This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells. Part 1 of 3.
Splitting human embryonic stem cells (hESCs) plated on mouse embryonic fibroblasts (MEFs)
Usually a confluent hESC plate can be split 1:6 to 1:10, depending on the particular hESC line. The split plate will become confluent again 5-7 days after splitting.
This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells. In the last step before plating, when the hESCs are resuspended, they should be of roughly uniform colony size and shape. Carefully pipette the hESC suspension up and down a few times to make the colonies smaller and more uniform, but not so much that single cells or very small colonies are generated.Immunofluorescence staining and microscopy or flow cytometry for hESC pluripotency markers, such as Oct-4 and SSEA-4, are needed to confirm maintenance of hESCs in an undifferentiated state during culture.
Human embryonic stem cell studies in the Teitell lab are supported by a California Institute for Regenerative Medicine (CIRM) seed grant RS1-00313. We thank members of the Broad Center of Regenerative Medicine and Stem Cell Research at UCLA, especially Dr. Amander Clark, Dr. Jerome Zack, and members of the UCLA Broad Institute Stem Cell Core Facility for their support of our studies.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Knockout Serum Replacer (KSR) | Reagent | Gibco | 10828-028 | |
DMEM/F12 | Reagent | Gibco | 11330-057 | |
Non-essential Amino Acids | Reagent | Gibco | 11140-050 | |
GlutaMax | Reagent | Gibco | 35050-061 | |
DMEM | Reagent | Gibco | 11995-065 | |
FBS | Reagent | Clontech | 631107 | |
L-glutamine | Reagent | Gibco | 25030-081 | |
BME | Reagent | Fisher | BP176-100 | |
bFGF | Reagent | R&D Systems | 233-FB-025 | |
Collagenase IV | Reagent | Gibco | 17104-019 | |
Dispase | Reagent | Stem Cell Technologies | 17105-041 | |
Penicillin / Streptomycin | Reagent | Gibco | 15140-122 | |
Gelatin | Reagent | Chemicon | ES-006-B | |
Matrigel | Reagent | BD Biosciences | 354277 | |
Oct-4 antibody | Reagent | Santa Cruz Biotechnology | SC-9081 | |
anti-h/mSSEA-4 Phycoerythrin Conjugated Mouse IgG3 | Reagent | R&D Systems | FAB1435P | |
FITCI-conjugated antirabbit IgG | Reagent | Jackson ImmunoResearch Laboratories. Inc. | 715-095-152 |