Single-Cell Western Analysis of iPSC-derived Neural€¦ · by providing insights into protein...
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Astrocytes GFAP/DAPI Neurons βIII Tubulin/DAPI Oligodendrocytes O4/DAPI Single-Cell Western Analysis of iPSC-derived Neural Progenitors Confirms High Differentiation Efficiency and Population Distribution Dynamics K. Flynn, E. Jabart, S. Luhowskyi, D. Galitz, J. Sabat, S. Stoesz, J. Cooper and J. Aho | Bio-Techne, 614 McKinley Place NE, Minneapolis, MN 55413 Abstract Human pluripotent stem cells, including embryonic (ES) and induced pluripotent stem (iPS) cells, offer an essentially unlimited source of neural cells that can be used to investigate mechanisms of human neurological disease and neural regeneration. A critical step during the derivation of neurons, astrocytes, or oligodendrocytes from pluripotent stem cells is generating a robust and homogeneous neural progenitor cell population, which ultimately impacts the efficiency of downstream differentiation protocols and helps control experimental reproducibility. Understanding population heterogeneity is an important step in the optimization of differentiation protocols, which is challenging with existing methods. This study demonstrates how Single-Cell Western blot analysis can complement traditional verification approaches by providing insights into protein expression both at the population and single-cell level. In this study, human iPS cell lines were differentiated into neural progenitor cells using the standardized protocol and reagents in the StemXVivo ® Neural Progenitor Differentiation Kit (R&D Systems). Combining our standardized differentiation protocol for neural progenitor cells with Milo ™ Single-Cell Western technology (ProteinSimple), we were able to assess population protein expression dynamics during the differentiation of iPS cells into neural progenitor cells. Using Oct-3/4 as a marker for pluripotent stem cells and Pax6 as a marker for neural progenitor cells we show, at the single-cell level, that during differentiation the cells shift from an exclusively Oct-3/4 expressing population to one that is robustly Pax6-positive. We were also able to identify subpopulations of neural progenitor cells that express Pax6 at high or low levels. Additionally, single-cell analysis also used to characterize the population distribution of terminally-differentiated neural cells following growth factor withdrawal-induced differentiation of iPS-derived neural progenitor cells. NPC Differentiation Efficiency Quantified Using High Content Imaging Single-Cell Western Analysis of Neural Progenitor Cell Differentiation Single-Cell Western Reveals iPSC and NPC Subpopulations Single-Cell Analysis of NPC-derived Neural Cell Cultures Kit-derived NPC Differentiate into Neural Cells Conclusions ▪ The StemXVivo ® Neural Progenitor Differentiation Kit efficiently differentiates human pluripotent cells into neural progenitor cells. ▪ The NPCs derived using this kit differentiate into neural cultures containing neurons, astrocytes, and oligodendrocytes. ▪ Single-Cell Western blotting shows NPC population homogeneity following differentiation. ▪ High and low expressing populations are identified within Oct-3/4-positive iPSCs and Pax6-positive neural progenitor cells. ▪ Single-cell Western and flow cytometry can be used to characterize cell type population distribution in iPSC-derived neural cell cultures. Differentiation Protocol Pluripotent Stem Cells Day 0 StemXVivo ® Neural Progenitor Differentiation Kit (Catalog # SC035) Days 7–14 Days 21+ NeuroXVivo ™ Cortical Neuron Culture Kit (Catalog # CDK011) Neuron Astrocyte Oligodendrocyte Neural Progenitor Cells Count Oct-3/4 Peak Area 0 10 20 30 40 50 10 4 10 5 10 6 Count Pax6 Peak Area 0 5 10 15 20 25 10 4 10 5 Undifferentiated SOX1 DAPI Pax6 DAPI StemXVivo ® Kit SOX1 Positive (%) 0 10 20 30 40 50 60 70 80 90 100 Undifferentiated StemXVivo ® Kit Pax6 Positive (%) 0 10 20 30 40 50 60 70 80 90 100 Undifferentiated StemXVivo ® Kit NPC Differentiation Efficiency Quantified Using High Content Imaging. The StemXVivo ® Neural Progenitor Differentiation Kit was used to differentiate human iPSK3 (A) or JOY6 (B) iPSCs into NPCs. (A) Immunostaining of iPSK3 cells before (Undifferentiated) and after (StemXVivo ® Kit) differentiation shows that kit-induced NPCs express characteristic neural progenitor markers, SOX1 (Catalog # AF3369) and Pax6 (Catalog # AF8150). (B) SOX1- and Pax6-positive cells were quantified in JOY6 cells both before (Undifferentiated) and after (StemXVivo ® Kit) differentiation using high content imaging (Operetta, Perkin-Elmer). Kit-differentiated cells were over 90% positive for SOX1 and over 75% positive for Pax6. In contrast, undifferentiated cells were less than 20% positive for SOX1 and 5% positive for Pax6. Protein Expression (Peak Area) Protein Expression (Peak Area) Protein Expression (Peak Area) 10 6 10 5 10 4 10 3 10 2 10 1 10 0 Undifferentiated Neural Progenitors 10 6 10 5 10 4 10 3 10 2 10 1 10 0 Undifferentiated Neural Progenitors 2000 4000 6000 8000 10000 12000 Undifferentiated Neural Progenitors 14000 16000 10 6 10 5 10 4 10 3 10 2 10 1 10 0 10 6 10 5 10 4 10 3 10 2 10 1 10 0 Oct-3/4 iPSCs NPCs iPSCs NPCs Oct-3/4 HSP60 Pax6 HSP60 Pax6 iPSCs NPCs SOX1 HSP60 SOX1 Distribution of Pax6 and SOX1 in Kit-derived NPCs. JOY6 iPSCs (Undifferentiated) and StemXVivo ® Neural Progenitor Differentiation Kit-derived NPCs (Neural Progenitors) were probed at the single-cell level for Oct-3/4, Pax6, and SOX1 expression using Milo ™ Single-Cell Western technology. Scatter plots demonstrate that undifferentiated iPSCs homogenously express Oct-3/4, while kit-derived NPCs homogenously express Pax6 and SOX1 while lacking lack Oct-3/4 expression. Each dot in the scatter plot represents protein expression level within one cell. Representative single- cell Western Blot images are shown for Oct-3/4, Pax6, and SOX1 in undifferentiated iPSCs and NPCs. HSP60 was included as a loading control. Biphasic Expression Patterns of Oct-3/4 and Pax6. Joy6 iPSCs and kit-derived neural progenitors were analyzed for Oct- 3/4 and Pax6 expression, respectively, using Milo ™ Single-Cell Western technology. A) Analysis of Oct-3/4 expression in iPSCs showed that 97% of the cells were Oct-3/4-positive. Population analysis of single-cell Oct-3/4 expression showed a biphasic pattern, with a large population of cells expressing high levels of Oct-3/4 and a smaller population expressing low levels of Oct-3/4. B) Analysis of Pax6 expression in kit-derived neural progenitor cells showed that 98% of the cells were Pax6-positive. Population analysis of single-cell Pax6 showed a biphasic expression pattern, with a populations of high Pax6-expressing cells and a low Pax6-expressing cells. Distribution of iPSC-derived Neurons, Astrocytes and Oligodendrocytes Using High Content Imaging. Joy6 iPSCs were subjected to NPC differentiation for 7 days followed by growth factor withdrawal and neural differentiation for 28 days and assessed for cell identity. Neurons were stained with Neuron-specific β-III Tubulin Antibody (Catalog # MAB1195); Astrocytes were stained with Human/Rat GFAP Antibody (Catalog # AF2594); and Oligodendrocytes were stained with Human Oligodendrocyte Marker O4 Antibody (Catalog # MAB1326). NorthernLights ™ (NL)493 Anti-Mouse IgG (Catalog # NL009), NL557 Anti-Mouse IgM (Catalog # NL019), and NL557 Anti-Sheep IgG (Catalog # NL010) Secondary Antibodies were used for visualization. A) High content imaging and analysis was performed using the Operetta ® system (Perkin Elmer). Note that under these conditions, the vast majority of cells are neuronal with less than 10% glia. B) Representative images of iPSC-derived neurons, astrocytes, and oligodendrocytes. Profiling NPC-derived Neural Cultures Using Flow Cytometry. Flow cytometry was performed using the BD Fortessa ™ following a 35 day neuronal differentiation of iPSC-derived neural progenitors. The primary antibodies, Neuron-specific β-III Tubulin Antibody (Catalog # MAB1195), Human/Rat GFAP Antibody (Catalog # AF2594), and Human Oligodendrocyte Marker O4 Antibody (Catalog # MAB1326) were used to detect neurons, astrocytes, and oligodendrocytes, respectively. Allophycocyanin (APC)-conjugated Anti-Mouse IgG (Catalog # F0101B), Phycoerythrin (PE)-conjugated Anti-Sheep IgG (Catalog # F0126), and Mouse APC-conjugated Anti-Mouse IgM (Catalog # F0117) were used as secondary antibodies. Neural populations (solid orange) were estimated to be 62.9% neurons, 33.9% astrocytes, and 10.8% oligodendrocytes. Single-Cell Western Analysis of β-III Tubulin in NPC-derived Neural Cell Cultures. Undifferentiated iPSCs and iPSC-derived neural cultures were analyzed for neural marker expression using Milo ™ Single-Cell Western technology. A) Scatter plot demonstrates that β-III Tubulin (Catalog # MAB1195) expression is observed in iPSC-derived neural cultures and at lower levels in undifferentiated iPSCs. B) Histograms demonstrate the distribution of β-III Tubulin expression intensity in single cells from undifferentiated iPSCs and iPSC-derived neural cultures. A. A. A. B. B. B. A. Induced Pluripotent Stem Cells B. Neural Progenitors Normalized to Mode 0 20 40 60 80 62.9% 33.9% 10.8% 100 10 3 β-III Tubulin - APC 10 4 10 5 10 0 Normalized to Mode 0 20 40 60 80 100 10 3 GFAP - PE 10 4 10 5 10 0 Normalized to Mode 0 20 40 60 80 100 10 3 04 - PE Neurons Astrocytes Oligodendrocytes 10 4 10 5 10 0 Percent (%) Positive 0 20 40 60 80 100 120 83.8% β-III Tubulin Neuron GFAP Astrocyte O4 Oligodendrocyte 8.3% 0.53% n = 331 cells Protein Expression 40000 20000 Undifferentiated iPSC-derived Neural Cultures 60000 80000 100000 120000 Count β-III Tubulin (Peak Area) Undifferentiated n=331 n=251 0 10 20 30 40 50 10 4 60 70 10 5 10 3 Count β-III Tubulin (Peak Area) iPSC-derived Neural Cells 0 5 10 15 20 25 10 4 30 35 10 5 10 3 β-III Tubulin 0 Trademarks and registered trademarks are the property of their respective owners. PS_SingleCellWesternAnalysis_24033
Transcript of Single-Cell Western Analysis of iPSC-derived Neural€¦ · by providing insights into protein...
Astrocytes
GFAP/DAPI
Neurons
βIII Tubulin/DAPI
Oligodendrocytes
O4/DAPI
Single-Cell Western Analysis of iPSC-derived Neural Progenitors Confirms High Differentiation Efficiency
and Population Distribution DynamicsK. Flynn, E. Jabart, S. Luhowskyi, D. Galitz, J. Sabat, S. Stoesz, J. Cooper and J. Aho | Bio-Techne, 614 McKinley Place NE, Minneapolis, MN 55413
Abstract
Human pluripotent stem cells, including embryonic (ES) and induced pluripotent stem (iPS) cells, offer an essentially unlimited source of neural cells that can be used to investigate mechanisms of human neurological disease and neural regeneration. A critical step during the derivation of neurons, astrocytes, or oligodendrocytes from pluripotent stem cells is generating a robust and homogeneous neural progenitor cell population, which ultimately impacts the efficiency of downstream differentiation protocols and helps control experimental reproducibility. Understanding population heterogeneity is an important step in the optimization of differentiation protocols, which is challenging with existing methods. This study demonstrates how Single-Cell Western blot analysis can complement traditional verification approaches by providing insights into protein expression both at the population and single-cell level. In this study, human iPS cell lines were differentiated into neural progenitor cells using the standardized protocol and reagents in the StemXVivo® Neural Progenitor Differentiation Kit (R&D Systems). Combining our standardized differentiation protocol for neural progenitor cells with Milo™ Single-Cell Western technology (ProteinSimple), we were able to assess population protein expression dynamics during the differentiation of iPS cells into neural progenitor cells. Using Oct-3/4 as a marker for pluripotent stem cells and Pax6 as a marker for neural progenitor cells we show, at the single-cell level, that during differentiation the cells shift from an exclusively Oct-3/4 expressing population to one that is robustly Pax6-positive. We were also able to identify subpopulations of neural progenitor cells that express Pax6 at high or low levels. Additionally, single-cell analysis also used to characterize the population distribution of terminally-differentiated neural cells following growth factor withdrawal-induced differentiation of iPS-derived neural progenitor cells.
NPC Differentiation Efficiency Quantified Using High Content Imaging
Single-Cell Western Analysis of Neural Progenitor Cell Differentiation
Single-Cell Western Reveals iPSC and NPC Subpopulations
Single-Cell Analysis of NPC-derived Neural Cell Cultures
Kit-derived NPC Differentiate into Neural Cells
Conclusions
▪ The StemXVivo® Neural Progenitor Differentiation Kit efficiently differentiates human pluripotent cells into neural progenitor cells.
▪ The NPCs derived using this kit differentiate into neural cultures containing neurons, astrocytes, and oligodendrocytes.
▪ Single-Cell Western blotting shows NPC population homogeneity following differentiation.▪ High and low expressing populations are identified within Oct-3/4-positive iPSCs and Pax6-positive neural
progenitor cells.▪ Single-cell Western and flow cytometry can be used to characterize cell type population distribution in
iPSC-derived neural cell cultures.
Differentiation Protocol
Pluripotent Stem Cells
Day 0 StemXVivo® Neural ProgenitorDifferentiation Kit (Catalog # SC035)
Days 7–14 Days 21+NeuroXVivo™ Cortical NeuronCulture Kit (Catalog # CDK011)
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NPC Differentiation Efficiency Quantified Using High Content Imaging. The StemXVivo® Neural Progenitor Differentiation Kit was used to differentiate human iPSK3 (A) or JOY6 (B) iPSCs into NPCs. (A) Immunostaining of iPSK3 cells before (Undifferentiated) and after (StemXVivo® Kit) differentiation shows that kit-induced NPCs express characteristic neural progenitor markers, SOX1 (Catalog # AF3369) and Pax6 (Catalog # AF8150). (B) SOX1- and Pax6-positive cells were quantified in JOY6 cells both before (Undifferentiated) and after (StemXVivo® Kit) differentiation using high content imaging (Operetta, Perkin-Elmer). Kit-differentiated cells were over 90% positive for SOX1 and over 75% positive for Pax6. In contrast, undifferentiated cells were less than 20% positive for SOX1 and 5% positive for Pax6.
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Distribution of Pax6 and SOX1 in Kit-derived NPCs. JOY6 iPSCs (Undifferentiated) and StemXVivo® Neural Progenitor Differentiation Kit-derived NPCs (Neural Progenitors) were probed at the single-cell level for Oct-3/4, Pax6, and SOX1 expression using Milo™ Single-Cell Western technology. Scatter plots demonstrate that undifferentiated iPSCs homogenously express Oct-3/4, while kit-derived NPCs homogenously express Pax6 and SOX1 while lacking lack Oct-3/4 expression. Each dot in the scatter plot represents protein expression level within one cell. Representative single-cell Western Blot images are shown for Oct-3/4, Pax6, and SOX1 in undifferentiated iPSCs and NPCs. HSP60 was included as a loading control.
Biphasic Expression Patterns of Oct-3/4 and Pax6. Joy6 iPSCs and kit-derived neural progenitors were analyzed for Oct-3/4 and Pax6 expression, respectively, using Milo™ Single-Cell Western technology. A) Analysis of Oct-3/4 expression in iPSCs showed that 97% of the cells were Oct-3/4-positive. Population analysis of single-cell Oct-3/4 expression showed a biphasic pattern, with a large population of cells expressing high levels of Oct-3/4 and a smaller population expressing low levels of Oct-3/4. B) Analysis of Pax6 expression in kit-derived neural progenitor cells showed that 98% of the cells were Pax6-positive. Population analysis of single-cell Pax6 showed a biphasic expression pattern, with a populations of high Pax6-expressing cells and a low Pax6-expressing cells.
Distribution of iPSC-derived Neurons, Astrocytes and Oligodendrocytes Using High Content Imaging. Joy6 iPSCs were subjected to NPC differentiation for 7 days followed by growth factor withdrawal and neural differentiation for 28 days and assessed for cell identity. Neurons were stained with Neuron-specific β-III Tubulin Antibody (Catalog # MAB1195); Astrocytes were stained with Human/Rat GFAP Antibody (Catalog # AF2594); and Oligodendrocytes were stained with Human Oligodendrocyte Marker O4 Antibody (Catalog # MAB1326). NorthernLights™ (NL)493 Anti-Mouse IgG (Catalog # NL009), NL557 Anti-Mouse IgM (Catalog # NL019), and NL557 Anti-Sheep IgG (Catalog # NL010) Secondary Antibodies were used for visualization. A) High content imaging and analysis was performed using the Operetta® system (Perkin Elmer). Note that under these conditions, the vast majority of cells are neuronal with less than 10% glia. B) Representative images of iPSC-derived neurons, astrocytes, and oligodendrocytes.
Profiling NPC-derived Neural Cultures Using Flow Cytometry. Flow cytometry was performed using the BD Fortessa™ following a 35 day neuronal differentiation of iPSC-derived neural progenitors. The primary antibodies, Neuron-specific β-III Tubulin Antibody (Catalog # MAB1195), Human/Rat GFAP Antibody (Catalog # AF2594), and Human Oligodendrocyte Marker O4 Antibody (Catalog # MAB1326) were used to detect neurons, astrocytes, and oligodendrocytes, respectively. Allophycocyanin (APC)-conjugated Anti-Mouse IgG (Catalog # F0101B), Phycoerythrin (PE)-conjugated Anti-Sheep IgG (Catalog # F0126), and Mouse APC-conjugated Anti-Mouse IgM (Catalog # F0117) were used as secondary antibodies. Neural populations (solid orange) were estimated to be 62.9% neurons, 33.9% astrocytes, and 10.8% oligodendrocytes.
Single-Cell Western Analysis of β-III Tubulin in NPC-derived Neural Cell Cultures. Undifferentiated iPSCs and iPSC-derived neural cultures were analyzed for neural marker expression using Milo™ Single-Cell Western technology. A) Scatter plot demonstrates that β-III Tubulin (Catalog # MAB1195) expression is observed in iPSC-derived neural cultures and at lower levels in undifferentiated iPSCs. B) Histograms demonstrate the distribution of β-III Tubulin expression intensity in single cells from undifferentiated iPSCs and iPSC-derived neural cultures.
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A. Induced Pluripotent Stem Cells B. Neural Progenitors
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n=331 n=251
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104
6070
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iPSC-derived Neural Cells
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104
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Trademarks and registered trademarks are the property of their respective owners. PS_SingleCellWesternAnalysis_24033
