Figure S1. Characterization of CD45.1 iPSC clones, related to figure 1: (A) Positive alkaline phosphatase staining (scale bar 200 μm) of CD45.1(1.1) iPSCs. (B).
Stem Cell Reports, Volume 7
Supplemental Information
Murine iPSC-Derived Macrophages as a Tool for Disease Modeling of Hereditary Pulmonary Alveolar Proteinosis due to Csf2rb Deficiency Adele Mucci, Jessica Kunkiel, Takuji Suzuki, Sebastian Brennig, Silke Glage, Mark P. Kühnel, Mania Ackermann, Christine Happle, Alexandra Kuhn, Axel Schambach, Bruce C. Trapnell, Gesine Hansen, Thomas Moritz, and Nico Lachmann
Figure S1
CD45.1 (1.1) iPSC-Mφ A
C
AP stain
brightfield
DAPI
50 μm
50 μm
Nanog
overlay
50 μm
50 μm
Oct4 100 μm brightfield
B
unstained
DAPI
CD45.1 (1.1)
overlay 50 μm Sox2
50 μm
% of max
50 μm
50 μm
50 μm
SSEA-1
CD45.1 (10.4) iPSC-Mφ D
F
AP stain
brightfield
50 μm
DAPI
50 μm
Nanog
overlay
50 μm
50 μm
Oct4 100 μm
brightfield
E
unstained
DAPI
CD45.1 (10.4)
overlay 50 μm Sox2
% of max
50 μm
50 μm
50 μm
50 μm
SSEA-1
Figure S2 F
unstained
OG2 ESC-Mφ
A
200 μm
F4/80
unstained CD45.1
unstained CD11b
unstained
unstained CD45.2
unstained
unstained CD11b
unstained
unstained CD45.2
unstained
unstained CD11b
unstained
unstained CD45.2
unstained
unstained CD11b
unstained
unstained CD45.2
unstained
unstained CD11b
unstained
unstained CD45.2
unstained
CD14
CD41
unstained
unstained
CD11c
unstained
Gr-1
CD3
unstained
B220
CD11c
unstained
Gr-1
CD3
unstained
B220
CD11c
unstained
Gr-1
CD3
unstained
B220
CD11c
unstained
Gr-1
CD3
unstained
B220
CD11c
unstained
Gr-1
CD3
unstained
B220
20 μm
G CD45.1 (1.1) iPSC-Mφ
B
200 μm
unstained
F4/80
unstained CD45.1
CD14
CD41
unstained
unstained
20 μm unstained
H CD45.1 (10.4) iPSC-Mφ
C
200 μm
F4/80
unstained CD45.1
CD14
CD41
unstained
unstained
20 μm unstained
I
lin-7-Mφ
D
200 μm
F4/80
unstained CD45.1
CD14
CD41
unstained
unstained
20 μm unstained
J
200 μm
20 μm
lin-9-Mφ
E
F4/80
unstained CD45.1
CD14
CD41
unstained
unstained
cell number [normalized to 1000 input cells] 25000
0 2x
lin-9 iPSC
30000
lin-7 iPSC
5000
CD45.1 iPSC (10.4)
10000
CD45.1 iPSC (1.1)
20000
CD45.1 iPSC (10.3)
35000
OG2-ESC
Figure S3
20x CD41+ cells Mφ
10x
15000
6x 2x 1x
Figure S4 A
Lung
BALF
C
CD45.1
brightfield
10 µm
autofluorescence (FITC)
DAPI
B CD68
10 µm
CD45.1
Figure S5 A
D
B
Sox2
10
Nanog
AP stain
E
unstained
miPAP2
ns
1
% of max
brightfield
2^-ΔΔCt [normalized to ESC]
ns ns
Oct4
0.100
0.010
C 0.001 OG2 ESC
miPAP2
F brightfield
DAPI
NANOG
brightfield
DAPI
OCT4
miPAP2
ectoderm
H
I
J
K
endoderm
mesoderm
unstained F4/80
unstained CD11b
unstained CD14
unstained CD45.1
unstained CD45.2
unstained
unstained
CD11c
unstained
Gr-1
CD3
unstained
B220
FSC
miPAP2 Mφ
CD41
G
SSEA-1
C3H MEF
CD41
unstained
Figure S1. Characterization of CD45.1 iPSC clones, related to figure 1: (A) Positive alkaline phosphatase staining (scale bar 200 μm) of CD45.1(1.1) iPSCs. (B) Representative flow cytometry plot revealing expression of the SSEA-1 surface marker (unstained in orange filled and SSEA-1 stained CD45.1(1.1) iPSCs in green). (C) NANOG, OCT4 and SOX2 expression by Immunofluorescence staining (scale bar 50 μm). (D) Positive alkaline phosphatase staining (scale bar 200 μm) of CD45.1(10.4) iPSCs. (E) Representative flow cytometry plot revealing expression of the SSEA-1 surface marker (unstained in orange filled and SSEA-1 stained CD45.1(10.4) iPSCs in green). (F) NANOG, OCT4 and SOX2 expression by Immunofluorescence staining (scale bar 50 μm). Figure S2. OG2 ESCs, CD45.1 iPSC clones as well as lin-7 and -9 iPSCs differentiate into macrophages, related to figure 2: (A) Representative pictures of OG2 ESC-Mφ, (B) CD45.1(1.1) iPSC-Mφ, (C) CD45.1(10.4) iPSC-Mφ, (D) lin-7-Mφ, (E) lin-9-Mφ in brightfield and in May-Gruenwald/GIEMSA stained cytospins (scale bar: 100 μm and 20 μm). (F-J) Exemplary flow cytometry plots for expression of surface markers F4/80, CD11b, CD14, CD11c, Gr-1, CD45.1, CD45.2, CD41, CD3 and B220 on OG2 ESC-Mφ, CD45.1(1.1) iPSC-Mφ, CD45.1(10.4) iPSC-Mφ, lin-7-Mφ and lin-9Mφ, respectively (orange filled for unstained and blue for stained). Figure S3. Macrophage differentiation efficiency of murine ESCs and several iPSC clones, related to figure 2: OG2 ESCs, three clones of CD45.1 iPSC (10.3, 1.1 and 10.4) as well as lin-7 and lin-9 iPSCs differentiation efficiency (indipendent experiments n=2-3, mean ±SD, X-fold increase compared to undifferentiated iPSCs). Figure S4. Engraftment of CD45.1 (10.3) iPSC-Mφ derived donor cells in the murine PAP model (Csf2rb-/- mice) following intratracheal transplantation, related to figure 3: (A) Detection of CD45.1 positive cells in lungs and bronchoalveolar lavage fluid (BALF) of Csf2rb-/- mouse transplanted intra-tracheally with 4x106 CD45.1(10.3) iPSC-Mφ (n=1). (B) Representative picture of MayGruenwald/GIEMSA-stained cytospin of fluorescence activated cell sorted for CD45.1 expression (scale bar 20μm). (C) Representative picture of cryopreserved lungs stained for CD45.1 (green), CD68 (red) and DAPI (blue) (scale bar 10μm). Figure S5. Characterization and differentiation of disease-specific iPSCs miPAP2
from Csf2rb-/- mice, related to figure 4: (A) ESC-like morphology in brightfield images and (B) positive alkaline phosphatase staining (scale bar 200 μm) of miPAP2 iPSCs. (C) NANOG, OCT4 and SOX2 expression by Immunofluorescence staining (scale bar 50 μm) as well as by (D) qRT-PCR using murine specific primers (independent experiments, n=3, mean ±SD). (E) Representative flow cytometry plot revealing expression of the SSEA-1 surface marker. (F) Representative pictures of miPAP2-derived teratomas containing tissues of all the three embryonic germ layers (scale bar 50 μm ectoderm and endoderm, 100 μm mesoderm). (G) Representative picture of EBs at day eight (scale bar 500 μm) and (H) representative flow cytometry plot for CD41 expression. (I) Representative picture in brightfield and (J) MayGruenwal/GIEMSA stained cytospins of miPAP2-Mφ (scale bar 100 μm and 20 μm). (K) Flow cytometry plots for surface marker expression (data for F4/80, CD11b, CD14, CD11c, Gr-1, CD45.1, CD45.2, CD41, CD3 and B220 given). (ns= not significant in 2way ANOVA test compared to ESCs)
