Journal: Cancer Science

Article Title: Integrated Transcriptomic and Functional Analyses Reveal lncRNA ‐ miRNA ‐ mRNA Axis in AML Relapse After Allo‐ HSCT

doi: 10.1111/cas.70417

Figure Lengend Snippet: Transcriptomic profiling of CD34 + cells from AML patients relapsing after allo‐HSCT reveals lncRNA‐driven ceRNA networks, with the SNHG8‐miR‐625‐ZC3H13/15 axis identified as a key relapse‐promoting module through CRISPR screening and single‐cell perturbation. Hypomethylating agents reverse this axis, linking clinical intervention to ceRNA regulation.

Article Snippet: MOLM13 or CD34 + cells from NC, CR, and RE cohorts (1 × 10 5 /well, 12‐well plates) were treated with AZA (MCE#HY‐10586) or DAC (MCE#HY‐A0004) for 72 h, with medium refreshed every 24 h. Control and treated cells were harvested for downstream RNA extraction and sequencing.

Techniques: CRISPR, Single Cell

Journal: Cancer Science

Article Title: Integrated Transcriptomic and Functional Analyses Reveal lncRNA ‐ miRNA ‐ mRNA Axis in AML Relapse After Allo‐ HSCT

doi: 10.1111/cas.70417

Figure Lengend Snippet: Transcriptomic alterations in CD34+ cells from AML‐HSCT relapse patients. (A) Bone marrow samples from relapsed (RE, n = 5), complete remission (CR, n = 5), and normal controls (NC, n = 4) were subjected to CD34+ sorting and whole‐transcriptome sequencing of long and short RNAs. (B) PCA of lncRNA, miRNA, and mRNA expression showing clear separation of RE from CR/NC groups. (C) Volcano plots highlighting differential expression of lncRNAs, miRNAs, and mRNAs across groups. (lncRNA 1664 [up 577, down 1087], miRNA 1206, mRNA 3173) (D) GO‐BP enrichment of mRNA differences between RE and CR. (E) Heatmap of the top 40 downregulated protein‐coding genes in RE relative to CR. (F) Hallmark GSEA showing the enriched Hallmark gene sets (CR vs. RE). (G) Differential pathway enrichment in RE, CR, and NC (Hallmark).

Article Snippet: MOLM13 or CD34 + cells from NC, CR, and RE cohorts (1 × 10 5 /well, 12‐well plates) were treated with AZA (MCE#HY‐10586) or DAC (MCE#HY‐A0004) for 72 h, with medium refreshed every 24 h. Control and treated cells were harvested for downstream RNA extraction and sequencing.

Techniques: Sequencing, Expressing, Quantitative Proteomics

Journal: Cancer Science

Article Title: Integrated Transcriptomic and Functional Analyses Reveal lncRNA ‐ miRNA ‐ mRNA Axis in AML Relapse After Allo‐ HSCT

doi: 10.1111/cas.70417

Figure Lengend Snippet: ce RNA regulatory networks inferred from correlation, KNN, and thermodynamic analyses. (A) Analytical pipeline integrating expression normalization, KNN distance computation, and ceRNA triplet assembly. (B) KNN‐based clustering across hierarchical layers: All RNAs, lnc‐miRNA, and lnc‐mi‐mRNA triplets. (C) UMAP visualization of lnc‐mi‐mRNA associations; top 30 triplets highlighted. (D) Sankey plot showing representative lnc‐mi‐mRNA regulatory relationships. (E) Thermodynamic modeling (RNA folding/binding ΔG eff ) supporting binding feasibility of miRNA‐target pairs. (F‐G) Ranked top 30 lnc‐miRNA (F) and mi‐mRNA (G) axes supported by correlation and energy integration. (H) Chord diagram illustrating the global ceRNA regulatory network in relapse CD34+ cells.

Article Snippet: MOLM13 or CD34 + cells from NC, CR, and RE cohorts (1 × 10 5 /well, 12‐well plates) were treated with AZA (MCE#HY‐10586) or DAC (MCE#HY‐A0004) for 72 h, with medium refreshed every 24 h. Control and treated cells were harvested for downstream RNA extraction and sequencing.

Techniques: Expressing, Binding Assay

Journal: Cancer Science

Article Title: Integrated Transcriptomic and Functional Analyses Reveal lncRNA ‐ miRNA ‐ mRNA Axis in AML Relapse After Allo‐ HSCT

doi: 10.1111/cas.70417

Figure Lengend Snippet: The SNHG8‐miR‐625‐5p‐ZC3H13/15 axis promotes AML relapse after HSCT. (A) qPCR analysis of CD34+ cells from respective donors (NC), AML at diagnosis (DX), and respective relapsed post‐HSCT patients (RE) with specific primers (B) Lentiviral shRNA‐mediated knockdown of SNHG8 in MOLM13 and THP‐1 cells reduced SNHG8 expression, increased miR‐625/7705, and decreased ZC3H13/15 and TSC2 mRNA. (C) CCK‐8 assays showing impaired proliferation after SNHG8 knockdown. (D) Cell cycle analysis demonstrating increased G0 and decreased G1/2 phase fractions. (E‐F) Apoptosis assays indicating increased apoptotic cell populations upon SNHG8 knockdown.

Article Snippet: MOLM13 or CD34 + cells from NC, CR, and RE cohorts (1 × 10 5 /well, 12‐well plates) were treated with AZA (MCE#HY‐10586) or DAC (MCE#HY‐A0004) for 72 h, with medium refreshed every 24 h. Control and treated cells were harvested for downstream RNA extraction and sequencing.

Techniques: Biomarker Discovery, shRNA, Knockdown, Expressing, CCK-8 Assay, Cell Cycle Assay

Journal: Cancer Science

Article Title: Integrated Transcriptomic and Functional Analyses Reveal lncRNA ‐ miRNA ‐ mRNA Axis in AML Relapse After Allo‐ HSCT

doi: 10.1111/cas.70417

Figure Lengend Snippet: Effects of hypomethylating agents (HMAs) on non‐coding and coding RNAs. (A) Experimental design: RE‐CD34+ and MOLM13 cells treated with AZA or DAC, followed by RNA‐seq. (B) Proportion of upregulated lncRNAs/mRNAs post‐treatment induced by DAC than AZA (C‐D) Scatter plots comparing baseline RE/CR differences (x‐axis) with AZA/DAC‐induced changes (y‐axis) in mRNA (C) and lncRNA (D). (E) Summary of lncRNAs/mRNAs reversed by AZA/DAC relative to RE/CR differences (F) Hallmark, Reactome, and GO‐BP enrichment showing different profiles of AZA and DAC effects on AML cells. (G) Pathway‐specific effects of AZA or DAC on MOLM13 or RE‐CD34+ cells Expression values were normalized to TPM/CPM. Comparisons between treatment groups were performed using an unpaired t‐test. Significance thresholds: * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: MOLM13 or CD34 + cells from NC, CR, and RE cohorts (1 × 10 5 /well, 12‐well plates) were treated with AZA (MCE#HY‐10586) or DAC (MCE#HY‐A0004) for 72 h, with medium refreshed every 24 h. Control and treated cells were harvested for downstream RNA extraction and sequencing.

Techniques: RNA Sequencing, Expressing

Journal: Cancer Science

Article Title: Integrated Transcriptomic and Functional Analyses Reveal lncRNA ‐ miRNA ‐ mRNA Axis in AML Relapse After Allo‐ HSCT

doi: 10.1111/cas.70417

Figure Lengend Snippet: HMA treatment dose‐dependently modulates the SNHG8‐miR‐625‐ZC3H13 ceRNA axis. (A) Quantitative RT‐PCR showing dose‐dependent effects of azacitidine (AZA; 250, 500, and 750 nM) on SNHG8, miR‐625‐5p, and ZC3H13 expression in MOLM13 cells. DMSO served as vehicle control. (B) Same as (A) but in relapse‐derived CD34+ (RE‐CD34+) cells (C) Dose‐dependent effects of decitabine (DAC; 50, 75, and 100 nM) on SNHG8, miR‐625‐5p, and ZC3H13 expression in MOLM13 cells. (D) Same as (C) but in RE‐CD34+ cells. Data are presented as mean ± SD; ns, not significant; ** p < 0.01; *** p < 0.001, by unpaired two‐tailed Student's t ‐test.

Article Snippet: MOLM13 or CD34 + cells from NC, CR, and RE cohorts (1 × 10 5 /well, 12‐well plates) were treated with AZA (MCE#HY‐10586) or DAC (MCE#HY‐A0004) for 72 h, with medium refreshed every 24 h. Control and treated cells were harvested for downstream RNA extraction and sequencing.

Techniques: Quantitative RT-PCR, Expressing, Control, Derivative Assay, Two Tailed Test

Journal: Cancer Science

Article Title: Integrated Transcriptomic and Functional Analyses Reveal lncRNA ‐ miRNA ‐ mRNA Axis in AML Relapse After Allo‐ HSCT

doi: 10.1111/cas.70417

Figure Lengend Snippet: HMA treatment reverses relapse‐associated ceRNA networks. (A) Heatmap of RNA‐seq log2FC values for the SNHG8 ceRNA axis members (SNHG8, miR‐625‐5p, ZC3H13, ZC3H15, TSC2) in MOLM13 and RE‐CD34+ cells treated with vehicle (Veh), AZA, or DAC. (B) Scatter plot of RNA‐seq versus qPCR log2FC values for SNHG8 axis genes across both cell types and treatment conditions (R 2 = 0.782, p < 0.01). Circle and square symbols denote MOLM13 and RE‐CD34+ cells, respectively; colors indicate AZA (pink) or DAC (yellow) treatment. (C) Reversal Index (RI) heatmap for the top 30 ceRNA triplets under AZA and DAC treatment. Yellow‐highlighted rows indicate SNHG8‐associated axes (SNHG8‐miR625‐ZC3H13 and SNHG8‐miR625‐ZC3H15). (D) Stacked bar plot summarizing the proportion of ceRNA triplets classified as reversed, unchanged, or strengthened by AZA and DAC treatment. (E) Scatter plot comparing Reversal Index values of AZA (x‐axis) versus DAC (y‐axis) for individual ceRNA triplets. Circle size reflects network centrality; colors denote pathway annotations (Cell Cycle, E2F/Epigenetic, Metabolism, PI3K‐AKT, TNF‐NFkB/Immune, and Other). The SNHG8 axis (yellow circle with border) falls in the “both reversed” quadrant. Wilcoxon signed‐rank test p = 0.0004.

Article Snippet: MOLM13 or CD34 + cells from NC, CR, and RE cohorts (1 × 10 5 /well, 12‐well plates) were treated with AZA (MCE#HY‐10586) or DAC (MCE#HY‐A0004) for 72 h, with medium refreshed every 24 h. Control and treated cells were harvested for downstream RNA extraction and sequencing.

Techniques: RNA Sequencing

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: Stem Cell Research & Therapy

Article Title: Generation and single-cell characterization of functional megakaryocytes derived from umbilical cord blood

doi: 10.1186/s13287-026-05047-9

Figure Lengend Snippet: CD34 + cells derived from UCB were induced to differentiate into MKs in vitro. A A schematic culture system of CD34 + HSPCs derived from UCB induced to differentiate into MKs. B The cell growth curve during the culture cycle, N = 3. C Representative flow cytometry analysis of CD34, CD41a, and CD42b expression in cells cultured at the indicated culture time points; N = 3. D The percentages of the CD34 − CD41a + CD42b + population at different culture times. N = 3. E Confocal images of UCB-derived MKs immunostained for α-tubulin and CD62P. Scale bar, 20 µm

Article Snippet: In brief, CD34 + cells were separated and enriched from UCB using a human CD34 MicroBead Kit (Miltenyi Biotec, GmbH, Germany) following the manufacturer's instructions.

Techniques: Derivative Assay, In Vitro, Flow Cytometry, Expressing, Cell Culture

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