cd34 enriched hspcs  (Miltenyi Biotec)

Journal: Journal of Translational Medicine

Article Title: Ex vivo long-term expansion of human hematopoietic stem and progenitor cells as a tool for modeling vector integration sites and clonality

doi: 10.1186/s12967-026-07700-6

Figure Lengend Snippet: Ex vivo expansion of HSPCs in A83-01, pomalidomide, and UM171 (APU). ( A ) Experimental scheme of the 7-day ex vivo expansion protocol. CD34 + CD38 − CD45RA − cells from umbilical cord blood (UCB) were seeded in low cell density for HSPC expansion and analyzed via flow cytometry for their HSC immune phenotype. ( B ) Representative plots for the gating strategy of CD34 + CD38 − CD45RA − CD90 + EPCR + cells in SFT3 (basal medium), UM171, and APU. ( C ) Surface marker expression of CD34, CD38, CD45RA, CD90, and EPCR in N = 3 UCB donors after 7-day expansion of CD34 + CD38 − CD45RA − cells in SFT3, UM171, and APU with 2 µM or 0.2 µM pomalidomide (Poma). ( D ) Expansion of the total cell number in one 96-well counted with Casy cell counter. ( E ) Fold change of expanded ST-HSCs after 7-days. The number of expanded CD34 + CD38 − CD45RA − CD90 + cells was divided by 1,000 (seeded cell number) and then normalized to SFT3. ( F ) Percentage of CD34 + CXCR4 + HSPCs after expansion. The mean is displayed with the SD by the error bars. Statistical analysis in D-F was performed with one-way ANOVA and Tukey’s test for multiple comparisons in N = 4 UCB donors (* = p < 0.05, ** = p < 0.01)

Article Snippet: Thus, CD34 + -enriched HSPCs were thawed and blocked with Fc receptor blocking reagent (Miltenyi Biotec) before staining with antibodies against CD34 (APC, clone HG12, catalog #345804, BD Biosciences, Franklin Lakes, NJ, Unites States), CD38 (FITC, clone HB7, catalog #11-0388-42, Thermo Fisher Scientific, Waltham, MA, USA), and CD45RA (BV605, clone HI100, catalog #304134, BioLegend, San Diego, CA, USA).

Techniques: Ex Vivo, Flow Cytometry, Marker, Expressing

Journal: Journal of Translational Medicine

Article Title: Ex vivo long-term expansion of human hematopoietic stem and progenitor cells as a tool for modeling vector integration sites and clonality

doi: 10.1186/s12967-026-07700-6

Figure Lengend Snippet: Single-cell RNA sequencing (scRNA seq) analysis reveals expression of HSC signature genes in APU-exp HSPCs. ( A ) Surface marker expression of CD34, CD38, CD45RA, CD90, and EPCR after 7-day expansion in SFT3 or SFT3 supplemented with single (A, P, U), minus-one (AP, AU, PU), and complete APU (APU with 2 µM or 0.2 µM Poma) compound conditions. ( B ) Experimental scheme for the scRNA seq experiment using the 10X Genomics Chromium technology and hashtag oligo (HTO)-labeled antibodies for sample multiplexing. UCB-derived CD34 + CD38 − CD45RA − HSPCs were analyzed uncultivated, 24 h cultivated in SFT, SFT + APU, or SFT + APU_0.2, and 7-day expanded in the conditions described in ( A ). ( C ) Uniform Manifold Approximation and Projection for Dimension Reduction (UMAP) displaying the integrated data of all samples with cluster annotation: Hematopoietic stem cells/multipotent progenitors (HSCs/MPPs), early and late cycling megakaryocytic-erythroid-mast cell progenitors (cMEMPs), granulocytes (neutrophils, eosinophils, basophils/mast cells), mast cells, monocytes and macrophages (Mono’s/Neutro’s), cycling MPPs (cMPPs), and cycling dendritic cells (cDCs). ( D ) Dot plot showing the average expression of the respective marker genes used in cluster annotation

Article Snippet: Thus, CD34 + -enriched HSPCs were thawed and blocked with Fc receptor blocking reagent (Miltenyi Biotec) before staining with antibodies against CD34 (APC, clone HG12, catalog #345804, BD Biosciences, Franklin Lakes, NJ, Unites States), CD38 (FITC, clone HB7, catalog #11-0388-42, Thermo Fisher Scientific, Waltham, MA, USA), and CD45RA (BV605, clone HI100, catalog #304134, BioLegend, San Diego, CA, USA).

Techniques: Single Cell, RNA Sequencing, Expressing, Marker, Labeling, Multiplexing, Derivative Assay

Journal: Journal of Translational Medicine

Article Title: Ex vivo long-term expansion of human hematopoietic stem and progenitor cells as a tool for modeling vector integration sites and clonality

doi: 10.1186/s12967-026-07700-6

Figure Lengend Snippet: Effect of APU on HSPCs after 24 h cultivation on the single-cell level. ( A ) UMAP of uncultivated and 24 h-cultivated HSPCs and their individual cluster abundances in the bar plot below. ( B ) Feature plots of the HSC signature genes HLF , MECOM , NKAIN1 , PROM1 , AVP , and CD34 . ( C) Dotplot comparing the average expression of HSC signature genes among the 24 h-cultivated HSPCs. ( D ) Volcano plot displaying the log2 fold changes (FC) and the -log10 p values of DEGs between HSPCs cultivated for 24 h in SFT + APU (APU_24h) compared to SFT (SFT_24h). Significant ( p > 0.05, Wilcoxon Rank Sum test) genes with an FC > 1 are colored in green (up in APU_24h) or yellow (up in SFT_24h). ( E ) Gene set enrichment analysis (GSEA) on the DEGs of APU_24h vs. SFT_24h using HSC-related gene sets from the Molecular Signatures Database (MSigDB), showing the normalized enrichment score (NES). ( F ) GSEA on the DEGs of APU_24h vs. SFT_24h and of SFT3 + APU_7d vs. SFT3_7d (HSC/MPP cluster) using TGFβ-related gene sets from the MSigDB

Article Snippet: Thus, CD34 + -enriched HSPCs were thawed and blocked with Fc receptor blocking reagent (Miltenyi Biotec) before staining with antibodies against CD34 (APC, clone HG12, catalog #345804, BD Biosciences, Franklin Lakes, NJ, Unites States), CD38 (FITC, clone HB7, catalog #11-0388-42, Thermo Fisher Scientific, Waltham, MA, USA), and CD45RA (BV605, clone HI100, catalog #304134, BioLegend, San Diego, CA, USA).

Techniques: Single Cell, Expressing

Journal: Journal of Translational Medicine

Article Title: Ex vivo long-term expansion of human hematopoietic stem and progenitor cells as a tool for modeling vector integration sites and clonality

doi: 10.1186/s12967-026-07700-6

Figure Lengend Snippet: Increased lentiviral transduction efficiency in APU-exp cells. ( A ) Graphical scheme of the combined transduction (td) and expansion protocol for UCB-derived CD34 + cells. Td was performed with 1 × 10 5 CD34 + 24 h post-thawing with a self-inactivating lentiviral vector encoding for mCherry driven by the CBX3.EFS promoter (SIN-LV.CBX3.EFS.mCherry) in APU/SFT3 medium supplemented with protamine sulfate and Synperonic ® F-108 in a U-bottom 96-well plate. On day 1 post-td, HSPCs were seeded for expansion. Td efficiency and stem cell phenotype were determined on day 6 post-td, and vector copy number (VCN) was determined on day 8 post-td. Each donor replicate of expanded cells was split for transplantation into three mice. ( B ) Number of cells after thawing CD34 + cells from N = 3 UCB donors (day − 1), the next day (day 0), and after expansion (day 6 post-td) counted with Casy. ( C ) HSPC immune phenotype of expanded cells 6 days post-td. Expression of CD34 + , CD34 + CD38 − CD45RA − CD90 + (labeled CD90 + ), and CD34 + EPCR + in expanded HSPCs. ( D ) Td efficiency in the bulk cell population, in CD34 + HSPCs, and CD34 + CD38 − CD45RA − CD90 + EPCR + (labeled as LT-HSC). ( E ) VCN per diploid cell determined by qPCR. Bars indicate the mean of n = 3 UCB donors with the SD displayed by the error bars; Statistical significances were determined by paired t-test; p < 0.05= *, p < 0.0001= ****

Article Snippet: Thus, CD34 + -enriched HSPCs were thawed and blocked with Fc receptor blocking reagent (Miltenyi Biotec) before staining with antibodies against CD34 (APC, clone HG12, catalog #345804, BD Biosciences, Franklin Lakes, NJ, Unites States), CD38 (FITC, clone HB7, catalog #11-0388-42, Thermo Fisher Scientific, Waltham, MA, USA), and CD45RA (BV605, clone HI100, catalog #304134, BioLegend, San Diego, CA, USA).

Techniques: Transduction, Derivative Assay, Plasmid Preparation, Transplantation Assay, Expressing, Labeling

Journal: Journal of Translational Medicine

Article Title: Ex vivo long-term expansion of human hematopoietic stem and progenitor cells as a tool for modeling vector integration sites and clonality

doi: 10.1186/s12967-026-07700-6

Figure Lengend Snippet: APU_0.2 allows long-term cultivation of CD34 + cells up to 5 weeks after lentiviral transduction. ( A ) Experimental setup of transducing expanded HSPCs. Purified CD34 + CD38 − CD45RA − cells were expanded for 7 days either in SFT3 or APU_0.2 medium before transduction (td) with the SIN-LV.SF.eGFP (SF) or the SIN-LV.EFS.eGFP (EFS) vector. 24 h post-td, HSPCs were either transplanted into immunocompromised mice or further cultivated. Long-term culture allowed downstream analyses. ( B ) Expansion factor after 7-day culture in SFT3 or APU_0.2 medium determined by Casy. Paired t-test revealed non-significant (ns = p > 0.05) differences. ( C ) Flow cytometric analysis of eGFP expression on day 7 post-td to determine the td efficiency. ( D ) Vector copy number (VCN) per diploid genome was determined via ddPCR. ( E ) Expansion factor over 5 weeks post-td either in SFT3 or APU_0.2 medium. Cell numbers were determined by Casy and divided by the seeded cell number from the week before. Paired t-test over the whole period of time between APU_0.2 and SFT3 to assess the expected mean difference (diff.) between the groups, the 95% confidence interval (CI), and the p value. ( F ) Microscopic images 3 weeks post-td. Imaging was performed with CellCyteX using 10x magnification. Scale bar length indicates 100 μm. ( G-H ) Monocytic markers (CD11b, CD14, CD33) were determined via flow cytometry 3, 4, and 6 weeks post-td of HSPCs cultivated in SFT3 ( G ) or APU_0.2 ( H ). ( I ) Dim and bright CD11b expression in HSPCs cultivated for 3 weeks post-td. Exemplary gating strategy of the CD11b + bright CD14 + population for SFT3 cultivated cells. ( J ) Percentage of CD34 + cells during the long-term culture in SFT3 or APU_0.2 up to 5 weeks post-td. Two-way ANOVA with Tukey’s multiple comparisons test. Individual values are biological replicates with N = 3 UCB donors. Error bars indicate the mean ± SD

Article Snippet: Thus, CD34 + -enriched HSPCs were thawed and blocked with Fc receptor blocking reagent (Miltenyi Biotec) before staining with antibodies against CD34 (APC, clone HG12, catalog #345804, BD Biosciences, Franklin Lakes, NJ, Unites States), CD38 (FITC, clone HB7, catalog #11-0388-42, Thermo Fisher Scientific, Waltham, MA, USA), and CD45RA (BV605, clone HI100, catalog #304134, BioLegend, San Diego, CA, USA).

Techniques: Transduction, Purification, Plasmid Preparation, Expressing, Imaging, Flow Cytometry

Journal: Cell reports

Article Title: Dual α-globin-truncated erythropoietin receptor knockin restores hemoglobin production in α-thalassemia-derived erythroid cells

doi: 10.1016/j.celrep.2024.115141

Figure Lengend Snippet: (A) Schematic of custom AAV6 DNA repair donors designed to integrate promoterless HBA-2A-YFP transgenes at the start codon of HBB . The table to the left indicates whether cassette integrated HBB , HBA1 , or HBA2 UTRs, as well as HBA1 or HBA2 transgene. (B) Schematic of Cas9/AAV6 genome editing workflow in primary HSPCs followed by in vitro erythroid differentiation. (C) Percentage of CD34 − /CD45 − HSPCs acquiring erythroid cell surface markers GPA and CD71 as determined by flow cytometry. Bars represent mean ± SEM. (D) Percentage of YFP + cells among CD34 − /CD45 − /CD71 + /GPA + cells was determined at day 14 of erythroid differentiation using flow cytometry. Bars represent mean ± SEM. ** p < 0.005 by unpaired two-tailed t test. (E) MFI of YFP + cells (from D) was determined by flow cytometry. Bars represent mean ± SEM. (F) At day 11 of erythroid cell differentiation, cells were stained for HSPC/erythroid markers and analyzed by flow cytometry. The percentage of YFP + cells are noted ( n = 1). (G) The percentage of CD34 − /CD45 − cells that acquired CD71 and GPA erythroid cell markers are plotted over the course of erythroid differentiation ( n = 1). (H) The percentage of YFP + or GFP + cells are plotted over the course of erythroid differentiation ( n = 1).

Article Snippet: Human CD34 + -enriched HSPCs derived from Plerixafor and/or G-CSF-mobilized peripheral blood from healthy donors , STEMCELL Technologies, AllCells, Fred Hutchinson Cancer Center Hematology Core , 70073.2.

Techniques: In Vitro, Flow Cytometry, Two Tailed Test, Cell Differentiation, Staining

Journal: Cell reports

Article Title: Dual α-globin-truncated erythropoietin receptor knockin restores hemoglobin production in α-thalassemia-derived erythroid cells

doi: 10.1016/j.celrep.2024.115141

Figure Lengend Snippet: (A) Schematic of custom AAV6 DNA repair donors designed to integrate promoterless HBA transgenes at the start codon of HBB . Table to the left indicates whether the cassette integrated HBB or HBA1 UTRs along with HBA1 transgene. (B) The percentage of edited alleles in WT HSPCs at day 14 of erythroid differentiation. Bars represent mean ± SEM. (C) Percentage of CD34 − /CD45 − αTM HSPCs acquiring erythroid cell surface markers as determined by flow cytometry. Bars represent mean ± SEM. (D) The percentage of edited alleles in αTM HSPCs at day 14 of erythroid differentiation. Bars represent mean ± SEM. (E) HPLC elution chromatogram displaying the hemoglobin tetramer profile from WT healthy control HSPCs following in vitro erythroid differentiation. Time displayed on the x axis represents retention time in minutes for each hemoglobin tetramer type to elute. Absorbance on the y axis indicates the concentration of a particular hemoglobin tetramer. (F–H) HPLC elution chromatograms displaying the hemoglobin tetramer profile from αTM HSPCs that have undergone editing and erythroid differentiation. Chromatograms represent two different donors (F and G) and a technical replicate from donor 2 (H) that was edited independently.

Article Snippet: Human CD34 + -enriched HSPCs derived from Plerixafor and/or G-CSF-mobilized peripheral blood from healthy donors , STEMCELL Technologies, AllCells, Fred Hutchinson Cancer Center Hematology Core , 70073.2.

Techniques: Flow Cytometry, Control, In Vitro, Concentration Assay

Journal: Cell reports

Article Title: Dual α-globin-truncated erythropoietin receptor knockin restores hemoglobin production in α-thalassemia-derived erythroid cells

doi: 10.1016/j.celrep.2024.115141

Figure Lengend Snippet: (A) Schematic of custom AAV6 donors designed to integrate promoterless HBA and HBA + tEPOR transgenes at the start codon of HBB . Both vectors are flanked by HBA1 UTRs. (B) The percentage of CD34 − /CD45 − WT HSPCs acquiring erythroid cell surface markers as determined by flow cytometry. Bars represent mean ± SEM. (C) The percentage of edited alleles in WT HSPCs over the course of erythroid differentiation. Bars represent mean ± SEM. * p = 0.01 comparing editing frequencies of HBA -edited cells at day 14 vs. HBA + tEPOR -edited cells at day 14 of differentiation by unpaired two-tailed t test; *** p = 0.0004 comparing editing frequencies of HBA + tEPOR -edited cells at day 0 vs. day 14 of differentiation by unpaired two-tailed t test. (D) The percentage of CD34 − /CD45 − αTM HSPCs acquiring GPA and CD71 as determined by flow cytometry. Bars represent mean ± SEM. (E) The percentage of edited alleles in αTM HSPCs over the course of erythroid differentiation. Bars represent mean ± SEM. * p = 0.02 comparing editing frequencies of HBA + tEPOR -edited cells at day 0 vs. day 14 of differentiation by unpaired two-tailed t test; ** p = 0.009 comparing editing frequencies of HBA -edited cells at day 14 vs. HBA + tEPOR -edited cells at day 14 of differentiation by unpaired two-tailed t test. (F) Cell count at day 14 of erythroid cell differentiation with fold change normalized to HBA . Bars represent mean ± SEM. ** p = 0.007 by unpaired two-tailed t test. (G–I) Hemoglobin tetramer HPLC plots from two different donors (G and H) and a technical replicate from donor 2 (I) that was edited independently. For comparison purposes, the HPLC plots for HBA -edited cells, originally shown in – , are also presented here. (J) Western blot of mock and edited αTM HSPCs at the end of erythroid cell differentiation compared to WT umbilical cord blood-derived erythroid cells. The western blot image was taken from a single gel that was cropped to place the WT control next to edited conditions, as indicated by the black line. Loading was standardized by using the same number of cells for input. (K) Ratio of α-globin to dimeric β-globin quantification from western blot. Bars represent mean ± SEM.

Article Snippet: Human CD34 + -enriched HSPCs derived from Plerixafor and/or G-CSF-mobilized peripheral blood from healthy donors , STEMCELL Technologies, AllCells, Fred Hutchinson Cancer Center Hematology Core , 70073.2.

Techniques: Flow Cytometry, Two Tailed Test, Cell Counting, Cell Differentiation, Comparison, Western Blot, Derivative Assay, Control

Journal: Cell reports

Article Title: Dual α-globin-truncated erythropoietin receptor knockin restores hemoglobin production in α-thalassemia-derived erythroid cells

doi: 10.1016/j.celrep.2024.115141

Figure Lengend Snippet:

Article Snippet: Human CD34 + -enriched HSPCs derived from Plerixafor and/or G-CSF-mobilized peripheral blood from healthy donors , STEMCELL Technologies, AllCells, Fred Hutchinson Cancer Center Hematology Core , 70073.2.

Techniques: Virus, Clinical Proteomics, Recombinant, Saline, Sequencing, Derivative Assay, Clone Assay, Purification, Software, Western Blot, Control, Flow Cytometry, Selection, Electroporation, Transfection, DNA Extraction