TITLE: A Monolayer Culture Method for Neural Induction of Human Pluripotent Stem Cells
AUTHORS: STEMCELL Technologies

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Multipotent neural progenitor cells (NPCs) generate the major cell types of the central nervous system (CNS): neurons, astrocytes and oligodendrocytes. Human pluripotent stem cells (hPSCs), including embryonic stem (ES) cells and induced pluripotent stem (iPS) cells, can be directed to differentiate into NPCs using a variety of methods. This process, known as ‘neural induction’, must be efficient and reliable, in order to generate high-quality NPCs for downstream applications. Although neural induction of hPSCs using embryoid body (EB) formation allows for visual assessment of induction success via the formation of neural rosettes, the whole process can take anywhere between 16 and 19 days to get single-cell neural progenitor cells (NPCs). As a result, monolayer culture-based neural induction methods have recently gained popularity, since they enable single-cell NPCs to be obtained in as few as six days. Here we describe a procedure for neural induction using STEMdiff™ Neural Induction Medium in a monolayer culture-based system to efficiently generate PAX6-positive NPCs.</div></div>

[STEPS]
?. [Preparation of Materials]
Before beginning the experiment, prepare Poly-Ornithine/Laminin- or Matrigel®-coated plates or coverslips.
?. [Procedure for Neural Induction]
Pre-warm STEMdiff™ Neural Induction Medium, Gentle Cell Dissociation Reagent, phosphate buffered saline (PBS) without Ca2+ and Mg2+, and DMEM/F-12 to .
37 °C
?. [Procedure for Neural Induction]
Estimate the volume of STEMdiff™ Neural Induction Medium required for initial seeding (see Table 1), and supplement with 10 μM Y-27632 (ROCK inhibitor).  ABCD1CulturewareVolume/Well (mL)Number of Cells Required2200,000 cells/cm^2250,000 cells/cm^236-well plate2.02.0 x 10^62.5 x 10^6412-well plate1.58.0 x 10^51.0 x 10^6524-well plate1.04.0 x 10^55.0 x 10^6
Cell density is critical for achieving success. For initial plating, seed hPSCs between 200,000 and 250,000 cells/cm2. If seeding at too low a density, the efficiency of neural induction will be reduced.
ABCD1CulturewareVolume/Well (mL)Number of Cells Required2200,000 cells/cm^2250,000 cells/cm^236-well plate2.02.0 x 10^62.5 x 10^6412-well plate1.58.0 x 10^51.0 x 10^6524-well plate1.04.0 x 10^55.0 x 10^6
?. [Procedure for Neural Induction]
Inspect each 10 cm2 plate of human ES or iPS cells (previously maintained in mTeSR™1 (Stemcell Technologies, catalog #85850) or TeSR™-E8™(Stemcell Technologies, catalog #05990)) and aspirate any areas of differentiated cells.
?. [Procedure for Neural Induction]
Rinse each plate once with 5 - 10 mL of sterile PBS.
?. [Procedure for Neural Induction]
Add 3 mL of Gentle Cell Dissociation Reagent and incubate at .
[for 8 - 10 minutes]
?. [Procedure for Neural Induction]
After incubation period, gently dislodge cells that are still attached, using a 5 mL pipet. Triturate cells by pipetting up and down 5 - 10 times.
?. [Procedure for Neural Induction]
Add 5 mL of DMEM/F-12 and collect cells into a 50 mL conical tube.
Optional: Add an additional 5 mL of DMEM/F-12 onto the 10 cm2 plate to rinse off any remaining cells and add to the 50 mL tube.
?. [Procedure for Neural Induction]
Count viable cells e.g. using Trypan Blue and a hemacytometer.
?. [Procedure for Neural Induction]
Centrifuge cells
?. [Procedure for Neural Induction]
Resuspend cells in an appropriate volume of STEMdiff™ Neural Induction Medium supplemented with 10 μM Y-27632 to achieve a seeding density of 200,000 or 250,000 cells/cm2, for ES cells and iPS cells respectively. See suggested volumes in Table 1.
?. [Procedure for Neural Induction]
Seed cells onto Poly-Ornithine/Laminin- or Matrigel®-coated plates or coverslips.
?. [Procedure for Neural Induction]
Replace medium daily with fresh STEMdiff™ Neural Induction Medium. Y-27632 is not required after seeding. If performing further differentiation to neuronal lineage, see Steps 15-16.
?. [Procedure for Neural Induction]
After six to nine days in STEMdiff™ Neural Induction Medium, cells will be OCT4-negative and PAX6-positive (Figures 1 - 2). At this point, cultures will be confluent and ready for passaging using ACCUTASE™.
Visual inspection of cultures is not a reliable method of confirming neural induction. We recommend assessing expression of markers for neural induction (PAX6) and/or undifferentiated ES or iPS cells (e.g. OCT4).
?. [Passaging]
Maintain neural progenitor cells (NPCs) in STEMdiff™ Neural Induction Medium until passage 3. At passage 3, transition cells into STEMdiff™ Neural Progenitor Medium.
After the first passage, NPCs should be passaged once they reach 70 - 80% confluency, and seeded in the next passage at a density between 125,000 and 200,000 cells/cm3.
Supplement STEMdiff™ Neural Induction Medium with Y-27632 for the first day after each passage. Y-27632 is not required when maintaining cells in STEMdiff™ Neural Progenitor Medium.
?. [Downstream Differentiation]
For differentiation to neuronal subtypes (e.g. dopaminergic, motor), inductive factors are added between days five and seven. Differentiation to glial subtypes requires NPCs at passage four or later.