This article describes a protocol for differentiating blood-derived pluripotent stem cells (BD-PSCs) into neuronal cells using specific culture substrates and media. The process involves coating glass coverslips with extracellular matrix proteins and incubating BD-PSCs to promote their differentiation into neural progenitor cells and subsequently into neurons.
Take a microplate with glass coverslips placed at the bottom of the wells.
Introduce a positively charged polymer to coat the coverslips and wash to remove any unbound polymer. Treat with an extracellular matrix protein to coat the coverslips and aspirate the unbound proteins.
The coating acts as a culture substrate that facilitates cell adhesion.
Add an induction medium to the wells and introduce a suspension of blood-derived pluripotent stem cells, or BD-PSCs. These cells, capable of differentiating into various cell types, are generated via reprogramming blood progenitor cells.
Incubate for the cells to adhere to the substrate. Growth factors and nutrients in the induction medium transform BD-PSCs into neural progenitor cells.
Introduce a differentiation medium containing neuronal growth factors and incubate, triggering differentiation of the neural progenitors into a neuronal phenotype, exhibiting extended cellular processes and cell-cell contacts.
Place glass coverslips in 4-well plates and coat them with 1-to-5 diluted poly-L-ornithine in double-distilled water. After incubating the coverslips at 37 degrees Celsius for one hour, wash them with double-distilled water.
Slowly thaw 0.5 to 2 milligrams per milliliter of laminin, and add it to the top of coverslips. Incubate them at 37 degrees Celsius for two hours, then remove excess laminin by pipetting, and add neuronal medium N2 to the culture dishes.
Culture BD-derived CD45 negative cells in N2 medium on laminin-ornithine-coated glass coverslips for two days in an incubator, with 5% carbon dioxide at 37 degrees Celsius to initiate a neuronal differentiation of newly BD generated cells. Next, culture cells in neuronal differentiation medium, and place the plates in an incubator at 37 degrees Celsius and 5% carbon dioxide for 16 days.
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