GSM8464613: Untreated 3D system mimicking bone marrow and containing CD117+ MSC, HMEC1 and primary CD34+ selected MNCs, scRNASeq; Homo sapiens; RNA-Seq
3D culture was performed in inserts with a membrane of 3 μm pores placed in a 6-well plate. Sterilized ceramic biphasic calcium phosphate particles (BCP) were immersed in PBS and loaded into the insert. BCP particles were incubated for 2 days in PBS with 10% FBS. Cells (1.4x106 of primary pAML MSCs and 0.7x106 HMEC-1) were resuspended in a 50/50 combination of osteoblastic differentiation medium (DMEM, 10% FBS, 1% penicillin-streptomycin, 0.1 M dexamethasone, 10 mM β-glycerophosphate, 250 µM ascorbic acid) and HMEC-1 medium (MCDB-131, 10% FBS, 1% penicillin-streptomycin, 1% glutamax 100X, 1 mg/L hydrocortisone, 10 μg/L epidermal growth factor) and added to the insert. Medium was changed twice a week for 3 weeks. Then, 1x106 of healthy pediatric BM mononuclear cells, positively-selected for CD34 (EasySepTMHuman CD34 Positive Selection Kit II following the manufacturer's instructions), were added. The medium was replaced by a mix of HMEC-1 medium and IMDM with 10% FBS, 1% penicillin-streptomycin, and 1% glutamax 100×. The next day, 1 µM of aracytidine was added or not. Medium was changed twice a week for 1 week. Disks formed in the insert were washed twice with PBS, and then retrieved by cutting the membrane of the insert. Disks were incubated for 10 min at 37°C in 5 mL of IMDM medium with 10% FBS, 1% penicillin–streptomycin, and collagenase (0.4 mg/mL), centrifuged, resuspended in 5 mL PBS and recentrifuged. The disks were then incubated 1 min in 300 μL of trypsin (in EDTA 0.05%) at 37°C. The remaining clots were finally dislocated by flushing. The reaction was stopped with 1 mL FBS. After centrifugation, cells were resuspended in 3 mL PBS containing 10% FBS and filtered through a 35 µm nylon mesh incorporated into the cap of a Falcon flow cytometry tube, and then centrifuged. Cells were resuspended in 50 µL PBS with 2% FBS. 35 µL were used for single-cell isolation. Gene expression and cellplex libraries were generated with the Chromium Single Cell 3′ v3.1 assay with feature barcoding (10X Genomics) and sequenced on the NovaSeq 6000 platform (Illumina, SP flow cell) to obtain around 100,000 reads per cell for gene expression and 5,000 reads per cell for cellplex