Supplementary Fig. 1 A schematic representation of this work. We demonstrate
that control of two opposing signalling pathways is a general method to regulate stem
cell expansion in tissue culture by studies on human embryonic, neural and pancreatic
stem cells. The relevance to medicine is strengthened by data showing that Notch
ligands promote stem cell expansion and in the adult brain. Rats with a stroke in a
specific region of their brain showed enhanced recovery when the Notch and FGF
receptors are stimulated by treatment with combinations of growth factors. These
results show that the Notch receptor controls stem cell growth in the lab and in the
body. Data from rats with damage to their brain shows that they respond to Dll4 by
increased numbers of stem cells and immature neurons. Treated animals also show
improved behavioural responses suggesting Notch activation may be important in
regenerative medicine. (Dll4, the Notch ligand Delta-4; CNS SC, central nervous
system stem cells; hES, human embryonic stem cells; pancreas identifies precursors
of the endocrine pancreas; 1-3, identify major manipulations of the signalling pathway
with - 1 activators of the Notch receptor – 2 and 3 inhibitors of the kinases Jak and
p38.)
Supplementary Fig. 2 Notch ligands activate second messenger signalling and
support NSC (E13.5) survival in vitro. (a) Dll4 (500ng/ml, 1h treatment) promotes
Notch1 cleavage. (b) Dll4 inhibits cell death in real-time cell lineage experiments
(red, death; black, survival). (c) Jag1 (500ng/ml) induces time-dependent
phosphorylation of mTOR. (d) Dll4 induces STAT3-Ser727 phosphorylation in a
dose-dependent manner. (e) DAPT and rapamycin (1M) block the Dll4-induced
phosphorylation of STAT3-Ser727 (2 hrs pre-treatment; 30 min Dll4 stimulation). (f)
Jag1 treatment (500ng/ml) induces the time-dependent phosphorylation of MSK-1
and LKB1
Supplementary Fig. 3 Notch activation promotes the generation of adult NSC in
vivo. (a) Dll4 (7 d treatment using pump) increases the number of BrdU positive cells
in the normal SVZ and areas surrounding the lateral ventricles in a dose-dependent
manner (7 d post-op). (b) Dll4 but not FGF2 increases the number of BrdU positive
cells in the ipsi- and contra-lateral SVZ of normal rats (7 d post-op). (c) Dll4 (7d
treatment, 42g/ml in osmotic pumps) increases the generation of BrdU positive cells
in the SVZ of the normal brain in the presence of FGF2 (7 days post-op). BrdU colocalizes with DCX in the SVZ of FGF2/Dll4-treated animals (7 days post-op; scale
bars, 50 m). (d) BrdU co-localizes with HU in the cortex of FGF2/Dll4-treated
normal animals (45 days post-op; scale bar 50 m). (e) FGF2 and Dll4 increase the
numbers of BrdU positive cells in the SVZ of ischemic rats (45 d post-op). (All error
bars: means ± SEM)
Supplementary Table 1 Notch ligands do not alter the differentiation potential of
embryonic (E13.5) CNS SC. Ratios of neurons (TUJ1+), astrocytes (GFAP+), and
oligodendrocytes (CNPase+) following Notch activation (5 d Notch + FGF2, 4 d withdrawal).
%
%Glia
%Oligodendrocytes
Neurons
(GFAP)
(CNPase)
(TUJ1)
FGF ► WD
42.37 ±
49.53 ±
9.12 ± 2.82
6.85
4.79
FGF+Dll4 ► WD
44.27 ±
47.02 ±
7.82 ± 4.32
9.83
5.15
FGF+Jag1 ► WD
41.15 ±
48.84 ±
8.14 ± 5.8
11.1
6.97
FGF+Dll4+Jag1
43.23 ±
45.08 ±
8.77 ± 4.45
► WD
4.63
6.89
Supplementary Table 2 Signal transduction manipulations promote colony formation in
hES cell cultures. Notch activation and JAK/p38 inhibition increase plating efficiency in
singly dissociated hES cell cultures (6 days; means ± SD).
Cell line/Treatment
Colony # (% of FGF2)
HSF6
FGF2
100±15
FGF2+JAK Inh.
568±70
FGF2+Dll4
172±34
FGF2+JAK Inh.+Dll4
617±76
FGF2+p38 Inh.
236±22
FGF2+JAK Inh.+p38 Inh
436±18
H1
FGF2
100±24
FGF2+JAK Inh.
448±57
H9
FGF2
100±5
FGF2+JAK Inh.
347±59
Supplementary Table 3 P-values corresponding to asterisks in figures. Figure and panel
numbers are shown. Per panel, p-values correspond to asterisks left to right.
Figure
p
Supplementary Figure
p
-8
1f
3.81x10
Supplementary Fig. 4a
0.001554
(top)
0.015707
0.011538
2a
0.015689
0.0000102
0.01049 Supplementary Fig. 4a
0.00000668
(bottom)
0.042739
0.00272
0.004535
0.008648
0.007327 Suppl. Fig. 4b (top)
0.009367
4b
2.44x10-8
0.019558
0.000236 Suppl. Fig. 4b (bottom)
0.000368
-9
5.28x10
0.007879
0.000125 Suppl. Fig. 4e
0.005812
0.000037
0.001083
4d
0.002276
0.003594
4e
0.004517
0.016263
0.000761
0.004177
0.009259
0.015811
0.012642
0.005852