cd34 micro bead kit  (Miltenyi Biotec)

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Harnessing DNA barcoding to enhance and sustain polyclonality in gene-edited hematopoietic stem cells

doi: 10.1016/j.omtm.2025.101615

Figure Lengend Snippet: Establishment and analysis of barcode library composition, diversity, and clonal distribution in in vitro culture and in vivo in post-transplant mice populations (A) Schematic representation of barcode library in the therapeutic donor: Top: AAV coRPK scheme. Middle: Zoom of variable and non-variable sequences. Bottom: Barcode library composition indicating N: T, A, C, G, and S: G and C, showing all possible combinations. (B) Top: scheme of the editing protocol for HSPCs. After purifying HSPCs, cells were pre-stimulated for 48 h. Following this pre-stimulation period, cells underwent electroporation and transduction with the appropriate virus. The transduction process lasted for 48 h. Subsequently, cells were washed and prepared for further analysis. Bottom: Media use for in vitro culture of human hematopoietic stem cells. Abbreviations: SFEM II StemSpan (P/S, penicillin/streptomycin; SCF, Stem Cell Factor; TPO, thrombopoietin; Flt3, FMS-like tyrosine kinase 3 ligand; IL-6, interleukin 6; IL-3, interleukin 3.) (C) Number and lineage distribution of colony forming units (CFUs) at different BC-AAV MOI. Smooth red indicates erythroid colonies (BFU-E), smooth blue indicates granulo-macrophage colonies (CFU-GM), mixed red and blue stripes denote myelo-erythroid mixed colonies (CFU-GEMM). (D) Percentage of gene integration in CFUs at different BC-AAV MOI, analyzed by in-out PCR. (E) Unique barcode sequences within the total population at different MOIs. (F) Top 10 more represented clones in relation to the entire population at day 14 of culture. Each bar in the graph represents a CB with an MOI. Within each bar, the groups represent the abundance of specific clones in relation to the total population. Each color represents a distinct clone within the bar. (G) Total number of clones within CD45 + human population in transplanted mice relative to 50,000 reads. (H) Shannon diversity index of human CD45 + cells at MOI 2000. (I) Abundance of the top 10 clones per mouse. (J) Dominance of the most prevalence clone in the BM of each mouse. (K) Representation of the top 10 major clones in relation to the entire population. Each bar in the graph represents an individual mouse. Within each bar, the groups represent the abundance of specific clones in relation to the total population, with each color representing a distinct clone within the bar. The boxplots show the median, range (from minimum to maximum values), and quartiles of the data distribution. ∗: p < 0.05; ∗∗: p < 0.01; ∗∗∗: p < 0.001.

Article Snippet: Purified CD34 + cells were obtained by immunoselection with the CD34 Micro-Bead kit (MACS; Miltenyi Biotech).

Techniques: In Vitro, In Vivo, Electroporation, Transduction, Virus, Clone Assay

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Harnessing DNA barcoding to enhance and sustain polyclonality in gene-edited hematopoietic stem cells

doi: 10.1016/j.omtm.2025.101615

Figure Lengend Snippet: Evaluation of engraftment, targeted alleles, and clonal distribution across different culture conditions (A) Summary table of the different tested conditions. The first column defines the nomenclature of the conditions, ranging from A to D. The second column indicates the cell concentration during prestimulation. The third column specifies whether SR1 was added or not. The fourth column shows the cell concentration during AAV transduction. Finally, the last column details the culture duration post-electroporation and AAV addition. (B) CFU number and distribution for cells transduced with the BC-AAV donor before transplantation. Smooth red indicates BFU-E, smooth blue indicates CFU-GM, and mixed red and blue stripes denote GEMM-CFU. Mean and SD are shown. (C) Percentage of targeted alleles in liquid culture (5 days post-transduction) assessed by ddPCR. (D) Percentage of human engraftment (hCD45 + ) in BM, spleen, and PB. (E) Percentage of targeted alleles in total BM and across different lineages measured by ddPCR at 60 days post-transplantation. CD45: hCD45 + ; CD34: hCD45 + hCD34 + ; CD33: hCD45 + hCD33 + ; CD19: hCD45 + hCD19 + . (F) Total number of clones within CD45 + populations, normalized to 50,000 reads. (G) Shannon diversity index per mouse. (H) Abundance of the top 10 clones per mouse. (I) Dominance of the most prevalent clone in the BM of each mouse. (J) Total number of clones within CD34 + populations, normalized to 50,000 reads. (K) Representation of the top 10 major clones relative to the entire population. Each bar represents an individual mouse, with groups within bars indicating the abundance of specific clones as a proportion of the total population. Colors across conditions are not directly comparable as analyses were conducted on a per-condition basis. Boxplots indicate median, range, and quartiles. A: 2.5 × 10 5 24 hpe +SR1; B: 2.5 × 10 5 /1 × 10 6 48 hpe +SR1; C: 2.5 × 10 5 /1 × 10 6 24 hpe -SR1; D: 2.5 × 10 5 /1 × 10 6 24 hpe +SR1. ∗: p < 0.05; ∗∗: p < 0.01; ∗∗∗: p < 0.001.

Article Snippet: Purified CD34 + cells were obtained by immunoselection with the CD34 Micro-Bead kit (MACS; Miltenyi Biotech).

Techniques: Concentration Assay, Transduction, Electroporation, Transplantation Assay, Clone Assay

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Harnessing DNA barcoding to enhance and sustain polyclonality in gene-edited hematopoietic stem cells

doi: 10.1016/j.omtm.2025.101615

Figure Lengend Snippet: Evaluation of engraftment, targeted alleles, and clonal distribution across different enhancers integration (A) Percentage of targeted alleles in liquid culture (5 days post-transduction) assessed by ddPCR in samples treated with different enhancers in comparison to those maintained under standard conditions across cord blood-CD34 + . (B) Fold increase in targeted alleles from A. (C) Percentage of human engraftment (hCD45 + ) in BM, spleen, and PB. (D) Percentage of targeted alleles in total BM and across different lineages measured by ddPCR at 60 days post-transplantation. CD45: hCD45 + ; CD34: hCD45 + hCD34 + ; CD33: hCD45 + hCD33 + ; CD19: hCD45 + hCD19 + . (E) Total number of clones within CD45 + populations, normalized to 50,000 reads. (F) Shannon diversity index per mouse. (G) Abundance of the top 10 clones per mouse. (H) Dominance of the most prevalent clone in the BM of each mouse. (I) Total number of clones within CD34 + populations, normalized to 50,000 reads. (J) Boxplot showing the percentage of shared clones between CD45 and CD34 populations within the same mouse. Boxplots indicate median, range, and quartiles. (K) Circular diagram representing the abundance of the top 10 major clones in different mice without AZD (left) and with the addition of AZD (right). The upper half corresponds to data obtained from the CD45 population, while the lower half corresponds to data obtained from the CD34 + population. Lines indicate the number of matches between the top 10 major clones from one mouse to another. (L) Heatmap showing the percentage of common clones within CD45 (left) and CD34 (right) populations across different mice. Each column and row in the heatmap represents an individual mouse, with a total of 5 for the standard-treated samples (top) and 6 for the AZD-treated samples (bottom). The color intensity reflects the percentage of shared clones, ranging from low (blue) to high (yellow), as indicated by the color scale bar. Conditions legend: D: 2.5 × 10 5 /1 × 10 6 24 hpe +SR1; E: 2.5 × 10 5 /1 × 10 6 24 hpe +SR1 +AZD; F: 2.5 × 10 5 /1 × 10 6 24 hpe +SR1 +AZD +TSA. ∗: p < 0.05; ∗∗: p < 0.01; ∗∗∗: p < 0.001.

Article Snippet: Purified CD34 + cells were obtained by immunoselection with the CD34 Micro-Bead kit (MACS; Miltenyi Biotech).

Techniques: Transduction, Comparison, Transplantation Assay, Clone Assay