Journal: Cell Stem Cell

Article Title: Nanoengineered 3D culture substrate enables superior persistence and polyclonal engraftment of genetically engineered hematopoietic stem cells

doi: 10.1016/j.stem.2025.12.016

Figure Lengend Snippet: Nichoids improve the functionality of ex vivo cultured HSPCs (A) Schematic representation with highlight of a single elementary unit (top) and scanning electron microscopy images (bottom) of the nichoid culture substrate. Scale bars: 30 or 90 μm. (B) Experimental workflow. Human CB-derived HSPCs were seeded on 2D or nichoids immediately after thawing and collected for downstream analyses on days 3 and 7. (C) Percentage of phenotypically defined HSPC subsets ( n = 9, 9, 10, 10). (D) Number of erythroid, myeloid, and mixed colonies generated in the CFU-C assay ( n = 6). Wilcoxon test. (E and F) Number of lineages (E) and cells (F) per colony generated by HSC-enriched single cells. More than 250 colonies were analyzed for each condition. Median ± 95% CI. Mann-Whitney test. (G–I) Percentage of human CD45+ (hCD45+) cells measured in the PB (G) over time and BM (H) and SP (I) at the endpoint ( n = 5). Mann-Whitney test (calculated at the last time point for PB). (J) Number of erythroid, myeloid, and mixed colonies generated by BM-derived CD34+ cells purified from mice in (H) ( n = 5). Mann-Whitney test. Unless otherwise specified, mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01. See also and .

Article Snippet: For immunophenotypic analyses of ex vivo cultured HSPCs , 3–10×10 4 cells were washed with 2% FBS in DPBS and stained with the following fluorescent-labeled antibodies (1:100 dilution, each) for 15 min at 4°C: anti-human CD34 PE (Miltenyi Biotec), anti-human CD133 PE-Vio 770 (Miltenyi Biotec), and anti-human CD90 APC (BD Biosciences).

Techniques: Ex Vivo, Cell Culture, Electron Microscopy, Derivative Assay, Generated, MANN-WHITNEY, Purification

Journal: Cell Stem Cell

Article Title: Nanoengineered 3D culture substrate enables superior persistence and polyclonal engraftment of genetically engineered hematopoietic stem cells

doi: 10.1016/j.stem.2025.12.016

Figure Lengend Snippet: Nichoids enable efficient GE across multiple platforms and superior long-term engraftment of HDR-edited HSPCs (A) Experimental workflow. Human CB- or mPB-derived HSPCs were seeded on 2D or 3D immediately after thawing and collected on day 3 for nucleofection. Specifically, cells were edited with Cas9/AAV6 alone or in the presence of GSE56 and Ad5-E4orf6/7 editing enhancers to insert a GFP reporter within the AAVS1 locus. HSPCs from both the 2D and 3D conditions were seeded in 2D after nucleofection and collected for downstream analyses on days 4 and 7. Transplantation was performed only for the Cas9/AAV6 plus GSE56 and Ad5-E4orf6/7 editing enhancers treatment using CB-derived HSPCs. After 15 weeks post-injection, CD34+ cells were purified from the BM of the mice and transplanted into secondary recipients. (B) Percentage of edited HSPCs by FACS analysis on day 7 ( n = 6). (C) Number of erythroid, myeloid, and mixed colonies generated in the CFU-C assay from edited HSPCs seeded on day 4 ( n = 6). Wilcoxon test. (D and E) Percentage of hCD45+ cells measured in the PB (D) over time and BM (E) at the endpoint of transplanted mice ( n = 5). Mann-Whitney test (calculated at the last time point for PB). (F) Percentage of GFP+ cells (within hCD45+ cells) in the PB over time from mice in (D) ( n = 5). (G) Number of erythroid, myeloid, and mixed colonies generated by BM-derived CD34+ cells purified from mice in (E) ( n = 5). Mann-Whitney test. (H and I) Percentage of hCD45+ cells measured in the PB (H) over time and BM (I) at the endpoint of transplanted secondary recipients ( n = 8, 6). Mann-Whitney test (calculated at the last time point for PB). (J and K) Percentage of GFP+ cells (within hCD45+ cells) in the PB (J) over time and BM (K) from mice in (H and I) ( n = 8, 6). Mann-Whitney test (calculated at the last time point for PB). (L) Experimental workflow. Human mPB-derived HSPCs were seeded on 2D or 3D immediately after thawing and collected on day 3 for nucleofection. Specifically, cells were edited with BE or PE to disrupt the B2M gene. HSPCs from both the 2D and 3D conditions were seeded in 2D after nucleofection and collected for downstream analyses on days 4 and 7. (M) Percentage of edited HSPCs by FACS analysis on day 7 ( n = 6). (N) Number of erythroid, myeloid, and mixed colonies generated in the CFU-C assay from edited HSPCs seeded on day 4 ( n = 6). Wilcoxon test. Mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. See also and and .

Article Snippet: For immunophenotypic analyses of ex vivo cultured HSPCs , 3–10×10 4 cells were washed with 2% FBS in DPBS and stained with the following fluorescent-labeled antibodies (1:100 dilution, each) for 15 min at 4°C: anti-human CD34 PE (Miltenyi Biotec), anti-human CD133 PE-Vio 770 (Miltenyi Biotec), and anti-human CD90 APC (BD Biosciences).

Techniques: Derivative Assay, Transplantation Assay, Injection, Purification, Generated, MANN-WHITNEY

Journal: Cell Stem Cell

Article Title: Nanoengineered 3D culture substrate enables superior persistence and polyclonal engraftment of genetically engineered hematopoietic stem cells

doi: 10.1016/j.stem.2025.12.016

Figure Lengend Snippet: Nichoids improve the engraftment and clonal output of HSPCs upon gene addition (A) Experimental workflow. Human mPB-derived HSPCs were seeded on 2D or 3D immediately after thawing and transduced on day 1 with a GFP-expressing LV vector in the presence of PGE2 alone or in combination with CsH. Specifically, transduction was performed directly on the scaffolds at MOI 100 for PGE2 alone and MOI 30 with CsH. After 14 h from transduction (day 2), HSPCs from both the 2D and 3D conditions were collected for downstream analyses on day 2 and seeded in 2D for VCN analyses on day 10. Transplantation was performed only for the CsH condition. (B) Percentage of GFP+ HSPCs by FACS analysis on day 10 ( n = 6). Wilcoxon test. (C) Number of erythroid, myeloid, and mixed colonies generated in the CFU-C assay from transduced HSPCs seeded on day 2 ( n = 6). Wilcoxon test. (D and E) Percentage of hCD45+ cells measured in the PB (D) over time and BM (E) at the endpoint of transplanted mice ( n = 7). Mann-Whitney test (calculated at the last time point for PB). (F) Percentage of GFP+ cells (within hCD45+ cells) in the PB over time from mice in (D) ( n = 7). (G) Number of erythroid, myeloid, and mixed colonies generated by BM-derived CD34+ cells purified from mice in (E) ( n = 7). Mann-Whitney test. (H) Representative plots of tracked ISs and their relative abundance over time from mice of the 2D (top, mouse A4) and 3D (bottom, mouse B12) conditions. Each colored bar univocally identifies an IS with >1% representation in at least one time point, with its abundance proportional to the height of the bar, and a colored ribbon connecting two neighboring time points for each recaptured clone. The total number of unique ISs is reported on each bar. (I and J) Estimated clonal population size (I) and diversity measured by Shannon index (J) normalized on VCN from IS analyses. Mann-Whitney test. (K) Representative plots of CISs from mice of the 2D (left, mouse A4) and 3D (right, mouse B12) conditions. Each dot represents a gene, labeled with the corresponding color if observed as significant. The dashed line is the alpha value 0.05. Grubbs test. Mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. See also and and .

Article Snippet: For immunophenotypic analyses of ex vivo cultured HSPCs , 3–10×10 4 cells were washed with 2% FBS in DPBS and stained with the following fluorescent-labeled antibodies (1:100 dilution, each) for 15 min at 4°C: anti-human CD34 PE (Miltenyi Biotec), anti-human CD133 PE-Vio 770 (Miltenyi Biotec), and anti-human CD90 APC (BD Biosciences).

Techniques: Derivative Assay, Expressing, Plasmid Preparation, Transduction, Transplantation Assay, Generated, MANN-WHITNEY, Purification, Labeling

Journal: International Journal of Molecular Sciences

Article Title: Induced Pluripotent Stem Cell-Derived Dendritic Cells Provide a Reliable In Vitro Platform for Functional Screening of Immunoregulatory Probiotics

doi: 10.3390/ijms27010303

Figure Lengend Snippet: Timeline and intermediate characterization of human-induced pluripotent stem cell (iPSC) differentiation into dendritic cells. ( a ) Timeline of human iPSC differentiation into dendritic cells. ( b ) Representative images of cellular morphology during differentiation at days 0, 12, 26, 31, and 32. Human iPSCs were cut into small pieces before seeding (Scale bar represents 200 μm). ( c ) Characterization of human iPSC-derived hematopoietic stem cells by flow cytometry. CD34 and CD45 were used as surface markers. ( d ) Characterization of human iPSC-derived monocytes by flow cytometry. CD14 was used as the surface marker.

Article Snippet: The antibodies used included: CD34-PE (Proteintech, Rosemont, IL, USA, #PE-65183), CD45-PE (BD Biosciences, Ashland, OR, USA, #555483), CD14-FITC (BD Biosciences, Ashland, OR, USA, #555397), CD11c-FITC (Proteintech, Rosemont, IL, USA, #FITC-65086), and CD83-PE (BD Biosciences, Ashland, OR, USA, #550634).

Techniques: Derivative Assay, Flow Cytometry, Marker

Journal: STAR Protocols

Article Title: Protocol to sequentially isolate mouse oligodendrocytes, microglia, endothelial cells, astrocytes, and neurons via magnetic cell sorting

doi: 10.1016/j.xpro.2025.104139

Figure Lengend Snippet: Establishment of flow cytometry gates using stained samples and unstained controls Representative scatter plots from a single experiment showing stained samples (black) and unstained negative control samples (pink) for each cell-specific antibody (O4 as a marker for oligodendrocytes, CD11b for microglia, CD34 for endothelial cells, and ACSA2 for astrocytes) used to set the negative and positive staining gates for the (A) oligodendrocyte, (B) microglia, (C) endothelial cell, (D) astrocyte, and (E) neuron fractions.

Article Snippet: CD34 antibody, anti-mouse, REAfinity, FITC, clone REA383 | RAM34 – 1:50 dilution , Miltenyi , 130-117-775.

Techniques: Flow Cytometry, Staining, Negative Control, Marker

Journal: Journal of Bone Oncology

Article Title: hMSCs-derived exosomal MIR17HG promotes follicular helper T cell differentiation and osteosarcoma progression via the miR-372-3p/BCL6 axis

doi: 10.1016/j.jbo.2025.100726

Figure Lengend Snippet: Morphological characterization of the hMSCs and hMSCs-derived exosomes. (A) The expression of CD34, CD44 and CD90 in hMSCs was determined by flow cytometry. (B)(C) Characterization of hMSCs-derived exosomes by TEM and NTA. (D) The expression of CD9, CD63 and CD81 in hMSCs-Exo were determined by flow cytometry. (E) The expression of the lncRNA MIR17HG in hMSCs was evaluated by qRT-PCR. Data were displayed as the mean ± SD. n = 3. oe-MIR17HG group compared with the oe-NC group, ***p < 0.001.

Article Snippet: The hMSCs were incubated with PE-labeled anti-CD34 antibody (Proteintech, PE-65183, China), FITC-labeled anti-CD44 antibody (Abcam, ab30405, USA) or FITC-labeled anti-CD90 antibody (Abcam, ab124527).

Techniques: Derivative Assay, Expressing, Flow Cytometry, Quantitative RT-PCR

Journal: Nature Communications

Article Title: Chromatin accessibility in stem cells unveils progressive transcriptional alterations in myelodysplastic syndrome

doi: 10.1038/s41467-025-65753-5

Figure Lengend Snippet: a Patient list. b Sorting gates. BM CD34 + CD38 – stem and CD34 + CD38 + progenitor cells, corresponding to the bottom 10% and top 30%, respectively, of Lin – CD34 + -gated cells, respectively, were sorted. c UMAP plots of the overall chromatin accessibility profiles of the stem and/or progenitor cells of each disease sample. d Venn diagrams showing overlaps of open and closed DARs (q < 0.05) in diseased stem and progenitor cells compared with their normal counterparts. e Number of open and closed DARs between stem and progenitor cells of normal controls ( n = 4), MDS-MLD ( n = 13), MDS-EB ( n = 15), and AML-MRC ( n = 4) (left). DARs between stem and progenitor cells in MDS-MLD and MDS-EB were also calculated using randomly selected four samples, and the calculation was repeated 40 times. The data are shown in box plots (right). The box plot represents the interquartile range (midline indicates the median) with whiskers (10th and 90th percentiles). + indicates mean value. Statistical significance was determined by two-sided Tukey-Kramer multiple comparison test for post-hoc analysis. Source data are provided as a Source Data file.

Article Snippet: Mononuclear cells were isolated from BM samples using Lymphoprep (STEMCELL Technologies) and then reacted with CD34 magnetic beads (Miltenyi Biotec) in the presence of Human Fc Receptor Blocking Solution (Miltenyi Biotec), followed by the purification of CD34-positive cells using MACS separation LS columns (Miltenyi Biotec).

Techniques: Comparison

Journal: Nature Communications

Article Title: Chromatin accessibility in stem cells unveils progressive transcriptional alterations in myelodysplastic syndrome

doi: 10.1038/s41467-025-65753-5

Figure Lengend Snippet: a Volcano plots showing DARs between normal CD34 + CD38 – HSCs and CD34 + CD38 + progenitor cells (Open/Close in normal progenitor cells compared to normal stem cells). b Distribution of open and closed DARs in normal progenitor cells compared to normal stem cells. TSS, transcription start site; TTS, transcription termination site; UTR, untranslated region. c Transcription factor-binding motifs enriched in open and closed DARs in normal progenitor cells compared to normal stem cells. d List of strictly defined Lin – CD34 + CD38 – HSCs (HSCs, MPPs, and MLPs) and Lin – CD34 + CD38 + progenitor cells (CMPs, GMPs, MEPs, and CLPs). e UMAP plots of normal HSPCs combined with MDS stem and progenitor pairs. f Heat map showing the enrichment of transcription factor binding motifs at the top 15% ATAC peaks of each HSPC fraction. The z-scores of the -logP values are shown as motif-enrichment scores. Each transcription factor was assigned to one of the HSPC fractions in which binding motif enrichment showed the highest scores and was at least 1.5-fold higher than the average of the other HSPC fractions. Source data are provided as a Source Data file.

Article Snippet: Mononuclear cells were isolated from BM samples using Lymphoprep (STEMCELL Technologies) and then reacted with CD34 magnetic beads (Miltenyi Biotec) in the presence of Human Fc Receptor Blocking Solution (Miltenyi Biotec), followed by the purification of CD34-positive cells using MACS separation LS columns (Miltenyi Biotec).

Techniques: Binding Assay

Journal: Nature Communications

Article Title: Chromatin accessibility in stem cells unveils progressive transcriptional alterations in myelodysplastic syndrome

doi: 10.1038/s41467-025-65753-5

Figure Lengend Snippet: a Heat map showing the enrichment of transcription factor binding motifs at the top 15% ATAC peaks of each disease stem and progenitor fraction. Z-scores of -logP values are shown as motif enrichment scores. Transcription factor-binding motifs whose -logP value of motif enrichment was higher than 30 at the DARs of any disease subtype, as shown in Supplementary Fig. , were selected. The HSPC fractions in which the binding motif of each transcription factor was most enriched in Fig. are indicated at the bottom. b Pearson correlation coefficients between motif enrichment scores and gene expression of the corresponding transcription factors in MDS stem (left) and progenitor (right) samples. Motif enrichment of the transcription factor-binding motifs was calculated by averaging the ATAC peak values of MDS DARs (DARs between MDS-MLD or MDS-EB and normal samples) with the corresponding binding motifs. c Correlation between the enrichment of transcription factor-binding motifs at DARs and the expression of corresponding transcription factor genes during disease progression. d Transcription factor networks of MDS stem (left) and progenitor cells (right) constructed by the GeneNet method of GeNeCK using the motif enrichment score of each sample indicated in Fig. 3b and visualized using Cytoscape. The color and width of the edge indicate the partial correlation coefficient, reflecting the strength of the linear relationship between two transcription factor motifs, independent of other motifs. The size of the oval node represents the maximum enrichment score in the disease group. As the AP-1 family motifs showed high enrichment scores at closed DARs, they are depicted as relatively large blue ovals. Motifs were selected based on the criteria described in Fig. 3a for MDS stem and progenitor cells. Source data are provided as a Source Data file.

Article Snippet: Mononuclear cells were isolated from BM samples using Lymphoprep (STEMCELL Technologies) and then reacted with CD34 magnetic beads (Miltenyi Biotec) in the presence of Human Fc Receptor Blocking Solution (Miltenyi Biotec), followed by the purification of CD34-positive cells using MACS separation LS columns (Miltenyi Biotec).

Techniques: Binding Assay, Gene Expression, Expressing, Biomarker Discovery, Construct

Journal: Nature Communications

Article Title: Chromatin accessibility in stem cells unveils progressive transcriptional alterations in myelodysplastic syndrome

doi: 10.1038/s41467-025-65753-5

Figure Lengend Snippet: a Correlation between IPSS-M scores and individual ATAC peak values. Pearson’s correlation coefficients (r) were also calculated (left panel) with biological replicates of MDS samples ( n = 28), and two-sided P values were obtained using the t-distribution with ( n − 2) degrees of freedom under the null hypothesis of zero correlation. Violin plot showing the distribution of significantly positively or negatively correlated peaks ( p < 0.05) in MDS stem and progenitor cells (right panel). The box plot represents the interquartile range (midline indicates the median) with whiskers (10th and 90th percentiles). + indicates mean. b Enrichment of transcription factor-binding motifs at the selected ATAC peaks in (a) showing significant positive and negative correlations with IPSS-M in MDS stem and progenitor cells. c Correlation between blast percentage and individual ATAC peak values as in (a) with biological replicates of MDS samples ( n = 28). Violin plot showing the distribution of significantly positively or negatively correlated peaks ( p < 0.05) in MDS stem and progenitor cells (right panel). The box plot represents the interquartile range (midline indicates the median) with whiskers (10th and 90th percentiles). + indicates mean. d Enrichment of transcription factor-binding motifs at the selected ATAC peaks in ( c ) showing significant positive and negative correlations with blast percentage in MDS stem and progenitor cells, as in ( b ). Source data are provided as a Source Data file.

Article Snippet: Mononuclear cells were isolated from BM samples using Lymphoprep (STEMCELL Technologies) and then reacted with CD34 magnetic beads (Miltenyi Biotec) in the presence of Human Fc Receptor Blocking Solution (Miltenyi Biotec), followed by the purification of CD34-positive cells using MACS separation LS columns (Miltenyi Biotec).

Techniques: Binding Assay

Journal: Nature Communications

Article Title: Chromatin accessibility in stem cells unveils progressive transcriptional alterations in myelodysplastic syndrome

doi: 10.1038/s41467-025-65753-5

Figure Lengend Snippet: a Definition of progenitor scores. MDS DARs, which are also differentially accessible during normal differentiation, were selected to define progenitor scores. DARs were divided into four subgroups depending on chromatin accessibility changes in the normal control, MDS stem, and progenitor cells, as shown schematically, and their numbers are indicated. The peak values of each DAR in normal stem and progenitor cells were arbitrarily set to 0 and 1, respectively. The peak values of each DAR in diseased samples were adjusted to the 0–1 scale relative to those in their normal counterparts, and the average of all DARs in disease stem and progenitor cells were taken as progenitor scores. b Violin plots showing progenitor scores of normal and disease stem and progenitor cells with biological replicates of normal ( n = 4), ICUS/MDS-MLD ( n = 15), MDS-EB ( n = 15) and AML-MRC samples ( n = 4). The box plot represents the interquartile range (midline indicates the median) with whiskers (10th and 90th percentiles). + indicates mean. Statistical significance was determined by the paired, two-tailed Student’s t -test. (c, d) Correlation of Progenitor scores with IPSS-M scores in MDS stem ( c ) and progenitor cells ( d ). e Progenitor score differences between progenitor and stem cells. The fitted least-squares linear regression (y ~ x) is shown as a solid line, with the 95% confidence band shaded ( c, d, e ). f Correlation of progenitor scores with survival. The cutoff values of IPSS-M scores and progenitor score differences to analyze their correlation with survival are indicated in ( e ). Kaplan-Meier survival curves of the IPSS-M high and low MDS groups (left), and progenitor score high and low MDS groups (right). Statistical significance was calculated using the log-rank test. g Evaluation of prognostic models in survival analysis. Concordance indexes (C-indexes) obtained with IPSS-M, progenitor score differences, and the combination of IPSS-M and progenitor score differences are depicted. Source data are provided as a Source Data file.

Article Snippet: Mononuclear cells were isolated from BM samples using Lymphoprep (STEMCELL Technologies) and then reacted with CD34 magnetic beads (Miltenyi Biotec) in the presence of Human Fc Receptor Blocking Solution (Miltenyi Biotec), followed by the purification of CD34-positive cells using MACS separation LS columns (Miltenyi Biotec).

Techniques: Control, Two Tailed Test

Journal: Nature Communications

Article Title: Chromatin accessibility in stem cells unveils progressive transcriptional alterations in myelodysplastic syndrome

doi: 10.1038/s41467-025-65753-5

Figure Lengend Snippet: a CEBP inhibition by expressing dominant-negative CEBP (DN-CEBP) in the MDS cell line MDS-L. b Volcano plots showing DARs (q < 0.05, FC > 2.0) in MDS-L cells expressing DN-CEBP ( n = 3 biological replicates) compared with parental cells ( n = 3 biological replicates). DARs with CEBP motifs are shown in magenta. c Changes in ATAC peak signal levels in MDS-L cells by CEBP inhibition at all ATAC peaks with CEBP motifs and at DARs with CEBP motifs that become open in MDS stem cells compared with normal stem cells. The box plot represents the interquartile range (midline indicates the median) with whiskers (10th and 90th percentiles). + indicates mean value. Statistical significance was determined by the paired, two-tailed Student’s t -test. Peak signals for the control and CEBP-DN were calculated as the mean of biological replicates ( n = 3). d Clustering of 889 DARs with CEBP motifs that become open in MDS/AML stem cells using non-negative matrix factorization (NMF). The DARs were subdivided into three clusters (DAR1-3). MDS was stratified into three subgroups (MDS1-3) based on the chromatin accessibility profiles at DAR1-3. e Gene ontology terms significantly enriched in DAR 1-3. f Correlation of DAR1-3 with IPSS-M in MDS stem cells ( n = 28 biological replicates). g Correlation of MDS subgroups 1-3 with progenitor scores in MDS stem cells ( n = 28 biological replicates). The box plot represents the interquartile range (midline indicates the median) with whiskers (10th and 90th percentiles). + indicates mean value. Statistical significance was evaluated using the Tukey–Kramer post hoc test (two-sided) (f, g). The p values of the Jonckheere-Terpstra trend test are also shown. h Schematic representation of chromatin accessibility changes at the target sites of major transcription factors in MDS stem and progenitor cells. Myeloid transcriptional networks are moderately activated in MDS stem cells, while some HSC-specific transcriptional networks are inactivated in MDS stem cells, suggesting aberrant myeloid programming in MDS stem cells. Source data are provided as a Source Data file.

Article Snippet: Mononuclear cells were isolated from BM samples using Lymphoprep (STEMCELL Technologies) and then reacted with CD34 magnetic beads (Miltenyi Biotec) in the presence of Human Fc Receptor Blocking Solution (Miltenyi Biotec), followed by the purification of CD34-positive cells using MACS separation LS columns (Miltenyi Biotec).

Techniques: Inhibition, Expressing, Dominant Negative Mutation, Two Tailed Test, Control