Gibco™ PSC Dopaminergic Neuron Differentiation Kit

The kit is a three-step system used over 35 days, with components that are sequentially added to specify PSCs to the midbrain floor plate, expand floor plate progenitor cells, and differentiate floor plate progenitor cells into functional midbrain dopaminergic neurons. Each component is performance tested to ensure generation of the correct phenotype, and the midbrain dopaminergic neurons have been functionally tested for the ability to produce spontaneous action potential and release of dopamine upon depolarization.

Step 1: Specification of PSCs into midbrain floor plate cells
During central nervous system development, midbrain dopaminergic neurons are derived from a distinct population of cells termed midbrain floor plate cells, which are formed during days 21–28 of gestation and located along the ventral midline of the developing neural tube. Recent reports have focused on the importance of differentiating hPSCs into cells from this region, rather than using a more generic neural stem cell population, in order to create authentic midbrain dopaminergic neurons. The first step in the creation of dopaminergic neurons using the PSC Dopaminergic Neuron Differentiation Kit is the specification of hPSCs into midbrain-specified floor plate progenitor (FP) cells in complete Floor Plate Specification Medium.

Step 2: Create a bankable population of floor plate progenitor cells
Standard methods of creating midbrain dopaminergic neurons involve lengthy protocols that are continuous and not scalable. Using the PSC Dopaminergic Neuron Differentiation Kit, a workable bank of floor plate progenitor cells that maintain midbrain identity is created.

The ability to bank progenitor cells in this step enables:
• A significant increase in resultant DA neuron purity
• The ability to scale experiments beyond current limits
• The ability to distribute cells among peers or thaw for later use

Step 3: Differentiation of floor plate precursor cells to midbrain dopaminergic neurons
The final step of the PSC Dopaminergic Neuron Differentiation Kit protocol involves differentiation of floor plate progenitor cells into midbrain dopaminergic neurons. Dopaminergic neurons generated during this step have been shown to maintain midbrain identity, exhibit a gene expression profile equivalent to that of neurons generated using published protocols, and exhibit functionality as shown by spontaneous action potential and release of dopamine upon depolarization.