Project description:To characterize transcriptional changes associated with gain of the transcription factor PLAG1-S that could contribute to transient ex vivo expansion of human cord-blood (CB) derived HSC we performed RNA-seq of 2 pools of Lin-CD34+ CB cells 72 hours following lentiviral introduction of ectopic PLAG1-S or firefly Luciferase (control).
Project description:To characterize transcriptional changes associated with loss of the transcription factor PLAG1-S that could contribute to PLAG1 essentiality in long-term human HSC we performed RNA-seq of 2 pools of Lin-CD34+ cord-blood derived hematopoietic stem and progenitor cells 72 hours following lentiviral introduction of shRNA against PLAG1 or non-targeting control.
Project description:To characterize genome-wide chromatin occupancy of the transcription factor PLAG1-S that could contribute to transient ex vivo expansion of human cord-blood (CB) derived HSC we performed CUT&RUN. 3 pools of Lin-CD34+ CB cells transduced to overexpress 3xFLAG-PLAG1-S were used for anti-FLAG or anti-IgG control enrichment of specific PLAG1-S DNA binding sites.
Project description:AML1-ETO expression in normal human umbilical cord blood CD34+ cells leads to long-term proliferation of an early self-renewing primitive progenitor cell with multilineage potential and stem cell ability, but these cells do not induce leukemia in immunodeficient mice. This comparative microarray study was initiated to determine the differences in the transcriptome of AML-ETO-expressing CD34+ cells after extended culture in vitro, using normal cord blood cells expanded for 6-8 weeks in vitro and subsequently purified for the CD34+ population as the control comparison. Keywords: Disease state analysis; comparison of changes in transcriptome due to long-term AML1-ETO expression in normal human hematopoietic CD34+ progenitor cells
Project description:Global gene expressions of human cord blood-derived 18Lineage-negative (18Lin-)CD34+CD38-CD133+GPI-80+ cells (CD34+ HSCs), 18Lin-CD34-CD133+GPI-80+ cells (CD34- HSCs) and 18Lin-CD34+CD133- cells (non-HSCs) were analyzed. Results provide an insight into the molecular mechanisms underlying the self-renewal, maintenance and differentiation of human cord blood-derived CD34+/- HSCs.
Project description:AML1-ETO expression in normal human umbilical cord blood CD34+ cells leads to long-term proliferation of an early self-renewing primitive progenitor cell with multilineage potential and stem cell ability, but these cells do not induce leukemia in immunodeficient mice. This comparative microarray study was initiated to determine the differences in the transcriptome of AML-ETO-expressing CD34+ cells after extended culture in vitro, using normal cord blood cells expanded for 6-8 weeks in vitro and subsequently purified for the CD34+ population as the control comparison. Experiment Overall Design: We have established a culture system whereby we retrovirally transduce human CD34+ cells, obtained from cord blood, with the leukemia fusion gene AML1-ETO. Cells expressing this fusion protein are able to proliferate long-term in vitro in a cytokine dependent manner. AML1-ETO-expressing cord blood cells have a large population of primitive self-renewing CD34+ cells with continued abnormal differentiation. We grow these cells in serum-free conditions using the BIT supplement from Stem Cell Technologies. For the current experiments we used cell cultures that had been proliferating in vitro for 8-12 weeks, in a cytokine cocktail of SCF, TPO, FLT3L, IL-6 all at 20 ng/mL and IL-3 at 10 ng/mL. Control cord blood samples that were CD34 purified were expanded for 5-8 weeks in the same culture media as used for AML1-ETO cells. All samples were magnetically selected for the CD34+ population, returned to culture, and one week later again selected for CD34+ cells and then lysed for RNA isolation.
