Project description:Reticular Dysgenesis (RD) is a rare but devastating form of severe combined immunodeficiency, characterized by a maturation arrest of the myeloid and lymphoid lineages paired with sensorineural hearing loss. RD is caused by biallelic loss-of-function mutations in the mitochondrial enzyme adenylate kinase 2 (AK2). To study the effect of AK2 depletion on HSPC differentiation, we developed a biallelic AK2 CRISPR knock-out model using human HSPCs. AK2 depleted HSPCs display severe proliferation and myeloid differentiation defects, recapitulating RD patient phenotype.
Project description:Severe congenital neutropenia (CN) is a pre-leukemia syndrome that, in the majority of patients, is caused by heterogeneous ELANE mutations encoding neutrophil elastase (NE). To study leukemogenesis associated with CN we generated CN and CN/AML patient-specific induced pluripotent stem cells (iPSCs). Additional mutations in leukemia-relevant genes, CSF3R and RUNX1, were introduced using CRISPR/Cas9 gene-editing. Consequently, we performed in vitro embryoid body (EB)-based hematopoietic and myeloid differentiation of generated iPSC lines. On day 14-17 of EB-based differentiation, iPSC-derived CD45+CD34+ cells were harvested and mRNA was isolated using RNeasy Mini- or Micro Kit (Qiagen). Sequencing libraries were prepared using the TruSeq RNA Sample Prep Kit (Illumina). Poly (A) selected single-read and pair-read sequencing libraries were sequenced on the Illumina platform in order to compare the transcriptomes of CN and CN/AML iPSCs-derived HSPCs from 2 CN/AML patients. Next, we identified that BAALC knockout resulted in a dramatic induction of granulocytic differentiation and a significant reduction in proliferation of CN/AML iPSC-derived HSPCs. To identify BAALC-dependent leukemia-associated gene expression, we compared the transcriptomes of CN/AML iPSCs before and after BAALC KO using a similar approach described above for CN and CN/AML iPSCs-derived HSPCs.
Project description:Treatment of healthy donors with G-CSF and dexamethasone, results in sufficient number of circulating granulocytes to prepare granulocyte concentrates for clinical purposes. Granulocytes obtained this way demonstrate relatively normal functional behavior combined with a prolonged life span. To study the influence of mobilizing agents on granulocytes, we used oligonucleotide microarrays to identify genes that are differentially expressed in mobilized granulocytes as compared to control ones. Keywords: mobilized granulocytes, G-CSF/dexamethasone treated granulocytes
Project description:Treatment of healthy donors with G-CSF and dexamethasone, results in sufficient number of circulating granulocytes to prepare granulocyte concentrates for clinical purposes. Granulocytes obtained this way demonstrate relatively normal functional behavior combined with a prolonged life span. To study the influence of mobilizing agents on granulocytes, we used oligonucleotide microarrays to identify genes that are differentially expressed in mobilized granulocytes as compared to control ones. Keywords: mobilized granulocytes, G-CSF/dexamethasone treated granulocytes Granulocytes were isolated from three individuals before (control situation) and 18 hours after treatment with G-CSF&dexamethasone; part of the control cells was cultured overnight in HBSS medium with or without addition of G-CSF and dexamethasone. Total RNA from each experimental condition was compared to pooled RNA of control granulocytes.
Project description:We characterized the genome wide occupancy of Med12 and p300 in mouse HSPCs. We also characterize p300 occupancy upon shRNA against control or Med12. ChIP-seq analysis of Med12 and/or p300 in untreated HSPCs
Project description:Using iChIP, we map H3K27Ac, H3K4me3 and H3K4me1 in small populations (HSPCs) in the presence or absence of Med12 and identify affected super-enhancers. iChIP was performed using previously published protocols (Lara-Astiaso et al, 2014 Immunogenetics. Chromatin state dynamics during blood formation - Science, 345, 6199) Examination of histone modification in mouse HSPCs with and without Med12