Midbrain dopamine neurons from hESCs

The Kriks et al. protocol has finally achieved what we believe are genuine midbrain, dopamine neurons. Perrier et al. (2004) described a method to make dopamine neurons that we initially thought were mDA neurons. Later work by developmental biologists, however, discovered additional markers of these neurons that the Perrier cells lacked (e.g., FOXA2/TH). The Perrier protocol used neural rosettes as a patterning intermediate to make dopamine neurons. Later work in vivo demonstrated that rodent mDA neurons are derived from the floor plate, a cell type at the base of the neural tube that is not normally neurogenic – its usual role is to produce SHH that helps pattern the neural tube. Fasano et al. (2010) demonstrated a protocol to make floor plate from hESCs but the protocol was not efficient at making mDA neurons. Floor plate is achieved by very early, pre-rosette exposure to high levels of SHH. The Kriks et al. protocol modified this protocol to include early WNT activation, and this turned out to be the key for driving the progenitors into mature mDA neurons. Importantly, these neurons can be transplanted into the brain and retain the mDA neuron phenotype: something that the Perrier et al. cells could not do.

at least two volumes of hESC to one volume of Accutase 3. Centrifuge for 5 minutes at 200 xg, room temperature.4. Gelatin treat a new tissue culture dish during the centrifugation.5. Resuspend cells in hESC media with 10 µM Y-27632.6. Aspirate gelatin from culture dish. 7. Add hESCs (from step 5) to gelatinized dish for 1 hour at 37˚C in the incubator.
MEFs preferentially attach to the gelatinized dish, giving a nearly pure population of hESCs after the incubation 8.While incubating, prepare a Matrigel-coated plate (1:20 in DMEM or hESC media).9.After the hour, collect the non-adherent cells from the incubator and gently wash the dish.Centrifuge cells as above.MEF conditioned media (CM) is harvested from MEF coated flasks.MEFs are plated at a density of 50,000 cells/cm2 in a T225 flask in MEF media.The next day, the cells are washed once with PBS before adding 100 mL of hESC media.Incubate media with MEFs for 24 hours before removal.T C for less than two weeks.Additional hESC media can be conditioned daily for up to ten days on the same flask of feeders.Just before using, FGF2 is added to CM to a final concentration of 10 ng/mL, hereafter called complete CM (cCM).

FAQs:
Q: How do I perform the "center plating" procedure?A: This is done to keep the local cell density high and to keep the monolayer from peeling off at the edge of the dish.To perform this procedure, aspire the fibronectin/laminin solution and "chase the rainbow" -move the aspirator just over the surface of the dish to enhance the drying.After the dish is completely dry, allow it to dry without the lid on in the hood for another 10-20 minutes.The cell suspension is placed in 1/10 of the media that will be used ultimately and is carefully and slowly added to the center of the dish.The media should "bead" on the surface of the dish, confining the cells to a small area in the center of the dish.After 10 minutes without moving the dish in the hood, the remaining 9/10 of media is carefully added around the perimeter of the dish, taking care not to disturb the drop.Carefully move the dish to the incubator Q: I see cell death during days 7-11.What am I doing wrong?A: Make sure your cells are mycoplasma negative.And, we have seen this with certain N2 batches.Recently, we've had more success using NeuroBasal/B27 (no RA) and N2 supplements.You might want to try this medium if you experience survival problems.
Q: I see cell death from days 16-20.What am I doing wrong?A: You might have to feed every day late in the differentiation to keep the culture robust.
23. Day 12 -no feed(aspirate poly-ornithine, wash 3 times with PBS, and add fibronectin/laminin overnight in incubator) 24.Day 13 -Passage 1:1 onto poly-L-ornithine/fibronectin/laminin-coated dishes with 30-45 minutes of Accutase treatment.Spin down in NB/B27, and resuspend in NB/B27 with BAGCT and DAPT (same as above without CHIR) 25.Day 14 -no feed 26.Day 15 -from here, keep the same media composition and feed every other day.27.Between D20-25 when cells become bipolar and make space on the dish, passage them again to poly-L-ornithine/fibronectin/laminin-coated dishes using Accutase.Replate 300-400K per 24 well/well or 2-3 million per 6 well/well with "center plating" (see FAQ) 10. Count cells and plate on Matrigel-treated dishes in hESC media conditioned on high-density MEFs with 10 ng/ml FGF2 and 10 µM Y-27632.Plate at 200,000 cells/cm2.At this density, cells should be confluent overnight.If they are not confluent, continue expansion in CM until they are and then induce differentiation.***Recently, the Studer and Tomishima labs have had problems with the supply chain for this media.This has caused us to explore new sources, and many are now using Neurobasal with N2 supplements and B27 (without retinoic acid) and Lglutamine.