Cymevene

Cells were infected with different multiplicity of infection (MOI) of HCMV when cells had reached a confluence of about 80–85% in 12-well plates. To account for the different volume due to different MOI, the viral inoculum was standardized to a final volume of 1 mL with growth medium. After a 2-h virus pre-adsorption at 37 °C, 1 mL of fresh medium was then added to each well. The cells and supernatant were harvested at 1-, 3- and 5-day post-infection (dpi) and in some experiments at 3-, 6-, 18-h before being subjected to total RNA and protein extraction/isolation or enzyme-linked immunosorbent assay (ELISA), respectively. The UV-irradiated HCMV was originated from the same virus stock used for infection experiment and was irradiated using Stratagene Stratalinker^® UV Crosslinker 1800 as described previously [53 (link)]. The same MOI of UV-irradiated HCMV was then used in parallel with the infection experiment. For GCV treatment, HUVEC were infected together with 1-, 10- and 50 µM of GCV (cymevene, Roche, Basel, Switzerland) for 5 days prior to samples being collected for analysis with a TaqMan real-time PCR assay.

Starting at 6 weeks of age, compound Rb-het/Sox2-TK mice were intraperitoneally injected with gancyclovir (GCV) at 100 mg/Kg in HBSS (Cymevene, Roche Pharmaceuticals) or vehicle (HBSS) every 2 weeks during the first 20 weeks of life, and then switched to single injections every 4 weeks, to complete a total of 12 injections. Animals were sacrificed when they were 42 weeks old, tissues were removed, and pituitaries were weighted before processing for immunohistochemistry.
For immunohistochemical analysis, tissue was fixed in formalin at 4ºC, embedded in paraffin wax, and sectioned at a thickness of 5 μm. Sections were stained with hematoxylin and eosin for pathological examination or processed for immunohistochemical analysis with antibodies against mouse Ki67 (Master Diagnostica, SP6), or Sox2 (CST #3728, C70B1).

All HSCT recipients were administered with intravenous tacrolimus (Prograf, Astellas, Tokyo, Japan) or cyclosporine (Sandimmun, Novartis, Basel, Switzerland) for the prevention of GVHD. On detecting CMV viremia, we initiated intravenous infusion of ganciclovir (Cymevene, Hoffmann‐La Roche, Basel, Switzerland) according to the protocol for risk-adapted preemptive therapy for CMV disease after HSCT [23 (link)]. However, in cases of severe neutropenia, anemia, or thrombocytopenia, we administered intravenous foscarnet (Foscavir, Fresenius Kabi, Graz, Austria) instead of ganciclovir. Oral valganciclovir (Valcyte, Hoffmann‐La Roche) was used for maintenance therapy [24 (link)].
All patients with CMV retinitis initially received an intravitreal injection of ganciclovir. In cases of ganciclovir resistance, we administered intravitreal foscarnet [25 (link), 26 (link)]. Patients with reduced CMV viremia who required further systemic treatment for CMV retinitis received the necessary treatment.

Six to eight weeks old SCID/bg mice were used in accordance with the institutional guidelines under the approved protocols. Lungs’ xenografts were induced by 1.5×10⁶ MDA-MB-231/EGFP cells resuspended in 100 μl PBS and administered into tail vein. Mice were randomly divided into groups according to the treatment. For evaluation of the ability of AT-MSC to colonize the lung tissue, 1×10⁶ of AT-MSC was fluorescently stained with Vybrant DiI solution (Invitrogen, Carlsbad, CA) in 1 ml of PBS for 20 min at 37°C.
Therapeutic (CD::UPRT-MSC, HSVtk-MSC or mixture of both in ratio 1:1) or control AT-MSC were administered intravenously into tail vein (1×10⁶ cells in 150 μl PBS) 9 days after the injection of the tumor cells. Treated animals received daily dose of 500 mg 5-FC/kg/day i.p (Ancotil, Valeant, Poland) and/or 50 mg/kg/day of GCV (Cymevene, Roche, Montreal, CA) for 14 days. Prodrugs were administered in two injections concomitantly. Mice were sacrificed on day 30–33 after the tumor cells administration, when the control – untreated animals had to be euthanized.

To evaluate the antitumoral activity, TK-cAd-MSCs were co-cultured with tumoral glioblastoma PG-U87, previously tested in some of our studies [7 (link), 10 (link), 23 (link)]. Both cell types were seeded in co-culture in 96-well plates at a 1:1 cell ratio. Half of the samples received GCV (0.004 μg/μL) (Cymevene; Roche). Every 2 days, the medium was replaced with a fresh medium. Triplicate samples of each co-culture condition were analyzed by BLI on days 1 (before starting GCV treatment), 5, 7, and 9. On day 9, cells were also visualized by fluorescent microscopy. The experiment was repeated three times.