scholarly journals Single-cell analysis defines the lineage plasticity of stem cells in cervix epithelium

2021 ◽  
Vol 10 (1) ◽  
Author(s):  
Zixian Zhao ◽  
Yujia Wang ◽  
Yingchuan Wu ◽  
Dandan Li ◽  
Ting Zhang ◽  
...  
Keyword(s):  
Single Cell ◽  
Progenitor Cell ◽  
Cell Function ◽  
Cultured Cells ◽  
Dynamic Change ◽  
Lineage Tracing ◽  
Genetic Lineage ◽  
Time Analysis ◽  
Cervical Epithelium

AbstractInformation about the dynamic change and post-injury regeneration of cervical epithelium is relatively rare, even though it is tightly related to gynecologic malignancy. Here, using a feeder cell-based culturing system, we stably cloned mouse and human P63 and KRT5 expressing cells from the adult cervix as putative cervical stem/progenitor cells (CVSCs). When subjected to differentiation, the cultured cells gave rise to mature cervical epithelium by differentiating into squamous or glandular cells. The ability of endogenous mouse CVSCs to reconstitute cervical epithelium after injury was also evident from the genetic lineage tracing experiments. Single-cell transcriptomic analysis further classified the CVSCs into three subtypes and delineated their bi-lineage differentiation roadmap by pseudo-time analysis. We also tracked the real-time differentiation routes of two representing single CVSC lines in vitro and found that they recapitulated the predicted roadmap in pseudo-time analysis. Signaling pathways including Wnt, TGF-beta, Notch and EGFR were found to regulate the cervical epithelial hierarchy and implicated the different roles of distinct types of cells in tissue homeostasis and tumorigenesis. Collectively, the above data provide a cloning system to achieve stable in vitro culture of a bi-lineage stem/progenitor cell population in the cervix, which has profound implications for our understanding of the cervix stem/progenitor cell function in homeostasis, regeneration, and disease and could be helpful for developing stem cell-based therapies in future.

TAMI-36. TAMEP ARE BRAIN TUMOR PARENCHYMAL CELLS CONTROLLING NEOPLASTIC ANGIOGENESIS AND PROGRESSION

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi205-vi206
Author(s):  
Roland Kälin ◽  
Linzhi Cai ◽  
Yuping Li ◽  
Ines Hellmann ◽  
Rainer Glass
Keyword(s):  
Brain Tumor ◽  
Brain Tumors ◽  
Single Cell ◽  
Disease Progression ◽  
Progenitor Cell ◽  
Local Environment ◽  
Lineage Tracing ◽  
Immunofluorescence Analysis ◽  
Progenitor Cell Population ◽  
Parenchymal Cells

Abstract Aggressive brain tumors like glioblastoma depend on support by their local environment and subsets of tumor-parenchymal cells may promote specific phases of disease-progression. We investigated the glioblastoma microenvironment with transgenic lineage-tracing models, intravital imaging, single-cell transcriptomics, immunofluorescence analysis as well as histopathology and characterized a previously unacknowledged population of tumor-associated cells with a myeloid-like expression profile (TAMEP) that transiently appeared during glioblastoma growth. TAMEP of mice and humans were identified with specific markers. Strikingly, TAMEP did not derive from microglia or peripheral monocytes but were generated by a fraction of CNS-resident, SOX2-positive progenitors. Abrogation of this progenitor cell-population, by conditional Sox2-knockout, drastically reduced glioblastoma-vascularization and -size. TAMEP manipulation profoundly altered vessel function and strongly attenuated the blood-tumor barrier. Hence, our data indicate TAMEP and their progenitors as new targets for glioblastoma therapy.

scholarly journals Single cell RNA sequencing of calvarial and long bone endocortical cells

2019 ◽  
Author(s):  
Ugur M. Ayturk ◽  
Joseph P. Scollan ◽  
Alexander Vesprey ◽  
Christina M. Jacobsen ◽  
Paola Divieti Pajevic ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Cultured Cells ◽  
Neutralizing Antibody ◽  
Long Bone ◽  
Appendicular Skeleton ◽  
Rna Seq ◽  
Isolated Cells

ABSTRACTSingle cell RNA-seq (scRNA-seq) is emerging as a powerful technology to examine transcriptomes of individual cells. We determined whether scRNA-seq could be used to detect the effect of environmental and pharmacologic perturbations on osteoblasts. We began with a commonly used in vitro system in which freshly isolated neonatal mouse calvarial cells are expanded and induced to produce a mineralized matrix. We used scRNA-seq to compare the relative cell type abundances and the transcriptomes of freshly isolated cells to those that had been cultured for 12 days in vitro. We observed that the percentage of macrophage-like cells increased from 6% in freshly isolated calvarial cells to 34% in cultured cells. We also found that Bglap transcripts were abundant in freshly isolated osteoblasts but nearly undetectable in the cultured calvarial cells. Thus, scRNA-seq revealed significant differences between heterogeneity of cells in vivo and in vitro. We next performed scRNA-seq on freshly recovered long bone endocortical cells from mice that received either vehicle or Sclerostin-neutralizing antibody for 1 week. Bone anabolism-associated transcripts were also not significantly increased in immature and mature osteoblasts recovered from Sclerostin-neutralizing antibody treated mice; this is likely a consequence of being underpowered to detect modest changes in gene expression, since only 7% of the sequenced endocortical cells were osteoblasts, and a limited portion of their transcriptomes were sampled. We conclude that scRNA-seq can detect changes in cell abundance, identity, and gene expression in skeletally derived cells. In order to detect modest changes in osteoblast gene expression at the single cell level in the appendicular skeleton, larger numbers of osteoblasts from endocortical bone are required.

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

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.

scholarly journals Single-cell RNA-seq analysis revealed long-lasting adverse effects of tamoxifen on neurogenesis in prenatal and adult brains

2020 ◽  
Vol 117 (32) ◽  
pp. 19578-19589 ◽  
Author(s):  
Chia-Ming Lee ◽  
Liqiang Zhou ◽  
Jiping Liu ◽  
Jiayu Shi ◽  
Yanan Geng ◽  
...  
Keyword(s):  
Adverse Effects ◽  
Single Cell ◽  
Molecular Mechanisms ◽  
Genetic Manipulation ◽  
Lineage Tracing ◽  
In Vitro Assays ◽  
Fetal Mortality ◽  
Neural Lineage ◽  
Cortical Neurogenesis

The CreER/LoxP system is widely accepted to track neural lineages and study gene functions upon tamoxifen (TAM) administration. We have observed that prenatal TAM treatment caused high rates of delayed delivery and fetal mortality. This substance could produce undesired results, leading to data misinterpretation. Here, we report that administration of TAM during early stages of cortical neurogenesis promoted precocious neural differentiation, while it inhibited neural progenitor cell (NPC) proliferation. The TAM-induced inhibition of NPC proliferation led to deficits in cortical neurogenesis, dendritic morphogenesis, synaptic formation, and cortical patterning in neonatal and postnatal offspring. Mechanistically, by employing single-cell RNA-sequencing (scRNA-seq) analysis combined with in vivo and in vitro assays, we show TAM could exert these drastic effects mainly through dysregulating the Wnt-Dmrta2 signaling pathway. In adult mice, administration of TAM significantly attenuated NPC proliferation in both the subventricular zone and the dentate gyrus. This study revealed the cellular and molecular mechanisms for the adverse effects of TAM on corticogenesis, suggesting that care must be taken when using the TAM-induced CreER/LoxP system for neural lineage tracing and genetic manipulation studies in both embryonic and adult brains.

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.

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

2019 ◽  
Vol 116 (45) ◽  
pp. 22754-22763 ◽  
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.

scholarly journals P-cresol, but not p-cresylsulphate, disrupts endothelial progenitor cell function in vitro

2012 ◽  
Vol 27 (12) ◽  
pp. 4323-4330 ◽  
Author(s):  
J.-z. Zhu ◽  
J. Zhang ◽  
K. Yang ◽  
R. Du ◽  
Y.-j. Jing ◽  
...  
Keyword(s):  
Endothelial Progenitor Cell ◽  
Progenitor Cell ◽  
Cell Function ◽  
Endothelial Progenitor

scholarly journals Ventricular, atrial and outflow tract heart progenitors arise from spatially and molecularly distinct regions of the primitive streak

2020 ◽  
Author(s):  
Kenzo Ivanovitch ◽  
Pablo Soro-Barrio ◽  
Probir Chakravarty ◽  
Rebecca A Jones ◽  
S. Neda Mousavy Gharavy ◽  
...  
Keyword(s):  
Single Cell ◽  
Heart Development ◽  
Primitive Streak ◽  
Outflow Tract ◽  
Lineage Tracing ◽  
Molecular Signature ◽  
Genetic Lineage ◽  
Cardiac Progenitors ◽  
Heart Field ◽  
Genetic Lineage Tracing

AbstractThe heart develops from two sources of mesoderm progenitors, the first and second heart field (FHF and SHF). Using a single cell transcriptomic assay in combination with genetic lineage tracing, we find the FHF and SHF are subdivided into distinct pools of progenitors in gastrulating mouse embryos at earlier stages than previously thought. Each subpopulation has a distinct origin in the primitive streak. The first progenitors to leave the primitive streak contribute to the left ventricle, shortly after right ventricle progenitor emigrate, followed by the outflow tract and atrial progenitors. Although cells allocated to the outflow tract and atrium leave the primitive streak at a similar stage, they arise from different regions. Outflow tract originate from distal locations in the primitive streak while atrial progenitors are positioned more proximally. Moreover, single cell RNA sequencing demonstrates that the primitive streak cells contributing to the ventricles have a distinct molecular signature from those forming the outflow tract and atrium. We conclude that cardiac progenitors are pre-patterned within the primitive streak and this prefigures their allocation to distinct anatomical structures of the heart. Together, our data provide a new molecular and spatial map of mammalian cardiac progenitors that will support future studies of heart development, function and disease.

scholarly journals Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma

2020 ◽  
Author(s):  
Karin D. Prummel ◽  
Helena L. Crowell ◽  
Susan Nieuwenhuize ◽  
Eline C. Brombacher ◽  
Stephan Daetwyler ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Single Cell Analysis ◽  
De Novo ◽  
Primordial Germ Cells ◽  
Gene Expression Signature ◽  
Time Lapse ◽  
Lineage Tracing ◽  
Genetic Lineage ◽  
Lateral Plate

AbstractThe mesothelium forms epithelial membranes that line the bodies cavities and surround the internal organs. Mesothelia widely contribute to organ homeostasis and regeneration, and their dysregulation can result in congenital anomalies of the viscera, ventral wall defects, and mesothelioma tumors. Nonetheless, the embryonic ontogeny and developmental regulation of mesothelium formation has remained uncharted. Here, we combine genetic lineage tracing, in toto live imaging, and single-cell transcriptomics in zebrafish to track mesothelial progenitor origins from the lateral plate mesoderm (LPM). Our single-cell analysis uncovers a post-gastrulation gene expression signature centered on hand2 that delineates distinct progenitor populations within the forming LPM. Combining gene expression analysis and imaging of transgenic reporter zebrafish embryos, we chart the origin of mesothelial progenitors to the lateral-most, hand2-expressing LPM and confirm evolutionary conservation in mouse. Our time-lapse imaging of transgenic hand2 reporter embryos captures zebrafish mesothelium formation, documenting the coordinated cell movements that form pericardium and visceral and parietal peritoneum. We establish that the primordial germ cells migrate associated with the forming mesothelium as ventral migration boundary. Functionally, hand2 mutants fail to close the ventral mesothelium due to perturbed migration of mesothelium progenitors. Analyzing mouse and human mesothelioma tumors hypothesized to emerge from transformed mesothelium, we find de novo expression of LPM-associated transcription factors, and in particular of Hand2, indicating the re-initiation of a developmental transcriptional program in mesothelioma. Taken together, our work outlines a genetic and developmental signature of mesothelial origins centered around Hand2, contributing to our understanding of mesothelial pathologies and mesothelioma.

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