Day2_r2
Homo sapiens
Protocols: In order to generate HuSGM3 CAR-T cells, newborn female or male NSG-SGM3 mice were sub lethally irradiated and injected intra-liver with 1x10^5 human cord blood-derived CD34+ hematopoietic stem cells. T cells developing in humanized NSG-SGM3 mice were retrieved from spleen and bone marrow, activated and transduced ex vivo to express CD19-targeting CAR molecule (HuSGM3 CD19 CAR-T). After transduction and expansion, HuSGM3 CD19 CAR-T cells were infused back into NSG-SGM3 mice previously co-engrafted with human cord blood-derived CD34+ hematopoietic stem cells and with ALL-CM leukaemia cells. After HuSGM3 CD19 CAR-T cell infusion, spleen from leukaemic and humanized SGM3 recipient was dissociated and cells subjects to FACS sorting. Human CD45+ cells (containing CB-derived normal hematopoiesis, ALL-CM leukaemic cells and HuSGM3 CAR-T cells) were recovered. CB CD34+ cells were supplied by commercial vendors (Lonza). HuSGM3 CAR-T cells generated as previously described. ALL-CM leukaemia cells were derived from patient with chronic myeloid leukaemia in lymphoid blast crisis.Spleen from leukemic HuSGM3 mice treated with CD19-28z chimeric antigen receptor-expressing T lymphocytes (CAR-T) cells (described here: https://www.cancer.gov/publications/dictionaries/cancer-drug/def/autologous-cd19-28z-chimeric-antigen-receptor-expressing-t-lymphocytes) was dissociated and cells subjected to FACS sorting. Human CD45+ cells were recovered. Droplet-based digital 3′ end scRNA-Seq was performed on a Chromium Single-Cell Controller (10X Genomics, Pleasanton, CA) using the Chromium Single Cell 3′ Reagent Kit v2 according to the manufacturer's instructions. Briefly, suspended single cells were partitioned in Gel Beads in Emulsion (GEMs) and lysed, followed by RNA barcoding, reverse transcription and PCR amplification (12-14 cycles). Sequencing-ready scRNA-Seq libraries were prepared according to the manufacturer's instructions, checked and quantified on 2100 Bioanalyzer (Agilent Genomics, Santa Clara, CA) and Qubit 3.0 (Invitrogen, Carlsbad, CA) instruments.
ENA-FIRST-PUBLIC
2019-01-31T17:02:08Z
ENA-LAST-UPDATE
2018-10-29T17:45:38Z
External Id
SAMEA5053921
INSDC center name
San Raffaele Telethon Institute for Gene Therapy.
INSDC first public
2019-01-31T17:02:08Z
INSDC last update
2018-10-29T17:45:38Z
INSDC status
public
Submitter Id
E-MTAB-6505:Day2_r2
broker name
ArrayExpress
cell type
leukocyte
common name
human
developmental stage
neonate
disease
normal
genotype
CD19.28z expression
growth condition
xenograft from HuSGM3 mouse infused with CD19.28z CAR-T cells
organism part
umbilical cord blood
phenotype
CD45+; CD19.28z+
sample name
E-MTAB-6505:Day2_r2
scientific_name
Homo sapiens