Journal: Blood Advances

Article Title: Novel humanized loss-of-function NF1 mouse model of juvenile myelomonocytic leukemia

doi: 10.1182/bloodadvances.2024015191

Figure Lengend Snippet: In vitro KO of human NF1 gene using CRISPR/Cas9. (A ) Schematic of in vitro NF1-KO in UCB derived HSPCs (UCB HSPCs − CD34 + cells) using CRISPR/Cas9 technology. (B) KO score quantification by sanger sequencing. (C) Functional read out of CFU capacity of NF1-KO cells alongside unedited controls in the presence of 2 ng/mL GM-CSF. Stacked bar graph represents quantification of CFU-GEMM (oligopotent), CFU-GM (granulocyte myeloid), CFU-E (erythroid) colonies. (D) NF1 protein (∼319 kDa) detection using WES in NF1-KO and control HSPCs. HSP90 (∼90 kDa) was run as housekeeping control. Chemiluminescence peaks are shown on the y-axis and molecular weights on the x-axis. Black arrows point toward the peaks expected for respective protein sizes. All values shown in bar graphs are mean ± standard deviation (SD). Student t test was performed for statistical analysis. ∗ P < .05; ∗∗ P < .001; ∗∗∗∗ P < .0001.

Article Snippet: Cells were filtered through a 70-μm strainer, and CD34 + HSPCs were purified using CD34 MicroBeads and magnetic-activated cell sorting columns (catalog no. 130-046-702; Miltenyi) following the manufacturer’s protocol.

Techniques: In Vitro, CRISPR, Derivative Assay, Sequencing, Functional Assay, Control, Standard Deviation

Journal: Journal of Translational Medicine

Article Title: Ex vivo long-term expansion of human hematopoietic stem and progenitor cells as a tool for modeling vector integration sites and clonality

doi: 10.1186/s12967-026-07700-6

Figure Lengend Snippet: Ex vivo expansion of HSPCs in A83-01, pomalidomide, and UM171 (APU). ( A ) Experimental scheme of the 7-day ex vivo expansion protocol. CD34 + CD38 − CD45RA − cells from umbilical cord blood (UCB) were seeded in low cell density for HSPC expansion and analyzed via flow cytometry for their HSC immune phenotype. ( B ) Representative plots for the gating strategy of CD34 + CD38 − CD45RA − CD90 + EPCR + cells in SFT3 (basal medium), UM171, and APU. ( C ) Surface marker expression of CD34, CD38, CD45RA, CD90, and EPCR in N = 3 UCB donors after 7-day expansion of CD34 + CD38 − CD45RA − cells in SFT3, UM171, and APU with 2 µM or 0.2 µM pomalidomide (Poma). ( D ) Expansion of the total cell number in one 96-well counted with Casy cell counter. ( E ) Fold change of expanded ST-HSCs after 7-days. The number of expanded CD34 + CD38 − CD45RA − CD90 + cells was divided by 1,000 (seeded cell number) and then normalized to SFT3. ( F ) Percentage of CD34 + CXCR4 + HSPCs after expansion. The mean is displayed with the SD by the error bars. Statistical analysis in D-F was performed with one-way ANOVA and Tukey’s test for multiple comparisons in N = 4 UCB donors (* = p < 0.05, ** = p < 0.01)

Article Snippet: Thus, CD34 + -enriched HSPCs were thawed and blocked with Fc receptor blocking reagent (Miltenyi Biotec) before staining with antibodies against CD34 (APC, clone HG12, catalog #345804, BD Biosciences, Franklin Lakes, NJ, Unites States), CD38 (FITC, clone HB7, catalog #11-0388-42, Thermo Fisher Scientific, Waltham, MA, USA), and CD45RA (BV605, clone HI100, catalog #304134, BioLegend, San Diego, CA, USA).

Techniques: Ex Vivo, Flow Cytometry, Marker, Expressing

Journal: Journal of Translational Medicine

Article Title: Ex vivo long-term expansion of human hematopoietic stem and progenitor cells as a tool for modeling vector integration sites and clonality

doi: 10.1186/s12967-026-07700-6

Figure Lengend Snippet: Single-cell RNA sequencing (scRNA seq) analysis reveals expression of HSC signature genes in APU-exp HSPCs. ( A ) Surface marker expression of CD34, CD38, CD45RA, CD90, and EPCR after 7-day expansion in SFT3 or SFT3 supplemented with single (A, P, U), minus-one (AP, AU, PU), and complete APU (APU with 2 µM or 0.2 µM Poma) compound conditions. ( B ) Experimental scheme for the scRNA seq experiment using the 10X Genomics Chromium technology and hashtag oligo (HTO)-labeled antibodies for sample multiplexing. UCB-derived CD34 + CD38 − CD45RA − HSPCs were analyzed uncultivated, 24 h cultivated in SFT, SFT + APU, or SFT + APU_0.2, and 7-day expanded in the conditions described in ( A ). ( C ) Uniform Manifold Approximation and Projection for Dimension Reduction (UMAP) displaying the integrated data of all samples with cluster annotation: Hematopoietic stem cells/multipotent progenitors (HSCs/MPPs), early and late cycling megakaryocytic-erythroid-mast cell progenitors (cMEMPs), granulocytes (neutrophils, eosinophils, basophils/mast cells), mast cells, monocytes and macrophages (Mono’s/Neutro’s), cycling MPPs (cMPPs), and cycling dendritic cells (cDCs). ( D ) Dot plot showing the average expression of the respective marker genes used in cluster annotation

Article Snippet: Thus, CD34 + -enriched HSPCs were thawed and blocked with Fc receptor blocking reagent (Miltenyi Biotec) before staining with antibodies against CD34 (APC, clone HG12, catalog #345804, BD Biosciences, Franklin Lakes, NJ, Unites States), CD38 (FITC, clone HB7, catalog #11-0388-42, Thermo Fisher Scientific, Waltham, MA, USA), and CD45RA (BV605, clone HI100, catalog #304134, BioLegend, San Diego, CA, USA).

Techniques: Single Cell, RNA Sequencing, Expressing, Marker, Labeling, Multiplexing, Derivative Assay

Journal: Journal of Translational Medicine

Article Title: Ex vivo long-term expansion of human hematopoietic stem and progenitor cells as a tool for modeling vector integration sites and clonality

doi: 10.1186/s12967-026-07700-6

Figure Lengend Snippet: Effect of APU on HSPCs after 24 h cultivation on the single-cell level. ( A ) UMAP of uncultivated and 24 h-cultivated HSPCs and their individual cluster abundances in the bar plot below. ( B ) Feature plots of the HSC signature genes HLF , MECOM , NKAIN1 , PROM1 , AVP , and CD34 . ( C) Dotplot comparing the average expression of HSC signature genes among the 24 h-cultivated HSPCs. ( D ) Volcano plot displaying the log2 fold changes (FC) and the -log10 p values of DEGs between HSPCs cultivated for 24 h in SFT + APU (APU_24h) compared to SFT (SFT_24h). Significant ( p > 0.05, Wilcoxon Rank Sum test) genes with an FC > 1 are colored in green (up in APU_24h) or yellow (up in SFT_24h). ( E ) Gene set enrichment analysis (GSEA) on the DEGs of APU_24h vs. SFT_24h using HSC-related gene sets from the Molecular Signatures Database (MSigDB), showing the normalized enrichment score (NES). ( F ) GSEA on the DEGs of APU_24h vs. SFT_24h and of SFT3 + APU_7d vs. SFT3_7d (HSC/MPP cluster) using TGFβ-related gene sets from the MSigDB

Article Snippet: Thus, CD34 + -enriched HSPCs were thawed and blocked with Fc receptor blocking reagent (Miltenyi Biotec) before staining with antibodies against CD34 (APC, clone HG12, catalog #345804, BD Biosciences, Franklin Lakes, NJ, Unites States), CD38 (FITC, clone HB7, catalog #11-0388-42, Thermo Fisher Scientific, Waltham, MA, USA), and CD45RA (BV605, clone HI100, catalog #304134, BioLegend, San Diego, CA, USA).

Techniques: Single Cell, Expressing

Journal: Journal of Translational Medicine

Article Title: Ex vivo long-term expansion of human hematopoietic stem and progenitor cells as a tool for modeling vector integration sites and clonality

doi: 10.1186/s12967-026-07700-6

Figure Lengend Snippet: Increased lentiviral transduction efficiency in APU-exp cells. ( A ) Graphical scheme of the combined transduction (td) and expansion protocol for UCB-derived CD34 + cells. Td was performed with 1 × 10 5 CD34 + 24 h post-thawing with a self-inactivating lentiviral vector encoding for mCherry driven by the CBX3.EFS promoter (SIN-LV.CBX3.EFS.mCherry) in APU/SFT3 medium supplemented with protamine sulfate and Synperonic ® F-108 in a U-bottom 96-well plate. On day 1 post-td, HSPCs were seeded for expansion. Td efficiency and stem cell phenotype were determined on day 6 post-td, and vector copy number (VCN) was determined on day 8 post-td. Each donor replicate of expanded cells was split for transplantation into three mice. ( B ) Number of cells after thawing CD34 + cells from N = 3 UCB donors (day − 1), the next day (day 0), and after expansion (day 6 post-td) counted with Casy. ( C ) HSPC immune phenotype of expanded cells 6 days post-td. Expression of CD34 + , CD34 + CD38 − CD45RA − CD90 + (labeled CD90 + ), and CD34 + EPCR + in expanded HSPCs. ( D ) Td efficiency in the bulk cell population, in CD34 + HSPCs, and CD34 + CD38 − CD45RA − CD90 + EPCR + (labeled as LT-HSC). ( E ) VCN per diploid cell determined by qPCR. Bars indicate the mean of n = 3 UCB donors with the SD displayed by the error bars; Statistical significances were determined by paired t-test; p < 0.05= *, p < 0.0001= ****

Article Snippet: Thus, CD34 + -enriched HSPCs were thawed and blocked with Fc receptor blocking reagent (Miltenyi Biotec) before staining with antibodies against CD34 (APC, clone HG12, catalog #345804, BD Biosciences, Franklin Lakes, NJ, Unites States), CD38 (FITC, clone HB7, catalog #11-0388-42, Thermo Fisher Scientific, Waltham, MA, USA), and CD45RA (BV605, clone HI100, catalog #304134, BioLegend, San Diego, CA, USA).

Techniques: Transduction, Derivative Assay, Plasmid Preparation, Transplantation Assay, Expressing, Labeling

Journal: Journal of Translational Medicine

Article Title: Ex vivo long-term expansion of human hematopoietic stem and progenitor cells as a tool for modeling vector integration sites and clonality

doi: 10.1186/s12967-026-07700-6

Figure Lengend Snippet: APU_0.2 allows long-term cultivation of CD34 + cells up to 5 weeks after lentiviral transduction. ( A ) Experimental setup of transducing expanded HSPCs. Purified CD34 + CD38 − CD45RA − cells were expanded for 7 days either in SFT3 or APU_0.2 medium before transduction (td) with the SIN-LV.SF.eGFP (SF) or the SIN-LV.EFS.eGFP (EFS) vector. 24 h post-td, HSPCs were either transplanted into immunocompromised mice or further cultivated. Long-term culture allowed downstream analyses. ( B ) Expansion factor after 7-day culture in SFT3 or APU_0.2 medium determined by Casy. Paired t-test revealed non-significant (ns = p > 0.05) differences. ( C ) Flow cytometric analysis of eGFP expression on day 7 post-td to determine the td efficiency. ( D ) Vector copy number (VCN) per diploid genome was determined via ddPCR. ( E ) Expansion factor over 5 weeks post-td either in SFT3 or APU_0.2 medium. Cell numbers were determined by Casy and divided by the seeded cell number from the week before. Paired t-test over the whole period of time between APU_0.2 and SFT3 to assess the expected mean difference (diff.) between the groups, the 95% confidence interval (CI), and the p value. ( F ) Microscopic images 3 weeks post-td. Imaging was performed with CellCyteX using 10x magnification. Scale bar length indicates 100 μm. ( G-H ) Monocytic markers (CD11b, CD14, CD33) were determined via flow cytometry 3, 4, and 6 weeks post-td of HSPCs cultivated in SFT3 ( G ) or APU_0.2 ( H ). ( I ) Dim and bright CD11b expression in HSPCs cultivated for 3 weeks post-td. Exemplary gating strategy of the CD11b + bright CD14 + population for SFT3 cultivated cells. ( J ) Percentage of CD34 + cells during the long-term culture in SFT3 or APU_0.2 up to 5 weeks post-td. Two-way ANOVA with Tukey’s multiple comparisons test. Individual values are biological replicates with N = 3 UCB donors. Error bars indicate the mean ± SD

Article Snippet: Thus, CD34 + -enriched HSPCs were thawed and blocked with Fc receptor blocking reagent (Miltenyi Biotec) before staining with antibodies against CD34 (APC, clone HG12, catalog #345804, BD Biosciences, Franklin Lakes, NJ, Unites States), CD38 (FITC, clone HB7, catalog #11-0388-42, Thermo Fisher Scientific, Waltham, MA, USA), and CD45RA (BV605, clone HI100, catalog #304134, BioLegend, San Diego, CA, USA).

Techniques: Transduction, Purification, Plasmid Preparation, Expressing, Imaging, Flow Cytometry

Journal: bioRxiv

Article Title: Interleukin-6 restricts pre-thymic T cell lineage commitment of progenitors driving loss of SIV control

doi: 10.64898/2026.01.29.702123

Figure Lengend Snippet: a, Schematic illustrating thymus-seeding progenitors (TSPs) arising from double-negative (DN; CD4⁻CD8⁻) CD34⁺CD7⁺ hematopoietic stem and progenitor cells (HSPCs) in the bone marrow and their migration to the thymus, where they undergo stepwise differentiation through CD4⁺CD3 − immature single-positive (ISP), CD4⁺CD8⁺ double-positive (DP), and single-positive (SP) CD4⁺ or CD8⁺ T-cell stages. b-c, Representative flow-cytometry plots showing (DN)CD34⁺CD7⁺ HSPCs in bone marrow (b) and thymus (c), with corresponding fluorescence-minus-one (FMO) controls. d, Frequency of (DN)CD34⁺CD7⁺ TSPs among bone-marrow and thymic HSPCs. e, Absolute numbers of (DN)CD34⁺CD7⁺ HSPCs per 10 6 total bone marrow mononuclear cells and thymocytes analyzed. f–i, Pearson correlation analyses between matched (N=8) bone-marrow and thymus derived CD4 − CD8 − double negative CD34 + progenitor populations, comparing frequencies of CD7 expressing (f), and non-expressing (g) DN CD34⁺ HSPCs. h, Pearson correlation between bone marrow derived CD34⁺CD38 − CD7⁺ (DN1) TSPs and thymus derived (DN)CD34+ (h), and (DN)CD34+CD7+TCF1+ (i) progenitors. Pearson correlation coefficients (r) and P values are shown. j, Schematic of the previously described Rhesus-specific nonanimal model of thymopoiesis known as Rhesus-artificial thymic organoid (RhATO) system , seeded with CD3 − (DN)CD34⁺CD7⁺ TSPs. k, Representative flow-cytometry plots illustrating T cell lineage committed differentiation intermediates representing frequencies of ISPs, and DPs early thymocyte intermediates, mature CD3⁺ T cells, as well as CD4⁺ and CD8⁺ single positive T cells, analyzed at week 4 of organoid establishment as previously described (k). l, Bar graph showing quantification of the frequencies of indicated thymocyte subsets generated in the RhATO-system (N=6 RhATOs). m, Schematic depicting uninfected (SIV⁻; N=15) and SIV-infected (SIV⁺; N=12) rhesus macaques (RMs) used to analyze bone marrow derived progenitors. n, Representative flow-cytometry plots showing (DN)CD34⁺CD7⁺ T cell committed progenitors in bone marrow of SIV⁻ and SIV⁺ RMs. o, Bar graph showing quantitation of the frequency of (DN)CD34⁺CD7⁺ TSPs among bone-marrow HSPCs in SIV⁻ and SIV⁺ RMs. p, Absolute numbers of (DN)CD34⁺CD7⁺ T-cell committed progenitors per 10⁶ bone-marrow mononuclear cells (BMMCs) analyzed. q, Absolute numbers of (DN)CD34⁺ HSPCs per 10⁶ BMMCs analyzed. r, Frequency of CD34⁺CD38 − CD7⁺ (DN1) TSPs among bone-marrow (DN)CD34+CD7+ progenitors in SIV⁻ and SIV⁺ animals. s, Experimental design showing RMs (N=6) infected with 300 TCID 50 SIVmac239M via intravenous route. Bone marrow aspirates (red dots) and blood (black dots) collected at the indicated timepoints starting with week 0 of infection. t, Line graph showing longitudinal plasma viral load (PVL) at the indicated timepoints. Geomean PVL indicated in black and individual data indicated in grey. u-v, Line graphs showing longitudinal frequencies of (DN)CD34⁺CD7⁺ (u) and CD34⁺CD38 − CD7⁺ (DN1) TSPs (v) TSPs at baseline (week 0) and at week 2, 4, 12, and 16 following SIV infection. w, Representative flow-cytometry plots and bar graph showing frequencies of T cells generated in RhATO cultures using 5000 CD3 − CD34 + HSPCs per RhATO isolated from bone marrow of RMs at week 16 of infection. x, Pearson correlation analysis between fold decrease of CD34 + CD38 − CD7 + (DN1) TSP population at week 2 of infection and frequency of T cells generated by CD3 − CD34 + HSPCs in RhATO culture. y–z, Pearson correlation analyses between total plasma viral burden until week 16 post infection represented as area under curve and fold decrease of CD34 + CD38 − CD7 + (DN1) TSP (y), and frequency of T cells generated in RhATO (z). Each symbol represents one animal. Statistical significance was determined using Mann Whitney unpaired two-tailed t-tests for unrelated data sets, Pearson correlation, or paired Wilcoxon test for related data sets; (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

Article Snippet: Isolation of human CD3-CD34+ HSPC: Total CD34+ HSPCs (Cat#BM34C-5) purchased from Charles River Laboratory Inc, Wilmington, MA, USA, were thawed and directly stained with live/dead stain, anti-CD3, and anti-CD34 antibodies.

Techniques: Migration, Flow Cytometry, Fluorescence, Derivative Assay, Expressing, Generated, Infection, Quantitation Assay, Clinical Proteomics, Isolation, MANN-WHITNEY, Two Tailed Test

Journal: bioRxiv

Article Title: Interleukin-6 restricts pre-thymic T cell lineage commitment of progenitors driving loss of SIV control

doi: 10.64898/2026.01.29.702123

Figure Lengend Snippet: a , Longitudinal plasma viral load (PVL) in SIV-infected rhesus macaques (RMs) measured before infection (D0) and at week (Wk) 2, 4 and 16 post SIV infection (N=6). b–f , Line graphs and corresponding heatmaps representing longitudinal plasma concentrations of IL-6 ( b ), IL-15 ( c ), TNF-β ( d ) IL-1β ( e ) and IP-10 (CXCL10) ( f ) across the same time points. ( g) , Pearson correlation matrices summarizing associations between CD34⁺CD38 − CD7⁺ (DN1) thymic seeding progenitors (TSPs) attrition, plasma viral load and circulating cytokines and chemokines during peak viremia (week 2 post SIV). Pearson correlation coefficients (r) is shown. ( h–k) , Pearson correlation between peak PVL and corresponding plasma concentrations of IL-6 ( h ), IL-15 ( i ), TNF-β ( j ) IL-1β ( k ) at week 2 post infection. l, Pearson correlation between (DN1) TSP attrition and plasma IL-6 concentrations at peak viremia (week 2) post infection. (m-n), Representative flow-cytometry plots illustrating RhATO-derived T cell developmental ontogeny from bone marrow CD3 − CD34 + HSPCs of RMs isolated at week 16 of infection, culture analyzed at week 4 of establishment. Flow plots show differentiation stages of thymocytes including CD4 + CD3 − immature single positive (ISP) early thymocyte, CD4 + CD8 + double positive (DP) intermediate thymocyte, (m) and mature CD3⁺ T cells (n). o-q, Pearson correlation analyses between plasma IL-6 at week 16 of infection and RhATO derived frequencies of ISPs (o), DPs (p), and CD3+ T cells (q) stages. r, Pearson correlation analyses between plasma IL-6 at week 16 of infection and frequency of CD3+ T cells in blood of RMs at week 16 post infection. s , Pearson correlation between fold decrease in frequency of bone marrow (DN1) TSPs and plasma IP-10 concentrations. Each symbol represents one animal; lines indicate longitudinal measurements from the same animal. Heat-map color scales indicate relative concentrations or viral load. Linear regression lines, Pearson correlation coefficients (r) and P value calculated by paired Wilcoxon test are shown where appropriate; (*P < 0.05; **P < 0.01).

Article Snippet: Isolation of human CD3-CD34+ HSPC: Total CD34+ HSPCs (Cat#BM34C-5) purchased from Charles River Laboratory Inc, Wilmington, MA, USA, were thawed and directly stained with live/dead stain, anti-CD3, and anti-CD34 antibodies.

Techniques: Clinical Proteomics, Infection, Flow Cytometry, Derivative Assay, Isolation

Journal: bioRxiv

Article Title: Interleukin-6 restricts pre-thymic T cell lineage commitment of progenitors driving loss of SIV control

doi: 10.64898/2026.01.29.702123

Figure Lengend Snippet: a, Cartoon illustrating early T cell lineage committed differentiation steps from CD4⁻CD8⁻CD34⁺CD38⁻CD7⁺ (DN1) TSPs to CD4 + CD3 − immature single-positive (ISPs) thymocyte stage, highlighting early phenotypic changes during T cell committed differentiation process. b, Experimental schematic illustrating isolation of CD3 − CD34 + hematopoietic stem and progenitor cells (HSPCs) from bone-marrow of SIV-uninfected (SIV⁻; N = 15) and SIV-infected (SIV⁺; N = 12) rhesus macaques (RMs) and their differentiation to ISP thymocyte in the rhesus-artificial thymic organoid (RhATO) system. c, Representative flow-cytometric plots showing frequencies of ISPs and corresponding CD45⁺ lymphocytes generated in RhATO culture at week 1 of culture as previously described. d, Bar graph showing quantitation of the frequency of ISP thymocytes for SIV⁻ (N=31 RhATOs) and SIV⁺ (N=34 RhATOs) conditions. e, Bar graph showing corresponding frequency of CD45⁺ lymphocytes. f, Bar graph showing ratio of the frequencies of ISP thymocytes to CD45⁺ lymphocytes generated in RhATO system. g, Schematic showing longitudinal ex-vivo tracking of the T cell lineage committed differentiation potential of the CD3 − CD34 + HSPCs isolated from bone marrow of six RMs at the indicated timepoints (red dots) post SIV infection. h, Representative flow-cytometric plot showing frequencies of the ISP thymocytes generated in RhATOs. Each plot represents each timepoint post infection T cell lineage committed potential of HSPCs were evaluated. i, Line graph showing geomean (dark green line) and individual data points (lighter green lines) of the frequencies of CD4⁺CD3⁻ ISP thymocytes generated in RhATOs from bone-marrow HSPCs collected at the indicated time points following SIV infection. j, Pearson correlation between drop in T cell lineage committed differentiation potential of bone marrow derived HSPCs (as estimated by frequency of ex-vivo generated ISPs in RhATO cultures at week 2 compared with day 0 post infection) and plasma IL-6 concentrations at week 2 post infection. k, Representative flow-cytometric plots identifying Lin⁻CD34⁺CD38⁻CD90⁻CD45RA⁺ lymphoid-primed multipotent progenitors (LMPPs) in bone marrow at baseline (SIV D0), week 2 and week 16 post-infection. l, Line graph showing geomean (dark blue) and individual values (lighter blue lines) frequencies of LMPPs in bone marrow at day 0, week 2 and 16 post infection. Each symbol represents an individual animal. Data are presented as geomean ± geomean standard deviation. Statistical significance was determined using Mann Whitney unpaired two-tailed t-tests for unrelated data sets, Pearson correlation, or paired Wilcoxon test for related data sets; (*P < 0.05; ****P < 0.0001; ns, not significant).

Article Snippet: Isolation of human CD3-CD34+ HSPC: Total CD34+ HSPCs (Cat#BM34C-5) purchased from Charles River Laboratory Inc, Wilmington, MA, USA, were thawed and directly stained with live/dead stain, anti-CD3, and anti-CD34 antibodies.

Techniques: Isolation, Infection, Generated, Quantitation Assay, Ex Vivo, Derivative Assay, Clinical Proteomics, Standard Deviation, MANN-WHITNEY, Two Tailed Test

Journal: bioRxiv

Article Title: Interleukin-6 restricts pre-thymic T cell lineage commitment of progenitors driving loss of SIV control

doi: 10.64898/2026.01.29.702123

Figure Lengend Snippet: a , Representative flow-cytometry plots showing frequencies of DN CD34⁺CD7 + T cell committed progenitors in RhATO cultures supplemented without (positive control) or with increasing concentrations of IL-6 (0.5, 5 or 50 ng ml⁻¹), analyzed at week 1 of culture establishment with bone marrow derived CD3 − CD34 + HSPCs from SIV naïve rhesus macaques (RMs) as previously described. DN progenitors were identified based on CD4 − CD8 − double negative (DN), CD34 and CD7 expression; numbers indicate frequencies within the gated populations. b , Bar graph showing quantification of the frequency of (DN)CD34⁺CD7⁺ TSPs across IL-6 concentrations, demonstrating a dose-dependent reduction in T cell progenitors. c, Schematic of the IL-6 receptor blockade experimental design. Bone marrow derived CD3 − CD34⁺ HSPCs from SIV naive RMs were FACS sorted and cultured RhATO system in the absence of IL-6 (grey), presence of IL-6 (green), and in the presence of IL-6 receptor binding antibody + IL-6 (blue), as indicated. d , Representative flow-cytometry plots showing (DN)CD34⁺CD7 + T cell progenitors in RhATO cultures at week 1 of culture establishment with bone marrow derived CD3 − CD34⁺ HSPCs from SIV naive RMs for above-described treatment conditions. e , Bar graph showing quantitation of the frequency of (DN)CD34⁺CD7⁺ progenitors across treatment conditions (N=6 RhATOs). f , Representative flow-cytometry plots showing T cell lineage committed differentiation of CD3 − CD34⁺ HSPCs marked by increase in the frequency of CD4 + CD3 − immature single positive thymocytes (ISPs) in RhATO. ISPs (early thymocyte intermediate) were identified based on CD45 + CD8 − CD3 − and CD4 + expression; numbers indicate frequencies within the gated populations. g , Bar graph showing quantification of the frequency of CD4 + CD3 − ISPs across treatment conditions. h , Schematic illustrating key early intermediates along the T cell committed differentiation (CD4 − CD8 − double negative, DN1 (CD38 − ), DN2/3(CD38 + ), immature single-positive thymocyte; ISP) and associated marker expression (CD34, CD7, CD38 and CD4). Arrows indicate relative changes in marker expression under IL-6⁻ and IL-6⁺ treatment conditions. i , Representative flow-cytometry plots showing frequencies of CD34 + CD38 − and CD34 + CD38 + fractions within CD4 − CD8 − (DN) HSPCs (SIV naïve) cultured in RhATO-system in the absence or presence of IL-6. J-k , Bar graphs showing quantitation of the frequencies of (DN)CD34⁺CD38⁻ (j) and (DN)CD34⁺CD38⁺(k) progenitors under IL-6⁻ and IL-6⁺ treatment conditions (N = 9 RhATOs). l , Representative flow-cytometry plots showing early T cell lineage committed differentiation to CD34⁺CD38 + CD7 + (DN2) TSPs in RhATO cultures under IL-6⁻ and IL-6⁺ treatment conditions (N = 9 RhATOs). m , Bar graph showing quantitation of the frequency of CD34⁺CD38 + CD7+ (DN2) TSPs under IL-6⁻ and IL-6⁺ treatment conditions. n-r , Analysis of the impact of IL-6 exposure on expansion of CD34+CD38+CD7+ (DN2) TSP intermediate in a human-specific nonanimal model of thymopoiesis. n, Bar graph showing quantitation of (DN)CD34⁺CD7⁺ T cell progenitors under IL-6⁻ (N = 6 hATOs) and IL-6⁺ (N = 18 hATOs) conditions. o, Representative flow-cytometry plots showing CD38 expression within CD34⁺CD7⁺ DN progenitors in hATO culture under IL-6⁻ and IL-6⁺ conditions. p-q , Bar graph showing quantitation of the frequencies of CD34⁺CD38⁻ (p) and CD34⁺CD38⁺ (q) progenitors within CD4 − CD8 − (DN) CD34 + HSPCs (HIV naïve) under IL-6⁻ and IL-6⁺ conditions. r , Bar graph showing quantitation of the frequencies of CD34⁺CD38⁺CD7⁺ (DN2) TSPs under IL-6⁻ and IL-6⁺ conditions. Each point represents an individual organoid. Bar graphs show geomean with geomean standard deviation. Statistical significance was assessed using repeated-measures ANOVA with multiple-comparison correction or Mann Whitney unpaired two-tailed t-tests for unrelated data sets; (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant).

Article Snippet: Isolation of human CD3-CD34+ HSPC: Total CD34+ HSPCs (Cat#BM34C-5) purchased from Charles River Laboratory Inc, Wilmington, MA, USA, were thawed and directly stained with live/dead stain, anti-CD3, and anti-CD34 antibodies.

Techniques: Flow Cytometry, Positive Control, Derivative Assay, Expressing, Cell Culture, Binding Assay, Quantitation Assay, Marker, Standard Deviation, Comparison, MANN-WHITNEY, Two Tailed Test

Journal: bioRxiv

Article Title: Interleukin-6 restricts pre-thymic T cell lineage commitment of progenitors driving loss of SIV control

doi: 10.64898/2026.01.29.702123

Figure Lengend Snippet: a–d, Analysis of early events of T cell lineage committed differentiation of bone marrow CD3 − CD34 + HSPCs in the absence or presence of IL-6 at week 1 of RhATO culture. a, Bar graph showing absolute number of DN CD34⁺CD7⁺ T cell progenitors per 5 × 10³ input HSPCs generated in the absence or presence of IL-6. b, Bar graph showing absolute numbers of DN CD34⁺CD38⁻ progenitors per 5 × 10³ input HSPCs in the absence or presence of IL-6. c, Bar graph showing absolute numbers of DN CD34⁺CD38⁺ progenitors per 5 × 10³ input HSPCs in the absence or presence of IL-6. d, Bar graph showing absolute numbers of CD34⁺CD38⁺CD7⁺ (DN2) TSPs per 5 × 10³ input HSPCs following IL-6 treatment. e–j, Analysis of early events of T cell lineage committed differentiation of human bone marrow derived primary CD3 − CD34 + HSPCs (HIV naïve) in the absence or presence of IL-6 at week 1 of hATO culture. e, Representative flow-cytometry plots showing frequency of (DN)CD34+CD7+ T cell progenitors in the absence or presence of IL-6 at week 1 of hATO culture. g, Bar graph showing absolute number of DN CD34⁺CD7⁺ T cell progenitors per 5 × 10³ input HSPCs generated in the absence or presence of IL-6. h, Bar graph showing absolute numbers of DN CD34⁺CD38⁻ progenitors per 5 × 10³ input HSPCs in the absence or presence of IL-6. i, Bar graph showing absolute number of DN CD34⁺CD38⁺ progenitors per 5 × 10³ input HSPCs in the absence or presence of IL-6. j, Bar graph showing absolute numbers of CD34⁺CD38⁺CD7⁺ (DN2) TSPs generated per 5 × 10³ input HSPCs in the absence or presence of IL-6. Each symbol represents an independent donor or biological replicate. Data are shown as geomean ± geomean s.d. Statistical significance was determined using Mann Whitney unpaired two-tailed t-tests. (**P < 0.01; **P < 0.0001).

Article Snippet: Isolation of human CD3-CD34+ HSPC: Total CD34+ HSPCs (Cat#BM34C-5) purchased from Charles River Laboratory Inc, Wilmington, MA, USA, were thawed and directly stained with live/dead stain, anti-CD3, and anti-CD34 antibodies.

Techniques: Generated, Derivative Assay, Flow Cytometry, MANN-WHITNEY, Two Tailed Test

Journal: bioRxiv

Article Title: Riboflavin drives nucleotide biosynthesis and iron-sulfur metabolism to promote acute myeloid leukemia

doi: 10.1101/2025.06.26.661633

Figure Lengend Snippet: A . Proliferation of NB4 and MOLM-13 cells in standard RPMI culture conditions and Plasmax complete over 6 days. No media changes were performed. Data representative of n =3 biological replicates. B . Proliferation of NB4 and MOLM-13 cells upon systematic removal of vitamins from Plasmax medium at day 3. Cell number is normalized to Plasmax complete medium. Cells cultured in Plasmax complete medium and treated with 1 μM all- trans retinoic acid (ATRA) served as an anti-proliferation/differentiation control. Data representative of n =4 pooled replicates from two independent biological experiments. C . Complete blood counts of mice fed adequate versus deficient riboflavin diets at start of experiment (baseline, day 0) and at 21 days. HCT, hematocrit test; MCV, mean corpuscular volume test; MCH, mean corpuscular hemoglobin test; MCHC, mean corpuscular hemoglobin concentration test; RDW, red cell distribution width test. D . Cell number of healthy CD34 + HSPC cells after 14 days of culture in Plasmax complete or Plasmax lacking the essential amino acid isoleucine (anti-proliferative control) or riboflavin. Cell number is normalized to Plasmax complete medium. Data representative of n =3 biological replicates. The experiment was repeated with two separate donors. Data are presented as the mean ± SD. ** P < 0.01 and **** P < 0.0001 by unpaired two-tailed Student’s t-test (A, C) and ordinary one-way ANOVA with Bonferroni’s multiple comparisons test (B, D).

Article Snippet: CD34 + hematopoietic stem/progenitor cells (HSPCs) were purchased from Lonza, and each vial contained 1 × 10 6 cells with greater than 90% CD34+ purity.

Techniques: Cell Culture, Control, Concentration Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Riboflavin drives nucleotide biosynthesis and iron-sulfur metabolism to promote acute myeloid leukemia

doi: 10.1101/2025.06.26.661633

Figure Lengend Snippet: A . Schema of vitamin depletion screens in AML cells. B . Proliferation of NB4 and MOLM-13 cells upon systematic removal of vitamins from Plasmax medium at day 6. Cell number is normalized to Plasmax complete medium. Cells cultured in Plasmax complete medium and treated with 1μM all- trans retinoic acid (ATRA) served as an anti-proliferation/differentiation control. Data representative of n =4 pooled replicates from two independent biological experiments. C . Representative histograms of cell surface CD11b-APC in NB4 and MOLM-13 cells upon systematic removal of vitamins from Plasmax medium at day 6. D . Quantification of CD11b geometric mean fluorescence intensity (MFI) in NB4 and MOLM-13 cells from C. at day 6. The average of 2 replicates per condition was normalized to the isotype control and compared to Plasmax complete. E . Cell death in NB4, MOLM-13, MV4-11 and OCI-AML2 cells as measured by Annexin V-APC cell surface staining at day 6 (NB4, OCI-AML2), day 8 (MV4-11) and day 11 (MOLM-13) post riboflavin withdrawal from Plasmax. Data representative of n =3 biological replicates. F . Schema of riboflavin depletion experiments in NSG mice. SST, succinylsulfathiazole; LC-MS/MS, liquid chromatography with tandem mass spectrometry. G . Quantification of riboflavin, FMN and FAD in blood plasma of mice fed adequate ( n =5) versus deficient ( n =4) riboflavin diets at 21 days. H . Average mouse mass of mice fed adequate ( n =5) versus deficient ( n =4) riboflavin diets over 21 days. I . Complete blood counts of mice fed adequate versus deficient riboflavin diets at start of experiment (baseline, day 0) and at 21 days. WBC, white blood cell count; RBC, red blood cell count; Hb, hemoglobin test; PLT, platelet count. J . Cell number of healthy CD34 + HSPC cells after 6 days of culture in Plasmax complete or Plasmax lacking isoleucine (anti-proliferative control) or riboflavin. Cell number is normalized to Plasmax complete medium. Data representative of n =3 biological replicates. The experiment was repeated with two separate donors. Data are presented as the mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001 and **** P < 0.0001 by ordinary one-way ANOVA with Bonferroni’s multiple comparisons test (B, J) and unpaired two-tailed Student’s t-test (E, G, I).

Article Snippet: CD34 + hematopoietic stem/progenitor cells (HSPCs) were purchased from Lonza, and each vial contained 1 × 10 6 cells with greater than 90% CD34+ purity.

Techniques: Cell Culture, Control, Fluorescence, Staining, Liquid Chromatography with Mass Spectroscopy, Liquid Chromatography, Mass Spectrometry, Clinical Proteomics, Cell Counting, Two Tailed Test

Journal: bioRxiv

Article Title: Riboflavin drives nucleotide biosynthesis and iron-sulfur metabolism to promote acute myeloid leukemia

doi: 10.1101/2025.06.26.661633

Figure Lengend Snippet: A . Schema of RFK perturbation via genetic, exogenous depletion and small molecule approaches in combination with the BCL-2 inhibitor venetoclax. B . Relative viability of doxycycline-inducible sgRNA-expressing NB4, MV4-11 and PDX16-01 cells targeting Rosa or RFK treated with venetoclax for 72 hours. Cells were treated with doxycycline for 4 days prior to venetoclax treatment to induce gene knockout. C . Relative viability of NB4 and MV4-11 cells cultured in Plasmax complete or riboflavin deficient medium and treated with venetoclax for 72 hours. Cells were pre-conditioned in appropriate medium for 4 days prior to venetoclax treatment. D . Chemical structure of roseoflavin, 8-dimethylaminoriboflavin. E . Relative viability of NB4 or MV4-11 cells treated with venetoclax alone, or venetoclax and two fixed concentrations of roseoflavin (3.13 μM, blue; or 6.25 μM, red) for 72 hours. F . NB4, MV4-11 and PDX16-01 cells were treated with escalating concentrations of venetoclax and roseoflavin for 72 hours to determine viability effects. The presence of treatment synergy was determined using SynergyFinder and the Bliss synergy index and is denoted as regions of red in the graphs. The mean of three biological replicates was used for each data point. G . Relative viability of CD34 + HSPC cells treated with escalating concentrations of roseoflavin for 72 hours. Concentrations used to sensitize NB4 and MV4-11 cells in E. are highlighted (3.13 μM, blue; or 6.25 μM, red). Data are presented as the mean ± SD.

Article Snippet: CD34 + hematopoietic stem/progenitor cells (HSPCs) were purchased from Lonza, and each vial contained 1 × 10 6 cells with greater than 90% CD34+ purity.

Techniques: Expressing, Gene Knockout, Cell Culture

Journal: bioRxiv

Article Title: Riboflavin drives nucleotide biosynthesis and iron-sulfur metabolism to promote acute myeloid leukemia

doi: 10.1101/2025.06.26.661633

Figure Lengend Snippet: A . CD34 + HSPC cells nucleofected with recombinant Cas9 and sgRNAs targeting Rosa or with two independent sgRNAs targeting RFK for 15 days. Colony formation capacity was determined (left) and colony number was quantified (middle). Effective editing of RFK by the two sgRNAs was determined using Tracking of Indels by Decomposition (TIDE)-sequencing (right). B . MOLM-13 cells were treated with escalating concentrations of venetoclax and roseoflavin for 72 hours to determine viability effects. The presence of treatment synergy was determined using SynergyFinder and the Bliss synergy index and is denoted as regions of red in the graphs. The mean of three biological replicates was used for each data point. Data are presented as the mean ± SD. **** P < 0.0001 by ordinary one-way ANOVA with Bonferroni’s multiple comparisons test.

Article Snippet: CD34 + hematopoietic stem/progenitor cells (HSPCs) were purchased from Lonza, and each vial contained 1 × 10 6 cells with greater than 90% CD34+ purity.

Techniques: Recombinant, Sequencing