Supplemental Material: Figure I

cells only isotype control

w/o VEGF

w/ VEGF

with VEGF

without VEGF

FN

FN

COLIV

COLIV

GFR matrigel

GFR matrigel

PE

-Flk

-1A

B

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20

30

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50

FN COLIV GFR matrigel

***

******

d5+0

# ###

§§ §§

Flk

-1+ c

ells

(%

)

Suppl. Figure I. MaGSCs, pre-differentiated for 5 days on OP9 cells as described in the Methods, were cultivated on either fibronectin (FN), collagen type IV (COLIV) or GFR matrigel for additional 5 days, with or without VEGF (10 ng/mL) supplementation, and analyzed for Flk-1 expression using flow cytometry. Representative dot blots (A) as well as the summary of the quantitative analysis in 3-6 independent experiments (B) are shown. Data represent mean values SD. ***P<0.001 vs. unstimulated cells; #P<0.05 and ###P<0.001 for the comparison of unstimulated cells cultivated on different ECM proteins; §§P<0.01 for the comparison of VEGF-stimulated cells cultivated on different ECM proteins.

Suppl Figure II. The expression of endothelial cell marker genes after cultivation of pre-differentiated maGSC on different ECM proteins in the presence of VEGF was compared using quantitative real-time PCR. These analyses revealed that cells cultured on COLIV expressed significantly higher amounts of Flk-1 (A), Tie-2 (B), and VE-cad (C), whereas the expression of vWF (D) was similar in all groups. Mean values SD of 3 independent experiments are shown. §P<0.05, §§P<0.01 and §§§P<0.001.

Supplemental Material: Figure II

A

d5+0 FN COLIV GFR matrigel0

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§ §§

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-1 /

GA

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50 µm

GFR matrigel

50 µm

Suppl. Figure III. (A) After cultivation of pre-differentiated maGSCs for additional 5 days on different ECM proteins, vWF-positive endothelial cells (green) were detected using immunocytochemistry, as described in the Methods. DAPI-positive cell nuclei appear blue. (B) Their angiogenic ability was analyzed using the spheroid angiogenesis assay. Representatives pictures as well as the quantitative analysis of 4 independent experiments are shown. Arrowheads point to sprouts, arrows point to tubular structures ‘connecting’ 2 spheroids. §§§P<0.001.

Supplemental Material: Figure III

vWF DAPI vWF DAPI vWF DAPI50 µm 50 µm

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50 µm

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FN COLIV GFR matrigel0

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Supplemental Material: Figure IV

Suppl. Figure IV. Gene expression pattern of FACS-sorted cell populations during endothelial differentiation: Quantitative analysis of the representative RT-PCR findings shown in Figure 3A. (A) Flk-1+ cells were examined at different time points of cultivation (i.e. on d5+8, d5+13 and d5+18). Compared to d5+8 (set at 1), the expression of Tie2, VE-cad, PECAM-1, vWF and eNOS, but not of SMA, was found to be upregulated with increasing cultivation time (n=3 independent experiments). (B) Comparison of FACS-sorted cell populations (n=3 independent experiments) on day 5+18 revealed a significantly lower expression of EC markers on the Flk1- and GFP+ cells compared to Flk-1+ cells (set at 1), whereas the expression of SMA was found to significantly increase in the GFP+ cells. *P<0.05, **P<0.01 and ***P<0.001.

A

B

Flk1+Flk

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GFP+

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2.0Flk1Tie2VE-cadPECAM1vWFeNOSSMA

*****

*** *

*

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*

mR

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exp

ress

ion

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ld in

crea

se o

ver

Flk

1+ )

d5+8

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ld in

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se o

ver

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8)

25.5%

neg. control

Supplemental Material: Figure V

Suppl. Figure V. Flow cytometry analysis of Flk-1 and PECAM-1 expression on maGSCs and maGSC-ECs. (A) In maGSCs, after 5 days of pre-differentiation on OP9 cells, 32.4% of the cells were positive for Flk1- and 6.5% for PECAM-1. (B) For comparison, 34.0% of maGSC-ECs, derived from Flk-1+ progenitors and cultivated in EDM in the presence of 50 ng/mL VEGF until passage 3, were positive for Flk-1, whereas the number of PECAM-1-positive cells had increased to 25.5%.

maGSCs (day 5) maGSC-ECs (passage 3)

6.5±2.0%

A B

Flk

-1

PECAM-1

2.8±1.0%

32.4±3.0%

Flk-1 PECAM-1

34.0%