Figure 4. TOFA suppresses abnormal differentiation of HSCs into myeloid cells.
(A) TOFA rescues stemness of cocultured WT HSPCs. Confluent WT, Fanca−/− or Fancd2−/− MSCs were pretreated with TOFA (8μM) for 48 hrs, and graded numbers of flow sorted WT LSK were plated on confluent stromal layers of WT, Fanca−/− or Fancd2−/− MSCs. Scoring of Cobblestone area as endpoint was determined after 7 days. (B) TOFA prevents abnormal expansion of donor myeloid cells in peripheral blood of irradiated recipient mice. Confluent WT, Fanca−/− or Fancd2−/− MSCs were pre-treated with TOFA (8μM) for 48 hrs, and flow sorted WT LSK were added to the cultures. Five days later, 1×105 WT output cells (CD45.2+) were collected and, along with 3×105 recipient BM cells (CD45.1+), 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). (C) TOFA prevents abnormal expansion of donor myeloid cells in the BM of irradiated recipient mice. Flow analysis of donor chimerism and lineage reconstitution in the BM of the recipients, described in (A), 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). (D) CFU of donor-derived (CD45.2+) bone marrow cells. 2×104 BMMCs isolated from transplant recipients, described in (A), at 4 months post-BMT were plated in triplicates (n=3–5 recipient mice). CFU is the total count of BFU-E, CFU-M, CFU-G, CFU-GM, CFU-GEMM, and Pre-B colonies. (E) CFU of progenitor lineages having significant difference. *P<0.05, **P<0.01, ***P<0.001, ns: not significant. Error bars represent mean ± SD.