This article describes a protocol for differentiating induced neural progenitor cells (iNPCs) into astrocytes. The process involves using specific media and growth factors to guide the differentiation and maturation of these cells over several weeks.
Take a culture of induced neural progenitor cells, or iNPCs, on an extracellular matrix protein-coated multi-well plate.
Replace the media with glial induction media and incubate. Growth factors and hormones in the media facilitate iNPC differentiation into glial precursor cells.
Change the media every other day for cell proliferation. Then, replace the media with astrocyte media and incubate.
Specific nutrients and growth factors in the media drive glial precursor cell differentiation into astrocytes.
Media changes every other day promote astrocyte maturation.
Change the medium to neuro-diff-medium when the cells are approximately 70% confluent and continue to change the medium every other day for three weeks. Do not split the cells during differentiation. After three weeks, cells should express the neuronal marker TuJ1.
To gain more mature neurons and neuronal subtypes, continue differentiation up to three months. For differentiation of iNPCs specifically towards a glial lineage, change the medium to glial induction medium, including 20 nanograms per milliliter of epidermal growth factor to induce differentiation into glial precursor cells. Remove half of the medium and replace with fresh medium every other day for about two weeks.
During this period, cells should be split 1 to 3 in the presence of 10 micromolar Rho-kinase inhibitor when 100% confluency is reached. After two weeks, differentiate the cells further into astrocytes by changing the medium to commercially available astrocyte medium when the cells are nearly confluent and by continuing to change the medium every second day. Approximately seven days later, cells start to express astrocyte markers.
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