Abstract
Groundbreaking studies from Dr. Yoshiki Sasai’s laboratory have recently introduced novel methods to differentiate mouse and human Embryonic Stem Cells (mESCs and hESCs) into organ-like 3D structures aimed to recapitulate developmental organogenesis programs (Eiraku et al., 2011; Eiraku and Sasai, 2012; Nakano et al., 2012; Kamiya et al., 2011). We took advantage of this method to optimize a 3D protocol to efficiently generate retinal progenitor cells and subsequently retinal neurons in vitro. This culture system provides an invaluable platform both to study early developmental processes and to obtain retinal neurons for transplantation approaches. The protocol described here has been successfully applied to several mouse ESC (including the R1, WD44 and G4 cell lines) and mouse induced-Pluripotent Stem Cell (iPSCs) lines.
Keywords: Retina, Mouse Embryonic Stem Cells, Embryoid Bodies, Developmental Biology, Organogenesis
Materials and Reagents
Equipment
Procedure
Recipes
Acknowledgments
This protocol was originally published as part of: La Torre et al. (2015). The author wishes to thank all present and past members of the Reh and Bermingham-McDonogh laboratories for many helpful discussions. Special thanks to Tom Reh and Akina Hoshino for invaluable help and advice, and NIH 1 PO1 GM081619 and the imaging core of the Vision Core Grant to the University of Washington, P30EY01730 (PI: Reh).
References
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Hi again! You can change only half the medium. After day 15 or so, the EBs are bigger and then, if you notice that the medium gets acidic quickly, you can do a full media change. The easiest way is to tilt the plate so all the EBs move to the bottom of one side and slowly (don't use suction! only a pipette) remove the media, add fresh media and you are done. Auguri!
Hi! We normally use a pipet with a 1000 tip. A pasteur will work too since it's big enough so the EBs are not broken during the transfer. From a 96 well plate, we move them to 3-4 wells of a 6 well plate. Therefore, we plate about 24-30 EBs per well but you don't need to count, just distribute them more or less evenly. Good luck with your cells! PS: I really want to learn Italian!
Dear Dr. La Torre, thanks so much for your feedback! Today is D2 of EBs and everything seems ok! Fingers crossed!P.S. we could share our knowledge: I in Italian and you in retinal differentiation :) ! Thanks again and have a good weekend.
Hi Min,You do NOT change media during the first few days. When are your cells dying? and also, what cell line are you using? you could try to move them to the bigger plates earlier (day 4-6) if your cell line tends to grow very quickly and that might be the issue. I hope this helps but don't hesitate to email me if you need any further info. Good luck!
I thought perhaps I should further clarify: if (a) your EBs develop well during the first few days (i.e. on Day 1 you see a well-defined ball of cells with smooth edges and on day 3 there is a clear epithelium developing all around the EB) and the media is changing color to yellowish before day 7, I would move the cells earlier to the 6 well plates. If (b) the EBs don't form well, they become darker, you don't see smooth edges from day1 or the neuroepithelium is not clearly developing from day3, then probably your dissociation is too aggressive and I would try to reduce the time for the enzymatic dissociation.
Thank you so much for the reply! We use E14 cell line. The EBs seem develop well on Day1 but we do not see clear edge on Day3. Then the EBs became darker and on Day7 we do not see any live cells. If we move the EBs to 6 well plate on Day4, do we need to add matrigel in the medium?
No need for more matrigel. The matrigel is only needed very early to promote polarization and trigger the right developmental program.
Thank you!