Download Automated Staining of Pluripotent Cells with Tra-1-60 and

TECHNICAL NOTE
Automated Cell Staining of Induced Pluripotent Stem
Cells on the C1™ Single-Cell Auto Prep System
StainAlive™ TRA-1-60 Antibody and CellTracker™ Orange CMRA
INTRODUCTION
Technical Background
A significant challenge in stem cell studies is ensuring
the identity, growth and status of a cell population.
For instance, in vitro embryonic and pluripotent stem
cells may differentiate spontaneously. While in directed
differentiated studies, a subset of the cell population
may choose alternate differentiation pathways. In both
models, tracking cell stemness, analyzing proliferation,
and monitoring viability are important to characterize
prior to drawing larger conclusions. In these situations,
surface antibody staining of live cells is an informative,
but onerous process to monitor heterogeneous cell
populations during cell sorting, purification, and
analysis of cell populations.
TRA-1-60 binds to a pluripotent stem cell specific
antigen; a neuraminidase-resistant carbohydrate
epitope expressed on the transmembrane glycoprotein,
podocalyxin. The antigen is expressed on the surface
of undifferentiated human embryonic stem cells and
embryonal carcinoma cells. The expression of Tra-1-60
on human embryonic stem cells is down regulated
upon differentiation (DeWolf, W.C., et al. 2007).
This technical note describes an automated cell staining
solution to assess single-cell heterogeneity and viability
using two stains in combination with the C1™ SingleCell Auto Prep System. StainAlive™ TRA-1-60 Antibody
(DyLight™ 488, Stemgent) is shown to successfully
identify human induced pluripotent stem cells (iPSC).
CellTracker™ Orange CMRA (Invitrogen), is stain for
live cell tracing, is shown to successfuly stain cells.
Using this methodology in the context of differentiation
studies, iPSCs are distinguishable from human neural
progenitor cells (NPCs).
When using the C1 System to study stem cell populations,
it is important to monitor differentiated cell states
to quickly assess and qualify isolated single cells. As
shown in the workflow in Figure 1, this can be done on
the C1 System using standard staining scripts. In order
to accommodate TRA-1-60 and CellTracker Orange, this
technical note addresses stain preparation, enabling
fast adoption of antibody stains with the C1 System.
When used in combination, the C1 System, TRA-1-60,
and CellTracker Orange enable a rigorous, standardized
method to monitor stem cell development and viability.
CellTracker Orange CMRA is a probe which freely
passes through cell membranes and is converted to
cell-impermeant fluorescent products. Because the cell
must be alive to convert the probe, CellTracker Orange is
both a viability and lineage stain. It is passed to daughter
cells through several generations, but not transferred
to adjacent cells in the population. CellTracker Orange
excites at 541 nm and emits at 565 nm (orange) making it
visually compatible with TRA-1-60 (DyLight™ excitation
at 493) emission at 518 nm (green). Using TRA-1-60 and
CellTracker Orange together on the C1 System with a
high resolution fluorescent microscope allows users to
visually verify undifferentiated cells while assessing cell
viability.
Experiment
This experiment compared cell capture and gene
expression analysis of human iPSCs and human iPSC
derived NPCs on the C1 System. Individual iPSCs and
NPCs were captured, washed, stained, lysed, and preamplified in a disposable C1 Integrated Fluidic Circuit
(IFC) for PreAmp. The resulting cDNA generated from
the stained cells was diluted and transferred to the
BioMark™ HD System for gene expression analysis
using a DELTAgene™ human pluripotency panel.
Methods
Human iPSCs were routinely passaged with 0.5 µM
EDTA and maintained with Essential 8™ medium from
Invitrogen on Matrigel™ (1:80 in DPBS). iPSCs were
then freshly dissociated into single-cell suspension
using Accutase for 5-7 minutes at 37˚C. Human NPCs
were differentiated from iPSCs in chemically defined
conditions using small molecules LDN-193189 and
SB431542 for dual-SMAD inhibition, as well as the
hedgehog-inhibitor cyclopamine (Chambers, S.M. et al.
2009). Success was confirmed by immune-staining for
PAX6, an NPC-specific marker.
A set of DELTAgene gene expression assays was used
to interrogate both iPSC and NPC samples. DELTAgene
assays use EvaGreen® dye, an intercalating, fluorescent
dye for quantitation (Real-Time PCR User Guide PN
68000088).The assays included six endogenous controls
and 90 pluripotency, differentiation, and stemness
genes commonly used to characterize undifferentiated
human stem cells (ISCI et al. 2007).
at 70:30 ratio with C1 Suspension Reagent and loaded
on a 10-17 µm C1 IFC (PN 100-5479). TRA-1-60 staining
solution (20 µL) was loaded into the staining inlet of the
same C1 IFC at a 1:50 dilution in C1 Cell Wash Buffer
(10 µg/mL). Dual staining with TRA-1-60 and CellTracker
Orange was performed in later IFC experiments using
10 µg/mL TRA-1-60 and a final concentration of 5 µM
CellTracker Orange.
Results
C1 System is compatible with TRA-1-60 Antibody
To study surface marker staining within the C1 System,
ten C1 IFCs were loaded with either Human iPSCs or NPCs
and stained with TRA-1-60. The capture sites of each
C1 IFC were scanned and imaged using a fluorescent
microscope with an automated stage. As seen in
Table 1, minimal fluorescence was observed in captured
NPCs. Captured iPSCs, however, demonstrated strong
fluorescence in at least 87% and up to 96% of cells.
Chip Run and
Cell Type
All C1 IFCs followed standard staining, lysing, reverse
transcription, and preamplification procedures as
described in the C1 System Protocol, using the C1
Single-Cell Auto Prep System to Capture Cells from Cell
Culture and Perform Preamplification (PN 100-4904).
All gene expression analysis was performed using the
BioMark HD System. Stain emission was observed by
fluorescence microscopy. Data analysis was performed
using the R-script package, SINGuLAR™ Analysis
Toolset (PN 100-6475). Cell suspensions and stain
solutions were optimized as described below.
Cell Suspension and Stain Solution Optimization
iPSCs and NPCs are more buoyant than the cell lines
(K562, BJ Fibroblasts, and HL60s) used to develop
the standard protocol. To adjust for this, 2000 single
iPSCs and NPCs in 10 µL media were resuspended
% Cell
Occupancy
% TRA-1-60 +
Cells
1. iPSC
94
87
2. iPSC
100
96
3. iPSC
93
96
4. iPSC
95
90
5. iPSC
93
96
6. iPSC
83
89
7. iPSC
91
87
8. iPSC
88
88
9. NPC
98
6
10. NPC
100
3
Table 1. TRA-1-60 Antibody Staining. In ten individual C 1 IFC
experiments, iPSCs and NPCs were stained with TRA-1-60. Cell occupancy
is the number of occupied capture sites containing a single cell. In IFCs
loaded with iPSCs, up to 96% of cells captured were TRA-1-60 positive. As
expected, IFCs loaded with NPCs showed a significantly lower percentage
of fluorescent cells.
Fast and Easy Workflow
Enrich and Dissociate
Load and Capture
TRA-1-60 Antibody
Accutase
C1™ Single-Cell Auto Prep System
Figure 1
Wash, Stain
and Image
Lyse, RT, Preamp
and Harvest
Observed by phase contrast microscopy in Figure 2,
capture occupancy can be confirmed while
simultaneously confirming pluripotency of the cell
captured. Cell lineage tracing and viability was later
confirmed with dual staining with CellTracker Orange,
discussed in the C1 System is capable of dual staining
with TRA-1-60 and CellTracker Orange section of this
technical note.
Hierarchical Clustering of iPSCs and NPCs
NPC
Human iPSC
iPSC
1
2
3
Human NPC
2
3
Figure 2. iPSC and NPC Capture Images. 1. Cells prior to single cell
dissociation. 2. An isolated single cell captured in the C1 IFC and imaged
under phase contrast microscopy. 3. The same cell imaged with
fluorescence microscopy for TRA-1-60 antibody signal.
Additionally, gene expression analysis (Figure 3)
confirms the undifferentiated state of iPSCs when
compared to NPCs studied. Principal component analysis
demonstrates iPSCs clustering together, away from
NPCs (Figure 4). Through gene expression profiling, rare
TRA-1-60+ NPCs (Table 1) can be characterized in order
to better correlate protein expression and mRNA gene
expression to distinguish differentiated cells. Principal
component analysis of genes SOX2, PAX6, OCT4, and
NANOG illustrate that the rare TRA-1-60+ NPCs cluster
with TRA-1-60- cells (Figure 4C). Thus, gene expression
analysis is still essential in characterizing individual cells
but staining can be used as an experimental checkpoint
to identify cell populations (Figure 5).
OCT4 &
NANOG
PAX6
Figure 3. Gene Expression Analysis of iPSC vs. NPC. Both iPSCs
and NPCs show similar expression of housekeeping genes, ACTB and
GAPDH. iPSCs have significantly higher expression levels of stemness
genes, OCT4 and NANOG, as expected: a 5.69 Ct difference for OCT4
and a 2.96 Ct difference for NANOG when averages of iPSCs and NPCs Ct
are compared. NPCs have significantly higher levels of PAX6 as expected,
expressing approximately 80x more copies, a 6.32 Ct difference.
Type
iPSC TRA-1-60 iPSC TRA-1-60 +
PC2
1
ACTB & GAPDH
Type
iPSC
IPSC Tube Control
NPC
Type
NPC TRA-1-60 NPC TRA-1-60 +
PC1
Figure 4. Principal Component Analysis of iPSCs and NPCs. A. Distinct
clusters display differentiated states of iPSC and NPC cell types. B. iPSCs
positive and negative for TRA-1-60 signal cluster together when profiled
for gene expression. C. NPCs both positive and negative for TRA-1-60
signal cluster together when profiled for gene expression.
A
Amplify and Detect
Differentiated
Differentiated
B
Differentiated
Differentiated
BioMark HD™ System
PC1
Fresh iPSC TRA-1-60 Fresh iPSC TRA-1-60 +
Thawed iPSC TRA-1-60 Thawed iPSC TRA-1-60 +
Figure 5. Differentiation Confirmed by Microscopy and Gene
Expression Profiling. A. An iPSC colony from a thawed sample shows
signs of differentiation under phase contrast microscopy. B. The same
iPSC colony stained with TRA-1-60 exhibits signal for undifferentiated
cells within the colony, while the differentiated regions indicated by the
arrows, do not express do not express the pluripotency marker. C. The
gene expression profile of thawed TRA-1-60 negative cells cluster away
from fresh and thawed TRA-1-60 positive cells, confirming differentiation.
The C1 System is capable of dual staining with
TRA-1-60 and CellTracker Orange
Staining with TRA-1-60 alone allows for differentiation
monitoring but does not facilitate cell tracking and
viability confirmation. With dual staining of TRA-1-60
and CellTracker Orange, cell state can be better
distinguished with the C1 System.
Because TRA-1-60 emits at 518 nm (green), CellTracker
Orange, emitting at 576 nm (orange), is a compatible
dual stain. Additionally, CellTracker Orange is a live
stain allowing for cell tracing. Lastly, demonstrated
in Figure 6, dual staining does not cause non-specific
staining in empty C1 IFC chambers. This ensures there
is no interference from background fluorescence when
performing analysis.
Conclusion
Due to divergent fluorescent signals, complementary
detection of cell differentiation and viability, as well as
mitigated non-specific staining, TRA-1-60 antibody and
CellTracker Orange are deemed compatible stains with
the C1 Single-Cell Auto Prep System.
These stains are alternative stains to LIVE/DEAD Calcein
AM Ethidium Homodimer that enable fast and easy
monitoring of stem cells. Both TRA-1-60 and CellTracker
Orange are compatible with the C1 System standard
procedures and chemistry. With the methodology and
guidelines presented in this technical note, these stains
can readily be implemented in the standard C1 workflow
for robust single-cell gene expression analysis in stem
cell biology research.
A
B
C
Figure 6. Dual Staining of a Captured iPSC. A. Phase contrast image
of an iPSC captured in the top capture site. The bottom site is an empty
capture site. B. TRA-1-60 signal is observed under confocal microscopy
indicating the presence and undifferentiated state of the iPSC cell. No
fluorescence is observed in the empty capture site. C. CellTracker™
Orange signal is observed confirming cell viability of the same captured
cell. No fluorescence is observed in the empty capture site.
References
Schopperle, W.M., and DeWolf, W.C. (2007) The TRA-160 and TRA-1-81 human pluripotent stem cell markers are
expressed on podocalyxin in embryonal carcinoma. Stem
Cells 25: 723-730.
Kroemer G, Santiago R, Cytofluorometric purification of
diploid and tetraploid cancer cells. Springer Protocols Cell
Cycle Synchronization Methods in Molecular Biology. 2011;
761:47-63.
Chambers SM, Fasano CA, Papapetrou EP, Tomishima M,
Sadelain M, Studer L. Highly efficient neural conversion of
human ES and iPS cells by dual inhibition of SMAD signaling.
Nat Biotechnol. 2009 Mar;27(3):275-80.
International Stem Cell Initiative et al., Characterization of
human embryonic stem cell lines. Nature Biotechnology,
2007 Jun; 17: 803-816.
© 2013 Fluidigm Corporation. All rights reserved. Fluidigm, the Fluidigm logo, BioMark, C1, DELTAgene, and SINGuLAR are trademarks or registered trademarks of
Fluidigm Corporation in the U.S. and/or other countries. All other trademarks are the property of their respective owners.
For Research Use Only. Not for use in diagnostic procedures.
100-6857 06/2013
Corporate Headquarters
7000 Shoreline Court, Suite 100
South San Francisco, CA 94080 USA
Toll-free: 1.866.FLUIDLINE | Fax: 650.871.7152
www.fluidigm.com
Sales
North America | +1 650 266 6170 | [email protected]
Europe/EMEA | +33 1 60 92 42 40 | [email protected]
Japan | +81 3 3662 2150 | [email protected]
China (including Hong Kong) | +86 21 3255 8368 | [email protected]
Asia | +1 650.266.6170 | [email protected]
Latin America | +1 650 266 6170 | [email protected]