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 (1mM) 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, 42mg/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 co-localizes with DCX in the SVZ of FGF2/Dll4-treated animals (7 days post-op; scale bars, 50 mm). (d) BrdU co-localizes with HU in the cortex of FGF2/Dll4-treated normal animals (45 days post-op; scale bar 50 mm). (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).

% Neurons
(TUJ1) / %Glia
(GFAP) / %Oligodendrocytes
(CNPase)
FGF ► WD / 42.37 ± 6.85 / 49.53 ± 4.79 / 9.12 ± 2.82
FGF+Dll4 ► WD / 44.27 ± 9.83 / 47.02 ± 5.15 / 7.82 ± 4.32
FGF+Jag1 ► WD / 41.15 ± 11.1 / 48.84 ± 6.97 / 8.14 ± 5.8
FGF+Dll4+Jag1 ► WD / 43.23 ± 4.63 / 45.08 ± 6.89 / 8.77 ± 4.45

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
1f / 3.81x10-8 / Supplementary Fig. 4a (top) / 0.001554
0.015707 / 0.011538
2a / 0.015689 / 0.0000102
0.01049 / Supplementary Fig. 4a (bottom) / 0.00000668
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
5.28x10-9 / 0.007879
0.000125 / Suppl. Fig. 4e / 0.005812
0.000037 / 0.001083
4d / 0.002276 / 0.003594
0.004517 / 0.015811
0.016263 / 0.012642
4e / 0.000761 / 0.005852
0.004177
0.009259