Human bone marrow mscs

Human Bone Marrow-MSCs (Lonza, Verviers, Belgium) were maintained in MSGM bullet kit (Lonza). They are positive for CD29, CD44, CD105, and CD166 and negative for markers of the hematopoietic lineage, such as CD14, CD34, and CD45. The human PCa-derived PC3 and DU145 cell lines (DSMZ, Braunschweig, Germany) were maintained in RPMI medium (Sigma-Aldrich, Milano, Italy) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Sigma-Aldrich), 0.2 mg/ml penicillin/streptomycin and 0.1% (w/v) L-glutamine (Sigma-Aldrich) at 37°C in a 5% CO₂ fully humidified atmosphere. The human breast cancer cell line MCF-7 (HTB-22) (ATCC, Rockville, MD, USA) was maintained in DMEM medium (Sigma-Aldrich) supplemented with 10% FBS; penicillin/streptomycin, L-glutamine, 1mM sodium piruvate (Gibco, Italy) and 1% MEM non-essential amino acid solution (Sigma-Aldrich).

GFP-labeled MSCs were prepared by labelling human bone marrow-MSCs (Lonza, Basel, Switzerland) with lentivirus GFP as previously described^{32 (link)}. GFP-labeled Muse cells were isolated as SSEA-3-positive cells from GFP-MSCs by fluorescence-activated cell sorting (FACS) as previously described^{10 (link),11 (link)}. We confirmed the viability of Muse cells and MSCs by trypan blue or PI staining every time, and found that ~ 98% of the cells were alive. The above GFP-MSCs and -Muse cells were frozen in a Beissel freezing container (Nihon Freezer, Tokyo, Japan), and kept in liquid nitrogen until use.

Nano-lantern-labeling with bioluminescence resonance energy transfer (BRET) efficacy is a bright luminescent protein that allows the detection of even a small number of transplanted cells^{35 (link)}. Human bone marrow-MSCs (Lonza) were labeled with Nano-lantern as previously described^{35 (link)}. Nano-lantern-labeled Muse cells were isolated from Nano-lantern-labeled MSCs as SSEA-3-positive cells by FACS. For ex-vivo dynamics of IV-injection, vehicle (HBSS), Nano-lantern-labeled-MSCs and -Muse cells (1.0 × 10⁵ cells/250 μl) were intravenously injected when mice were 14 weeks old. After 7 days, animals were sacrificed under deep anesthesia and analyzed as described previously^{24 (link)}.