Submitted by: Gordon Keller
Validated by Sunita D’Souza
Title | Mesoderm Differentiation Hematopoiesis |
Date Submitted | January 5th 2012 |
Submitted by - | Kennedy, Marion (mkennedy@uhnresearch.ca) and Keller, Gordon (gkeller@uhnresearch.ca) |
Adapted from - |
|
Contributors - | |
Affiliation(s) - | McEwen Centre for Regenerative Medicine, University Health Network, Toronto, Ontario, Canada.University/ Institute |
*This next section will be on the side and is only relevant for website programmers*
Score | =Likes/(Likes+Dislikes)+Likes/Downloads or some other algorithm… Suggestions??? I can also come up with something better in due time. |
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Status | [Validated (>=3, In Progress (=1-2), Not Validated (none)] |
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The protocol outlined below describes the method for generating hematopoiesis from hESCs, specifically H1 cells. Generally the first hematopoietic progenitors start appearing at T5-7 and are mainly primitive erythroid with a few macrophages. We have defined stages by cell surface markers and colony morphology, in our differentiation cultures, that may reflect the emergence of yolk sac, fetal and definitive hematopoiesis
The formation of EBs is the first important step in the differentiation of hESC. This is best achieved by culturing small aggregates of hESCs in miminal amounts of BMP-4 for
24 hours. At this stage, BMP-4 functions to promote the survival of the hESCs.
COLLAGENASE (FOR DISSOCIATION) (SIGMA# C-0130)
Collagenase Type 1 is mainly used for dissociation of the embryoid bodies.
Final Conc. |
For 1 Liter |
||
---|---|---|---|
Collagenase |
(SIGMA# C-0130) | 0.2% |
2g |
FCS |
|
200mL | |
PBS (with Ca2+, Mg2 | (Cellgro# 21-030-CM) | 800mL |
Note: final concentration is 0.2% or 2 mg/mL in 20% FCS/
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.
CYTOKINES
All cytokines are stored lyophilized, at -20oC, except Erythropoietin which is stored at 4oC
Cytokine |
Buffer |
Stock Conc. |
|
---|---|---|---|
hBMP-4 |
(R&D Systems# 314-BP) | H20, 4mM HCL, 0.1%BSA |
10ug/mL |
hbFGF | (R&D Systems# 234-FSE) | PBS, 0.1%BSA,1mM DTT | 10ug/mL |
hVEGF | (R&D Systems# 293-VE) | PBS, 0.1%BSA | 5ug/mL |
hSCF | (R&D Systems# 255-SC) | PBS, 0.1%BSA | 50ug/mL |
hErythropoietin | Janssen-Ortho Inc (EPREX) | PBS, 0.1%BSA | 2000units/mL |
hIL-6 |
(R&D Systems# 206-IL) | PBS, 0.1%BSA |
5ug/mL |
hIL-11 | (R&D Systems# 218-IL) | PBS, 0.1%BSA | 5ug/mL |
STEMPRO 34 (Invitrogen# 10639-011)
Stempro 34 is sold as a kit with 2 components. The supplement is kept at -20oC and the liquid media at 4oC. When combined, the media is unstable, therefore, we use it for a maximum of 2 weeks. If not used right away, we store the medium as 50mL aliquots and supplement them as needed. The supplement is frozen as 1.3mL aliquots which is the amount required for 50mL. of medium
Final Conc. |
For 500mL |
||
---|---|---|---|
STEMPRO 34 Kit |
(Invitrogen 10639-011) |
|
500 mL |
Penicillin/Streptomycin P/S | (Gibco# 15070-063) | 1% | 5 mL |
*MATRIGEL (REDUCED FACTOR) (BD# 356 230)
Each batch of matrigel has its own unique levels of endotoxin and protein concentrations. We find that the endotoxin levels should not be higher than 2 endotoxin units/mL and the protein levels should range between 7 to 10 mg/mL. If the protein levels are higher than this you may need to dilute the matrigel more than 1:1. This is determined by observing the hESC colony morphology and the ability of the hESCs to differentiate into the lineage required of them.
Caution: When working with matrigel, all tubes, plates and pipettes should be prechilled, as matrigel solidifies at room temperature.
MATRIGEL1:1 PREPARATION
The formation of EBs is the first important step in the differentiation of hESC. This is best achieved by culturing small aggregates of hESCs in the presence of BMP-4 for 24 hours. At this stage, BMP-4 functions to promote the survival of the hESCs.
A. COLLAGENASE B (Roche# 11 088 831 001)
Collagenase B is routinely used for dissociation and passaging of undifferentiated hESCs
Final Conc. |
For 1 Liter |
||
---|---|---|---|
Collagenase B |
(Sigma# T-4799) | 0.25% |
1 g |
DMEM/F12 | (Cellgro# 10-092-CV) | 1 mM | 1000 mL |
B. DNASE I (VWR, Cat # 80510-412, 10MU)
C. TRYPSIN-EDTA
Final Conc. |
For 1 Liter |
||
---|---|---|---|
Trypsin |
(Sigma# T-4799) | 0.25% |
2.5 g |
EDTA 0.5 M (pH 8) | 1 mM | 2 mL | |
PBS (without Ca2+, Mg2+) | (Cellgro# 21-031-CM) | 1000 mL |
D. STOP MEDIUM
Final Conc. |
For 40 mL |
||
---|---|---|---|
hESC WASH Medium |
50% |
20 mL | |
WASH Medium 50% 20 mL |
50% | 20 mL | |
+/- Matrigel (1:1)* | (BD# 356 230) | 1:800 | 100 uL |
E. hESC WASH MEDIUM
Final Conc. |
For 500 mL |
||
---|---|---|---|
Supplemented DMEM/F12 |
|
475 mL | |
KnockoutTM Serum Replacement | (Gibco# 10828-028) | 5% | 25 mL |
SUPPLEMENTED DMEM/F12
Final Conc. |
For 500 mL |
||
---|---|---|---|
DMEM/F12 |
(Cellgro# 10-092-CV) |
|
500 mL |
Penicillin/Streptomycin | (Gibco# 15070-063) | 1% | 5 mL |
INDUCTION / DIFFERENTIATION MEDIA
F. AGGREGATION MEDIUM
Stock Conc. | Final Conc. |
per mL |
For 50 ml |
|
---|---|---|---|---|
STEMPRO 34 |
|
50ml | ||
Glutamine | 100x | 1% | 10uL | 500ul |
Transferrin | 300mg/ml | 150ug/mL | 5uL | 250ul |
Ascorbic Acid | 5mg/ml | 50ng/mL | 10uL | 500ul |
MTG | 26 λ/2mls | 3uL | 150ul | |
BMP4* | 10ug/ml | 10ng/mL | 1uL | 50ul |
G. INDUCTION 1 MEDIUM
Stock Conc. | Final Conc. |
per mL |
For 50 ml |
|
---|---|---|---|---|
STEMPRO 34 |
|
50ml | ||
Glutamine | 100x | 1% | 10uL | 500ul |
Transferrin | 300mg/ml | 150ug/mL | 5uL | 250ul |
Ascorbic Acid | 5mg/ml | 50ng/mL | 10uL | 500ul |
MTG | 26 λ/2mls | 3uL | 150ul | |
BMP4* | 10ug/ml | 10ng/mL | 1uL | 50 |
ActA** | 10ug/ml | 0.3ng/ml | 0.03uL | 1.5ul |
bFGF | 10ug/ml | 5ng/ml | 0.5uL | 25ul |
* Bmp4 and ActA**concentration may vary according to lot# or the hES cell line used…these concentrations are for H1 cells.
H. IMDM+10%FCS
Final Conc. |
For 500 mL |
||
---|---|---|---|
Iscove's Modified Dulbecco's Medium |
(Cellgro# 15-016-CV) |
|
450 mL |
FCS (batch tested) |
(Gibco# 10828-028 ) | 10% |
50 mL |
I. INDUCTION 2 MEDIUM
Stock Conc. | Final Conc. |
per mL |
For 50 ml |
|
---|---|---|---|---|
STEMPRO 34 |
|
50ml | ||
Glutamine | 100x | 1% | 10uL | 500ul |
Transferrin | 300mg/ml | 150ug/mL | 5uL | 250ul |
Ascorbic Acid | 5mg/ml | 50ng/mL | 10uL | 500ul |
MTG | 26 λ/2mls | 3uL | 150ul | |
VEGF | 5ug/ml | 10ng/mL | 1uL | 100ul |
Dkk | 50ug/ml | 150ng/mL | 3uL | 150ul |
bFGF | 10ug/ml | 5ng/ml | 0.5uL | 25ul |
J. INDUCTION 2 MEDIUM (Feed T6)
Stock Conc. | Final Conc. |
per mL |
For 50 ml |
|
---|---|---|---|---|
STEMPRO 34 |
|
50ml | ||
Glutamine | 100x | 1% | 10uL | 250ul |
Transferrin | 300mg/ml | 150ug/mL | 5uL | 100ul |
Ascorbic Acid | 5mg/ml | 50ng/mL | 10uL | 500ul |
MTG | 26 λ/2mls | 3uL | 150ul | |
VEGF | 5ug/ml | 10ng/mL | 2uL | 100ul |
bFGF | 10ug/ml | 5ng/mL | 0.5uL | 50ul |
hIL6 | 10ug/ml | 10ng/ml | 2ul | 100ul |
hSCF | 50ug/ml | 50ng/ml | 2ul | 100ul |
hIL-11 | 5ug/ml | 5ng/ml | 2ul | 100ul |
hEPO | 2000units/ml | 2units/ml | 2ul | 100ul |
hIGF-1 | 25ug/ml | 25ng/ml | 2ul | 100ul |
Bold type indicates a 2x concentration of the cytokine as the media will be diluted by 1/2
K. HEMATOPOIETIC EXPANSION MEDIUM
Stock Conc. | Final Conc. |
per mL |
For 50 ml |
|
---|---|---|---|---|
STEMPRO 34 |
|
50ml | ||
Glutamine | 100x | 1% | 10uL | 500ul |
Transferrin | 300mg/ml | 150ug/mL | 5uL | 250ul |
Ascorbic Acid | 5mg/ml | 25ng/mL | 5uL | 250ul |
MTG | 26 λ/2mls | 2uL | 100ul | |
hTPO | 10ug/ml | 50ng/mL | 5uL | 250ul |
hIL-3 | 10ug/ml | 50ng/mL | 5uL | 250ul |
hFLT3L | 10ug/ml | 20ng/ml | 2ul | 100ul |
hSCF | 50ug/ml | 100ng/ml | 2ul | 100ul |
hIL-11 | 5ug/ml | 5ng/ml | 1ul | 50ul |
hEPO | 2000units/ml | 2units/ml | 1ul | 50ul |
hIGF-1 | 25ug/ml | 25ng/ml | 1ul | 50ul |
HEMATOPOIETIC MEC MIX
Mec Mix |
||||||||
---|---|---|---|---|---|---|---|---|
Reagent |
Stock/ml | Final |
10ml | 14ml | 18ml | 24ml | 32ml | 45ml |
MEC | 100% | 55% | 5.5ml | 7.7ml | 10ml | 13ml | 17.6ml | 25ml |
PDS | 100% | 15% | 1.5ml | 2.1ml | 2.7ml | 3.6ml | 4.8ml | 6.8ml |
PFHM-11 | 100%l | 5% | 0.5ml | 0.7ml | 0.9ml | 1.2ml | 1.6ml | 1.3ml |
Glutamine | 100% | 1x | 100ul | 140ul | 180ul | 240ul | 320ul | 450ul |
Transferrin | 30mg | 150ug/ml | 50ul | 70ul | 90ul | 120ul | 160ul | 275ul |
TPO | 10ug | 50ng/mL | 50ul | 70ul | 90ul | 120ul | 160ul | 225ul |
IL-3 | 10ug | 50ng/ml | 50ul | 70ul | 90ul | 120ul | 160ul | 225ul |
VEGF | 5ug | 10ng/ml | 20ul | 28ul | 36ul | 48ul | 64ul | 90ul |
SCF | 50ug | 100ng/ml | 20ul | 28ul | 36ul | 48ul | 64ul | 90ul |
EPO | 2000units/ml | 4units | 20ul | 28ul | 36ul | 48ul | 64ul | 90ul |
IL-6 | 5ug/ml | 10ng | 20ul | 28ul | 36ul | 48ul | 64ul | 90ul |
IGF-1 | 25ug | 50ng | 20ul | 28ul | 36ul | 48ul | 64ul | 90ul |
IL-11 | 5ug | 5ng | 10ul | 14ul | 18ul | 24ul | 32ul | 45ul |
GM-CSF | 1ug | 1ng | 10ul | 14ul | 18ul | 24ul | 32ul | 45ul |
IMDM | 100% | 2.2ml | 3ml | 3.9ml | 5.2ml | 7ml | 9.7ml |
T0: Generation of embryoid bodies (EBs)
T1: Feeding Aggregates and expansion
Add 2mls of AGGREGATION MEDIUM (F) supplemented with 10ng/ml bFGF to the cultures. The final concentration of bFGF in the well will be 5ng/ml.
Please note low concentration of BMP4 (1-2ng/ml) at D0, followed by 10ng of BMP4 at D1-D4 and 4-5ng of ActA from D1-D4 led to a drastic increase in the amount of erythroid progenitors. All other cytokines are as described.
T1.75: Induction 1
There will be some debris in the cultures. We separate the aggregates from the debris by harvesting 3 wells into one 15ml round bottomed tube and then allowing them to settle for 30min in an 37°C,5%CO2,5%O2 incubator
While the aggregates are settling, make INDUCTION 1 MEDIUM (G) so that there will be 2mL for each well harvested. After the aggregates have settled, aspirate the supernatant and then add INDUCTION 1 MEDIUM . Dispense the aggregates into a new 6 well Locluster plate at 2mL per well. Distribute the aggregates evenly.
Notes
Mesoderm induction and hemangioblast specification in the EBs should be evaluated by flow cytometric analysis, monitoring the cells for expression of KDR and CD117 (c-kit). As each hESC line has its own unique kinetics, it is best to define the hemangioblast stage based on the profile seen below, rather than by time in culture.
The hemangioblast stage is defined by the appearance of a population that expresses medium levels of KDR and are CD117neg . EBs at this stage also contain a KDRneg CD117pos population. This profile is detected in H1-derived EBs at day 3 and in HES2-derived EBs at day 4. The window for hemangioblast development is narrow and occurs before CD31 is expressed on the cells.
T3-4: Harvest for staining
Facs Profiles
T4-8: Induction 2: Specifying Blood from the EBs
Notes
If one monitors the cultures at this time with FACS, the KDR/CD117 and KDR/CD31 profiles should be the same as the Post-Hemangioblast profiles above.
T8: Expanding Precursors
Settle and wash the EBs as above at T4
Resuspend and distribute as above in HEMATOPOIETIC EXPANSION MEDIUM (K) At this time cocktails of cytokines may vary as well as culture methods according to the lineage one desires. For general expansion of hematopoietic lineages we maintain our cultures in suspension changing the media every 4 days. The cultures at this time can be grown in ambient oxygen conditions at 37° and 5%CO2
Harvest to single cells for FACs, Sort, Counts or Reaggregations
Dissociation T1-8 (Trypsin-EDTA)
Dissociation T8+(Collagenase)
IMAGES OF HEMATOPOIETIC COLONIES IN METHYLCELLUOSE COLONIES
MAST COLONY – 20X
MAST COLONY – 10X
MAC/MONO – 10X
MAC/MONO – 10X
MACROPHAGE – 10X
MACROPHAGE – 20X
ERY-MYELO – 10X
MACROPHAGE – 10X
ERYTHROID – 10X
Granulocyte/Macrophage
Blood. 2010 Apr 8;115(14):2769-76. Epub 2010 Jan 11.
Directed differentiation of hematopoietic precursors and functional osteoclasts from human ES and iPS cells.
Grigoriadis AE, Kennedy M, Bozec A, Brunton F, Stenbeck G, Park IH , Wagner EF, Keller GM.
Department of Craniofacial Development and Orthodontics, Guy's Hospital, King's College London, London, UK. agi.grigoriadis@kcl.ac.uk
This page was last modified on October 18, 2012