scholarly journals Single cell PCR from archival stained bone marrow slides: A method for molecular diagnosis and characterization

2004 ◽  
Vol 18 (3) ◽  
pp. 176-181 ◽  
Author(s):  
Stefanie Zanssen
Keyword(s):  
Bone Marrow ◽  
Single Cell ◽  
Molecular Diagnosis ◽  
Single Cell Pcr

IL7 Counteraction with Notch Signaling Followed by EBF1 Expression Marks the B-Cell Commitment in CLP Stage

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2452-2452
Author(s):  
Sasan Zandi ◽  
Panagiotis Tsapogas ◽  
Robert Månsson ◽  
Mikael Sigvardsson
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
B Cell ◽  
Single Cell ◽  
Reporter Gene ◽  
B Cell Development ◽  
Expression Level ◽  
Single Cell Pcr ◽  
Cell Commitment

Abstract Development of B-cell lineage from hematopoietic Stem cells in bone marrow is a stepwise process associated with a gradual loss of myeloid and T cell potential. This process involves a complex interaction of transcription factors like EBF1 and E2A, and extrinsic signals including IL7. It has been suggested that IL7 plays an inductive role in B-cell commitment through EBF activation in early B-Cell development. Mice deficient in Il-7 signaling show a dramatic reduction in the number of B-cell progenitors and reduced expression of EBF1 in the common lymphoid progenitor (CLP) compartment and ectopic expression of EBF can partially rescue the B-cell phenotype. However, the rather limited ability of EBF1 to rescue the phenotype as well as the powerful function of Il-7 in the expansion of committed cells creates a complex situation with an inherent difficulty to separate instructive and permissive actions of Il-7. Using transgenic mice carrying a reporter gene under the control of the EBF1 dependent Igll1 promoter, we were able to identify a B220−CD19− committed B-cell progenitor likely to represent the earliest committed population in the mouse bone marrow. This has opened the possibility to investigate lineage commitment in cells not expressing classical surface markers creating increased possibilities to study lineage choices. In order to investigate the inductive role of Il-7 we crossed the Igll1 reporter mice to Il-7 deficient mice. Analysis of reporter gene expression, gene expression by multiplex single cell PCR as well as functional analysis by in vitro differentiation assays, all supported that the committed lineage negative population was dramatically decreased in the absence of Il-7. These data all support the idea that Il-7 is critical not only for expansion of B-lineage progenitors but also for commitment per se. Investigation of the expression of EBF-1 by Real time and single cell PCR suggested that the expression level of EBF was on an average 50% lower in Il-7 deficient progenitors as compared to wild type cells. This expression level was recapitulated in mice heterozygote for a mutation in the EBF1 gene but since the formation of the early committed cells was not as dramatically effected in these mice, we found a need to look for a further function of Il-7 in its instructive role in B-cell development. This prompted us to investigate a potential function of Il-7 in the modulation of Notch signals known to counteract B-cell commitment and EBF function. This revealed that the addition of Il-7 largely inhibits the Notch response in pro-B cells in vitro. Therefore we suggest that Il-7 is directly involved in B-cell commitment and that this function is achieved by modulation of EBF1 both at the transcriptional and functional level.

scholarly journals IMMU-27. SINGLE CELL RNA-SEQUENCING IDENTIFIES NOVEL BONE MARROW DERIVED MYELOID CELLS IN GLIOBLASTOMA ASSOCIATED WITH TUMOR AGGRESSION

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii110-ii110
Author(s):  
Christina Jackson ◽  
Christopher Cherry ◽  
Sadhana Bom ◽  
Hao Zhang ◽  
John Choi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Single Cell ◽  
Tumor Cells ◽  
Rna Sequencing ◽  
Metabolic Pathways ◽  
Myeloid Cells ◽  
Tumor Grade ◽  
Sequencing Data ◽  
Single Cell Rna Sequencing ◽  
Two Populations

Abstract BACKGROUND Glioma associated myeloid cells (GAMs) can be induced to adopt an immunosuppressive phenotype that can lead to inhibition of anti-tumor responses in glioblastoma (GBM). Understanding the composition and phenotypes of GAMs is essential to modulating the myeloid compartment as a therapeutic adjunct to improve anti-tumor immune response. METHODS We performed single-cell RNA-sequencing (sc-RNAseq) of 435,400 myeloid and tumor cells to identify transcriptomic and phenotypic differences in GAMs across glioma grades. We further correlated the heterogeneity of the GAM landscape with tumor cell transcriptomics to investigate interactions between GAMs and tumor cells. RESULTS sc-RNAseq revealed a diverse landscape of myeloid-lineage cells in gliomas with an increase in preponderance of bone marrow derived myeloid cells (BMDMs) with increasing tumor grade. We identified two populations of BMDMs unique to GBMs; Mac-1and Mac-2. Mac-1 demonstrates upregulation of immature myeloid gene signature and altered metabolic pathways. Mac-2 is characterized by expression of scavenger receptor MARCO. Pseudotime and RNA velocity analysis revealed the ability of Mac-1 to transition and differentiate to Mac-2 and other GAM subtypes. We further found that the presence of these two populations of BMDMs are associated with the presence of tumor cells with stem cell and mesenchymal features. Bulk RNA-sequencing data demonstrates that gene signatures of these populations are associated with worse survival in GBM. CONCLUSION We used sc-RNAseq to identify a novel population of immature BMDMs that is associated with higher glioma grades. This population exhibited altered metabolic pathways and stem-like potentials to differentiate into other GAM populations including GAMs with upregulation of immunosuppressive pathways. Our results elucidate unique interactions between BMDMs and GBM tumor cells that potentially drives GBM progression and the more aggressive mesenchymal subtype. Our discovery of these novel BMDMs have implications in new therapeutic targets in improving the efficacy of immune-based therapies in GBM.

scholarly journals Comprehensive network modeling from single cell RNA sequencing of human and mouse reveals well conserved transcription regulation of hematopoiesis

BMC Genomics ◽  
2020 ◽  
Vol 21 (S11) ◽  
Author(s):  
Shouguo Gao ◽  
Zhijie Wu ◽  
Xingmin Feng ◽  
Sachiko Kajigaya ◽  
Xujing Wang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Transcription Factors ◽  
Single Cell ◽  
Rna Sequencing ◽  
Transcription Regulation ◽  
Regulatory Networks ◽  
Stem Cell Differentiation ◽  
Hematopoietic Stem ◽  
Single Cell Rna Sequencing ◽  
Human And Mouse

Abstract Background Presently, there is no comprehensive analysis of the transcription regulation network in hematopoiesis. Comparison of networks arising from gene co-expression across species can facilitate an understanding of the conservation of functional gene modules in hematopoiesis. Results We used single-cell RNA sequencing to profile bone marrow from human and mouse, and inferred transcription regulatory networks in each species in order to characterize transcriptional programs governing hematopoietic stem cell differentiation. We designed an algorithm for network reconstruction to conduct comparative transcriptomic analysis of hematopoietic gene co-expression and transcription regulation in human and mouse bone marrow cells. Co-expression network connectivity of hematopoiesis-related genes was found to be well conserved between mouse and human. The co-expression network showed “small-world” and “scale-free” architecture. The gene regulatory network formed a hierarchical structure, and hematopoiesis transcription factors localized to the hierarchy’s middle level. Conclusions Transcriptional regulatory networks are well conserved between human and mouse. The hierarchical organization of transcription factors may provide insights into hematopoietic cell lineage commitment, and to signal processing, cell survival and disease initiation.

scholarly journals Single-Cell RNA-Seq of Bone Marrow-Derived Mesenchymal Stem Cells Reveals Unique Profiles of Lineage Priming

PLoS ONE ◽  
2015 ◽  
Vol 10 (9) ◽  
pp. e0136199 ◽  
Author(s):  
Brian T. Freeman ◽  
Jangwook P. Jung ◽  
Brenda M. Ogle
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Single Cell ◽  
Rna Seq ◽  
Lineage Priming

scholarly journals SiglecF(HI) Marks Late‐Stage Neutrophils of the Infarcted Heart: A Single‐Cell Transcriptomic Analysis of Neutrophil Diversification

2021 ◽  
Vol 10 (4) ◽  
Author(s):  
David M. Calcagno ◽  
Claire Zhang ◽  
Avinash Toomu ◽  
Kenneth Huang ◽  
Van K. Ninh ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Single Cell ◽  
Negative Selection ◽  
Quantitative Polymerase Chain Reaction ◽  
Chain Reaction ◽  
Cell Sorter ◽  
Infarcted Heart ◽  
Single Cell Rna Sequencing ◽  
Polymerase Chain ◽  
Deficient Mice

Background Neutrophils are thought to be short‐lived first responders to tissue injuries such as myocardial infarction (MI), but little is known about their diversification or dynamics. Methods and Results We permanently ligated the left anterior descending coronary arteries of mice and performed single‐cell RNA sequencing and analysis of >28 000 neutrophil transcriptomes isolated from the heart, peripheral blood, and bone marrow of mice on days 1 to 4 after MI or at steady‐state. Unsupervised clustering of cardiac neutrophils revealed 5 major subsets, 3 of which originated in the bone marrow, including a late‐emerging granulocyte expressing SiglecF, a marker classically used to define eosinophils. SiglecF HI neutrophils represented ≈25% of neutrophils on day 1 and grew to account for >50% of neutrophils by day 4 post‐MI. Validation studies using quantitative polymerase chain reaction of fluorescent‐activated cell sorter sorted Ly6G + SiglecF HI and Ly6G + SiglecF LO neutrophils confirmed the distinct nature of these populations. To confirm that the cells were neutrophils rather than eosinophils, we infarcted GATA‐deficient mice (∆dblGATA) and observed similar quantities of infiltrating Ly6G + SiglecF HI cells despite marked reductions of conventional eosinophils. In contrast to other neutrophil subsets, Ly6G + SiglecF HI neutrophils expressed high levels of Myc‐regulated genes, which are associated with longevity and are consistent with the persistence of this population on day 4 after MI. Conclusions Overall, our data provide a spatial and temporal atlas of neutrophil specialization in response to MI and reveal a dynamic proinflammatory cardiac Ly6G + SigF + (Myc + NFϰB + ) neutrophil that has been overlooked because of negative selection.

scholarly journals Single Cell Phenotyping Reveals Heterogeneity among Haematopoietic Stem Cells Following Infection

2016 ◽  
Author(s):  
Adam L MacLean ◽  
Maia A Smith ◽  
Juliane Liepe ◽  
Aaron Sim ◽  
Reema Khorshed ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Single Cell ◽  
Life History Theory ◽  
Cell Function ◽  
Trichinella Spiralis ◽  
Haematopoietic Stem Cell ◽  
Haematopoietic Stem Cells ◽  
Hsc Niche

AbstractThe haematopoietic stem cell (HSC) niche provides essential micro-environmental cues for the production and maintenance of HSCs within the bone marrow. During inflammation, haematopoietic dynamics are perturbed, but it is not known whether changes to the HSC-niche interaction occur as a result. We visualise HSCs directly in vivo, enabling detailed analysis of the 3D niche dynamics and migration patterns in murine bone marrow following Trichinella spiralis infection. Spatial statistical analysis of these HSC trajectories reveals two distinct modes of HSC behaviour: (i) a pattern of revisiting previously explored space, and (ii) a pattern of exploring new space. Whereas HSCs from control donors predominantly follow pattern (i), those from infected mice adopt both strategies. Using detailed computational analyses of cell migration tracks and life-history theory, we show that the increased motility of HSCs following infection can, perhaps counterintuitively, enable mice to cope better in deteriorating HSC-niche micro-environments following infection.Author SummaryHaematopoietic stem cells reside in the bone marrow where they are crucially maintained by an incompletely-determined set of niche factors. Recently it has been shown that chronic infection profoundly affects haematopoiesis by exhausting stem cell function, but these changes have not yet been resolved at the single cell level. Here we show that the stem cell–niche interactions triggered by infection are heterogeneous whereby cells exhibit different behavioural patterns: for some, movement is highly restricted, while others explore much larger regions of space over time. Overall, cells from infected mice display higher levels of persistence. This can be thought of as a search strategy: during infection the signals passed between stem cells and the niche may be blocked or inhibited. Resultantly, stem cells must choose to either ‘cling on’, or to leave in search of a better environment. The heterogeneity that these cells display has immediate consequences for translational therapies involving bone marrow transplant, and the effects that infection might have on these procedures.

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