Midbrain dopaminergic (DA) neurons derived from human pluripotent stem cells (hPSCs) provide an excellent alternative to primary human neurons for disease modeling and drug screening for Parkinson’s disease, characterized by degradation of dopaminergic neurons found in the substantia nigra. The PSC Dopaminergic Neuron Differentiation Kit enables the differentiation of pluripotent stem cells (PSCs) to midbrain dopaminergic neurons. Unlike other protocols or commercially available solutions to differentiate PSCs to dopaminergic neurons which can be biologically restrictive, lengthy, or ill-defined, the PSC Dopaminergic Neuron Differentiation Kit allows you to differentiate PSCs to dopaminergic neurons with increased flexibility, speed, and scalability, all while retaining proper biological relevance. 

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Product overview and workflow:

During differentiation, hPSCs are first induced in Floor Plate Specification Medium into midbrain-specified floor plate progenitor (FP) cells. Next, FP cells are expanded as adherent cultures in Floor Plate Cell Expansion Medium and then cultured in suspension to form spheres. Finally, the spheres are differentiated into mature dopaminergic neurons in Dopaminergic Neuron Maturation Medium. The entire differentiation workflow takes approximately 35 days (Figure 1).

hPSC workflow

Figure 1. Simplified workflow diagram. Pluripotent stem cells cultured in Essential 8 medium can be specified to the midbrain floor plate, expanded and banked, then matured to midbrain dopaminergic neurons in 35 days.

  • Start with PSCs cultured in Essential 8 medium
  • Begin specification when cells are up to 80% confluent
  • Change medium every 2 days
  • Split cells and re-seed in expansion medium
  • Feed cells every 2 days
  • Cells can be frozen and banked at day 16 or continued to day 20 for maturation
  • Split cells and re-seed in maturation medium
  • Feed cells every 2 days
  • Neurons can be seen as early as day 30; optimal results at day 35

During this step, PSCs are patterned toward the midbrain floorplate.

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 developing neural tube. Recent reports have focused on identifying the appropriate in vitro conditions to differentiate hPSCs to properly regionalized floor plate precursors, rather than a more general neural stem cell population, in order to create authentic DA neurons. The first step of dopaminergic neuron differentiation is the specification of hPSCs into midbrain-specified floor plate progenitor (FP) cells in complete Floor Plate Specification Medium on vitronectin-coated plates. During specification, spent medium is replaced every other day with fresh complete specification medium, and the FP cells are harvested on day 10 to begin the expansion stage. 

Figure 2. Marker expression of induced floor plate progenitor (FP) cells. hPSCs were treated with complete Floor Plate Specification Medium for 7 days, and the cells were analyzed for the key phenotypic markers of the human dopaminergic neuron lineage. (A–C) After floor plate specification of hPSCs, the cells express FP marker FOXA2 (green) and rostral marker OTX2 (red). (D–E) The specified FP cells are positive for the DA progenitor marker LMX1A (green), but negative for the neural stem cell marker SOX1 (red). The expression of FOXA2 and OTX2 (Figure 2), key markers for the midbrain floor plate progenitor phenotype, can be examined as early as day 7 of specification. For image-based analysis of intermediate floor plate progenitors, we recommend using the Human Dopaminergic Neuron Immunocytochemistry Kit, which includes a complete set of primary and secondary antibodies, a nuclear DNA stain, and premade buffers for an optimized staining experiment.

This step allows creation of workable banks of precursor cells and significantly increases resultant DA neuron purity.

Standard methods of creating midbrain dopaminergic neurons involve lengthy protocols which are continuous and not scalable. The ability to bank precursor cells in this step enables:

  • Significant increase in resultant DA neuron purity through enrichment (Figure 3)
  • Creation of workable banks of precursor cells for distribution or later use (Figure 4)
  • Pausing of an experiment to be resumed later upon thawingExpansion of midbrain floorplate precursor cells
 

Figure 3. Expansion of midbrain floorplate precursor cells significantly increases purity of resultant DA neurons.

Figure 4. Maintenance of midbrain markers after expansion: expanded floor plate cells maintain their phenotype. (A) Phase image of passaged floor plate cells induced from Gibco episomal induced pluripotent stem cells. (B–F) Expanded cells maintained phenotype markers of FOXA2, LMX1A, and OTX2. 84.90 ± 6.95% cells were double-stained with FOXA2 and OTX2.

This step matures midbrain floor plate cells to TH-positive 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. The phenotypes of cells generated during this step can be confirmed using the PSC Dopaminergic Neuron ICC Kit, and the cells can then be used in downstream assays.  

Figure 5. Representative images of mature DA neurons. The following images were obtained from cells stained with reagents provided in the Human Dopaminergic Neuron Immunocytochemistry Kit 14 days after the addition of complete Dopaminergic Neuron Maturation Medium. The majority of the TH-expressing neurons also co-expressed FOXA2. (A) Anti-TH (green). (B) Anti-FOXA2 (red) and NucBlue stain (a DAPI nuclear DNA stain) (blue). (C) Merged image with anti-TH and anti-FOXA2 (green and red).

Figure 6. Gene expression profile of key genes were comparable to neurons generated using published protocol (Kriks day 48 after differentiation vs. DA neuron differentiation on day 35 after differentiation).

During this step, PSCs are patterned toward the midbrain floorplate.

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 developing neural tube. Recent reports have focused on identifying the appropriate in vitro conditions to differentiate hPSCs to properly regionalized floor plate precursors, rather than a more general neural stem cell population, in order to create authentic DA neurons. The first step of dopaminergic neuron differentiation is the specification of hPSCs into midbrain-specified floor plate progenitor (FP) cells in complete Floor Plate Specification Medium on vitronectin-coated plates. During specification, spent medium is replaced every other day with fresh complete specification medium, and the FP cells are harvested on day 10 to begin the expansion stage. 

Figure 2. Marker expression of induced floor plate progenitor (FP) cells. hPSCs were treated with complete Floor Plate Specification Medium for 7 days, and the cells were analyzed for the key phenotypic markers of the human dopaminergic neuron lineage. (A–C) After floor plate specification of hPSCs, the cells express FP marker FOXA2 (green) and rostral marker OTX2 (red). (D–E) The specified FP cells are positive for the DA progenitor marker LMX1A (green), but negative for the neural stem cell marker SOX1 (red). The expression of FOXA2 and OTX2 (Figure 2), key markers for the midbrain floor plate progenitor phenotype, can be examined as early as day 7 of specification. For image-based analysis of intermediate floor plate progenitors, we recommend using the Human Dopaminergic Neuron Immunocytochemistry Kit, which includes a complete set of primary and secondary antibodies, a nuclear DNA stain, and premade buffers for an optimized staining experiment.

This step allows creation of workable banks of precursor cells and significantly increases resultant DA neuron purity.

Standard methods of creating midbrain dopaminergic neurons involve lengthy protocols which are continuous and not scalable. The ability to bank precursor cells in this step enables:

  • Significant increase in resultant DA neuron purity through enrichment (Figure 3)
  • Creation of workable banks of precursor cells for distribution or later use (Figure 4)
  • Pausing of an experiment to be resumed later upon thawingExpansion of midbrain floorplate precursor cells
 

Figure 3. Expansion of midbrain floorplate precursor cells significantly increases purity of resultant DA neurons.

Figure 4. Maintenance of midbrain markers after expansion: expanded floor plate cells maintain their phenotype. (A) Phase image of passaged floor plate cells induced from Gibco episomal induced pluripotent stem cells. (B–F) Expanded cells maintained phenotype markers of FOXA2, LMX1A, and OTX2. 84.90 ± 6.95% cells were double-stained with FOXA2 and OTX2.

This step matures midbrain floor plate cells to TH-positive 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. The phenotypes of cells generated during this step can be confirmed using the PSC Dopaminergic Neuron ICC Kit, and the cells can then be used in downstream assays.  

Figure 5. Representative images of mature DA neurons. The following images were obtained from cells stained with reagents provided in the Human Dopaminergic Neuron Immunocytochemistry Kit 14 days after the addition of complete Dopaminergic Neuron Maturation Medium. The majority of the TH-expressing neurons also co-expressed FOXA2. (A) Anti-TH (green). (B) Anti-FOXA2 (red) and NucBlue stain (a DAPI nuclear DNA stain) (blue). (C) Merged image with anti-TH and anti-FOXA2 (green and red).

Figure 6. Gene expression profile of key genes were comparable to neurons generated using published protocol (Kriks day 48 after differentiation vs. DA neuron differentiation on day 35 after differentiation).


Functional midbrain dopaminergic neurons

The function of the resultant midbrain dopaminergic neurons was determined (Figures 7 and 8).

Figure 7. Evaluation of spontaneous and depolarization-induced dopamine release in differentiated neurons by HPLC. The function of differentiated neurons was evaluated to measure spontaneous and depolarization-induced dopamine release by HPLC. Midbrain FP cells and rosette derived neural stem cells were differentiated for 2 weeks. Medium conditioned (day12 to 14) was collected for the measurement of spontaneous dopamine release. Depolarization-induced dopamine release was achieved by incubating DA neurons with Hank’s balanced salt solution (HBSS) or with HBSS containing 56 mM KCl for 15 min at 37˚C.  Differentiation DA neurons can not only produce dopamine (conditioned medium) but also have capacity to release dopamine upon voltage gated stimulation by adding excess K+.

Figure 8. Evaluation of spontaneous action potential in differentiated neurons, by MEA (multi-electrode array). iPSC-derived midbrain FP cells were plated on MEA plates and matured for more than 10 days. (A) Activity Map displays spike magnitude detected. (B) Spikes Pane displays voltage waveforms for spikes detected. Spontaneous action potential was recorded by multi-electrode array (Axion), indicating that population have electrophysiologically active neurons. 

Characterization of dopaminergic neurons

For the image-based analysis of mature DA neurons, we recommend using the Human Dopaminergic Neuron Immunocytochemistry Kit, which includes a complete set of primary and secondary antibodies, a nuclear DNA stain, and premade buffers for an optimized staining experiment.

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