Primocin

After NITB sample collection, sample was incubated in Advanced DMEM/F12 (Life Technologies; 12634‐034) supplemented with GlutaMax (Life Technologies; 12634‐034), HEPES (Life Technologies; 15630‐056), penicillin (10,000 IU/ml) and streptomycin (10,000 IU/ml) (Life Technologies; 15140‐122) (AdDF+++) with 100 µg/ml Primocin (InvivoGen; ant‐pm1) for transport. Samples were shipped at 4°C overnight to Utrecht, The Netherlands. Human airway cells were isolated, processed and cultured as described previously (Sachs et al, 2019 (link)). In short, cells were spun down and supernatant was removed. Cells clumps were mechanically sheared using narrowed glass pipette to remove mucus. Cells were resuspended in AdDF+++ supplemented with 100 µg/ml Primocin, 1.25 mM NAc (Sigma‐Aldrich; A9165), 0.15% Pronase E (Sigma‐Aldrich; 7433‐2) and 0.5 mg/ml collagenase (Sigma‐Aldrich, C9407) and incubated for 20 min at 37°C. Samples were washed twice with AdDF+++ with Primocin and spun down at 300× g for 5 min. The resulting pellet was resuspended in ice‐cold 70% 10 mg/ml cold Cultrex growth factor reduced BME type 2 (Trevigen; 3533‐010‐02) in airway organoid medium.

Colonoid monolayers were established from colonoid cultures harvested after 7 days. To establish a monolayer, approximately 200 colonoids were completely disaggregated with the help of a 27‐gauge needle. Approximately 100 μl of cell suspension in IntestiCult^™ Organoid Growth medium (Human) (Stemcell^™ Technologies) with 10 μM Y‐27632 and 100 μg/ml Primocin (InvivoGen) were seeded on permeable polyester filter insert (Corning Transwell, pore size 0.4 μM) coated with 10 μg/ml human collagen IV (Sigma‐Aldrich) solution, in 24‐well plates.²⁷ An additional 50 and 600 μl of IntestiCult^™ Organoid Growth medium with Y‐27632 and Primocin was added to the Transwell filter and the well, respectively, and kept in culture for 3 days. Then, expansion medium was substituted with differentiation medium, where 50% v/v IntestiCult (Component B) was substituted by DMEM/Ham's F‐12. The differentiation medium was replaced with fresh differentiation medium after 2 days and colonoid monolayers were then kept in culture for one additional day to reach 3 days of differentiation.

Early (2p.–7p.) and late (15p.–19p.) passages of endometrial and menstrual blood mesenchymal stem cells were selected to undergo decidualization in vitro. Decidualization was induced by culturing cells with 0.5 μM dibutyryl cyclic-AMP (db-cAMP) (BioGems International, Inc., Westlake Village, CA, USA) and 1 μM medroxyprogesterone acetate (MPA) (Cayman Chemical Company, Ann Arbor, MI, USA) agents at 1.5 × 10⁴ cells/cm² density. Decidualization medium, used for whole differentiation period consisted of: RPMI (phenol free) medium (Corning Incorporated, NY, USA), 2% Charcoal Stripped Fetal Bovine Serum (Gibco, Thermo Scientific, NY, USA) and 50 μg/ml primocin (Sigma Aldrich, MO, USA). Selected decidualization time was 3 and 6 days, cells were cultured under selected conditions of 37°C, 5% CO₂ in an incubator. During decidualization every other day the medium was replaced with the same fresh medium and filtered through 0.2 μm filter. Undifferentiated EndSCs and MenSCs were used as a control group in later experiments.

Mouse skeletal myoblast C2C12 cells, purchased from American Tissue Culture Collection (Manassas, VA), were maintained as previously described17 (link). FACS sorted mouse skeletal muscle satellite cells, obtained from Eric Olson (UT Southwestern Medical Center) and prepared as previously described53 (link), were maintained with daily replenishment of medium consisting of Ham’s F-10 containing 20% FBS with 0.1 mg/ml Primocin (Invitrogen) and 5 ng/mL bFGF (Gibco) on Matrigel (BD Biosciences)-coated plates. To induce differentiation, myoblasts were cultivated in DMEM containing 2% horse serum (with 0.1 mg/ml Primocin for mouse muscle satellite cells) with or without 10 μg/mL insulin (Sigma Aldrich, St. Louis, MO) (DM or DM + I). All cell culture products were purchased from Gibco (Grand Island, NY) unless otherwise noted. Rapamycin and PD 0332991 (Selleckchem, Houston, TX) were resuspended in DMSO and used at concentrations as indicated.

Isolation and purification of hair follicle melanocytes (HFMs) from scalp hairs was carried out as originally previously described in Tobin, et al.^{62 (link)} with modifications^{63 (link)}. Cell lineage identity was confirmed by GP100 and TRP-1 expression. Melanocytes were maintained in full 2:1 melanocyte growth media (2 parts minimal essential medium (MEM) supplemented with 1% foetal bovine serum (FBS), 1% nonessential amino acids, 100 µg/ml Primocin, 2 mM Glutamax, 5 ng/ml basic fibroblast growth factor, and 5 ng/ml endothelin-1 (Sigma, Dorset, UK) mixed with 1 part keratinocyte serum-free medium (K-SFM) supplemented with 25 µg/ml bovine pituitary extract (BPE), 0.2 ng/ml rEGF, 100 µg/ml Primocin (antibiotic), and 2 mM Glutamax in humidified incubators at 37 °C with 5% CO₂ unless stated otherwise.