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. Author manuscript; available in PMC: 2016 Nov 1.
Published in final edited form as: Stem Cells. 2015 Jul 24;33(11):3382–3396. doi: 10.1002/stem.2100

Figure 1. Fanca−/− and Fancd2−/− MSCs impairs WT HSPC self-renewal and induces myeloid expansion.

Figure 1

(A) Schematic representation of the ex vivo coculture experiments. WT LSK (Lin-Sca1+c-kit+) cells isolated by FACS were cultured on confluent stromal layers of WT, Fanca−/− or Fancd2−/− MSCs followed by in CAFC or BM transplantation (BMT). (B) Limited dilution analysis of CAFC assay. Assay was conducted in a flat bottom 96 well plate with confluent MSCs before plating the sorted LSK cells. Cultures were maintained in 40% methyl cellulose medium for two weeks and the colonies were counted on week 1 and 2. Group of at least 6 phase dim cells were counted as one colony. (C) Abnormal myeloid expansion of WT HSPCs cocultured on Fanca−/− or Fancd2−/− MSCs in peripheral blood of irradiated recipient mice. 1×105 WT output cells (CD45.2+) collected after coculturing on WT, Fanca−/− or Fancd2−/− MSCs for five days, along with 3×105 recipient BM cells (CD45.1+), were injected into each lethally irradiated recipient mouse. Donor chimerism and lineage reconstitution in peripheral blood of the recipients were examined at 4 months post transplantation. Representative flow plots (Left) and quantifications (Right) are shown. Results are means plus or minus SD of 3 independent experiments (n=9 per group). (D) Abnormal myeloid expansion of WT HSPCs cocultured on Fanca−/− or Fancd2−/− MSCs in the BM of irradiated recipient mice. Flow analysis of donor chimerism and lineage reconstitution in the BM of the recipients, described in (C), at 4 months post-BMT. Representative flow plots (Left) and quantifications (Right) are shown. Results are means plus or minus SD of 3 independent experiments (n=9 per group). *P<0.05, **P<0.01, ***P<0.001, ns: not significant. Error bars represent mean ± SD.