Differentiating Induced Neural Progenitor Cells into Astrocytes

1. Differentiation of iNPCs

1. Differentiation towards neuronal lineage​
   1. Culture cells on laminin-coated plates as described above. Change media to neuro-diff-media (DMEM/F-12, 1x N2, 1x B27, 300 ng/ml cAMP, 200 µM vitamin C, 10 ng/ml BDNF, 10 ng/ml GDNF) when cells are approximately 70% confluent. Change media every other day for three weeks. Do not split cells during differentiation.
   2. After three weeks, cells should express the neuronal marker TUJ1. To gain more mature neurons and neuronal subtypes, continue differentiation upto 3 months.
2. Differentiation towards glial lineage
   1. Change media to glial induction media (1:1 DMEM/F-12 : Neurobasal, 1x N2, 1x B27, 10 µM β-mercaptoethanol, 100 µM non-essential amino acids, 1.5 mM L-Glutamine, 5 µg/ml insulin, 40 ng/ml T3) including 20 ng/ml EGF to induce differentiation into glial precursor cells. Remove half of the media and replace with fresh media every other day for about 2 weeks. During this period, cells should be split 1:3 in the presence of 10 µM ROCK inhibitor Y27632 when 100% confluency is reached.
   2. After 2 weeks, differentiate cells further into astrocytes: when cells are nearly confluent, change media to commercially available astrocyte media, change media every second day. After about 7 days, cells start to express astrocyte markers (e.g., GFAP). If cells get 100% confluent during the differentiation protocol, split them in a 1:2 ratio in the presence of 10 µM ROCK inhibitor Y27632.