scholarly journals Triple-cell lineage tracing by a dual reporter on a single allele

2019 ◽  
Vol 295 (3) ◽  
pp. 690-700 ◽  
Author(s):  
Kuo Liu ◽  
Muxue Tang ◽  
Hengwei Jin ◽  
Qiaozhen Liu ◽  
Lingjuan He ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Fate ◽  
Cell Function ◽  
Cell Lineage ◽  
Lineage Tracing ◽  
Cell Populations ◽  
Genetic Lineage ◽  
Traffic Light ◽  
Reporter System

Genetic lineage tracing is widely used to study organ development and tissue regeneration. Multicolor reporters are a powerful platform for simultaneously tracking discrete cell populations. Here, combining Dre-rox and Cre-loxP systems, we generated a new dual-recombinase reporter system, called Rosa26 traffic light reporter (R26-TLR), to monitor red, green, and yellow fluorescence. Using this new reporter system with the three distinct fluorescent reporters combined on one allele, we found that the readouts of the two recombinases Cre and Dre simultaneously reflect Cre+Dre−, Cre−Dre+, and Cre+Dre+ cell lineages. As proof of principle, we show specific labeling in three distinct progenitor/stem cell populations, including club cells, AT2 cells, and bronchoalveolar stem cells, in Sftpc-DreER;Scgb1a1-CreER;R26-TLR mice. By using this new dual-recombinase reporter system, we simultaneously traced the cell fate of these three distinct cell populations during lung repair and regeneration, providing a more comprehensive picture of stem cell function in distal airway repair and regeneration. We propose that this new reporter system will advance developmental and regenerative research by facilitating a more sophisticated genetic approach to studying in vivo cell fate plasticity.

scholarly journals Signalling codes for the maintenance and lineage commitment of embryonic gastric epithelial progenitors

Development ◽  
2020 ◽  
Vol 147 (18) ◽  
pp. dev188839
Author(s):  
Sergi Sayols ◽  
Jakub Klassek ◽  
Clara Werner ◽  
Stefanie Möckel ◽  
Sandra Ritz ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cell Fate ◽  
Ex Vivo ◽  
Cell Lineage ◽  
Lineage Tracing ◽  
Gastric Epithelium ◽  
Genetic Lineage ◽  
Genetic Lineage Tracing ◽  
Notch Pathways

ABSTRACTThe identity of embryonic gastric epithelial progenitors is unknown. We used single-cell RNA-sequencing, genetic lineage tracing and organoid assays to assess whether Axin2- and Lgr5-expressing cells are gastric progenitors in the developing mouse stomach. We show that Axin2+ cells represent a transient population of embryonic epithelial cells in the forestomach. Lgr5+ cells generate both glandular corpus and squamous forestomach organoids ex vivo. Only Lgr5+ progenitors give rise to zymogenic cells in culture. Modulating the activity of the WNT, BMP and Notch pathways in vivo and ex vivo, we found that WNTs are essential for the maintenance of Lgr5+ epithelial cells. Notch prevents differentiation of the embryonic epithelial cells along all secretory lineages and hence ensures their maintenance. Whereas WNTs promote differentiation of the embryonic progenitors along the zymogenic cell lineage, BMPs enhance their differentiation along the parietal lineage. In contrast, WNTs and BMPs are required to suppress differentiation of embryonic gastric epithelium along the pit cell lineage. Thus, coordinated action of the WNT, BMP and Notch pathways controls cell fate determination in the embryonic gastric epithelium.

scholarly journals Universality of clonal dynamics poses fundamental limits to identify stem cell self-renewal strategies

2020 ◽  
Author(s):  
Cristina Parigini ◽  
Philip Greulich
Keyword(s):  
Stem Cell ◽  
Cell Fate ◽  
Adult Stem Cells ◽  
Cell Lineage ◽  
Universality Class ◽  
Lineage Tracing ◽  
Self Renewal ◽  
Fundamental Limits ◽  
Challenges And Opportunities

How adult stem cells maintain self-renewing tissues is in vivo commonly assessed by analysing clonal data from cell lineage tracing assays. To identify strategies of stem cell self-renewal requires that different models of stem cell fate choice predict sufficiently different clonal statistics. Here we show that models of cell fate choice can, in homeostatic tissues, be categorized by exactly two ‘universality classes’, whereby models of the same class predict, under asymptotic conditions, the same clonal statistics. Those classes relate to generalizations of the canonical asymmetric vs. symmetric stem cell self-renewal strategies and are differentiated by a conservation law. This poses both challenges and opportunities to identify stem cell self-renewal strategies: while under asymptotic conditions, self-renewal models of the same universality class cannot be distinguished by clonal data only, models of different classes can be distinguished by simple means.

scholarly journals Universality of clonal dynamics poses fundamental limits to identify stem cell self-renewal strategies

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Cristina Parigini ◽  
Philip Greulich
Keyword(s):  
Stem Cell ◽  
Cell Fate ◽  
Adult Stem Cells ◽  
Cell Lineage ◽  
Universality Class ◽  
Lineage Tracing ◽  
Self Renewal ◽  
Fundamental Limits ◽  
Challenges And Opportunities

How adult stem cells maintain self-renewing tissues is commonly assessed by analysing clonal data from in vivo cell lineage-tracing assays. To identify strategies of stem cell self-renewal requires that different models of stem cell fate choice predict sufficiently different clonal statistics. Here, we show that models of cell fate choice can, in homeostatic tissues, be categorized by exactly two ‘universality classes’, whereby models of the same class predict, under asymptotic conditions, the same clonal statistics. Those classes relate to generalizations of the canonical asymmetric vs. symmetric stem cell self-renewal strategies and are distinguished by a conservation law. This poses both challenges and opportunities to identify stem cell self-renewal strategies: while under asymptotic conditions, self-renewal models of the same universality class cannot be distinguished by clonal data only, models of different classes can be distinguished by simple means.

scholarly journals Profiling proliferative cells and their progeny in damaged murine hearts

2018 ◽  
Vol 115 (52) ◽  
pp. E12245-E12254 ◽  
Author(s):  
Kai Kretzschmar ◽  
Yorick Post ◽  
Marie Bannier-Hélaouët ◽  
Andrea Mattiotti ◽  
Jarno Drost ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Function ◽  
Cardiac Fibroblasts ◽  
Cardiac Injury ◽  
Cardiac Regeneration ◽  
Cell Types ◽  
Postnatal Growth ◽  
Lineage Tracing ◽  
Genetic Lineage ◽  
Proliferating Cells

The significance of cardiac stem cell (CSC) populations for cardiac regeneration remains disputed. Here, we apply the most direct definition of stem cell function (the ability to replace lost tissue through cell division) to interrogate the existence of CSCs. By single-cell mRNA sequencing and genetic lineage tracing using two Ki67 knockin mouse models, we map all proliferating cells and their progeny in homoeostatic and regenerating murine hearts. Cycling cardiomyocytes were only robustly observed in the early postnatal growth phase, while cycling cells in homoeostatic and damaged adult myocardium represented various noncardiomyocyte cell types. Proliferative postdamage fibroblasts expressing follistatin-like protein 1 (FSTL1) closely resemble neonatal cardiac fibroblasts and form the fibrotic scar. Genetic deletion of Fstl1 in cardiac fibroblasts results in postdamage cardiac rupture. We find no evidence for the existence of a quiescent CSC population, for transdifferentiation of other cell types toward cardiomyocytes, or for proliferation of significant numbers of cardiomyocytes in response to cardiac injury.

Regulating Fibrocartilage Stem Cells via TNF-α/Nf-κB in TMJ Osteoarthritis

2021 ◽  
pp. 002203452110372
Author(s):  
R. Bi ◽  
K. Chen ◽  
Y. Wang ◽  
X. Luo ◽  
Q. Li ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Lineage ◽  
Cartilage Degeneration ◽  
Lineage Tracing ◽  
Sprague Dawley ◽  
Etanercept Treatment ◽  
Tnf Α ◽  
Temporomandibular Joint Osteoarthritis

In this study, we investigate harnessing fibrocartilage stem cell (FCSC) capacities by regulating tumor necrosis factor α (TNF-α) signaling for cartilage repair in temporomandibular joint osteoarthritis (TMJOA). Stem cell specifics for FCSCs were characterized in the presence of TNF-α. Etanercept as a TNF-α inhibitor and BAY 11-7082 as an Nf-κB inhibitor were used to study TNF-α regulation of FCSCs. Lineage tracing was performed in Gli1-CreERT+;Tmfl/fl mice when etanercept (1 mg/kg, every 3 d) or isometric vehicle was subcutaneously injected to trace specific changes in FCSCs. Surgically induced TMJOA Sprague-Dawley rats were generated with BAY 11-7082 (5 mg/kg, every 3 d) or vehicle subcutaneous injection to investigate the functional role of TNF-α/Nf-κB in TMJOA. Anterior disc displacement (ADD) rabbits were used to analyze the therapeutic effect of etanercept as a TMJOA intra-articular treatment with etanercept (0.02 mg in 100 μL, every 2 wk) or isometric vehicle. In vitro, TNF-α inhibited proliferation of FCSCs and increased FCSC apoptosis. TNF-α activation interfered with osteogenic and chondrogenic differentiation of FCSCs, while etanercept could partially recover FCSC specificity from TNF-α. FCSC lineage tracing in Gli1-CreERT+;Tmfl/fl mice showed that the chondrogenic capacity of Gli1+ cell lineage was markedly suppressed in osteoarthritis cartilage, the phenotype of which could be significantly rescued by etanercept. Specifically blocking the Nf-κB pathway could significantly weaken the regulatory effect of TNF-α on FCSC specificity in vitro and in TMJOA rats in vivo. Finally, intra-articular etanercept treatment efficiently rescued TMJ cartilage degeneration and growth retardation in ADD rabbits. Inhibition of TNF-α signaling reduced Nf-κB transcripts and recovered FCSC specificities. In vivo, etanercept treatment effectively rescued the osteoarthritis phenotype in TMJOA mice and ADD rabbits. These data suggest a novel therapeutic mechanism whereby TNF-α/Nf-κB inhibition promotes FCSC chondrogenic capacity for cartilage transformation in TMJOA.

scholarly journals Curculigoside Ameliorates Bone Loss by Influencing Mesenchymal Stem Cell Fate in Aging Mice

2021 ◽  
Vol 9 ◽  
Author(s):  
Na Wang ◽  
Ziyi Li ◽  
Shilun Li ◽  
Yukun Li ◽  
Liu Gao ◽  
...  
Keyword(s):  
Stem Cell ◽  
Bone Loss ◽  
Cell Fate ◽  
Cell Lineage ◽  
Binding Motif ◽  
Osteoporosis Therapy ◽  
Senile Osteoporosis ◽  
Age Related

Senile osteoporosis is characterized by increased bone loss and fat accumulation in marrow. Curculigoside (CCG) is the major bioactive component of Curculigo orchioides, which has been used as anti-osteoporosis therapy for elder patients since antiquity. We aimed to investigate the underlying mechanisms by which CCG regulated the bone-fat balance in marrow of aging mice. In our study, CCG treatment was identified to interfere with the stem cell lineage commitment both in vivo and in vitro. In vivo, CCG promoted the transcriptional co-activator with PDZ-binding motif (TAZ) expression to reverse age-related bone loss and marrow adiposity. In vitro, proper concentration of CCG upregulated TAZ expression to increase osteogenesis and decrease adipogenesis of bone marrow mesenchymal stem cells (BMSCs). This regulating effect was discounted by TAZ knockdown or the use of MEK-ERK pathway inhibitor, UO126. Above all, our study confirmed the rescuing effects of CCG on the differential shift from adipogenesis to osteogenesis of BMSCs in aging mice and provided a scientific basis for the clinical use of CCG in senile osteoporosis.

scholarly journals aPKCλ controls epidermal homeostasis and stem cell fate through regulation of division orientation

2013 ◽  
Vol 202 (6) ◽  
pp. 887-900 ◽  
Author(s):  
Michaela T. Niessen ◽  
Jeanie Scott ◽  
Julia G. Zielinski ◽  
Susanne Vorhagen ◽  
Panagiota A. Sotiropoulou ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Fate ◽  
Lineage Tracing ◽  
Premature Aging ◽  
Temporary Increase ◽  
Proliferative Potential ◽  
Cell Dynamics ◽  
Asymmetric Divisions

The atypical protein kinase C (aPKC) is a key regulator of polarity and cell fate in lower organisms. However, whether mammalian aPKCs control stem cells and fate in vivo is not known. Here we show that loss of aPKCλ in a self-renewing epithelium, the epidermis, disturbed tissue homeostasis, differentiation, and stem cell dynamics, causing progressive changes in this tissue. This was accompanied by a gradual loss of quiescent hair follicle bulge stem cells and a temporary increase in proliferating progenitors. Lineage tracing analysis showed that loss of aPKCλ altered the fate of lower bulge/hair germ stem cells. This ultimately led to loss of proliferative potential, stem cell exhaustion, alopecia, and premature aging. Inactivation of aPKCλ produced more asymmetric divisions in different compartments, including the bulge. Thus, aPKCλ is crucial for homeostasis of self-renewing stratifying epithelia, and for the regulation of cell fate, differentiation, and maintenance of epidermal bulge stem cells likely through its role in balancing symmetric and asymmetric division.

scholarly journals Visualization of stem cell activity in pancreatic cancer expansion by direct lineage tracing with live imaging

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Takahisa Maruno ◽  
Akihisa Fukuda ◽  
Norihiro Goto ◽  
Motoyuki Tsuda ◽  
Kozo Ikuta ◽  
...  
Keyword(s):  
Stem Cell ◽  
Epithelial Mesenchymal Transition ◽  
Cell Activity ◽  
Live Imaging ◽  
Lineage Tracing ◽  
Ductal Adenocarcinoma ◽  
Genetic Lineage ◽  
Mesenchymal Transition ◽  
Stem Cell Activity

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease. Although rigorous efforts identified the presence of ‘cancer stem cells (CSCs)’ in PDAC and molecular markers for them, stem cell dynamics in vivo have not been clearly demonstrated. Here we focused on Doublecortin-like kinase 1 (Dclk1), known as a CSC marker of PDAC. Using genetic lineage tracing with a dual-recombinase system and live imaging, we showed that Dclk1+ tumor cells continuously provided progeny cells within pancreatic intraepithelial neoplasia, primary and metastatic PDAC, and PDAC-derived spheroids in vivo and in vitro. Furthermore, genes associated with CSC and epithelial mesenchymal transition were enriched in mouse Dclk1+ and human DCLK1-high PDAC cells. Thus, we provided direct functional evidence for the stem cell activity of Dclk1+ cells in vivo, revealing the essential roles of Dclk1+ cells in expansion of pancreatic neoplasia in all progressive stages.

scholarly journals The Second Oncogenic Hit Determines the Cell Fate of ETV6-RUNX1 Positive Leukemia

2021 ◽  
Vol 9 ◽  
Author(s):  
Guillermo Rodríguez-Hernández ◽  
Ana Casado-García ◽  
Marta Isidro-Hernández ◽  
Daniel Picard ◽  
Javier Raboso-Gallego ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Fate ◽  
Expression Profiles ◽  
Lymphoblastic Leukemia ◽  
Cell Lineage ◽  
Gene Expression Profiles ◽  
Genetic Alterations ◽  
Lineage Tracing ◽  
Genetic Lineage ◽  
Second Hit

ETV6-RUNX1 is almost exclusively associated with childhood B-cell acute lymphoblastic leukemia (B-ALL), but the consequences of ETV6-RUNX1 expression on cell lineage decisions during B-cell leukemogenesis are completely unknown. Clinically silent ETV6-RUNX1 preleukemic clones are frequently found in neonatal cord blood, but few carriers develop B-ALL as a result of secondary genetic alterations. The understanding of the mechanisms underlying the first transforming steps could greatly advance the development of non-toxic prophylactic interventions. Using genetic lineage tracing, we examined the capacity of ETV6-RUNX1 to instruct a malignant phenotype in the hematopoietic lineage by cell-specific Cre-mediated activation of ETV6-RUNX1 from the endogenous Etv6 gene locus. Here we show that, while ETV6-RUNX1 has the propensity to trigger both T- and B-lymphoid malignancies, it is the second hit that determines tumor cell identity. To instigate leukemia, both oncogenic hits must place early in the development of hematopoietic/precursor cells, not in already committed B-cells. Depending on the nature of the second hit, the resulting B-ALLs presented distinct entities that were clearly separable based on their gene expression profiles. Our findings give a novel mechanistic insight into the early steps of ETV6-RUNX1+ B-ALL development and might have major implications for the potential development of ETV6-RUNX1+ B-ALL prevention strategies.

scholarly journals Epithelial Cell Lineage and Signaling in Murine Salivary Glands

2019 ◽  
Vol 98 (11) ◽  
pp. 1186-1194 ◽  
Author(s):  
M.H. Aure ◽  
J.M. Symonds ◽  
J.W. Mays ◽  
M.P. Hoffman
Keyword(s):  
Salivary Gland ◽  
Epithelial Cells ◽  
Cell Fate ◽  
Cell Lineage ◽  
Cell Types ◽  
Lineage Tracing ◽  
Genetic Lineage ◽  
Recent Advances ◽  
Epithelial Development ◽  
Gland Development

Maintaining salivary gland function is critical for oral health. Loss of saliva is a common side effect of therapeutic irradiation for head and neck cancer or autoimmune diseases such as Sjögren’s syndrome. There is no curative treatment, and current strategies proposed for functional regeneration include gene therapy to reengineer surviving salivary gland tissue, cell-based transplant therapy, use of bioengineered glands, and development of drugs/biologics to stimulate in vivo regeneration or increase secretion. Understanding the genetic and cellular mechanisms required for development and homeostasis of adult glands is essential to the success of these proposed treatments. Recent advances in genetic lineage tracing provide insight into epithelial lineage relationships during murine salivary gland development. During early fetal gland development, epithelial cells expressing keratin 14 (K14) Sox2, Sox9, Sox10, and Trp63 give rise to all adult epithelium, but as development proceeds, lineage restriction occurs, resulting in separate lineages of myoepithelial, ductal, and acinar cells in postnatal glands. Several niche signals have been identified that regulate epithelial development and lineage restriction. Fibroblast growth factor signaling is essential for gland development, and other important factors that influence epithelial patterning and maturation include the Wnt, Hedgehog, retinoic acid, and Hippo signaling pathways. In addition, other cell types in the local microenvironment, such as endothelial and neuronal cells, can influence epithelial development. Emerging evidence also suggests that specific epithelial cells will respond to different types of salivary gland damage, depending on the cause and severity of damage and the resulting damaged microenvironment. Understanding how regeneration occurs and which cell types are affected, as well as which signaling factors drive cell lineage decisions, provides specific targets to manipulate cell fate and improve regeneration. Taken together, these recent advances in understanding cell lineages and the signaling factors that drive cell fate changes provide a guide to develop novel regenerative treatments.

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