Journal: Molecular Therapy Advances

Article Title: Ferrostatin-1 and hinokitiol supplementation enhance human hematopoietic stem cell expansion in a chemically defined medium

doi: 10.1016/j.omta.2026.201711

Figure Lengend Snippet: Effect of antioxidant compounds on human CB CD34 + cell expansion and immunophenotype (A) Relative proliferation of CB CD34 + cells at day 14 in 3a medium supplemented with each of the compounds in SCREEN-WELL REDOX library. Data from three independent experiments, each performed with unique CB donor, are represented as relative luminescence (%) to DMSO-treated cells. Each row represents a compound. Well ID is shown on the left. (B and C) Fold change in HSPC (B) and HSC (C) fraction under each treatment condition relative to DMSO control group at day 14 of ex vivo culture. Representative data from 3 independent experiments, each performed with unique CB donor.

Article Snippet: Human CB CD34 + cells were purchased from HemaCare (CB34C-3) and STEMCELL Technologies (200-0001).

Techniques: Control, Ex Vivo

Journal: Molecular Therapy Advances

Article Title: Ferrostatin-1 and hinokitiol supplementation enhance human hematopoietic stem cell expansion in a chemically defined medium

doi: 10.1016/j.omta.2026.201711

Figure Lengend Snippet: Effect of selected compounds on CD34 + cell expansion in 3a medium Human CB CD34 + cells were seeded in 96-well plates at 10,000 cells per well in 3a medium with selected compounds. (A) Relative cell proliferation (fold increase) in the presence of selected compounds at day 14 compared to untreated control cells. Cell proliferation was assessed using CellTiter-Glo Luminescent cell viability assay. (B) Immunophenotypic analysis showing HSC percentage within HSPCs. (C) Lipid peroxidation levels at day 14 following treatment with selected compounds analyzed by BODIPY 581/591 C11 staining. Data show BODIPY-ox negative portion (%) in HSPCs. (D) Cellular ROS levels at day 14 following treatment with selected compounds measured with CellROX Deep Red. Data show negative portion (%) in HSPCs. Representative data from three independent experiments, each performed with unique CB donor in duplicate, are shown. (B–D) Mean ± SD, statistical analyses were conducted between Fer-1 versus DMSO vehicle control by t test, ∗ p < 0.05.

Article Snippet: Human CB CD34 + cells were purchased from HemaCare (CB34C-3) and STEMCELL Technologies (200-0001).

Techniques: Control, Cell Viability Assay, Staining

Journal: Molecular Therapy Advances

Article Title: Ferrostatin-1 and hinokitiol supplementation enhance human hematopoietic stem cell expansion in a chemically defined medium

doi: 10.1016/j.omta.2026.201711

Figure Lengend Snippet: Effect of selected compounds and Fer-1 combination on human CB CD34 + cell expansion Human CB CD34 + cells were seeded in 96-well plates at 10,000 cells per well in 3a medium with selected compounds with or without Fer-1. (A) Relative cell proliferation (fold change) at day 14 compared to untreated cells. Pooled data from three independent experiments, each performed with unique CB donor cells, are shown. (B and C) HSPC (B) and HSC (C) percentage in live cells with indicated compounds with or without Fer-1. Data represent three independent experiments, each performed with unique CB donor cells.

Article Snippet: Human CB CD34 + cells were purchased from HemaCare (CB34C-3) and STEMCELL Technologies (200-0001).

Techniques:

Journal: Molecular Therapy Advances

Article Title: Ferrostatin-1 and hinokitiol supplementation enhance human hematopoietic stem cell expansion in a chemically defined medium

doi: 10.1016/j.omta.2026.201711

Figure Lengend Snippet: Effect of Fer-1 and hinokitiol combination (FHK) on human CB CD34 + cell expansion Human CB CD34 + cells were seeded in 6-well plates at 300,000 cells per well in 3a medium with DMSO (vehicle control), or Fer-1 (10 μM) plus hinokitiol (0.5 μM) (FHK). (A) Relative cell proliferation at day 14 compared to DMSO. Pooled data from 3 independent experiments performed in duplicate. Mean ± SEM, ∗∗ p < 0.01 by t test. (B and C) Immunophenotypic analysis showing percentage of human HSPC (B) and HSC (C) in ex vivo expanded CB CD34 + cells at day 14. Pooled data from 3 independent experiments with CD34 + cells from 4 unique CB donors, performed in duplicate. (D and E) Lipid peroxidation (D) and intracellular ROS (E) levels in ex vivo expanded CD34 + CD45RA − cells measured by BODIPY 581/591 C11 and CellROX Deep Red staining, respectively. Representative overlaid histograms (left) and mean fluorescence intensity (MFI, right) are shown. Pooled data from 2 independent experiments with CD34 + cells from 2 unique CB donors, performed in duplicate. (F and G) Colony forming unit (CFU) activity in ex vivo expanded human CB CD34 + cells cultured for 14 days. Colony count of total progenitors (F) and various types of colonies (G) were quantified. Pooled data from 3 independent experiments with CD34 + cells from 4 unique CB donors, performed in duplicate. Mean ± SEM. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗∗ p < 0.0001 by one-way ANOVA with Tukey’s multiple-comparison test except (G). Each CB donor is represented by a unique symbol.

Article Snippet: Human CB CD34 + cells were purchased from HemaCare (CB34C-3) and STEMCELL Technologies (200-0001).

Techniques: Control, Ex Vivo, Staining, Fluorescence, Activity Assay, Cell Culture, Comparison

Journal: Molecular Therapy Advances

Article Title: Ferrostatin-1 and hinokitiol supplementation enhance human hematopoietic stem cell expansion in a chemically defined medium

doi: 10.1016/j.omta.2026.201711

Figure Lengend Snippet: Expanded human CB cells engraftment and chimerism after transplantation (A) Schematic outlining the xenotransplantation experiment in NOG-EXL mice. Schematic created in BioRender.com. (B) Human blood cell chimerism (CD45 + cell percentage) in peripheral blood at indicated time point (left: pooled data, right: individual mouse data. (C–E) Human blood cell lineage distribution (chimerism ratio) at week 24 in peripheral blood (C), spleen (D), and bone marrow (E) of transplanted mice. Pooled data from two independent experiments performed with expanded CD34 + cells from two unique CB donors (represented by unique symbol ● and▲). Fresh cells from each donor were used as control for comparison. Experiment#1, N = 3 mice per group. Experiment#2, N = 4 (fresh), 4(DMSO), and 5(FHK). Mean ± SEM, two-way ANOVA with Tukey’s multiple-comparison test (right). ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. (F) Bone marrow analyses at week 24 post-transplantation showing human CD45 + cells, lineage - cells, HSPCs, and HSC distribution. Data from one transplantation experiment are shown. Mean ± SEM, one-way ANOVA with Tukey’s multiple-comparison test. ∗∗∗ p < 0.001.

Article Snippet: Human CB CD34 + cells were purchased from HemaCare (CB34C-3) and STEMCELL Technologies (200-0001).

Techniques: Transplantation Assay, Control, Comparison

Journal: Biomolecules

Article Title: CTCF Regulates Erythroid Differentiation Through Control of Core Erythroid Transcription Factors

doi: 10.3390/biom16040549

Figure Lengend Snippet: Constitutive and inducible CTCF downregulation inhibits erythroid differentiation in CD34 + cells. ( A ) CTCF expression was analyzed by RT-qPCR in CD34 + cells following infection with pLKO empty vector (EV) or pLKO shCTCF lentiviruses and puromycin selection for two days. Expression levels were normalized to RPS14 . Data represent mean ± SD ( n = 6). *** p < 0.001 by two-tailed one-sample t -test. ( B ) Benzidine staining of CD34 + cells infected with pLKO empty vector (EV) or pLKO shCTCF and treated with 3 U/mL erythropoietin (EPO) for 10 days. In each experiment, a minimum of 200 cells were counted, and the percentage of benzidine-positive cells is shown. Data represent mean ± SD ( n = 3). ** p < 0.01, *** p < 0.001, **** p < 0.0001 by two-way ANOVA followed by Tukey’s post hoc multiple-comparisons test. ( C ) Percentage of glycophorin A-positive cells analyzed by flow cytometry after infection of CD34 + cells with pLKO empty vector (EV) or pLKO shCTCF and treatment with 3 U/mL erythropoietin (EPO) for 5 days. Data represent mean ± SD ( n = 3). ** p < 0.01 by two-way ANOVA. ( D ) Protein expression of γ-globin and GATA1 was analyzed by Western blot in CD34 + cells following infection with pLKO empty vector (EV) or pLKO shCTCF and treatment with 3 U/mL erythropoietin (EPO) for 5 days. Protein signal quantification was normalized to the loading control (actin). ( E ) CTCF expression was analyzed by RT-qPCR in CD34 + cells following infection with inducible pTRIPZ shCTCF lentiviruses, two days of puromycin selection, and two days of induction with 2 µg/mL doxycycline. Expression levels were normalized to RPS14. Data represent mean ± SD ( n = 3). * p < 0.05 by two-tailed one-sample t -test. ( F ) Benzidine staining of CD34 + cells infected with inducible pTRIPZ empty vector (EV) or pTRIPZ shCTCF, as described in ( E ), and treated with 3 U/mL erythropoietin (EPO) for 5 days. In each experiment, a minimum of 200 cells were counted, and the percentage of benzidine-positive cells is shown. Data represent mean ± SD ( n = 3). * p < 0.05 by two-way ANOVA. ( G ) Protein expression of γ-globin and GATA1 was analyzed by Western blot in CD34 + cells following infection with inducible pTRIPZ empty vector (EV) or pTRIPZ shCTCF, as described in ( E ), and treatment with erythropoietin (EPO) for 5 days. Protein signal quantification was normalized to the loading control (actin). All original Western blot images can be found in the .

Article Snippet: Primary CD34 + cells were obtained from human umbilical cord blood kindly donated by the Blood and Tissue Bank of Cantabria with approval from the local ethics committee (Ethics Committee on Clinical Research of Cantabria; Santander, Spain).

Techniques: Expressing, Quantitative RT-PCR, Infection, Plasmid Preparation, Selection, Two Tailed Test, Staining, Flow Cytometry, Western Blot, Control

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: Nature Communications

Article Title: ATRX loss couples genome instability at a G-rich repeat to dysregulation of human alpha-globin expression

doi: 10.1038/s41467-026-69169-7

Figure Lengend Snippet: a Schematic representation of human α-globin locus on chromosome 16p13.3. From the telomeric end, the locus contains the ζ-globin gene HBZ , the minor μ-globin gene HBM , the two α-globin genes HBA2 and HBA1 , and lastly the minor θ-globin gene HBQ1 . Two pseudogenes are present: ψζ HBZP1 between HBZ and HBM , and ψα1 HBAP1 between HBM and HBA2 . Four distal enhancers (R1-R4) are situated upstream of the genes. b Schematic overview of the experimental workflow to generate ATRX knockout erythroid cells from healthy donor CD34 + HSPCs, followed by downstream analysis. c Western blot analysis of ATRX expression in day 7 differentiated cells, confirming efficient knockout. Quantification is presented on the right (n = 3 biological replicates, data shown as mean ± SEM). ** p = 0.009, two-tailed unpaired Welch’s t-test. d Relative expression of HBA, HBM and α to β-like globin ratios by RT-qPCR analysis in day 10 and day 13 differentiated erythroid cells (n = 3 biological replicates, data shown as mean ± SEM). Differences between AAVS1 and ATRX KO were compared using a two-tailed unpaired Welch’s t-test on log₂-transformed fold-change values. e Genotyping summary of the collected 351 BFU-E colonies derived from ATRX KO samples showing genotype distribution. f Three-dimensional plot showing Biomark gene expression analysis of ATRX, HBA and HBM in single BFU-E colony. Each dot represents one colony, and the edited AAVS1 colonies are shown in blue; ATRX KO colonies with frameshift mutations (ATRX_FS) are shown in red. AAVS1 controls gather around the internal space whereas the majority of the ATRX KO colonies assemble around the origin indicating low expression of ATRX, HBM and HBA . g Quantified gene expression analyses of BFU-E colonies show statistical differences between AAVS1 (n = 15) and ATRX KO groups (n = 57) (Mann-Whitney U test). Individual colonies are selected from two biological repeats. *** p = 1.493 × 10 -5 for ATRX expression, ** p = 0.003632 for HBM expression, and p = 0.9083 for HBA expression. ns: not significant. Source data are provided as a Source Data file.

Article Snippet: 1-3 × 10 5 of CD34 + cells were transfected with RNPs (3.2 μg sgRNA (synthesised by Synthego) + 6 μg SpCas9 nuclease (IDT)) using Amaza P3 Primary cell 4D-Nucleofector X Kit (Lonza, Cat No V4XP-3032) and 4D-Nucleofector X Unit device (Lonza).

Techniques: Knock-Out, Western Blot, Expressing, Two Tailed Test, Quantitative RT-PCR, Transformation Assay, Derivative Assay, Gene Expression, MANN-WHITNEY