Journal: Journal of Translational Medicine

Article Title: Considerations in the development of circulating tumor cell technology for clinical use

doi: 10.1186/1479-5876-10-138

Figure Lengend Snippet: CTC Assays and Technologies

Article Snippet: Lymphoprep™ (Ficoll-Isopaque) , Axis-Shield PoC, Oslo, Norway , Enrichment , ---------- , Separates mononuclear cells from other cells in whole blood based on cell density..

Techniques: Incubation, Isolation, Multiplex Assay, Fluorescence, Field Flow Fractionation, FACS, Microscopy, Microarray, Expressing, Activation Assay, Staining, Diagnostic Assay, Immunofluorescence, Marker, Immunohistochemistry, Selection, Amplification, Lysis, Cytometry, Cell Culture, Imaging, Flow Cytometry, Suction Filtration, In Vivo, Injection, Spectroscopy, Irradiation, Immunocytochemistry, Real-time Polymerase Chain Reaction, Plasmid Preparation, Transfection, Functional Assay

Journal: Frontiers in Oncology

Article Title: The Prognostic Role of Circulating Tumor Cells (CTCs) in Lung Cancer

doi: 10.3389/fonc.2018.00311

Figure Lengend Snippet: The Summary of different Circulating Tumor Cell isolation methods currently used in research.

Article Snippet: Density-based Filtration , Cells are separated based on different densities after centrifugation. , Cells separated into distinct layers , CTC size and density not uniform CTCs may get lost in plasma or by formation of CTC aggregates Poor sensitivity , Ficoll Lymphoprep (Stem Cell Technologies, Vancouver, Canada) ( ) OncoQuick (VWR, Radnor, PA) Accucyte ( ) .

Techniques: Cell Isolation, Clinical Proteomics, Selection, Biomarker Discovery, Expressing, Filtration, Isolation, Centrifugation, Shear, Adsorption, Electrophoresis, Imaging, Fluorescence

Journal: Frontiers in Oncology

Article Title: The Prognostic Role of Circulating Tumor Cells (CTCs) in Lung Cancer

doi: 10.3389/fonc.2018.00311

Figure Lengend Snippet: Summary of long-term Circulating Tumor Cell culture in Lung cancer.

Article Snippet: Density-based Filtration , Cells are separated based on different densities after centrifugation. , Cells separated into distinct layers , CTC size and density not uniform CTCs may get lost in plasma or by formation of CTC aggregates Poor sensitivity , Ficoll Lymphoprep (Stem Cell Technologies, Vancouver, Canada) ( ) OncoQuick (VWR, Radnor, PA) Accucyte ( ) .

Techniques: Cell Culture, Isolation, Expressing, Biomarker Discovery

Journal: bioRxiv

Article Title: Natural and age-related variation in circulating human hematopoietic stem cells

doi: 10.1101/2023.11.30.569167

Figure Lengend Snippet: a experimental design, 1b – annotated 2D UMAP projection of our metacell manifold following filtration of metacells with low CD34 expression. Symmetric ( 1c ) and asymmetric ( 1d ) regulation of specific HSC markers upon bifurcation to the CLP (right) and MEBEMP (left) lineages. Each panel shows the expression of one gene (Y axis). Metacells in all panels are ordered (left to right) by increasing AVP expression in the MEBEMP lineage, and decreasing AVP expression in the CLP lineage. Units for gene expression in all the figure panels are log2 of each gene’s fractional expression. 1e – the BEMP-E metacell population of interest (dotted line) linking BEMPs to their MEBEMP-L precursors. 1f – positively and negatively regulated TFs involved in early BEMP differentiation. 1g – gene-gene plot of IRF8 against TCF7 expression as hallmark markers of DC and T cell differentiation respectively. The high ACY3 NKTDP metacell population of interest is depicted (dotted line). This population exhibits high expression of both T and dendritic cell regulators, forming a gradient consisting of NK/T cell-like progenitors exhibiting a high TCF7/IRF8 expression ratio along with high expression of other T cell hallmarks such as CD7 , MAF , IL7R , TRBC2 , and DC-like progenitors exhibiting a low TCF7/IRF8 expression ratio, along with high expression of other DC hallmarks, such as the myeloid TF PU.1 and the MHC class II gene CD74 ( 1h ).

Article Snippet: 1 ml of blood was used for DNA production, and the remaining volume was used for PBMC isolation via Ficoll, using Lymphoprep filled Sepmate tubes (StemCell technologies), followed by CD34 magnetic bead-based enrichment using the EasySep human CD34 positive selection kit II (StemCell technologies).

Techniques: Filtration, Expressing, Cell Differentiation

Journal: bioRxiv

Article Title: Natural and age-related variation in circulating human hematopoietic stem cells

doi: 10.1101/2023.11.30.569167

Figure Lengend Snippet: 1a – age distribution (decimals) of studied population by sex. 1b – 2D UMAP projection of our metacell model prior to CD34- metacell filtering. 1c - relative expression heatmap of cell states (columns) and markers used for cell state annotation (rows). 1d – Filtering metacells with low CD34 expression. 1e - gene-gene expression plot of DNTT and RUNX3 , showing early CLP differentiation and their bifurcation into late CLPs and NKTDPs. 1f –expression plot of MPO and GATA1/VPREB1 expression showing all 3 differentiation routes (GMPs, CLPs, MEBEMPs) from HSCs, and highlighting the GMP’s bifurcation from MPP / MEBEMP. All gene expression values are obtained by normalizing gene expression to sum to 1 and taking log2.

Article Snippet: 1 ml of blood was used for DNA production, and the remaining volume was used for PBMC isolation via Ficoll, using Lymphoprep filled Sepmate tubes (StemCell technologies), followed by CD34 magnetic bead-based enrichment using the EasySep human CD34 positive selection kit II (StemCell technologies).

Techniques: Expressing

Journal: bioRxiv

Article Title: Natural and age-related variation in circulating human hematopoietic stem cells

doi: 10.1101/2023.11.30.569167

Figure Lengend Snippet: 2a – 2D UMAP projection of a non- CD34 -enriched BM metacell model from the Human Cell Atlas , colored by a BM-specific cell type annotation. 2b - projection of our PB CD34 + derived metacells on the non- CD34 enriched BM metacell model. 2c – projection of BM CD34 + derived metacells [Setty et al.] on the non- CD34 enriched BM metacell model. 2d - gene-gene expression plots comparing PB CD34 + derived metacells with their BM CD34 + counterparts for all differentiation trajectories. Panels (left to right, top to bottom) represent CLP differentiation, MEBEMP differentiation, GMP differentiation, BEMP differentiation, DC differentiation, and MPO/CLP/MEBEMP trifurcation from HSCs. The first 4 differentiation panels represent similar PB and BM behaviors, while the last 2 show dissimilarities between them. PB / BM metacells are colored by PB / BM annotations, respectively. 2e – gene-gene expression plots comparing PB CD34 + derived metacells with their BM CD34 + counterparts for markers and regulators of CLP differentiation and bifurcation. 2f – relative expression heatmap of the megakaryocytic markers PF4 and PPBP and cell type specific markers, across metacells with high megakaryocytic signature. The figure shows an abnormally high doublet rate involving megakaryocytes.

Article Snippet: 1 ml of blood was used for DNA production, and the remaining volume was used for PBMC isolation via Ficoll, using Lymphoprep filled Sepmate tubes (StemCell technologies), followed by CD34 magnetic bead-based enrichment using the EasySep human CD34 positive selection kit II (StemCell technologies).

Techniques: Derivative Assay, Expressing

Journal: bioRxiv

Article Title: Natural and age-related variation in circulating human hematopoietic stem cells

doi: 10.1101/2023.11.30.569167

Figure Lengend Snippet: 4a - factors positively and negatively regulated in the early stages of BEMP specification. 4b – gene-gene expression plots of DNTT and ACY3 comparing CD34 -enriched and non-enriched BM (top , ), as well as non-enriched and partially enriched PB (bottom 34 ] to our CD34+ PB model. Metacells are color-coded by SYT2 expression (log2 transformed). The SYT2 high, ACY3 high, DNTT intermediate population clearly seen in our data is completely lacking from the BM datasets. 4c – anti-correlation of the DC IRF8 -MHC-II coupled dynamics and the T cell regulator TCF7, involved in the bifurcation of the NKTDP cell state to its sub-populations.

Article Snippet: 1 ml of blood was used for DNA production, and the remaining volume was used for PBMC isolation via Ficoll, using Lymphoprep filled Sepmate tubes (StemCell technologies), followed by CD34 magnetic bead-based enrichment using the EasySep human CD34 positive selection kit II (StemCell technologies).

Techniques: Expressing, Transformation Assay

Journal: bioRxiv

Article Title: Natural and age-related variation in circulating human hematopoietic stem cells

doi: 10.1101/2023.11.30.569167

Figure Lengend Snippet: 5a,b – Individual-specific differential gene expression after controlling for each individual’s distribution across the CD34 + PB manifold in MEBEMPs (top) and CLPs (bottom).

Article Snippet: 1 ml of blood was used for DNA production, and the remaining volume was used for PBMC isolation via Ficoll, using Lymphoprep filled Sepmate tubes (StemCell technologies), followed by CD34 magnetic bead-based enrichment using the EasySep human CD34 positive selection kit II (StemCell technologies).

Techniques: Expressing

Journal: bioRxiv

Article Title: Natural and age-related variation in circulating human hematopoietic stem cells

doi: 10.1101/2023.11.30.569167

Figure Lengend Snippet: a characterization of inter-individual HSPC compositional state variation (scheme). 2b – boxplots of cell state frequency distributions across individuals (logarithmic scale). Percents calculated out of CD34 + population. Boxplot centers, hinges and whiskers represent median, first and third quartiles and 1.5× interquartile range, respectively. Numbers represent mean +/- SD for each distribution. 2c - correlation of cell state frequencies across individuals. 2d ( top ) - individual cell state frequency profiles over the HSC-MEBEMP and HSC-CLP differentiation gradients of 6 subjects (colored lines), each representing one of six archetypes (classes) of HSPC composition in healthy individuals. Dashed lines represent the median (black) and 5 th and 95 th percentiles (grey) of the studied population. 2d ( bottom ) cell state enrichment map over 15 differentiation bins (rows), for all studied individuals (columns) clustered into 6 classes. Classes I & II represent individuals relatively enriched in lymphoid progenitors, whereas classes V & VI represent individuals with relative depletion of lymphoid progenitors. Individuals are sorted by stemness in each class. Age and sex bins are denoted for each individual (top). 2e – CBC correlations to cell type frequencies: %Lym (from WBC, calculated for entire cohort, left), HCT (males, center), RDW (males, right). Missing individuals lacked sufficient cells for analysis. Permutation test p values are displayed for each correlation. 2f – boxplots of CLP frequency distributions in individuals with (right) and without (left) clonal hematopoiesis. 2g – Relative cell state frequencies in mutant (right) and non-mutant (left) cells following GoT of sample #122 (DNMT3A mutated, VAF = 0.07). 2h – CH frequency (by gene) in age- and sex-matched high (red) and low (black) RDW individuals.

Article Snippet: 1 ml of blood was used for DNA production, and the remaining volume was used for PBMC isolation via Ficoll, using Lymphoprep filled Sepmate tubes (StemCell technologies), followed by CD34 magnetic bead-based enrichment using the EasySep human CD34 positive selection kit II (StemCell technologies).

Techniques: Mutagenesis

Journal: bioRxiv

Article Title: Natural and age-related variation in circulating human hematopoietic stem cells

doi: 10.1101/2023.11.30.569167

Figure Lengend Snippet: a diagnostic approach to leukemia analysis using our HSPC reference atlas (scheme): 1. scRNAseq on CD34-enriched PB and construction of a patient-specific metacell model, 2. Projection of patient derived metacells on the healthy reference atlas - compositional variance and differential gene expression analysis, 3. Mutational and CNV analysis using targeted DNA sequencing and RNA-based karyotyping, 4. RNA-based clonal hierarchy and population substructure analysis using: 4.1 individual cell state frequency profiles over the HSC-MEBEMP and HSC-CLP differentiation gradients, 4.2 sub-population identification of AML cells with CLP, HSC and MEBEMP characteristics 4.3 de-novo identification of clonal specific gene clusters and signatures. 5b – density plot of the number of differentially-expressed genes (≥2-fold) per metacell as compared to its projection counterpart on our healthy HSPC atlas, for 2 healthy (left), 2 MDS (middle), and 2 AML (right) patients. 5c – projection of metacells derived from 2 MDS (left) and 2 AML (right) patients on our healthy HSPC reference metacell model. 5d – individual cell state frequency profiles over the HSC-MEBEMP and HSC-CLP differentiation gradients for 2 MDS cases (red lines). Dashed lines represent the median (black) and 5 th and 95 th percentiles (grey) of the healthy population, and MDS-2’s initial profile (red, right panel, 8 month prior to current profiling). 5e – each of the 4 panels refers to a different cell state gene signature as noted on the x-axis. Top - boxplots of gene module expression distributions for different cell states in our reference atlas. Bottom - Gene signature expression density plots for each of the AML subclones. Reference gene signature distributions (top) were used to identify subpopulations of AML cells with CLP, HSC and MEBEMP characteristics (bottom). Dashed lines represent the threshold for expressing a gene signature, and the fraction of cells expressing a signature per AML clone is listed. 5f - left – correlation heatmap of differentially expressed gene signatures for AML-1. The malignant state is characterized by multiple novel gene expression signatures in addition to aberrant expression of “healthy” differentiation-related modules, right – UMAP projection of the metacell model of AML-1, colored by relative expression of differentially expressed genes. Overexpression of BCL2 in AML-1-2 compared to AML-1-1 can be seen on the top left panel. 5g – same as 5f for AML-2.

Article Snippet: 1 ml of blood was used for DNA production, and the remaining volume was used for PBMC isolation via Ficoll, using Lymphoprep filled Sepmate tubes (StemCell technologies), followed by CD34 magnetic bead-based enrichment using the EasySep human CD34 positive selection kit II (StemCell technologies).

Techniques: Diagnostic Assay, Derivative Assay, Expressing, DNA Sequencing, Over Expression