Lineage tracing reveals the dynamic contribution of Id2+ progenitor cells to branching morphogenesis

OBJECTIVES/GOALS: Our lab previously identified a population of vascular smooth muscle (SMC)-derived progenitor cells (AdvSca1-SM) which expand robustly in response to disease and can differentiate into multiple cell types. We now aim to define the role of these AdvSca1-SM cells in atherosclerotic plaque progression. METHODS/STUDY POPULATION: Goal one uses SMC lineage tracing mice and a model of atherosclerosis to track reprogramming of SMCs to AdvSca1-SM cells in the setting of disease. Arteries are analyzed using flow cytometry and immunofluorescence to quantify changes in number of mature SMCs and AdvSca1-SM cells. Goal two uses AdvSca1-SM lineage tracing mice with high cholesterol-induced atherosclerosis and plaque neovascularization. Arteries are analyzed to quantify expansion of AdvSca1-SM cells, subsequent re-differentiation into mature SMC, endothelial cells, or macrophages, and contribution to plaque neovascularization. Mechanistic findings from both goals are being investigated in diseased human coronary arteries. RESULTS/ANTICIPATED RESULTS: Flow cytometry from SMC lineage tracing mice revealed a 7- to 13-fold expansion of AdvSca1-SM cells in carotid arteries (p<0.001) and aortas (p = 0.03) after 6 weeks of western diet; no differences in macrophage numbers were observed. Additional SMC and AdvSca1-SM cell lineage tracing mice are on atherogenic diets to assess early and advanced atherosclerosis. We predict that AdvSca1-SM cells will contribute to macrophage accumulation as well as plaque neovascularization in the setting of severe atherosclerosis. Translational relevance of mechanisms driving SMC reprogramming and AdvSca1-SM cell contribution to plaque progression are being applied to studies of diseased human coronary arteries. DISCUSSION/SIGNIFICANCE OF IMPACT: Our data suggest a role for AdvSca1-SM cells in atherosclerosis. Ongoing work will clarify the mechanisms driving plaque-associated AdvSca1-SM expansion and define the ultimate fates of these cells. In vivo modulation of this process could provide the basis for future anti-atherosclerotic therapies. CONFLICT OF INTEREST DESCRIPTION: AD - CCTSI TOTTS TL1TR002533; SL - 18POST34030397 from the American Heart Association; AJ – no conflicts; KS - 1F31HL147393 from the National Heart, Lung, and Blood Institute, NIH; MM – no conflicts; RT – no conflicts; KSM – no conflicts; RAN - R01CA236222 from the National Cancer Institute, NIH, and 2018-03 from the Lungevity Foundation; and MCMW-E - R01 HL121877 from the National Heart, Lung, and Blood Institute, NIH, and 25A8679 from the Chernowitz Foundation.

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Abstract 305: Single Cell RNA Sequencing of Adult Cardiac c-kit+ Cells in a Murine Lineage Tracing Model

Circulation Research ◽

10.1161/res.117.suppl_1.305 ◽

2015 ◽

Vol 117 (suppl_1) ◽

Author(s):

Bryan D Maliken ◽

Onur Kanisicak ◽

Jeffery D Molkentin

Keyword(s):

Single Cell ◽

Progenitor Cells ◽

Rna Sequencing ◽

Cardiac Injury ◽

Mesenchymal Cells ◽

Lineage Tracing ◽

Tyrosine Kinase Receptor ◽

Early Differentiation ◽

Gfp Reporter ◽

Single Cell Rna Sequencing

A subset of adult cardiac resident cells defined by the stem cell factor tyrosine kinase receptor termed c-kit, are believed to have myogenic potential and are now being delivered via intracoronary infusion to presumably promote cardiac regeneration and improve ventricular function after ischemic cardiac injury. However, recent studies have shown that despite these benefits, c-kit+ progenitor cells in the adult murine heart are more inclined to take on an endothelial rather than cardiomyocyte lineage. To better define the factors involved in early differentiation of these resident cardiac progenitor cells and to distinguish distinct cell subpopulations, we performed single cell RNA sequencing on c-kit+ cells from Kit-Cre lineage traced GFP reporter mice versus total mesenchymal cells from the heart that were CD31- and CD45-. Cells were isolated by cardiac digestion and FACS was performed, positively sorting for the c-kit+ lineage while negatively sorting for CD31 and CD45 to eliminate endothelial and leukocyte progenitor contamination, respectively. Following this isolation, cells were examined to determine GFP reporter status and then submitted for single cell RNA sequencing using the Fluidigm A1 system. Clustering of 654 genes from this data identified 6 distinct subpopulations indicating various stages of early differentiation among CD31- and CD45-negative cardiac interstitial cells. Furthermore, comparison of GFP+ c-kit cells to the total non-GFP mesenchymal cells identified 75 differentially expressed transcripts. These unique gene signatures may help parse the genes that underlie cellular plasticity in the heart and define the best molecular lineages for transdifferentiation into cardiac myocytes.

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Chief cell plasticity is the origin of metaplasia following acute injury in the stomach mucosa

Gut ◽

10.1136/gutjnl-2021-325310 ◽

2021 ◽

pp. gutjnl-2021-325310

Author(s):

Brianna Caldwell ◽

Anne R Meyer ◽

Jared A Weis ◽

Amy C Engevik ◽

Eunyoung Choi

Keyword(s):

Progenitor Cells ◽

Mucosal Injury ◽

Chief Cell ◽

Lineage Tracing ◽

Acute Injury ◽

Specific Gene ◽

Cell Plasticity ◽

Chief Cells ◽

Stomach Mucosa

ObjectiveMetaplasia arises from differentiated cell types in response to injury and is considered a precursor in many cancers. Heterogeneous cell lineages are present in the reparative metaplastic mucosa with response to injury, including foveolar cells, proliferating cells and spasmolytic polypeptide-expressing metaplasia (SPEM) cells, a key metaplastic cell population. Zymogen-secreting chief cells are long-lived cells in the stomach mucosa and have been considered the origin of SPEM cells; however, a conflicting paradigm has proposed isthmal progenitor cells as an origin for SPEM.DesignGastric intrinsic factor (GIF) is a stomach tissue-specific gene and exhibits protein expression unique to mature mouse chief cells. We generated a novel chief cell-specific driver mouse allele, GIF-rtTA. GIF-GFP reporter mice were used to validate specificity of GIF-rtTA driver in chief cells. GIF-Cre-RnTnG mice were used to perform lineage tracing during homoeostasis and acute metaplasia development. L635 treatment was used to induce acute mucosal injury and coimmunofluorescence staining was performed for various gastric lineage markers.ResultsWe demonstrated that mature chief cells, rather than isthmal progenitor cells, serve as the predominant origin of SPEM cells during the metaplastic process after acute mucosal injury. Furthermore, we observed long-term label-retaining chief cells at 1 year after the GFP labelling in chief cells. However, only a very small subset of the long-term label-retaining chief cells displayed the reprogramming ability in homoeostasis. In contrast, we identified chief cell-originating SPEM cells as contributing to lineages within foveolar cell hyperplasia in response to the acute mucosal injury.ConclusionOur study provides pivotal evidence for cell plasticity and lineage contributions from differentiated gastric chief cells during acute metaplasia development.

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Basal stem cell fate specification is mediated by SMAD signaling in the developing human lung

10.1101/461103 ◽

2018 ◽

Cited By ~ 3

Author(s):

Alyssa J. Miller ◽

Qianhui Yu ◽

Michael Czerwinski ◽

Yu-Hwai Tsai ◽

Renee F. Conway ◽

...

Keyword(s):

Single Cell ◽

Progenitor Cells ◽

Cell Fate ◽

Basal Cell ◽

Human Lung ◽

Branching Morphogenesis ◽

Basal Cells ◽

Smad Signaling ◽

Cell Fate Decisions ◽

Cell Transition

AbstractBasal stem cells (basal cells), located in the bronchi and trachea of the human lung epithelium, play a critical role in normal airway homeostasis and repair, and have been implicated in the development of diseases such as cancer1-4. Additionally, basal-like cells contribute to alveolar regeneration and fibrosis following severe injury5-8. However, the developmental origin of basal cells in humans is unclear. Previous work has shown that specialized progenitor cells exist at the tips of epithelial tubes during lung branching morphogenesis, and in mice, give rise to all alveolar and airway lineages9,10. These ‘bud tip progenitor cells’ have also been described in the developing human lung11-13, but the mechanisms controlling bud tip differentiation into specific cell lineages, including basal cells, are unknown. Here, we interrogated the bud tip-to-basal cell transition using human tissue specimens, bud tip progenitor organoid cultures11, and single-cell transcriptomics. We used single-cell mRNA sequencing (scRNAseq) of developing human lung specimens from 15-21 weeks gestation to identify molecular signatures and cell states in the developing human airway epithelium. We then inferred differentiation trajectories during bud tip-to-airway differentiation, which revealed a previously undescribed transitional cell state (‘hub progenitors’) and implicated SMAD signaling as a regulator of the bud tip-to-basal cell transition. We used bud tip progenitor organoids to show that TGFT1 and BMP4 mediated SMAD signaling robustly induced the transition into functional basal-like cells, and these in vitro-derived basal cells exhibited clonal expansion, self-renewal and multilineage differentiation. This work provides a framework for deducing and validating key regulators of cell fate decisions using single cell transcriptomics and human organoid models. Further, the identification of SMAD signaling as a critical regulator of newly born basal cells in the lung may have implications for regenerative medicine, basal cell development in other organs, and understanding basal cell misregulation in disease.

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Heterogeneity of murine periosteum osteochondroprogenitors involved in fracture healing

10.1101/2020.06.24.169003 ◽

2020 ◽

Author(s):

Brya G Matthews ◽

Francesca V Sbrana ◽

Sanja Novak ◽

Jessica L. Funnell ◽

Ye Cao ◽

...

Keyword(s):

Fracture Healing ◽

Progenitor Cells ◽

Progenitor Cell ◽

Callus Formation ◽

Lineage Tracing ◽

Fracture Callus ◽

Stem And Progenitor Cells ◽

Periosteal Cells ◽

Osteoblast Lineage

AbstractThe periosteum is the major source of cells involved in fracture healing. We sought to characterize differences in progenitor cell populations between periosteum and other bone compartments, and identify periosteal cells involved in fracture healing. The periosteum is highly enriched for progenitor cells, including Sca1+ cells, CFU-F and label-retaining cells. Lineage tracing with αSMACreER identifies periosteal cells that contribute to >80% of osteoblasts and ~40% of chondrocytes following fracture. A subset of αSMA+ cells are quiescent long-term injury-responsive progenitors. Ablation of αSMA+ cells impairs fracture callus formation. In addition, committed osteoblast-lineage cells contributed around 10% of osteoblasts, but no chondrocytes in fracture calluses. Most periosteal progenitors, particularly those that form osteoblasts, can be targeted by αSMACreER. We have demonstrated that the periosteum is highly enriched for skeletal stem and progenitor cells and there is heterogeneity in the populations of cells that contribute to mature lineages during periosteal fracture healing.

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Murine HSCs contribute actively to native hematopoiesis but with reduced differentiation capacity upon aging

eLife ◽

10.7554/elife.41258 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 22

Author(s):

Petter Säwen ◽

Mohamed Eldeeb ◽

Eva Erlandsson ◽

Trine A Kristiansen ◽

Cecilia Laterza ◽

...

Keyword(s):

Stem Cell ◽

Steady State ◽

Progenitor Cells ◽

Blood Cells ◽

Lineage Tracing ◽

Compensatory Mechanism ◽

Fetal Origins ◽

Hematopoietic Stem ◽

Differentiation Capacity ◽

Hsc Transplantation

A hallmark of adult hematopoiesis is the continuous replacement of blood cells with limited lifespans. While active hematopoietic stem cell (HSC) contribution to multilineage hematopoiesis is the foundation of clinical HSC transplantation, recent reports have questioned the physiological contribution of HSCs to normal/steady-state adult hematopoiesis. Here, we use inducible lineage tracing from genetically marked adult HSCs and reveal robust HSC-derived multilineage hematopoiesis. This commences via defined progenitor cells, but varies substantially in between different hematopoietic lineages. By contrast, adult HSC contribution to hematopoietic cells with proposed fetal origins is neglible. Finally, we establish that the HSC contribution to multilineage hematopoiesis declines with increasing age. Therefore, while HSCs are active contributors to native adult hematopoiesis, it appears that the numerical increase of HSCs is a physiologically relevant compensatory mechanism to account for their reduced differentiation capacity with age.

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Osteocalcin expressing cells from tendon sheaths in mice contribute to tendon repair by activating Hedgehog signaling

eLife ◽

10.7554/elife.30474 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 12

Author(s):

Yi Wang ◽

Xu Zhang ◽

Huihui Huang ◽

Yin Xia ◽

YiFei Yao ◽

...

Keyword(s):

Progenitor Cells ◽

Hedgehog Signaling ◽

Progenitor Cell ◽

Tendon Repair ◽

Tendon Sheath ◽

Lineage Tracing ◽

Specific Marker ◽

Molecular Function ◽

Hh Signaling ◽

Necessary And Sufficient

Both extrinsic and intrinsic tissues contribute to tendon repair, but the origin and molecular functions of extrinsic tissues in tendon repair are not fully understood. Here we show that tendon sheath cells harbor stem/progenitor cell properties and contribute to tendon repair by activating Hedgehog signaling. We found that Osteocalcin (Bglap) can be used as an adult tendon-sheath-specific marker in mice. Lineage tracing experiments show that Bglap-expressing cells in adult sheath tissues possess clonogenic and multipotent properties comparable to those of stem/progenitor cells isolated from tendon fibers. Transplantation of sheath tissues improves tendon repair. Mechanistically, Hh signaling in sheath tissues is necessary and sufficient to promote the proliferation of Mkx-expressing cells in sheath tissues, and its action is mediated through TGFβ/Smad3 signaling. Furthermore, co-localization of GLI1+ and MKX+ cells is also found in human tendinopathy specimens. Our work reveals the molecular function of Hh signaling in extrinsic sheath tissues for tendon repair.

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Targeted mobilization of Lrig1+ gastric epithelial stem cell populations by a carcinogenic Helicobacter pylori type IV secretion system

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1903798116 ◽

2019 ◽

Vol 116 (39) ◽

pp. 19652-19658 ◽

Cited By ~ 8

Author(s):

Lydia E. Wroblewski ◽

Eunyoung Choi ◽

Christine Petersen ◽

Alberto G. Delgado ◽

M. Blanca Piazuelo ◽

...

Keyword(s):

Helicobacter Pylori ◽

Stem Cell ◽

Progenitor Cells ◽

Intrinsic Factor ◽

Secretion System ◽

Lineage Tracing ◽

Host Cells ◽

Dependent Manner ◽

Cell Marker ◽

H Pylori

Helicobacter pylori-induced gastritis is the strongest risk factor for gastric adenocarcinoma, a malignancy preceded by a series of well-defined histological stages, including metaplasia. One microbial constituent that augments cancer risk is the cag type 4 secretion system (T4SS), which translocates the oncoprotein CagA into host cells. Aberrant stem cell activation is linked to carcinogenesis, and Lrig1 (leucine-rich repeats and Ig-like domains 1) marks a distinct population of progenitor cells. We investigated whether microbial effectors with carcinogenic potential influence Lrig1 progenitor cells ex vivo and via lineage expansion within H. pylori-infected gastric mucosa. Lineage tracing was induced in Lrig1-CreERT2/+;R26R-YFP/+ (Lrig1/YFP) mice that were uninfected or subsequently infected with cag+H. pylori or an isogenic cagE− mutant (nonfunctional T4SS). In contrast to infection with wild-type (WT) H. pylori for 2 wk, infection for 8 wk resulted in significantly increased inflammation and proliferation in the corpus and antrum compared with uninfected or mice infected with the cagE− mutant. WT H. pylori-infected mice harbored significantly higher numbers of Lrig1/YFP epithelial cells that coexpressed UEA1 (surface cell marker). The number of cells coexpressing intrinsic factor (chief cell marker), YFP (lineage marker), and GSII lectin (spasmolytic polypeptide-expressing metaplasia marker) were increased only by WT H. pylori. In human samples, Lrig1 expression was significantly increased in lesions with premalignant potential compared with normal mucosa or nonatrophic gastritis. In conclusion, chronic H. pylori infection stimulates Lrig1-expressing progenitor cells in a cag-dependent manner, and these reprogrammed cells give rise to a full spectrum of differentiated cells.

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Mutations in thyroid hormone receptor α1 cause premature neurogenesis and progenitor cell depletion in human cortical development

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1908762116 ◽

2019 ◽

Vol 116 (45) ◽

pp. 22754-22763 ◽

Cited By ~ 6

Author(s):

Teresa G. Krieger ◽

Carla M. Moran ◽

Alberto Frangini ◽

W. Edward Visser ◽

Erik Schoenmakers ◽

...

Keyword(s):

Thyroid Hormone ◽

Progenitor Cells ◽

Hormone Receptor ◽

Progenitor Cell ◽

Thyroid Hormone Receptor ◽

Cortical Development ◽

Neural Progenitor Cell ◽

Cortical Layer ◽

Lineage Tracing

Mutations in the thyroid hormone receptor α 1 gene (THRA) have recently been identified as a cause of intellectual deficit in humans. Patients present with structural abnormalities including microencephaly, reduced cerebellar volume and decreased axonal density. Here, we show that directed differentiation of THRA mutant patient-derived induced pluripotent stem cells to forebrain neural progenitors is markedly reduced, but mutant progenitor cells can generate deep and upper cortical layer neurons and form functional neuronal networks. Quantitative lineage tracing shows that THRA mutation-containing progenitor cells exit the cell cycle prematurely, resulting in reduced clonal output. Using a micropatterned chip assay, we find that spatial self-organization of mutation-containing progenitor cells in vitro is impaired, consistent with down-regulated expression of cell–cell adhesion genes. These results reveal that thyroid hormone receptor α1 is required for normal neural progenitor cell proliferation in human cerebral cortical development. They also exemplify quantitative approaches for studying neurodevelopmental disorders using patient-derived cells in vitro.

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