This application protocol describes an in-process quality control assay to evaluate purity and identity of midbrain dopaminergic neurons differentiated from pluripotent stem cells (PSC-mDA neurons). The step-by-step protocol is based on a flow cytometric approach using the PSC-mDA Neuron Analysis Kit, anti-human, REAfinity™ (# 130-127-439).
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 cultivation1–3 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:
Table 1: CHIR99201 and hSHH concentrations for cellular controls. Concentrations of CHIR99201 and hSHH used in the above-mentioned example protocol to obtain different cellular populations for cellular controls.
|Cellular control||CHIR99201 concentration||hSHH concentration|
|Pluripotent stem cells||None. Cells grown in StemMACS iPS-Brew XF, human.||None. Cells grown in StemMACS iPS-Brew XF, human.|
|Ventral forebrain cells||0 µM||600 ng/mL|
|Dorsal forebrain cells||0 µM||0 ng/mL|
|Ventral hindbrain cells||2 µM||600 ng/mL|
▲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.
If differentiated cells were frozen prior to analysis, thawing can be performed according to the following protocol:
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).
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
Table 2: Overview cell samples for flow cytometry. Samples for flow cytometer set up and compensation, samples for gate definition, samples for analysis of mDA neurons, and the required buffer and antibody volumes for intracellular staining are displayed.
|#||Sample||Purpose||Cell number||Volume of 1× Permeabilization Buffer||Antibody volume|
|Samples for flow cytometer set up and compensation|
|1||(Optional) Unfixed viable mDA neurons||Propidium iodide staining to determine the viability of mDA neurons||105|
|2||Fixed mDA neurons||Unstained; for flow cytometer set up||5×105|
|3||FoxA2-APC (REA506) single staining of fixed mDA neurons||Compensation||5×105||98 µL||2 µL|
|4||OTX2-Vio® B515 (REA1178) single staining of fixed mDA neurons||Compensation||5×105||98 µL||2 µL|
|5||PAX-6-PE (REA507) single staining of fixed dorsal forebrain cells||Compensation||5×105||98 µL||2 µL|
|Samples for gate definition|
|6||Oct3/4-APC (REA622) single staining of fixed PSCs||Definition of a gate for Oct3/4-APC (REA622)||5×105||98 µL||2 µL|
|7||OTX2-Vio B515 (REA1178) single staining of fixed ventral hindbrain cells||Definition of a gate for OTX2-Vio B515 (REA1178)||5×105||98 µL||2 µL|
|8||FoxA2-APC (REA506) and PAX-6-PE (REA507) staining of fixed dorsal forebrain cells||Definition of a gate for FoxA2-APC (REA506) and PAX-6-PE (REA507)||5×105||96 µL||2 µL each|
|9||Sox1-PE (REA698) and TTF-1-Vio B515 (REA1090) staining of fixed dorsal forebrain cells||Definition of a gate for Sox1-PE (REA698)||5×105||96 µL||2 µL each|
|10||TTF-1-Vio B515 (REA1090) and Sox1-PE (REA698) staining of fixed ventral forebrain||Definition of a gate for TTF-1-Vio B515 (REA1090)||5×105||96 µL||2 µL each|
|Samples for analysis of mDA neurons|
|11||Staining 1 of fixed mDA neurons||Staining with Oct3/4-APC (REA622)||5×106*||98 µL||2 µL|
|12||Staining 2 of fixed mDA neurons||Staining with FoxA2-APC (REA506), OTX2-Vio B515 (REA1178), and PAX-6-PE (REA507)||5×105||94 µL||2 µL each|
|13||Staining 3 of fixed mDA neurons||Staining with TTF-1-Vio B515 (REA1090) and Sox1-PE (REA698)||5×105||96 µL||2 µL each|
*For reliable detection of remnant Oct3/4 positive cells, it is recommended to run three independent samples.
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.
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