Journal: Frontiers in Immunology

Article Title: Mice with humanized immune system as novel models to study HIV-associated pulmonary hypertension

doi: 10.3389/fimmu.2022.936164

Figure Lengend Snippet: (A) Human spleen and liver organoids in the mouse kidney capsule. NSG BLT mice were engrafted with fetal liver, thymus and hematopoietic cells; human engraftments were confirmed by gross inspections of the mouse kidney capsules at euthanasia, where organoids develop in humanized mice. (B) Experimental timeline. NSG mice were engrafted with either human hematopoietic CD34+ cells (cord-blood derived) or with human liver, thymus, and hematopoietic stem cells (fetal-derived). After confirmation of human engraftments by flow cytometry, humanized mice were infected with HIV and either placed in hypoxia or injected with SU5416 to induce PAH. Animals were monitored for any signs of disease including graft-vs-host disease, labored breathing or wasting throughout the course of the study. Mice were subjected to open-chest right heart catheterizations under anesthesia; specimens were collected for further analyses.

Article Snippet: Mice humanized with CD34 + stem cells were purchased from The Jackson Laboratory and showed a 31-48% level of human CD45 + cell engraftment.

Techniques: Capsules, Derivative Assay, Flow Cytometry, Infection, Injection

Journal: Cell Stem Cell

Article Title: Precise Gene Editing Preserves Hematopoietic Stem Cell Function following Transient p53-Mediated DNA Damage Response

doi: 10.1016/j.stem.2019.02.019

Figure Lengend Snippet: p53-Dependent Transcriptional Response Is Predominant upon AAVS1 and IL2RG Editing (A and B) Venn diagram of DEGs in ZFN AAVS1 or IL2RG versus −DSB in CD34 + CD133 + CD90 + (A) and CD34 + CD133 + CD90 − (B) sorted HSPCs. (C) Heatmap of regularized log-normalized read counts for genes showing differential expression with adjusted p values < 0.05 across the indicated conditions in HSPC subpopulations; genes (listed in B) are sorted according to hierarchical clustering, and colors represent the read count values scaled per row ( Z scores). (D) Heatmap showing NES from GSEA against the hallmark gene sets of the Molecular Signatures Database (MSigDB), starting from the list of genes ranked by Log2FC. Terms are sorted according to NES. (E and F) Volcano plot showing significant down- (green) and up- (red) regulated genes in primitive cells upon DSB at IL2RG (E) and AAVS1 (F) loci. DEGs in proximity of the targeted locus and top 10 p53 target genes ranked by false discovery rate (FDR) are indicated.

Article Snippet: CB-CD34 , Lonza , Cat# 2C-101.

Techniques: Expressing

Journal: Cell Stem Cell

Article Title: Precise Gene Editing Preserves Hematopoietic Stem Cell Function following Transient p53-Mediated DNA Damage Response

doi: 10.1016/j.stem.2019.02.019

Figure Lengend Snippet: Transient p53 Inhibition Increases the Clonogenic and In Vivo Repopulating Capacity of Edited HSPCs (A) Percentage of HDR and NHEJ 72 h post-editing with AAVS1 or IL2RG HS RNP in presence or not of GSE56 (n = 9); Wilcoxon matched-pairs signed rank test. (B) Percentage of GFP+ cells 72 h after AAVS1 or IL2RG gene editing in presence or not of GSE56 (n = 15); paired t test. (C) Colonies from sorted CD133 + CD90 + or CD133 + CD90 − edited cells (n = 11); Mann-Whitney test. (D–F) Percentage of human CD45 + cells in the peripheral blood (PB) of NSG mice transplanted with (D) HSPCs treated with control mRNA or GSE56 or IL2RG ZFN+AAV6 cells with or without GSE56 (n = 4, 3, 4, and 4), (E) IL2RG HS RNP+AAV6 cells with or without GSE56 (n = 6 and 5), and (F) HSPCs edited as in (E) at AAVS1 site (n = 5 and 5); nonparametric longitudinal data analysis. (G–I) Percentage of GFP+ cells measured in PB of mice transplanted with cells from (D) in (G), from (E) in (H), and from (F) in (I), respectively. (J) Percentage of human CD45 + GFP+ cells in hematopoietic organs of mice from (D)–(F) (HS/AAV6: n = 14; HS/AAV6+GSE56: n = 15); Mann-Whitney test. (K) Human PB engraftment after secondary transplant of CD34 + HSPCs sorted from BM of mice in (E) and (F) (n = 6 and 6). (L) Percentage of GFP+ cells measured within human graft of mice from (K). ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. Lines indicate median values.

Article Snippet: CB-CD34 , Lonza , Cat# 2C-101.

Techniques: Inhibition, In Vivo, MANN-WHITNEY

Journal: Cell Stem Cell

Article Title: Precise Gene Editing Preserves Hematopoietic Stem Cell Function following Transient p53-Mediated DNA Damage Response

doi: 10.1016/j.stem.2019.02.019

Figure Lengend Snippet:

Article Snippet: CB-CD34 , Lonza , Cat# 2C-101.

Techniques: Blocking Assay, Purification, Recombinant, CRISPR, Electroporation, Negative Control, Staining, SYBR Green Assay, Multiplex Assay, Flow Cytometry, Software, Plasmid Preparation, Real-time Polymerase Chain Reaction