NextSeq 550 paired end sequencing; Combined Functional and Genomic Single clonal Stem Cell Analysis Pinpointed at Diagnosis Leukemic Clones  Responsible for Acute Myeloid Leukemia Relapse
Combined Functional and Genomic Single clonal Stem Cell Analysis Pinpointed at Diagnosis Leukemic Clones  Responsible for Acute Myeloid Leukemia Relapse
CD34+ cells were isolated from PB or BM (following mononuclear cell separation) of AML patients by positive selection using anti-human CD34 microbeads. Enriched CD34+ cells were sorted by FACSariaII (BD bioscience, CA, USA) into four subpopulations of HSPCs according to their surface markers and CD33 expression. The sorted fractions were collected either into Iscove's modified Dulbecco's medium (IMDM) +20% fetal bovine serum (FBS) (Biological Industries #02-023-1A) to be further used in the colony forming unit (CFU) assay or into PBS+2%FBS to be used in the xenotransplantation assay. The CFU assay was adjusted to the generation of single cell-derived subclones. HSPCs were seeded in 1% standard methylcellulose medium supplemented with 37% FBS, 1.2% bovine serum albumin (BSA), 1% L-glutamine, 10-6M b-mercaptoethanol, 1 IU/ml of  human erythropoietin (EPO), 50 ng/ml of human stem cell factor (SCF), 25 ng/ml of human FLT3 ligand, 10 ng/ml  of interleukin 3 (IL-3), 10 ng/ml of interleukin 6 (IL-6), 10 ng/ml of granulocytecolony stimulating factor (G-CSF), 5 ng/ml  of granulocyte-macrophage colony-stimulating factor (GM-CSF), 10 ng/ml of human thrombopoietin (TPO), 100 IU ml penicillin and 100 mg/ml streptomycin. Colonies were counted after 14 days and each of them was picked under sterile conditions. Every individual colony was divided into two cell suspensions to perform both genotyping analysis and xenotransplantation into NSG mice. xenograft protocol: NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice (The Jackson Laboratory, Bar Harbor, Maine, USA) were maintained and bred under specific pathogen-free conditions at the Biomedical Core Facility (Faculty of Medicine, the Technion, Haifa, Israel). Twenty-four hours prior to transplantation, 8-12-week-old female NSG mice were sub-lethally irradiated with 225 cGy. Primary human cells were intra-femorally injected. The total number of cells transplanted per mouse depended on the number of viable cells obtained from the BM/PB of a patient [ranged from 1 colony (a single cell-derived subclone) to 106 cells]. The animals were sacrificed 8-30 weeks after transplantation or if tumors developed in the injected knee. The BM was then harvested. Human engraftment was evaluated by flow cytometry using the following anti-human specific antibodies: anti-CD45-APC Cyanine7 (APC-Cy7) (BioLegend #304014 clone H130); anti-CD33-BV510 (BioLegend #366610); anti-CD34-BV421 (BioLegend #343610); anti-CD38-FITC (BD #555459); anti-CD15 APC (BD #551376); anti-CD19 PE-CY7 (BD #557835); Cell viability was obtained using Propidium Iodide – PI (BioLegend #421301). Analyses were performed using FlowJo software v.10 (Treestar, OR, USA), and the expression of markers was assessed and presented as the percentage of stained cells. The threshold for engraftment detection was 0.1% of human CD45+ cells. All flow cytometry analyses were done using an LSR II (BD Bioscience, CA, USA) or FACSariaII (BD Bioscience, CA, USA) device. Engraftment of >5% of hCD45+ cells with the major CD33+ population (>60% of the hCD45+ cells) was classified as overt leukemia; the presence of 0.1-5% of hCD45+ cells or the presence of >5% of hCD45+ cells with <60% of the CD33+ population in the hCD45+ cells was classified as non-overt leukemia; and the presence of <0.1% of human CD45+ (hCD45+) population was considered as non-engraftment. hCD45+ cells were sorted and underwent whole genome amplification (WGA) for further genotyping assays. CD34+ cells were isolated from PB or BM (following mononuclear cell separation) of AML patients by positive selection using anti-human CD34 microbeads. Enriched CD34+ cells were sorted by FACSariaII (BD bioscience, CA, USA) into four subpopulations of HSPCs according to their surface markers and CD33 expression. The sorted fractions were collected either into Iscove's modified Dulbecco's medium (IMDM) +20% fetal bovine serum (FBS) (Biological Industries #02-023-1A) to be further used in the colony forming unit (CFU) assay or into PBS+2%FBS to be used in the xenotransplantation assay. The CFU assay was adjusted to the generation of single cell-derived subclones. HSPCs were seeded in 1% standard methylcellulose medium supplemented with 37% FBS, 1.2% bovine serum albumin (BSA), 1% L-glutamine, 10-6M b-mercaptoethanol, 1 IU/ml of  human erythropoietin (EPO), 50 ng/ml of human stem cell factor (SCF), 25 ng/ml of human FLT3 ligand, 10 ng/ml  of interleukin 3 (IL-3), 10 ng/ml of interleukin 6 (IL-6), 10 ng/ml of granulocytecolony stimulating factor (G-CSF), 5 ng/ml  of granulocyte-macrophage colony-stimulating factor (GM-CSF), 10 ng/ml of human thrombopoietin (TPO), 100 IU ml penicillin and 100 mg/ml streptomycin. Colonies were counted after 14 days and each of them was picked under sterile conditions. Every individual colony was divided into two cell suspensions to perform both genotyping analysis and xenotransplantation into NSG mice One µl of DNA template was added to a hybridization mix together with an MIP pool (final concentration of 0.05pM per probe) in 1x Ampligase buffer (Epicentre). The mix was incubated in a thermal cycler according to the following program: 98°C for 3 minutes, 85°C for 30 minutes, 60°C for 60 minutes and 56°C for 1 or 2 overnight incubations. The product was mixed with (final concentration in brackets): dNTPs (Larova, 15pM), Betaine (375 mM, Sigma-Aldrich,), NAD+ (1 mM, New England Biolabs), additional Ampligase buffer (0.5x), Ampligase (total of 1.25U, Epicentre) and Phusion HF (0.16U, New England Biolabs). The mix was incubated at 56°C for 60 minutes followed by 72°C for 20 minutes. Enzymatic digestion of linear probes was performed by adding Exonuclease I (4U, New England Biolabs) and Exonuclease III (25U, New England Biolabs). The mix was incubated at 37°C for 2 hours, followed by 80°C for 20 minutes. The final product was amplified using iProof HF Master Mix (Biorad). Samples were pooled and concentrated using AMPure XP beads (Beckman Coulter) at 1.3x volumetric concentration, size-selected (190-370bp) using Blue Pippin (Sage scientific), and sequenced in either NextSeq or Novaseq6000 (Illumina) 2X151bp paired-end run using custom primers as described in the past(Biezuner et al., 2022)