scholarly journals Single-Cell Gene Expression Analysis Reveals Gene Regulatory Networks Driving Proliferation in Pituitary Stem and Endocrine Cells

2021 ◽  
Vol 5 (Supplement_1) ◽  
pp. A509-A510
Author(s):  
Leonard Cheung ◽  
Alexandre Daly ◽  
Michelle Brinkmeier ◽  
Sally Ann Camper
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Stem Cells ◽  
Stem Cell ◽  
Single Cell ◽  
Regulatory Networks ◽  
Endocrine Cells ◽  
Postnatal Period ◽  
Pituitary Cells ◽  
Cell Gene

Abstract A fundamental question for pituitary development and disease is to understand the mechanisms that regulate proliferation, quiescence, and differentiation of stem cells and endocrine lineage-committed precursor cells. Pituitary stem cells, marked by expression of SOX2, are highly proliferative during development and the early postnatal period. This pool becomes quiescent over time, but stem cells retain the ability to re-enter the cell cycle and differentiate into nascent endocrine cells of all lineages in response to physiological demands. The rodent pituitary gland undergoes substantial growth in the postnatal period, and much of this increase in size is due to proliferation of committed progenitors, such as Pou1f1-expressing cells. We performed single-cell RNA transcriptomics analyses of over 8,000 male and female 4-day-old mouse pituitary cells in order to assess stem cell heterogeneity and to identify novel pituitary stem cell biomarkers. We identified a number of factors enriched in pituitary stem cells relative to differentiating cells, including the transcription factors TGIF1 and NR4A3 as well as several members of the nuclear factor I family (NFIA, NFIB, NFIX). We also detected stem cell-specific expression of the cortisol synthesizing enzyme HSD11B1 and folate receptor FOLR1, suggesting novel roles for these pathways in pituitary stem cells. There were few transcriptomic differences between proliferating and non-proliferating stem cells. However, proliferating stem cells and proliferating committed progenitors shared expression of cell-cycle associated genes and novel transcription factors such as BRCA1 and E2F1. Furthermore, single-cell gene network inference and clustering (SCENIC) analyses demonstrated activation of common gene regulatory networks in both proliferating stem and proliferating endocrine populations, including both the E2f1 and Brca1 regulons. RNA velocity, trajectory, and phylogenetic analyses and find that proliferating stem and endocrine cells are likely independent cellular states. Instead, they support the idea that proliferating stem cells become quiescent and transition to committed progenitors that re-enter the cell cycle and then subsequently activate hormone gene expression. In conclusion, our single-cell gene expression analyses of early postnatal pituitary cells have shed light on the developmental trajectory from proliferating stem cell to quiescent differentiated hormone-producing cells.

Cell Cycle Related Stem Cell Gene Expression.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4155-4155
Author(s):  
Gerri J. Dooner ◽  
Gerald A. Colvin ◽  
Mark S. Dooner ◽  
Peter J. Quesenberry
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Stem Cells ◽  
Stem Cell ◽  
Rank Order ◽  
Continuum Theory ◽  
Surface Expression ◽  
Stem Cell Regulation ◽  
Cell Gene Expression ◽  
Cell Gene

Abstract We have previously reported that marrow stem cells show changes in engraftment (Habibian, et al J Ex. Hem, 188:393–398, 1998), homing (Cerny et al., J Hematother Stem Cell Res11:913–922, 2002) and differentiation (Colvin et al., J Cell Phys199:20–31, 2004) phenotype as they transit a cytokine-driven cell cycle. mRNA and surface expression of adhesion proteins also change (Becker et al., Exp Hematol27:533–541, 1999). We have evaluated gene expression by Real-time PCR of murine lineage negative, Sca+ (Lin-Sca+) stem cells stimulated by Il-3, Il-6, Il-11 and Steel factor (at 0, 24 and 48h) and lineage negative Rhodamine low, Hoescht low (LRH) stem cells stimulated by TPO, Flt-3 and Steel at various points in cell cycle transit (0,32,40,48h). In Lin-Sca+ cells (4experiments, time 0) expression of the following genes in descending order was as follows: IKAROS, L-selectin, Pu-1, Gata-2, Pecam, Cd84, Rock-1, c-fms, FOG, Cxcr4, c-kit, Cd4. The following were either not expressed or expressed at very low levels: Il-11, Ccr4, Sdf-1, Gata-1, P-selectin and Vecam. A pattern of depressed gene expression in S-phase (24h) with subsequent recovery (48h) was seen with c-fms and c-kit. With LRH cells (2 experiments, time 0) approximate descending rank order of gene expression was Cd45r, Cd34, G-CSFR, Mac-1, GM-CSFR and Flt-3. Il7r was not detected. With cycle progression Cd34 and Sca-1 were markedly elevated while Mac-1 and c-mpl were decreased. The expression of GM-CSFR, G-CSFR, Cd45r and Cd4 showed variable fluctuation. Il-7r was negative throughout. These data show that primitive marrow stem cells express a wide variety of “hematopoietic genes”, that expression modulates with cell cycle transit and perhaps most importantly that observed changes in gene expression are reversible. This is consistent with the continuum theory of stem cell regulation.

scholarly journals Single-cell molecular analysis defines therapy response and immunophenotype of stem cell subpopulations in CML

Blood ◽  
2017 ◽  
Vol 129 (17) ◽  
pp. 2384-2394 ◽  
Author(s):  
Rebecca Warfvinge ◽  
Linda Geironson ◽  
Mikael N. E. Sommarin ◽  
Stefan Lang ◽  
Christine Karlsson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Single Cell ◽  
Gene Expression Analysis ◽  
Therapy Response ◽  
Cell Heterogeneity ◽  
Key Points ◽  
Cell Gene Expression ◽  
Cell Subpopulations ◽  
Cell Gene

Key Points Single-cell gene expression analysis reveals CML stem cell heterogeneity and changes imposed by TKI therapy. A subpopulation with primitive, quiescent signature and increased survival to therapy can be high-purity captured as CD45RA−cKIT−CD26+.

Loss Of p53 Rescues The Defective Function Of Foxo3-/- Hematopoietic Stem Cells But Enhances Their Predisposition To Malignancy

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4199-4199 ◽  
Author(s):  
Carolina L. Bigarella ◽  
Pauline Rimmele ◽  
Rebeca Dieguez-Gonzalez ◽  
Raymond Liang ◽  
Brigitte Izac ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Stem Cells ◽  
Dna Repair ◽  
Stem Cell ◽  
Gene Expression Profile ◽  
Double Mutant ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Anti Oxidant

Abstract Leukemic stem cells (LSCs) share many of the same properties of normal hematopoietic stem cells (HSCs) including their highly quiescent state, capacity to self-renew, low levels of reactive oxygen species (ROS) and enhanced DNA repair program. These properties make the efficient and specific eradication of these cells challenging. Foxo3 and p53 are two transcription factors essential for the modulation of HSC quiescence and self-renewal. While Foxo3 is inhibited by signaling from several oncoproteins but crucial for the maintenance of the LSCs in both chronic and acute myeloid leukemia (CML and AML respectively), mutations of p53 although rare, are associated with poor prognosis in advanced stages of these diseases. In vivo ROS-mediated activation of p53 is known to lead to loss of quiescence, alterations of cell cycle and exhaustion of the Foxo3-/- HSC pool. Seeking to understand the contribution of p53 to Foxo3-/- HSC cycling defects, we crossed p53+/- and Foxo3+/- mice. To our surprise we found the bone marrow (BM) frequency of both p53+/-Foxo3-/- and p53-/-Foxo3-/- LSK (Lin-Sca1+cKit+) and long-term-HSC (LT-HSC, LSK Flk2-CD34-) populations greatly increased as compared to their Foxo3-/- counterparts (n=5 mice per genotype; p<0.05). Using Ki67 and DAPI staining we found that loss of one or both alleles of p53 gradually rescued the cell cycle defect of Foxo3-/- HSC and increased the frequency of LSK cells in Go by 2-fold. Loss of p53 also rescued the impaired capacity of Foxo3-/- LSK cells to competitively repopulate multilineage blood over 16 weeks, as shown by the higher frequency of p53+/-Foxo3-/- and p53-/-Foxo3-/- donor-derived cells in the peripheral blood of recipient animals (∼47% recipients of double-mutant cells versus 20% in Foxo3-/- recipients, n=5 per group). Furthermore, loss of p53 significantly improved the compromised self-renewal of Foxo3 mutant HSC in serial BM transplantations. In our quest to identify mechanisms whereby p53 depletion improves Foxo3-/- HSC function, we noticed that the DNA damage accumulated in Foxo3-/- HSC at the steady-state was remarkably ameliorated by removal of one or both alleles of p53 from Foxo3-/- HSCs, as measured by flow cytometry levels of phospho-H2AX (gamma-H2AX) and DNA breaks by comet assay (n=3, p<0.05). Unexpectedly, ROS levels were also significantly reduced by 30% in p53+/-Foxo3-/- in comparison to Foxo3-/- LSK cells, while ROS levels in p53+/- LSK cells were similar to that in WT cells. Consistent with these results, the expression of several anti-oxidant enzymes including Sod1, Sod2, Catalase, Gpx1, Sesn1 and Sesn2 (n≥2), was highly upregulated while a number of genes implicated in mitochondrial generation of ROS were significantly deregulated as a result of loss of one or both alleles of p53. These combined findings suggest that a switch from anti-oxidant to pro-oxidant activity of p53 contributes to Foxo3-/- HSC defects. Despite their apparent normal stem cell function, p53+/-Foxo3-/- HSC were highly altered in their gene expression profile. Interestingly, Gene Set Enrichment Analysis (GSEA) of the microarray analysis (Illumina bead chip mouse-Ref8) of WT, p53+/-, Foxo3-/-, and p53+/-Foxo3-/- LSK cells showed that a cluster of genes associated with fatty acid metabolism was highly enriched in p53+/-Foxo3-/- HSCs (ES=0.746; p<0.01). In addition, from 3976 genes exclusively deregulated in p53+/-Foxo3-/- LSK cells, 201 (out of 1051) overlapped with genes downregulated, while 9 (out of 14) overlapped with genes exclusively upregulated in a LSC-gene signature. To evaluate whether this pre-leukemic profile was associated with increased susceptibility to malignancy, we compared the potential and timeline of BCR-ABL-transformed p53+/-Foxo3-/- HSC as compared to controls in establishing CML in mice. We found a shorter time to the onset of the disease and decreased survival of the recipients of p53+/-Foxo3-/- transformed HSCs (n=4 per group, p<0.05) as compared to WT and Foxo3-/- controls. We propose that the p53+/-Foxo3-/- double-mutant HSCs are enriched for preleukemic stem cells based on their quiescence and self-renewal capacity, low ROS, robust DNA repair, susceptibility to transformation and aberrant gene expression profile. These findings raise the possibility that the coordinated Foxo3 and p53 regulation of ROS wires together the stem cell program. Disclosures: No relevant conflicts of interest to declare.

Defining Cellular States, Differentiation Trajectories, and Regulatory Networks Through Single Cell Profiling

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. SCI-2-SCI-2
Author(s):  
Bertie Gottgens
Keyword(s):  
Stem Cells ◽  
Transcription Factor ◽  
Stem Cell ◽  
Single Cell ◽  
Regulatory Networks ◽  
Conflicts Of Interest ◽  
Blood Stem Cell ◽  
Single Cell Genomics ◽  
Blood Stem Cells ◽  
Combinatorial Interactions

Abstract Transcription factor proteins have long been recognized as key regulators of blood stem cell function. They are thought to act as components of wider regulatory networks, with combinatorial interactions responsible for directing blood stem cell fate choices. However, the complexity of these networks coupled with the infrequency of stem cells have presented formidable challenges for past research efforts aiming to define blood stem cell regulatory networks. Importantly, recent innovations in single cell genomics and computational network inference technologies offer new opportunities to better understand the regulatory network control of blood cell development. The Göttgens group uses a combination of experimental and computational approaches to study how transcription factor networks control the function of blood stem cells and how mutations that perturb such networks cause leukemia. This integrated approach has resulted in the discovery of new combinatorial interactions between key blood stem cell regulators, as well as experimentally validated computational models for blood stem cells. Current research focuses on single cell genomics of early blood development and the development of computer models to chart the transcriptional landscape of blood stem and progenitor cell differentiation. Disclosures No relevant conflicts of interest to declare.

Single-cell gene profiling of planarian stem cells using fluorescent activated cell sorting and its “index sorting” function for stem cell research

2010 ◽  
Vol 52 (1) ◽  
pp. 131-144 ◽  
Author(s):  
Tetsutaro Hayashi ◽  
Norito Shibata ◽  
Ryo Okumura ◽  
Tomomi Kudome ◽  
Osamu Nishimura ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Single Cell ◽  
Stem Cell Research ◽  
Cell Sorting ◽  
Gene Profiling ◽  
Cell Gene ◽  
Index Sorting

Metabolic suppression during mesodermal differentiation of embryonic stem cells identified by single-cell comprehensive gene expression analysis

2015 ◽  
Vol 11 (9) ◽  
pp. 2560-2567 ◽  
Author(s):  
Yuanshu Zhou ◽  
Ikuma Fujisawa ◽  
Kosuke Ino ◽  
Tomokazu Matsue ◽  
Hitoshi Shiku
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Single Cell ◽  
Expression Analysis ◽  
Gene Expression Analysis ◽  
Embryonic Stem ◽  
Metabolic Suppression ◽  
Cell Gene Expression ◽  
Cell Gene

Metabolic suppression has been revealed during mesodermal differentiation by using single-cell gene expression analysis.

scholarly journals The role of proto-oncogene GLI1 in pituitary adenoma formation and cell survival regulation

2015 ◽  
Vol 22 (5) ◽  
pp. 793-803 ◽  
Author(s):  
Katharina Lampichler ◽  
Patricio Ferrer ◽  
Greisa Vila ◽  
Mirjam I Lutz ◽  
Florian Wolf ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Stem Cells ◽  
Stem Cell ◽  
Pituitary Adenoma ◽  
Cell Survival ◽  
Pituitary Adenomas ◽  
Target Gene ◽  
Human Pituitary ◽  
Hh Signaling

The Hedgehog (Hh) pathway is an important regulator of early tissue patterning and stem cell propagation. It was found to be aberrantly activated in numerous types of human cancer and might be relevant in cancer stem cells. The identification of adult stem cells in the pituitary raised the question if tumor-initiating cells and Hh signaling are involved in pituitary adenoma formation. The present study aimed at the evaluation of Hh signaling in relation to stem cell and cell cycle markers in 30 human pituitary adenomas and in cultured murine adenoma cells. Therefore, expression levels of components of the Hh pathway, stem cell marker SOX2, cell cycle regulator tumor-protein 53 (TP53), proliferation marker Ki67 (MKI67) and superoxide dismutase 1 (SOD1) were evaluated in 30 human pituitary adenomas in comparison to control tissue. Modulation of cell function and target gene expression by the inhibition and activation of the Hh pathway were studied in murine adenoma cells. We show that transcription factor glioma-associated oncogene 1 (GLI1) is overexpressed in 87% of all pituitary adenomas. The expression of GLI1 significantly correlated with that of SOX2, TP53, MKI67 and SOD1. Inhibition of GLI1 resulted in the downregulation of the above genes and severe cell death in mouse adenoma cells. On the other hand, activation of the Hh pathway increased cell viability and target gene expression. In conclusion, our findings point toward an alternative, ligand-independent Hh pathway activation with GLI1 playing a major role in the cell survival of pituitary adenoma cells.

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