Flow cytometry-based quality control assay for PSC-derived midbrain dopaminergic neurons

Pluripotent stem cell (PSC) differentiation is a core aspect of PSC research. In the last decade, protocols for the generation of PSC-derived midbrain dopaminergic (mDA) neurons have been established and optimized. Nevertheless, differences in the manufactured cell lots cannot be avoided and can be influenced by slight variations (e.g. user-dependent handling variations, cell density, variations in starting cell composition, substance concentration, and activity). 

Therefore, a quality control step that allows a reliable in vitro evaluation of the efficiency of the differentiation is pivotal. Such a quality control assay would need to reliably and efficiently assess the identity of the cells, the cell number of the different sub-populations, and to detect non-differentiated cells that might contaminate the culture.

The PSC-mDA Neuron Analysis Kit, anti-human, REAfinity has been developed as flow cytometry-based quality control assay for in vitro phenotyping of the identity and purity of the culture during differentiation of human PSCs in mDA neurons by combination of antibodies specific for positive and negative mDA markers. The kit allows the detection of early expressed and specific regional markers and enables to assess the identity of the cells, the cell number of the different sub-populations, and to detect non-differentiated cells that might contaminate the culture. 

FoxA2- and OTX2-specific antibodies are included as positive markers for ventral midbrain dopaminergic progenitors. In addition, the panel includes markers that indicate contamination by other cell populations. The PAX-6-specific antibody specifically detects cells of a dorsal phenotype, whereas a caudal phenotype is indicated by loss of OTX2 expression. Moreover, SOX-1 is a neural ectoderm marker and identifies contamination of different neural cell populations. TTF-1 (also known as Nkx2.1) is a ventral forebrain marker but can be partially expressed by the target cells depending on the cell clone. Finally, Oct3/4 serves as marker to detect residual pluripotent cells (figure 1). 

Cellular controls specific to other brain regions, such as ventral and dorsal forebrain cells, ventral hindbrain cells, as well as remnant PSC, are also evaluated for reliable gate definition and detection of contaminating cell populations.

To date, many different protocols are available to differentiate mDA neurons from PSCs. Independent of the protocol of choice, correctly differentiated cells will start to express mDA neuron- and brain region-specific markers.

For the quality control assay of this application protocol, a protocol based on embryoid body cultivation^1–3  was adapted to an adherent differentiation protocol as shown in figure 2.

The adapted protocol is based on dual SMAD inhibition for neural conversion and regionalization by inhibition of glycogen synthase kinase 3 (GSK3) by CHIR99021. This leads to induction of WNT signaling for rostro-caudal patterning and activation of hSHH and bone morphogenetic protein (BMP) signaling for dorso-ventral patterning. Different media are optimized for neural induction (NIM), neural proliferation (NPM), and neural differentiation (NDM) as depicted in figure 2.

Cellular controls, such as pluripotent stem cells, ventral and dorsal forebrain cells, and ventral hindbrain cells, are necessary to provide an internal control of the differentiation process as well as to ensure the best gate definition for the flow cytometry-based quality control analysis (table 1). This is pivotal for reliable detection of contaminating cell populations.

PSCs can be taken from normal PSC culturing conditions (e.g. maintained in StemMACS™ iPS-Brew XF, human). Ventral and dorsal forebrain and ventral hindbrain cellular controls are obtained by following the same differentiation protocol described above but with CHIR99201 and hSHH concentrations modified as follows:

▲Note: It is possible to generate a cell bank of each cellular control and freeze down aliquots that can then be thawed and analyzed at the timepoint of the quality control assay.

• Differentiated cells are harvested with 0.05% trypsin/EDTA and Soybean Trypsin Inhibitor to obtain single cells for flow cytometry analysis, replating, and for further differentiation. Please refer to the respective data sheet for more information.

1. Aspirate differentiation medium.
2. Wash each well with D-PBS without Ca²⁺ and Mg²⁺, e.g., use 1–2 mL for one well of a 6-well plate.
3. Add 0.05% Trypsin/EDTA. Gently tilt the plate to ensure even distribution of the solution.
4. Incubate for 5 minutes at 37 °C.
5. Add Soybean Trypsin Inhibitor to stop the reaction.
6. Carefully pipet up and down using a 5 mL serological pipette and dissociate to a single-cell suspension.
7. Determine cell number.
8. Prepare two aliquots, one for replating and further cultivation and one for the quality control assay.
9. For replating and further differentiation, transfer the desired cell number into a new tube. Centrifuge at 300×g for 5 minutes at room temperature. Aspirate supernatant completely. Resuspend the cells in new culture ware and place the plate into the incubator (37 °C, 5% CO₂).
10. For the quality control assay, proceed with chapter 3.3 Immunofluorescent staining.

• Differentiated cells can be cryopreserved and analyzed at a later timepoint. 

1. Transfer the desired cell number into a 15 mL conical tube. Calculate with 10⁶ cells per 1 mL aliquot.
2. Centrifuge cells for 5 minutes at 200×g at room temperature. Aspirate supernatant completely.
3. Resuspend the cell pellet in StemMACS™ Cryo-Brew to 10⁶ cells per mL.
4. Quickly transfer the cell suspension into cryogenic vials (1 mL per vial).
5. Place the vials into an isopropanol freezing container and immediately store at –80 °C.
6. After 24 hours transfer the vials into a liquid nitrogen tank for long-term storage.          

If differentiated cells were frozen prior to analysis, thawing can be performed according to the following protocol:

1. Take a vial with cells out of the liquid nitrogen container.
2. Incubate the vial in a 37 °C water bath until only a little lump of ice is left.
3. Quickly transfer cell suspension into a 15 mL conical tube and dropwise add 10 mL of medium (MACS® Neuro Medium + MACS NeuroBrew w/o Vitamin A (1:50) or Gibco™ Neurobasal™ Medium + Gibco™ B-27™ Supplement (50X) w/o Vitamin A (1:50) at room temperature).
4. Centrifuge cells at 300×g for 5 minutes at room temperature. Aspirate supernatant completely.
5. Add 1 mL of PEB buffer and determine cell number.
6. Centrifuge cells at 300×g for 5 minutes at room temperature. Aspirate supernatant completely.
7. Proceed with the chapter 3.3 Immunofluorescent staining.

• Always prepare reagents freshly. Failure to do so may lead to suboptimal results.
• Total buffer volumes should be calculated beforehand and will depend on the number of samples.

Fixation/Permeabilization Solution
To achieve the appropriate working concentration for safe fixation and permeabilization of cells, the Fixation/Permeabilization Solution 1 must be diluted 1:4 with the Fixation/Permeabilization Solution 2 (i.e. for 10⁶ cells use 0.25 mL of Fixation/Permeabilization Solution 1 and 0.75 mL of Fixation/Permeabilization Solution 2).

Permeabilization <<buffer
To achieve the appropriate working concentration for safe permeabilization of cells, the 10× Permeabilization Buffer must be diluted 1:10 with deionized or distilled water before use (i.e. 1 mL of 10× Permeabilization Buffer and 9 mL of deionized/distilled water).

The PSC-mDA Neuron Analysis Kit, anti-human, REAfinity contains six REAfinity Antibody conjugates that ensure low background and high staining specificity and are directed against intracellular markers (table 2). The analysis can be carried out at day 11 and 16 of the differentiation process. 

Cells are first fixed and permeabilized for intracellular staining using the Fixation/Permeabilization Solution 1, Fixation/Permeabilization Solution 2, and Permeabilization Buffer that are prepared freshly as described above. To set up the instrument and compensate for spectral overlap, single stainings of FoxA2⁺ differentiated mDA neurons, OTX2⁺ mDA neurons, and PAX-6⁺ dorsal forebrain cells and an unstained cell sample are required. The unstained cell sample does not contain antibodies. It should be treated, e.g., fixed and permeabilized, in the same way as the stained samples.

Furthermore, control stainings are required to define the appropriate gates for the different markers. 

Table 2  gives an overview about the different cell samples that are required for flow cytometric analysis

1. Determine the cell number.
2. Prepare samples as described in chapter 3.2 Sample preparation.
   ▲ Note: For reliable detection of remnant Oct3/4⁺ cells, it is recommended to run three independent samples.
3. Centrifuge cell suspension at 300×g for 5 minutes at room temperature. Aspirate supernatant completely.
4. Resuspend cell pellet in 1 mL of cold Fixation/Permeabilization Solution.
5. Mix well and incubate for 30 minutes in the dark in the refrigerator (2−8 °C).
6. Wash cells by adding 1−2 mL of buffer and centrifuge at 300×g for 5 minutes at 4 °C. Aspirate supernatant completely.
7. Resuspend cell pellet in cold 1× Permeabilization Buffer according to table 2.
8. Add antibodies according to table 2.
9. Mix well and incubate for 30 minutes in the dark in the refrigerator (2−8 °C). 
   ▲ Note:  Higher temperatures and/or longer incubation times may lead to non-specific cell labeling. Working on ice requires increased incubation times.
10. Wash cells by adding 1 mL of cold 1× Permeabilization Buffer and centrifuge at 300×g for 5 minutes at 4 °C. Aspirate supernatant completely.
11. Resuspend cell pellet in a suitable amount of buffer for analysis by flow cytometry.

• Please refer to the MACSQuant Instrument user manual and software guide for detailed information on using the MACSQuant Analyzer.
• Before starting to acquire the samples make sure that the MACSQuant Analyzer has been carefully cleaned. Perform an extended wash after each sample. It is recommended to acquire the samples with medium flow rate and to detect >500,000 events for staining 1 and >200,000 events for staining 2 or 3.

1. Set up the scatter settings and voltages for all channels with the unstained fixed cell sample (sample # 2, table 2).
2. Set the trigger and exclude debris from analysis based on scatter signals (figure 3A). Subsequently, exclude doublets (figure 3B).
3. Use single stained samples (samples # 3−5, table 2) to adjust the voltage of APC, Vio® B515, and PE. Define the compensation to avoid spectral overlaps, e.g., by displaying PE vs. Vio B515 (figure 3C and D).
4. Start flow cytometric data acquisition of samples for analysis of mDA neurons (samples # 6−13, table 2).

• Cellular controls according to table 2 (sample # 6−10) are used to define reliable gates for each marker.
• In case of markers that are expected to be expressed on mDA neurons (FoxA2, OTX-2) a negative control is chosen, whereas in case of markers that are not supposed to be expressed on mDA neurons (Oct3/4, Pax-6, Sox1, TTF-1) the gate is defined according to a positive cellular control as indicated in figure 4.

The results obtained from the flow analysis can be used to establish the overall success of the differentiation protocol and evaluate the presence of undifferentiated cells. By performing a qualitative and quantitative analysis it is also possible to establish cut-offs and minimal criteria for the differentiation performances to ensure consistency between different rounds of differentiation.

An example of the results that can be obtained after this paradigm is shown in figure 5 for three human iPSC lines after 16 days of differentiation. Combination of the markers FoxA2, OTX2, PAX-6, TTF-1, Sox1, and Oct3/4 allows to determine the identity and purity of the cell product in a flow cytometry assay as shown in the example below.

iPSC-derived mDA neurons were generated and analyzed after 16 days of differentiation. For the analysis, gates were defined using cellular controls. First of all, Oct3/4 showed no measurable contamination of residual pluripotent cells (staining 1). FoxA2- and OTX2-specific antibodies were included as positive markers for ventral midbrain dopaminergic progenitors. Thereby, the percentage of FoxA2⁺OTX2⁺ cells showed the purity of the target mDA neurons, which was 87.1% (staining 2).

In addition, the kit includes markers that indicate contamination by other cell populations.

The PAX-6-specific antibody specifically detects cells of a dorsal phenotype, whereas a caudal phenotype is indicated by loss of OTX2 expression. Furthermore, SOX-1 is a neural ectoderm marker and identifies contamination of different neural cell populations. Percentage of PAX-6⁺, OTX2^–, and Sox1⁺ cells was very low as shown in staining 2 and 3. Around 34% of cells showed expression of TTF-1, which is a ventral forebrain marker but can be partially expressed by the target cells.

¹Kirkeby, A. et al. (2012). Generation of regionally specified neural progenitors and functional neurons from human embryonic stem cells under defined conditions. Cell Rep. 1(6): 703-714.

²Kirkeby, A. et al. (2013). Generating regionalized neuronal cells from pluripotency, a step-by-step protocol. Front Cell Neurosci. 6: 64. 

³Kirkeby A. et al. (2017). Predictive Markers Guide Differentiation to Improve Graft Outcome in Clinical Translation of hESC-Based Therapy for Parkinson's Disease. Cell Stem Cell. 20(1): 135-148.