human cd34 hematopoietic stem cells  (Jackson Laboratory)

Journal: Blood Advances

Article Title: CAR T cells targeting CCR4 selectively deplete human Tregs ex vivo and in vivo

doi: 10.1182/bloodadvances.2025017573

Figure Lengend Snippet: Tregs are depleted by the CCR4-CAR in a humanized mouse model. (A) Experimental design. NSG-SGM3-IL15 engrafted with CD34 + hematopoietic stem cells were injected with 1 million CAR + CCR4-CARTs IV. Blood was collected on days 0, 3, 5, and 8, and mice were euthanized on day 11. (B) Representative flow plots showing the proportion of human CD45 (hCD45) and mCD45 leukocytes at baseline. (C) Proportion of Tregs, CD4 + non-Treg, and CD4 − cells of hCD45 percent at baseline. (D) Percentage of Tregs, non-Treg, and CD4 − cells that are CCR4 + at baseline. (E) Representative flow plots showing the CCR4 + and FOXP3 + expression on the CD4 + population before and after CART administration gated on CD4 + cells. (F-K) Proportions of Tregs, CD4 + non-Tregs, and CD4 − cells over time. Significance was determined using t tests corrected for multiple comparisons, with comparison to baseline indicated on graph; ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001; ∗∗∗∗ P < .0001. M1, Mouse 1; M2, Mouse 2; M3, Mouse 3; mCD45, mouse CD45.

Article Snippet: Mice were engrafted with human cord blood–derived CD34 + hematopoietic stem cells, and females were available for use at age 13 to 18 weeks after evaluation of engrafted human cell populations by flow cytometry at The Jackson Laboratory.

Techniques: Injection, Expressing, Comparison

Journal: PLOS Pathogens

Article Title: Mechanistic insights and in vivo HIV suppression by the BRD4-targeting small molecule ZL0580

doi: 10.1371/journal.ppat.1013449

Figure Lengend Snippet: (A) Experimental design and timeline for the in vivo study using HIV-infected CD34 + Hu-mice. At week 0, 12 Hu-mice were intravenously inoculated with a single dose of HIV-1 JR-CSF (10 ng HIV p24 antigen per animal). Plasma samples were collected at weeks 2 and 3 after HIV inoculation for confirmation of HIV infection by RT-qPCR and HIV p24 ELISA. At week 3, 11 out of the 12 Hu-mice were confirmed with systemic HIV infection (the one without infection was removed from the study). The 11 Hu-mice with successful HIV infection were randomized into four treatment groups at week 3: Empty Vehicle (EV) (n = 2), ART (n = 3), ZL0580 (n = 3), and ART + ZL0580 (n = 3). Treatment regimens were initiated at week 3 and administered daily by intraperitoneal until week 7. Plasma viral loads were closely monitored during the treatments at weeks 5, 6, 7. From week 7, all animals were subjected to treatment interruption (TI) and plasma viral loads were closely monitored once every 2 weeks until week 15. (B) Kinetics of plasma viral loads (HIV RNA copies/ml) post HIV inoculation for each mouse in EV (black circles), ART (orange triangles), ZL0580 (blue squares), and ART + ZL0580 (red diamonds) treatment groups. The shaded areas denote the phase of establishing systemic HIV infection (orange), treatment phase (blue), and treatment interruption (TI) monitoring phase (green). RT-qPCR was performed in duplicate and the mean of viral RNA copies for each sample at different time points was shown. (C) Summary data for kinetics of plasma viral loads over weeks post HIV inoculation for all animals in the four treatment groups: EV (black circles), ART (orange triangles), ZL0580 (blue squares), and ART + ZL0580 (red diamonds). Data are presented as mean ± SEM. Statistical comparison of viral loads among the four groups at each time point was performed using one-way ANNOVA. * at week 7 and *** at week 13 denote significant difference of the individual treatment group (ART, ZL0580, or ART + ZL0580) as compared to the EV group. The dashed line indicates the limit of detection (LOD). (D) Comparison of area under the curve (AUC) among the four groups after treatment interruption (TI) between weeks 7 and 15. Statistical comparison was performed using one-way ANNOVA. (E) Percentage change in body weight over days after treatment for all treatment groups. Data are presented as mean ± SEM. In this figure: * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: To assess in vivo HIV-suppressive activity of ZL0580, we utilized a humanized mouse model (Hu-mice) generated by engrafting human CD34 + hematopoietic stem cells (HSCs) into the immunodeficient NOD SCID gamma (NSG) mice (the Jackson Laboratory).

Techniques: In Vivo, Infection, Clinical Proteomics, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Cell Reports Medicine

Article Title: Lentiviral vectors for hematopoietic stem cell gene therapy restore α-globin expression in α-thalassemia red blood cells

doi: 10.1016/j.xcrm.2025.102362

Figure Lengend Snippet: α-Globin transgene expression in differentiated erythroid cells derived from healthy donor (HD) CD34 + HSPCs CD34 + HSCPs from three healthy donors (HD) were transduced with the α-globin EVs at 2E7 TU/mL and cultured for 12 days under erythroid differentiation conditions and analyzed on day 14. (A) VCN analysis. (B) Gene expression analysis of transgene-derived α∗-globin mRNA and endogenous β-globin mRNA to determine α∗/β-globin ratio. (C) α∗/β-globin mRNA ratio normalized to the corresponding VCN. Following transduction, some cells were cultured in myeloid conditions to assess off-lineage transgene expression. (D) Assessment of α∗-globin mRNA expression in short-term myeloid cells cultured under myeloid conditions. Each data point represents a technical replicate, and error bars represent median, and median with 95% confidence interval in (C).

Article Snippet: Mobilized CD34 + Stem/Progenitor Cells; GCSF; 1 × 10e6 cells, Cryo , Charles River Laboratories , N/A.

Techniques: Expressing, Derivative Assay, Transduction, Cell Culture, Gene Expression

Journal: Cell Reports Medicine

Article Title: Lentiviral vectors for hematopoietic stem cell gene therapy restore α-globin expression in α-thalassemia red blood cells

doi: 10.1016/j.xcrm.2025.102362

Figure Lengend Snippet: Evaluation of candidate vector potency and toxicity in CD34 + HSPCs from human bone marrow (BM) and mobilized peripheral blood (mPB) (A) Experimental setup for transduction and erythroid differentiation. BM-derived CD34 + from four HDs were transduced with EV-α and EV-α-UV vectors and analyzed on day 14. (B–D) BM-derived CD34 + cells were analyzed for (B) VCN, (C) α∗/β-globin ratio, and (D) α∗/β-globin ratio normalized by VCN. (E–G) mPB stem cells (mPBSCs) from three HDs were transduced with increasing doses of EV-α (1E6 to 6E7 TU/mL; MOIs: 2, 6, 13, 45, and 150) using 1 mg/mL Lentiboost. Analysis includes (E) VCN, (F) α∗/β-globin mRNA ratio, and (G) normalized gene expression. Shaded blue and red regions indicate α/β-globin mRNA threshold for blood-transfusion-free and transfusion-dependent individuals (<0.2), respectively. The ranges of α/β-globin mRNA ratios expected in patients with different α-thalassemia genotypes are shown on the right axis. (H and I) To assess potential cytotoxicity from the EV-α-LV at high VCN, CD34 + mPBSC from additional HDs were transduced at 1E8 TU/mL, and cells were analyzed for (H) VCN in short-term myeloid cells and, (I) CFU assay to quantify erythroid (red) and myeloid (blue) colonies produced in non-transduced (NTD) and high dose EV-α transduced cells (EV-α). Each symbol in (B–D) represents an independent biological replicate (different donors); repeated symbols for the same donor indicate technical replicates. In (E–G), different colors represent different donors, and each dot indicates a technical replicate. Error bars represent mean and mean with standard deviation.

Article Snippet: Mobilized CD34 + Stem/Progenitor Cells; GCSF; 1 × 10e6 cells, Cryo , Charles River Laboratories , N/A.

Techniques: Plasmid Preparation, Transduction, Derivative Assay, Gene Expression, Colony-forming Unit Assay, Produced, Standard Deviation

Journal: Cell Reports Medicine

Article Title: Lentiviral vectors for hematopoietic stem cell gene therapy restore α-globin expression in α-thalassemia red blood cells

doi: 10.1016/j.xcrm.2025.102362

Figure Lengend Snippet: Long-term assessment of transduced HSPCs engrafted in NSG mice (A) Healthy donor CD34 + mPBSCs were transduced with EV-α and EV-α-UV at low (6E6 TU/mL, MOI: 6), medium (2E7 TU/mL, MOI: 20), and high (4E7 or 6E7 TU/mL, MOIs: 20 and 32, respectively) doses and transplanted into NSG mice. At 16 weeks post-transplant, mice were euthanized, and bone marrow (BM) hCD45 + cells were analyzed. Erythroid differentiation of BM hCD45 + was performed ex vivo to assess α-globin transgene expression. To evaluate VCN distribution, a colony forming unit assay using methylcellulose cultures was conducted using BM hCD45 + . (B–D) BM analysis for (B) engraftment (percentage of hCD45 + cells out of total [human + mouse] CD45 + cells), (C) lineage distribution, and (D) bulk VCN of engrafted cells. (E and F) Ex vivo RBC differentiation for (E) α∗/β-globin mRNA ratio and (F) normalized gene expression to VCN. (G and H) Transduction profiles of engraftment HSPCs using CFU assay, showing (G) percentage of VCN+ colonies and (H) VCN values in single colonies. (I) Vector integration site analysis (VISA), performed by ligation-mediated PCR and next generation-sequencing, and aligned to the human genome, showing the relative abundance of the top 10 most abundant clones, shown in different colors. Lower abundance clones are shown in gray. Total number of unique integrants shown on top of each bar. (J) Word cloud summarizing nearest genes to integration sites, indicating transcription units (∗), within 50 kb of a cancer-related gene (∼), and the nearest gene associated with lymphoma (!). Bars represent mean, and error bars represent mean with standard deviation. Analysis of NSG assay was based on ANOVA (analysis of variance) across all arms with selected statistics shown, with p < 0.05 considered significant. Non-significance (ns) was set at p < 0.05. Each data point represents an individual biological replicate (one mouse).

Article Snippet: Mobilized CD34 + Stem/Progenitor Cells; GCSF; 1 × 10e6 cells, Cryo , Charles River Laboratories , N/A.

Techniques: Transduction, Ex Vivo, Expressing, Colony-forming Unit Assay, Gene Expression, Plasmid Preparation, Ligation, Next-Generation Sequencing, Clone Assay, Standard Deviation

Journal: Cell Reports Medicine

Article Title: Lentiviral vectors for hematopoietic stem cell gene therapy restore α-globin expression in α-thalassemia red blood cells

doi: 10.1016/j.xcrm.2025.102362

Figure Lengend Snippet: Restoration of α-globin expression in differentiated erythroid cells derived from CD34 + HSPCs from three patients with ATM (A) Experimental setup for transduction, with EV-α or EV-α-UV, and differentiation of bone marrow (BM), cord-blood (CB), and fetal liver (FL) CD34 + HSPCs. BM cells were transduced at doses ranging from 2E6 to 2E7 TU/mL, CB at 7E4 TU/mL, and FL at 1E4 TU/mL, then analyzed for vector copy number (VCN), mRNA expression, and protein production via HPLC on days 14 and 21 (BM: blue, CB: orange, FL: green). (B) VCN analysis. (C) α/β-globin mRNA ratio. (D) Normalized mRNA expression. Shaded regions represent the threshold for transfusion-free (blue) and transfusion-dependent (red) individuals (α/β-globin mRNA ratio <0.2). The ranges of α/β-globin mRNA ratios expected in patients with different α-thalassemia genotypes are shown on the right axis. (E) α/β-globin chain ratio in BM cells. (F) α/(β + γ 1 + γ 2 )-globin chain ratio. (G) HbA and HbF production as a percentage of total HPLC peaks. (H and I) BM cells analyzed from single-cell RNA-seq. (H) UMAP showing HBA2 mRNA expression and distribution in in vitro differentiated erythroid cells for each condition. Each plot displays levels of HBA2 expression, indicated by the color scale to the right of each figure. (I) Heatmap of top 15 most differentially expressed genes. Each data point in (B–G) represents a replicate measurement on BM, CB, and FL CD34 + HSPCs. For BM samples (blue), data include technical replicates as well as independent experiments performed at different transduction doses using cells from the same donor. Colors indicate donor source. Bars represent mean with standard deviation. See Table 1 for detailed α/β-globin ratios.

Article Snippet: Mobilized CD34 + Stem/Progenitor Cells; GCSF; 1 × 10e6 cells, Cryo , Charles River Laboratories , N/A.

Techniques: Expressing, Derivative Assay, Transduction, Plasmid Preparation, RNA Sequencing, In Vitro, Standard Deviation