Dysregulation of breathing control through distinct molecular mechanisms of the brainstem cell population in neonatal sepsis

Abstract RUNX1 (AML1) is a transcription factor critically involved in normal haematopoiesis. Inactivating RUNX1 mutations have been frequently described in a variety of myeloid neoplasms, including high-risk myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Here, we aimed to functionally and molecularly define the actions of a dominant negative mutant by in vitro and in vivo experiments and RNA- and ChIP-sequencing approaches. Overexpression of the RUNX1 mutant S291fs300X in cord blood (CB) CD34+ cells caused a decline in erythroid colony formation (p= 0.01) while the CFU-GM colonies showed enhanced replating capacity compared to control (>3 times). It appeared that the replating potential was restricted to CD14-/CD15- progenitor cells. Long-term suspension cultures with myeloid growth factors (IL-3, SCF) of RUNX1 S291fs300X CB CD34+ cells provided a rather homogenous cell population after 10 weeks of culture. These cells are growth factor dependent and are phenotypically defined by CD34+/CD38+/CD33+/IL1-RAP+/CD45RA+/CD123+ resembling a GMP phenotype which can be propagated for approximately 20 weeks in suspension. Comparable results were obtained with normal bone marrow CD34+cells transduced with the RUNX1 S291fs300X. Karyotype analyses demonstrated no abnormalities while integration site analysis showed a variety of different integration sites and differences between individual samples, suggesting that the myeloid differentiation block is related to the RUNX1 S291fs300X mutation.Long-term MS5 stromal co-cultures of transduced RUNX1 S291fs300X CB CD34+ cells showed after 8-10 weeks a rather homogenous cell population with limited potential to expand and localized under the stromal layer. This cell population is phenotypically defined by CD34+/CD38-. The interactions with the stroma appear to prevent proliferation but retain quiescence, indicating that sufficient niche-cell interactions might be crucial for transformation. NSG mice experiments are performed to test the reproducibility of these findings in vivo. Q-PCR studies demonstrated reduced expression of C/EBPα in RUNX1 S291fs300X CB CD34+ cells, one of the key targets in myeloid differentiation. Therefore, week 10 RUNX1 S291fs300X CB CD34+ cells were transduced with a retroviral C/EBPα overexpression vector. The re-expression of C/EBPα resulted in a reduction in cell proliferation, decline of undifferentiated blasts and an increase in CD15 expression. RNA- and ChIP-sequencing data revealed a decreased expression of crucial RUNX1 target genes including C/EBPα and Cited2 and also a retained binding of mutant RUNX1 on these loci in conjunction with a decrease of H3K27ac. Further research into the molecular mechanisms by which this RUNX1 S291fs300Xderegulates gene-expression is in progress. Our results implicate that overexpression of RUNX1 S291fs300X mutant leads to impaired erythroid differentiation and a strong differentiation block of the myeloid lineage resulting in the expansion and maintenance of a GMP-like cell population. Disclosures No relevant conflicts of interest to declare.

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ON SIMULATING THE GENERATION OF MOSAICISM DURING MAMMALIAN CEREBRAL CORTICAL DEVELOPMENT

Journal of Biological System ◽

10.1142/s0218339009002740 ◽

2009 ◽

Vol 17 (01) ◽

pp. 27-62 ◽

Cited By ~ 1

Author(s):

MICHAEL A. CASE ◽

HUGH R. MACMILLAN

Keyword(s):

Decision Making ◽

Single Cell ◽

Cell Fate ◽

Cell Population ◽

Cellular Response ◽

Molecular Mechanisms ◽

Cortical Development ◽

Coarse Grained ◽

Biological Organization ◽

Cellular Processes

Renewed calls for a systems biology reflect the hope hat enduring biological questions at single-cell and cell-population scales will be resolved as modern molecular biology, with its reductionist program, approaches a nearly-complete characterization of the molecular mechanisms of specific cellular processes. Due to the confounding complexity of biological organization across these scales, computational science is sought to complement the intuition of experimentalists. However, with respect to the molecular basis of cellular processes during development and disease, a gulf between feasible simulations and realistic biology persists. Formidable are the mathematical and computational challenges to conducting and validating cell population-scale simulations, drawn from single-cell level and molecular level details. Nonetheless, in some biological contexts, a focus on core processes crafted by evolution can yield coarse-grained mathematical models that retain explanatory potential despite drastic simplification of known biochemical kinetics. In this article, we bring this modeling philosophy to bear on the nature of neural progenitor cell decision making during mammalian cerebral cortical development. Specifically, we present the computational component to a research program addressing developmental links between (i) the cellular response to endogenous DNA damage, (ii) primary mechanisms of neuronal genetic heterogeneity, or mosaicism, and (iii) the cell fate decision making that defines the population kinetics of neurogenesis.

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Isolation and Characterization of Murine Clonogenic Osteoclast Progenitors by Cell Surface Phenotype Analysis

Blood ◽

10.1182/blood.v91.4.1272 ◽

1998 ◽

Vol 91 (4) ◽

pp. 1272-1279 ◽

Cited By ~ 23

Author(s):

Yukari Muguruma ◽

Minako Y. Lee

Keyword(s):

Cell Surface ◽

Cell Population ◽

Progenitor Cell ◽

Molecular Mechanisms ◽

Osteoclast Differentiation ◽

Tartrate Resistant Acid Phosphatase ◽

Hematopoietic Progenitors ◽

Surface Phenotype ◽

Isolation And Characterization ◽

Progenitor Cell Population

Abstract Osteoclasts are bone resorbing cells of hematopoietic origin; however, a progenitor cell population that gives rise to mature osteoclasts remains elusive. We have characterized a unique cell surface phenotype of clonogenic osteoclast progenitors (colony-forming unit–osteoclast [CFU-O]) and obtained a marrow cell population selectively enriched for these progenitors. Whole bone marrow cells were sequentially separated based on physical and cell surface characteristics, and the presence of CFU-O and other hematopoietic progenitors was examined. CFU-O was enriched in a nonadherent, low-density, lineage-marker–negative (Lin−), Thy1.2-negative (Thy1.2−), Sca1-negative (Sca1−), and c-kit–positive (c-kit+) population, as were the progenitors that were responsive to macrophage–colony-stimulating factor(CSF; CFU-M), granulocyte-macrophage-CSF (CFU-GM), and stem cell factor (CFU-SCF). When the Lin−Thy1.2−Sca1−population was divided into c-kithigh and c-kitlow populations based on c-kit fluorescence, over 88% of CFU-M, CFU-GM, and CFU-SCF were found in the c-kithighpopulation. In relation to the above mentioned hematopoietic progenitors, CFU-O was significantly higher in the c-kitlowpopulation: 80% of progenitors present in the c-kitlowpopulation were CFU-O. The CFU-O in both c-kithigh and c-kitlow populations showed key features of the osteoclast: multinucleated tartrate-resistant acid phosphatase–positive cell formation, expressions of vitronectin receptors, c-src and calcitonin receptors, and bone resorption. We have identified a progenitor cell population in the earliest stage of the osteoclast lineage so far described and developed a method to isolate it from other hematopoietic progenitors. This should help pave the way to understand the molecular mechanisms of osteoclast differentiation.

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Alpaca (Vicugna pacos), the first nonprimate species with a phosphoantigen-reactive Vγ9Vδ2 T cell subset

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1909474117 ◽

2020 ◽

Vol 117 (12) ◽

pp. 6697-6707 ◽

Cited By ~ 7

Author(s):

Alina S. Fichtner ◽

Mohindar M. Karunakaran ◽

Siyi Gu ◽

Christopher T. Boughter ◽

Marta T. Borowska ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Population ◽

Molecular Mechanisms ◽

Mammalian Species ◽

Cell Subset ◽

Γδ T Cells ◽

Vicugna Pacos ◽

A Genome ◽

Vγ9vδ2 T Cells

Vγ9Vδ2 T cells are a major γδ T cell population in the human blood expressing a characteristic Vγ9JP rearrangement paired with Vδ2. This cell subset is activated in a TCR-dependent and MHC-unrestricted fashion by so-called phosphoantigens (PAgs). PAgs can be microbial [(E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate, HMBPP] or endogenous (isopentenyl pyrophosphate, IPP) and PAg sensing depends on the expression of B7-like butyrophilin (BTN3A, CD277) molecules. IPP increases in some transformed or aminobisphosphonate-treated cells, rendering those cells a target for Vγ9Vδ2 T cells in immunotherapy. Yet, functional Vγ9Vδ2 T cells have only been described in humans and higher primates. Using a genome-based study, we showed in silico translatable genes encoding Vγ9, Vδ2, and BTN3 in a few nonprimate mammalian species. Here, with the help of new monoclonal antibodies, we directly identified a T cell population in the alpaca (Vicugna pacos), which responds to PAgs in a BTN3-dependent fashion and shows typicalTRGV9- andTRDV2-like rearrangements. T cell receptor (TCR) transductants andBTN3-deficient human 293T cells reconstituted with alpaca or human BTN3 or alpaca/human BTN3 chimeras showed that alpaca Vγ9Vδ2 TCRs recognize PAg in the context of human and alpaca BTN3. Furthermore, alpaca BTN3 mediates PAg recognition much better than human BTN3A1 alone and this improved functionality mapped to the transmembrane/cytoplasmic part of alpaca BTN3. In summary, we found remarkable similarities but also instructive differences of PAg-recognition by human and alpaca, which help in better understanding the molecular mechanisms controlling the activation of this prominent population of γδ T cells.

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Single-cell meta-analysis of cigarette smoking lung atlas

10.1101/2021.12.09.472029 ◽

2021 ◽

Author(s):

Jun Nakayama ◽

Yusuke Yamamoto

Keyword(s):

Gene Expression ◽

Cigarette Smoking ◽

Single Cell ◽

Cell Population ◽

Molecular Mechanisms ◽

Meta Analysis ◽

Fundamental Properties ◽

Patient State ◽

Cell Gene Expression ◽

Public Datasets

Single-cell RNA-seq (scRNA-seq) technologies have been broadly utilized to reveal the molecular mechanisms of respiratory diseases and physiology at single-cell resolution. Here, we constructed a cigarette smoking lung atlas by integrating data from 8 public datasets, including 104 lung scRNA-seq samples with patient state information. The cigarette smoking lung atlas generated by this single-cell meta-analysis (scMeta-analysis) revealed early carcinogenesis events and defined the alterations of single-cell gene expression, cell population, fundamental properties of biological pathways, and cell-cell interactions induced by cigarette smoking. In addition, we developed two novel scMeta-analysis methods incorporating clinical metadata: VARIED (Visualized Algorithms of Relationships In Expressional Diversity) and AGED (Aging-related Gene Expressional Differences). VARIED analysis revealed the expressional diversity associated with smoking carcinogenesis in each cell population. AGED analysis revealed differences in gene expression related to both aging and smoking states. Our scMeta-analysis provided new insights into the effects of smoking and into cellular diversity in the human lung at single-cell resolution.

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Generation, selection and transcriptomic profiling of human neuromesodermal and spinal cord progenitors in vitro

10.1101/182279 ◽

2017 ◽

Cited By ~ 2

Author(s):

Laure Verrier ◽

Lindsay Davidson ◽

Marek Gierliński ◽

Kate G. Storey

Keyword(s):

Spinal Cord ◽

Cell Population ◽

Molecular Mechanisms ◽

Scale Up ◽

Embryonic Stem ◽

Paraxial Mesoderm ◽

Model Organisms ◽

Human Spinal Cord ◽

Distinct Cell

AbstractRobust protocols for directed differentiation of human pluripotent cells are needed to establish the extent to which mechanisms operating in model organisms are relevant to our own development. Recent work in vertebrate embryos has identified neuromesodermal progenitors as a bipotent cell population that contributes to paraxial mesoderm and spinal cord. However, precise protocols for in vitro differentiation of human neuromesodermal progenitors are lacking. Informed by signalling activities during spinal cord generation in amniote embryos, we show here that transient dual-SMAD inhibition, together with retinoic acid (dSMADi-RA), provides rapid and reproducible induction of human spinal cord progenitors from neuromesodermal progenitors. We use CRISPR-Cas9 to engineer a GFP-reporter for a neuromesodermal progenitor-associated transcription factor Nkx1.2 in human embryonic stem cells, to facilitate selection of this cell population. RNA-sequencing (RNA-Seq) was then used to identify human and conserved neuromesodermal progenitor transcriptional signatures, validate this differentiation protocol and implicate new pathways and processes in human neural differentiation. This optimised protocol, novel reporter line and transcriptomic data are useful resources with which to dissect cellular and molecular mechanisms regulating the generation of human spinal cord, allow scale-up of distinct cell populations for global analyses, including proteomic, biochemical and chromatin interrogation and open up translational opportunities.

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Identification of An Abnormal JAK2 WT CD34+ Cell Population Characterized by High Bcl-Xl Expression Levels Both in JAK2 Positive and Negative PV, ET and PMF Patients,

Blood ◽

10.1182/blood.v118.21.3472.3472 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3472-3472

Author(s):

Daniela Cilloni ◽

Valentina Rosso ◽

Marina Podestà ◽

Enrico Bracco ◽

Federica Benvenuto ◽

...

Keyword(s):

Progenitor Cells ◽

Cell Population ◽

Protein Level ◽

Molecular Mechanisms ◽

Jak2v617f Mutation ◽

Cell Populations ◽

Double Negative ◽

Wild Type ◽

Jak2 Mutation ◽

Cd34 Cells

Abstract Abstract 3472 Background: The JAK2V617F gene mutation is a common feature of Essential Thrombocythemia (ET), Polycythemia Vera (PV) and Primary Myelofibrosis (PMF); however the disease can occur without the JAK2 mutation suggesting that ET, PV and PMF share a common molecular mechanisms which is still unknown. In addition, in patients with the JAK2V617F mutation a substantial proportion of progenitor cells do not bear the JAK2V617F mutation and it is still unclear whether this population is normal or not. Methods: 64 patients affected by MPNs and 12 healthy subjects were included in the study. Twenty three patients were affected by ET, 25 by PV and 22 by PMF. We analyzed the expression, of the anti-apoptotic gene Bcl-xL, both at mRNA and protein level by qRT-PCR and immunofluorescence assay with specific antibodies. Erythroblasts, CD34+ and CD34+CD38-cell populations were selected and analyzed. Moreover, we designed specific PNA (Peptide Nucleic Acid) for JAK2V617F and for Bcl-xL isoform to identify their presence/expression at single CD34+ cell level. Results: Bcl-xL protein and mRNA were found progressively over-expressed in erythroblasts, in CD34+ and in CD34+ CD38- cells from ET, PV and PMF patients without any differences between JAK2V617F and wild type (WT) patients. The median protein level in erythroblasts increased from 22 Relative Units (RU) in ET, to 68,5 in PV and to 85 in PMF. Similarly, in CD34+ Bcl-xL increased from 30 RU in ET, to 89 RU in PV, and to 135 RU in PMF. Finally, CD34+CD38-increased from 17.4 RU in ET, to 45.5 RU in PV, to 101.7 RU in PMF. In addition, analysis by PNA identified distinct progenitor cells population. In patients bearing the JAK2 mutation, three CD34+ cell sub-populations were found: (a) one JAK2 positive and with high levels of Bcl-xL; (b) one JAK2 negative and with high levels of Bcl-xL. Thus, the cell population showing high levels of Bcl-xL is always greater than the JAK2 positive one; (c) one double negative population. In patients without JAK2 mutation, two CD34+ cell populations are identified: (a) one characterized by high levels of Bcl-xL and (b) one double negative. The distribution of these cell subpopulations is different in the distinct MPN. Conclusions: We have identified a previously undisclosed CD34+ cell population which is characterized by high level of Bcl-xL expression and represents the majority of CD34+ cells both in wild type and mutated samples. Interestingly, in mutated samples, this population encompasses the JAK2 mutated one. These findings suggest that Bcl-xL plays an important role in MPN, although it remains undisclosed whether this activation is dependent from an upstream oncogenetic signal. Finally, with this technique we can now identify a putative residual normal CD34+ cells in MPN which may be useful for monitoring patients undergoing targeted therapy with inhibitors. Altogether, these findings disclose unfolded aspects of pathophysiology of MPN and may contribute to a better understanding of the pathogenesis of MPN. Disclosures: Barosi: Novartis: Membership on an entity's Board of Directors or advisory committees. Saglio:Novartis Pharmaceutical: Consultancy, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Speakers Bureau; Pfizer: Consultancy.

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Cranial Suture Mesenchymal Stem Cells: Insights and Advances

Biomolecules ◽

10.3390/biom11081129 ◽

2021 ◽

Vol 11 (8) ◽

pp. 1129

Author(s):

Bo Li ◽

Yigan Wang ◽

Yi Fan ◽

Takehito Ouchi ◽

Zhihe Zhao ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Stem Cell ◽

Distribution Pattern ◽

Cell Population ◽

Molecular Mechanisms ◽

Long Bone ◽

Stem Cell Population ◽

Repair Capacity ◽

Injury Repair

The cranial bones constitute the protective structures of the skull, which surround and protect the brain. Due to the limited repair capacity, the reconstruction and regeneration of skull defects are considered as an unmet clinical need and challenge. Previously, it has been proposed that the periosteum and dura mater provide reparative progenitors for cranial bones homeostasis and injury repair. In addition, it has also been speculated that the cranial mesenchymal stem cells reside in the perivascular niche of the diploe, namely, the soft spongy cancellous bone between the interior and exterior layers of cortical bone of the skull, which resembles the skeletal stem cells’ distribution pattern of the long bone within the bone marrow. Not until recent years have several studies unraveled and validated that the major mesenchymal stem cell population of the cranial region is primarily located within the suture mesenchyme of the skull, and hence, they are termed suture mesenchymal stem cells (SuSCs). Here, we summarized the characteristics of SuSCs, this newly discovered stem cell population of cranial bones, including the temporospatial distribution pattern, self-renewal, and multipotent properties, contribution to injury repair, as well as the signaling pathways and molecular mechanisms associated with the regulation of SuSCs.

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Ultrastructural Changes in the Mammary Epithelial Cell Population During Neoplastic Development Induced by A Chemical Carcinogen.

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100091238 ◽

1976 ◽

Vol 34 ◽

pp. 250-251 ◽

Cited By ~ 2

Author(s):

J. Russo ◽

W. Isenberg ◽

M. Ireland ◽

I.H. Russo

Keyword(s):

Mammary Gland ◽

Cell Population ◽

Virgin Female ◽

Histological Change ◽

Mammary Epithelial ◽

Female Rats ◽

Ultrastructural Changes ◽

Post Inoculation ◽

Chemical Carcinogen ◽

Sprague Dawley

The induction of rat mammary carcinoma by the chemical carcinogen DMBA is used as a model for the study of the human disease (1). We previously described the histochemical changes that occur in the mammary gland of DMBA treated animals before the earliest manifested histological change, the intraductal proliferation (IDP), was observed (2). In the present work, we demonstrate that a change in the stable cell population found in the resting mammary gland occurs after carcinogen administration.Fifty-five day old Sprague-Dawley virgin female rats were inoculated intragastrically with 20mg of 7,12-dimethylbenz(a)anthracene (DMBA) in 1ml sesame oil. Non-inoculated, age-matched females were used as controls. Mammary glands from control and inoculated rats were removed weekly from the time of inoculation until 60 days post-inoculation. For electron microscopy, the glands were immersed in Karnovsky's fixative, post-fixed in 1% OsO4, dehydrated, and embedded in an Epon-Araldite mixture. Thick (lμ) sections were stained with 1% toluidine blue and were used for selecting areas for ultrastructural study.

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Transcription factor/DNA interactions visualized by electron spectroscopic imaging

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100122654 ◽

1992 ◽

Vol 50 (1) ◽

pp. 450-451

Author(s):

David P. Bazett-Jones ◽

Mark L. Brown

Keyword(s):

Transcription Factor ◽

Dna Sequences ◽

Transcription Initiation ◽

Molecular Mechanisms ◽

Phosphorus Content ◽

Spectroscopic Imaging ◽

Electron Spectroscopic Imaging ◽

Dna Backbone ◽

Two Parameters ◽

High Level

A multisubunit RNA polymerase enzyme is ultimately responsible for transcription initiation and elongation of RNA, but recognition of the proper start site by the enzyme is regulated by general, temporal and gene-specific trans-factors interacting at promoter and enhancer DNA sequences. To understand the molecular mechanisms which precisely regulate the transcription initiation event, it is crucial to elucidate the structure of the transcription factor/DNA complexes involved. Electron spectroscopic imaging (ESI) provides the opportunity to visualize individual DNA molecules. Enhancement of DNA contrast with ESI is accomplished by imaging with electrons that have interacted with inner shell electrons of phosphorus in the DNA backbone. Phosphorus detection at this intermediately high level of resolution (≈lnm) permits selective imaging of the DNA, to determine whether the protein factors compact, bend or wrap the DNA. Simultaneously, mass analysis and phosphorus content can be measured quantitatively, using adjacent DNA or tobacco mosaic virus (TMV) as mass and phosphorus standards. These two parameters provide stoichiometric information relating the ratios of protein:DNA content.

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