Last, T21-FL GATA1s grafts showed increased
infiltration of myeloid lineage cells into the
spleen compared with N-FL GATA1s (fig. S4R).
To investigate whether the observed lineage
shifts and engraftment patterns were associated
with development of preleukemia or malignant
transformation to full leukemia, we assessed
xenografts for the presence of immature blast
cells as assessed from cytomorphology. There
was a dramatic increase in blasts to ~30 to
40% in T21-FL GATA1s but not in N-FL GATA1s
xenografts(Fig.2,FandG,andfig.S6N).This
was further confirmed through histology,
which showed active blast infiltration with-
in the BM of T21-FL GATA1s grafts but not in
N-FL GATA1s xenografts (fig. S5D). Higherblast percentages of ~50 to 80% were observed
in both N-FL and T21-FL GATA1s/STAG2ko
grafts. Subsequently, we carried out a detailed
flow cytometric analysis of lineage markers
on large nongranulated cells in the blast gate
(fig. S7A). N-FL and T21-FL control and N-FL
GATA1s grafts had no enrichment of this
gated population. The blast population ofWagenblastet al.,Science 373 , eabf6202 (2021) 9 July 2021 4 of 13
ABN-FLBone marrow T21-FLBone marrow C N-FL Bone marrowControlGATA1s
ST
AG2ko020406080100B-Lymphoid-Myeloid-Erythroid-
T-cell-% Lineage marker distributionMegakaryocytic-Marker* * ****ControlGATA1sSTAG2koGATA1s/STAG2ko020406080% CD45+ EngraftmentControlGATA1s
STAG2koGATA1s/STAG2koGATA1s/STAG2ko020406080% CD45+ Engraftment****DT21-FLBone marrowControlGATA1sSTAG2koGATA1s/STAG2ko020406080100B-Lymphoid-Myeloid-Erythroid-
T-cell-Megakaryocytic-% Lineage marker distributionMarker**** ***FControlGATA1sGATA1s/STAG2koN-FL T21-FLBlasts
Myeloid cells (at different maturation stages)
Lymphocytes****
**
*
***** * ***
***
H IGATA1sSTAG2koGATA1s/STAG2koControlEGT21-FL H&E CD45 CD61Control GATA1s
STAG2ko
GATA1s/STAG2koControl GATA1sSTAG2koGATA1s/STAG2ko020406080100% Human cellsN-FL T21-FLUnknownBlast
Myeloid cells (not blast-like)Erythroid precursorsLymphocytesN-FLT21-FLControl
GATA1s
STAG2ko
GATA1s/STAG2koControl
GATA1s
STAG2ko
GATA1s/STAG2ko0 3060 90 120 150 180 210
020406080100Time [d]Percent survivalMedian
Survival:
GATA1s/
STAG2ko
120d (n=5)JK(^003060) 90 120 150 180 210
20
40
60
80
100
Time [d]
Percent survival
Median
Survival:
GATA1s/
STAG2ko
88d (n=5)
Stem-
Lymphoid-
Myeloid-
Erythroid-
Meg-
Markers
Stem-
Lymphoid-
Myeloid-
Erythroid-
Meg-
Markers
Pre- or
GATA1s/ Leukemia
out of CD45+: STAG2- Patient
% CD34+ 14.0 14.3 8.7 –/+
% CD117+ 4.1 4.0 84.2 +
% CD41+ 1.2 4.6 24.9 +
% CD42b+ 0.5 0.6 9.0 +
% CD61+ 0.3 1.0 6.1 +
% CD36+ 8.7 11.2 40.8 +
% CD71+ 2.7 1.9 66.4 +
% GlyA+ 4.6 2.9 30.5 –
% CD4+ 21.0 20.5 88.7 +
% CD7+ 9.0 7.3 77.6 +
% HLA-Dr+ 85.7 87.3 17.4 –
% CD56+ 3.3 1.5 2.7 –/+
% CD33+ 20.7 21.4 91.2 +
% CD11b+ 8.3 9.12 4.4 +
% CD13+ 2.8 2.5 0.4 –/+
% CD14+ 7.6 7.5 1.6 –
Control GATA1s
N-FL Pre- or
GATA1s/ Leukemia
out of CD45+: STAG2- Patient
% CD34+ 4.7 29.5 21.3 –/+
% CD117+ 8.8 84.1 88.7 +
%CD41 1.3 31.0 12.4 +
%CD42b 1.6 8.1 21.4 +
%CD61 0.2 5.3 1.4 +
%CD36 4.9 3.4 7.1 +
%CD71 8.4 66.9 84.0 +
%GlyA 24.3 78.8 96.0 –
%CD4 26.6 65.3 72.4 +
%CD7 15.3 80.2 89.8 +
%HLA-Dr 63.5 25.9 1.6 –
%CD56 19.6 12.5 10.1 –/+
%CD33 29.0 87.6 88.7 +
%CD11b 9.3 2.8 3.7 +
%CD13 3.5 0.5 0.1 –/+
%CD14 9.3 1.6 0.3 –
Control GATA1s
T21-FL
Fig. 2. T21 is required for preleukemia initiation but dispensable for leukemia
development.(A) Engraftment of N-FL LT-HSC grafts in NSG mice. Engraftment
was assessed on the basis of human CD45+expression in BM (only mice with >1% of
CD45+cells in BM and >90% CRISPR/Cas9 efficiency were taken for the analysis;
n= 3 cohorts). (B) Engraftment as described in (A) for T21-FL (n= 4 cohorts).
(C) Lineage marker distribution based on cell surface markers in N-FL grafts in NSG
mice. (D) Lineage marker distribution as described in (C) for T21-FL.P< 0.05
for T21-FL control myeloid cells versus N-FL control myeloid cells, andP< 0.05 for
T21-FL control lymphoid cells versus N-FL control lymphoid cells. (E) Hematoxylin
and eosin (H&E) and IHC stainings for human CD45 and human megakaryocytic
marker CD61 in humeri of T21-FL grafts. Scale bar, 50mm. (F) Morphological
analysis of human cells in primary xenografts of N-FL and T21-FL grafts.
Human cells were prepared by using cytospin and stained with Giemsa
(100× magnification). Scale bar, 10mm. (G) Quantification of cell morphology as
seen in (F) (n= 400 cells per condition). (H) Percent expression of cell surface
markers within the CD45+blast population in N-FL grafts in NSG mice. Data
are from pooled samples of multiple xenografts. (I) Percent expression of cell
surface markers as described in (H) for T21-FL. Data are from pooled samples of
multiple xenografts. (J) Survival curve of N-FL LT-HSC grafts in NSGW41 mice
(n= 5 mice per condition). (K) Survival curve as described in (J) for T21-FL
(n= 5 mice per condition). Unpaired Student’sttest: *P< 0.05; *P< 0.001;
**P< 0.0001; error bars indicate standard deviation.
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