NK cell differentiation

2.1.10.1 EL08.1D2 co-culture

CD34+ cells isolated from cord blood samples or hiPSC-generated CD34+ cells were co-cultured on a monolayer of irradiated EL08.1D2 murine feeder cells in 96-well plates. 500 CD34+ cells were plated per well with the following cocktail of cytokines: IL-3 (week one), IL-15, IL-7, SCF in weeks 1-3 and IL-15 only for week 4 and 5. Fresh medium was added weekly and cells were collected at different time points during differentiation for cell counts and flow cytometry analysis.

2.1.10.2 Feeder-free NK cell differentiation with cytokines

CD34+ cells isolated from cord blood samples or hiPSC-generated CD34+ cells were cultured at a density of 1x105 cells/ml in GBGM (Glycostem basal growth medium) expansion medium (GBGM, 10% Human serum AB, 1x LDC (low-dose cytokine) supplemented with 25 ng/ml SCF, 25 ng/ml Flt3L, 25ng/ml TPO, 25 ng/ml IL-7 and 20 µg/ml Clivarin). After 10 days of expansion, suspension cells were collected and re-plated in GBGM differentiation medium I (GBGM, 10% Human serum AB, 1x LDC mix, 25 ng/ml SCF, 25 ng/ml Flt3L, 20 ng/ml IL-15, 25 ng/ml IL-7 and 20 µg/ml Clivarin) at a density of at least 1x106 cells/ml. On day 14 of differentiation, cells were re-plated in GBGM differentiation medium (GBGM 2% Human serum AB, 1x LDC mix, 20 ng/ml SCF, 20 ng/ml IL-15, 20 ng/ml IL-7 and 1000 U/ml IL-2) at a density of at least 2-3x106 cells/ml. Cell culture medium was refreshed every 2-3 days by replacing 500 µl with fresh medium. 1x LDC mix contained: 10 pg/ml GM-CSF, 250 pg/ml G-CSF and 50 pg/ml IL-6. Cells at different time points during differentiation were collected for cell counts, RNA extraction and flow cytometry analysis.

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Phenotypic analysis by flow cytometry 2.2

Cells were prepared as a single cell suspension in PBS/0.1% FBS and were labelled with a combination of the following fluorochrome-conjugated monoclonal antibodies (mABs): CD16-APC, CD34-APC, CD43-FITC, CD45-PE, CD56-PE, CD94-PerCPCy5.5, CD133-PE, NKp46-APC and NKG2D-APC all obtained from BD Biosciences and CD117-PerCPCy5.5 from Beckman Coulter. Tra-1-60 and Tra-1-81 antibodies were non-conjugated mouse monoclonal IgM antibodies obtained from Santa Cruz Biotechnology used in combination with an AlexaFluor®488 goat anti- mouse IgG (Thermofischer Scientific). Samples were analysed using a BD FACSVerseTM flow cytometer with BD FACSuiteTM and FlowJo software. Control staining with appropriate isotype controls was performed using APC mouse IgG1 κ, FITC mouse IgG1 κ and PE mouse IgG1 κ.

Antibody Specificity Clone Fluorochrome Company Catalogue number

TRA-1-85 Mouse anti- human TRA-1-85 PE BD Pharmingen 563021 CD3 Mouse anti- human SK7 APC-Cy7 BD Pharmingen 557832 CD4 Mouse anti- human SJ25C1 APC-Cy7 BD Pharmingen 557791 CD14 Mouse anti- human HCD14 PerCP-Cy5.5 BioLegend 325621 CD16 Mouse anti- human

VEP13 VioGreen Miltenyi Biotec 130-098-095

CD16 Mouse anti- human 3G8 APC-Cy7 BioLegend 302017 CD34 Mouse anti- human 581 APC BD Pharmingen 555824

69 CD43 Mouse anti- human 1G10 FITC BD Pharmingen 555475 CD45 Mouse anti- human HI30 PE BD Pharmingen 555483 CD45 Mouse anti- human

5B1 VioBlue Miltenyi Biotec 130-098-136

CD56 Mouse anti- human B159 PE-Cy7 BD Pharmingen 557747 CD73 Mouse anti- human AD2 PE-Cy7 BD Pharmingen 561258 CD90 Mouse anti- human 5E10 PE-Cy7 BD Pharmingen 561558 CD94 Mouse anti- human DX22 APC BioLegend 305508 CD133 Mouse anti- human

AC133 PE Miltenyi Biotec 130-080-801

NKp30 (CD337) Mouse anti- human P30-15 APC BioLegend 325209 NKp46 (CD335) Mouse anti- human

9E2 VioBright FITC Miltenyi Biotec 130-104-567

NKp44 (CD336) Mouse anti- human P44-8 PE BioLegend 325107 NKG2A (CD159a) Mouse anti- human

131411 PE R&D Systems FAB1059P

NKG2C (CD159c)

Mouse anti- human

134591 FITC R&D Systems FAB138G

NKG2D (CD314)

Mouse anti- human

1D11 PerCP-Cy5.5 BioLegend 320817

CD107a Mouse anti- human

H4A3 PE BD

Pharmingen

70 Pan KIR2D Mouse anti-

human

NKVFS1 VioBlue Miletnyi Biotec 130-099-042

IgG2a, к Mouse G155-178 PE BD

Pharmingen

556653

IgG1, к Mouse MOPC-21 APC-Cy7 BD

Pharmingen

557873

IgG1, к Mouse MOPC-21 PE BioLegend 400111

IgG1, к Mouse MOPC-21 APC BioLegend 400119

IgG1, к Mouse MOPC-21 FITC BD

Pharmingen

555909

IgG1, к Mouse MOPC-21 PerCP/Cy5.5 BioLegend 400149

IgG1 Mouse IS5-21F5 VioBlue Miltenyi Biotec 130-094-670

IgG1 Mouse IS5-21F5 VioBright FITC Miltenyi Biotec 130-104-562

IgG2a Mouse S43.10 VioBlue Miltenyi Biotec 130-098-898

IgM Mouse IS5-20C4 VioGreen Miltenyi Biotec 130-100-010

IgG1, к Mouse MOPC-21 PE-Cy7 BD

Pharmingen

557872

Table 2.2 Antibodies used for Flow cytometry.

Colony forming unit assay 2.3

Colony forming unit (CFU) assays were performed using the StemMACS HSC-CFU media obtained from Miltenyi Biotec. The medium is based on methylcellulose in IMDM (1%), supplemented with FBS (30%) and the following cytokines: stem cell factor (SCF – 50ng/ml), GM-CSF (20ng/ml), G-CSF (20ng/ml), IL-3 (20ng/ml), IL-6 (20ng/ml) and Erythropoietin (Epo- 3U/ml). It supports the clonal progeny of a

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single-cell to differentiate and grow in distinct colonies including BFU-E, CFU-E, CFU-GEMM, CFU-G, CFU-M and CFU-GM. 2x103 CD34+ cells were added to 2ml StemMACS HSC-CFU media, vortexed vigorously until the cells were well suspended and let tubes sit for 10mins to allow air bubbles to rise. 1ml of the cell suspension was plated in a well of a 12-well plate at a final density of 1x103 cells/well. The outer wells were filled with PBS to avoid evaporation. Cells were incubated at 37°C/5% CO2 for 12-14 days.

CD107a degranulation assay 2.4

NK cells were incubated with K562 cells in a 1:1 effector: target ratio for 2 hours at 37°C/5% CO2 in RPMI/10% FCS. Cells were labelled with a CD107a-PE conjugated antibody from BD and expression was determined via flow cytometry.

Molecular Biology 2.5

Name Purpose Company Catalogue number

TRIzol® reagent RNA isolation Life technologies 015596-018

SuperScript®III First-Strand

cDNA synthesis Life technologies

18080-051

Fast SYBR® Green master mix

qPCR Thermo Scientific 4385612

Table 2.3 Reagents used in Molecular Biology

2.5.1 RNA isolation

Cells for RNA isolation were collected as described in passaging of adherent/suspension cells and pelleted by centrifugation. Supernatant was removed

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and cell pellets stored at -80˚C until RNA extraction. For homogenization, pelleted cells were resuspended in 1ml TRIzol® reagent and incubated at RT for 5 minutes. 0.2ml of chloroform was added per 1ml TRIzol® reagent. Samples were shaken vigorously by hand for 15 seconds. Samples were incubated for 2-3 minutes at RT and centrifuged at 12,000xg for 15 minutes at 4˚C. The aqueous phase was transferred into a new tube and RNA precipitated by the addition of 0.5mL of 100% isopropanol. After incubation at RT for 10 minutes, samples were centrifuged at 12,000xg for 10 minutes at 4˚C. The supernatant was removed and the gel-like RNA pellet was washed with 75% ethanol, by vortexing briefly and centrifugation at 7,500 x g for 5 minutes at 4˚C. After 2-3 washes, the RNA pellet was air-dried for 10-15 minutes at RT and resuspended in 20-50 µl of nuclease-free water. RNA absorbance was measured using nanodrop.