Multimarker Analysis Methods for
the Rapid Characterization of
Pluripotent, Multipotent, and
Differentiating Stem Cells
Christian Carson, PhD
BD Biosciences
R&D Scientist
Stem Cell Research
23-11033-00
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Stem Cell
Research
Overview
2
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Multimarker
Analysis
Methods
Overview
Applications
MSCs
NSCs
Applications
Pluripotent
Stem Cells
Q&A
Session
Stem Cells
Embryonic
iPS
Oct3/4
Sox2
Klf4
c-Myc
Nanog
LIN28
Adult
Schematic adapted from Wobus AM and Boheler, KR. Physiol. Rev. 2005; 85:635-678.
3
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Stem Cell Research
Schematic adapted from http://stemcells.nih.gov/index.asp
4
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Challenges in Stem Cell Research
• Characterize cell types and stages of differentiation
by identifying cell-type specific biomarkers
• Identify and isolate cells of interest from a
heterogeneous pool
• Analyze cells for quality and purity
• Analyze cell function
5
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
BD Biosciences
6
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Stem Cell
Research
Overview
7
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Multimarker
Analysis
Methods
Overview
Applications
MSCs
NSCs
Applications
Pluripotent
Stem Cells
Q&A
Session
Immunophenotyping
Immunophenotyping is a cellular analysis method for the identification of biomarkers and
their expression/co-expression profiles using directly- or indirectly-fluorochrome conjugated
antibodies and an analyzer such as a flow cytometer or imaging system.
The identification of biomarkers using immunophenotyping methods facilitates the
characterization of a cell type, its identification, and its isolation.
• Identification of cell subpopulations:
– Cells suitable for transplantation
– Tumor initiating cells
• Isolation of pure cell populations for downstream assays:
– Arrays/sequencing
– In vitro disease models
– Biochemistry
– Transplantation
• Development of quality control assays for cell preparations:
– Assessing purity
– Identification of contaminants
8
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Tools for Immunophenotyping
BD LyoplateTM Screening Panels support the rapid, cost-effective
immunophenotyping of stems cells and stem cell–derived cells.
BD Lyoplate
Screening Panel
Contents
Applications
Human Cell
Surface Markers
• 242 CD Markers*
• Isotype Controls
• Alexa Fluor® 647
Second Step
• Flow
Cytometry
• Bioimaging
Available
September
2009
Mouse Cell
Surface Markers
• 200+ CD Markers
• Isotype Controls
• Alexa Fluor® 647
Second Step
• Flow
Cytometry
• Bioimaging
Available
Fall 2009
Cat. No.
*CD and other cell surface molecules. One marker per well.
• Plate-based format is compatible with automation and multichannel pipetting
• The proprietary lyophilized format allows for room temperature storage, long shelf life
• Open wells permit the use of additional markers of choice
• Compatible with BFP, CFP, GFP, YFP, OFP, and RFP expressing cells
9
Alexa Fluor® is a registered trademark of Molecular Probes, Inc. The Alexa Fluor® dye antibody conjugates in this product are sold under
license from Molecular Probes, Inc., for research use only, excluding use In combination with microarrays, or as analyte specific reagents.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Tools for Immunophenotyping
BD FACS™ CAP (Combinatorial Antibody Profile) is a custom multicolor
immunophenotyping and analysis service. The in-depth analysis service
provides an inventory of receptors and markers present on the cell
surface, yielding an information-rich “fingerprint.”
• 212 fluorescently-labeled anti-human
antibodies
• Multicolor cocktails in each well of a
96-well plate
• Flexibility to integrate researcher’s
specific markers
• Analysis supported by proprietary
software
In addition, the BD Custom Technology Team helps researchers to create
multicolor antibody cocktails for in-house flow cytometry immunophenotyping.
Optimized multicolor cocktails streamline sample preparation, acquisition and
analysis, and improve standardization between experiments.
10
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Stem Cell
Research
Overview
Multimarker
Analysis
Methods
Overview
Applications
MSCs
NSCs
Applications
Pluripotent
Stem Cells
Q&A
Session
• Screening of MSCs
• Screening, isolation and analysis of hESC-derived NSCs,
glia, and neurons
11
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Mesenchymal Stem Cells (MSCs)
12
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
•
MSCs are the progenitors
of multiple mesenchymal
lineages
– Bone, cartilage,
muscle, fat tissue, and
marrow stroma
•
MSCs can be isolated and
cultured in vitro
•
Promises of MSCs
– Regenerative medicine
– In vitro models of
human diseases
• Drug screening
• Toxicology
• Basic research
Characterization of MSCs
Objective: correlate CD marker profile to lineage capacity and multipotency
CD-marker lib plate A
CD-marker lib plate B
600000
fluoresence intensity
fluoresence intensity
600000
500000
400000
300000
200000
100000
400000
300000
200000
100000
0
0
1
A
B
Collaboration with Paul Coffer
and Koen Braat
University Medical Center
Utrecht, Netherlands
500000
2
3
4
5
6
7
8
9
10
11
12
1
2
3
4
5
6
7
8
buffer
CD10
CD21
CD33
CD44
CD49d
CD61
CD72
CD1a
CD11a
CD22
CD34
CD45
CD49e
CD62E
CD73
CD1b
CD11b
CD23
CD35
CD45RA
CD49e
CD62L
CD74
CD1d
CD11c
CD24
CD36
CD45RB
CD50
CD62P
CD77
CD2
CD13
CD25
CD37
CD45RO
CD51/61
CD63
CD79b
CD3
CD14
CD26
CD38
CD46
CD53
CD64
CD80
CD4
CD15
CD27
CD40
CD47
CD54
CD66
CD81
CD5
CD15s
CD28
CD41a
CD48
CD55
CD66b
CD83
CD6
CD16
CD29
CD41b
CD49a
CD56
CD66f
CD84
CD7
CD18
CD30
CD42a
CD49b
CD57
CD69
CD85j
buffer
CD103
CD137L
CD154
CD181
CD235a
HLA-DR
P-glycop
CD88
CD104
CD138
CD158a
CD182
CD244
HLA-DR
Siglec-6
CD89
CD106
CD140a
CD158b
CD183
CLIP
InvNKT
Siglec-7
CD90
CD108
CD140b
CD161
CD184
CMRF-44
LAIR-1
Vbeta5
CD91
CD109
CD141
CD162
CD195
CMRF-56
CD220
Vbeta8
CD94
CD110
CD142
CD163
CD200
EGFR
mu-Calp
INTb7
CD95
CD117
CD146
CD164
CD206
F11R
MUC2
PRR2
CD97
CD123
CD147
CD165
CD209
fMLPr
NGFR
ABCG2
CD98
CD130
CD150
CD166
CD221
gdTCR
NKB1
abTCR
CD99
CD99R
CD134 CD135
CD151 CD152
CD172b CD177
CD226 CD227
HLA-ABC HLA-A2
NKG2D NKP46
B7-H2 beta2-m
<7000
>7000
>20000
>50000
CD8
CD19
CD31
CD42b
CD49b
CD58
CD70
CD86
9
10
11
CD9
CD20
CD32
CD43
CD49c
CD59
CD71
CD87
CD100
CD137
CD153
CD180
CD229
HLA-DQ
NPM-ALK
BldGrpA
12
Functional characterization
Multipotency?
Osteogenic capacity?
Immunosuppression?
control
BM-MSC MSC-hTERT
H11
>100000 >200000 >300000 >400000 >500000
Experimental results using a beta version of the BD LyoplateTM Human Cell Surface Marker Screening Panel
13
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Neural Stem Cells (NSCs)
•
Found during embryonic development and
in restricted regions of the adult brain
•
NSCs can be isolated and cultured in vitro
– Fetal and adult brain
– Differentiated from hESCs
•
Promises of NSCs
– Transplantation therapy
– In vitro models of human development
– In vitro models of human diseases
• Drug screening
• Toxicology
• Basic research
(NSC)
14
.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
For Research Use Only. Not for use in diagnostic or therapeutic procedures
Neural Stem Cell Research
Differentiation of hESCs into neural stem cells
Day 0
7
hESCs
mTeSR™1
EBs
withdraw bFGF
17
Rosettes
N2
20
NSCs
N2, B27, bFGF
35
Neurons, Glia
N2, B27, dbcAMP
The field needs:
– Robust standardized methods for isolating NSCs to eliminate batchto-batch variability
– Isolation of pure populations of NSCs, glia, and neurons for
downstream assays: arrays/sequencing, biochemistry, in vitro assays
15
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Objective
Day 0
7
hESCs
mTeSR™1
EBs
withdraw bFGF
17
Rosettes
N2
20
NSCs
N2, B27, bFGF
35
Neurons, Glia
N2, B27, dbcAMP
– Define cell surface signatures of hESC, hESC-derived NSCs, glia, and
neurons
– Develop standardized methods for isolating these cell types by flow
cytometry
Collaboration with Larry Goldstein
University of California, San Diego (UCSD) /
Howard Hughes Medical Institute (HHMI)
16
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Materials and Methods
hESCs
EBs
Rosettes
NSCs
Neurons, Glia
Cell Surface Marker Screen to Identify Markers for Sorting Neurons and Glia from Differentiating NSCs
Generate hESC-derived NSCs, glia, and neurons
Identify markers
Cells were screened using a beta version of the BD LyoplateTM Human Cell Surface Marker
Screening Panel by flow cytometry and bioimaging to identify a unique cell surface signature
for neurons. Signatures were validated by multicolor flow cytometric analysis.
Detect and isolate cells of interest from heterogeneous pool
Cell were isolated using a BD FACSAriaTM II cell sorter.
Analyze for purity of sorted cells
Expression of cell-type specific markers was confirmed by multicolor flow cytometric analysis.
17
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Multicolor Analysis of hESC-derived NSCs
Sox2 Nestin Ki67 Hoechst
Sox2 PE
Oct3/4 Alexa Fluor® 488
18
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
99%
Sox2 PE
99%
Nestin Alexa Fluor® 647
Characterization of Naïve and Differentiated hESCs
Experimental results using a beta version of the BD LyoplateTM Human Cell Surface Marker Screening Panel
hESC (H9)
80
80
60
40
60
40
20
65
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
40
85.2
22.5
0
0
60
20
77.5
68.8
APC-A
0
10
2
10
3
10
4
10
5
60
40
20
14.8
97.9
0
APC-A
10
0
10
1
APC-A
10
2
10
3
10
4
0
80
80
40
20
1.55
98.5
10
2
10
3
10
4
10
5
0
10
2
10
3
10
4
10
5
40
99.3
4.87
0
APC-A
60
20
95.1
1.71
0
0
40
20
98.3
0
60
% of Max
80
% of Max
80
% of Max
80
% of Max
100
% of Max
100
40
0
10
2
10
3
10
4
10
5
98.7
10
0
10
1
10
2
10
3
10
4
0
80
80
80
20
20
12.8
87.2
20
98.5
0
10
2
10
3
10
4
10
5
0
10
2
10
3
10
4
10
5
40
98.7
18.1
0
APC-A
60
20
81.9
1.47
0
0
40
% of Max
80
% of Max
80
% of Max
100
% of Max
100
% of Max
100
40
0
10
2
10
3
10
4
10
5
10
0
10
1
10
2
10
3
10
4
0
80
0.57
99.4
0
20
88.8
10 2
10 3
10 4
APC-A
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
10 5
10 2
10 3
APC-A
10 4
10 5
40
99.8
0
0
60
20
0.22
11.2
0
0
40
% of Max
80
% of Max
80
% of Max
80
% of Max
80
% of Max
100
20
16.7
10 2
10 3
APC-A
10 4
10 5
4
10
5
10
2
10
3
10
4
10
5
60
40
20
83.3
45.1
0
0
10
APC-A
100
40
3
2.88
APC-A
60
10
97.1
100
20
2
40
100
40
10
0
APC-A
60
1.3
60
100
60
5
20
1.27
0
APC-A
10
APC-A
100
40
4
40
APC-A
60
10
0
APC-A
60
3
60
100
60
10
20
0.7
0
APC-A
2
APC-A
100
60
10
APC-A
100
60
2.1
0
100
20
19
40
20
31.2
0
0
60
% of Max
80
% of Max
80
% of Max
80
% of Max
100
0
CD marker “D”
NSCs
100
35
CD marker “C”
EB 3
100
20
CD marker “B”
EB 2
100
% of Max
CD marker “A”
EB 1
100
54.9
0
10 0
10 1
10 2
APC-A
10 3
10 4
0
10 2
10 3
APC-A
10 4
10 5
Isolation of Neurons by Flow Cytometry
NSCs were differentiated 2 weeks prior to sorting
BD FACSAria II sorter, 20 psi, 100-µm nozzle
Sorted
Ki-67 Nestin Map2b Hoechst
Presort
Ki-67 Nestin Map2b Hoechst
Sort gating
NSC, Glia
Neurons
20
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Isolation of Neurons by Flow Cytometry
Presort
Sort gating
46%
45%
Ki-67 Alexa Fluor® 488
Sorted
94%
Nestin Alexa Fluor® 647
Nestin Alexa Fluor® 647
NSCs were differentiated 2 weeks prior to sorting
BD FACSAria II sorter, 20 psi, 100-µm nozzle
NSC, Glia
5%
8%
90%
Ki-67 Alexa Fluor® 488
21
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Neurons
Conclusions
hESCs
EBs
Rosettes
NSCs
Neurons, Glia
– Further defined the cell surface signature for hESCs, NSCs, neurons,
and glia using flow cytometry and imaging
– Used signatures to develop methods for identifying and isolating
these cell types by flow cytometry
– These methods will enable:
• More standardized and robust isolation of hESC-derived neural
stem cells
• Downstream applications requiring consistent or pure cell
populations
22
– Immunophenotyping can be applied to address similar problems in
other stem cell fields
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Stem Cell
Research
Overview
Multimarker
Analysis
Methods
Overview
Applications
MSCs
NSCs
Applications
Pluripotent
Stem Cells
Q&A
Session
– Intracellular and cell surface marker analysis of pluripotent
stem cells
– Methods for sorting hESCs
23
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Challenges of Sorting Pluripotent Stem Cells
• Are sorted hESCs viable?
– Fong, et al. Stem Cell Rev. 2009
– Nicholas, et al. Stem Cells Dev. 2007
– Sidhu, et al. Stem Cells Dev. 2006
• Do sorted cells still express
markers of pluripotency?
• Are sorted cells capable of
further differentiation?
24
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Multicolor Flow Cytometric Immunophenotyping Kits
BD StemFlowTM Kits are comprehensive,
ready-to-use systems designed to increase
productivity and minimize assay-to-assay
variability.
9
Human Pluripotent Stem Cell
Transcription Factor Analysis Kit
(Cat. No. 560589)
Mouse Pluripotent Stem Cell
Transcription Factor Analysis Kit
(Cat. No. 560585)
9
9
9
9
9
Hu
hNanog PE
Oct3/4 PerCP-Cy™5.5
Sox2 Alexa Fluor® 647
9
9
9
Ms
mNanog PE
Oct3/4 PerCP-Cy™5.5
Sox2 Alexa Fluor® 647
9
9
9
• All kits contain BD™ CompBead Plus compensation particles, matched isotype controls, and verified protocols
• Intracellular analysis kits contain fix and perm buffers
• 50 tests
25
Cy™ is a trademark of Amersham Biosciences Corp. Cy™ dyes are subject to proprietary rights of Amersham Biosciences Corp and Carnegie
Mellon University and are made and sold under license from Amersham Biosciences Corp only for research and in vitro diagnostic use. Any
other use requires a commercial sublicense from Amersham Biosciences Corp, 800 Centennial Avenue, Piscataway, NJ 08855-1327, USA.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
GFP
Hu/Ms
SSEA-1 PE
Oct3/4 PerCP-Cy™5.5
SSEA-4 Alexa Fluor® 647
Drop-ins
Drop-
Human and Mouse Pluripotent Stem Cell
Analysis Kit
(Cat. No. 560477)
Sorting
Cell Surface
Analysis
9
Intracellular
Analysis
Antibodies
Hu
SSEA-1 FITC
SSEA-3 PE
Tra-1-81 Alexa Fluor® 647
Species
Human Pluripotent Stem Cell
Sorting and Analysis Kit
(Cat. No. 560461)
Materials and Methods
•
Cell surface markers (BD StemFlow Human Pluripotent Stem Cell
Sorting and Analysis Kit)
– SSEA-1 negative (differentiation)
– SSEA-3 positive (pluripotency)
– Tra-1-81 positive (pluripotency)
•
Cell sorting with BD FACSAria II system
– 20 psi, 100-µm nozzle
•
hESC
Media
Surface
Enzyme
H9
mTeSR™1
BD MatrigelTM
Accutase or Dispase
H9
KOSR
MEFs
Collagenase IV
H7
KOSR
MEFs
Collagenase IV
HUES9
HUES
MEFs
Trypsin
Accutase used to dissociate cells in single-cell suspension
mTeSR is a trademark of StemCell Technologies.
26
KnockOut™ Serum Replacement is a trademark of Invitrogen.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Sorting is Possible Under Feeder and Feeder-Free
Culturing Conditions
27
H9 day 3 post-sort
HUES9 day 4 post-sort
H7 day 10 post-sort
mTeSRTM1, BD Matrigel
HUES, MEF
Goldstein Lab, UCSD
KOSR, MEF
KnockOut™ Serum Replacement is a trademark of Invitrogen.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Sorted hESCs Express Pluripotency Markers
Experimental results using the BD StemFlowTM Human and Mouse Pluripotent Stem Cell Analysis Kit
H9 P6 post-sort
SSEA-4 Oct-3/4 SSEA-1 Hoechst
93.8%
1.1%
5.2%
Oct3/4 Percp-Cy5.5
0.0%
28
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
SSEA-4 Alexa Fluor® 647
SSEA-4 Alexa Fluor® 647
TRA1-81 SSEA-1 Hoechst
3.6%
95.3%
0.1%
1.0%
SSEA-1 PE
Sorted hESCs Retain Differentiation Potential
H9 P7 post-sort
Mesoderm
GATA4 Hoechst
29
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Ectoderm
Sox1 Hoechst
Endoderm
FOXA2 Hoechst
Conclusions
• Sorted hESCs are viable
and recoverable
• Sorted cells express
markers of pluripotency,
are able to differentiate,
and maintain normal
karyotype
• Standardized method for
sorting hESCs by flow
cytometry
30
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Spectral Overlap
Uncompensated
Median
31
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Compensated
Median
•
Always need a positivestained control
•
Wastes cells
•
Cumbersome when
assaying for markers that
might or might not be
expressed on cells
•
Solution = compensation
beads
•
Standard BD CompBead
particles work on small
cells found in blood, but
are not ideal for larger
cells
BD CompBead Plus
Overlays of unstained cells and cells and beads stained with SSEA-4 conjugates
H9 hESC
CompBead
CompBead Plus
SSEA-4 FITC
SSEA-4 PE
SSC-A
•
•
•
•
FSC-A
32
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
SSEA-4 Alexa Fluor® 647
Autofluorescence of beads tracks hESCs
Facilitates scatter setup
Compensation for any mouse or rat antibody
Negative Control (BSA) particles included
BD CompBead Plus Anti-Mouse Ig Set
Catalog No. 560497
BD CompBead Plus Anti-Rat Ig Set
Catalog No. 560499
BD Biosciences
33
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
BD Biosciences Technologies to Support
Stem Cell Research Applications
•
•
•
Extracellular Matrices (eg, BD Matrigel)
Media and media supplements
Cell cultureware, growth factors, cytokines
•
Validated antibodies and kits
– Flow cytometry
– Imaging
– Western blot
•
Flow cytometry systems
– Cell sorting (live cells)
– Cell analysis (fixed cells)
•
Automated high-content imaging systems
– Cell analysis (fixed cells and live cells)
•
Custom media, surfaces, reagents, instruments
34
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Acknowledgments
Stem Cell Research
Bob Balderas
Jeanne Elia
Nil Emre
Jody Martin
Rosanto Paramban
Jurg Rohrer
Jason Vidal
TAS
Sue Reynolds
35
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
UCSD
Goldstein Lab:
Jessica Flippin
Rhiannon Nolan
Shauna Yuan
R&D Cytometry Lab
Andrea Nguyen
Dennis Sasaki
Stem Cell
Research
Overview
36
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Multimarker
Analysis
Methods
Overview
Applications
MSCs
NSCs
Applications
Pluripotent
Stem Cells
Q&A
Session
To alert the presenter you have a question
press 1, then 0.
If you have further questions:
• Contact your BD Biosciences Reagent Sales Account Manager
or email Applications Support
• ResearchApplications@bd.com
Visit our BD Stem Cell Research page:
bdbiosciences.com/stemcellsource
37
For Research Use Only. Not for use in diagnostic or therapeutic procedures.