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Stem Cell Information

Subcloning Analysis

View the subcloning protocol

Cell Line MEF density # cells
plated
# clones
after 7 days (%)
# clones
cultured
BG01(1) 0.375 x 106/well
6 well plate
100/well
2 wells
45 (~23%) 11
0.187 x 106/well 140/well
6 wells
62 (~7.4%)
TE03 0.187 x 106/well
6 well plate
200/well 97 (~8%) 8
TE06 0.375 x 106/well
6 well plate
200/well
6 wells
8 (~1%) 2
UC06 0.187 x 106/well
6 well plate
100/well
6 wells
67 (~11%)

13 (~2%)
13 (~2%)
56 (~9%)

6

4
4
NA

WA01 0.187 x 106/well
6 well plate
100/well
6 wells
4 (<1%) 3
WA07 0.75 x 106/10cm dish 125/dish
4 dishes
23 (~5%) 12
WA09 0.187 x 106/well
6 well plate
100/well
6 wells
141 (~23%) 5

NA = Not applicable

For most cell lines, these numbers represent a single experiment. Replicates are in progress.

  • BG01(1)—There appear to be two distinct morphologies that vary in their immunoreactivity to SSEA-1 antibody. One clone has tightly packed cells and is SSEA-1–negative, whereas the other has more flattened, larger cells and is SSEA-1–positive. There was no difference in other undifferentiated marker expression.
  • WA01—No difference in morphology, immunostaining with undifferentiated markers or Oct4 FACS analysis.
  • WA07—No difference in morphology or in immunostaining with undifferentiated markers.
  • WA09—No difference in morphology or in immunostaining with undifferentiated markers.
  • UC06(1)—(in progress)

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Subcloning Protocol

We use an adapted form of the feeder-free protocols detailed in Xu et al. (Nature Biotechnology 19:971–974, 2001) and outlined below.

  • Conditioned medium is prepared by incubating mitotically inactivated MEFs with hESC medium at a ratio of 1ml per 100,000 MEFs. This medium is collected every day up to 10 days and may be stored frozen for up to 1 month.
  • For subcloning plates, mitotically inactivated MEFs are plated at a density of 0.75 x 105/ml - 1.5 x 105/ml with 2.5ml per well of a gelatin-coated 6-well dish.
  • hESC are collected by sedimentation, washed once in PBS, and dissociated with Trypsin-EDTA (Invitrogen Cat #25300-054) or prewarmed Cell Dissociation Buffer (Invitrogen Cat #13150-016). Cells are centrifuged at 200g for 5 minutes, resuspended in conditioned medium containing 8ng/ml bFGF, counted, and serially diluted for plating at a concentration of 100 cells per well.
  • Conditioned medium is replaced every day until colonies begin to appear (3 to 7 days), at which time normal hESC medium containing 4ng/ml bFGF is used.
  • The number of colonies formed is assessed on day 7 of culture (cloning efficiency).
  • When colonies reach approximately 100 to 200 cells, they are individually transferred to new wells.