Fig 2: Single-cell genotyping using TARGET-seq (related to Fig. 3). (A) Variant allele frequency (VAF) and coverage of PTPN11 c.226G>A amplicon sequenced from patient ID17 (total of 384 single cells sorted, 365 cells passed QC, coverage cutoff 100). (B) VAF and coverage of PTPN11 c.1508G>C amplicon sequenced from patient ID5 (total of 384 cells single sorted, 280 cells passed QC, coverage cutoff 30). (C) Single-cell mutation profiling: cells plotted as per FACS index data and colored according to their mutational status. Plots showing Lin-CD34+CD38- cells from patient ID17. (D) Single-cell mutation profiling: cells plotted as per FACS index data and colored according to their mutational status. Plots showing Lin-CD34+CD38- cells from patient ID5. (E) Single-cell mutation profiling: cells plotted as per FACS index data and colored according to their mutational status. Plots showing Lin-CD34+CD38+ cells from patient ID17. (F) Single-cell mutation profiling: cells plotted as per FACS index data and colored according to their mutational status. Plots showing Lin-CD34+CD38+ cells from patient ID5. Data acquired with two independent experiments (one per biological replicate).

Fig 3: Expression of Cell Surface G6B, a Cell Surface Protein, Identifies Mutant Clone-Derived HSPCs in Myelofibrosis(A) Left: expression of 6 megakaryocyte markers from a panel of 20 HSPC and megakaryocyte cell surface antigens assayed by mass spectrometry time of flight (CyTOF) shows expression of G6B on CD34+ HSPCs from patients with primary myelofibrosis (PMF), post-essential thrombocythemia myelofibrosis (PET-MF), and post-polycythaemia vera myelofibrosis (PPV-MF) with either JAK2V617F (JAK2+) or calreticulin (mutCALR) driver mutations. Histograms show cell count (y axis) by expression level (x axis). Right: viSNE dimensionality reduction plots on a representative control and myelofibrosis sample for CD9 and G6B, illustrating more substantial differential expression of G6B than CD9 in myelofibrosis versus control cells(B) Flow cytometric analysis of G6B expression on CD34+ Lin- HSPCs showing significant increase in G6B+ cells in myelofibrosis (% GFP+ cells, 28.8% ± 5.5% versus 2.4% ± 1.0%); chart shows mean + SEM (left) and example plot (right) shown, illustrating expression in both CD41+ and negative cells. **p = 0.01 (t test). Controls (N = 8); myelofibrosis (N = 11).(C) Immunohistochemical staining for G6B (diaminobenzidine, DAB brown) of bone marrow biopsy sections from controls and myelofibrosis patients with JAK2V617F and mutCALR-positive myelofibrosis showing marked expansion of G6B+ megakaryocytes and progenitors in myelofibrosis.(D) Mononuclear cells from healthy donors and patients with JAK2V617F+ myelofibrosis were combined and 50 cell “mini-bulk” replicates were sorted from the G6B+ and G6B- fractions for Taqman qRT-PCR to quantify expression of JAK2V617F mutated and wild-type JAK2. Chart shows JAK2V617F relative to wild-type JAK2 expression for all mini-bulks from 3 replicate experiments.(E) Internalization of a CD34 × G6B bi-specific antibody and isotype control antibody conjugated to a pH-sensitive cyanine CypHer5E dye that fluoresces at an acidic pH following internalization. Left: representative images show clear intracellular fluorescence for CD34 × G6B bi-specific but not isotype control. Right: mean fluorescence intensity of cells measured by flow cytometry 30 min after addition of antibody with/without two endocytosis inhibitors, Dynasore and Pitstop 2. Data shown using SET-2 cells, chart shows mean + SEM, ** - P < 0.01 n= 3. See also Figure S7.

Fig 4: CDK6-AS1 is associated with a more immature phenotype(A) Unsupervised hierarchical clustering of healthy hematopoietic samples (GSE98633) (Schwarzer et al., 2017) with the 337-gene signature, subdivided into immature Lin-/CD34+/CD38- of different origins, myeloid monocyte, and granulocyte progenitors (Lin-/CD34+/CD38+/CD123+ CD45RA- CMP and Lin-/CD34+/CD38+/CD123+ CD45RA+ GMP) and more mature hematopoietic cells (CD15+/CD66+ granulocytes, CD36+/GlyA+ erythroblasts, CD14+ monocytes and CD41+/CD42b+ megakaryocytes). Samples were also subdivided into CDK6-AS1 quartiles.(B) t-SNE plot of the healthy CD34+, CMP, GMP, and more committed cell pools from healthy donors clustered according to the expression values of the 337-gene signature.(C) Expression of CD34 antigen in blasts of bone marrow samples of 13 patients with Q1-AML versus 16 patients with Q4-AML at diagnosis. Data are represented as mean ± SEM.

Fig 5: Multipotent Myelofibrosis Hematopoietic Stem and Progenitor Cells (HSPCs) Are Biased for Megakaryocyte Differentiation(A) Left: model of classically defined CD34+ lin- HSPC subpopulations, in which multi-potent cells (HSCs, hematopoietic stem cells; MPPs, multi-potent progenitor cells; LMPPs, lymphoid-primed multi-potent progenitors) are CD38- and down-stream progenitors (CMPs, common myeloid progenitors; MEPs, megakaryocyte-erythroid progenitors; GMPs, granulocyte-monocyte progenitors) are CD38+. CD45RA+ populations (LMPP/GMP) do not have erythroid or megakaryocyte potential. Middle: % of each classically defined HSPC population in the CD34+ lin- compartment, demonstrating increased MPPs and reduced LMPPs in myelofibrosis (MF) compared to controls. Right: % cells expressing CD41, a surface antigen previously shown to identify cells with increased potential for megakaryocyte differentiation, is increased in both CD38- CD45RA- (HSC/MPP) and CD38+ CD45RA- (CMP/MEP) compartments in myelofibrosis (MF patients, N = 23; controls, N = 14, see also Table S1).(B) Representative FACS plot of a healthy donor control and myelofibrosis patient showing gating strategies.(C) Left: FACS analysis of CD41- HSC (top), CD41- MPP (middle), and CD41+ HSC/MPP (bottom) from healthy donors cultured in megakaryocyte differentiation media (with added recombinant human TPO and stem cell factor [SCF]). CD41+ HSC/MPP demonstrate increased potential for megakaryocyte differentiation, with faster acquisition of the mature megakaryocyte antigen CD42 at an early time point (day 6). Right: images of cultures showing enlarged cell size and proplatelet formation (red star) indicative of accelerated megakaryocyte differentiation from CD41+ HSC/MPP. Representative examples of 3 replicate experiments shown.(D) FACS analysis of CD41- HSC, CD41- MPP and CD41+ HSC/MPP from healthy donors cultured for 12–14 days in megakaryocyte (MK), erythroid (Ery), or myeloid (Mye) differentiation media. CD41+ HSC/MPP showed a higher % of mature CD41+42+ megakaryocytes and glycophorin A+ CD71+ erythroblasts and equivalent CD11b/CD14+ myeloid cells versus CD41- fractions. Representative examples of 3 replicate experiments shown. % of total live (7AAD-), single cells shown.(E) Summary chart (left) and representative FACS plots (right) showing percentage of myelofibrosis and control CD41- HSC/MPP cultured in “bi-potent” erythroid and megakaryocyte differentiation media that give rise to megakaryocyte versus erythroid progeny 6 days after plating (gated on live cells). (controls, n = 7; myelofibrosis [MF], n = 8). Charts show mean + SEM,***p < 0.001; **p = 0.01; *p < 0.05). See also Figure S1.