ABSTRACT: The donor AML patient (unsorted) that was used to generate OCI-AML22 was expanded for the indicated number of days (Donor: 0 day, 12d: 12 days, 2m: 2 months, 3m: 3 months), then sorted for the indicated fraction (CD34p: CD34+; CD34m: CD34-, CD38m: CD38-, CD38m: CD38-).
Project description:The donor AML patient (unsorted) that was used to generate OCI-AML22 was expanded for 3-4 months, then sorted for the indicated fraction (CD34p: CD34+; CD34m: CD34-)
Project description:Donor_OCIAML22_bulk: The donor (unsorted) that was used to generate OCI-AML22.
OCI-AML22_bulk_1month_inVitro: The donor (unsorted) that was used to generate OCI-AML22 was expanded for 1 month in vitro, then sequenced.
OCI-AML22_graft_CD34minus_WGS: The donor (unsorted) that was used to generate OCI-AML22 was expanded for 1 month in vitro, then injected in NSG-SGM3 mice. 21 weeks after injection, cells from 5 mice injected with 5 mice injected with 1475 cells per mice and 4 mice injected with 469 cells per mice were sorted. The human CD34- fraction was sequenced (CD45h+CD45m-7aad-annexinV-CD34-)
OCI-AML22_graft_CD34positive_WGS: The donor (unsorted) that was used to generate OCI-AML22 was expanded for 1 month in vitro, then injected in NSG-SGM3 mice. 21 weeks after injection, cells from 5 mice injected with 5 mice injected with 1475 cells per mice and 4 mice injected with 469 cells per mice were sorted. The human CD34+ fraction was sequenced (CD45h+CD45m-7aad-annexinV-CD34+)
Project description:ATAC-Seq on OCIAML-22 CD34+, CD34-, and Bulk Fractions
RNA-Seq on OCIAML-22 CD34+/CD38-, CD34+/CD38+, CD34-/CD38+, CD34-/CD38- Fractions
WGS on Donor Bulk, OCIAML-22 Bulk, and CD34+ and CD34- Fractions out of OCIAML-22 Xenografts
Project description:Comparison of the CD34+CD38- versus CD34+CD38+ fractions of human umbilical cord blood and comparison of the slow-dividing fraction versus the fast dividing fraction of the CD34+/CD38- population.
Project description:Differences in chemo-sensitivity of subpopulations of AML stem cells could have important clinical implications. Using in vitro cytotoxicity, xenograft models and colony forming assays, we compared chemotherapy sensitivity between Lineage (Lin-)CD34-CD38-, Lin-CD34-CD38+, Lin-CD34+CD38- and Lin-CD34+CD38+ populations from 26 primary AMLs (19 paediatric and 7 adult). We identified a common recurring pattern of chemo-response associated with a poor clinical outcome: In each of 16/26 (62%) AMLs, Lin-CD34-CD38- cells were the most chemoresistant of the four subpopulations to daunorubicin in vitro. Cytarabine-resistant colonies formed only from Lin-CD34-CD38- populations following tertiary passages through both NOG mice and methylcellulose in these AMLs The presence of chemo-resistant Lin-CD34-CD38- populations was signficantly associated with reduced relapse-free survival in childhood AML. Consistently, CD34 negativity was significantly associated with an increased risk of relapse in a larger retropsective cohort (n=89). Samples enriched for chemo-resistant Lin-CD34-CD38- LSCs with a stem cell profile and an undifferentiated genotype revealed pathways likely to confer chemo-resistance, These strongly indicated dependence of chemo-resistant Lin-CD34-CD38- LSCs on their niche environment as well as deregulated DNA damage responses, lipid and Notch1 signalling, Our findings have major implications for the risk stratification of childhood AML and could lead to the development of novel therapeutic approaches.
Project description:Differences in chemo-sensitivity of subpopulations of AML stem cells could have important clinical implications. Using in vitro cytotoxicity, xenograft models and colony forming assays, we compared chemotherapy sensitivity between Lineage (Lin-)CD34-CD38-, Lin-CD34-CD38+, Lin-CD34+CD38- and Lin-CD34+CD38+ populations from 26 primary AMLs (19 paediatric and 7 adult). We identified a common recurring pattern of chemo-response associated with a poor clinical outcome: In each of 16/26 (62%) AMLs, Lin-CD34-CD38- cells were the most chemoresistant of the four subpopulations to daunorubicin in vitro. Cytarabine-resistant colonies formed only from Lin-CD34-CD38- populations following tertiary passages through both NOG mice and methylcellulose in these AMLs The presence of chemo-resistant Lin-CD34-CD38- populations was signficantly associated with reduced relapse-free survival in childhood AML. Consistently, CD34 negativity was significantly associated with an increased risk of relapse in a larger retropsective cohort (n=89). Samples enriched for chemo-resistant Lin-CD34-CD38- LSCs with a stem cell profile and an undifferentiated genotype revealed pathways likely to confer chemo-resistance, These strongly indicated dependence of chemo-resistant Lin-CD34-CD38- LSCs on their niche environment as well as deregulated DNA damage responses, lipid and Notch1 signalling, Our findings have major implications for the risk stratification of childhood AML and could lead to the development of novel therapeutic approaches. 3 subpopulations of leukemia stem cells from 9 patients with primary childhood AML were analysed. We compared gene expression profiles of Lin-CD34+CD38- (Q1), Lin-CD34+CD38+ (Q2) and Lin-CD34-CD38- (Q3) cells between 3 AMLs in which the CD34+ cells were most chemo-resistant (AML-1P, AML-6P and AML-10P) and the same cells from the remaining AMLs in which the Lin-CD34-CD38- cells were the most chemo-resistant population. We also compared the gene expression profiles of Lin-CD34-CD38- (Q3) cells in the 3 samples with the highest LC50 values (AML-15P, AML-17P and AML-19P) with that of the 3 AMLs exhibiting the lowest LC50 values (AML-2P, AML-5P and AML-11P).
Project description:Cord blood (CB) samples from normal donors were obtained with informed consent. Fresh CB samples were processed within 18-34h after collection. Mononuclear cells were isolated and CD34+ fraction was separated. CB CD34+ enriched fraction was lineage depleted by staining with purified anti-human CD2, CD3, CD4, CD7, CD8a, CD11b, CD14, CD19, CD20, CD56, CD235a followed by Qdot 605 conjugated goat F(ab')2 anti-mouse IgG (H+L). Cells were also stained with anti-human CD38-FITC, CD45RA-PE or -BV650, CD123-PE Cy7, CD90-biotin, CD34- PerCP and CD10-APC. Finally, cells were incubated with streptavidin-conjugated APC-eF780 and Hoechst 33258 (Invitrogen, final concentration: 1 g/ml). Populations were defined, as follows: HSC - Lin-CD34+CD38-CD90+CD45RA-CD10-, MPP - Lin-CD34+CD38-CD90-CD45RA-CD10-, LMPP - Lin-CD34+CD38-CD90-/loCD45RA+CD10-, MLP - Lin-CD34+CD38-CD90-/loCD45RA+CD10+, GMP - Lin-CD34+CD38+CD123+CD45RA+CD10-, CMP - Lin-CD34+CD38+CD123+CD45RA-CD10-, MEP - Lin-CD34+CD38+CD123-CD45RA-CD10-.
Project description:Triplicate samples of donor-matched native neutrophils (purified through fluorescence-assisted flow cytometry using CD66b as a marker) and 18-day in vitro cultured neutrophils (derived from CD34+ cells) were analysed through TMT-based quantitative proteomics to examine possible protein abundance changes underlying neutrophil cell culture.
Project description:In an attempt to identify novel markers and immunologic targets in leukemic stem cells (LSC) in acute myeloid leukemia (AML) and chronic myeloid leukemia (CML), we screened samples of patients with AML, CML, and controls for expression of cell surface antigens on CD34+/CD38− and CD34+/CD38+ cells by multi-color flow cytometry. In addition, we examined mRNA expression profiles in highly purified CD34+/CD38− and CD34+/CD38+ cells by gene array- and qPCR analyses. Aberrantly expressed markers were identified in all patient cohorts examined.
