In vivo reconstitution of RSs from MDS-RS HSCs. (A) Representative FACS gating strategy used for analysis of human myeloid (CD33/CD66b/CD15⁺) and lymphoid (CD19⁺) engraftment in xenotransplanted nonobsese diabetic/LtSz-scid IL2Rγc^−/− (NSG) mice transplanted with healthy or SF3B1-mutated MDS-RS human stem and progenitor cells. The number of purified HSCs, CMPs, GMPs, and MEPs transplanted into NSG mice was according to their relative ratios in the patient BM. Patient 4: 1400, 5000, 21 000, and 1400 cells; patient 5: 25 000, 55 000, 49 300, and 25 200 cells, respectively. Top panels show gating in a nontransplanted NSG mice (negative control); bottom panels show analysis in an NSG mouse transplanted with MDS-RS HSCs from patient 4. (B) In vivo human B-lymphoid and myeloid engraftment in BM of NSG mice 20 to 22 weeks posttransplantation of FACS-purified HSCs or indicated progenitors (mean values from 2 or 3 mice per cell population per patient) from 2 SF3B1-mutated MDS-RS. (C) Mean VAF in myeloid cells derived from patient 4 HSCs transplanted in NSG mice. (D) Prussian blue stains from sections of paraffin-embedded BM tissue from NSG mice with human reconstitution from healthy (top left) or SF3B1-mutated MDS-RS HSCs (top right; patient 5). The bottom left panel shows positive control from BM of MDS-RS patient; scale bar, 50 μm. The bottom right panel shows characteristic Prussian blue–positive cells in BM of NSG mice transplanted with MDS-RS HSCs from patient 5; scale bar, 10 μm. (E) Cytospins of erythroid cells purified from BM of patient 5 (supplemental Figure 6), stained with Prussian blue; scale bar, 20 μm. (F) Percentage of RSs of total nucleated BM cells in BM of NSG mice transplanted with purified HSCs. PAT, patient.