Lmwha

Human EnSC were isolated from endometrial biopsies as described previously.²⁸ Briefly, endometrial biopsies were subjected to enzymatic digestion using 500 µg/mL of collagenase type Ia (Sigma‐Aldrich, Poole, UK) and 100 µg/mL of DNase I (Lorne Laboratories Ltd, Reading, UK) for 1 hour at 37°C. Digested tissue was filtered through a 40 µM cell strainer to remove glandular cell clumps, and the flow‐through collected and cultured in DMEM/F12 (Thermo Scientific, Loughborough, UK) containing 10% dextran‐coated charcoal‐treated fetal bovine serum (DCC‐FBS), 1 × antibiotic‐antimycotic mix, 10 µM of L‐glutamine (Thermo Scientific), 1 nM of estradiol, and 2 μg/mL of insulin (Sigma‐Aldrich). Cells were lifted with 0.05% trypsin and re‐seeded as required. To induce decidual transformation, confluent EnSC monolayers were downregulated in phenol‐free DMEM/F‐12 media containing 2% DCC‐FBS and decidualized with 10 µM of medroxyprogesterone acetate (MPA) and 0.5 mM of 8‐bromo‐cAMP (C+M treatment) (Sigma‐Aldrich). To study the effect of exogenous HA, decidualizing EnSC were treated with either 100 µg/mL of HMWHA (molecular mass ~1320 kDa) or LMWHA (molecular mass ~33.0 kDa), purchased from Bio‐Techne (USA), as described elsewhere.²⁹, ³⁰ Medium was refreshed every 2 days. All experiments were performed at passage 2.

The uNK cells killing assay has been described in detail previously.⁸, ¹⁵ Briefly, EnSC were seeded at a density of 50 000 cells/well in 96‐well plates and decidualized for 6 days. Isolated uNK cells from whole endometrial tissue (n = 25 000) were then added to the cultures in the presence of C+M. Cultures continued for a further 2 days before harvesting for either RNA extraction or SA‐β‐Gal activity measurement. For experiments investigating the expression of CLU and IL1RL1, experiments were scaled to 6‐well plates. Conditioned media was collected prior to lysis of cells. To block CD44, isolated uNK cells were incubated with the CD44 neutralizing antibody Hermes‐1 (Thermo Scientific, MA4400, 10 μg/ml, 30 min, RT) prior to co‐culture assay. Where appropriate, 100 µg/mL of HMWHA or LMWHA (Bio‐Techne, MN, USA) or BCM were added simultaneously with uNK cells at the indicated timepoints. When indicated, HMWHA or BCM^‐ was pre‐incubated with 300 IU/mL of recombinant HYAL2 (Sigma‐Aldrich) for 30 minutes prior to the addition to co‐cultures.