Collagenase 2 solution

On day 8 after MSC/IL-12 cells administration tumors were excised, single-cell suspension was obtained using collagenase II solution (500 U/ml; Gibco BRL, Paisley, UK). Red blood cells were lysed using 0.15 M ammonium chloride (Sigma Aldrich, St Louis, MO, USA). Dead cells were removed by centrifugation using Histopaque-1077 gradient (Sigma Aldrich, USA). To identify the subpopulations of macrophages, antibodies against the following antigens were used: CD45, F4/80, CD206 and CD86 (eBioscience, USA) or isotype-matched control antibodies. All FACS-analyzed (BD FACSCanto, BD, USA) populations were gated in a 7-AAD window to enrich for viable cells. 7-AAD^-CD45⁺ F4/80⁺ CD86⁺ cells were considered as M1 macrophages, 7-AAD^-CD45⁺ F4/80⁺ CD206⁺ cells were considered as M2 macrophages.

Flow cytometry was used to determine the subsets of macrophages in the ischemic muscles. Seven days after the artery ligation and hADSCs, mADSCs, or PBS⁻ administrations, the muscles were collected, minced, and digested with collagenase II solution for 1 h at 37 °C (500 U/mL; Gibco). The red blood cells were lysed using 0.15 M ammonium chloride solution (Sigma Aldrich). The cell suspensions were filtered through 70-μm cell strainer. The dead cells were removed by centrifugation using Histopaque-1077 gradient (Sigma Aldrich). Cells were incubated with rat anti-mouse CD16/CD32 blocking antibody (eBioscience) and then incubated with anti-CD45 (BioLegend), anti-F4/80 (eBioscience), anti-CD206 (Bio-Rad), and anti-CD86 (Biologend) antibodies for 30 min. All FACS-analyzed populations were gated in a 7-AAD (eBioscience) window to enrich for live cells. To analyze the macrophage phenotype, 7-AAD-viable muscle derived cells and CD45 (to identify lymphoid cells) were gated and then F4/80 (to identify macrophages) and CD206 (to identify CD45⁺/ F4/80⁺/ CD206⁺; M2 macrophages) and CD86 (to identify CD45⁺/ F4/80⁺/ CD86⁺, M1 macrophages). Gates dividing negative from positive cells were based on the isotype antibody control probes in the flow cytometric analysis (BD FACSCanto, BD) [23 (link)].

At 90 dpi, 5 hearts per group were collected for flow cytometry. Hearts were washed with PBS to remove peripheral blood, minced, and incubated in collagenase II solution (Thermo Fisher Scientific, Waltham, MA, USA) for 4 h at 37 °C. The tissue homogenate was then strained through a 40-micron cell strainer, layered on Lympholyte-M (Cedarlane Laboratories Ltd. Burlington, Canada), and centrifuged at 500× g for 20 min. Lymphocytes were collected from the interface of the liquid layers and filtered again through a 40-micron strainer. Lymphocytes were pooled in each group and stained with CD3-FITC, CD4-PerCP-Cy5.5, and CD8-BV711 antibodies (Becton Dickinson, Franklin Lakes, NJ, USA). All fluorophores were standardized with isotype controls (Becton Dickinson, Franklin Lakes, NJ, USA) and compensation using fluorescence minus one (FMO) standards. Additionally, a PE-conjugated IE1-specific tetramer (H-2L^d, YPHFMPTNL) synthesized by the National Institutes of Health Tetramer Core Facility at Emory was used to determine MCMV-specific CD8+ T cells. Flow cytometry was conducted on a Fortessa X-20 machine (Becton Dickinson, Franklin Lakes, NJ, USA), and data were analyzed using FlowJo software (Becton Dickinson, Franklin Lakes, NJ, USA).

Primary osteoblasts were isolated from long bones according to the protocol of Bakker et al. [48 (link)]. Briefly, femur and tibia are scrapped with a scalpel, epiphyses are cutted and bone marrow was flushed out with PBS using a 5 ml syringe and a 25 gauge needle. The clean diaphyses were then cutted into 1-2 mm² pieces and incubated in 4 ml collagenase II solution (2 mg/ml, Gibco, Thermo Fisher Scientific) for 2h at 37°C in a shaking water-bath to remove remaining soft tissue and adherent cells. After washing, the bone pieces were transferred in a 25 cm² flask in complete medium consisting of alpha-MEM (Lonza) supplemented with 10% fetal calf serum (Hyclone, Thermo Fisher Scientific), glutamine (2mM, Sigma-Aldrich), ascorbic acid (50 μg/mL, Sigma-Aldrich). After 3 weeks of culture, cells were seeded in differentiation medium consisting of complete medium supplemented with CaCl₂ (1.4 mM, Sigma-Aldrich) and μ-glycerophosphate (50 mg/mL, Sigma-Aldrich).

A 37-micron reversible strainer was placed in each AggreWell™ 400 (Sigma, St. Louis, United States) well from which EBs were harvested, the EB suspension was transferred to a filter, and the well’s surface was rinsed with DMEM/F12 (Sigma, St. Louis, United States) to collect any remaining EBs. Flip the 37-micron mesh and place it on a clean centrifuge tube. Embryoid bodies collected on the filter were resuspended in a medium and incubated for 2 days. Half-chance the medium with 2.5 mL/well of EB Medium B every 2–3 days. Collagenase II solution was prepared (2,500 U/mL, #12,604-013; Thermo Fisher, United States). The EBs were resuspended in Collagenase II solution. Then, 3 mL of TrypLETMExpress (Thermo Fisher Scientific, United States) was added and mixed, and incubation was continued. After that, the cells were centrifuged at 200–300 g for 3–5 min. The cells were resuspended in RoboSepTM buffer (Stemcell, #20104) and incubated according to the EasySepTM human CD34 positive selection kit II (Stemcell, #17856) technical manual for CD34 positive cell sorting and purification.