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: Science Advances

Article Title: KRAS G12V /HLA-A*02:01–targeted chimeric antigen receptor T cells exhibit potent preclinical activity against solid tumors

doi: 10.1126/sciadv.aea2511

Figure Lengend Snippet: ( A ) Specificity screening of 11 positive clones against KRAS(WT)-HLA-A*02:01 and KRAS(G12V)-HLA-A*02:01 tetramers using enzyme-linked immunosorbent assay (ELISA; n = 3 independent experiments). ( B ) Antibody titration curves of selected clones, monoclonal antibody (mAb) A4 and B9, were measured by ELISA ( n = 3 independent replicates). OD 450 , optical density at 450 nm. ( C ) Schematic of CAR constructs showing CD19-, A4-, and B9-specific architectures. ( D ) Transduction efficiency was assessed on the basis of CD34 expression using flow cytometry ( n = 3 biological replicates). ( E ) Representative flow cytometry histograms showing CAR-positive populations after magnetic enrichment. ( F ) Frequency quantification of the indicated cell subsets by flow cytometry ( n = 3 biological replicates; ≥20,000 events analyzed per replicate). Data represent the means ± SD. Significance by two-way analysis of variance (ANOVA) with Tukey’s post hoc test: *** P < 0.001. TTE, terminally differentiated effector T cells; TEM, effector memory T cells; TCM, central memory T cells.

Article Snippet: Antibodies used in this study included human CD34 PE-conjugated antibody (R&D Systems, FAB7227P; RRID: AB_10973177), mouse immunoglobulin G1 (IgG1) PE-conjugated isotype control (R&D Systems, IC002P; RRID:AB_357242), fluorescein isothiocyanate (FITC) anti-human CD45RA (BioLegend, 983002; RRID: AB_2650650), APC anti-human CD197 (CCR7) (BioLegend, 353213; RRID: AB_10915474), FITC mouse IgG2b κ isotype control (BioLegend, 402207; RRID: AB_3097051), APC mouse IgG2a κ isotype control (BioLegend, 981906; RRID: AB_3097032), and FITC anti-human HLA-A2 (BioLegend, 343303; RRID: AB_1659246).

Techniques: Clone Assay, Enzyme-linked Immunosorbent Assay, Titration, Construct, Transduction, Expressing, Flow Cytometry

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: Cells

Article Title: IMG-A1: A Novel Immortalized Granulosa Cell Line for Investigating FSH-Dependent Folliculogenesis and Ovarian Pathophysiology

doi: 10.3390/cells14241940

Figure Lengend Snippet: Basic Characterization of the IMG-A1 Granulosa Cell Line. ( a , b ) Phase-contrast micrographs of the IMG-A1 cell culture. ( c ) Staining for senescence-associated β-galactosidase activity in passage 18 cells. ( d – f ) Immunocytochemical staining for the proliferation marker Ki67, showing a phase-contrast image ( d ), Ki67 immunofluorescence ( e ), and a merged image with DAPI nuclear counterstain ( f ). ( g , h ) Representative metaphase spreads showing a normal diploid karyotype (40 chromosomes) ( g ) and a near-tetraploid karyotype (78 chromosomes) from the IMG-A1 line ( h ). ( i – k ) Ploidy analysis by flow cytometry. Control blood cells ( i ) and primary granulosa cells ( j ) show characteristic diploid (2n) and tetraploid (4n, G2/M phase) peaks. The IMG-A1 culture ( k ) displays a predominantly near-tetraploid and near-octaploid cell population. ( l ) Flow cytometry analysis confirming the homogeneity of the IMG-A1 culture (passages 10 and 40) based on staining for the stromal marker CD29 and the absence of hematopoietic markers CD45, CD34, and the fibroblast marker CD90. Quadrant gates were set based on unstained controls. Scale bars = 100 µm.

Article Snippet: PE CD34 Rat mAb [RAM34] , Elabscience Biotechnology Co., Ltd., Wuhan, China , E-AB-F1284D.

Techniques: Cell Culture, Staining, Activity Assay, Marker, Immunofluorescence, Flow Cytometry, Control