CD34+ cell reprogramming using episomal vectors
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Title |
CD34+ cell reprogramming using episomal vectors |
Date Submitted |
May 5, 2012 |
Submitted by - |
Olga Momcilovic, Zeng Lab omomcilovic@buckinstitute.org |
Adapted from - |
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Contributors - |
Olga Momcilovic |
Affiliation(s) - |
Zeng Lab Buck Institute |
Introduction:
Protocol:
- Prime CD34+ cells
- Thaw CD34+ cells using Lonza’s protocol and culture for 4-5 days (http://www.lonzabio.com/uploads/tx_mwaxmarketingmaterial/Lonza_ManualsProductInstructions_Procedure_for_Thawing_Poietics_Cells.pdf)
- Day 1: nucleoporate 1x10^6 hCD34+ cells with single (up to 10 µg), or combination of plasmids (8 µg C5 + 2 µg Tg) by Amaxa using program U-008
- Days 1 and 2: culture nucleoporated cells in one well of a 12-well plate in the CD34+ medium with cytokines
- Day 3: transfer nucleoporated cells to 3 wells of MEF coated 12-well plate and culture in MEF medium for one day
- Once cells are seeded into wells, spin plates at 100xg for 30 min to help cells attach to MEF coated wells
- Day 4: replace MEF medium with hESC medium (supplemented with 10 ng/ml FGF2)
- OPTIONAL: collect MEF medium and spin it down at 100xg for 5 min; aspirate medium and resuspend cell pellet in hESC medium with 10 ng/ml FGF2 – some CD34+ cells may not attach during the first day, so save them and replate them
- Change hESC medium every other day for total of 6 days
- OPTIONAL: add valporic acid (0.5 mM) or Na-butyrate (0.25 mM)
- Switch to MEF-CM with 10 ng/ml FGF2 one week after transfer onto MEF coated wells
- Two weeks after nucleoporation, perform TRA1-60 staining on live cells to identify most likely iPSC clones
- With cord blood CD34+ cells expect to see colonies appearing 7-11 days post-nucleoporation
- With adult bone marrow and peripheral blood CD34+ cells colonies start appearing 11-14 days post-nucleoporation
- Manually dissect each TRA1-60 positive colony and transfer to a separate well of a 12-well plate: each colony becomes a clone
- OPTIONAL: add 10 µM ROCK inhibitor and/or hESC cloning and recovery supplement to improve survival and attachment of dissected colonies
- For the first 2 – 3 passages keep clones in 12-well plates, then expand to 35 mm dishes
- Manually passage clones for the first 6 – 10 passages, then switch to 1 mg/ml collagenase (depending on whether clones remain undifferentiated when enzymatically passaged). In instances when less than 10% of colonies are differentiated, remove differentiated cells manually and proceed to enzymatic passage; if more than 10% colonies are differentiated, continue with manual passaging
- Gradually reduce FGF2 concentration in MEF-CM to 4 ng/ml and switch to hESC medium by mixing MEF-CM and hESC medium in order to adopt iPSC clones to hESC medium with 4 ng/ml of FGF2.
Materials:
Product |
Company |
Catalogue number |
MEF, mitomycin C treated |
Millipore |
PMEF-N |
DMEM, high glucose |
Gibco |
11995 |
FBS |
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KNOCKOUT™ DMEM/F12 |
Gibco |
12660 |
NEAA |
Gibco |
11140 |
Anti-Anti |
Gibco |
15240 |
KNOCKOUT™ Serum Replacer |
Gibco |
10828 |
2-mercaptoethanol |
Gibco |
21985 |
GlutaMAX™-1 |
Gibco |
35050 |
CD34+ cells |
Lonza |
2C-101 |
HPGM™ |
Lonza |
PT-3926 |
DNase I |
Sigma |
D4513 |
SCF |
Peprotech |
AF-300-07 |
TPO |
Peprotech |
AF-300-18 |
FL |
Peprotech |
AF-300-19 |
FGF2 |
Stemgent |
03-0002 |
Nucleofector kit for CD34+ cells |
Lonza |
VPA-1003 |
ROCK inhibitor, Y27632 |
Stemgent |
04-0012 |
hESC cloning and recovery supplement |
Stemgent |
01-0014-500 |
Na-butyrate |
Stemgent |
04-0005 |
Valporic acid |
Stemgent |
04-0007 |
TRA1-60 antibody |
eBioscience |
13-8863-83 |
C5 – EBNA1 carrying OCT4, SOX2, KLF4, LIN28, cMYC |
Addgene |
http://www.addgene.org/28213/ |
TG – EBNA1 carrying SV-40 Large T antigen |
Addgene |
http://www.addgene.org/28220/ |
MEF medium
90% DMEM
10% FBS
1% Anti Anti
hESC/hiPSC medium
KNOCKOUTTM DMEM/F12
20% KNOCKOUTTM Serum Replacer
1% GlutaMAXTM-1
1% NEAA
1% Anti/Anti
4 – 10 ng/ml FGF2
0.1 mM 2-mercaptoethanol
CD34+ cell medium (recommended by Lonza)
HPGM™ Hematopoietic Progenitor Growth Medium supplemented with the following concentrations of cytokines:
FL – 50 ng/ml
TPO – 50 ng/ml
SCF – 25 ng/ml
All cytokines are from Peprotech and are diluted in trechalose at concentration of 100 ng/µl.
Abbreviations
MEF = mouse embryonic fibroblasts
FBS = fetal bovine serum
NEAA = non-essential amino acids
FL = Flt3 ligand
SCF = stem cell factor
TPO = Thrombopoietin
MEF-CM = hESC medium conditioned for 24 hrs on MEF
Troubleshooting:
References: