Journal: Oncoimmunology

Article Title: Ex vivo-generated conventional dendritic cells type 1 and type 2 from blood progenitors induce potent antigen-specific T-cell immunity

doi: 10.1080/2162402X.2026.2695692

Figure Lengend Snippet: CD34-derived cDC1s and cDC2s can be generated from blood progenitors. CD34-positive cells isolated from blood were differentiated into cDC1s and cDC2s in a 17-day protocol in which progenitors initially expand and afterward undergo skewing towards cDC1 and cDC2 differentiation. (A) Schematic representation of the 17-day protocol for DC generation. (B) Bar graph showing the percentage of cDC1s and cDC2s ± SEM at the end of the culture on day 17 ( n = 10). (C) Representative dot plot showing the presence of cDC1s and cDC2s based on the expression of CD141/CLEC9A and CD1c, respectively, after gating on viable single cells. (D) Violin plot showing the absolute number of cDC1s and cDC2s obtained with the current protocol, depending on the initial amount of cultured CD34-positive cells. Each dot represents the value of expanded cDC1s and cDC2s obtained from one independent donor ( n = 12). (E) Bar graphs show the percentage of positive DCs ± SEM for the given marker ( n = 3). CD34-derived cDC1s and cDC2s were characterized by flow cytometry to determine their lineage marker expression.

Article Snippet: CD34 + cells were first eluted through an LS-column (130-042-401, Miltenyi Biotec), followed by an enrichment step on an MS-column (130-042-201, Miltenyi Biotec).

Techniques: Derivative Assay, Generated, Isolation, Expressing, Cell Culture, Marker, Flow Cytometry

Journal: Oncoimmunology

Article Title: Ex vivo-generated conventional dendritic cells type 1 and type 2 from blood progenitors induce potent antigen-specific T-cell immunity

doi: 10.1080/2162402X.2026.2695692

Figure Lengend Snippet: CD34-derived cDC1s and cDC2s are endocytic and exhibit antigen-presenting capacity. (A) Dot plots showing the percentage of cDC1s and cDC2s positive for CTV signal, indicative of CTV-labeled tumor cell uptake. (B) The scatter dot plot shows the frequency of tumor cell uptake positive DCs (identified as the percentage of CTV positive cDC1s and cDC2s) (mean ± SEM). (C) Representative histograms showing the percentage of cDC1s and cDC2s positive for Alexa647, indicative of active Dextran-Alexa647 uptake. (D) Scatter dot plot displaying the active dextran uptake (mean ± SEM). (E) Representative dot plots showing the frequency of proliferating autologous CFSE-labeled pan T-cells (indicated by the low CFSE expression) upon coculture with either untreated, TT-stimulated, or CMV-stimulated cDC1s for 6 d. (F) Scatter dot displaying the mean ± SEM of the percentage of autologous proliferating T-cells upon coculture with cDC2s (blue) and cDC1s (red) for 6 d under the indicated antigenic stimuli. (G) Scatter dot showing the mean ± SEM of the raw concentration of IFNγ and IL-2 detected by Luminex assay on supernatants collected after 6 d of cDC1 and cDC2 coculture with autologous T-cells together with the indicated antigenic stimuli. Statistical significance during uptake assays was calculated with either an unpaired t-test or a Mann–Whitney test. Statistical significance across antigen-specific assays was determined with Kruskal–Wallis test for the multiple comparison. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: CD34 + cells were first eluted through an LS-column (130-042-401, Miltenyi Biotec), followed by an enrichment step on an MS-column (130-042-201, Miltenyi Biotec).

Techniques: Derivative Assay, Labeling, Expressing, Concentration Assay, Luminex, MANN-WHITNEY, Comparison

Journal: Oncoimmunology

Article Title: Ex vivo-generated conventional dendritic cells type 1 and type 2 from blood progenitors induce potent antigen-specific T-cell immunity

doi: 10.1080/2162402X.2026.2695692

Figure Lengend Snippet: CD34-derived cDC1s and cDC2s induce tumor-derived antigen-specific CD8 T-cell clones. To address the capacity of these DCs to prime the induction of a T-cell clone specific against the tumor antigen MART1, matured CD34-derived pan-DCs (combined cDC1s and cDC2s) loaded with the MART1-derived peptide were cultured with autologous naive CD8 T-cells. (A) Schematic representation of the experimental layout. (B) Representative dot plot showing the frequency of MART1-positive CD8 T-cells after the induction protocol with CD34-derived pan-DCs, as indicated by the abundance of dextramer-PE positive CD8 T-cell. (C) Before–after dot plots showing the frequency of MART1-specific dextramer-stained CD8 T-cells for each donor compared to a negative control dextramer. Each dot represents an individual donor ( n = 4). (D) Schematic representation of the rechallenge experimental layout. To address the responsiveness of induced MART1-specific T-cells to a secondary antigen rechallenge, MART1-loaded autologous moDCs were cocultured overnight with the earlier primed CD8 T-cell pool containing the MART1-specific CD8 T-cells. (E) Representative dot plots showing the expression levels of CD137 and CD25 on CD8 T-cells cocultured with either unloaded or MART1-loaded moDCs. (F) Before–after dot plots showing the frequency of positive CD8 T-cells for the simultaneous expression of CD25 and CD137. (G) Before–after dot plots showing the frequency of positive CD8 T-cells for the activation marker CD25, CD137, CD69, CD107a, and the intracellular cytokines IFNγ, IL-2, and TNFα after coculture with either unloaded or MART1-loaded moDCs. Each dot represents an individual donor ( n = 4). Statistical significance was calculated using either a paired t-test or a Wilcoxon test. P- values are numerically depicted in the graph.

Article Snippet: CD34 + cells were first eluted through an LS-column (130-042-401, Miltenyi Biotec), followed by an enrichment step on an MS-column (130-042-201, Miltenyi Biotec).

Techniques: Derivative Assay, Clone Assay, Cell Culture, Staining, Negative Control, Expressing, Activation Assay, Marker

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