Mesencult stimulatory supplement

UC-MSC were isolated from the umbilical cords of healthy neonates using the direct plastic adherence method. Briefly, the UC samples were sheared into long segments and washed to remove residual cord blood. Then, blood vessels and clots were scraped and washed away, and UC tissue was minced and digested using collagenase (Nordmark Biochemicals, German) and hyaluronidase (Sigma-Aldrich, Germany). Cells were then plated and incubated in a 5% CO2 atmosphere at 37 °C in MesenCult^™ Basal Medium (STEMCELL Technologies, France) supplemented with 10% (v/v) MesenCult^™ Stimulatory Supplement (STEMCELL Technologies, France) and 1% antibiotic-antifungal (v/v, Sigma-Aldrich, Germany). UC-MSC propagated to the 3rd–8th passages were characterized using accepted MSC-positive markers anti-CD73, CD90, and CD105 (BD Pharmingen, BD Biosciences, CA, USA), negative hematopoietic markers anti-CD45, CD34, CD11b, CD19, and HLA-DR (BD Stemflow PE hMSC Negative Cocktail, BD Biosciences, CA, USA) and cellular viability (BD 7-AAD, BD Biosciences, CA, USA) by flow cytometry (BD FACSCalibur™ Flow Cytometer; BD Biosciences, CA, USA) (data not shown).

MesenCult MSC Basal Medium and MesenCult Stimulatory Supplements were obtained from StemCell Technologies Inc. (Vancouver, Canada). Plasmid DNA coding luciferase enzyme (pGL-3) and Cx43-siRNA was obtained from Sangon Biotechnology Co. Ltd. (Shanghai, China). Plasmid DNA coding for HSV-tk was purchased from InvivoGen Biotechnology Co. (San Diego, USA). GCV was obtained from Haikou Qili Pharmaceutical Co., Ltd. (Hainan, China). 1,1'-Dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (CM-Dil) was obtained from Invitrogen Life Technologies (Carlsbad, USA). Calcein acetoxymethyl ester (calcein-AM), 1% crystal violet and 4',6-diamidino-2-phenylindole (DAPI) were purchased from Yeasen Biotechnology Co. Ltd. (Shanghai, China). Dulbecco's modified Eagle's medium (DMEM) and foetal bovine serum (FBS) were purchased from Gibco BRL (Gaithersburg, USA). Iron (III) acetylacetonate (99%) and 4-biphenylcarboxylic acid (95%) were purchased from Acros Organics (Geel, Belgium). Polycaprolactone (MW = 14,000), oleic acid (90%), and branched polyethylenimine (PEI, MW = 25,000) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, USA). Chloroform and tetrahydrofuran in chemically pure were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

Bone marrow cells were seeded at 2.5 × 10⁴ cells/cm² and maintained in MesenCult MSC Basal Medium supplemented with MesenCult Stimulatory Supplements (STEMCELL Technologies, Vancouver, BC, Canada) in accordance with the manufacturer’s instructions for 2 weeks, after which the number of CFU-F was scored. Clusters of ≥32 cells were considered colonies. Thirty-two cells were chosen because this represents a population of cells derived from at least five population doublings of a single cell, thereby discounting transit-amplifying cells [19 (link)]. GFP fluorescence was detected using an EVOS FL microscope (Life Technologies, Carlsbad, CA, USA).