EB Neurons

Procedures for neuroepithelia and forebrain neuron differentiation

Making iPS aggregates/”Embryoid bodies” (Day 1-4)

  1. Prepare Dispase @ 1 mg/ml in DMEM/F12 (Collagenase can also be used at the same concentration). Warm in a 37°C water bath to dissolve (7-15 min) and filter sterilize with a Steri-flip.
  2. Aspirate iPS media off iPS cells and add Dispase (0.5 ml/well of a 6 well plate).  iPS cells should be grown to the same density as those prior to passaging/splitting.
  3. Incubate and wait 2-5 minutes until the edges of cell colonies begin to curl off of the plate.  Tap or swirl the plate to dislodge colonies.
  4. Add 3 mls of iPS media and gently pipette all of the iPS colonies from one entire 6 well plate and transfer them to a 15 ml tube. Gently triturate 3-5 times to break cell colonies into smaller clusters.  Clusters should be roughly twice the size as clusters for passaging iPS cells. 
  5. Allow the iPS cell clusters to settle to the bottom of the tube (2-3min).  Aspirate off the medium with caution so as to not aspirate the entire pellet. 
  6. Wash the cells once by adding 5-6 mls of fresh iPS media and then centrifuge for 2 minutes at 200g.
  7. Aspirate off supernatant and resuspend cells in iPS media (for 6 wells use 60 ml iPS medium) and transfer to flasks. 

            Cell aggregates will initially look unhealthy from shock of separation from feeders.  To speed cell recovery, feed for the first time within ~12 hours and replace most of the medium to remove debris.  Switching cells to a new flask is also useful to remove MEF that may have attached during the first 12 hours.

  1. Continue feeding with iPS medium, no exogenous factors every day for four days.

            When feeding, use a 5 ml pipette to gently pull aggregates up and then blow them back into the medium 2-3 times.  This will help clean dead cells off the aggregate surface.  Let the clusters settle to the bottom in a standing flask and aspirate off the medium.

            See the alternative procedures section for a discussion of iPS aggregates vs. embryoid bodies

 

Differentiating to Primitive Neuroepithelia (Day 5-10)

After free floating culture in iPS medium for 4 days, the aggregates are ready for further differentiation.

  1. Collect the iPS cell clusters, centrifuge for 1 min at 200g and wash once with 5 mls of Neural Induction medium (no exogenous factors.) 
  2. Re-suspend cells in 50-60 mls of Neural Induction medium (N2) and transfer to a new flask. 

Cells in Neural Induction medium can be fed every other day.

  1. After 2 days, aggregates should be bright and clear and are ready for attachment.  Induce attachment by plating cells on a laminin coated substrate.  Use 20 μg/ml mouse or human laminin in DMEM/F12 or Neural Induction medium on either plastic or plain glass or glass coated with Polyornithine (see alternative procedures for more information) (I find that in my hands matrigel worked best for all plating steps.)

Laminin coated surfaces should be incubated at 37°C overnight for best results. 

  1. When plating aggregates, provide enough space for colonies to grow out without contacting one another.  Aggregates should be transferred to a 15 ml tube and agitated gently with a 5 ml pipette to remove loose cells.  20-30 aggregates should be deposited in fresh Neural Induction medium in each well of a 6 well plate or 2-4 aggregates /coverslip.  If plating on a 6 well plate, shake the plate on the incubator shelf up and down twice and then left and right twice gently to evenly distribute the clusters, the same method as for splitting iPS cells.  Cells should attach overnight (minimize jarring plates, including frequent incubator closing and opening to improve attachment; an alternative method for attachment is to add 10% FBS in neural induction medium for overnight). 
  2. Attached aggregates will collapse to form a monolayer colony after 1-2 days.  Continue feeding with Neural Induction medium (no exogenous factors) every other day. 
  3. After 10-11 total days of differentiation (4-5 days following attachment), over 95% of the colonies should take on a morphology in which the center cells exhibit an elongated, columnar morphology .  After 10-11 days of differentiation, primitive neuroepithelia is receptive to neural patterning signals.  Attempts to add patterning signals (notably retinoic acid) prior to this time point can lead to differentiation to non-neural fates.  At this point cells can continue to be cultured in Neural Induction medium or alternatively switched to conditions designed to regionally specify the neuroepithelia to more specific cell fates (such as the addition of SHH for ventralization).

Generating Definitive Neuroepithelia (day 11-17)

  1. Scratch off any colonies that do not contain any primitive neuroepithelia cells (this should be less than 10% of colonies, see the troubleshooting section for additional help if this is not the case).  The “bad” colonies can be marked by an objective marker lens under a phase contrast scope and then scraped away with a pipette tip in a sterile hood.
  2. Neuroepithelial cells should be fed with the same medium every other day and cultured for 7 days (no exogenous factor).  During this period starting at day 14-15 the columnar neuroepithelia cells will further compact and proliferate, often forming ridges or rings of cells outlining a distinct lumen.  The overall morphology is reminiscent of the neural tube and cells at this stage are often referred to as neural tube-like rosettes.
  3. After 17-18 days of differentiation under these conditions the neuroepithelia that makes up the rosettes will stain positive for the definitive neural tube stage marker Sox1 (dilute at 1:500).

Isolating Definitive Neuroepithelia (day 17-18)

To increase the purity of neuroepithelia cells generated, we have added a sub-culture step after the formation of neural tube-like rosettes (see notes on neuroepithelia isolation timing in the alternative procedures section). 

(We use STEMdiff™ Neural Rosette Selection Reagent at this point now, per manufacturer’s instructions.)

  1. Treat the culture with 0.5 mg/ml dispase in DMEM/F12 or Neural Induction medium. Incubate at 37°C ~2-3 minutes, monitoring closely for when inner neuroepithelia start to peel away from the flat peripheral cells at the edges of colonies.
  2. Once the rosettes start to peel off, tap the plate to speed the process while trying to keep flat peripheral cells attached.
  3. Once the neuroepithelial cells are detached, collect the neuroepithelial cell clusters in a 15 ml centrifuge tube.

Use gentle pipetting during rosette isolation as the cells separate easily and it is best to not break clusters of neuroepithelia up much initially.

  1. Spin at 100g for 2 min and wash once with fresh Neural Induction medium.
  2. Aspirate the medium and resuspend the clusters of definitive neuroepithelia in 5 ml of Neural Induction medium supplemented with N2/B27 .
  3. Over the next 24 hours the rosette aggregates will roll up to form round spheres while any flat non-rosette peripheral cells will usually attach to the culture vessel. After this period rosette aggregates should be switched to a new flask with Neural Induction medium N2/B27. At this stage, cAMP (final concentration at 1 uM) and IGF (10 ng/ml) could be added to improve the survival/proliferation.
  4. After several days in Neural Induction medium and B27, neuroepithelial aggregates (neurospheres) are ready for further differentiation to neural cells.

 

Differentiation of forebrain neurons from human iPS cells 

  1. Collect the neurospheres in the flask to a 15 ml tube, centrifuge at 1000 rpm for 2 min
  2. Wash once with DMEM-F12
  3. Plate neurospheres on laminin/ Polyornithine (matrigel) coated coverslips (96-well plates) in the presence of Neural Differentiation medium supplemented with B27, cAMP (1 μM), and BDNF, GDNF, and IGF1(10ng/ml).  
  4. Please note: If the spheres are too big, they could be broken using glass pipette at 1 day before plating. Another way is to dissociate the neurospheres to small clusters using Accutase (2-5 minutes).
  5. For specifying the ventral telencephalic progenitors, SHH (R&D, 100 ng/ml) will be added to the neuroepithelial cells at day 10 (day 10 to 24).  After plating the neurospheres, cells are cultured in Neural Differentiation medium supplemented with cAMP (1 μM), and BDNF, GDNF, and IGF1( 10ng/ml).  The amount of SHH is reduced to 10 ng/ml.

 

Alternative Procedures

Alternatives to laminin-enhanced adhesion:  Attachment of iPS aggregates or neuroepithelial clusters can be accomplished with a variety of different adhesion molecules including fibronectin and Matrigel.  Another quick and cost effective method to facilitate adhesion is to supplement Neural Induction medium with 10% fetal bovine serum for 12-24 hours.  The serum should then be washed away after the aggregates have attached.  Although serum should be avoided for neuroepithelial differentiation, this short-exposure to enhance adhesion does not significantly reduce the overall efficiency of the culture system for generating neuroepithelia.  It should be noted that the use of serum may affect some gene expression patterns. 

 

Time for neuroepithelia isolation:  Neuroepithelia cells can technically be isolated at any point after the primitive neuroepithelia stage at 10 days of differentiation and grown in suspension.  The benefit of isolating neuroepithelia at the primitive stage is that it allows for early selection of neuroepithelia and limits cell death and differentiation that result from high density culture.  However, culture of neuroepithelia as free floating clusters as opposed to a monolayer affects the exposure of cells to patterning signals and mitogens.  If after 17-18 days of culture, neural tube-like rosettes are difficult to observe and there are too many non-neuroepithelial cells in culture, try enzymatically isolating neuroepithelia at the primitive neuroepithelial stage (day 10).  Grow cells as aggregates for 1-2 days in Neural Induction medium and then re-plate the cells at a lower density.  Take care to not break neuroepithelia cluster up too much.  If neuroepithelia clusters attach and form monolayer colonies of larger flat cells like the ones seen at the edges of colonies at 10 days the clusters are too small.  Breaking neuroepithelia clusters less initially or keeping clusters in culture longer will allow more cell proliferation and should solve this problem.

Mechanical neuroepithelia isolation:  If you are having trouble enzymatically isolating neuroepithelia at the primitive (10 day) or definitive (17-18 day) stage from the flat surrounding cells, try isolating the neuroepithelia cells mechanically.  Very gentle pipetting with a 1000 µl tip can usually dislodge the neuroepithelia which is denser in the center of colonies, as opposed to the flat, tightly bound cells at the periphery.

 

Reagents

 

Item

Supplier

Catalogue #

  L-Glutamine solution            (200 mM)

Sigma, St. Louis, MO

G-7513

MEM non-essential amino acids solution

Gibco-BRL, Rockville, MD

11140-050

Kockout serum replacer

Gibco-BRL

10828-028

Dulbecco’s modified eagle medium: Nutrient mixture F-12 1: 1 (DMEM/F12)

Gibco-BRL

11330-032

Dulbecco’s modified eagle medium (DMEM)

Gibco-BRL

11965-092

Neurobasal medium

Gibco-BRL

21103-049

β-Mercaptoethanol (1000x)

Invitrogen

21985023

N2 supplement

Gibco-BRL

17502-048

Laminin from human placenta

Sigma

L6274

       Bovine serum albumin  (BSA)

Sigma

A-7906

Cyclic AMP

Sigma

D-0260

Sonic hedgehog (SHH)

R&D

1845-SH

Dispase

Gibco-BRL

17105-041

Acutase

Innovative Cell Technologies

AT104

Trypsin Inhibitor (1mg/ml dissolved in DMEM/F12 & sterile filtered)

Gibco-BRL

17075-029

Heparin

Sigma

H3149

Recombinant human BDNF

PeproTech Inc

450-02

Recombinant human GDNF

PeproTech Inc

450-10

Recombinant human IGF1

PeproTech Inc

100-11

Polyornithine

Sigma

P-3655

Fetal bovine serum

Sigma

F-2006

       

 

iPS cell Medium

500 ml

Component

Amount

 Final Concentration

DMEM/F-12

392.5 ml

 

Serum Replacer (KOSR)

100 ml

20%

MEM Non-Essential Amino Acids Solution

5 ml

0.1 mM

Beta-Mercaptoethanol (14.3 M)

3.5  µl

0.1 mM

L-Glutamine (200 mM)

2.5 ml

1 mM

Sterile filter with a 0.22 µM filter, add 4ng/ml bFGF just prior to feeding cells, Medium is stored at 4°C for up to 2 w.

 

Neural Induction Medium (N2)

500 ml

Component

Amount

 Final Concentration

DMEM/F12

490 ml

 

N2

5 ml

1X

MEM Non-Essential Amino Acids Solution

5 ml

0.1 mM

Heparin (2 mg/ml)

500 µl

2 μg/ml

Sterile filter with a 0.22 µM filter, add cytokines and signaling molecules (such as FGFs) just prior to feeding cells.

 

Neural Induction Medium (N2/B27)

500 ml

Component

Amount

 Final Concentration

DMEM/F12

480 ml

 

N2

5 ml

1X

B27

10 ml

1X

MEM Non-Essential Amino Acids Solution

5 ml

0.1 mM

Heparin (2 mg/ml)

500 µl

2 μg/ml

Sterile filter with a 0.22 µM filter, add cytokines and signaling molecules (such as FGFs) just prior to feeding cells.

 

Neuronal Differentiation Medium

500 ml

Component

Amount

 Final Concentration

Neurobasal medium

480 ml

 

N2

5 ml

1X

B27

10 ml

1X

MEM Non-Essential Amino Acids Solution

5 ml

0.1 mM

Sterile filter with a 0.22 µM filter, add cytokines and signaling molecules just prior to feeding cells

 

Dispase solution 10 ml

Component

Amount

 Final Concentration

Dispase

10 mg

1 mg/ml

DMEM/F12

10 ml

 

Leave in a 37°C water bath for 15 min and filter sterilize the dispase solution with a 50 ml-Steri-flip before use.

 

NE cells could be isolated by treated the culture with 0.5 mg/ml dispase in DMEM/F12 or Neural Induction medium. Incubate at 37°C ~2-3 minutes, monitoring closely for when inner neuroepithelia start to peel away from the flat peripheral cells at the edges of colonies.

Once the rosettes start to peel off, tap the plate to speed the process while trying to keep flat peripheral cells attached.

Mechanical neuroepithelia isolation:  If you are having trouble enzymatically isolating neuroepithelia at the primitive (10 day) or definitive (17-18 day) stage from the flat surrounding cells, try isolating the neuroepithelia cells mechanically.  Very gentle pipetting with a 1000 µl tip can usually dislodge the neuroepithelia which is denser in the center of colonies, as opposed to the flat, tightly bound cells at the periphery.