Figure 3: Engraftment and genotype of human RARS-originated and control haematopoietic cells in the bone marrow of NOD/SCID/IL2rγ^−/− mice.

(a) Schematic representation of the xenograft model. Right-hand side panel shows the flow cytometry profile of bone marrow cells recovered from one human-cell-engrafted mice. The majority of human CD45-expressing cells were positive for a myeloid marker CD33⁺ in all analysed cases in this study. (b) Percentage of human CD45⁺ cells in the bone marrow of NSG mice at 6 and 18–20 weeks after transplantation (MDS1, n=3; MDS2, n=1; MDS3, n=3 and MDS4, n=1). (c) Percentages of human haematopoietic cells and ratio of CD19⁺/CD33⁺ isolated from the bone marrow of mice engrafted with either MDS-RS (n=4 patients and transplanted in a total eight mice) or healthy controls CD34⁺ cells (n=3 healthy donors and transplanted in a total of 11 mice) or congenital sideroblastic anaemia patient (n=1 patient transplanted in two mice). The y axis (left) represents the percentage of the human CD45⁺ cells present in the total mouse bone marrow. The y axis (right) represents the ratio of the human CD19⁺ versus human CD33⁺ cells within the human CD45⁺ cells recovered following xenotransplant. MDS xenografts showed a significant skewing towards myeloid lineage. *P<0.05, ****P<0.0001 (t-test). (d) Targeted mutational analysis shows the presence of concordant SF3B1 mutations in primary CD34⁺ bone marrow sample (grey) and xenograft (black) in all analysed cases. Three independent PCR/sequencing experiments were performed to confirm/determine the mutant allele burden throughout the experiments. The sequencing coverage across the SF3B1 amplicons was ≥1,000 reads. *P<0.01 (t-test).