CENTER FOR REGENERATIVE MEDICINE of BOSTON UNIVERSITY and BOSTON MEDICAL CENTER
670 Albany Street
nd
2 Floor
Boston, MA 02118
Tel: 617-414-2969
http://www.bumc.bu.edu/stemcells/
iPSC CORE
Establishing iPSC lines from a reprogramming experiment
When performing the reprogramming protocol to establish hiPSC lines from PBMCs or
fibroblasts, around 30 to 40 days after infecting the cells with STEMCCA or Sendai viruses,
the colonies exhibit hESC-like morphology and are ready to be picked. Usually 3 clones per
patient are isolated and only 1 clone is characterized (immunostaining and qPCR for
pluripotency markers, karyotyping, DNA fingerprinting) and has the STEMCCA excised (in
case the clone was generated with STEMCCA-Loxp vector).
1. Manually pick 6 individual colonies and place them in 6 wells of a 96-well plate
containing 100 ul of hESC medium.
2. For each well, pipet up and down a few times (usually 5 times) to break the colony in
small-medium size clumps (~50-200 cells) and plate the 100 ul in a well of a 12-well
plate containing feeders and 1ml of hESC + 10 uM Rock Inhibitor.
3. Next day, add fresh medium without Rock Inhibitor. Add fresh medium daily for about
seven days.
4. Split cells (see protocol for splitting hiPSCs with Collagenase) at ~1:5-1:10 dilution into
a new 12-well feeder plate and use the remaining cells for gDNA extraction for
STEMCCA copy number verification assay (see Derek/Jean’s protocol).
5. After around a week, select three clones that showed only one integration of the
STEMCCA and split them with Collagenase to wells of a 6-well plate. Usually, 1 well of
a 12-well plate is transferred to 1 well of a 6-well plate. The best clone is usually
designated as clone 1.
6. For the next split, plate clones 2 and 3 in 3 wells of a 6-well plate, and clone 1 in 4 wells
of a 6-well plate. When the colonies are large enough, freeze down 3 vials for each
clone (1 well per vial/total: 9 vials). The extra well of clone 1 should be split and the
clone should be kept in culture until all the characterization assays are performed.
7. The following wells should be plated for the characterization assays:
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4 wells of a 24-well plate for immunostaining (Tra-1-81, Oct4, SSEA4, SSEA1)
1 T25 flask for karyotyping and DNA fingerprinting **
670 Albany Street, 2nd Floor. Boston, MA 02118. 617-414-2969.
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1 well of a 6-well plate for RNA (cDNA synthesis and qPCR)
1 well of a 6-well plate for gDNA (positive control for PCR screening for Cre
excision)
3 wells of a 6-well plate with 3 different dilutions for Cre excision (see protocol
for Cre excision)
** Plate cells in the T25 flask at 1:6-1:10 dilution.
** Ideally, the cells should be plated in the T25 flask on a Thursday or a Friday and shipped to
Cell Line Genetics on a Monday.
** Follow the instructions from Cell Line Genetics for preparing and shipping the cells
(https://www.clgenetics.com/wp/wp-content/uploads/2014/06/Mailing-Live-Cultures4.17.14.pdf).
** When the T25 flask is sent to Cell Line Genetics for karyotyping and DNA fingerprinting
analysis, gDNA from the donor sample (fibroblasts or PBMCs) should be sent along.
670 Albany Street, 2nd Floor. Boston, MA 02118. 617-414-2969.