Journal: Research and Practice in Thrombosis and Haemostasis

Article Title: PIK3R3 is a candidate regulator of platelet count in people of Bangladeshi ancestry

doi: 10.1016/j.rpth.2023.100175

Figure Lengend Snippet: Detailed view of the Bangladeshi rs946528 association interval. LocusZoom plot of associations in the 562 kB association interval for the chromosome 1 index variant rs946528, defined as variants within r 2 ≥ 0.5 calculated using linkage disequilibrium (LD) reference data from the Bengali from Bangladesh 1000 Genomes dataset. Variants with P > .05 are excluded to enable visualization of the recombination peaks. Indicated variants are the index rs946528 and the 3 variants associated with platelet count in prior East Asian ancestry–specific genome-wide association studies. Beneath and aligned with this are the 8 UCSC Genome Browser–annotated genes within the interval; positions of the 95% credible set of 343 variants and annotated regions of epigenetic activity from CD34 − , CD41 + , and CD42 + megakaryocytes, the progenitor cell for circulating platelets, which were provided by the BLUEPRINT Epigenomics Project . Megakaryocyte chromatin immunoprecipitation sequencing (ChIP-seq) data are also shown indicating relevant transcription factor binding sites . The expanded view shows the relationship between the significantly associated variant rs1707303 and a putative regulatory region surrounding the first exon of the candidate gene PIK3R3.

Article Snippet: Beneath and aligned with this are the 8 UCSC Genome Browser–annotated genes within the interval; positions of the 95% credible set of 343 variants and annotated regions of epigenetic activity from CD34 − , CD41 + , and CD42 + megakaryocytes, the progenitor cell for circulating platelets, which were provided by the BLUEPRINT Epigenomics Project [ ].

Techniques: Variant Assay, GWAS, Activity Assay, ChIP-sequencing, Binding Assay

Journal: PLoS Genetics

Article Title: Differential effects of RASA3 mutations on hematopoiesis are profoundly influenced by genetic background and molecular variant

doi: 10.1371/journal.pgen.1008857

Figure Lengend Snippet: (A) Expression of Rasa3 in flow sorted adult B6J HSPCs (X ± SEM, n = 3 pools of cells from 6 mice for all cell types). (B) E12.5-E13.5 Vav-Cre ; Rasa3 control and (C) mutant embryos. (D) E12.5-E13.5 Tek-Cre; Rasa3 control and mutant (E) embryos. Mutant embryos recapitulate the germline null phenotype with severe hemorrhage and decreased fetal liver erythropoiesis (arrows). HSC, hematopoietic stem cells; MPP, multipotent progenitors; CMP, common myeloid progenitor; MEP, Megakaryocyte-erythroid progenitor; BFU-E, burst forming unit-erythroid; CFU-E, colony forming unit-erythroid. *p < 0.05, **p < 0.01, ***p < 0.001 vs . BFU-E/CFU-E.

Article Snippet: Human CD34 + HSPCs were isolated from de-identified cord blood in accordance with Institutional Review Board approval (Northwell Health).

Techniques: Expressing, Control, Mutagenesis

Journal: PLoS Genetics

Article Title: Differential effects of RASA3 mutations on hematopoiesis are profoundly influenced by genetic background and molecular variant

doi: 10.1371/journal.pgen.1008857

Figure Lengend Snippet: (A) Total, CD45 - erythroid, and CD45 + non-erythroid cell counts are significantly depleted in Mx1-Cre; Rasa3 mutant bone marrow. n = 8 (B,C) Terminal erythroid differentiation progresses normally in mutant bone marrow compared to both non-anemic (n = 8) and phlebotomized anemic (n = 6) controls. (D) Total counts of all hematopoietic progenitor cell types are significantly decreased in Mx1-Cre; Rasa3 mutant bone marrow vs . control (n = 8). LT- and ST-HSCs do not significantly differ. (E) Decreased BFU-E and CFU-E colony formation capacity in mutant bone marrow. n = 3. Equal numbers of mutant and control were plated in colony forming assays. LT-HSC, long term hematopoietic stem cell; ST-HSC, short term hematopoietic stem cell; LMPP, lymphoid-primed multipotent progenitor; CLP, common lymphoid progenitor; CMP, common myeloid progenitor; GMP, granulocyte monocyte progenitor; MEP, Megakaryocyte-erythroid progenitor. All values X ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Human CD34 + HSPCs were isolated from de-identified cord blood in accordance with Institutional Review Board approval (Northwell Health).

Techniques: Mutagenesis, Control

Journal: PLoS Genetics

Article Title: Differential effects of RASA3 mutations on hematopoiesis are profoundly influenced by genetic background and molecular variant

doi: 10.1371/journal.pgen.1008857

Figure Lengend Snippet: (A) Schematic of the three-phase erythroid differentiation culture system utilized with cord blood-derived CD34+ cells. (B) qRT-PCR demonstrates RASA3 kinetics of expression during human erythropoiesis. (C) RASA3 expression levels in sh Luciferase control versus knockdown cultures. (D) Quantification of shRASA3-39 mediated knockdown of RASA3 (n = 4). (E) Representative flow cytograms of terminal erythroid differentiation of glycophorin A (GPA) positive cells, monitored by progressive loss of α4-integrin and acquisition of Band 3, in shRASA3 versus shLuciferase through Day 16. Boxes represent early (top) and late (bottom) cells quantified in F. (F) Quantification of erythroblast population percentages in sh Luciferase and shRASA3-39 at days 8, 11, and 16 in vitro (n = 3, 7, 3, respectively).

Article Snippet: Human CD34 + HSPCs were isolated from de-identified cord blood in accordance with Institutional Review Board approval (Northwell Health).

Techniques: Derivative Assay, Quantitative RT-PCR, Expressing, Luciferase, Control, Knockdown, In Vitro

Journal: Stem Cells Translational Medicine

Article Title: Enhanced Genetic Modification of Adult Growth Factor Mobilized Peripheral Blood Hematopoietic Stem and Progenitor Cells With Rapamycin

doi: 10.5966/sctm.2014-0010

Figure Lengend Snippet: Rapamycin dose effect on transduction efficiency, cell viability, and cell growth. (A): LV2 transduction of hematopoietic stem and progenitor cells (HSPCs) (percentage of GFP expression) over a range of rapamycin concentrations (n = 3). (B): Cell growth and viability of the same samples shown in (A). (C): Incremental in vitro HPSC proliferation following transduction with or without rapamycin (n = 7). (D): Phenotypic analysis of GFP expression in subpopulations of HSPCs 7 days after transduction in the presence or absence of rapamycin (n = 9 donors for CD34+ and CD34+/CD90+, n = 3 for CD90+/CD49f+). Height of bars represents the average value, and error bars represent SEM. ∗, p < .05; ∗∗, p < .01; ∗∗∗, p < .001; ∗∗∗∗, p < .0001. Abbreviations: D, days; GFP, green fluorescent protein; HSPC, hematopoietic stem-progenitor cells; LV, lentivirus; ns, not significant; Rapa, rapamycin.

Article Snippet: CD34+ HSPCs were isolated from G-CSF-mobilized peripheral blood from healthy donors provided by Progenitor Cell Therapy (Allendale, NJ, http://pctcelltherapy.com ).

Techniques: Transduction, Expressing, In Vitro

Journal: Stem Cells Translational Medicine

Article Title: Enhanced Genetic Modification of Adult Growth Factor Mobilized Peripheral Blood Hematopoietic Stem and Progenitor Cells With Rapamycin

doi: 10.5966/sctm.2014-0010

Figure Lengend Snippet: Rapamycin enhances genetic modification of adult hematopoietic stem and progenitor cells (HSPCs) with multiple lentiviral vectors. (A): Enhancement of genetic modification of adult HSPCs in the presence of rapamycin with multiple vectors. GFP+ percentage analysis from LV1 (n = 9) and LV3 (n = 3) transduced cells (left y-axis) and transgene copy number analysis by quantitative polymerase chain reaction from LV4 (n = 3) transduced cells (right y-axis) after 7 days of expansion culture. (B): Increase in transduction of individual donor hematopoietic stem and progenitor cells in response to rapamycin treatment. CD34+ cells from six donors were transduced with LV1 and analyzed for GFP+ expression after 7 days of expansion culture. Treatment groups were analyzed for significance using the Student’s paired, two-tailed t test. Height of bars represents the average value, and error bars represent SEM. ∗∗∗, p < .001; ∗, p < .05. Abbreviations: Avg, average; GFP, green fluorescent protein; HSPC, hematopoietic stem/progenitor cells; LV, lentivirus; Rapa, rapamycin.

Article Snippet: CD34+ HSPCs were isolated from G-CSF-mobilized peripheral blood from healthy donors provided by Progenitor Cell Therapy (Allendale, NJ, http://pctcelltherapy.com ).

Techniques: Modification, Real-time Polymerase Chain Reaction, Transduction, Expressing, Two Tailed Test

Journal: Stem Cells Translational Medicine

Article Title: Enhanced Genetic Modification of Adult Growth Factor Mobilized Peripheral Blood Hematopoietic Stem and Progenitor Cells With Rapamycin

doi: 10.5966/sctm.2014-0010

Figure Lengend Snippet: Rapamycin effect on hematopoietic potential. CFU assays were conducted as described in Materials and Methods. (A, B): 500 cells from each sample were plated in methylcellulose (in triplicate) 1 day after transduction with LV1 (A) or after 7 days of expansion in SFT6 + SR-1 (B). Colony number for each colony type was scored on day 14. The average number of colonies from each sample is shown with a bar for each colony type. Statistical analysis was performed using paired t tests (n = 3 donors). Height of bars represents the average value, and error bars represent SEM. ∗∗∗, p < .001. Abbreviations: B/CFU-E, late erythrocyte; CFU, colony-forming unit; G, granulocytes; GEMM, granulocyte, erythrocyte, monocyte, megakaryocyte; GM, granulocyte-macrophage progenitor; LV, lentivirus; M, monocytes; Rapa, rapamycin; SFT6, stem cell factor, flt-3 ligand, thrombopoietin, and interleukin-6; Untd, untransduced.

Article Snippet: CD34+ HSPCs were isolated from G-CSF-mobilized peripheral blood from healthy donors provided by Progenitor Cell Therapy (Allendale, NJ, http://pctcelltherapy.com ).

Techniques: Transduction

Journal: Stem Cells Translational Medicine

Article Title: Enhanced Genetic Modification of Adult Growth Factor Mobilized Peripheral Blood Hematopoietic Stem and Progenitor Cells With Rapamycin

doi: 10.5966/sctm.2014-0010

Figure Lengend Snippet: Analysis of colony-forming potential and transgene copy number following transduction in rapamycin. (A): Rapamycin enhanced colony-forming potential of genetically modified cells. CD34+ cells were transduced with LV1 (multiplicity of infection, 10) after overnight prestimulation in SFT6 followed by 7 days of expansion in SFEM SFT6 SR1. GFP+ and GFP− cells were sorted and plated for CFU. Statistical analysis was performed with paired t test (n = 3). (B): Methylcellulose colonies of LV1-transduced cells with or without rapamycin treatment were picked, lysed, and analyzed by quantitative polymerase chain reaction with WPRE- and apolipoprotein B (ApoB)-specific primers as described in Materials and Methods. The ratio of transgene copy number determined by WPRE and cell number determined by ApoB were calculated and presented. The data sets were analyzed for statistical significance by paired t test. (C, D): Stability of transgene average copy number per cell in 4 weeks differentiation cultures of transduced hematopoietic stem and progenitor cells using LV1 (C) and LV4 (D). Height of bars represents the average value, and error bars represent SEM. ∗, p < .05; ∗∗, p < .01; ∗∗∗, p < .001; and ∗∗∗∗, p < .0001. Abbreviations: CFU, colony-forming unit; GFP, green fluorescent protein; LV, lentivirus; Rapa, rapamycin; WPRE, woodchuck hepatitis virus post-translational regulatory element.

Article Snippet: CD34+ HSPCs were isolated from G-CSF-mobilized peripheral blood from healthy donors provided by Progenitor Cell Therapy (Allendale, NJ, http://pctcelltherapy.com ).

Techniques: Transduction, Genetically Modified, Infection, Real-time Polymerase Chain Reaction, Virus

Journal: Stem Cells Translational Medicine

Article Title: Enhanced Genetic Modification of Adult Growth Factor Mobilized Peripheral Blood Hematopoietic Stem and Progenitor Cells With Rapamycin

doi: 10.5966/sctm.2014-0010

Figure Lengend Snippet: Rapamycin-enhanced gene marking in differentiated lineage progenies. Phenotypic analysis of GFP+ cells in CD33+, CD14+, and CD15+ myeloid cells, as well as glycophorin A+ erythroid population. CD34+ cells were prestimulated overnight in SFT6 and transduced with LV3 (multiplicity of infection, 10) in the absence (top) and presence (bottom) of rapamycin during transduction. Transduced cells were harvested after overnight transduction and plated in myeloid differentiation bulk culture as described in Materials and Methods. The plots shown result from day 14 of culture. Abbreviation: GFP, green fluorescent protein; LV, lentivirus; SFT6, stem cell factor, flt-3 ligand, thrombopoietin, and interleukin-6.

Article Snippet: CD34+ HSPCs were isolated from G-CSF-mobilized peripheral blood from healthy donors provided by Progenitor Cell Therapy (Allendale, NJ, http://pctcelltherapy.com ).

Techniques: Transduction, Infection

Journal: Stem Cells Translational Medicine

Article Title: Enhanced Genetic Modification of Adult Growth Factor Mobilized Peripheral Blood Hematopoietic Stem and Progenitor Cells With Rapamycin

doi: 10.5966/sctm.2014-0010

Figure Lengend Snippet: Rapamycin treatment on CD34+ cells greatly enhanced engraftment of GFP+ cells in NSG mice spleen and bone marrow at 16 weeks. Cells were prestimulated overnight in SFT6 StemSpan SFEM medium before 24 hours of transduction, and then they were transplanted. (A): Fluorescence-activated cell sorting phenotypic analysis of spleens from NSG mice transplanted with 1 × 106 nontransduced CD34+ cells, transduced CD34+ cells without rapamycin, or transduced CD34+ cells with rapamycin treatment. Upper row: CD45+/GFP+ cells. Lower row: CD4+/GFP+ cells. (B): Statistical analysis of spleens and bone marrows from NSG mice transplanted with 1 × 106 transduced CD34+ cells without rapamycin (n = 10) or with rapamycin treatment (n = 11). The data were analyzed with unpaired two-tailed Student’s t test. ∗∗∗, p < .001; ∗∗∗∗, p < .0001. Abbreviations: GFP, green fluorescent protein; NSG, NOD.Cg-Prkdcscid IL2rgtm1Wjl/SzJ; Rapa, rapamycin; SFEM, serum free expansion media; SFT6, stem cell factor, flt-3 ligand, thrombopoietin, and interleukin-6; Tx, transduction.

Article Snippet: CD34+ HSPCs were isolated from G-CSF-mobilized peripheral blood from healthy donors provided by Progenitor Cell Therapy (Allendale, NJ, http://pctcelltherapy.com ).

Techniques: Transduction, Fluorescence, FACS, Two Tailed Test

Journal: Cells

Article Title: Therapy Development by Genome Editing of Hematopoietic Stem Cells

doi: 10.3390/cells10061492

Figure Lengend Snippet: Overview of diseases addressed by gene editing in human primary hematopoietic cells.

Article Snippet: NCT03432364 , Ex vivo , I/II , USA , CD34+ HSPCs , ZFN , mRNA , Sangamo Therapeutics.

Techniques: Modification, CRISPR, TALENs, Disruption, Non-Homologous End Joining, Expressing, Plasmid Preparation, Electroporation, CpG Methylation Assay

Journal: Cells

Article Title: Therapy Development by Genome Editing of Hematopoietic Stem Cells

doi: 10.3390/cells10061492

Figure Lengend Snippet: Overview of HSC-based clinical studies using gene editing.

Article Snippet: NCT03432364 , Ex vivo , I/II , USA , CD34+ HSPCs , ZFN , mRNA , Sangamo Therapeutics.

Techniques: Ex Vivo, Modification, CRISPR, Electroporation, In Vivo, Disruption

Journal: Nature biotechnology

Article Title: Homology-driven genome editing in hematopoietic stem and progenitor cells using zinc finger nuclease mRNA and AAV6 donors

doi: 10.1038/nbt.3408

Figure Lengend Snippet: Mobilized blood CD34 + HSPCs ( a , b ) or fetal liver CD34+ HSPCs ( c , d ) were transduced with increasing doses of GFP-expressing AAV vectors of the indicated serotypes, and GFP expression was determined at 2 days (fetal liver) or 3–5 days (mobilized blood) post-transduction by flow cytometry. The vector panel for each cell type were from independent manufacturing sources, and the doses used were 1 × 10 3 , 3 × 10 3 , 1 × 10 4 , 3 × 10 4 and 1 × 10 5 vector genomes (vg)/cell for mobilized blood HSPCs, and 1 × 10 2 , 5 × 10 2 , 1 × 10 3 , 5 × 10 3 and 1 × 10 4 vg/cell for fetal liver HSPCs. Data from representative experiments at doses of 1 × 10 4 vg/cell are shown ( a,c ), together with the mean data +/− SD from 2 (mobilized blood) and 3 (fetal liver) experiments using independent HSPC donor sources ( b , d ).

Article Snippet: Leukopaks containing G-CSF mobilized peripheral blood CD34 + HSPCs were purchased (Apheresis Care Group, Inc. San Francisco, CA) and CD34 + HSPCs purified by magnetic bead selection using a CliniMACS cell selection device (Miltenyi Biotec, Auburn, CA).

Techniques: Transduction, Expressing, Flow Cytometry, Plasmid Preparation

Journal: Nature biotechnology

Article Title: Homology-driven genome editing in hematopoietic stem and progenitor cells using zinc finger nuclease mRNA and AAV6 donors

doi: 10.1038/nbt.3408

Figure Lengend Snippet: ( a ) Schematic showing use of AAV vector as a template for homology directed repair (HDR) of a double-strand break (DSB), as induced by target-specific nucleases. ( b ) Schematic of AAV vector genomes containing CCR5 homology donors. R and L refer to CCR5 genomic sequences, comprising 1431 and 473 bp respectively, and inserted in antisense orientation when compared to the AAV genome . Vector CCR5-RFLP contains an additional Xho1 restriction site and vector CCR5-GFP contains a promoter GFP cassette with a polyadenylation (pA) sequence. ( c ) Mobilized blood CD34 + HSPCs were transduced with AAV6 vectors carrying the CCR5-RFLP donor at indicated doses for 16 hours, then electroporated with CCR5 ZFN mRNA (120μg/ml). Cells were analyzed 3–5 days post-electroporation by deep sequencing to measure the efficiency of genome modification (% indels and site-specific RFLP insertions). Results from one representative of 3 experiments using 3 different HSPC donors are shown. ( d ) Dose-dependent insertion of XhoI site at CCR5 , confirmed by RFLP analysis. One representative experiment is shown, with % HDR quantitation for any sample greater than background. ( e ) Mobilized blood HSPCs were treated as described, but using CCR5-GFP donor vectors, with and without CCR5 ZFN mRNA electroporation. Cells were collected 3–6 days post-transduction and analyzed by flow cytometry for % GFP + . Results were combined from 5 experiments using 4 different donors and show mean +/− SD. * p<0.05, ** p<0.01, unpaired t-test. ( f ) Flow cytometry plots from one representative experiment using 3,000 vg/cell CCR5-GFP donor, at 6 days post-electroporation. ( g ) Confirmation of targeted integration of GFP expression cassette at the CCR5 locus by semi-quantitative In-Out PCR, for one representative experiment. Ctrl. is a PCR that serves as a genomic DNA loading control. The % HDR-mediated insertion of GFP was estimated following normalization and by comparison to standards, and numbers are shown for any samples greater than background. Uncropped images of all gels in this figure are available in .

Article Snippet: Leukopaks containing G-CSF mobilized peripheral blood CD34 + HSPCs were purchased (Apheresis Care Group, Inc. San Francisco, CA) and CD34 + HSPCs purified by magnetic bead selection using a CliniMACS cell selection device (Miltenyi Biotec, Auburn, CA).

Techniques: Plasmid Preparation, Genomic Sequencing, Sequencing, Transduction, Electroporation, Modification, Quantitation Assay, Flow Cytometry, Expressing, Control, Comparison

Journal: Nature biotechnology

Article Title: Homology-driven genome editing in hematopoietic stem and progenitor cells using zinc finger nuclease mRNA and AAV6 donors

doi: 10.1038/nbt.3408

Figure Lengend Snippet: ( a ) Schematic of AAV vectors used, which all contain a GFP expression cassette. Vector CCR5-GFP additionally contains 1431 and 473 bp sequences with homology to the CCR5 locus, while vector AAVS1-GFP contains 801 and 840bp of sequences with homology to the AAVS1 ( PPP1R12C ) genomic locus . ( b ) Fetal liver HSPCs were mock treated (Mock), or transduced with 1,000 vg/cell of the indicated AAV vectors for 24 hours, then electroporated with CCR5 ZFN mRNA. Cells were analyzed for GFP by flow cytometry at day 1 and day 10 post-electroporation. Shown is data from one representative experiment, and ( c ) mean +/− SD GFP + for n=2 fetal liver tissues. ( d ) Evaluation of targeted integration of GFP at the CCR5 locus by semi-quantitative In-Out PCR, for one representative experiment. The Ctrl. PCR serves as a loading control. Quantitation for the single sample above background control is shown. An uncropped image of this gel is available in . ( e ) Mobilized blood HSPCs were transduced without or with AAVS1-GFP or CCR5-GFP donors at 10,000 vg/cell for 16 hours, and/or electroporated with AAVS1 or CCR5 ZFN mRNA. Cells were analyzed 8 days post-electroporation by flow cytometry. Shown is data from one representative experiment, and ( f ) mean ± SD GFP + expression from n=3 samples, except that the no donor, AAVS1 ZFN and no donor, CCR5 ZFN treatments were n=1. * p < 0.05, one-way ANOVA.

Article Snippet: Leukopaks containing G-CSF mobilized peripheral blood CD34 + HSPCs were purchased (Apheresis Care Group, Inc. San Francisco, CA) and CD34 + HSPCs purified by magnetic bead selection using a CliniMACS cell selection device (Miltenyi Biotec, Auburn, CA).

Techniques: Expressing, Plasmid Preparation, Transduction, Flow Cytometry, Electroporation, Control, Quantitation Assay

Journal: Nature biotechnology

Article Title: Homology-driven genome editing in hematopoietic stem and progenitor cells using zinc finger nuclease mRNA and AAV6 donors

doi: 10.1038/nbt.3408

Figure Lengend Snippet: ( a ) Mobilized blood CD34 + HSPCs were transduced with 1,000 vg/cell CCR5-RFLP donor, and/or electroporated 16 hrs later with 40 μg/ml of CCR5 ZFN mRNA. Mock treated HSPCs were cultured as a control. Cells were counted at 1–9 days post-electroporation. Results are shown from one representative experiment from a total of 3 independent experiments using 3 different HSPC donors. ( b ) Cells were also subjected to colony formation assay at 24 hours post-electroporation, with CFUs evaluated 14 days later. Mean +/− SD from duplicated samples are shown. No significant differences were detected among the 4 treatment conditions (p>0.05, one-way ANOVA). ( c ) CFUs were also genotyped by deep sequencing to detect rates of insertion of the XhoI site. Between 23 and 88 validated individual colonies were picked for each colony type. Mean +/− SD from 2 combined experiments using different HSPC donors are shown. No significant differences were detected (p>0.05, one-way ANOVA). ( d ) Mobilized blood HSPCs treated with CCR5-GFP donor and CCR5 ZFN mRNA were used for colony formation assays. Representative GFP + erythroid and myeloid colonies are shown. The white bars represent 100μm.

Article Snippet: Leukopaks containing G-CSF mobilized peripheral blood CD34 + HSPCs were purchased (Apheresis Care Group, Inc. San Francisco, CA) and CD34 + HSPCs purified by magnetic bead selection using a CliniMACS cell selection device (Miltenyi Biotec, Auburn, CA).

Techniques: Transduction, Cell Culture, Control, Electroporation, Colony Assay, Sequencing

Journal: Nature biotechnology

Article Title: Homology-driven genome editing in hematopoietic stem and progenitor cells using zinc finger nuclease mRNA and AAV6 donors

doi: 10.1038/nbt.3408

Figure Lengend Snippet: ( a ) Representative flow cytometry plot showing different subsets within fetal liver CD34 + HSPCs, defined as primitive (P) early (E) and committed (C) progenitors. ( b ) Fetal liver HSPCs were treated with 1,000 vg/cell CCR5-GFP vectors for 24 hours and then electroporated with 40 μg/ml CCR5 ZFN mRNA. One day later the cells were sorted into the indicated populations based on expression of CD133 and CD90. Control cells received only the CCR5-GFP vectors. Cells were cultured for a further 7 days followed by analysis for GFP expression. Representative day 7 flow cytometry plots are shown from both the unsorted and sorted populations in each treatment arm, and ( c ) Mean +/− SD GFP + cells at day 7, for n=3 independent CD34 + sources. No significant differences (p>0.05) were observed between subgroups C, E and P in the CCR5-GFP plus CCR5 ZFN treated groups, one-way ANOVA. * p<0.05, unpaired t-test. ( d ) In-Out PCR showing levels of GFP insertion at the CCR5 locus in the indicated subsets of fetal liver CD34 + cells. The Ctrl. PCR serves as a loading control. Numbers for values above background controls are shown. ( e ) Mobilized blood CD34 + HSPCs were transduced with 10,000 vg/cell CCR5-GFP, with or without electroporation 16 hours later with 60 μg/ml CCR5 ZFN mRNA. Eight days later, cells were analyzed for GFP expression by flow cytometry in the indicated subsets. The gating strategy used to isolate the P, E and C populations from the bulk CD34+ population is shown in . ( f ) Mobilized blood CD34 + HSPCs were treated as in (e), but using 10,000 vg/cell AAVS1-GFP and 40 μg/ml AAVS1 mRNA. ( g ) In-Out PCR to detect GFP insertion at the CCR5 locus in the indicated subsets and treatments. Numbers for values above background controls are shown. ( h ) In-Out PCR to detect GFP insertion at the AAVS1 locus in the indicated subsets and treatments. Numbers for values above background controls are shown. Uncropped images of all gels in this figure are available in .

Article Snippet: Leukopaks containing G-CSF mobilized peripheral blood CD34 + HSPCs were purchased (Apheresis Care Group, Inc. San Francisco, CA) and CD34 + HSPCs purified by magnetic bead selection using a CliniMACS cell selection device (Miltenyi Biotec, Auburn, CA).

Techniques: Flow Cytometry, Expressing, Control, Cell Culture, Transduction, Electroporation

Journal: Nature biotechnology

Article Title: Homology-driven genome editing in hematopoietic stem and progenitor cells using zinc finger nuclease mRNA and AAV6 donors

doi: 10.1038/nbt.3408

Figure Lengend Snippet: ( a ) Neonatal NSG mice were engrafted with fetal liver HSPCs, either mock treated, or treated with AAV6 donors (CCR5-GFP or CCR5-RFLP) plus CCR5 ZFN mRNAs, using 2 different donor tissues. Genome editing levels in these input HSPCs were 9.1% and 12% for CCR5-GFP treated cells, by flow cytometry, and 6.7% and 11.2% for CCR5-RFLP treated cells, by deep sequencing. Peripheral blood of the mice was analyzed at weeks 8, 12, and 16 for the % of human CD45 + cells, and bone marrow (BM) and spleen were analyzed at 16 weeks. Shown is the combined data from the two separate cohorts of mice. No significant differences were found in the levels of human cells in the blood or tissues between mock and treated samples (two-way ANOVA). ( b ) Rates of genome modification in human cells were measured in blood and tissue samples from individual mice by flow cytometry for GFP insertions, or deep sequencing for RFLP insertions; na, not available due to high background autofluorescence of cells. Actual numbers are available in . Cells from mock-treated mice gave only background levels in all assays (not shown). ( c ) Representative examples of In-Out PCR showing GFP addition at the CCR5 locus in peripheral blood, bone marrow, and spleen from individual mice at 16 weeks post engraftment. Numbers for any samples above the levels of the background controls are shown. ( d ) Bone marrow was isolated at 16 weeks post-engraftment from 2 mice each from the CCR5-GFP or CCR5-RFLP cohorts and was pooled. The levels of human CD45 + cell engraftment and gene modification (GFP + by flow cytometry, RFLP insertion by deep sequencing) were measured in the pooled cell populations. Each primary BM pool was used to transplant a separate adult female NSG mouse and, 20 weeks later, bone marrow was isolated from the secondary transplant recipients and analyzed for human CD45 + content and levels of genome modification in the same way. ( e ) Mobilized blood HSPCs were treated with AAVS1-GFP vectors, with and without AAVS1 ZFN, and used to engraft NSG mice. The frequency of GFP + cells in the input HSPCs, measured at 5 days post-transfection in culture, were 0.72%, 20.5% and 30.7% respectively, for cells receiving 10,000 vg/cell AAVS1-GFP alone, 3,000 vg/cell AAVS1-GFP plus ZFNs and 10,000 vg/cell AAVS1-GFP plus ZFNs. At 20 weeks, bone marrow was isolated and analyzed for human CD45 + leucocytes. ( f ) Detection of GFP insertion at the AAVS1 locus in bone marrow samples from individual mice, measured by In-Out PCR. Numbers for any samples above the levels of the background controls are shown. Uncropped images of all gels in this figure are available in .

Article Snippet: Leukopaks containing G-CSF mobilized peripheral blood CD34 + HSPCs were purchased (Apheresis Care Group, Inc. San Francisco, CA) and CD34 + HSPCs purified by magnetic bead selection using a CliniMACS cell selection device (Miltenyi Biotec, Auburn, CA).

Techniques: Flow Cytometry, Sequencing, Modification, Isolation, Transfection