Figure 3
Treatment of c-Kit-enriched murine BM cells with p18SMI compounds enhances HSC expansion.
(A) The average number of total cells, HSPCs (Lin−Sca1+) and long term (LT)-HSCs (Lin-Sca1+CD48−CD150+) were analyzed by flow cytometry. (B) Single CD34−LKS cell were deposited into each well of a 96-well plate, along with differentiation medium plus different concentrations of p18SMI compounds to show the dose-response effect of p18SMI compounds. (C) CAFC-LDA assay of cells treated with XIE18-6, compound 40 and DMSO to reveal the frequency of primitive HSCs. Above data (A–C) is mean ± SEM for all experiments of two or more and were performed in duplicate or triplicate. (D) Experimental design for transplantation (n = 10). CD34-LKS cells were sorted into 96-well plates at 50 cells/well and cultured for 3 days with cytokine plus 20 μM of compounds or DMSO. Cells from each well were mixed with 2 × 106 freshly isolated bone marrow MNCs and co-transplanted by tail-vein injection into lethally irradiated CD45.2 recipient mice. 16 weeks after transplantation, bone marrow MNCs were stained to determine the engraftment level of donor cells. Bone marrow MNCs were also stained for lineage analysis. Multi-lineage differentiation was examined by flow cytometry. GM, T and B indicate lineages for myeloid, T and B cells, respectively. (E,F) The engraftment levels at 16 weeks after primary transplantation and multi-lineage differentiation profile. Data is mean ± SEM for all experiments (***p < 0.001, **p < 0.01).
