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: 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