Title | McEwen - Pancreatic differentiation |
Date Submitted | 04/2012 |
Submitted by | Sunita D’Souza |
Adapted from | Stage-specific signaling through TGFβ family members and WNT regulates patterning and pancreatic specification of human pluripotent stem cells |
Contributors |
|
Affiliation(s) | University Health Network, Toronto |
Table of Contents
Introduction
Flow Chart
Materials and Reagent Preparation
Protocol
Acknowledgements
Contributors
Pancreatic – Insulin producing β cells protocol (adapted from Nostro et al., 2011)
The generation of insulin-producing β-cells from human pluripotent stem cells is dependent on efficient endoderm induction and appropriate patterning and specification of this germ layer to a pancreatic fate. In this study, we elucidated the temporal requirements for TGFβ family members and canonical WNT signaling at these developmental stages and show that the duration of nodal/activin A signaling plays a pivotal role in establishing an appropriate definitive endoderm population for specification to the pancreatic lineage. WNT signaling was found to induce a posterior endoderm fate and at optimal concentrations enhanced the development of pancreatic lineage cells. Inhibition of the BMP signaling pathway at specific stages was essential for the generation of insulin-expressing cells and the extent of BMP inhibition required varied widely among the cell lines tested. Optimal stage-specific manipulation of these pathways resulted in a striking 250-fold increase in the levels of insulin expression and yielded populations containing up to 25% C-peptide+ cells.
REAGENT LIST
Reagents
Reagent | Company | Catalogue # | |
---|---|---|---|
1. |
anti-CXCR4-phycoerythrin (1:100) |
BD |
551966 |
2. |
anti-CD31-phycoerythrin (1:100) |
BD |
553373 |
3. |
anti-CD117-allophycocyanin (1: 100) |
Caltag |
CD11705 |
4. |
anti-KDR–allophycocyanin (1: 10) |
R&D |
FAB357A |
5. |
HPi3 (1:20) |
Novus Biologicals |
NBP1-18947 |
6. |
HPα2 (1:20) |
Novus Biologicals |
NBP1-18950 |
7. |
HPx1 (1:20) |
Novus Biologicals |
NBP1-18951 |
8. |
HPd1 (1:20) |
Novus Biologicals |
NBP1-18953 |
9. |
anti-mouse IgG-phycoerythrin (1:100) |
Jackson ImmunoResearch |
715-006-150 |
10. |
goat anti-human SOX17 (1:40) |
R&D |
BAF1924 |
11. |
goat anti-FOXA2 , clone M20 (1:50) |
SantaCruz |
SC6554 |
12. |
rat anti-human C-peptide (AB1921) (1:300) |
BCBC consortium |
AB1921 |
13. |
mouse anti-GCG (1:500) |
Sigma |
G2654 |
14. |
donkey anti-goat IgG-Alexa 488 (1:400), |
Invitrogen |
A11055 |
15. |
goat anti-mouse allophycocyanin (1:200). |
R&D |
F0101B |
16. |
16% paraformaldehyde |
Electron Microscopy Sciences |
50980487 |
17. |
rat anti-human C-peptide, AB1921, 1:1000 |
Beta Cell Biology Consortium |
AB1921 |
18. |
goat anti-human glucagon, C-18, 1:500 |
Santacruz |
Sc-7779 |
19. |
mouse anti-SST (AB1985; 1:500 |
Beta Cell Biology Consortium |
AB1985 |
20. |
goat IgG (Sigma) |
Jackson ImmunoResearch |
005-000-003 |
21. |
mouse, rabbit or rat IgG |
Jackson ImmunoResearch |
015-000-003 / 005-000-003 / 012-000-003 |
22. |
goat anti-mouse IgG-PE |
Jackson ImmunoResearch |
115-115-208 |
23. |
donkey anti-rat IgG-Cy3 |
Jackson ImmunoResearch |
712-166-153 |
24. |
rabbit anti-mouse Alexa 350, 1:200 |
Invitrogen |
A-21062 |
REAGENT PREPARATION
STOP MEDIUM
|
|
Final Conc. |
For 40 mL |
hESC WASH Medium |
|
50% |
20 mL |
FCS |
|
50% |
20 mL |
+/- Matrigel (1:1) * |
(BD# 356 230) |
1:800 |
100 uL |
DNASE I (VWR, Cat # 80510-412, 10MU)
10MU | X | 1X106 U | X | 1mg | = 153mg |
1 MU | 65150 U |
L-ASCORBIC ACID (AA) (SIGMA # A-4544)
Prepare a stock solution of 5 mg/mL in cold TC-H2O. Leave on ice and vortex periodically until completely dissolved. Filter sterilize, aliquot and store at -20ºC. Use once and discard
MONOTHIOGLYCEROL (MTG) (SIGMA# M-6145)
The amounts of MTG indicated in our protocols are recommended concentrations. However, it is important to test each new batch of MTG as there is variability between them. MTG should be aliquoted (1 mL) and stored frozen (-20ºC). When aliquots are thawed, they can be used for several experiments and then discarded. Aliquoting of MTG is strongly recommended as it minimizes the amount of oxidation due to repeated opening
TRANSFERRIN (ROCHE# 10 652 202 )
The amounts of Transferrin indicated in our protocols are recommended concentrations. However, it is important to test each new batch of transferrin as there is variability between them. It should be aliquoted (2 mL) and stored at 4ºC.
L-Glutamine (Gibco# 25030)
TRYPSIN-EDTA (Invitrogen# 25-053-CL)
Company | Catalogue # | Buffer | Stock conc. | ||
---|---|---|---|---|---|
1. |
Wnt3a |
R&D Systems |
|
PBS, 0.1%BSA, 0.5% CHAPS, 1mM EDTA |
50ug/mL |
2. |
ActivinA |
R&D Systems |
338-AC/CF |
PBS, 0.1%BSA |
10ug/mL |
3. |
hBMP-4 |
R&D Systems |
314-BP |
H20, 4mM HCL, 0.1%BSA |
10ug/mL |
4. |
hbFGF |
R&D Systems |
233-FB |
PBS, 0.1%BSA,1mM DTT |
10ug/mL |
5. |
hVEGF |
R&D Systems |
293-VE |
PBS, 0.1%BSA |
5ug/mL |
6. |
hFGF10 |
R&D Systems |
345FG/CF |
PBS, 0.1%BSA |
100ug/mL |
7. |
hNOGGIN |
R&D Systems |
3344/NG |
PBS, 0.1%BSA |
100ug/mL |
8. |
Dorsomorphin |
Sigma |
P5499 |
DMSO |
1mM |
9. |
SB431542 |
Sigma |
S4317 |
DMSO |
20mM |
10. |
L-685,458 |
ToCris |
2627 |
DMSO I think now it is R&D |
10mM |
11. |
RA |
Sigma |
R2625 |
DMSO |
1mM |
Serum Free Differentiation (SFD) Media
Reagent | Stock conc. | Working conc. | Per ml | 1000ml |
---|---|---|---|---|
IMDM |
Cellgro |
10-016-CV |
75% |
750ml |
Ham’s F12 |
Cellgro |
10-080-CV |
25% |
250ml |
Penicillin/Streptomycin P/S |
Invitrogen |
15070-063 |
1% |
10ml |
N2 Supplement |
Invitrogen |
17502-048 |
1% |
5ml |
B27 Supplement |
Invitrogen |
12587-010 |
1% |
10ml |
7.5% BSA in PBS |
10ug/ml |
A9647 |
0.05% |
6.66ml |
RPMI (Gibco# 31800) supplemented with antibiotics, 10mM HEPES and 1mM Pyruvate.
(A) Day 0 Stage 1 MEDIUM
Reagent | Stock conc. | Working conc. | Per ml |
---|---|---|---|
RPMI |
1X |
1X |
1ml |
Glutamine |
100X |
1% |
10ul |
MTG |
26λ/2mLs |
3ul/ml |
3ul |
Ascorbic acid |
5mg/ml |
50ug/ml |
10ul |
BMP4 |
10ug/ml |
0.25ng/mL |
0.025 |
bFGF |
10ug/ml |
5ng/mL |
0.5ul |
ActA |
50ug/mL |
100ng/mL |
2ul |
VEGF |
5ug/mL |
10ng/mL |
2ul |
(B) Day 1-2 Stage 1 MEDIUM
Reagent | Stock conc. | Working conc. | Per ml |
---|---|---|---|
RPMI |
1X |
1X |
1ml |
Glutamine |
100X |
1% |
10ul |
MTG |
26λ/2mLs |
3ul/ml |
3ul |
ActA |
5ug/mL |
100ng/mL |
2ul |
WNT3a |
50ug/mL |
25ng/mL |
0.5ul |
(C) Day 3,5 Stage 2 MEDIUM
Reagent | Stock conc. | Working conc. | Per ml |
---|---|---|---|
SFD |
1X |
1X |
1ml |
Glutamine |
100X |
1% |
10ul |
MTG |
26λ/2mLs |
3ul/ml |
3ul |
FGF10 |
100ug/mL |
50ng/mL |
0.5ul |
WNT |
50ug/mL |
3ng/mL |
0.06ul |
Dorsomorphin |
1mM |
0.75uM |
0.75ul |
* Dorsomorphin is required for H1 and H9 differentiation. Other human pluripotent stem cell lines may not require Dorsomorphin at stage 2.
H21 (Gibco# 12800) with high D-glucose (4,500mg/L).
(D) Day 6,8 Stage 3 MEDIUM
Reagent | Stock conc. | Working conc. | Per ml |
---|---|---|---|
H21 (high glucose) |
1X |
1X |
1ml |
Glutamine |
100X |
1% |
10ul |
B27 |
100X |
1% |
10ul |
Ascorbic acid |
5mg/ml |
50ug/ml |
10ul |
Cyclopamine |
0.1mM |
0.25uM |
2.5ul |
RA |
10mM |
2uM |
0.2ul |
Noggin |
100ug/ml |
50ng/ml |
0.5ul |
FGF10 |
100ug/mL |
50ng/mL |
0.5ul |
(E) Day 9,11 Stage 4 MEDIUM
Reagent | Stock conc. | Working conc. | Per ml |
---|---|---|---|
H21 (high glucose) |
1X |
1X |
1ml |
Glutamine |
100X |
1% |
10ul |
B27 |
100X |
1% |
10ul |
Ascorbic acid |
5mg/ml |
50ug/ml |
10ul |
SB431542 |
20mM |
6uM |
0.3ul |
Noggin |
100ug/ml |
50ng/ml |
0.5ul |
(F) Day 12-20 Stage 5 MEDIUM
Reagent | Stock conc. | Working conc. | Per ml |
---|---|---|---|
SFD |
1X |
1X |
1ml |
Glutamine |
100X |
1% |
10ul |
Glucose |
26λ/2mLs |
3ul/ml |
3ul |
Ascorbic acid |
5mg/ml |
50ug/ml |
10ul |
SB431542 |
20mM |
6uM |
0.3ul |
Noggin |
100ug/ml |
50ng/ml |
0.5ul |
à Secretase Inhibitor |
10mM |
1uM |
1ul |
Including Glucose present in SFD, the final Glucose concentration is 40mM.
** We find that the concentration of γsecretase Inhibitor (L-685,458) can be dropped to 0.25uM.
Methods
This protocol is designed for H1 and H9 human embryonic stem cell lines. Other lines may not
require Dorsomorphin at stage 2 of differentiation.
hESC maintenance
Our lab routinely adapts hESCs to trypsin passage as this allows for easy passage and the maintenance and production of large numbers of cells. Successful maintenance of healthy undifferentiated hESCs is dependent on the appropriate concentrations of feeder cells and hESCs.
Mouse Embryonic Feeder Cells (MEFs). should be approximately 80% confluent and fresh, ideally cultured for only 24 hours prior to use. If the density of the MEFs is too high (confluent) the hESCs do not form discrete colonies but rather grow as disperse groups of cells, forming a monolayer. MEFs that are too sparce (<50%) do not provide adequate support for hESC maintenance. We routinely freeze irradiated MEFs at 2x10^6 cells per vial. Each vial contains enough cells for 18-24 wells of a twelve-well plate. The plating efficiency of each batch of MEFs needs to be tested.
hESCs. hESCs should be cultured at a density that allows the growth of distinct colonies with sharp borders within 4-5 days of culture. If the cells are too dense, the developing colonies grow into each other and form a monolayer. When too few cells are cultured, they can differentiate and tend to grow slower. Our stock of hESCs are frozen at 2x10^6 cells per vial. This concentration can be used for 6-24 wells of a twelve-well plate. The number of wells that can be cultured is dependent on the hESC line as well as the extent to which they are adapted to trypsin passage. Under optimal conditions with well adapted hESCs, you should be able to reach 70% confluency 4-5 days after plating, at this stage cells are ready to be differentiated.
Note: The protocol described below is designed to be carried out in a 12-well plate format.
Day 0: Stage 1 Endoderm Progenitors
Day 1-2: Stage 1 Endoderm Progenitors
Day 3: Harvest for Flow Cytometry
Day 3, 5 : Stage 2 Foregut/Midgut Endoderm
Day 6-8: Stage 3 Pancreatic Endoderm
Day 9, 11: Stage 4 Endocrine Progenitors
Day 13-20: Stage 5 Endocrine Cells
Stage-specific signaling through TGFβ family members and WNT regulates patterning and pancreatic specification of human pluripotent stem cells.
M. Cristina Nostro,1 Farida Sarangi,1 Shinichiro Ogawa,1 Audrey Holtzinger,1 Barbara Corneo,2 Xueling Li,3 Suzanne J. Micallef,3 In-Hyun Park,4 Christina Basford,5 Michael B. Wheeler,5 George Q. Daley,6 Andrew G. Elefanty,3 Edouard G. Stanley,3 and Gordon Keller1*
Development. 2011 March 1; 138(5): 861–871. doi: 10.1242/dev.055236 PMCID: PMC3035090
This page was last modified on October 23, 2012