The PSC-mDA Neuron Phenotyping Kit, human is a flow cytometry-based quality control assay for
in vitro
phenotyping of the identity and purity of the culture during differentiation of human PSCs in midbrain dopaminergic (mDA) neurons.
The PSC-mDA Neuron Phenotyping Kit:
  • Allows a qualitative and quantitative analysis: 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. Moreover, it enables to establish numerical cut-offs for the differentiation performances, to ensure consistency between different

Data and images for PSC-mDA Neuron Phenotyping Kit, human

Figures

Schematic drawing and region-specific marker expression of a human fetal brain

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During development expression of region-specific markers arises in response to different concentration gradients of key patterning molecules. Rostro-caudal and dorso-ventral patterning are driven by increasing CHIR99021 and human sonic hedgehog (hSHH) concentrations, respectively.
This differential marker expression allows to distinguish between different cellular populations and provides the basis of the flow cytometry-based quality control assay for PSC-mDA neurons using the PSC-mDA Neurons Phenotyping Kit, human.
During development expression of region-specific markers arises in response to different concentration gradients of key patterning molecules. Rostro-caudal and dorso-ventral patterning are driven by increasing CHIR99021 and human sonic hedgehog (hSHH) concentrations, respectively.
This differential marker expression allows to distinguish between different cellular populations and provides the basis of the flow cytometry-based quality control assay for PSC-mDA neurons using the PSC-mDA Neurons Phenotyping Kit, human.

Figure 2

Example of immunofluorescent staining and analysis using the PSC-mDA Neuron Phenotyping Kit, human:
Combination of the markers included in the PSC-mDA Neurons Phenotyping Kit, human (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. iPSC-derived mDA neurons were generated and analyzed after 16 days of differentiation. For the analysis, gates were defined using cellular controls. Staining 1 shows no measurable contamination of residual Oct3/4
+
pluripotent stem cells. Staining 2 shows the purity of the target mDA neurons (FoxA2
+
OTX2
+
) as well as contamination by other cell populations. Specifically, the PAX-6-specific antibody detects cells of a dorsal phenotype, whereas a caudal phenotype is indicated by loss of OTX2 expression. Finally, staining 3 shows expression levels of SOX-1, a neural ectoderm marker that identifies contamination of different neural cell populations, and expression of TTF-1, which is a ventral forebrain marker but can be partially expressed by the target cells.
Staining 1
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Figure 2

Example of immunofluorescent staining and analysis using the PSC-mDA Neuron Phenotyping Kit, human:
Combination of the markers included in the PSC-mDA Neurons Phenotyping Kit, human (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. iPSC-derived mDA neurons were generated and analyzed after 16 days of differentiation. For the analysis, gates were defined using cellular controls. Staining 1 shows no measurable contamination of residual Oct3/4
+
pluripotent stem cells. Staining 2 shows the purity of the target mDA neurons (FoxA2
+
OTX2
+
) as well as contamination by other cell populations. Specifically, the PAX-6-specific antibody detects cells of a dorsal phenotype, whereas a caudal phenotype is indicated by loss of OTX2 expression. Finally, staining 3 shows expression levels of SOX-1, a neural ectoderm marker that identifies contamination of different neural cell populations, and expression of TTF-1, which is a ventral forebrain marker but can be partially expressed by the target cells.
Staining 2
View details

Figure 2

Example of immunofluorescent staining and analysis using the PSC-mDA Neuron Phenotyping Kit, human:
Combination of the markers included in the PSC-mDA Neurons Phenotyping Kit, human (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. iPSC-derived mDA neurons were generated and analyzed after 16 days of differentiation. For the analysis, gates were defined using cellular controls. Staining 1 shows no measurable contamination of residual Oct3/4
+
pluripotent stem cells. Staining 2 shows the purity of the target mDA neurons (FoxA2
+
OTX2
+
) as well as contamination by other cell populations. Specifically, the PAX-6-specific antibody detects cells of a dorsal phenotype, whereas a caudal phenotype is indicated by loss of OTX2 expression. Finally, staining 3 shows expression levels of SOX-1, a neural ectoderm marker that identifies contamination of different neural cell populations, and expression of TTF-1, which is a ventral forebrain marker but can be partially expressed by the target cells.
View details

Figure 2

Example of immunofluorescent staining and analysis using the PSC-mDA Neuron Phenotyping Kit, human:
Combination of the markers included in the PSC-mDA Neurons Phenotyping Kit, human (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. iPSC-derived mDA neurons were generated and analyzed after 16 days of differentiation. For the analysis, gates were defined using cellular controls. Staining 1 shows no measurable contamination of residual Oct3/4
+
pluripotent stem cells. Staining 2 shows the purity of the target mDA neurons (FoxA2
+
OTX2
+
) as well as contamination by other cell populations. Specifically, the PAX-6-specific antibody detects cells of a dorsal phenotype, whereas a caudal phenotype is indicated by loss of OTX2 expression. Finally, staining 3 shows expression levels of SOX-1, a neural ectoderm marker that identifies contamination of different neural cell populations, and expression of TTF-1, which is a ventral forebrain marker but can be partially expressed by the target cells.
View details

Figure 2

Example of immunofluorescent staining and analysis using the PSC-mDA Neuron Phenotyping Kit, human:
Combination of the markers included in the PSC-mDA Neurons Phenotyping Kit, human (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. iPSC-derived mDA neurons were generated and analyzed after 16 days of differentiation. For the analysis, gates were defined using cellular controls. Staining 1 shows no measurable contamination of residual Oct3/4
+
pluripotent stem cells. Staining 2 shows the purity of the target mDA neurons (FoxA2
+
OTX2
+
) as well as contamination by other cell populations. Specifically, the PAX-6-specific antibody detects cells of a dorsal phenotype, whereas a caudal phenotype is indicated by loss of OTX2 expression. Finally, staining 3 shows expression levels of SOX-1, a neural ectoderm marker that identifies contamination of different neural cell populations, and expression of TTF-1, which is a ventral forebrain marker but can be partially expressed by the target cells.

Specifications for PSC-mDA Neuron Phenotyping Kit, human

Overview

The PSC-mDA Neuron Phenotyping Kit, human is a flow cytometry-based quality control assay for
in vitro
phenotyping of the identity and purity of the culture during differentiation of human PSCs in midbrain dopaminergic (mDA) neurons.
The PSC-mDA Neuron Phenotyping Kit:
  • Allows a qualitative and quantitative analysis: 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. Moreover, it enables to establish numerical cut-offs for the differentiation performances, to ensure consistency between different rounds of differentiation.
  • Enables a fast in-process quality control assay: being based on flow cytometry it represents a faster approach compared to traditional IHC methods.
  • Based on REAfinity™ Antibody conjugates: ensures superior lot-to-lot consistency, low background, and high staining specificity.

Detailed product information

Background information

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.
The PSC-mDA Neuron Phenotyping Kit, human has been developed as a 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 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.
The PSC-mDA Neuron Phenotyping Kit, human takes advantage of the fast approach that flow analysis provides and combines it with the high staining specificity and low background of REAfinity™ Antibodies. In summary, this assay results in a quick and reliable qualitative and quantitative analysis during the differentiation process.

Applications

  • Flow cytometry-based quality control kit for in vitro phenotyping of identity and purity of PSC-derived mDA neurons during differentiation.

Resources for PSC-mDA Neuron Phenotyping Kit, human

References for PSC-mDA Neuron Phenotyping Kit, human

Publications

  1. Kirkeby, A. et al. (2012) Generation of regionally specified neural progenitors and functional neurons from human embryonic stem cells under defined conditions. Cell Rep 1: 703-714
  2. Kirkeby, A. et al. (2013) Generating regionalized neuronal cells from pluripotency, a step-by-step protocol. Front Cell Neurosci 6: 64
  3. Kirkeby, 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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