Project description:Intense immunosuppression followed by autologous hematopoietic stem cell transplantation (aHSCT) is a potential treatment for patients suffering from aggressive multiple sclerosis (MS). However it remains unresolved whether autologous CD34+ hematopoietic progenitor cells of MS patients show gene expression differences prior to aHSCT that indicate a preset proinflammatory state, which would then also predispose to or predetermine recurrence of the autoimmune disease. To approach this key point we compared the micro RNA gene expression signature of CD34+ cells collected from MS patients and healthy donors (HD). Gene expression of CD34+ cells was analysed with the Human miRNA Microarray (Agilent-Technologies) chip. No substantial alterations in the gene expression profile of CD34+ HPCs in MS were observed.
Project description:Intense immunosuppression followed by autologous hematopoietic stem cell transplantation (aHSCT) is a potential treatment for patients suffering from aggressive multiple sclerosis (MS). However it remains unresolved whether autologous CD34+ hematopoietic progenitor cells of MS patients show gene expression differences prior to aHSCT that indicate a preset proinflammatory state, which would then also predispose to or predetermine recurrence of the autoimmune disease. To approach this key point we compared the micro RNA gene expression signature of CD34+ cells collected from MS patients and healthy donors (HD). Gene expression of CD34+ cells was analysed with the Human miRNA Microarray (Agilent-Technologies) chip. No substantial alterations in the gene expression profile of CD34+ HPCs in MS were observed. Samples of CD34+ cells were obtained from 4 female MS patients and 4 age matched healthy donors (3 female) mobilized by G-CSF (2x5μg/kg/day). White blood cells, containing the CD34+ cell fraction, were collected by leucocytapheresis from peripheral blood, frozen and stored in liquid nitrogen. All samples were thawed and processed at one center and CD34+ HPCs purified by magnetic bead separation using the autoMACS system (Miltenyi). Purity and viability of CD34+ cells was analyzed by Fluorescence Activated Cell Sorter (FACS). Total cellular RNA were extracted with TRIzol reagent and analyzed with the Human miRNA Microarray (Agilent-Technologies).
Project description:Intense immunosuppression followed by autologous hematopoietic stem cell transplantation (aHSCT) is a potential treatment for patients suffering from aggressive multiple sclerosis (MS). However it remains unresolved whether autologous CD34+ hematopoietic progenitor cells of MS patients show gene expression differences prior to aHSCT that indicate a preset proinflammatory state, which would then also predispose to or predetermine recurrence of the autoimmune disease. To approach this key point we compared the gene expression signature of CD34+ and CD34- cells collected from MS patients and healthy donors (HD). Whole genome gene expression of CD34+ and CD34- cells was analysed with the Human 4x44K Design Array (Agilent-Technologies). As main observation we found only minor differences in the gene expression signature of MS patients compared to HD. Only a single gene, troponin-type-1 (TNNT1), reached statistical significance after correction for multiple comparisons (logFC=3.1, p<0.01). There was a decreased expression of several protease genes, myeloperoxidase (MPO), neutrophil-elastase (ELA2), cathepsin-G (CTSG) and serine-protease 21 (PRSS21) in HPCs of MS patients, albeit not reaching statistical significance. In summary we did not detect substantial alterations in the gene expression profile of CD34+ HPCs in MS. Our data support the use of autologous hematopoietic stem cell transplantation for treatment of an autoimmune disease.
Project description:Intense immunosuppression followed by autologous hematopoietic stem cell transplantation (aHSCT) is a potential treatment for patients suffering from aggressive multiple sclerosis (MS). However it remains unresolved whether autologous CD34+ hematopoietic progenitor cells of MS patients show gene expression differences prior to aHSCT that indicate a preset proinflammatory state, which would then also predispose to or predetermine recurrence of the autoimmune disease. To approach this key point we compared the gene expression signature of CD34+ and CD34- cells collected from MS patients and healthy donors (HD). Whole genome gene expression of CD34+ and CD34- cells was analysed with the Human 4x44K Design Array (Agilent-Technologies). As main observation we found only minor differences in the gene expression signature of MS patients compared to HD. Only a single gene, troponin-type-1 (TNNT1), reached statistical significance after correction for multiple comparisons (logFC=3.1, p<0.01). There was a decreased expression of several protease genes, myeloperoxidase (MPO), neutrophil-elastase (ELA2), cathepsin-G (CTSG) and serine-protease 21 (PRSS21) in HPCs of MS patients, albeit not reaching statistical significance. In summary we did not detect substantial alterations in the gene expression profile of CD34+ HPCs in MS. Our data support the use of autologous hematopoietic stem cell transplantation for treatment of an autoimmune disease. Samples of CD34+ cells were obtained from 4 female MS patients and 4 age matched healthy donors (3 female) mobilized by G-CSF (2x5M-NM-<g/kg/day) and 4 MS patients (2 female) mobilized by G-CSF (5M-NM-<g/kg/day) plus Cyclophosphamide (Cy, 4g/m2). White blood cells, containing the CD34+ cell fraction, were collected by leucocytapheresis from peripheral blood, frozen and stored in liquid nitrogen. All samples were thawed and processed at one center and CD34+ HPCs purified by magnetic bead separation using the autoMACS system (Miltenyi). Purity and viability of CD34+ cells was analyzed by Fluorescence Activated Cell Sorter (FACS). Total cellular RNA were extracted with TRIzol reagent and analyzed with the Human 4x44K Design Array (Agilent-Technologies).
Project description:The comparative characterization of hematopoietic stem cells from healthy stem cell donors and patients with acute myeloid leukemia on a proteome level has the potential to reveal differentially regulated proteins which might be candidates for specific immunotherapy target molecules. Exemplarily, we analyzed the proteome of the cytosolic and the membrane fraction of CD34 and CD123 co-expressing FACS-sorted leukemic progenitors from five patients with acute myeloid leukemia employing mass spectrometry. As a reference, CD34+CD123+ normal hematopoietic progenitor cells from five healthy stem cell donors were analyzed. In this TMT 10-plex labeling based approach 2068 proteins were identified with 256 proteins differentially regulated in one or both cellular compartments. This study demonstrates the feasibility of a mass spectrometry based proteomic approach to detect differentially expressed proteins in two compartment fractions of leukemic stem cells as compared to their healthy stem cell counterparts.
Project description:miRNA expression in a patient with AML comparing with pooled CD34 hematopoietic progenitor cells from 5 healthy volunteers RNA from bone marrow of a patient with AML with more than 90% blast and RNA pooled from 5 volunteers with CD34+ cells selected by automacs from bone marrow
Project description:We applied a novel approach of parallel transcriptional analysis of multiple, highly fractionated stem and progenitor populations from patients with acute myeloid leukemia (AML) and a normal karyotype. We isolated phenotypic long-term HSC (LT-HSC), short-term HSC (ST-HSC), and committed granulocyte-monocyte progenitors (GMP) from individual patients, and measured gene expression profiles of each population, and in comparison to their phenotypic counterparts from age-matched healthy controls. Bone marrow samples from AML patients with normal karyotype and age-matched healthy controls were used in this study. Hematopoietic stem and progenitor compartments were purified by multiparameter-high speed fluorescence-activated cell sorting (FACS) from CD34+ enriched bone marrow to isolate LT-HSC (Lin-/CD34+/CD38-/CD90+), ST-HSC (Lin-/CD34+/CD38-/CD90-), and GMP (Lin-/CD34+/CD38+/CD123+/CD45R+).