scholarly journals Induction of Apoptosis Without Differentiation by Retinoic Acid in PLB-985 Cells Requires the Activation of Both RAR and RXR

Blood ◽  
1997 ◽  
Vol 90 (9) ◽  
pp. 3345-3355 ◽  
Author(s):  
Yury Monczak ◽  
Michel Trudel ◽  
William W. Lamph ◽  
Wilson H. Miller
Keyword(s):  
Retinoic Acid ◽  
Cell Surface ◽  
Transcriptional Activation ◽  
Surface Marker ◽  
Cell Surface Marker ◽  
Retinoid Receptor ◽  
Color Flow ◽  
Nb4 Cells ◽  
Induction Of Apoptosis ◽  
Stimulation Of

Abstract Retinoic acid (RA) induces differentiation, followed by apoptosis in acute promyelocytic leukemia (APL) cells, both in vitro and in patients. One problem in understanding these mechanisms is to distinguish molecular events leading to differentiation from those leading to apoptosis. We have identified a leukemic cell line, PLB-985, where RA directly induces apoptosis with no morphologic, genetic, or cell-surface marker evidence of differentiation. These cells differentiate following dimethyl sulfoxide (DMSO), but not RA, treatment. Two-color flow cytometry showed no alteration of the cell cycle after RA treatment, and cell-surface marker analysis of CD11a, CD11b, and CD13 showed no modulation typical of differentiating cells. RNA expression of myeloblastin and transglutaminase, genes regulated by RA-induced differentiation in NB4 cells, was unchanged by RA treatment. Instead, RA induced apoptosis, as shown by typical apoptotic morphological features, genomic DNA laddering, and positive labeling in the TUNEL assay. We found that induction of apoptosis in this model requires a different pattern of retinoid receptor binding and transcriptional activation than is seen in APL cells. As previously described, treatment with retinoid receptor-selective ligands showed that stimulation of RAR alone is sufficient to induce differentiation and apoptosis in NB4 cells, and that stimulation of RXR has no effect on the parameters analyzed. In PLB-985 cells, on the other hand, apoptosis was induced only upon costimulation of both RAR and RXR. Stimulation of either receptor alone had no effect on the cells. Consistent with these findings, bcl-2 RNA and protein levels were downregulated after stimulation of both RAR and RXR, but not with an RAR-specific ligand alone, as in NB4 cells. The expression of several other bcl-2 family members (bcl-X, ich-1, bax, bag, and bak ) and retinoid receptors (RARα, RXRα, and RXRβ) was not affected by treatment with RAR- and/or RXR-activating retinoids; RARβ RNA was undetectable before and after retinoid treatment. Thus, our cell model provides a useful tool in determining the genetic events mediating apoptosis as a response to RA, unobscured by events implicated in differentiation.

Atlas of the immune cell repertoire in human atherosclerotic plaques characterized by single cell RNA-sequencing and multi-color flow cytometry

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
H Horstmann ◽  
A Lindau ◽  
S Hansen ◽  
P Stachon ◽  
I Hilgendorf ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Cell Surface ◽  
Immune Cell ◽  
Surface Marker ◽  
Cell Surface Marker ◽  
Atherosclerotic Plaques ◽  
Color Flow ◽  
Single Cell Rna Sequencing ◽  
Human Carotid

Abstract Rationale Atherosclerosis is a chronic inflammatory disease that is driven by the accumulation of pro- and anti-inflammatory leukocytes in the intima of affected arteries. Yet, the cellular composition of human atherosclerotic plaques is only poorly understood. Here, we characterized immune cells to human carotid atherosclerotic plaques by multi-color flow cytometry and scRNAseq. Methods and results First, we compared a set of previously reported digestion protocols to liberate leukocytes from human carotid plaques after surgical thrombendarteriectomy. One digestion cocktail, containing Collagenase IV and DNase I, was superior regarding cell survival and cell surface marker preservation. Second, leukocytes from 56 surgical specimen were characterized by flow cytometry with a set of 16 parameters and cell surface markers capable of identifying principal hematopoietic leukocyte lineages. This protocol allowed to extract and analyze on average 4x103 viable CD45+ leukocytes from a mean of 988 mg plaque tissue. Surprisingly, we found that atherosclerotic plaques were dominated by T cells with 33.7±2.2% CD4+ T-helper cells and 25.6±2.5% CD8+ cytotoxic T cells. CD11b+ myeloid cells, including monocytes and macrophages, represented only 20.2±4.0% of all CD45+ leukocytes. CD19+B cells and CD56+ NK-cells accounted for 3.9±1.2 and 3.3±0.5%, respectively. TCR-g/d+ T cells and neutrophils were undetectable in atherosclerotic plaques. This cellular composition differed significantly from peripheral blood, but was not relevantly changed between different plaque locations, indicating that macrophage-rich necrotic cores mostly contain dead cells. We confirmed the principal composition of human plaques by single-cell RNA-sequencing from six patients. To allow an estimation of cellular heterogeneity independent of classical cell surface marker assignment, we performed an unsupervised cluster detection algorithm by t-distributed stochastic neighbor embedding (tSNE) and found more than 16 leukocyte clusters with unique cell surface marker expression, suggesting an unexpected high diversity of plaque leukocytes. Conclusion We developed an immune cell phenotyping protocol optimized for human carotid plaques. The definition of phenotypes and frequencies in atherosclerotic plaques will allow to build clinical associations between the immune cell composition and clinical outcomes in future. Funding Acknowledgement Type of funding source: None

scholarly journals Isolation of Neural Progenitor Cells From the Human Adult Subventricular Zone Based on Expression of the Cell Surface Marker CD271

2014 ◽  
Vol 3 (4) ◽  
pp. 470-480 ◽  
Author(s):  
Miriam E. van Strien ◽  
Jacqueline A. Sluijs ◽  
Brent A. Reynolds ◽  
Dennis A. Steindler ◽  
Eleonora Aronica ◽  
...  
Keyword(s):  
Cell Surface ◽  
Progenitor Cells ◽  
Subventricular Zone ◽  
Neural Progenitor Cells ◽  
Neural Progenitor ◽  
Surface Marker ◽  
Cell Surface Marker ◽  
Human Adult

scholarly journals Epha1 is a cell surface marker for neuromesodermal progenitors and their early mesoderm derivatives

2019 ◽  
Author(s):  
Luisa de Lemos ◽  
André Dias ◽  
Ana Nóvoa ◽  
Moisés Mallo
Keyword(s):  
Cell Surface ◽  
Transcriptional Profiling ◽  
Surface Marker ◽  
Cell Surface Marker ◽  
Paraxial Mesoderm ◽  
Molecular Signature ◽  
Tail Bud ◽  
Molecular Fingerprint ◽  
Simple Method ◽  
Cell Subpopulations

ABSTRACTThe vertebrate body is built during embryonic development by the sequential addition of new tissue as the embryo grows at its caudal end. During this process, the neuro-mesodermal progenitors (NMPs) generate the postcranial neural tube and paraxial mesoderm. Recently, several approaches have been designed to determine their molecular fingerprint but a simple method to isolate NMPs from embryos without the need for transgenic markers is still missing. We isolated NMPs using a genetic strategy that exploits their self-renew properties, and searched their transcriptome for cell surface markers. We found a distinct Epha1 expression profile in progenitor-containing areas of the mouse embryo, consisting of two cell subpopulations with different Epha1 expression levels. We show that Sox2+/T+ cells are preferentially associated with the Epha1 compartment, indicating that NMPs might be contained within this cell pool. Transcriptional profiling showed enrichment of high Epha1-expressing cells in known NMP and early mesoderm markers. Also, tail bud cells with lower Epha1 levels contained a molecular signature suggesting the presence of notochord progenitors. Our results thus indicate that Epha1 could represent a valuable cell surface marker for different subsets of axial progenitors, most particularly for NMPs taking mesodermal fates.

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