Science - USA (2021-07-09)

(Antfer) #1

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


Control Chr21 miRNAs

FSC-A

SSC-A

CD34-BV421

CD117-APC

N-FL N-FL

CD45+ Cells:

Out of CD45+:

ABSort LT-HSCs
from N-FL

Lentiviral transduction of
miR-99a, miR-125b-2 and miR-155
(Chr21 miRNAs)

Xenotransplantation
(12 weeks)

Differentiation analysis:
flow cytometry


  • Megakaryocytic

  • Erythroid

  • Myeloid

  • Lymphoid


SFFV mOrange

CD

F H

J

E

I K

GSort LT-HSCs
from T21-FL

Xenotransplantation
(20 weeks)

Electroporation of RNPs

Cas9
GATA1s
STAG2ko

Cas9
miR-99a KO
miR-125b-2 KO
miR-155 KO

Leukemic analysis:
flow cytometry + genotyping

+


  • Blast


24h

T21-FL
Control KO
GATA1s

T21-FL
Control KO
GATA1s/STAG2ko

T21-FL
Chr21 miRNAs KO
GATA1s

FSC-A

SSC-A

CD34-BV421

CD117-PE

CD45+ Blast:

T21-FL
Chr21 miRNAs KO
GATA1s/STAG2ko
Out of CD45+:

ControlGAT

A1s

GATA1s/STAG2ko

ControlGATA1s

GATA1s/STAG2ko

0

20

40

60

% CD45+ Engraftment

Control
KO

Chr21
miRNAs KO

Control

Chr21 miRNAs

Control

Chr21 miRNAs

0

20

40

60

80

100

B-Lymphoid-

Myeloid-

Erythroid-
T-cell-

Megakaryocytic-

% Lineage marker distribution

Marker

Bone
marrowSpleen

N-FL

****

***

Control
Chr21 miRNAs

Control
Chr21 miRNAs

0

20

40

60

80

100

% CD45+mOrange+ Engraftment

Bone
marrowSpleen

N-FL

**** ****

ControlGATA1s

GATA1s/STAG2ko

ControlGATA1s

GATA1s/STAG2ko

0

20

40

60

80

% CD117+CD45+ (out of blast gate)

Control
KO

Chr21
miRNAs KO

ControlGATA1s

GATA1s/STAG2ko

ControlGATA1s

GATA1s/STAG2ko

0

20

40

60

80

100

% Human cells
Unknown

Blast
Myeloid cells
(not blast-like)

Erythroid
precursors

Control
KO

Lymphocytes

Chr21
miRNAs KO

Control

Chr21 miRNAs

0

20

40

60

80

100

% Human cells

N-FL

Unknown

Erythroid
precursors

Lymphocytes

Myeloid cells
(not blast-like)

Blast

miR-99a-5p
miR-125b-2-5p

miR-155-5p

let-7c-5p

1

2

4

8

16

32

Relative expression to N-FL

(normalized by

RNU48

)

T21-FL
LT-HSC

* **

Fig. 5. Chr21 miRNAs predispose to preleukemia.(A) Relative expression of
Chr21 miRNAs in T21-FL LT-HSCs compared with N-FL as measured with reverse-
transcription quantitative PCR (n= 3 replicates per condition). (B) Overview
of lentiviral mediated overexpression of Chr21 miRNAs in N-FL LT-HSCs used for
primary xenotransplantation into NSG and NSGW41 mice. (C) Engraftment of
transduced control and Chr21 miRNAs in N-FL LT-HSCs transplanted into NSG
mice (only mice with >1% CD45+cells in BM were analyzed,n= 9 or 10 mice per
condition). (D) Lineage marker distribution based on cell surface markers of
engrafted NSG mice in (C). (E) Quantification of cell morphology of human cells
prepared by using cytospin in grafts described in (C) (n= 400 cells per
condition). (F) Flow cytometry plots out of CD45+cells in primary xenografts
described in (C). (G) Experimental overview of sorting T21-FL LT-HSCs for
CRISPR/Cas9 editing with miR-99a, miR-125b-2, and miR-155 gRNAs and


subsequently with GATA1s and STAG2 gRNAs for primary xenotransplantation
into NSG mice. (H) Engraftment of control knockout (KO) and Chr21 miRNAs
KO in T21-FL LT-HSCs transplanted into NSG mice. Each subgroup was
additionally edited with control, GATA1s, and GATA1s/STAG2 gRNAs (only mice
with >1% CD45+cells in BM and >90% CRISPR/Cas9 efficiency are depicted,
n= 5 to 10 mice per condition). (I) Quantification of cell morphology of
human cells prepared by using cytospin in transplanted NSG mice described
in (H) (n= 400 cells per condition). (J) Flow cytometry plots showing blast
populations out of CD45+cells in primary xenografts described in (H).
(K) Quantification of CD117+CD45+blasts of transplanted NSG mice described
in (H) (§§§ indicate significance in relation to GATA1s control KO). Unpaired
Student’sttest: *P< 0.05; **P< 0.01; ***/§§§P< 0.001; ****P< 0.0001;
error bars indicate standard deviation.

RESEARCH | RESEARCH ARTICLE

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