FABp4

ctr E-MSCs0

10

20

30

AU

OC

ctr E-MSCs0

10

20

30

AU

Col2a1

ctr E-MSCs0

10

20

30

AU

A B

C

control E-MSCs 7d control E-MSCs 7d

Figure 1SMultipotency and differentiation capacity of E-MSCs is preserved. Staining with Alizarin Red for osteoblasts (A) or Oil Red O for adipocytes (B) of control MSCs or E-MSCs retrieved from capsules 7 days post encapsulation (E-MSCs 7d) and induced to differentiate, scale bars 500m and 30m respectively. C) mRNA expression of differentiation-related genes, such as fatty acid binding protein 4 (FABP4), Osteocalcin (OC) and Collagen2a1 (Col2a1) was measured by quantitative real-time PCR in control (monolayer culture) vs. E-MSCs 7d decapsulated cells. Encapsulated MSCs 1 month after subcutaneous injection in naïve mice : D) a representative image of capsules stained with FDA viability assay (scale bar 200m); E) expression of differentiation-related mRNAs of encapsulated MSCs in comparison with control MSCs (monolayer culture).

FABp4

ctr E-MSCs0

10

20

30

AU

OC

ctr E-MSCs0

10

20

30

AU

Col2a1

ctr E-MSCs0

10

20

30

AU

ED

Fluo

resc

ein

/ PI

9

CCL8

syn ctr GVHD + E-MSCs0

1000

2000

3000

4000

ng/m

lsTNFr1

syn ctr GVHD + E-MSCs0.0

1.0

2.0

3.0

4.0

5.0

ng/m

l

B

Figure 2SAdministration of E-MSCs does not affect CCL8 (A) and sTNFr1 (B) serum levels in mice with GVHD. Serum was collected from mice with established GVHD at day 6. Livers were collected from the same mice at day 6, processed for paraffin embedding , sectioned for histologic examination with hematoxylin/eosin and semiquantitative scoring of lobular inflammation (C-D). Two representative sections of liver stained with haematoxylin and eosin (E) or immunostained (brown) for CD3 (F) of GVHD animals without (left) or with E-MSCs treatment (right column). Scale bar 100m, 5 mice per group.

A

D

E GVHD E-MSCs0.0

0.5

1.0

1.5

2.0

Lobu

lar i

nfilt

ratio

n sc

ore

F

GVHD + E-MSCs02468

1012

Cum

ulat

ive

scor

e

C

10

Syngeneic ctr

GVHDGVHD + E-MSCs s.c.

GVHD+ free MSCs.c.

Figure 3SCell encapsulation does not modify the immuno-regulatory properties of MSCs.BALB/c mice irradiated with 700 rad were inoculated intravenously with 107 bone marrow cells (BMC) and 15x106 spleen cells derived from C57 donor mice while mice indicated as syngeneic controls received bone marrow and spleen cells derived from BALB/c donors.After 2 days, one group of mice received a s.c. injection of 400.000 either encapsulated or free MSCs. Survival curves for the experimental groups are shown. Data from two experiments were pooled (syngeneic control: n=4; GVHD: n=16; E-MSCs treated: n=19; free MSCs: n=13). The results are presented as Kaplan-Meier survival curves. Statistical difference was demonstrated between GVHD-positive controls and mice treated with E-MSCs or free MSCs s.c. (P < 0.0001). No difference was found between mice treated with E-MSCs or free MSCs s.c..

0 5 10 15 20 25 300

102030405060708090

100110

days

Per

cent

sur

viva

l

11