysis of single-cell lineage tracing data.

Single-cell lineage tracing permits the labeling of individual cells with a heritable marker to follow the fate of each cell’s progeny. Over the last twenty years, several single-cell lineage tracing methods have emerged, enabling major discoveries in developmental biology, oncology and gene therapies. Analytical tools are needed to draw meaningful conclusions from lineage tracing measurements, which are characterized by high variability, sparsity and technical noise. However, the single cell lineage tracing field lacks versatile and easy-to-use tools for standardized and reproducible analyses, in particular tools accessible to biologists. Here we present CellDestiny, a RShiny app and associated web application developed for experimentalists without coding skills to perform visualization and analysis of single cell lineage-tracing datasets through a graphical user interface. We demonstrate the functionality of CellDestiny through the analysis of (i) lentiviral barcoding datasets of murine hematopoietic progenitors; (ii) published integration site data from Wiskott-Aldrich Symdrome patients undergoing gene-therapy treatment; and (iii) simultaneous barcoding and transcriptomic analysis of murine hematopoietic progenitor differentiation in vitro. In summary, CellDestiny is an easy-to-use and versatile toolkit that enables biologists to visualize and analyze single-cell lineage tracing data.

Clonal behaviour of myogenic precursor cells throughout the vertebrate lifespan.

ABSTRACTTo address questions of stem cell diversity during skeletal myogenesis, a Brainbow-like genetic cell lineage tracing method, dubbed Musclebow2, was derived by enhancer trapping in zebrafish. It is shown that, after initial formation of the primary myotome, at least 15 muscle precursor cells (mpcs) seed each somite, where they proliferate but contribute little to muscle growth prior to hatching. Thereafter, dermomyotome-derived mpc clones rapidly expand while some progeny undergo terminal differentiation, leading to stochastic clonal drift within the mpc pool. No evidence of cell-lineage-based clonal fate diversity was obtained. Neither fibre nor mpc death was observed in uninjured animals. Individual marked muscle fibres persist across much of the lifespan indicating low rates of nuclear turnover. In adulthood, early-marked mpc clones label stable blocks of tissue comprising a significant fraction of either epaxial or hypaxial somite. Fusion of cells from separate early-marked clones occurs in regions of clone overlap. Wounds are regenerated from several local mpcs; no evidence for specialised stem mpcs was obtained. In conclusion, our data indicate that most mpcs in muscle tissue contribute to local growth and repair and suggest that cellular turnover is low in the absence of trauma.

Abstract 6032: Establishment of a bladder cancer model using organoids derived from bladder epithelium in genetically engineered mice

Abstract Despite recent advances in molecular profiling of bladder cancer, molecular mechanisms for urothelial carcinogenesis have not fully understood. It is partly because the lack of faithful disease models that accurately recapitulate human bladder cancer. In our previous study, we have analyzed mutation landscape of chemical-induced mouse bladder cancer and human bladder cancer by exome-sequence and found that some components of histone methyltransferase complex like Kmt2c, Kmt2d, Kdm6a were highly prevalent as common mutations and associated with Trp53. Additionally, the downregulation of tumor suppressor gene PTEN is known in human muscle invasive bladder cancer (MIBC). We also investigated cell of origin of chemical-induced mouse bladder cancer using lineage tracing method and found that Krt5 expressing cells with Trp53 alteration efficiently give a rise of high-grade MIBC with squamous differentiation. Based on these results, we have tried to make MIBC mouse model using genetically engineered mouse in which Cas9 and target gene mutation express specifically in Krt5 expressing cells by Cre-LoxP system, combined with CRISPR/Cas9 gene editing technique and 3D organoid culture system. Eight-week-old mice harboring Krt5CreERT2/+; LSL-Cas9; LSL-Trp53+/R172H were used in this study. After introducing gene recombination by administration of tamoxifen, we generated organoids from bladder epithelial cells. For further gene editing, we infected adeno-associated virus with sgPten and sgMll3 to the organoids, which successfully edited the target genes. There seemed to be no significant differences in growth of the organoids in culture between the wild-type and gene-edited organoids. However, gene-edited organoids showed tumorigenesis in vivo when inoculated in subcutaneous space, renal subcapsular space or bladder submucosa of athymic mice, whereas wild-type organoids did not show tumorigenesis in vivo. Histologically, the organoid-derived tumors showed morphological similarity to human urothelial carcinoma with squamous differentiation and intra-tumoral heterogeneity. Furthermore, tumor derived organoid has a potential of tumor development in immune-competent mice. These tumors also maintain squamous phenotype and tumor heterogeneity. These findings suggest that the technique is a promising approach toward the establishment of a useful model of human bladder cancer for the elucidation of bladder carcinogenesis including driver genes. In addition, we think our syngeneic mouse model may be useful tool for further research of tumor immunity and immune checkpoint inhibitor drugs. Citation Format: Akihiro Hamada, Yuki Kita, Hideaki Takada, Kenji Nakamura, Toru Sakatani, Takeshi Sano, Takayuki Goto, Atsuro Sawada, Shusuke Akamatsu, Takashi Kobayashi. Establishment of a bladder cancer model using organoids derived from bladder epithelium in genetically engineered mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6032.

Genetic loss of function of Ptbp1 does not induce glia-to-neuron conversion in retina.

Direct reprogramming of glia into neurons is a potentially promising approach for the replacement of neurons lost to injury or neurodegenerative disorders. Knockdown of the polypyrimidine tract-binding protein Ptbp1 has been recently reported to induce efficient conversion of retinal Mϋller glia into functional neurons. Here, we use a combination of genetic lineage tracing, single-cell RNA sequencing (scRNA-seq), and electroretinogram analysis to show that selective induction of either heterozygous or homozygous loss-of-function mutants of Ptbp1 in adult retinal Mϋller glia does not lead to any detectable level of neuronal conversion. Only a few changes in gene expression are observed in Mϋller glia following Ptbp1 deletion, and glial identity is maintained. These findings highlight the importance of using genetic manipulation and lineage-tracing methods in studying cell-type conversion.

In preprints: the fast-paced field of single-cell lineage tracing

In preprints: the fast-paced field of single-cell lineage tracing

Dynamic Epicardial Contribution to Cardiac Interstitial c-Kit and Sca1 Cellular Fractions.

Background: The cardiac interstitial cellular fraction is composed of multiple cell types. Some of these cells are known to express some well-known stem cell markers such as c-Kit and Sca1, but they are no longer accepted to be true cardiac stem cells. Although their existence in the cardiac interstitium has not been disputed, their dynamic throughout development, specific embryonic origin, and potential heterogeneity remain unknown. In this study, we hypothesized that both c-KitPOS and Sca1POS cardiac interstitial cell (CIC) subpopulations are related to the Wilms’ tumor 1 (Wt1) epicardial lineage. Methods: In this study, we have used genetic cell lineage tracing methods, immunohistochemistry, and FACS techniques to characterize cardiac c-KitPOS and Sca1POS cells. Results: Our data show that approximately 50% of cardiac c-KitPOS cells are derived from the Wt1-lineage at E15.5. This subpopulation decreased along with embryonic development, disappearing from P7 onwards. We found that a large proportion of cardiac c-KitPOS cells express specific markers strongly suggesting they are blood-borne cells. On the contrary, the percentage of Sca1POS cells within the Wt1-lineage increases postnatally. In accordance with these findings, 90% of adult epicardial-derived endothelial cells and 60% of mEFSK4POS cardiac fibroblasts expressed Sca1. Conclusion: Our study revealed a minor contribution of the Wt1-epicardial lineage to c-KitPOS CIC from embryonic stages to adulthood. Remarkably, a major part of the adult epicardial-derived cell fraction is enriched in Sca1, suggesting that this subpopulation of CICs is heterogeneous from their embryonic origin. The study of this heterogeneity can be instrumental to the development of diagnostic and prognostic tests for the evaluation of cardiac homeostasis and cardiac interstitium response to pathologic stimuli.

Repressing PTBP1 fails to convert reactive astrocytes to dopaminergic neurons in a 6-hydroxydopamine mouse model of Parkinson's disease.

Lineage reprogramming of resident glial cells to dopaminergic neurons (DAns) is an attractive prospect of the cell-replacement therapy for Parkinson's disease (PD). However, it is unclear whether repressing polypyrimidine tract binding protein 1 (PTBP1) could efficiently convert astrocyte to DAns in the substantia nigra and striatum. Although reporter-positive DAns were observed in both groups after delivering the adeno-associated virus (AAV) expressing a reporter with shRNA or CRISPR-CasRx to repress astroglial PTBP1, the possibility of AAV leaking into endogenous DAns could not be excluded without using a reliable lineage-tracing method. By adopting stringent lineage-tracing strategy, two other studies show that either knockdown or genetic deletion of quiescent astroglial PTBP1 fails to obtain induced DAns under physiological condition. However, the role of reactive astrocytes might be underestimated because upon brain injury, reactive astrocyte can acquire certain stem cell hallmarks that may facilitate the lineage conversion process. Therefore, whether reactive astrocytes could be genuinely converted to DAns after PTBP1 repression in a PD model needs further validation. In this study, we used Aldh1l1-CreERT2-mediated specific astrocyte-lineage-tracing method to investigate whether reactive astrocytes could be converted to DAns in a 6-hydroxydopamine (6-OHDA) mouse model of PD. However, we found that no astrocyte-originated DAn was generated after effective and persistent knockdown of astroglial PTBP1 either in the substantia nigra or in striatum, while AAV 'leakage' to nearby neurons was easily observed. Our results confirm that repressing PTBP1 does not convert astrocytes to DAns, regardless of physiological or PD-related pathological conditions.

Recent Advances in Single-Cell View of Mesenchymal Stem Cell in Osteogenesis.

Osteoblasts continuously replenished by osteoblast progenitor cells form the basis of bone development, maintenance, and regeneration. Mesenchymal stem cells (MSCs) from various tissues can differentiate into the progenitor cell of osteogenic lineage and serve as the main source of osteoblasts. They also respond flexibly to regenerative and anabolic signals emitted by the surrounding microenvironment, thereby maintaining bone homeostasis and participating in bone remodeling. However, MSCs exhibit heterogeneity at multiple levels including different tissue sources and subpopulations which exhibit diversified gene expression and differentiation capacity, and surface markers used to predict cell differentiation potential remain to be further elucidated. The rapid advancement of lineage tracing methods and single-cell technology has made substantial progress in the characterization of osteogenic stem/progenitor cell populations in MSCs. Here, we reviewed the research progress of scRNA-seq technology in the identification of osteogenic markers and differentiation pathways, MSC-related new insights drawn from single-cell technology combined with experimental technology, and recent findings regarding the interaction between stem cell fate and niche in homeostasis and pathological process.