diamond cd34 magnetic bead isolation kit  (Miltenyi Biotec)

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Impact of aging on CD146 + mesenchymal stromal cells-mediated regulation of bone marrow CD34 + hematopoietic stem/progenitor cell mobilization

doi: 10.3389/fbioe.2026.1802093

Figure Lengend Snippet: In vitro testing of the impact of age on human BMSC-mediated CD34 + HSPCs migration. (A) Schematic representation of the experimental setup, including (i) a timeline of the principal experimental steps, (ii) a graphic description of the different experimental conditions and (iii) a representative image showing migrated CD34 + HSPCs (red) in the different experimental conditions. (B) Age-dependent effects of mobilizing agents on CD146 + BMSC-mediated CD34 + HSPC transmigration; each graph represents an individual donor with its technical replicates, express as percentage of migrated cells relative to the internal reference (without CD146 + BMSCs). N = 3 subjects per group, with 3 technical replicates per subject. Statistical analysis was performed using the Kruskal–Wallis test. * p < 0.05; ** p < 0.01. Negative control, NC; hematopoietic stem progenitor cells, HSPCs; bone marrow stromal cells, BMSCs; untreated control, CTRL; specific CXCR4 inhibitor, AMD3100; granulocyte colony-stimulating factor, G-CSF; norepinephrine, NE. (Panel A (ii) and (iii) created in https://BioRender.com ).

Article Snippet: BM CD34 + HSPCs were isolated from one subject (female, 64 years old) using the Diamond CD34 + Magnetic Bead Isolation Kit (Miltenyi Biotec, cat # 130-094–531) according to the manufacturer’s instructions.

Techniques: In Vitro, Migration, Transmigration Assay, Negative Control, Control

Journal: Nature Genetics

Article Title: Stable clonal contribution of lineage-restricted stem cells to human hematopoiesis

doi: 10.1038/s41588-025-02405-w

Figure Lengend Snippet: a . Flow cytometry gating strategy for isolation of distinct HSPC populations in BM from HDs. The following populations were sorted: hematopoietic stem cells (HSCs, Lineage − CD34 + CD38 − CD90 + CD45RA − ), erythroid progenitors (EPs, Lineage − CD34 + CD38 + CD41a − CD123 − CD45RA − ), megakaryocyte progenitors (MkPs, Lineage − CD34 + CD38 + CD41a + ) and ProB-cells (Lineage − CD34 + CD19 + ). Representative profiles from one HD. Percentages indicate the mean frequency ± standard error of the mean (SEM) for all donors used for clonal tracking of lineage contributions, of the gated stem/progenitor cells relative to total CD34 + cells (n = 56 for HSCs, EPs, and MkPs and n = 21 for ProB-cells). b . Flow cytometry gating strategy for isolation of mature blood cell lineages from BM MNCs from healthy donors. The following populations were sorted: myeloid-cells (CD14 + CD33 + CD3 − CD19 − CD56 − ), B-cells (CD19 + CD3 − CD33 − CD56 − ), T-cells (CD3 + CD8a + CD4 − CD19 − CD33 − CD56 − and CD3 + CD4 + CD8a − CD19 − CD33 − CD56 − ).

Article Snippet: BM CD34 + or CD34 + CD19 − cells were obtained by CD34 magnetic bead enrichment according to the manufacturer’s instructions (130-046-702, Miltenyi Biotec) or flow cytometry, respectively.

Techniques: Flow Cytometry, Isolation

Journal: Nature Genetics

Article Title: Stable clonal contribution of lineage-restricted stem cells to human hematopoiesis

doi: 10.1038/s41588-025-02405-w

Figure Lengend Snippet: MCFs (dots) in different blood cell lineages and their 95% credible intervals estimated by Bayes using the number of ddPCR events and number of cells analyzed (error bars) of clones defined by specified CH-driver mutations not confidently detected in Lineage − CD34 + CD38 − CD90 + CD45RA − HSCs (n = 4) are shown. Clones observed specifically only in T cells are indicated by solid lines (n = 3).

Article Snippet: BM CD34 + or CD34 + CD19 − cells were obtained by CD34 magnetic bead enrichment according to the manufacturer’s instructions (130-046-702, Miltenyi Biotec) or flow cytometry, respectively.

Techniques: Clone Assay

Journal: Nature Genetics

Article Title: Stable clonal contribution of lineage-restricted stem cells to human hematopoiesis

doi: 10.1038/s41588-025-02405-w

Figure Lengend Snippet: a , Mutations with BM MNC MCF ≥ 2% for CH driver mutations and ≥1% for other CH-US mutations were used for clonal lineage tracing. Panel shows MCF of the CH driver (left, n = 51) and CH-US (right, n = 10) clonal somatic mutations in Lineage − CD34 + CD38 − CD90 + CD45RA − HSCs compared to BM MNCs. Mutations not confidently detected in HSCs ( n = 4) are shown with open symbols. R 2 values adjusted for the number of predictors for a linear model fit and P values for Pearson correlation analysis (two-sided test) adjusted with Benjamini–Hochberg method correction for multiple comparisons are shown. b , Phylogenetic trees mapping 17 clonal mutations targeted to HSCs in ten HDs (HDs 01, 02, 03, 05, 07, 08, 09, 13, 24 and 65) constructed from WGS results of in vitro expanded clones from single HSPCs without (gray) and with (colored) CH mutations identified through ECTS. Branches that share the same CH mutation are highlighted in bold. The position and length of each branch represent the estimated time period when the mutation occurred. The y axis in the trees is scaled based on donor age, starting (top of the phylogenetic tree) at the time of conception (40 weeks before birth) and ending (bottom of the tree) at the time of collection of the investigated BM sample. The 95% confidence intervals of the age (years) of branches are shown in parentheses next to the CH mutation names. WGS analysis confirmed that the clone marked by the synonymous CH-US mutation TET2 p.P1460P in HD03 was not accompanied by other known CH driver mutations.

Article Snippet: BM CD34 + or CD34 + CD19 − cells were obtained by CD34 magnetic bead enrichment according to the manufacturer’s instructions (130-046-702, Miltenyi Biotec) or flow cytometry, respectively.

Techniques: Construct, In Vitro, Clone Assay, Mutagenesis

Journal: Nature Genetics

Article Title: Stable clonal contribution of lineage-restricted stem cells to human hematopoiesis

doi: 10.1038/s41588-025-02405-w

Figure Lengend Snippet: a . Number of cells subjected to mutational ddPCR analysis for all 33 cases at the first visit analyzed: Lineage − CD34 + CD38 − CD90 + CD45RA − HSCs, Lineage − CD34 + CD38 + CD41 + MkP for the platelet (P) lineage, Lineage − CD34 + CD38 + CD41 − CD123 − CD45RA − EP for the erythroid (E) lineage, CD33 + CD14 + myeloid-cells (M), CD19 + B-cells (B) and CD4 or CD8 single positive T-cells (T). The center lines and boxes denote median values and the first and third quartiles, respectively. Whiskers indicate maximum and minimum values within 1.5× interquartile ranges. b . HSC clones with unique lineage replenishment patterns are shown in the same manner as Fig. . Each pattern was observed only for a single HSC clone.

Article Snippet: BM CD34 + or CD34 + CD19 − cells were obtained by CD34 magnetic bead enrichment according to the manufacturer’s instructions (130-046-702, Miltenyi Biotec) or flow cytometry, respectively.

Techniques: Clone Assay

Journal: Nature Genetics

Article Title: Stable clonal contribution of lineage-restricted stem cells to human hematopoiesis

doi: 10.1038/s41588-025-02405-w

Figure Lengend Snippet: a , Presence or absence of mutations originating in HSCs across platelet, erythroid, myeloid, B and T cell lineages, presented as those involving all lineages (Multi), all lineages except T cells and all lineages except B and T cells. HSCs (Lineage − CD34 + CD38 − CD90 + CD45RA − ) and MkPs (Lineage − CD34 + CD38 + CD41 + ) for the platelet lineage; EPs (Lineage − CD34 + CD38 + CD41 − CD123 − CD45RA − ) for the erythroid lineage; myeloid cells (CD33 + CD14 + ), B cells (CD19 + ) and T cells (CD4 or CD8 single positive). Positivity was judged using the overlapped area of posterior distributions by Bayesian inference using the number of sorted cells and the number of events by droplet digital polymerase chain reaction (ddPCR) between the targeted sample and the normal control (see for details). No other lineage replenishment patterns than those shown were reproducibly observed. b , Multipotent balanced (PEMBT), lineage-biased (mapped by CH driver mutations, solid lines with red or orange; other CH-US mutations, dashed blue colors) and lineage-restricted (driver, solid purple; CH-US, dashed green) contribution patterns observed from single-HSC-derived clones based on percent contribution (percent mean MCF ± 95% credible interval estimated with the Bayes method using the number of ddPCR events and the number of cells analyzed; ) to the PEMBT cell lineages. Lineage bias patterns were categorized by comparing the ratio of MCF in each lineage (see for definition of bias patterns). Only HSC lineage bias patterns observed for more than one HSC clone are shown. Four HSC clones with unique patterns (each observed only once) are shown in Extended Data Fig. . c , HSC MCF (%) for somatic CH driver and CH-US mutations associated with PEMBT ( n = 15), PEMB ( n = 25) and PEM ( n = 13) lineage replenishment patterns. Lineage-biased and -restricted PEMB and PEM clones were pooled. Center lines and boxes denote median values and the first and third quartiles, respectively. Whiskers indicate maximum and minimum values within 1.5× interquartile range. Mutated driver genes are indicated with different colors. Statistical tests were performed using two-sided Wilcoxon rank-sum test, and P values were adjusted with the Benjamini–Hochberg method. d , MCF ratio for all mutations shown in c for the indicated lineages compared to HSCs for PEMBT ( n = 15), PEMB ( n = 25) and PEM ( n = 13) clones. Lineage-biased and -restricted PEMB and PEM clones were pooled. Individual mutations are indicated with a colored bubble; bubble size represents the MCF in HSCs, and the color indicates the targeted gene, as indicated in the legends. Center lines and boxes denote median values and the first and third quartiles, respectively. Whiskers indicate maximum and minimum values within 1.5× interquartile range, and outliers beyond these are plotted. Statistical tests were performed using two-sided Wilcoxon rank-sum test, and P values were adjusted with the Benjamini–Hochberg method separately for each lineage. e , Distribution of CH driver and CH-US mutations in PEM, PEMB and PEMBT HSC clones. Lineage-biased and -restricted PEMB and PEM clones were pooled. The distribution of mutated genes in each lineage pattern was compared against that of the overall distribution of mutations for the frequency of (1) DNMT3A , (2) TET2 and (3) other mutations (combined) by two-sided Fisher’s exact test. Mutational distribution within PEM and PEMB patterns was also compared. P values were adjusted with the Benjamini–Hochberg method.

Article Snippet: BM CD34 + or CD34 + CD19 − cells were obtained by CD34 magnetic bead enrichment according to the manufacturer’s instructions (130-046-702, Miltenyi Biotec) or flow cytometry, respectively.

Techniques: Digital PCR, Control, Derivative Assay, Clone Assay

Journal: Nature Genetics

Article Title: Stable clonal contribution of lineage-restricted stem cells to human hematopoiesis

doi: 10.1038/s41588-025-02405-w

Figure Lengend Snippet: a , MCF in BM Lineage − CD34 + CD19 + pro-B cells of individual HSC-derived clones without ( n = 4) and with ( n = 15) mature B cell contribution. Center lines and boxes denote median values and the first and third quartiles, respectively. Whiskers indicate 1.5× interquartile ranges, and outliers beyond these bounds are plotted. P value was evaluated with the two-sided Wilcoxon rank-sum test. b , Correlation between contribution to mature B cells and BM Lineage − CD34 + CD19 + pro-B cells of individual HSC-derived clones without ( n = 4) and with ( n = 15) mature B cell contribution. The green line and gray shading represent the regression line and its 95% confidence interval, respectively. R 2 value (adjusted for the number of predictors for the linear model fit) and P value for Pearson correlation analysis (two-sided test) are provided. c – e , Contribution (percent MCF) of individual HSC-derived clones to myeloid and B cell lineages in the BM of HDs 07, 13 and 10 (gray triangles) and corresponding data from the BM of NSG mice (each circle represents a separate mouse) 10–14 weeks after transplantation of the corresponding donor CD34 + (HD07 and HD13) or CD34 + CD19 − (HD10) cells. Only mice in which the tracked clone (mapped by the indicated mutation) was confidently detected in myeloid cells are shown. Multiple B cell samples (2,000 cells per sample, n = 2–3) were isolated from each NSG BM sample and used as technical replicates for each mouse, and the average values are plotted. y.o., years old. Horizontal bar indicates the mean for all mice. P value provided at the bottom was assessed with a binomial mixed-effect model (two sided) to evaluate the association of B cell and myeloid cell positivity before and after transplantation (see for details).

Article Snippet: BM CD34 + or CD34 + CD19 − cells were obtained by CD34 magnetic bead enrichment according to the manufacturer’s instructions (130-046-702, Miltenyi Biotec) or flow cytometry, respectively.

Techniques: Derivative Assay, Clone Assay, Transplantation Assay, Mutagenesis, Isolation

Journal: Nature Genetics

Article Title: Stable clonal contribution of lineage-restricted stem cells to human hematopoiesis

doi: 10.1038/s41588-025-02405-w

Figure Lengend Snippet: a . Flow cytometry gating strategy for identification and isolation of mature human myeloid (human (h)CD45 + hCD33 + hCD19 − hCD235 − mouse (m)Ter119 − mCD41 − mCD45 − ) and human B (hCD45 + hCD19 + hCD33 − hCD235 − mTer119 − mCD41 − mCD45 − ) cells in the BM of NSG mice 10–14 weeks after transplantation of human CD34 + /CD34 + CD19 − HSPCs. b . Percentage of human (h)CD45 reconstitution of total (human+mouse) CD45 + cells (upper panel) and distribution of human myeloid and B-cells among human CD45 + cells (lower panel) in BM of NSG mice 10–14 weeks after transplantation of human CD34 + /CD34 + CD19 − hematopoietic stem/progenitor cells from HDs 7, 13 and 10. Each bar represents data from one mouse.

Article Snippet: BM CD34 + or CD34 + CD19 − cells were obtained by CD34 magnetic bead enrichment according to the manufacturer’s instructions (130-046-702, Miltenyi Biotec) or flow cytometry, respectively.

Techniques: Flow Cytometry, Isolation, Transplantation Assay