Journal: eBioMedicine

Article Title: Effectorless Fc-fusion improves FLT3L drug-like properties for cancer immunotherapy combinations

doi: 10.1016/j.ebiom.2025.105822

Figure Lengend Snippet: In vitro characterisation of FLT3L-Fc NG2LH activity . Representative sensorgrams of FLT3L-Fc NG2LH binding to recombinant human monomeric FLT3-monoFc (a) and dimeric FLT3-Fc (b). Black solid lines are curves fit to the data using a 1:1 binding model, and coloured lines are raw data. The tested recombinant human FLT3-monoFc concentrations (from bottom to top) are 62.5, 125, 250, 500, 1000, and 2000 nM; recombinant human dimeric FLT3-Fc concentrations (from bottom to top) are 3.75, 7.5, 15, 30, 60, and 120 nM. The sensorgrams were generated after in-line reference cell correction followed by buffer sample subtraction. The experiments were conducted at 37 °C. (c) Binding affinities of recombinant human monomeric FLT3-monoFc and dimeric FLT3-Fc proteins to captured FLT3L-Fc NG2LH. k a association rate constant; k d dissociation rate constant; K D equilibrium dissociation constant. Data for FLT3L-Fc NG2LH binding to recombinant human FLT3-monoFc were averaged from three independent experimental Biacore runs. Data for FLT3L-Fc NG2LH binding to recombinant human dimeric FLT3-Fc were averaged from two independent experimental Biacore runs. (d) ADCP for FLT3L-Fc NG2LH and its variants. Data shown are a representative set taken from one of three independent experiments. Average (SD) of duplicate values are shown. (e) Proliferation of OCI-AML5 cells in vitro. A representative dose response curve is shown, Average (SD) of triplicate values. Data were normalised to 10 μg/mL FLT3L-Fc NG2LH as the maximum (100%) response and the assay media alone control as the minimum (0%) response. (f) Human cDC1 differentiation from cord blood stem cells. Frequency of cDC1 at the end of culture is shown (percent of live cells). Results were obtained from 2 donors. (g) In vitro proliferation of mouse bone marrow cells in response to mFLT3L or mFLT3L-Fc. Average (SD) of duplicate values are shown.

Article Snippet: CD34+ human cord blood stem cells (StemCell Technologies) were seeded at 5000 cells per well in 96-well U-bottom plates (Costar) in 100 μL expansion media (StemSpan media–StemCell Technologies, 10% FBS, 40 ng/mL IL-3, 200 ng/mL SCF, 100 ng/mL TPO–cytokines from Peprotech).

Techniques: In Vitro, Activity Assay, Binding Assay, Recombinant, Generated, Control

Journal: STAR Protocols

Article Title: Protocol for optimizing culture conditions for ex vivo activation during CRISPR-Cas9 gene editing in human hematopoietic stem and progenitor cells

doi: 10.1016/j.xpro.2025.103722

Figure Lengend Snippet: Representative images of different types of CD34 + derived colonies

Article Snippet: Human cord blood CD34+ stem/progenitor cells , Lonza , Cat#2C-101.

Techniques: Derivative Assay

Journal: STAR Protocols

Article Title: Protocol for optimizing culture conditions for ex vivo activation during CRISPR-Cas9 gene editing in human hematopoietic stem and progenitor cells

doi: 10.1016/j.xpro.2025.103722

Figure Lengend Snippet: Gating strategy for flow cytometry analysis Representative gating strategy relative to cell viability, GFP expression, and lineage composition analyses in Blood and SP (A) or BM (B). (A) Human CD45+ cells were detected gating on live cells (7AAD-). Within this gate, GFP+ cells and My, B, and T cells were distinguished: My cells were identified as CD45+/CD13+; B cells as CD45+/CD19+; T cells as CD45+/CD19-/CD13-/CD3+ and CD19-/CD13-/CD3+/CD8+. CD19-/CD13-/CD3-/CD8- cells were defined as “Other”. (B) Human CD45+ cells were detected gating on live cells (7AAD-). Within this gate, GFP+ cells and My, B, T cells, and HSPCs were distinguished: My cells were identified as CD45+/CD13+; B cells as CD45+/CD19+; T cells as CD45+/CD19-/CD13-/CD3+; and HSPCs as CD45+/CD19-/CD13-/CD3-/CD34+. CD45+/CD19-/CD13-/CD3-/CD34- cells were defined as “Other”.

Article Snippet: Human cord blood CD34+ stem/progenitor cells , Lonza , Cat#2C-101.

Techniques: Flow Cytometry, Expressing

Journal: STAR Protocols

Article Title: Protocol for optimizing culture conditions for ex vivo activation during CRISPR-Cas9 gene editing in human hematopoietic stem and progenitor cells

doi: 10.1016/j.xpro.2025.103722

Figure Lengend Snippet: Representative gating strategy relative to cell viability, subpopulation composition, and GFP expression analyses of GE-HSPCs Alive cells: 7AAD − /Annexin V − ; early apoptotic cells: 7AAD − /Annexin V + ; late apoptotic cells: 7AAD + /Annexin V + ; necrotic cells: 7AAD + /Annexin V − . CD34 + cells were identified within alive cells, and within this gate, GFP + cells and HSPC subpopulations were distinguished as CD34 + CD133 − CD90 − , CD34 + CD133 + CD90 − , or CD34 + CD133 + CD90 + . The percentage of GFP + cells was then detected within each population.

Article Snippet: Human cord blood CD34+ stem/progenitor cells , Lonza , Cat#2C-101.

Techniques: Expressing

Journal: STAR Protocols

Article Title: Protocol for optimizing culture conditions for ex vivo activation during CRISPR-Cas9 gene editing in human hematopoietic stem and progenitor cells

doi: 10.1016/j.xpro.2025.103722

Figure Lengend Snippet:

Article Snippet: Human cord blood CD34+ stem/progenitor cells , Lonza , Cat#2C-101.

Techniques: Blocking Assay, Recombinant, CRISPR, Staining, Lysis, Western Blot, Saline, Bicinchoninic Acid Protein Assay, Software, Single Cell Gel Electrophoresis, Cell Culture, Bioassay, Membrane

Journal: STAR Protocols

Article Title: Protocol for optimizing culture conditions for ex vivo activation during CRISPR-Cas9 gene editing in human hematopoietic stem and progenitor cells

doi: 10.1016/j.xpro.2025.103722

Figure Lengend Snippet: BM human chimerism and lineage composition

Article Snippet: Human cord blood CD34+ stem/progenitor cells , Lonza , Cat#2C-101.

Techniques:

Journal: STAR Protocols

Article Title: Protocol for optimizing culture conditions for ex vivo activation during CRISPR-Cas9 gene editing in human hematopoietic stem and progenitor cells

doi: 10.1016/j.xpro.2025.103722

Figure Lengend Snippet: Cellular and Molecular assays

Article Snippet: Human cord blood CD34+ stem/progenitor cells , Lonza , Cat#2C-101.

Techniques:

Journal: Cell Reports Medicine

Article Title: A p38 MAPK-ROS axis fuels proliferation stress and DNA damage during CRISPR-Cas9 gene editing in hematopoietic stem and progenitor cells

doi: 10.1016/j.xcrm.2024.101823

Figure Lengend Snippet: Ex vivo activation is a prerequisite for efficient HDR-based correction, although it heightens DDR activation and affects GE HSPC repopulation capacity (A) Experimental workflow for short and standard protocols in human CB-derived HSPCs. Indicated treatments were performed at day 1 (short) or day 3 (standard) post-thawing, while in vitro analyses were conducted at 24 or 96 h post-treatments (corresponding to day 2 or 4, and day 5 or 7, respectively). (B) 53BP1 foci distribution (24 h: n = 6,8,5,8,6,8; 96 h: n = 6, 6, 6, 4, 6, 7). Mann-Whitney test. (C) Relative expression of CDKN1A (24 h: n = 5, 6, 7, 7, 6, 7; 96 h: n = 6, 5, 6, 6, 6, 5). For each time point, fold change was calculated relatively to HSPCs treated with an RNP loaded with a gRNA with no specificity against the human genome. Mann-Whitney test. (D) Percentage of wild-type and HDR- or NHEJ-edited alleles measured 96 h post-editing (HS: n = 5; HS+AAV6: n = 2, 2, 6, 4, 4, 6). Wilcoxon test. (E) Percentage of GFP + cells within HSPC subpopulations (CD34 + CD133 - , CD34 + CD133 + , and CD34 + CD133 + CD90 + cells) at 96 h post-treatments ( n = 5). Kruskal-Wallis test. (F) Percentage of HSPCs in indicated cell-cycle phases measured at day 1 (short: n = 5) and day 3 (standard: n = 4). Mann-Whitney test. (G) Number of colonies generated by HSPCs plated at 96 h post-treatment (HS Cas9: n = 6; HS+AAV6: n = 6; LS Cas9: n = 4; IRR: n = 6). Wilcoxon test for intra-treatment comparisons or Mann-Whitney test for inter-treatment measurements. (H) Absolute number of CD3 + TCRα/β + cells in ATO seeded with HSPCs after the indicated treatment ( n = 6). Wilcoxon test. (I) Percentage of GFP + cells measured by flow cytometry in ATO seeded with HS+AAV6-edited HSPCs ( n = 6). Wilcoxon test. (J–L) Percentage of human CD45 + cells in the (J) PB, (K) BM, and (L) SP of mice transplanted with HSPCs edited as indicated ( n = 10, 8, 10, 9). Mann-Whitney test (calculated at the last time point for PB). (M) Percentage of B cells, T cells, myeloid, and other cells within the human graft (PB) ( n = 10, 8, 10, 9). Mann-Whitney test for short vs. standard comparisons. (N–P) Percentage of GFP + cells (within hCD45 + ) measured in the (N) PB, (O) BM, and (P) SP of mice transplanted with HS+AAV6 HSPCs ( n = 10, 9). Mann-Whitney test (calculated at the last time point for PB). Mean ± SEM and, unless otherwise specified, lines indicate median values. ns > 0.05; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001.

Article Snippet: Human cord blood CD34 + Stem/Progenitor cells , Lonza , Cat#2C-101.

Techniques: Ex Vivo, Activation Assay, Derivative Assay, In Vitro, MANN-WHITNEY, Expressing, Generated, Flow Cytometry

Journal: Cell Reports Medicine

Article Title: A p38 MAPK-ROS axis fuels proliferation stress and DNA damage during CRISPR-Cas9 gene editing in hematopoietic stem and progenitor cells

doi: 10.1016/j.xcrm.2024.101823

Figure Lengend Snippet: p38 MAPK activation mediates excessive proliferation through mitogenic ROS (A) Quantification of phospho-p38 by flow cytometry analysis in ex vivo cultured HSPCs ( n = 4, 6, 5). Mann-Whitney test. (B) Experimental workflow indicating in vitro treatments with DMSO (vehicle) or 4 μM p38i at day 1 and 2 post-thawing of human CB-derived HSPCs. In vitro analyses were conducted on days 1, 3, and 7 post-thawing. (C and D) Quantification of (C) cytosolic ROS detected by CM-H 2 DCFDA and of (D) mitochondrial superoxides measured by MitoSOX (C: n = 3, 5, 5, 4, 4; D: n = 3, 7, 7, 7, 4). Mann-Whitney test. (E and F) Quantification of (E) basal oxygen consumption rate (OCR) or (F) spare respiratory capacity (SRC) after 7 days of culture ( n = 4). Mann-Whitney test. (G and H) Quantification of (G) extracellular acidification rate (ECAR) or (H) glycolytic capacity (GC) after 7 days of culture ( n = 5). Wilcoxon test. (I) Representative plot of CellTrace dilution and division index of CD34 + cells measured at day 7 ( n = 6). Wilcoxon test. (J and K) Cellular confluence of (J) bulk CD34 + or (K) CD34 + CD133 + CD90 + CD45RA − HSPCs reported as fold change (FC) to the first time point ( n = 3). Mann-Whitney test. (L) Duration (hours) of cell-cycle phases measured in single HSPCs expressing the Fucci2a reporter. Live imaging started on day 2 after p38 inhibitor treatment; each dot represents individual cells. Mann-Whitney test. (M) Division index of HSPCs measured at day 7 ( n = 6). As a control, the DMSO condition from (I) is reported. Wilcoxon test. (N) Duration (hours) of cell-cycle phases measured in single HSPCs expressing the Fucci2a reporter. Live imaging started on day 2 after NAC treatment; each dot represents individual cells. As a control, the DMSO condition from (L) is reported. Mann-Whitney test. Mean ± SEM. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗∗ p < 0.0001.

Article Snippet: Human cord blood CD34 + Stem/Progenitor cells , Lonza , Cat#2C-101.

Techniques: Activation Assay, Flow Cytometry, Ex Vivo, Cell Culture, MANN-WHITNEY, In Vitro, Derivative Assay, Expressing, Imaging, Control

Journal: Cell Reports Medicine

Article Title: A p38 MAPK-ROS axis fuels proliferation stress and DNA damage during CRISPR-Cas9 gene editing in hematopoietic stem and progenitor cells

doi: 10.1016/j.xcrm.2024.101823

Figure Lengend Snippet: p38 MAPK/ROS triggers proliferation stress and heightens DNA damage (A) Quantification of 8-oxo-2′-deoxyguanosine by flow cytometry ( n = 5). Mann-Whitney test. (B) Quantification of SSBs and DSBs by comet assay; each point represents a single cell from 3 independent experiments (up to 250 cells were analyzed). Mann-Whitney test. Whiskers represent 10–90 percentile. (C) Quantification of SSBs and DSBs by comet assay at day 7 of culture; each point represents a single cell from 3 independent experiments (up to 250 cells were analyzed). Mann-Whitney test. Whiskers represent 10–90 percentile. (D and E) Percentage of (D) γH2AX- or (E) pRPA-positive HSPCs (γH2AX: n = 9, 7, 3, 7, 4; pRPA = n = 6, 6, 3, 6, 3). Mann-Whitney test. (F) Western blot analysis of pATR (left) and pCHK1 (right) at day 7 ( n = 4). Histone H3 was used as a loading control, and fold change relative to control is reported. One-sample t test. (G) Percentage of HSPC subpopulations (CD34 + CD133 - , CD34 + CD133 + , and CD34 + CD133 + CD90 + cells) ( n = 7). Wilcoxon test. Unless otherwise specified, mean ± SEM. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001.

Article Snippet: Human cord blood CD34 + Stem/Progenitor cells , Lonza , Cat#2C-101.

Techniques: Flow Cytometry, MANN-WHITNEY, Single Cell Gel Electrophoresis, Western Blot, Control

Journal: Cell Reports Medicine

Article Title: A p38 MAPK-ROS axis fuels proliferation stress and DNA damage during CRISPR-Cas9 gene editing in hematopoietic stem and progenitor cells

doi: 10.1016/j.xcrm.2024.101823

Figure Lengend Snippet: The functionality of gene-edited HSPCs is enhanced by p38 inhibitor treatment (A) Experimental workflow indicating in vitro treatments with vehicle (DMSO) or p38i at day 1 and 2 post-thawing of human CB- or mPB-derived HSPCs. Cells were GE at day 3 by nucleofection of HS Cas9 in the presence or not of AAV6. In vitro analyses were conducted on days 4 and 7 (24 and 96 h post-editing, respectively). (B) Percentage of HSPC subpopulations (CD34 + CD133 - , CD34 + CD133 + , and CD34 + CD133 + CD90 + cells) at 96 h post-nucleofection (HS Cas9: n = 7, 7, 7; HS+AAV6: n = 8, 8, 8). Wilcoxon test. (C) Percentage of HDR-corrected alleles at 96 h post-editing in CB-sorted CD34 + CD133 + CD90 + CD45RA − ( n = 3). Mann-Whitney test. (D and E) Quantification of (D) cytosolic ROS detected by CellROX and of (E) mitochondrial superoxides measured by MitoSOX in HS+AAV6 CB-derived HSPCs (D: n = 3, 4, 3, 4; E: n = 4, 4, 3, 3). Mann-Whitney test. (F) Quantification of SSBs and DSBs by comet assay in HS+AAV6 CB-derived HSPCs; each point represents a single cell from 2 independent experiments (up to 165 cells were analyzed). Mann-Whitney test. Whiskers represent 10–90 percentile. (G) Western blot analysis of pATR (left) and pCHK1 (right) at 96 h post-nucleofection in HS+AAV6-edited HSPCs (pATR: n = 5; pCHK1: n = 4). Histone H3 was used as a loading control, and fold change relative to control is reported. One-sample t test. (H) Percentage of micronuclei-positive HSPCs at 96 h post-editing (HS+AAV6: n = 6). Wilcoxon test. (I) Number of colonies generated by CB-derived HSPCs plated in methylcellulose 24 h post-treatments (HS Cas9: n = 7; HS+AAV6: n = 12). Wilcoxon test for intra-treatment comparisons or Mann-Whitney test for inter-treatment measurements were performed. (J) Number of erythroid, myeloid, and mixed colonies from (I). Wilcoxon test. (K) Number of colonies generated after secondary replating in methylcellulose ( n = 6). Mann-Whitney test. Mean ± SEM. ns > 0.05; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001.

Article Snippet: Human cord blood CD34 + Stem/Progenitor cells , Lonza , Cat#2C-101.

Techniques: In Vitro, Derivative Assay, MANN-WHITNEY, Single Cell Gel Electrophoresis, Western Blot, Control, Generated

Journal: Cell Reports Medicine

Article Title: A p38 MAPK-ROS axis fuels proliferation stress and DNA damage during CRISPR-Cas9 gene editing in hematopoietic stem and progenitor cells

doi: 10.1016/j.xcrm.2024.101823

Figure Lengend Snippet: Single-cell transcriptomic analysis reveals replication stress mitigation and stemness maintenance by p38 inhibition across conditions (A) Schematic representation of the single-cell RNA transcriptomic analysis experimental workflow: at day 1 and 2 post-thawing human CB-derived HSPCs were treated with vehicle (DMSO) or p38i. At day 3, cells were nucleofected with HS Cas9 in the presence or not of AAV6. A not edited control was included. At day 4 (24 h post-editing), HSPCs were sorted and collected for subsequent analyses. (B) Uniform manifold approximation and projection (UMAP) of annotated populations with cells belonging to all conditions. (C and D) Barplots showing the distribution of (C) all the cells retrieved and (D) CD34 + CD133 + CD90 + CD45RA − assigned to each subpopulation. (E) Enriched terms obtained performing GSEA of intra-condition comparisons of p38i vs. vehicle. Rows report terms enriched in at least one of the comparisons (columns) (false discovery rate < 0.1) from the gene ontology (GO), biological processes (BP), and the hallmark signatures (MSigDB). Colors indicate negative/positive normalized enrichment score (NES). (F) Percentage of cell-cycle distribution across all the cell subpopulations (left) or the HSC cluster (right). (G) Module score of DNA replication initiation and response to DNA damage/replication stress signatures as in Jacobs et al . Wilcoxon test (∗∗∗ p < 0.001). (H) GSEA analysis of intra-condition comparisons of p38i vs. vehicle of HSPC-related categories retrieved from the literature (see ).

Article Snippet: Human cord blood CD34 + Stem/Progenitor cells , Lonza , Cat#2C-101.

Techniques: Inhibition, Derivative Assay, Control

Journal: Cell Reports Medicine

Article Title: A p38 MAPK-ROS axis fuels proliferation stress and DNA damage during CRISPR-Cas9 gene editing in hematopoietic stem and progenitor cells

doi: 10.1016/j.xcrm.2024.101823

Figure Lengend Snippet: Repopulating capacity and clonal repertoire of GE HSPCs are ameliorated by p38 inhibitor administration (A) Experimental workflow showing the time of in vitro treatments, GE, and in vivo injection of human CB- or mPB-derived HSPCs into NSG mice, followed by blood and organ analyses. (B and C) Percentage of human CD45 + cells in the (B) PB and (C) BM of NSG mice transplanted with CB-derived HSPCs edited as indicated ( n = 12, 14, 16, 18). Mann-Whitney test (calculated at the last time point for PB). (D and E) Percentage of GFP + cells (within hCD45 + cells) in the (D) PB and (E) BM of mice from (B and C) ( n = 16, 18). Mann-Whitney test. (F) Number of colonies formed by BM-derived CD34 + cells purified from mice in (C) (HS Cas9: n = 8, 11; HS + AAV6: n = 16, 12). Mann-Whitney test. (G) Number of erythroid, myeloid, and mixed colonies from samples in (F). Mann-Whitney test. (H) Number of unique indels in human BM-derived cells (HS Cas9: n = 3, 5; HS+AAV6: n = 8, 10). Median values are reported. Mann-Whitney test. (I) Number of unique BARs in HS+AAV6 human BM-derived cells ( n = 19, 24). Median values are reported. Mann-Whitney test. (J) Schematic representation of the secondary transplantation: BM-derived CD34 + cells recovered from primary mice were injected into secondary recipients. Hematopoietic organs were analyzed at the endpoint (13 weeks). (K and L) Percentage of hCD45 + cells in the (K) PB and (L) BM of secondary-transplanted mice ( n = 9, 11). Mann-Whitney test. (M and N) Percentage of GFP + cells (within hCD45 + cells) in the (M) PB and (N) BM of mice in (K and L) ( n = 9, 11). Mann-Whitney tests. (O) Percentage of B cells, T cells, myeloid, and other cells within hCD45 + cells in the PB of mice in (K) ( n = 9, 11). Mann-Whitney test. Mean ± SEM and, unless otherwise specified, lines indicate median values. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001.

Article Snippet: Human cord blood CD34 + Stem/Progenitor cells , Lonza , Cat#2C-101.

Techniques: In Vitro, In Vivo, Injection, Derivative Assay, MANN-WHITNEY, Purification, Transplantation Assay

Journal: Cell Reports Medicine

Article Title: A p38 MAPK-ROS axis fuels proliferation stress and DNA damage during CRISPR-Cas9 gene editing in hematopoietic stem and progenitor cells

doi: 10.1016/j.xcrm.2024.101823

Figure Lengend Snippet:

Article Snippet: Human cord blood CD34 + Stem/Progenitor cells , Lonza , Cat#2C-101.

Techniques: Blocking Assay, Purification, Recombinant, CRISPR, Negative Control, Staining, Lysis, cDNA Synthesis, SYBR Green Assay, Software