Science - USA (2021-07-09)

(N-FL and T21-FL, respectively) obtained at
16 to 19 weeks of gestation and carried out
phenotypic analysis of the HSPC hierarchy
(Fig. 1A and fig. S1B). T21 karyotype of sorted
HSPCs was confirmed by means of droplet
digital polymerase chain reaction (PCR) with
a set of probes against Chr21 (fig. S1C). In
addition, error-corrected targeted sequencing

at 7500× coverage did not reveal any preexisting mutations of GATA1 exon 2 in T21-FL–derived HSPCs (table S1); although rare, preexisting mutations in exon 3 would have been missed ( 24 ).TheimpactofT21ontheHSPChierarchy of fetal liver revealed a 30% increase in the percentage of total phenotypic long-term HSCs (LT-HSCs) and a simultaneous decrease

in short-term HSCs (ST-HSCs) compared with that in N-FL. In addition, an expansion of megakaryocyte-erythroid progenitors (MEPs) wasseeninT21-FLcomparedwithN-FL,as previously reported (figs. S1, D to E, and S2A) ( 25 ). Quantification ofGATA1and its isoforms revealed comparable expression in N-FL and T21-FL HSPC subpopulations, with a gradual

Wagenblastet al.,Science 373 , eabf6202 (2021) 9 July 2021 2 of 13

A Sort HSPC subpopulations from N-FL and T21-FL

Electroporation of RNPs against GATA1/STAG2

Single cell differentiation/ proliferation (17 days)

Differentiation analysis: flow cytometry + genotyping

Cas9 +

Megakaryocytic

Erythroid

Myeloid

BC 230 180

116

66

40

GAT

A1s Control

kDa

ControlSTAG2ko

180

116

66

40

kDa

GAPDH:

ControlGATA1sSTAG2ko

GATA1s/STAG2ko

GATA1s/ST

AG2koControlGATA1sSTAG2ko

GAT

A1s/S

TAG2koControlGAT

A1s STAG2ko

GAT

A1s/ST

AG2ko

0

20,000

40,000

60,000

80,000

100,000

Number of CD45+ cells/well

N-FL:LT-HSC ST-HSC CMP MEP *** *** p=0.07 ****

n = 202n = 68n = 140n = 26n = 118n = 63n = 129n = 38n = 181n = 69n = 119n = 29n = 117n = 69n = 58n = 28

**** **

MEP

ControlGAT

A1s STAG2ko

GAT

A1s/ST

AG2koControlGATA1sSTAG2ko

GATA1s/STAG2ko

0

20,000

40,000

60,000

80,000

100,000

T21-FL: LT-HSC ST-HSC CMP ****** *** *** p=0.08

GATA1s/STAG2ko

0

500

1,000

1,500

2,000

2,500

N-FL LT-HSCs * ** *** *

T21-FL LT-HSCs

n = 26n = 13n = 22n = 13n = 33n = 23n = 27n = 8

GH

Near-clonal xenotransplantation (20 weeks)

Differentiation analysis: flow cytometry + genotyping + morphology

Secondary xenotransplantation (12 weeks)

+

Megakaryocytic

Erythroid

Myeloid

Lymphoid +

DEF

Megakaryocytic (Meg) containing colonies:

Control GATA1s STAG2ko

GATA1s/STAG2ko

GAT

A1s/S

TAG2koControl GATA1s STAG2ko

GAT

A1s/S

TAG2ko

0

20

40

60

80

100

% Lineage Output E M

E, M

Meg, E

Meg, M

Meg, Erythroid (E), Myleoid (M)

LT-HSC

n = 203n = 68n = 144

CD71+, M

Meg, CD71+, M

ST-HSC CMP MEP

n = 26n = 118n = 63n = 130n = 38n = 181n = 69n = 121n = 29n = 126n = 69n = 73n = 30

N-FL:

GATA1s/STAG2ko

GATA1s/ST

AG2koControl GATA1s STAG2ko

GATA1s/STAG2ko

0

20

40

60

80

100

% Lineage Output

LT-HSC ST-HSC CMP MEP

T21-FL:

Fig. 1. GATA1s induces a megakaryocytic bias in hematopoietic stem and
progenitor cells.(A) Experimental overview of in vitro single-cell differentiation/
proliferation assay and near-clonal xenotransplantation. (B) Western blot assay
of GATA1 in combined N-FL CMP and MEP cells, which were CRISPR/Cas9–edited
with control and GATA1s gRNAs (n= 2 experiments). (C) Western blot assay
of STAG2 in combined N-FL CMP and MEP cells, which were CRISPR/Cas9–edited
with control and STAG2ko gRNAs (n= 2 experiments). (D) Proliferation capacity
assessed by the overall number of CD45+cells from in vitro single-cell assay of
individual CRISPR/Cas9–edited cells for N-FL. Numbers of single-cell colonies with
appropriate positive genotype are indicated for each condition (n= 2 experiments).
(E) Proliferation capacity described in (D) for T21-FL (P< 0.0001 for

combined T21-FL versus N-FL,n= 2 or 3 experiments). (F) Proliferation capacity assessed as the number of CD41+megakaryocytic cells in all megakaryocyte- containing colonies from the single-cell assays described in (D) and (E). (G) Lineage output from in vitro single-cell assay described in (D) [P< 0.05 for Meg in GATA1s versus control,P= 0.12 for Meg in GATA1s/STAG2ko versus control, and P< 0.01 for (E) in GATA1s versus control among all cell types,n= 2 experiments]. (H) Lineage output from in vitro single-cell assay as described in (G) for T21-FL [P< 0.001 for Meg in GATA1s versus control,P< 0.01 for Meg in GATA1s/STAG2ko versus control, andP= 0.16 for (E) in GATA1s versus control among all cell types, n= 2 or 3 experiments]. Unpaired Student’sttest: *P< 0.05; **P< 0.01; ***P< 0.001; ****P< 0.0001; error bars represent standard error of the mean.

RESEARCH | RESEARCH ARTICLE

Science - USA (2021-07-09)

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