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.
Project description:Expression Profiling of CD133 and CD34 positive Hematopoietic Stem Cells imuno-magnetically isolated from Bone Marrow and Umbilical Cord Blood
Project description:Gene expression profiles of CD34+CD38- stem cells and more differentiated CD34+CD38+ progenitor cells were compared. Comparison of expression profiles of hematopoietic stem cells from fetal liver, umbilical cord blood, bone marrow and mobilized pheripheral blood allowed us to identify a unique set of genes with conserved expression during ontogeny. Experiment Overall Design: CD34+CD38- en CD34+CD38+ cell populations were isolated by cell sorting from human Bone Marrow, mobilized peripheral blood, umbilical cord blood and fetal liver. Total RNA was isolated from each cell population followed by the synthesis of biotinylated cRNA. After fragmentation the biotinylated cRNA was hybridized to affymetrix U133A chips.
Project description:In order to revealed the multiple protein functional modules and kinases networks of human early erythropoiesis. We isolated the CD34+ cells drived from Human umbilical cord blood samples and induced to undergo erythropoiesis in vitro. Then, the cultured erythroid cells were sorted with FACS and sampled for proteome and phosphoproteome analysis.
Project description:KMT2A-MLLT3, a fusion protein formed by the t(9;11) translocation in acute myeloid leukemia, is an epigenetic transcription factor that is known to regulate a unique leukemic gene expression profile. We show that rearranged during transfection (RET) proto-oncogene is highly overexpressed in the KMT2A-MLLT3 subgroup. In addition to biochemical studies, the RNA-seq and ATAC seq analyses have been performed in KMT2A-MLLT3 positive MOLM-13 AML cell line, compared to the umbilical cord blood-driven CD34+ hematopoietic stem progenitor cells.
Project description:MLL-AF9 expression in normal human umbilical cord blood CD34+ cells leads to long-term proliferation of a myeloid progenitor cell with leukemogenic potential. Expression of a Core Binding Factor leukemia fusion (AML1-ETO or CBFbeta-SMMHC) in human CD34+ cells results in self-renewal of primitive progenitor cells 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 how faithful these cell cultures are to the transcriptome of patient samples expressing each of these different fusion proteins, and to analyze the signaling pathways that are unique to CBF cultures and MLL-fusion cultures, with the hope of determining why the MLL-fusion cells are leukemogenic while the CBF cells are not. Keywords: Disease state analysis; comparison of leukemia fusion gene expression in normal human hematopoietic progenitor cells
Project description:The 8p11 myeloproliferative syndrome (EMS), also referred to as the stem cell leukemia/lymphoma syndrome, is a chronic myeloproliferative disorder that rapidly progresses into an acute leukemia. Molecularly, EMS is characterized by fusion of various partner genes to the FGFR1 gene, resulting in constitutive activation of the tyrosine kinase activity within FGFR1. The two most common fusion genes in human EMS are ZMYM2/FGFR1 (previously known as ZNF198/FGFR1) and BCR/FGFR1. To study the transcriptional programs becoming deregulated by the FGFR1 fusion genes, global gene expression analysis on human CD34+ cord blood cells expressing either of the fusion oncogenes ZMYM2/FGFR1 and BCR/FGFR1 was performed. As a reference gene we also included the more studied BCR/ABL1 fusion oncogene associated with chronic myeloid leukemia. We found that the 3 different fusion oncogenes had in common the upregulation of several genes involved in the JAK/STAT signalling pathway and also other sets of genes. However, the gene expression profiles were not identical, suggesting that both the tyrosine kinase containing gene and the partner gene would affect the transcription of downstream target genes. Bicistronic retroviral murine stem cell virus (MSCV) vectors expressing ZMYM2/FGFR1, BCR/FGFR1or P210 BCR/ABL1 and GFP were used. The MIG control vector expressed GFP only. Two days post transfection of human CD34+ umbilical cord blood cells, GFP-sorted cells were collected in three biological replicates and RNA was isolated immediately. In total, 12 samples were hybridized and scanned.