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Stem Cell Information

Generation of Neural Precursors from Pluripotent Stem Cells

(Kozhich et al., 2012. Stem Cell Reviews and Reports)


Materials and Equipment

hESC culture medium

  • 400ml DMEM:F12 (Invitrogen #11330-032)
  • 100ml Knockout Serum Replacer (Invitrogen #10828-028)
  • 5ml Non essential amino acids 100x (Invitrogen #11140-050)
  • 2.5ml L-Glutamine (1mM; Invitrogen #25030-081)
  • 3.5µl β-mercaptoethanol (0.1mM; Sigma #7522)
  • 4ng/ml Fibroblast growth factor-2 (bFGF, R&D Systems Inc. #233-FB). A stock solution of 10µg/ml is prepared in D-PBS containing 0.1% bovine serum albumin. Working aliquots are stored at -20oC and upon thaw should be stored at 4oC and used within 2 weeks.
  • Storage at 4oC, not to exceed 2 weeks

Neural Precursor Medium

  • 48.5ml DMEM (Invitrogen #11965-044)
  • 1ml B27 supplement minus Vitamin A (Invitrogen #12587-010)
  • 0.5ml L-Glutamine (2mM; Invitrogen #25030-081)
  • Storage at 4oC, not to exceed 2 weeks

ITSFn Medium

  • 49ml DMEM/F12 (Invitrogen #10565-018)
  • 0.5ml Insulin-Transferrin-Selenium-A Supplement (100X) (Invitrogen #51300-044)
  • 0.5ml L-glutamine (1mM; Invitrogen #25030-081)
  • 5µg/ml human fibronectin (Roche Applied Sciences #11080938001)
  • Storage at 4oC, not to exceed 2 weeks

Reagents

  • D-PBS (Invitrogen #14190-250)
  • Collagenase IV (Invitrogen #17104-019). A stock solution of 1.5mg/ml is prepared in DMEM:F12 (Invitrogen #11330-032), and frozen in working aliquots at -20oC. Aliquots are thawed immediately prior to use.
  • Accutase (Innovative Cell Technologies, Inc. #AT104)
  • Y27632 (ROCK inhibitor - Selective inhibitor of Rho-associated protein kinase p160ROCK, EMD Biosciences #688000). A stock solution of 5mM is prepared by dissolving 0.5mg in 296µl of sterile distilled water. Working aliquots are stored at -20oC and upon thaw should be stored at 4oC and used within 2 weeks.
  • Recombinant human Noggin (R&D Systems #6057-NG-100): A stock solution of 100µg/ml is prepared in D-PBS containing 0.1% bovine serum albumin. Working aliquots are stored at -20oC and upon thaw should be stored at 4oC and used within 2 weeks.
  • Epidermal growth factor (EGF, R&D Systems #236-EG): A stock solution of 20µg/ml is prepared in D-PBS containing 0.1% bovine serum albumin. Working aliquots are stored at -20oC and upon thaw should be stored at 4oC and used within 2 weeks.
  • Brain derived neurotrophic factor (BDNF, R&D Systems #248-BD): A stock solution of 10µg/ml is prepared in D-PBS containing 0.1% bovine serum albumin. Working aliquots are stored at -20oC and upon thaw should be stored at 4oC and used within 2 weeks.
  • Glial cell derived neurotrophic factor (GDNF, R&D Systems #212-GD): A stock solution of 20µg/ml is prepared in D-PBS containing 0.1% bovine serum albumin. Working aliquots are stored at -20oC and upon thaw should be stored at 4oC and used within 2 weeks.
  • Distilled water (Mediatech #25-055-CM)
  • Poly-D-ornithine (Sigma #P365509). A stock solution of 10mg/ml is prepared in sterile distilled water. Working aliquots are stored at -20oC and upon thaw should be stored at 4oC and used within 2 weeks.
  • Mouse laminin (Sigma #L2020)
  • Neurobasal medium (Invitrogen #21103-049)
  • mTeSR1 medium (Stem Cell Technologies #05850) ENStem-A™ Neuronal Differentiation Medium (Millipore #SCM017)
  • ENStem-ATM Neural Freezing Medium (Millipore #SCM011)

Equipment

  • Cell scrapers (Costar #3010)
  • AggreWellTM400 plate (Stem Cell Technologies #27845)
  • Low attachment 6-well culture dishes (Corning #3471)
  • Tissue culture-treated 6-well plates
  • Benchtop low speed centrifuge
  • Microplate carrier for centrifuge
  • Tissue culture incubator – 37oC/5% CO2

Methods


Embryoid Body (EB) Formation

  1. If starting from colonies grown on feeders, wash cells once with D-PBS and add 1ml pre-warmed collagenase IV (1.5mg/ml) per well of a 6-well plate. If starting with cells on Matrigel go directly to step 6.
  2. Incubate at 37oC for 20 mins at which time the plate should be tapped sharply. If the colonies do not detach at this time the plate should be incubated for a further 10-20mins and the process repeated. If the majority of colonies have not detached within 1 hour they may be removed using a cell scraper.
  3. Remove colonies and transfer to a 15ml tube, wash each well with 1-2ml DMEM:F12 and add wash to the 15ml tube.
  4. Allow to sediment for 3 minutes, aspirate down to the pellet, wash with 5ml DMEM:F12 without disrupting the colonies and allow to sediment again.
  5. Wash with 5ml D-PBS and allow to sediment.
  6. Add 1ml Accutase and incubate at 37oC for 10 mins, agitating by swirling the tube after 5 mins.
  7. Add 5ml DMEM:F12 per 1ml Accutase used and pipet up and down gently.
  8. Centrifuge at 200g for 5 mins.
  9. Remove wash and resuspend in hESC culture medium to a density of 1.2x106 cells/ml in hESC medium containing 10µM Y27632 (ROCK inhibitor). This should generate approximately 1200 EBs of size 1000 cells per EB.
  10. Prepare the AggreWell plate according to the manufacturer’s instructions using hESC culture medium containing 10µM Y27632 (ROCK inhibitor) and add 1ml of cell suspension per well.
  11. Spin at 200g for 5 mins and incubate in the tissue culture incubator for 24 hours
  12. To harvest, the EBs are dislodged from the AggreWell by forceful ejection of the culture medium present in the well using a 1ml pipetman and transferred to a 15ml tube using a 2ml pipette. Do not triturate.
  13. A further 1.5-2ml of medium is added and step 12 repeated.
  14. Repeat until the majority of EBs are removed – no more than 5 times total.
  15. Allow the EBs to sediment for 5 mins and aspirate the supernatant.
  16. Transfer EBs from one well of the AggreWell plate to one well of a low attachment 6-well culture dish in 3ml hESC medium per well and return to the incubator.

Generation of Neural Precursors

  1. Twenty-four hours after harvest, transfer the EBs to a 15ml tube and allow to sediment for 5 mins.
  2. Aspirate the medium and replace with NPM containing 500ng/ml noggin and 20ng/ml bFGF. Return EBs to the low-attachment well.
  3. Change the medium by sedimentation every 2-3 days for 2 weeks.
  4. Change the medium to ITSFn and transfer to standard tissue culture plasticware at a ratio of 1 well EBs to 1 well of a 6-well culture dish. EBs should attach and neuroepithelial-like cells should emerge.
  5. Change the medium every other day for 7-8 days.
  6. Wash the cells once with D-PBS, add 1ml of Accutase and incubate at 37oC for 10mins.
  7. Remove all detached lumps and single cells to a 15ml tube and wash with NPM. Allow the biggest aggregates to sediment briefly (30secs), then remove the upper cell suspension to a new 15ml tube.
  8. Spin the cell suspension at 200g for 5 mins.
  9. Resuspend in NPM containing 20ng/ml each of bFGF and EGF and plate on pre-coated poly-D-ornithine/laminin plates at 1.5-2x106 cells per well of a 6-well dish. This is considered p0 for neural precursors.
  10. Cells may be passaged 1:2 or 1:3 (first passage only) with Accutase for expansion or 1:12 for differentiation.
  11. Cells may be frozen using ENStem-ATM Neural Freezing Medium. We recommend the addition of 10µM Y27632 (ROCK inhibitor) upon thawing.

Poly-D-ornithine/Laminin Coated Plates

  1. Add 2ml of filter-sterilized 20µg/ml poly-D-ornithine in distilled water per well of a tissue culture-treated 6-well plate.
  2. Incubate overnight at 37oC.
  3. Wash once with distilled water and add 2ml of 10µg/ml laminin diluted in D-PBS.
  4. Incubate overnight at 37oC.
  5. To use, aspirate laminin immediately prior to plating cells. Do not wash.
  6. For culture on glass, the concentrations of poly-D-ornithine and laminin should be adjusted to 50µg/ml and 20µg/ml respectively.
  7. Precoated plates may be stored at -20oC after incubation is complete – do not remove the laminin.

Differentiation of Neural Precursor Cells to Neurons

  1. Passage the neural precursor cells 1:12 onto poly-D-Ornithine/laminin coated plates in NPM containing 20ng/ml each of bFGF and EGF.
  2. After 24 hours, exchange the medium for NPM without growth factors.
  3. After a further 2 days, exchange the medium for Neurobasal medium supplemented with 2mM glutamine, 1x B27 supplement, 10ng/ml BDNF and 2ng/ml GDNF.
  4. Exchange medium every two to three days for up to 10 days or longer if cultures remain healthy.

Notes

This protocol describes a robust, reproducible method for the generation of embryoid bodies using the AggreWellTM400 system. Neural precursors may also be generated from colony-derived EBs, which does not require the use of the AggreWellTM400 plate and associated reagents. Detached colonies may be cultured directly in NPM containing noggin and bFGF (Generation of Neural Precursors step 2.). Similarly, single cell suspensions may be aggregated without the use of the AggreWellTM400 plate using 24-well low attachment dishes. In 24-well plates, 1.5x106 cells are plated per well in hESC medium containing 10µM Y27632 (ROCK inhibitor) and incubated overnight at which time EBs from all wells may be combined for step 2 of Generation of Neural Precursors. Under these conditions, however, the uniformity and reproducibility of the AggreWellTM is lost. It should be noted that high quality cultures with a low proportion of spontaneous differentiation are essential for success with any approach.

For differentiation, cells may also be cultured in mTeSR1 medium (Stem Cell Technologies) or in Neuronal Differentiation Medium (NDM: Millipore). Cells differentiated in mTeSR1 appear to maintain some proliferative capacity and serial passaging may enrich for neuronal precursors. After initial differentiation in mTeSR1, medium may be exchanged for other differentiation media such as NDM or Neurobasal medium containing B27, BDNF and GDNF. Cells at this stage may also be differentiated on 1.25% Matrigel (BD Biosciences) instead of poly-D-Ornithine/laminin. Precoated poly-D-Ornithine/laminin plates from BD may also be used but, in our hands, do not perform as well as self-coated plates.