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: 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