GoKawiilEthics statement
Human CB samples were obtained with informed consent from Trillium Health, Credit Valley and William Osler Hospitals according to procedures approved by the University Health Network (UHN) Research Ethics Board (REB# 02-0763). The investigation of data from the Ontario Health Study was approved by the University of Toronto Research Ethics Board (protocol #00033112). Consent for use of data and blood samples was previously collected through the Ontario Health Study63 and CARTaGENE64 regional cohorts within the Canadian Partnership for Tomorrow’s Health65. All research using human material was performed in accordance with relevant guidelines and regulations.
Human CB sample processing
Mononuclear cells from pools of male and female CB (approximately 1–8 donors) were obtained by centrifugation over a density barrier of Lymphoprep (Multicell). After red cell lysis with ammonium chloride (STEMCELL technologies), mononuclear cells were enriched for HSPCs by positive selection with a CD34 Microbead kit (Miltenyi) per the manufacturer’s instructions, or by lineage depletion as previously described66. Resulting HSPC-enriched CB cells were cryopreserved in 50% PBS, 40% fetal bovine serum (FBS) and 10% DMSO and stored at −80 °C short term or −150 °C long term.
CB LT-HSC scRNA-seq
Human immunophenotypic LT-HSCs were purified from pooled CB samples based on a Lin–CD34+CD38−CD45RA−CD90+CD49f+ immunophenotype (see FACS section below). CB LT-HSCs were purified and submitted to the Hospital for Sick Children Genomics Core for scRNA-seq profiling on the BD Rhapsody platform.
Mice
Animal experiments were done in accordance with institutional guidelines approved by the UHN Animal Care Committee. The following mouse strains (The Jackson Laboratory) were used in this study: NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG; strain 005557), NSG-Tg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ (NSG-SGM3; strain 013062) and NSG-Kitem1Mvw/SzJ (NSGW41). All in vivo experiments were done with 8–12-week-old female or male mice. NSG and NSG-SGM3 were conditioned with 225 cGy of gamma radiation; NSGW41 females were not irradiated. All mice were housed at the animal facility (ARC) at Princess Margaret Cancer Centre in a room designated only for immunocompromised mice with individually ventilated racks equipped with complete sterile microisolator caging (IVC), on corncob bedding and supplied with environmental enrichment in the form of a red house or tube and a cotton nestlet. Rooms were maintained at 21–22 °C and 30–40% humidity with a 12 h–12 h light–dark cycle with sunset and sunrise. Cages were changed at least once a week under a biological safety cabinet. Health status was monitored using a combination of environmental monitoring and evaluation of soiled bedding from sentinel mice.
FACS and flow cytometry analyses
HSPC-enriched human CB cells were thawed via slow dropwise addition of X-VIVO 10 media (Lonza) with 50% FBS and 100 μg ml–1 DNaseI (Roche). Cells were centrifuged at 400g for 10 min, then resuspended in PBS + 5% FBS. The populations sorted for individual experiments are indicated; populations are designated based on the full stem and progenitor hierarchy as previously described37,67. Cells were resuspended at less than 107 cells per millilitre and stained in one or two subsequent rounds for 15 min at room temperature each. See Supplementary Table 22 for antibodies. Cells were washed following staining and resuspended in PBS + 2% FBS and filtered through a 35-μm nylon mesh for sorting or analyses. To isolate xenografted human cells, bone marrow from three to five mice per experimental group was thawed, pooled, mouse depleted using a MACS-based kit (Miltenyi) and stained for cell sorting. Cells were isolated with BD FACSAria Fusion, BD FACSAria III or BD Symphony S6 cell sorters, or analysed on BD FACSCelesta or BD Symphony A1 coupled to a high-throughput system; instruments were controlled using BD FACSDiva software (v9). Flow cytometry data were evaluated using FlowJo (v10.8–10.10).
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