Stem Cell Research Analysis and Isolation of Stem Cells Using Flow Cytometry
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Stem Cell Research Analysis and Isolation of Stem Cells Using Flow Cytometry
2 Cover Illustration: Fairman Studios, LLC.
Flow cytometry is a powerful methodology for
characterizing, analyzing, and isolating stem cells
and their derivatives.
Recent discoveries in stem cell biology have amplified the importance
of pluripotent stem cells in therapeutics and have improved researchers’
understanding of normal and disease processes. Still, the inherent
heterogeneous nature of differentiating cultures remains a primary
challenge in stem cell research.
Flow cytometry is unique in its ability Researchers can use fluorochrome-
to investigate large cell populations conjugated antibodies to either cell surface
at the single-cell level. In contrast to or intracellular biomarkers to verify that
methods such as Western blot and cellular stem cells have maintained pluripotency.
imaging, multicolor flow cytometry Since stem cells differentiate into the three
enables researchers to interrogate primary germ layers and into differentiated
heterogeneous cell populations and analyze tissue, antibodies can monitor their
their subpopulations. Using multiple changing expression patterns. Analysis based
fluorescent-labeled antibodies, researchers on cell surface markers can preserve cell
can obtain robust, multiparametric viability for use in additional experiments.
data and population-based statistics on BD Lyoplate™ cell surface marker screening
differentiating stem cell cultures—and panels provide a powerful method for
can isolate stem cells and their derivatives discovering surface marker signatures that
from primary tissue and diverse in vitro can be used to explore these cells in depth.
populations.
BD Biosciences offers a diverse set of
Flow cytometry has been used for decades tools including high-quality antibodies,
by biologists studying hematopoietic buffers, protocols, and instrumentation to
stem cells to address the challenge of support stem cell research. This evolving
heterogeneity. New methods and tools toolset combines the power of advanced
are enabling researchers to employ this technologies and world-class service to
powerful technique to make key discoveries support investigators in characterizing,
about other stem cell types and their analyzing, and sorting heterogeneous stem
respective lineages. cell populations.
3Isolation of live cell populations
Cell surface staining for analysis
and sorting of live cells
Each type of stem cell or derivative expresses characteristic digestion process, preventing antibody labeling, they must
surface and intracellular proteins that can be used for be evaluated for each surface marker being measured.
identification (Tables 1 and 2). Because intracellular analysis BD Accutase tends to be more broadly applicable than
requires permeabilization, surface markers are essential trypsin and yields a more consistent single-cell suspension
when researchers want to isolate live cell populations for than does scraping.
further analysis. Fluorescence-activated cell sorting, or FACS, Monoclonal antibodies
can be used to sort cells of interest in bulk or in single-cell Once cells are harvested and the dissociation buffer is
depositions for downstream applications. To analyze cells removed, the cells are ready to be stained with antibodies.
for surface marker expression, a single-cell suspension must BD Biosciences offers an extensive reagent selection
be stained with fluorescent-labeled antibodies and analyzed of antibodies against hundreds of stem cell markers
or sorted on a flow cytometer. conjugated to a variety of fluorochromes for flexibility in
Sample preparation experimental design. For analysis of rare events and low-
Stem cells tend to be adherent and can grow as three- density antigens, the BD Horizon™ Brilliant Violet™ family
dimensional structures. To prepare a single-cell suspension of reagents can increase brightness and resolution. For ease
for flow cytometric analysis, enzymatic digestion (with of use, BD Stemflow™ kits and cocktails contain standard
BD™ Accutase cell detachment solution or trypsin) or antibody panels for analysis or sorting of different stem cell
mechanical scraping can be used. Since enzymatic methods types, as shown in Table 1.
might cleave or modify some protein epitopes during the
Human Markers Mouse Markers BD Stemflow™ or other Kit Cat. No.
Pluripotent Stem Cells (ESCs and iPSCs)
Positive: Alkaline Phosphatase, SSEA-4, Positive: SSEA-1 Human iPSC Sorting and Analysis Kit 562626
SSEA-3, TRA-1-81, TRA-1-60
Human Pluripotent Stem Cell Sorting and 560461
Negative: SSEA-1
Analysis Kit
Human and Mouse Pluripotent Stem Cell 560477
Analysis Kit
Hematopoietic Stem Cells (HSCs)
Positive: CD34, CD49f, CD90 Positive: CD150, c-Kit, Sca1 BD Pharmingen™ Human Lineage Cocktail 4 562722
Table 1.
Negative: CD38, CD45RA, Lineage* Negative: CD34, CD41, CD48,
Representative surface markers of selected Lineage Mouse Hematopoietic Stem Cell Isolation Kit 560492
stem cells and derivatives.
Mesenchymal Stem Cells (MSCs)
Positive: CD44, CD73, CD90, CD105, Positive: CD29, CD44, CD90, Human MSC Analysis Kit 562245
CD146, CD271 CD105, CD106, Sca-1
Human Mesenchymal Stem Cell Lineage 562530
Negative: CD11b, CD19, CD31, CD34, Negative: CD11b, CD31, Antibody Cocktail
CD45, CD144, HLA-DR CD45, Ter-119
Neural Stem Cells (NSCs)
Positive: CD15mid, CD24, CD184 – Human Neural Cell Sorting Kit 562271
Negative: CD44, CD271
Neurons
Positive: CD15low, CD24 – Human Neural Cell Sorting Kit 562271
Negative: CD44, CD184
Cancer
CD15, CD24, CD34, CD44, CD45, CD49f, – – –
CD166, CD326, CD338, Her-2/Neu, Lgr5
*Human lineage (lin) markers: CD2, CD3, CD4, CD7, CD8,
CD10, CD11b, CD14, CD19, CD20, CD56, CD235a
4HETEROGENEITY
A B
(x 1,000)
250
5
10
200
4
10
7-AAD
150
SSC-A
Hematopoietic stem cell phenotyping
3
10
Cells of the hematopoietic system are well characterized Lineage-Viable
100
with respect to surface marker expression, which is often
used to isolate and characterize subsets of cells during
2
10
50
hematopoiesis. Hematopoietic stem cells (HSCs), the source
0
Scatter
of these hematopoietic cells, are currently a focus area in
-124
50 100 150 200 250 101 102 103 104 105
stem cell biology because they can be used to replenish FSC-A (x 1,000)
0
FITC Lineage Cocktail 4
C D
normal bone marrow function.
5
5
10
Historically, among a pool of cells, HSCs were identified
10
as lineage-negative cells that expressed CD90 and CD34.1
CD90+CD45RA-
Recently, researchers have used additional markers to
4
10
BV421 CD38
BV605 CD90
4
10
enrich pools of long-term HSCs (LT-HSCs) capable of self-
renewal. These markers include CD38,2 CD45RA,3 and most
3
10
recently CD49f.4 Reportedly, about 10% of cells with a
3
10
Lin–CD34+CD38–CD90+CD45RA–CD49f+ phenotype are able to
provide long-term repopulating capacity in mouse models.4
2
-10 0 10
2
2
10
CD34+CD38-
-531
102 103 104 105 -102 0102 103 104 105
-650
APC CD34 APC-H7 CD45RA
E F
5
10
Tube: enriched
4
10
Population #Events %Parent %Total
BV605 CD90
All Events 201,698 #### 100.0
Scatter 145,671 72.2 72.2
Lineage-Viable 48,194 33.1 23.9
CD34+CD38- 4,650 9.6 2.3
3
10
CD90+CD45RA- 879 18.9 0.4
CD49f+ 184 20.9 0.1
CD49f+
2
-10 0 10
-5312
-105 0 102 103 104 105
PE CD49f
Gating strategy for LT-HSCs.
Frozen human cord blood mononuclear cells (Stem Cell Technologies)
were enriched using the BD IMag™ human lineage cell depletion
set – DM (Cat. No. 560030), stained with antibodies, and acquired and
analyzed on a BD LSRFortessa™ flow cytometer. A. First, cells were
gated based on light-scatter properties to screen out debris. B. Next,
viable, lineage-negative (CD2, CD3, CD4, CD7, CD8, CD10, CD11b,
CD14, CD19, CD20, CD56, CD235a) cells were gated based on the
viability dye 7-AAD and the BD Pharmingen human lineage cocktail 4
kit (Cat. No. 562722). To identify a highly enriched LT-HSC population,
(C) cells were gated on CD34+CD38–; (D) then (in a child gate) on
CD90+CD45RA–; and (E) finally on CD49f+. This combination of cell
surface markers, summarized in (F) the complete gating hierarchy,
results in a population rich in LT-HSCs.
5Study of differentiation pathways
Intracellular staining for
transcription factor analysis
Intracellular staining allows researchers to extend the Sample preparation
speed and statistical relevance of flow cytometry to the For intracellular staining, as with surface staining, a
investigation of functional proteins inside the cell. It can be single-cell suspension must be prepared using enzymatic
used in combination with surface staining to identify critical or mechanical methods. BD Accutase is recommended
time points, markers, and proportions of cells moving along since it helps to prevent cell clumping and can preserve
particular differentiation pathways. Intracellular staining surface proteins for simultaneous analysis. After optional
protocols require the cells to be fixed and permeabilized so surface staining, cells must be fixed and permeabilized to
that antibodies can access the cytoplasm and nucleus. Since enable antibodies to enter. The cells are then stained with
fixation effectively kills the cells, intracellular staining is not fluorescent-labeled antibodies to intracellular antigens and
compatible with live-cell sorting. analyzed on a flow cytometer.
To optimize permeabilization and staining conditions, BD
has developed several kits for the detection of key stem cell
transcription factors. The kits contain optimized antibodies
and buffer systems to characterize stem cells as well as their
differentiation into various lineages.
Table 2. Cell Type Intracellular Markers BD Stemflow Kit Cat. No.
Representative intracellular markers of Embryonic stem cells (ESCs) Nanog, Oct3/4, Sox2 Human Pluripotent Stem Cell 560589
selected stem cells and derivatives. Induced pluripotent stem cells (iPSCs) Transcription Factor Analysis Kit
Mouse Pluripotent Stem Cell 560585
Transcription Factor Analysis Kit
Neural stem cells (NSCs) Nestin, Pax6, Sox1, Sox2 Human Neural Lineage Analysis Kit 561526
Astrocytes GFAP Human Neural Lineage Analysis Kit 561526
Neurons Doublecortin Human Neural Lineage Analysis Kit 561526
Early pancreatic endoderm FoxA2, Pax6, Pdx1, Sox17 Human Definitive and Pancreatic 562496
Endoderm Analysis Kit
Late pancreatic endoderm NeuroD1, Nkx6.1 – –
Cardiac cTNI, GATA4, Islet-1, Myosin Heavy – –
Chain
Hepatic AFP, GATA4 – –
6INTRACELLULAR
Day 0: H9 hESCs
A
5
5
10
10
4
4
10
10
Sox17+FoxA2+
Sox17
Sox17
1%
3
3
10
10
Nanog+
92%
2
2
10
10
1
1
10
10
0
0
-63
-63
2 3 4 5 2 3 4 5
-68 0 10 10 10 10 -149 0 10 10 10 10
FoxA2 Nanog
Stem cell differentiation
B Day 3: Definitive Endoderm
The ability of human pluripotent stem cells to differentiate
into various cell lineages is a central topic in developmental
5
5
10
10
biology and has applications for regenerative medicine
and cellular therapy. As pluripotent cells differentiate into
4
10
4
Sox17+FoxA2+
10
different lineages, the expression of transcription factors Sox17
57%
Sox17
and other proteins can change. Multiparametric flow
3
3
10
10
cytometry is an excellent method for determining Nanog+
the relative numbers of cells expressing markers of interest, 24%
2
2
10
10
and can be used to optimize, quantitate, and compare
1
1
10
10
0
0
differentiation protocols and differentiation potential.
-52
-52
1 2 3 4 5 2 3 4 5
-42 10
0 10 10 10 10 -113 0 10 10 10 10
For example, in mammalian embryonic development, the
FoxA2 Nanog
definitive endoderm generates the liver, pancreas, and
intestine.5 During lineage specification into definitive Changes in transcription factors in definitive endoderm
endoderm, the levels of transcription factors Sox17 and development.
FoxA2 increase, while pluripotency markers such as Nanog H9 hESCs (WiCell) were differentiated into definitive endoderm
according to D’Amour, et al6 and monitored on a BD LSRFortessa
decrease.6
flow cytometer using the BD Stemflow™ definitive and pancreatic
The differentiation of neural stem cells to neural lineages endoderm analysis kit (Cat. No. 562496). As embryonic stem cells (A)
differentiated toward definitive endoderm (B), more cells expressed
can also be monitored using multicolor flow analysis. As
both Sox17 and FoxA2, while Nanog expression decreased.
neural stem cells (NSCs) differentiate into neurons, they
gradually express less Nestin and more of the early neuronal
marker doublecortin (DCX). A subpopulation of cells that
continues to express Nestin further delineates into a glial
cell population that expresses CD44.
A Day 0 Day 14 Day 27
17.3% 40.0%
5
5
5
10
10
10
6.5%
4
4
4
10
10
10
3
3
3
10
10
10
Changes in intracellular and surface
2
90.5%
2
10
2
-10 0 10
10
DCX
markers in neural cell differentiation. 73.7% 49.8%
2
0
1
010
NSCs derived from H9 hESCs using the
-384
-124
-384
M 2 3 4 5 1 2 3 4 5 2 3 4 5
0 10 10 10 10 10 10 10 10 10 0 10 10 10 10
-42 -3 -42
serum-free embryoid body (SFEB) method
were differentiated into neurons and glia B Nestin
and monitored on a BD LSRFortessa flow
5
5
5
cytometer using the BD Stemflow human
10
10
10
neural lineage analysis kit (Cat. No. 561526).
5.3% 35.4%
4
4
4
10
10
10
A. As differentiation progressed, cells
expressed less Nestin and more DCX.
3
3
3
10
10
10
B. Nestin+ cells were further delineated into
a glial cell population that expressed both
CD44
2
2
2
-10 0 10
10
10
CD44 and Nestin over time.
2
0
0
-384
-124
-384
M 2 3 4 5 1 2 3 4 5 2 3 4 5
0 10 10 10 10 10 10 10 10 10 0 10 10 10 10
-42 -3 -42
Nestin
7Unique multimarker signatures
Rapid and efficient
surface marker screening
A major challenge facing stem cell biology is the Both the human (Cat. No. 560747) and mouse (Cat. No.
heterogeneous nature of cultures and differentiations. To 562208) panels contain three plates. Each well contains
sort viable cells to purify for use in later experiments, one lyophilized, purified antibody to one cell surface marker
must know the cell surface signature for the particular or isotype control. Following reconstitution, the cellular
cell of interest. Since different types of related cells may samples are stained with purified antibodies, and detection
share markers, researchers must find a unique multimarker reagents included with the panel are added. Finally,
signature for each, while other markers may be useful in samples are analyzed by flow cytometry or imaging.
distinguishing subpopulations of a particular type of cell.
To provide flexibility while simplifying workflow, open
BD Lyoplate cell surface marker screening panels provide wells allow the panel to be expanded to include additional
a comprehensive and efficient solution for profiling stem markers. Powerful BD Biosciences analysis tools facilitate
cells and their derivatives for hundreds of human or mouse data mining and heatmap generation.
cell surface markers by flow cytometry or cellular imaging.
Deciphering the cell surface proteome enables researchers
to define strategies for the analysis and isolation of Table 3.
targeted cells from heterogeneous populations for Considerations for flow cytometry vs image screening with
BD Lyoplate panels.
functional studies, drug screening, in vivo animal studies,
and cell therapy research. Property of sample Flow cytometry Imaging
Suspension cells X
The hundreds of monoclonal antibodies in each panel
Rare cell populations X
constitute one of the most cost-effective screening tools
Subpopulation analysis
available for cellular analysis. To simplify the transition X
Co-staining with multiple markers
to more targeted, larger-scale experiments, all antibodies Reporter lines X X
included in the screening panels are available in the Specific morphology changes X
BD Biosciences catalog. Limited number of cells X
A Hits FACS
Using surface marker screening to Heterogeneous Flow screen
(-) t
te an
et in
iii
neural induction
+)
i i
characterize and enrich neural stem
os m
e(
-r ta
-r 9)
CD184
SC tt
CD44
CD24
EB con
EB (H
N ose
SC
SC
10% of
its
cells and neurons.
hE
N
H
ii total
% +ve CD184 pos
0
CD15 pos
A. H9 hESCs were induced using the SFEB Flow 5
10
15 CD54 neg
FSC-A CD271 CD15
method,7 and the BD Lyoplate human cell screen 20
25
30
CD29
CD71
35
40 CD49c
surface marker screening panel (Cat. No.
45
50 CD49e
55
60 CD63
560747) was used to screen the resulting 65
70
75
CD57
CD47
80
heterogeneous neural cultures on a 85
90
95
CD81
CD59
100
BD™ LSR II flow cytometer to identify a CD58
CD56
CD46
surface marker signature for NSCs. After CD24 pos
CD55
identifying potential hits, NSC cell surface CD271neg
CD50
phenotypes were verified and a CD184+CD44–
CD271–CD24+CD15mid sorting profile was used
on a BD FACSAria™ II cell sorter to obtain a
Enriched NSCs
near-pure subpopulation of NSCs.
8 FACS
Hits
P3
P5DISCOVERY
Screening of neural populations In panel B, the purified NSCs were differentiated into
Neural cell populations derived from pluripotent stem neuronal and glial cell populations, which were screened by
cells are important for studying human disease and imaging using the same panel to identify surface markers
development. Pluripotent stem cells can be differentiated for isolating neurons. An imaging screen was chosen due
into self-renewing NSCs, which can be further differentiated to the unique morphology of neurons and the ability
into heterogeneous populations of neurons and glia.7 A key to co-stain with a neuronal-specific marker. A potential
to further research is to identify surface marker signatures neuronal surface phenotype of CD44–CD184–CD24+CD15low
for each of these cell types. was verified by flow cytometry and used to purify neurons.
In the example, the BD Lyoplate human cell surface marker In addition to neural cells, the BD Lyoplate human cell
screening panel (Cat. No. 560747) was used to identify surface marker screening panel has also been used to
cell surface phenotypes for NSCs and neurons. In panel A, identify cell surface markers of cardiomyocytes derived
heterogeneous neural induction cultures were screened by from pluripotent stem cells.8 Most recently, this powerful
flow cytometry, and potential NSC markers were identified methodology was used to develop a human stem cell model
on a heatmap. A resulting NSC cell surface phenotype of Alzheimer’s disease.9
of CD184+CD44–CD271–CD24+CD15mid was verified using
intracellular NSC markers. The surface phenotype was used
to sort a near-pure subpopulation of NSCs, the ability of
which to differentiate both in vivo and in vitro was later
Hits FACS
confirmed. Heterogeneous Flow screen
(-) t
te an
et in
iii
neural induction
+)
i i
os m
e(
-r ta
-r 9)
CD184
SC tt
CD44
CD24
EB con
EB (H
N ose
SC
SC
10% of
its
hE
N
H
ii total
% +ve CD184 pos
0
CD15 pos
Flow 5
10
15 CD54 neg
FSC-A CD271 CD15
screen 20
25
30
CD29
CD71
35
40 CD49c
45
50 CD49e
55
60 CD63
65 CD57
70
75 CD47
80
85 CD81
90
95 CD59
100
CD58
CD56
CD46
CD24 pos
CD55
CD271neg
CD50
Enriched NSCs
B
FACS
B. The purified NSCs were differentiated Hits
P3
into a mixed culture of neuronal and glial
P5
Heterogeneous neuronal
CD44
cell populations, which was screened using differentiation
CD24
P4
the same panel to identify a surface marker
signature for neurons. An imaging screen Image
was chosen due to the unique morphology screen
M
CD184 CD15
of neurons and the ability to co-stain with a
neuron-specific marker. Potential hits were
verified by flow cytometry, and a CD44–
CD184–CD24+CD15low sorting profile was used
to purify neurons.7
Near-pure neurons
9SERVICES
Services and Support
For more than 25 years, BD has actively worked with Technical Applications Support
stem cell researchers to develop tools that help improve BD Biosciences technical applications support specialists
workflow, ease of use, and performance. This in-depth are available to provide field- or phone-based assistance
knowledge and experience is available to customers and advice. Expert in a diverse array of topics, BD technical
through comprehensive training, application and technical application specialists are well equipped to address
support, and expert field service. customer needs in both instrument and application support.
Training Field Service Engineers
Held at BD training centers worldwide, BD Biosciences BD Biosciences field service engineers are located across
flow cytometry training courses combine theory and the world. When instrument installation or service is
hands-on practice to provide participants with the skills required, a BD Biosciences Technical Field Service Engineer
and experience they need to take full advantage of the can be dispatched to the customer site. On-site service and
capabilities of their instrument. maintenance agreements are available to provide long-term
support.
Special Order Research Products
In addition to other services, BD instruments can be
customized to me et specific customer requirements via the
special order research program.
Custom Services
Mobilizing technology for research applications requires
close collaboration. The Custom Technology Team (CTT) at
BD Biosciences works with customers to provide solutions
through custom reagents, panels, or assay protocols. Staffed
by leading scientists with a breadth and depth of scientific
and technical expertise, the CTT team will coordinate
with researchers to study the problem at hand, make
recommendations, and help implement solutions. In this
way, BD Biosciences technical know-how is translated into
practical solutions that allow customers to focus on research.
References
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AS, Buckle AM, Peault B. Isolation of a A, Jurisica I, Dick JE. Isolation of single surface marker signatures for the
candidate human hematopoietic stem- human hematopoietic stem cells capable isolation of neural stem cells, glia and
cell population. Proc Natl Acad Sci U S A. of long-term multilineage engraftment. neurons derived from human pluripotent
1992;89:2804-2808. Science. 2011;333:218-221. stem cells. PLoS One. 2011;6:e17540.
2. B
hatia M, Wang JC, Kapp U, Bonnet 5. Murry CE, Keller G. Differentiation of 8. Uosaki H, Fukushima H, Takeuchi A, et
D, Dick JE. Purification of primitive embryonic stem cells to clinically relevant al. Efficient and scalable purification of
human hematopoietic cells capable of populations: lessons from embryonic cardiomyocytes from human embryonic
repopulating immune-deficient mice. development. Cell. 2008;132:661-680. and induced pluripotent stem cells by
Proc Natl Acad Sci U S A. 1997;94:5320- VCAM1 surface expression. PLoS One.
6. D’Amour KA, Agulnick AD, Eliazer S,
5325. 2011;6:e23657.
Kelly OG, Kroon E, Baetge EE. Efficient
3. M
ajeti R, Park CY, Weissman IL. differentiation of human embryonic 9. Israel MA, Yuan SH, Bardy C, et al.
Identification of a hierarchy of stem cells to definitive endoderm. Nat Probing sporadic and familial Alzheimer’s
multipotent hematopoietic progenitors Biotechnol. 2005;23:1534-1541. disease using induced pluripotent stem
in human cord blood. Cell Stem Cell. cells. Nature. 2012;482:216-220.
2007;1:635-645.
1011
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