In the last blog we gave an overview on how to
characterize iPS cells. Today, we dive in and explain how we
test the cells for pluripotency, the potential to turn into different cell
types of the human body.
Alex: Hi, it's
been a while, but it's summer and I heard
Lauren has accepted a new position!
Birgitt: Yes, we'll miss Lauren and we wish her the best of luck for her future.
Alex: So what's next?
Birgitt: I'll explain how we test your cells to see if they have the potential to
show "yes we can" "Change" into other cells. To test
proteins and markers in the cell you need to visualize them. This can be done
with a method called
immunostaining. Immunostaining is an
antibody-based method
that detects specific proteins on the surface or inside of the cells. The picture
below is an example of how we can immunostain the cells. We use an antibody
specific for a certain marker on the cell, called
epitope. Once this antibody
binds to the epitope we then incubate with a fluorescently labeled antibody
that binds to the first antibody. We can then visualize this fluorescence with
a fluorescent microscope and take the colorful images you will see in the next
few blogs.
More info about immunostaining
here
Alex: Hmm sounds a lot like
2008. I posted
some pretty fluorescence pictures on my Facebook page that you shared with me
in the past. I am looking forward to understanding more about these immunostaining
techniques.
Birgitt: We used
three different antibodies to detect whether or not your stem cells are
pluripotent. The three markers we used were OCT4, SOX2, and SSEA4, these are
proteins that are expressed in pluripotent cells and therefore called
pluripotency markers. These are abbreviations for protein names octamer-binding
transcription factor 4 (OCT4), sex
determining region Y-box 2 (SOX2), and stage-specific embryonic 4
(SSEA4). Since these protein names are so complicated we usually only use the
abbreviations.
See the results in the three images below.
The blue color is the nuclear stain or
counterstain which shows where the nucleus of the cell is located. The green
fluorescence corresponds one of the pluripotency markers.
|
iPS
colony stained with SSEA-4 antibody |
|
iPS
colony stained with OCT4 antibody |
|
iPS
colony stained with SOX2 antibody |
Alex: I hope we can talk about this more next time and I am also very excited to come out to San Francisco for the Festival of Genomics on November 5
th, 2015 to chat with you on stage about my Parkinson’s disease, stem cells, and more (
http://www.festivalofgenomicscalifornia.com/).
Birgitt: I am thrilled to have the opportunity to talk with you on stage, can't wait! And maybe the topic of pluripotency will be reelected in the next post.