Competitive Repopulating Unit Assay Protocol

Competitive repopulating unit (CRU) assays were performed as previously described^{29 (link)} using limiting numbers of BMNCs from CD45.1 donor mice. Freshly isolated BMNCs were immediately used for BM transplantation, or BMNCs were co-cultured with rMSCs for 6 days in StemSpan SFEM media (STEMCELL Technologies) supplemented with 10 % KnockOut Serum Replacement (Thermo Fisher Scientific), Pen/Strep, 10ng/ml of TPO (PROSPEC) and 20ng/ml of stem cell factor (SCF; PEPROTEC), then collected and CD45⁺ cells were isolated by MACS column (Miltenyi Biotec), and finally a fraction of the cultured cells corresponding to the indicated number (1,000, 2,500, 5,000, 12,500 and 25,000) of initial BMNCs was subjected to BM transplantation. The test cells (CD45.1) were transplanted together with 2 x 10⁵ competitor BMNCs (CD45.2) into CD45.2 recipient mice lethally irradiated (12 Gy, two split doses). The percentage of donor (CD45.1⁺) cells in the PB was determined 16 weeks later by flow cytometry. Mice with more than 1 % in all three lineages (myeloid, B and T cells) were considered positive and the others were defined as negative mice. The CRU frequency was calculated using L-Calc software (STEMCELL Technologies).