In vitro multi-lineage differentiation assay
CD19-APCR700, CD10-BV510
In vitro assay was performed in non-tissue culture-treated 96-well flat bottom plate (Falcon). 2 h before cell seeding, plates were coated with StemSpan Differentiation Coating Material (Stem Cell Technologies) according to manufacturer specifications. Primitive subpopulations (Lin- CD34+CD38- CD90+CD45RA- and Lin- CD34+CD38- CD90- CD45RA-) derived from either BM or MPB sources, were seeded in SFEM II medium (Stem Cell Technologies) supplemented with hSCF (100 ng/ml), hFLT3 (10 ng/ml), hIL-7 (100 ng/ml), hIL-2 (10 ng/ml) (Novartis), hTPO (75 ng/ml), hIL-6 (40 ng/ml), hIL-3 (10 ng/ml), hIL-11 (50 ng/ml), hEPO (0.1 U/ml) (Peprotech), hIL-4 (10 ng/ml) (Miltenyi Biotec), hLDL (4 µg/ml) (Stem Cell technologies). Medium change was performed every 3-4 days. After 3 weeks of culture, cells were harvested and labeled with the following anti-human antibodies:Mouse anti-human CD235a-PE (Clone: GA-R2/HIR2; BD Biosciences, 561051), Verified Reactivity: Human (QC Testing); Application: Flow cytometry (Routinely Tested) 1:100Mouse anti-human CD33-BB515 (Clone: WM53; BD Biosciences, 564588), Verified Reactivity: Human (QC Testing); Application: Flow cytometry (Routinely Tested). Dilution: 1:50Mouse anti-human CD7-BB700 (Clone: M-T701; BD Biosciences, 566488), Verified Reactivity: Human (QC Testing), Rhesus, Cynomolgus, Baboon (Reported); Application: Flow cytometry (Routinely Tested). Dilution: 1:50Mouse anti-human CD71-BV711 (Clone: M-A712; BD Biosciences, 563767), Verified Reactivity: Human (QC Testing); Application: Flow cytometry (Routinely Tested). Dilution: 1:20Mouse anti-human CD19-APCR700 (Clone: SJ25C1; BD Biosciences, 659121), Verified Reactivity: Human; Application: Flow cytometry. Dilution: 1:20Mouse anti-human CD3-BV605 (Clone: OKT3; Biolegend, 317322), Verified Reactivity: Human; Application: Flow cytometric analysis of antibody surface-stained cells. Dilution: 1:50Mouse anti-human CD56-PC5 (Clone: 5.1H11; Biolegend, 362516), Verified Reactivity: Human; Application: Flow cytometric analysis of antibody surface-stained cells. Dilution: 1:50Mouse anti-human CD34-BV421 (Clone: 561; Biolegend, 343610), Verified Reactivity: Human; Application: Flow cytometric analysis of antibody surface-stained cells. Dilution: 1:20Mouse anti-human CD1a-APC (Clone: HI149; BD Biosciences, 561755), Verified Reactivity: Human (QC Testing); Application: Flow cytometry (Routinely Tested) 1:33Mouse anti-human CD5-BUV737 (Clone: UCHT2; BD Biosciences, 612842), Verified Reactivity: Human (QC Testing); Application: Flow cytometry (Routinely Tested) 1:33Mouse anti-human CD42b-BV786 (Clone: HIP1; BD Biosciences, 740976), Verified Reactivity: Human (Tested in Development); Application: Flow cytometry (Qualified) 1:20Mouse anti-human CD41-PC7 (Clone: HIP8; Biolegend, 303718), Verified Reactivity: Human; Reported Reactivity: African Green, Baboon, Capuchin Monkey, Cynomolgus, Rhesus; Application: Flow cytometric analysis of antibody surface-stained cells. 1:50Mouse anti-human CD10-BV510 (Clone: HI10a; Biolegend, 312219), Verified Reactivity: Human, Cynomolgus, Rhesus; Reported Reactivity: African Green, Baboon, Capuchin monkey, Chimpanzee; Application: Flow cytometric analysis of antibody surface-stained cells. 1:100Mouse anti-human CD15-APCfire750 (Clone: W6D3; Biolegend, 323041), Verified Reactivity: Human; Application: Flow cytometric analysis of antibody surface-stained cells. 1:50All the antibodies were purchased from Biolegend and BD Biosciences and they are well characterized and validated by providers. The stained samples were acquired through BD FACS Symphony A5 (BD Biosciences) cytofluorimeter after Rainbow bead (Spherotech) calibration.Technically validated results were always included in the analyses, and we did not apply any exclusion criteria for outliers.