Neural induction - Dual SMAD inhibition

Dual SMAD inhibition takes a confluent, feeder free culture of hPSCs and rapidly differentiates them into early neurectoderm (Chambers et al., 2009). This rapid differentiation is caused by blocking the two signaling pathways that utilize SMADs for transduction: BMP and TGFB. Oct4 is extinguished and Pax6 expression has begun by around day 7–8, depending on the line used. This neurectoderm can be passaged to become rosettes or can be patterning to become many other types of neural cells (for example, see Fasano et al., 2010).

This protocol is based on Chambers et al. 2009.
[1 ml for a 6 well dish, 2 ml for a 6 cm dish, or 5 ml for a 10 cm dish.There is no need for a PBS wash.] 2. Place in the incubator (37 C) for about 30 minutes -colonies will become single cells.3. Pipette cells a few times to make a single cell suspension.Filter through a 45 μm strainer to remove any remaining clumps.4. Wash and centrifuge cells (200xg for 5 min) two times in hESCM to remove Accutase.5.While washing, prepare Matrigel-coated dishes.Add 19 ml of hESCM to a nearly-thawed Matrigel aliquot and pipette until completely thawed and homogeneous.Work quickly and do not let the Matrigel warm up or it will polymerize.6.After washing hPSCs, resuspend the cells in hESCM with 10 μM Y-27632 and place on a gelatin-coated dish of the same size as used to grow the hPSCs in step 1. 7. Place dish in incubator (37 C) for 30 min.8. Collect the non-adherent cells and gently wash the dish with PSCM with 10 μM Y-27632 and centrifuge the cells (200xg for 5 min).hPSCs are rounded and therefore non-adherent.Contaminating MEFs are flat and more adherent.This step eliminates many MEFs by leaving them stuck to the dish.9. Resuspend the cells in complete conditioned media (cCM) with 10 μM Y-27632.10.Determine the cell concentration using a hemocytometer and add cCM with 10 μM Y-27632 to the appropriate cell concentration for desired density (see chart at end).11.Aspirate the Matrigel solution and plate cells directly on dish.
[There is no need to wash the dish though some do.]12.Twenty-four hours after plating, aspirate media and add fresh cCM with 10 μM Y-27632.13.Forty-eight hours after plating, aspirate the media and add fresh cCM -from this point on, Y-27632 is no longer necessary.14.Cells can be maintained for additional days in cCM until the desired density is reached (~90-95% for CNS or about 60% for a mixture of neural crest and CNS).

! [Neural induction]
1. To initiate differentiation, add KSR containing 10 μM SB431542 and 200 ng/ml Noggin.Defined as Day 0 of the induction.100 nM LDN 193189 can be used instead of Noggin for version 3.0, 9/26/2014 the protocol as well, although we do not currently understand possible downstream changes in cell fate.2. On day 1 of differentiation, aspirate the KSR and add fresh KSR with 10μM SB431542 and 200 ng/ml Noggin.3. On day 2 of differentiation, aspirate the KSR and add fresh KSR with 10μM SB431542 and 200 ng/ml Noggin.4. On day 4, aspirate the KSR and add a mixture of KSR/N2 media (3:1) with 10μM SB431542 and 200 ng/ml Noggin (final concentration).5. On day 6, aspirate the KSR and add a mixture of KSR/N2 (1:1) with 10μM SB431542 and 200 ng/ml Noggin (final concentration).! 6.On day 8, aspirate the KSR and add a mixture of KSR/N2 (1:3) with 10μM SB431542 and 200 ng/ml Noggin (final concentration).7. On day 10, cells can be passaged en bloc or as single cells onto Matrigel-coated dishes.

! [Variation 1: Passage of neural cells en bloc]
1. Mechanically dissociate thickened neurectoderm using a 200 μL pipette into small pieces, or cut monolayer of cells using a StemProEZ Passage tool (Invitrogen) 2. Plate the blocks of tissue onto Matrigel-coated dishes in N2 containing the appropriate growth factors at 2:1 or 3:1.Note that this is 2:1, not 1:2 -you want less surface area because not all of the pieces will attach and you need very high cell density to get good progression of CNS neural cells.

! [Variation 2: Passage of neural cells as single cells]
1. Aspirate differentiation media and add minimal Accutase to coat the dish and let sit at 37 C until all cells are rendered to single cells (approximately 30 minutes).2. Avoiding bubbles, triturate the cells in the dish using a pipette with additional N2 media until the cells are in single cell suspension and filter using a 45 μm cell strainer.3. Wash and centrifuge cells (200 x g for 5 minutes) twice in N2 media.4. Resuspend the cells in N2 media.5. Determine the cell concentration using a hemocytometer.6. Resuspend the cells in N2 media to a cell concentration of 5 x 106/mL.7. Prepare Matrigel-coated dish by carefully aspirating all liquid from the dish, taking care not to touch the surface.Let dish dry for 15 minutes before plating drops.8. Spot plate with 20 μL drops of the cell suspension (1x105 cells) and let stand in the hood for 20 minutes before slowly adding N2 containing the appropriate growth factors.Move dish carefully to the incubator.

! ! [Differentiation into neurons]
1. Minimal supportive media for differentiating neural cells into neurons is N2 containing 20 ng/ mL of BDNF and 200 μM ascorbic acid.Differentiations are usually carried out for at least one(and sometimes two) weeks with media changes every two to three days.
! SHH can be purchased that is either mouse or human (although they are 92% identical at the amino acid level).Furthermore, it can be purchased with engineered isoleucines on the N-terminus: these modifications make the protein more hydrophobic and likely act as a membrane tether and intercellular transport mechanism.The engineered modifications phenocopy the cholesterol and palmitate modifications that occur in mammalian cells and are necessary since bacterial expression of the protein does not provide these mammalian modifications.Most of our previous work uses conventional SHH (such as R&D Systems cat.# 461-SH) but we have found that it is more economical to use 10-fold less of the isoleucine-modified version. !
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).