Cells expressing PAX8 are the main source of homeostatic regeneration of adult mouse endometrial epithelium and give rise to serous endometrial carcinoma

ABSTRACTHumans and mice have cyclical regeneration of the endometrial epithelium. It is expected that such regeneration is ensured by tissue stem cells, but their location and hierarchy remain debatable. A number of recent studies have suggested the presence of stem cells in the mouse endometrial epithelium. At the same time, it has been reported that this tissue can be regenerated by stem cells of stromal/mesenchymal or bone marrow cell origin. Here, we describe a single-cell transcriptomic atlas of the main cell types of the mouse uterus and epithelial subset transcriptome and evaluate the contribution of epithelial cells expressing the transcription factor PAX8 to the homeostatic regeneration and malignant transformation of adult endometrial epithelium. According to lineage tracing, PAX8+ epithelial cells are responsible for long-term maintenance of both luminal and glandular epithelium. Furthermore, multicolor tracing shows that individual glands and contiguous areas of luminal epithelium are formed by clonal cell expansion. Inactivation of the tumor suppressor genes Trp53 and Rb1 in PAX8+ cells, but not in FOXJ1+ cells, leads to the formation of neoplasms with features of serous endometrial carcinoma, one of the most aggressive types of human endometrial malignancies. Taken together, our results show that the progeny of single PAX8+ cells represents the main source of regeneration of the adult endometrial epithelium. They also provide direct experimental genetic evidence for the key roles of the P53 and RB pathways in the pathogenesis of serous endometrial carcinoma and suggest that PAX8+ cells represent the cell of origin of this neoplasm.

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PAX8 expressing epithelial cells are a cancer-prone source of clonal cyclical regeneration of endometrial epithelium

10.1101/853994 ◽

2019 ◽

Author(s):

Dah-Jiun Fu ◽

Andrea J. De Micheli ◽

Mallikarjun Bidarimath ◽

Lora H. Ellenson ◽

Benjamin D. Cosgrove ◽

...

Keyword(s):

Stem Cells ◽

Epithelial Cells ◽

Endometrial Carcinoma ◽

Marrow Cell ◽

Lineage Tracing ◽

Cell Of Origin ◽

Pax8 Expression ◽

Endometrial Epithelium ◽

Cell Transcriptome ◽

Single Cell Transcriptome

AbstractHumans and mice have cyclical regeneration of the endometrial epithelium. It is expected that such regeneration is ensured by tissue stem cells. However, their location remains debatable. A number of recent studies have suggested the presence of stem cells in the mouse endometrial epithelium. At the same time, it has been reported this tissue can be regenerated by stem cells of stromal/mesenchymal or bone marrow cell origin. Here we show that cells expressing transcription factor PAX8 are the main contributor to the homeostatic regeneration of endometrial epithelium. In the uterus, based on a single-cell transcriptome and immunostaining analyses, PAX8 expression is limited to the endometrial epithelium. According to lineage tracing, PAX8 positive (PAX8+) epithelial cells are responsible for long-term maintenance of the epithelium. Furthermore, multicolor tracing shows that individual glands are formed by clonal expansion of cells labeling both glandular and luminal epithelial components. Inactivation of tumor suppressor genes Trp53 and Rb1 in PAX8+ cells but not FOXJ1+ cells leads to formation of neoplasms with features of serous endometrial carcinoma, the most aggressive type of human endometrial malignancy. Taken together, our results show that a progeny of single PAX8+ cells represent the main source of cyclical regeneration of the endometrial epithelium. They also provide direct experimental genetic evidence for the key roles of the P53 and RB pathways in the pathogenesis of serous endometrial carcinoma, and suggest that PAX8+ cells represent the cell-of-origin of this neoplasm.

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Mouse telomerase reverse transcriptase (mTert) expression marks slowly cycling intestinal stem cells

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1013004108 ◽

2010 ◽

Vol 108 (1) ◽

pp. 179-184 ◽

Cited By ~ 337

Author(s):

Robert K. Montgomery ◽

Diana L. Carlone ◽

Camilla A. Richmond ◽

Loredana Farilla ◽

Mariette E. G. Kranendonk ◽

...

Keyword(s):

Stem Cells ◽

Reverse Transcriptase ◽

Tissue Injury ◽

Cell Types ◽

Lineage Tracing ◽

Intestinal Stem Cells ◽

Intestinal Cell ◽

Stem Cell Population ◽

Telomerase Reverse Transcriptase

The intestinal epithelium is maintained by a population of rapidly cycling (Lgr5+) intestinal stem cells (ISCs). It has been postulated, however, that slowly cycling ISCs must also be present in the intestine to protect the genome from accumulating deleterious mutations and to allow for a response to tissue injury. Here, we identify a subpopulation of slowly cycling ISCs marked by mouse telomerase reverse transcriptase (mTert) expression that can give rise to Lgr5+ cells. mTert-expressing cells distribute in a pattern along the crypt–villus axis similar to long-term label-retaining cells (LRCs) and are resistant to tissue injury. Lineage-tracing studies demonstrate that mTert+ cells give rise to all differentiated intestinal cell types, persist long term, and contribute to the regenerative response following injury. Consistent with other highly regenerative tissues, our results demonstrate that a slowly cycling stem cell population exists within the intestine.

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Origin of Myofibroblasts in Lung Fibrosis

Current Tissue Microenvironment Reports ◽

10.1007/s43152-020-00022-9 ◽

2020 ◽

Vol 1 (4) ◽

pp. 155-162

Author(s):

CF Hung

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Epithelial Cells ◽

Lung Fibrosis ◽

Cell Types ◽

Mesenchymal Cells ◽

Lineage Tracing ◽

Alveolar Epithelial Cells ◽

Multiple Sources ◽

Alveolar Epithelial

Abstract Purpose of Review In this brief review, we will highlight important observational and experimental data in the literature that address the origin of scar-forming cells in lung fibrosis. Recent Findings Several cellular sources of activated scar-forming cells (myofibroblasts) have been postulated including alveolar epithelial cells; circulating fibrocytes; and lung stromal cell subpopulations including resident fibroblasts, pericytes, and resident mesenchymal stem cells. Recent advances in lineage-tracing models, however, fail to provide experimental evidence for epithelial and fibrocyte origins of lung myofibroblasts. Resident mesenchymal cells of the lung, which include various cell types including resident fibroblasts, pericytes, and resident mesenchymal stem cells, appear to be important sources of myofibroblasts in murine models of lung injury and fibrosis. Summary Lung myofibroblasts likely originate from multiple sources of lung-resident mesenchymal cells. Their relative contributions may vary depending on the type of injury. Although lineage-tracing experiments have failed to show significant contribution from epithelial cells or fibrocytes, they may play important functional roles in myofibroblast activation through paracrine signaling.

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Bipotent stem cells support the cyclical regeneration of endometrial epithelium of the murine uterus

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1814597116 ◽

2019 ◽

Vol 116 (14) ◽

pp. 6848-6857 ◽

Cited By ~ 14

Author(s):

Shiying Jin

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cell Population ◽

Lineage Tracing ◽

Tissue Loss ◽

Stem Cell Population ◽

Epithelial Stem Cells ◽

Mouse Uterus ◽

Epithelial Regeneration ◽

Endometrial Epithelium

The endometrial epithelium of the uterus regenerates periodically. The cellular source of newly regenerated endometrial epithelia during a mouse estrous cycle or a human menstrual cycle is presently unknown. Here, I have used single-cell lineage tracing in the whole mouse uterus to demonstrate that epithelial stem cells exist in the mouse uterus. These uterine epithelial stem cells provide a resident cellular supply that fuels endometrial epithelial regeneration. They are able to survive cyclical uterine tissue loss and persistently generate all endometrial epithelial lineages, including the functionally distinct luminal and glandular epithelia, to maintain uterine cycling. The uterine epithelial stem cell population also supports the regeneration of uterine endometrial epithelium post parturition. The 5-ethynyl-2′-deoxyuridine pulse-chase experiments further reveal that this stem cell population may reside in the intersection zone between luminal and glandular epithelial compartments. This tissue distribution allows these bipotent uterine epithelial stem cells to bidirectionally differentiate to maintain homeostasis and regeneration of mouse endometrial epithelium under physiological conditions. Thus, uterine function over the reproductive lifespan of a mouse relies on stem cell-maintained rhythmic endometrial regeneration.

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Stem Cells in T-ALL Have a Primitive CD133+/CD34+/CD7- Phenotype.

Blood ◽

10.1182/blood.v104.11.1885.1885 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1885-1885

Author(s):

Charlotte V. Cox ◽

Roger S. Evely ◽

Nicholas J. Goulden ◽

Allison Blair

Keyword(s):

Stem Cells ◽

Cell Types ◽

Scid Mice ◽

Cell Of Origin ◽

Proliferating Cells ◽

Self Renewal ◽

Proliferative Ability

Abstract The cell of origin of childhood acute lymphoblastic leukaemia (ALL) has been the subject of conflicting reports in recent years. One model suggests that many haemopoietic cell types are susceptible to transformation and the level of commitment of the target cell influences the characteristics of the resulting blast cell population. A second model suggests that primitive haemopoietic cells are the targets for transformation, with some differentiation occurring subsequent to the transformation event. This model suggests a hierarchy of progenitors may exist in ALL. In support of this latter model, we have demonstrated that leukaemic stem cells in B-ALL have a primitive CD34+/CD10−/CD19− phenotype and T-ALL cells with NOD/SCID engrafting capacity are CD34+/CD4−. In this investigation we have attempted to further purify and characterise leukaemic stem cells from children with T-ALL. Cells from 7 patients were sorted for expression of CD34 and CD7 and the sorted subfractions evaluated for long-term proliferative ability in vitro using a serum free suspension culture assay and in the NOD/SCID mouse model. In this group of patients, the CD34+/CD7+ fraction represented 7±6% of cells at sorting, 6±4% were CD34+/CD7− and the majority were CD34−/CD7+ (60±12%). After 3 weeks in culture, the majority of proliferating cells were derived from the CD34+/CD7− subfraction (53±16%). By week 6, >70% of proliferating cells were derived from the CD34+/CD7− subfraction. Unsorted ALL cells and the sorted subfractions from 4 of these patients, were evaluated for their ability to engraft sublethally irradiated NOD/SCID mice. In each case, engraftment was achieved using 105–106 unsorted cells (25–80% CD45+) and with the CD34+/CD7− subfraction only (4–84% CD45+ with 3x103–8x104 cells). There was no engraftment with the other subfractions despite injecting up to 100 fold more cells. The engrafted cells had the same karyotype as the patient at diagnosis and expressed high levels of CD2, CD4 and CD7 implying they had differentiated in vivo. The self-renewal capacity of the CD34+/CD7− cells was evaluated by secondary transplantation. CD45+ cells from NOD/SCIDs engrafted with CD34+/CD7− cells successfully engrafted secondary recipients with equivalent levels of human cell engraftment, demonstrating these cells were capable of self-renewal. These findings suggest that cells with a more primitive phenotype may be the targets for transformation in T-ALL, rather than committed lymphocytes. To further investigate this hypothesis, we sorted cells from 4 of these patients for expression of CD133 and CD7 and evaluated their proliferative ability as described above. Results to date indicate that the CD133+/CD7− fraction represents only 0.35% of nucleated cells at sorting. However, after 3 weeks in culture, 48±9% of proliferating cells were derived from this subfraction and by week 6, 58±20% of cells were derived from the CD133+/CD7− subfraction. In vivo analyses completed in 2 patients to date have shown that only the CD133+/CD7− subfraction was capable of engrafting NOD/SCID mice (0.5–54% CD45+ using 3x103–105 cells). These results demonstrate that T-ALL cells with long-term proliferative and NOD/SCID repopulating capacity express the primitive haemopoietic cell antigens CD133 and CD34 and lack expression of T-lineage markers. These findings add further support to the concept of a common cell of origin for acute leukaemias.

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Short-read and long-read RNA sequencing of mouse hematopoietic stem cells at bulk and single-cell levels

Scientific Data ◽

10.1038/s41597-021-01078-4 ◽

2021 ◽

Vol 8 (1) ◽

Author(s):

Xiuran Zheng ◽

Dan Zhang ◽

Mengying Xu ◽

Wanqin Zeng ◽

Ran Zhou ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Single Cell ◽

Rna Sequencing ◽

Cell Types ◽

Lineage Tracing ◽

Self Renewal ◽

Hematopoietic Stem ◽

Long Read

AbstractHematopoietic stem cells (HSCs) lie at the top of the differentiation hierarchy. Although HSC and their immediate downstream, multipotent progenitors (MPP) have full multilineage differentiation capacity, only long-term (LT-) HSC has the capacity of long-term self-renewal. The heterogeneity within the HSC population is gradually acknowledged with the development of single-cell RNA sequencing and lineage tracing technologies. Transcriptional and post-transcriptional regulations play important roles in controlling the differentiation and self-renewal capacity within HSC population. Here we report a dataset comprising short- and long-read RNA sequencing for mouse long- and short-term HSC and MPP at bulk and single-cell levels. We demonstrate that integrating short- and long-read sequencing can facilitate the identification and quantification of known and unannotated isoforms. Thus, this dataset provides a groundwork for comprehensive and comparative studies on transcriptional diversity and heterogeneity within different HSC cell types.

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Stability of Imprinting and Differentiation Capacity in Naïve Human Cells Induced by Chemical Inhibition of CDK8 and CDK19

Cells ◽

10.3390/cells10040876 ◽

2021 ◽

Vol 10 (4) ◽

pp. 876

Author(s):

Raquel Bernad ◽

Cian J. Lynch ◽

Rocio G. Urdinguio ◽

Camille Stephan-Otto Attolini ◽

Mario F. Fraga ◽

...

Keyword(s):

Stem Cells ◽

Pluripotent Stem Cells ◽

Primordial Germ Cell ◽

Normal Karyotype ◽

Cell Types ◽

Dna Demethylation ◽

Starting State ◽

Teratoma Formation

Pluripotent stem cells can be stabilized in vitro at different developmental states by the use of specific chemicals and soluble factors. The naïve and primed states are the best characterized pluripotency states. Naïve pluripotent stem cells (PSCs) correspond to the early pre-implantation blastocyst and, in mice, constitute the optimal starting state for subsequent developmental applications. However, the stabilization of human naïve PSCs remains challenging because, after short-term culture, most current methods result in karyotypic abnormalities, aberrant DNA methylation patterns, loss of imprinting and severely compromised developmental potency. We have recently developed a novel method to induce and stabilize naïve human PSCs that consists in the simple addition of a chemical inhibitor for the closely related CDK8 and CDK19 kinases (CDK8/19i). Long-term cultured CDK8/19i-naïve human PSCs preserve their normal karyotype and do not show widespread DNA demethylation. Here, we investigate the long-term stability of allele-specific methylation at imprinted loci and the differentiation potency of CDK8/19i-naïve human PSCs. We report that long-term cultured CDK8/19i-naïve human PSCs retain the imprinting profile of their parental primed cells, and imprints are further retained upon differentiation in the context of teratoma formation. We have also tested the capacity of long-term cultured CDK8/19i-naïve human PSCs to differentiate into primordial germ cell (PGC)-like cells (PGCLCs) and trophoblast stem cells (TSCs), two cell types that are accessible from the naïve state. Interestingly, long-term cultured CDK8/19i-naïve human PSCs differentiated into PGCLCs with a similar efficiency to their primed counterparts. Also, long-term cultured CDK8/19i-naïve human PSCs were able to differentiate into TSCs, a transition that was not possible for primed PSCs. We conclude that inhibition of CDK8/19 stabilizes human PSCs in a functional naïve state that preserves imprinting and potency over long-term culture.

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The evolving definition of salivary gland stem cells

npj Regenerative Medicine ◽

10.1038/s41536-020-00115-x ◽

2021 ◽

Vol 6 (1) ◽

Author(s):

Cecilia Rocchi ◽

Lara Barazzuol ◽

Rob P. Coppes

Keyword(s):

Stem Cells ◽

Salivary Gland ◽

Adult Stem Cell ◽

Cell Types ◽

Research Field ◽

New Therapeutic Approaches ◽

Recent Developments ◽

Radiation Induced ◽

Definition Of

AbstractDysfunction of the salivary gland and irreversible hyposalivation are the main side effects of radiotherapy treatment for head and neck cancer leading to a drastic decrease of the quality of life of the patients. Approaches aimed at regenerating damaged salivary glands have been proposed as means to provide long-term restoration of tissue function in the affected patients. In studies to elucidate salivary gland regenerative mechanisms, more and more evidence suggests that salivary gland stem/progenitor cell behavior, like many other adult tissues, does not follow that of the hard-wired professional stem cells of the hematopoietic system. In this review, we provide evidence showing that several cell types within the salivary gland epithelium can serve as stem/progenitor-like cells. While these cell populations seem to function mostly as lineage-restricted progenitors during homeostasis, we indicate that upon damage specific plasticity mechanisms might be activated to take part in regeneration of the tissue. In light of these insights, we provide an overview of how recent developments in the adult stem cell research field are changing our thinking of the definition of salivary gland stem cells and their potential plasticity upon damage. These new perspectives may have important implications on the development of new therapeutic approaches to rescue radiation-induced hyposalivation.

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Hlf Expression Marks Early Emergence of Hematopoietic Stem Cell Precursors With Adult Repopulating Potential and Fate

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.728057 ◽

2021 ◽

Vol 9 ◽

Author(s):

Wanbo Tang ◽

Jian He ◽

Tao Huang ◽

Zhijie Bai ◽

Chaojie Wang ◽

...

Keyword(s):

Stem Cells ◽

Mouse Model ◽

Hematopoietic Stem Cells ◽

Fetal Liver ◽

Lineage Tracing ◽

Genetic Lineage ◽

Hematopoietic Stem ◽

Genetic Lineage Tracing

In the aorta-gonad-mesonephros (AGM) region of mouse embryos, pre-hematopoietic stem cells (pre-HSCs) are generated from rare and specialized hemogenic endothelial cells (HECs) via endothelial-to-hematopoietic transition, followed by maturation into bona fide hematopoietic stem cells (HSCs). As HECs also generate a lot of hematopoietic progenitors not fated to HSCs, powerful tools that are pre-HSC/HSC-specific become urgently critical. Here, using the gene knockin strategy, we firstly developed an Hlf-tdTomato reporter mouse model and detected Hlf-tdTomato expression exclusively in the hematopoietic cells including part of the immunophenotypic CD45– and CD45+ pre-HSCs in the embryonic day (E) 10.5 AGM region. By in vitro co-culture together with long-term transplantation assay stringent for HSC precursor identification, we further revealed that unlike the CD45– counterpart in which both Hlf-tdTomato-positive and negative sub-populations harbored HSC competence, the CD45+ E10.5 pre-HSCs existed exclusively in Hlf-tdTomato-positive cells. The result indicates that the cells should gain the expression of Hlf prior to or together with CD45 to give rise to functional HSCs. Furthermore, we constructed a novel Hlf-CreER mouse model and performed time-restricted genetic lineage tracing by a single dose induction at E9.5. We observed the labeling in E11.5 AGM precursors and their contribution to the immunophenotypic HSCs in fetal liver (FL). Importantly, these Hlf-labeled early cells contributed to and retained the size of the HSC pool in the bone marrow (BM), which continuously differentiated to maintain a balanced and long-term multi-lineage hematopoiesis in the adult. Therefore, we provided another valuable mouse model to specifically trace the fate of emerging HSCs during development.

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Enzymatic treatment of long-term human marrow cultures reveals the preferential location of primitive hemopoietic progenitors in the adherent layer

Blood ◽

10.1182/blood.v62.2.291.291 ◽

1983 ◽

Vol 62 (2) ◽

pp. 291-297 ◽

Cited By ~ 175

Author(s):

L Coulombel ◽

AC Eaves ◽

CJ Eaves

Keyword(s):

Stem Cells ◽

Marrow Cell ◽

Point Of View ◽

Adherent Cell ◽

Reproducible Method ◽

Preferential Location ◽

Very High ◽

Marrow Cultures

Abstract Recent studies with long-term mouse marrow cultures have indicated the importance of the adherent layer as a primary reservoir of the most primitive stem cells, from which derivative stem cells and more differentiated progenitors are continuously generated. We have now examined the role of the adherent cell layer in long-term human marrow cultures from this point of view. Prerequisite to such an undertaking was the development of a nontoxic and reproducible method for detaching the adherent layer and making it into a single-cell suspension suitable for characterization by colony assays. Both trypsin and collagenase could be used to obtain suspensions that met these criteria. Lack of toxicity was demonstrated by the preservation of CFU-E, BFU-E, and CFU- C plating efficiency in fresh human marrow cell suspensions exposed to the same enzymatic treatments. Collagenase treatment of long-term marrow culture adherent layers was considered superior because it freed all hemopoietic colony-forming cells but left some of the other cells still adherent. Using this method, we found that CFU-C, BFU-E, and CFU- G/E were consistently detectable in the adherent layer for at least 8 wk, with the majority of the BFU-E and CFU-G/E being located in the adherent layer (70%-75% after 2–3 wk and more than 90% by 7–8 wk). Although corresponding numerical differences in adherent and nonadherent CFU-C populations were not observed, the colonies derived from them showed marked differences in the size they achieved; the adherent layer being the exclusive site of CFU-C, with a very high proliferative capacity. These findings emphasize the importance of assessing the progenitor content of the adherent layer of long-term human marrow cultures and provide an appropriate methodology.

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