Multicolor Lineage Tracing Research Articles

44Endogenous cardiomyocyte-derived cardiac regeneration occurs slowly after myocardial infarction and continues into the late phase: multicolor lineage tracing mice model

44Endogenous cardiomyocyte-derived cardiac regeneration occurs slowly after myocardial infarction and continues into the late phase: multicolor lineage tracing mice model

Intestinal cancer stem cells marked by Bmi1 or Lgr5 expression contribute to tumor propagation via clonal expansion

Although the existence of cancer stem cells in intestine tumors has been suggested, direct evidence has not been yet provided. Here, we showed, using the multicolor lineage-tracing method and mouse models of intestinal adenocarcinoma and adenoma that Bmi1- or Lgr5- positive tumorigenic cells clonally expanded in proliferating tumors. At tumor initiation and during tumor propagation in the colon, the descendants of Lgr5-positive cells clonally proliferated to form clusters. Clonal analysis using ubiquitous multicolor lineage tracing revealed that colon tumors derived from Lgr5-positive cells were monoclonal in origin but eventually merged with neighboring tumors, producing polyclonal tumors at the later stage. In contrast, the origin of small intestine tumors was likely polyclonal, and during cancer progression some clones were eliminated, resulting in the formation of monoclonal tumors, which could merge similar to colon tumors. These results suggest that in proliferating intestinal neoplasms, Bmi1- or Lgr5-positive cells represent a population of cancer stem cells, whereas Lgr5-positive cells also function as cells-of-origin for intestinal tumors.

Bmi1-positive cells in the lingual epithelium could serve as cancer stem cells in tongue cancer.

We recently reported that the polycomb complex protein Bmi1 is a marker for lingual epithelial stem cells (LESCs), which are involved in the long-term maintenance of lingual epithelial tissue in the physiological state. However, the precise role of LESCs in generating tongue tumors and Bmi1-positive cell lineage dynamics in tongue cancers are unclear. Here, using a mouse model of chemically (4-nitroquinoline-1-oxide: 4-NQO) induced tongue cancer and the multicolor lineage tracing method, we found that each unit of the tumor was generated by a single cell and that the assembly of such cells formed a polyclonal tumor. Although many Bmi1-positive cells within the tongue cancer specimens failed to proliferate, some proliferated continuously and supplied tumor cells to the surrounding area. This process eventually led to the formation of areas derived from single cells after 1–3 months, as determined using the multicolor lineage tracing method, indicating that such cells could serve as cancer stem cells. These results indicate that LESCs could serve as the origin for tongue cancer and that cancer stem cells are present in tongue tumors.

Single-Cell Analysis Uncovers Clonal Acinar Cell Heterogeneity in the Adult Pancreas

Acinar cells make up the majority of all cells in the pancreas, yet the source of new acinar cells during homeostasis remains unknown. Using multicolor lineage-tracing and organoid-formation assays, we identified the presence of a progenitor-like acinar cell subpopulation. These cells have long-term self-renewal capacity, albeit in a unipotent fashion. We further demonstrate that binuclear acinar cells are terminally differentiated acinar cells. Transcriptome analysis of single acinar cells revealed the existence of a minor population of cells expressing progenitor markers. Interestingly, a gain of the identified markers accompanied by a transient gain of proliferation was observed following chemically induced pancreatitis. Altogether, our study identifies a functionally and molecularly distinct acinar subpopulation and thus transforms our understanding of the acinar cell compartment as a pool of equipotent secretory cells.

Identification of normal and neoplastic stem cells by the multicolor lineage tracing methods.

Adult stem cells and embryonic (ES) and induced pluripotent stem (iPS) cells are two major focus areas of stem cell research. Studies on adult stem cells are important not only as sources for regenerative medicine but for analyzing the mechanisms of tissue homeostasis, tissue repair after injury, cancinogenesis, and aging. On the other hand, ES and iPS cells are mainly important for regenerative medicine. However, many adult stem cells, especially those in low-turnover tissues, have remained unidentified. We have been working on the development of methods using multiple fluorescent markers, to improve the accuracy of lineage-tracing analyses of adult stem cells and their fetal progenitors. With this method, we were able to identify lingual epithelial stem cells (LESCs). By using the same strategy, we could potentially identify candidate cancer stem cells. In this review, we would like to introduce how the multicolor lineage tracing method could be used in various stem cell studies.

Quantitative lineage tracing strategies to resolve multipotency in tissue-specific stem cells

Lineage tracing has become the method of choice to study the fate and dynamics of stem cells (SCs) during development, homeostasis, and regeneration. However, transgenic and knock-in Cre drivers used to perform lineage tracing experiments are often dynamically, temporally, and heterogeneously expressed, leading to the initial labeling of different cell types and thereby complicating their interpretation. Here, we developed two methods: the first one based on statistical analysis of multicolor lineage tracing, allowing the definition of multipotency potential to be achieved with high confidence, and the second one based on lineage tracing at saturation to assess the fate of all SCs within a given lineage and the "flux" of cells between different lineages. Our analysis clearly shows that, whereas the prostate develops from multipotent SCs, only unipotent SCs mediate mammary gland (MG) development and adult tissue remodeling. These methods offer a rigorous framework to assess the lineage relationship and SC fate in different organs and tissues.

Dynamics of Lgr6+ Progenitor Cells in the Hair Follicle, Sebaceous Gland, and Interfollicular Epidermis

SummaryThe dynamics and interactions between stem cell pools in the hair follicle (HF), sebaceous gland (SG), and interfollicular epidermis (IFE) of murine skin are still poorly understood. In this study, we used multicolor lineage tracing to mark Lgr6-expressing basal cells in the HF isthmus, SG, and IFE. We show that these Lgr6+ cells constitute long-term self-renewing populations within each compartment in adult skin. Quantitative analysis of clonal dynamics revealed that the Lgr6+ progenitor cells compete neutrally in the IFE, isthmus, and SG, indicating population asymmetry as the underlying mode of tissue renewal. Transcriptional profiling of Lgr6+ and Lgr6− cells did not reveal a distinct Lgr6-associated gene expression signature, raising the question of whether Lgr6 expression requires extrinsic niche signals. Our results elucidate the interrelation and behavior of Lgr6+ populations in the IFE, HF, and SG and suggest population asymmetry as a common mechanism for homeostasis in several epithelial skin compartments.

Differentiation potential of individual olfactory c-Kit+ progenitors determined via multicolor lineage tracing.

Olfactory tissue undergoes lifelong renewal, due to the presence of basal neural stem cells. Multiple categories of globose basal stem cells have been identified, expressing markers such as Lgr5, Ascl1, GBC-2, and c-Kit. The differentiation potential of individual globose cells has remained unclear. Here, we utilized Cre/loxP lineage tracing with a multicolor reporter system to define c-Kit+ cell contributions at clonal resolution. We determined that reporter expression permitted identification of c-Kit derived progeny with fine cellular detail, and that clones were found to be comprised by neurons only, microvillar cells only, microvillar cells and neurons, or gland/duct cells. Quantification of reporter-labeled cells indicated that c-Kit+ cells behave as transit amplifying or immediate precursors, although we also found evidence for longer-term c-Kit+ cell contributions. Our results from the application of multicolor fate mapping delineate the clonal contributions of c-Kit+ cells to olfactory epithelial renewal, and provide novel insight into tissue maintenance of an adult neuroepithelium.

Concise Review: Understanding Clonal Dynamics in Homeostasis and Injury Through Multicolor Lineage Tracing

Lineage tracing is an essential tool to study stem cell fate. Although traditional lineage tracing techniques have considerably advanced our understanding of stem cell behavior, they pose significant limitations for identification and longitudinal tracking of the progeny of individual stem cells, to compare their behaviors. This is of importance given the well-established heterogeneity among stem cells both in terms of potentialities and proliferative capacities. The recent development of multicolor genetic reporters addressable to specific cell populations largely overcomes these issues. These new "rainbow" technologies provide increased resolution in clonal identification and offer the possibility to study the relative distribution, contacts, tiled arrangement, and competitive interactions among cells or groups of cells of the same type.

Macula densa progenitor cells contribute to nephron formation during kidney development (692.6)

Cells of the macula densa (MD) in the adult kidney are strategically localized in the distal tubule at the glomerular vascular entrance, which mirrors the position of nephron‐committed progenitor cells in the developing kidney, at the interface between the ureteric bud and the metanephric mesenchyme. Indirect evidence suggests that MD cells contribute to nephrogenesis. Here we aimed to assess the stem/progenitor identity of MD cells via in vivo lineage tracing. We developed MD‐Confetti mice with tamoxifen‐inducible expression of the confetti fluorescent reporter construct (CFP/GFP/YFP/RFP) specifically in MD cells by crossing nNOS‐CreERT2 and Confetti/fl mice. Confetti expression was induced by ip tamoxifen injections on postnatal days 2 and 3, and kidneys were harvested three weeks later. MD cells were identified by one of the four Confetti colors, expressing either membrane‐targeted CFP, nuclear‐GFP, cytosolic YFP or RFP. MD plaques usually contained individual MD cells of all four colors. However, in vivo multi‐color lineage tracing identified MD cells as a stem/progenitor population within nascent nephrons dedicated to generating the thick ascending limb (TAL) of Henle’s loop. In addition to multicolor cell labeling in MD plaques, fluorescence image analysis revealed the presence of several, exclusively clonal (i.e. single‐color) multi‐cell tracing units in TALs. Our results highlight the new role and important contribution of individual MD stem/progenitor cells to nephron formation during kidney development.

Examining estrogen regulation of cancer stem cells through multicolor lineage tracing.

We have designed a dual-color fluorescent reporter that can track microRNA expression in vitro, which can be used for lineage tracing experiments. We have used this system to track miR-140 promoter activity in breast cancer cells and to follow the impact of estrogen signaling in cancer stem cell subpopulations.

Establishment of a Novel Lingual Organoid Culture System: Generation of Organoids Having Mature Keratinized Epithelium from Adult Epithelial Stem Cells

Despite the strong need for the establishment of a lingual epithelial cell culture system, a simple and convenient culture method has not yet been established. Here, we report the establishment of a novel lingual epithelium organoid culture system using a three-dimensional matrix and growth factors. Histological analyses showed that the generated organoids had both a stratified squamous epithelial cell layer and a stratum corneum. Very recently, we showed via a multicolor lineage tracing method that Bmi1-positive stem cells exist at the base of the epithelial basal layer in the interpapillary pit. Using our new culture system, we found that organoids could be generated by single Bmi1-positive stem cells and that in the established organoids, multiple Bmi1-positive stem cells were generated at the outermost layer. Moreover, we observed that organoids harvested at an early point in culture could be engrafted and maturate in the tongue of recipient mice and that the organoids generated from carcinogen-treated mice had an abnormal morphology. Thus, this culture system presents valuable settings for studying not only the regulatory mechanisms of lingual epithelium but also lingual regeneration and carcinogenesis.

Multicolor lineage tracing methods and intestinal tumors

The generation of chimeras, which is now a standard technology for producing gene modified mutant mice, was originally developed as a tool for developmental biology. However, the application of conventional single marker chimeric mice for developmental study was initially limited. This situation has been dramatically changed by development of multicolor chimeric mice using various kinds of fluorescent proteins. Now using our technology, up to ten different clones could be distinguished by their colors, which enable us to perform more accurate statistical analyses and lineage tracing experiments than by conventional methods. This method could be applied to visualize not only cell turnover of normal stem cells but also cancer development of live tissues in vivo. In the present review, we will discuss how these methods have been developed and what questions they are now answering by mainly focusing on intestinal stem cells and intestinal tumors.