Supplementary Figure 1 (Page1)

Transcription

1 Supplementary Figure 1 (Page1) A B FSC-A 45 Fitc-A 133 APC-A 19 APC-Cy7-A 34 PE-A 45 Fitc-A FSC-A 45 Fitcs-A C 38 PE-Cy7-A 45 Fitc-A 45 Fitc-A 7AAD FSC-A

2 Supplementary Figure 1 (Page2) D (1) (6) (2) (3) 133 APC-A (4) (5) 10 Alexa Fluor 405-A 19 APC-Cy7-A 133 APC-A 133 APC-A 7 PE-Texas Red-A 10 Alexa Fluor 405-A 10 Alexa Fluor 405-A 45RA BV510-A FSC-A 34 PE-A (7) (8) 34 PE-A 34 PE-A 133 APC-A 133 APC-A (9) (10) 34 PE-A 34 PE-A 33 APC-R700-A 33 APC-R700-A

3 Supplementary Figure 1 (Page3) E F 3 PerCP-eFluor710-A 45 Fitc-A 19 APC-Cy7-A 3 PerCP-eFluor710-A 3 PerCP-eFluor710-A 19 APC-Cy7-A 19 APC-Cy7-A 19 APC-Cy7-A 34 PE-A

4 Supplementary Figure 1 (Page4) G Linkages

5 Supplementary Figure 1 Detailed description of the gating strategies to define beads (A), viable CD34 + cells (B, C), viable WBC (C), CD34 subtypes (D) and mature T- and B-lymphocytes (E, F). The hierarchical gating structure and the linkages used are shown in (G). Beads (A and G): Starting from a plot depicting all events acquired, beads were defined by serial gating in the APC vs SSC and in the APC-Cy7 vs Fitc plots. The invert beads command will depict all events except for beads (first plot in B and C; named WBC in G). Viable CD34 + cells (B and G): Starting from WBC, CD34 + cells were defined by their CD34 positivity, followed by their weak expression of CD45, their typical light scattering properties (FSC vs SSC plot), their negativity for 7AAD (third plot in C), and their CD38 expression (see gate in B, plot 4). Cells with very high CD38 expression were considered non-specific and excluded. Linking 7AAD- cells with those in the CD38 gate resulted in the purified CD34+ cells termed 34 viable (G) which were the basis for CD34 subtyping (D). Viable WBC (C): They were defined by their CD45 expression, their negativity for 7AAD and their FSC/SSC properties. The linkage 45+ OR 34 total adds together viable CD34 + cells (red dots in C) and viable WBC. Together they are named WBC total (G). CD34 subtypes (D): Based on the 34 viable cells (1), typical co-expression patterns are depicted. (2): expression of CD133 is rather weak, but positive and negative cells can be separated, particularly when using contour plots (see (2) vs (7)). (3): The proportion of CD7 + cells was always low, and distinct populations could never be defined. Since non-specific positivity could not be excluded, this subtype was not pursued any further. (4) and (5): In contrast to CD10, CD19 always showed a poor separation of positive and negative cells. (6): CD45RA always allowed separating earlier CD45RA - from more committed CD45RA + cells. These two cell fractions were then further subtyped in CD133 vs CD10 contour plots (7 and 8), resulting in the six CD34 subtypes also shown in Figure 2. The last two plots depict the CD33 vs CD133 expression of CD45RA negative (9) and CD45RA positive cells (10). CD33 expression is higher among the more committed CD45RA + cells. T-cells and CD34 - B-lymphocytes (E and F): CD3 + WBC (E) were defined by their strong CD45 expression. After linkage with 7AAD - cells, they were finally gated in a CD3 vs CD19 plot. CD19 + B-lymphocytes (F) were also gated in a CD3 vs CD19 plot. CD34 co-expressing cells were excluded.

6 Supplementary Figure 2 A (1) (2) (3) 45RA-133+; 45RA (4) 45RA-133-; 45RA (5) 45RA ; 45RA Supplementary Figure 2A: Example representative of auto PBSC after G-CSF mobilization. CD133 vs CD10 contour plots depicting viable CD34 + cells gated for 45RA - (1) and for 45RA + events (2). Histograms: (3) MPP (green) and LMPP (blue), (4) EMP (green) and late GMP (pink), (5) MLP (blue) and BLP (pink).

7 B (1) (2) (3) 45RA-133+; 45RA (4) 45RA-133-; 45RA (5) 45RA ; 45RA Supplementary Figure 2B: Example representative of BMd. CD133 vs CD10 contour plots depicting viable CD34 + cells gated for 45RA - (1) and for 45RA + events (2). Histograms: (3) MPP (green) and LMPP (blue), (4) EMP (green) and late GMP (pink), (5) MLP (blue) and BLP (pink).

8 C (1) (2) (3) 45RA-133+; 45RA (4) 45RA-133-; 45RA (5) 45RA ; 45RA Supplementary Figure 2C: Example representative of patient BM1y. CD133 vs CD10 contour plots depicting viable CD34 + cells gated for 45RA - (1) and for 45RA + events (2). Histograms: (3) MPP (green) and LMPP (blue), (4) EMP (green) and late GMP (pink), (5) MLP (blue) and BLP (pink).

9 Supplementary Table 1: Antibodies and dyes used for CD34 subtyping Antibody/Reagent Fluorochrome Clone Company catalog number Vol. per test b 7AAD a Aminoactinomycin D BD Biosciences, San Jose, CA, USA µl CD45 FITC/ Fluorescein isothiocyanate/ 2D1/ CD34 PE a Phycoerythrin 8G12 BD Biosciences, San Jose, CA, USA µl CD3 PerCP-eFluor 710 Peridinin chlorophyll-efluor 710 SK7 ebioscience, San Diego, CA, USA µl CD7 PE-CF594 Phycoerythrin-CF594 M-T701 BD Biosciences, San Jose, CA, USA ,5µl CD10 BV421 Brilliant Violet 421 HI10a BD Biosciences, San Jose, CA, USA µl CD19 APC-Cy7 Allophycocyanin-Cyanin 7 SJ25C1 BD Pharmingen, San Diego, CA, USA µl CD33 APC-R700 Allophycocyanin-R700 P67-6 BD Biosciences, San Jose, CA, USA µl CD38 PE-Cy7 Phycoerythrin-cyanin-7 HB-7 BD Biosciences, San Jose, CA, USA µl CD45RA BV510 Brilliant violet 510 HI100 BD Biosciences, San Jose, CA, USA µl AC133-1 APC Allophycocyanin AC Miltenyi Biotec, Bergisch Gladbach, Germany ,5µl a BD Stem Cell Enumeration Kit b cell concentration= 5-15x10E6 WBC/ml; sample size= 100µl cell suspension per test