Post on 10-Jan-2022
23-10679-00
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Isolation and Analysis of Pluripotent, Neural, and Hematopoietic Stem Cells
Christian CarsonBD BiosciencesR&D ScientistStem Cell
3For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Overview
• Introduction– Challenges in stem cell research– Antibody portfolio of stem cell markers
• Cell sorting of neural stem cells (NSCs) and neurons– BD Lyoplate™ CD screening panel– BD FACS™ CAP service
• Cell sorting of human embryonic stem cells (hESCs)• Flow cytometry kits for pluripotent stem cell research
– Compensation beads for larger cells and bright markers• Mouse hematopoietic stem cell (mHSC) isolation• Overview of stem cell flow kits
4For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Stem Cell Background
Schematic adapted from Wobus AM. and Boheler, KR., Physiol. Rev.2005; 85:635-678.
Embryonic
Adult
iPS
Oct3/4Sox2Klf4
c-MycNanogLIN28
5For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Stem Cell Background
Schematic adapted from http://stemcells.nih.gov/index.asp
6For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Challenges in Stem Cell Research
• Identify and isolate cells of interest from a heterogeneous pool
• Analyze cells for quality and purity
• Analyze cell function
7For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Biomarkers are Crucial for Stem Cell Analysis and Isolation
Undifferentiated Stem Cell Progenitor Cell Differentiated Cell
Growth Factors & Cytokines Growth Factors & Cytokines
Biomarker IdentificationDNA/RNA microarrays, sequencing, PCR, antibody arrays
Isolate stem cells of interest using flow
cytometry or magnetic beads Biomarkers to define differentiation status:
cell analysis using flow cytometry, imaging, WB, IHC
Defined media, surfaces, and supplement to control cell fate
Undifferentiated Stem Cell Progenitor Cell Differentiated Cell
Growth Factors & Cytokines Growth Factors & Cytokines
Functional in vitro assays (apoptosis, cell cycle, cell proliferation, BD™ Phosflow) In vivo animal studiesCell therapy Research
8For Research Use Only. Not for use in diagnostic or therapeutic procedures.
BD Biosciences
First to commercialize tools for isolating and analyzing hematopoietic stem cells, now bringing this expertise to the
broader stem cell field
9For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Portfolio of High Quality Antibodies
For Research Use Only. Not for use in diagnostic or therapeutic procedures .
10For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Purified mAbs for Stem Cell Research
human Nanog Nestin GATA4
hESC hESC-derived neural stem cells
hESC-derived cardiomyocytes
11For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Fluorochrome-Conjugated mAbs for Imaging
MergeOct3/4 AlexaFluor® 555
Sox2 AlexaFluor® 647
HoechstSSEA-4 AlexaFluor® 488
H9 grown on BD Matrigel™ hESC-qualified matrix with mTeSRTM1
12For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Fluorochrome-Conjugated mAbs for Imaging
SSEA-1 (CD15): Multi-lineageCdx2: TrophectodermOct3/4: PluripotencyGATA4: Endoderm
Cdx2 SSEA-1 Oct3/4 Hoechst SSEA-1 GATA4 Cdx2 HoechstSSEA-1 Hoechst
Differentiated H9 hESCs
13For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Fluorochrome-conjugated mAbs for Flow Cytometry
H9 hESCs grown on BD Matrigel™ hESC-qualified matrix with mTeSRTM1
Oct3/4 PE
SSEA
-4 A
lexa
Fluo
r®48
8
Sox2
Ale
xaFl
uor®
647
98.9%98.9%
14For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Fluorochrome-conjugated mAbs for Flow Cytometry
SSEA-1 (CD15): Multi-lineageCdx2: TrophectodermOct3/4: Pluripotency
13%
12%
75% 83% 13%
2.2%1.8%
Oct
3/4
PE
Cdx2 Alexa Fluor® 6 47SS
EA-1
Ale
xaFl
uor®
488
Differentiated H9 hESCs
15For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Neural Stem Cells: Background
(NSC)
Neural progenitor (NP)
Glial progenitor (GP)
Neuron
• 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
For Research Use Only. Not for use in diagnostic or therapeutic procedures .
16For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Day 0 17 20 357
mTeSR™1 withdraw bFGF N2 N2, B27, bFGF N2, B27, dbcAMP
Rosettes NSCsEBshESCs Neurons, Glia
Generation of Neural Cells from hESCs
17For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Closer Look at hESC-derived NSCs
Sox2
PE
Oct3/4 Alexa Fluor® 488
Sox2
PE
Nestin Alexa Fluor® 647
Sox2 Nestin Ki67 Hoechst
57%
39%
Sox2: hESCs, NSCsNestin: NSCsOct3/4: hESCsKi67: proliferation
99%99%
Ki-6
7 A
lexa
Fluo
r®48
8
Sox2 Alexa Fluor® 647
18For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Day 0 17 20 357
mTeSR™1 withdraw bFGF N2 N2, B27, bFGF N2, B27, dbcAMP
Rosettes NSCsEBshESCs Neurons, Glia
• Current Challenges– Protocols are difficult– Batch-to-batch variability of NSCs– Neuronal differentiation is heterogeneous
• Need pure populations for applications: transplantation, arrays/sequencing, in vitro disease models
Generation of Neural Cells from hESC
19For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Generation of Neural Cells from hESCs
• Performed a screen using 192 human CD markers by flow cytometryand bioimaging
– Define cell surface signatures of hESCs, NSCs, NPs (neural progenitors), neurons, and glia
– Develop a method to isolate near-pure populations of NSCs, neurons, and glia
Day 0 17 20 357
mTeSR™1 withdraw bFGF N2 N2, B27, bFGF N2, B27, dbcAMP
Rosettes NSCsEBshESCs Neurons, Glia
20For Research Use Only. Not for use in diagnostic or therapeutic procedures.
-0.07 -0.2221053 -0.9382092 -0.3600478 0.04848257 -0.6427204 -0.0965072 -0.5051265 0.31062201 -0.9596719 0.00442925 13.0942584
36.9160629 -0.7527683 0.17649351 0.17426521 0.82253589 0.21145591 -2.7629528 -1.1505126 -0.0209159 -0.159877 -0.9562543 0.00154477
-0.204648 -0.0449692 0.24057416 -198.89747 -0.4811347 8.47799043 -0.3847163 -0.2909979 -7.8067669 -0.4183869 0.0936432 0.20322625
1.75365687 -3.0534518 -0.0094327 -0.3755981 -0.1430554 -0.8189337 0.262365 0.12179768 -0.5470267 0.05440191 0.55347232 0.24019139
45.4773753 -0.0830485 -0.628298 -0.5515379 -0.5363636 -32.985099 -39.462133 -0.9709501 11.610663 0.36158578 -2.438756 -1.1947368
-11.588038 -31.27054 -22.872317 -0.0099453 -9.8008886 -0.4522898 -2.5502392 5.2406015 -55.738414 -23.849009 -25.71702 -45.414286
-6.701162 -0.6466165 0.08086124 0.20902939 -10.451059 -0.3137389 -0.2993165 -0.1642174 1.19215995 -0.1988859 0.20556391 6.70792891
-0.1276213 -23.091729 -1.6487355 0.10385509 -0.1257348 -0.4111347 -85.518934 -0.0548189 -0.0901982 -0.059877 -0.1657348 -0.225974
NSC
hESC
Cell Surface Marker Screening with BD Lyoplate™ Human CD Marker Panel
CD marker screening in 96-well format by flow cytometry
21For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Cell Surface Marker Screening with BD Lyoplate™ Human CD Marker Panel
CD Marker Tuj1 Merge
CD marker screening in 96-well format by imaging
22For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Generation of Neural Cells from hESCs
• Performed a screen using 192 human CD markers by flow cytometryand bioimaging
– Define cell surface signatures of hESCs, NSCs, NPs (neural progenitors), neurons, and glia
– Develop a method to isolate near-pure populations of NSCs, neurons, and glia
Day 0 17 20 357
mTeSR™1 withdraw bFGF N2 N2, B27, bFGF N2, B27, dbcAMP
Rosettes NSCsEBshESCs Neurons, Glia
23For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Isolation of Neural Subtypes from hESC-derived NSCs
SSC-A
FSC
-A
NSCs were differentiated 2 weeks prior to sorting
BD FACSAria™ II sorter70 PSI, 70-µm nozzle
70% 20%
9%
46%
45%
8.5%
1%
29%70%
Sox2
Ki-6
7
3.5%94%
1%
Presort Sorted: P2 Sorted: P3
Ki-6
7N
estin
Map
2bH
oech
st
24For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Isolation of Neural Subtypes from Differentiating hESC-derived NSCs
Glia
Neurons
GFAP Hoechst
Tuj1 Hoechst
NSCs were differentiated 4 weeks prior to sortingBD FACSAria II sorter, 25 PSI, 100-µm nozzle
25For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Generation of Neural Cells from hESCs
• Performed a screen using 192 human CD markers by flow cytometryand bioimaging
– Define cell surface signatures of hESCs, NSCs, NPs (neural progenitors), neurons, and glia
– Develop a method to isolate near-pure populations of NSCs, neurons, and glia
Day 0 17 20 357
mTeSR™1 withdraw bFGF N2 N2, B27, bFGF N2, B27, dbcAMP
Rosettes NSCsEBshESCs Neurons, Glia
26For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Isolation of NSCs from Embryoid Bodies by Flow Cytometry
Sox2 Nestin HoechstNSCs Sorted from EBs
Map2b Tuj1 Ki-67 Hoechst Map2b Tuj1 GFAP HoechstDifferentiation of NSCs Neurons Sorted from Diff NSCs
Co-cultured on Astrocytes
EB
27For Research Use Only. Not for use in diagnostic or therapeutic procedures.
BD™ Lyoscreen Human Cell Surface Kit
• Approximately 260 human cell surface markers in 96-well plates
• Each kit contains five tests of each antibody
• Customer dilutes antibodies to desired concentration into daughter plates
• Other kit components– Fluorchrome-conjugated secondary
antibodies for flow cytometry and imaging
– Isotype controls– Analysis template
1
2
3
28For Research Use Only. Not for use in diagnostic or therapeutic procedures.
BD FACS™ CAP Cell Surface Phenotyping Service
• Custom service leverages BD’s expertise as the world leader in flow cytometry
• Flexible format can integrate customer’s specific markers
• Continuous addition of new important monoclonal antibodies
• Delivers flow cytometry-based combinatorial antibody profile
CD3 CD34CD10
CAP = Combinatorial Antibody Profile
29For Research Use Only. Not for use in diagnostic or therapeutic procedures.
BD FACS™ CAP Service Luminal-like cells and basal-like cells show distinct cell surface marker expression
BD FACS™ CAP technology may be used on a variety of humancell types
23-10679-00
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Pluripotent Stem Cell Research
31For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Sorting and Analysis of Pluripotent Stem Cells
• Are sorted hESCs viable?– Fong et al. Stem Cell Rev. 2009– Bajpai et al. Mol Reprod Dev. 2008– 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?
• No commercial, standardized methods for sorting or analysis by flow cytometry.
32For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Sorting Experimental Design
• Cell surface markers– SSEA-1 negative (differentiation)– SSEA-3 positive (pluripotency)– Tra-1-81 positive (pluripotency)
• Cell sorting with BD FACSAria II system– 25 PSI, 100-µm nozzle
• hESCs used:– H9 P48
• Grown on BD Matrigel hESC-qualified Matrix with mTeSR™1– H9 P41
• Cultured in KOSR on MEFs– HUES9
• Cultured in HUES on MEFs (Goldstein Lab, UCSD)
33For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Sorting Based on Markers for Pluripotency and Differentiation
TRA-1-81 Alexa Fluor® 647SSEA-3 PE
SSEA
-3 P
E
SSEA
-1 F
ITC
34For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Sorting is Possible Under Feeder and Feeder-free Culturing Conditions
H9 P41, P5 Day 3 post-sort HUES9 Day 4 post-sortH9 P48 day 2 post-sort
mTeSRTM1, BD Matrigel, Accutase KOSR, MEF, Coll IV HUES, MEF, TrypsinGoldstein Lab, UCSD
35For Research Use Only. Not for use in diagnostic or therapeutic procedures.
TRA1-81 SSEA-1 Hoechst SSEA-4 Oct-3/4 SSEA-1 Hoechst
Sorted H9 hESCs Express Pluripotency Markers
H9 P42 P6 post-sort
0.0%
5.2%1.1%
93.8%
SSEA-4 Alexa Fluor® 647
Oct
3/4
Perc
p-C
y5.5
95.3%
0.1%1.0%
3.6%
SSEA-1 PE SS
EA-4
Ale
xaFl
uor®
647
36For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Sox17 Hoechst
EndodermEctoderm
H9 P43, P7 sort
Mesoderm
EBs
Sorted H9 hESCs Retain Differentiation Potential
37For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Sorting and Analysis of Pluripotent Stem Cells
• Are sorted hESCs viable?– Fong et al. Stem Cell Rev. 2009– Bajpai et al. Mol Reprod Dev. 2008– 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?
• No commercial, standardized methods for sorting or analysis by flow cytometry.
38For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Flow Cytometry Kits for Pluripotent Stem Cell Research• Comprehensive, easy-to-use
– Compensation beads– Verified protocols and software analysis guidelines
• Analysis and sorting• Multicolor
– Pre-conjugated antibodies to markers for self-renewal and differentiation• Open, modular
– Compatible for “dropping-in” additional antibodies to cell surface markers, transcription factors, cytokines, and phosphorylated proteins
• Compatible with GFP-expressing cells
39For Research Use Only. Not for use in diagnostic or therapeutic procedures.
BD™ CompBead Plus
SSEA-4 FITC SSEA-4 PE
SSEA-4 AlexaFluor® 647 SSEA-4 BD Horizon™ V450SS
C-A
FSC-A
• Autofluorescence of beads tracks hESCs
• Facilitate scatter setup• Compensation for any mouse
or rat antibodyH9 hESCCompBeadCompBead Plus
Overlays of unstained cells and cells and beads stained with SSEA-4 conjugates
40For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Dropping in Additional Antibodies
• Drop-ins add to the number and type of markers to analyzeper sample
• Enable detailed analysis of cell fate and function of single cells:– Correlation of marker expression (up regulation and down regulation)– Simultaneous analysis of transcription factors, cell surface markers,
cellular processes (cell cycle, cell signaling, cell death)
• Example:– Mouse ES (E14) 5-day differentiation time-course (10 μM RA)
• Analysis of mNanog, Oct3/4, Sox2 (Mouse Pluripotency Analysis Kit – TF) + GATA4- Alexa Fluor® 488 drop-in
41For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Dropping in Additional Antibodies
Sox2 Alexa Fluor® 647Oct3/4 PerCP-Cy5.5 Nanog PE
GA
TA4
Ale
xaFl
uor®
488
Day 0
Day 2
Day 5
Sox2 Alexa Fluor® 647Oct3/4 Percp-Cy5.5 Nanog PE
Sox2 Alexa Fluor® 647Oct3/4 PerCP-Cy5.5 Nanog PE
14.5% 1.8%
0.4%83.3%
12.6% 3.2%
1.2%83%
14.6% 1.0%
1.0%83.5%
0.9% 0.8%
12.8%85.5%
0.2% 1.1%
91.6%7.0%
0.5% 1.6%
56.3%41.6%
0.0% 2.2%
97.4%0.3%
0.7% 0.8%
11.5%87%
0.4% 0.7%
88.2%10.6%
Mouse ES (E14) 5-day differentiation time course (10 μM RA)
42For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Dropping in Additional Antibodies
hNanog PE Oct3/4 PerCP-Cy5.5 GATA4 Alexa Fluor®488
Day 0
Day 2
Day 5 4.6%
Sox2 Alexa Fluor®647
4.5% 5.2% 15.0%
14.3% 55.3% 19% 1.4%
93.2%
99.2%
92.6% 0.7%
Mouse ES (E14) 5-day differentiation time course (10 μM RA)
44For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Mouse HSC Isolation Kit
• Contents:– APC Lineage Cocktail– PE c-Kit – FITC CD34– PE-Cy™7 Sca-1– Matched Isotype
Controls– CD16/CD32 (Fc III/II
Rec)– 7-AAD vital dye– BD CompBeads– Verified protocols
• Utility:– Sorting CD34+/- KLS from mouse
bone marrow– 100 mice ~ 10 sorts– Compatible with magnetic
enrichment– Compatible with side population
KLS (SPKLS)
LT-HSC: CD34-, SCA-1+, C-kit+
ST-HSC: CD34+, SCA-1+, C-kit+
MPP: CD34+, SCA-1+, C-kit+
45For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Mouse HSC Isolation Kit
C57 BM magnetic bead-enriched
Lin
cock
tail
APC
C-kit PE Sca-1 PE-Cy7
C-k
it PE
CD34lo/- CD34hi
SSC
-A
FSC-A
CD34 FITC
Cou
nt
46For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Mouse HSC Isolation Kit
Cobblestone cells
Mitomycin-C treated M2-10B4 stromal cells
Unfractionated Bone Marrow KLS CD34hi KLS CD34lo/-
Cobblestone cells
47For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Mouse HSC Isolation Kit
Cobblestone cells
KSL CD34hi
Colony forming assay
CFU
48For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Mouse HSC Isolation Kit
Lin-
APC
C-Kit PE
Viable Cells
Sca-1 PE-Cy7C
-Kit
PE
Lin-
CD34 FITC
Sca-
1 PE
-Cy7
Lin-
Hoechst Red
Hoe
chst
Blu
eAll Cells
CFU-G
CFU-M
CFU-GM
Side population, Kit+, Sca-1+, Lin-, CD34- (SPKLS)
49For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Kits for Stem Cell Research
• All kits contain BD™ CompBead Plus, matched isotype controls, and verified protocols• IC Analysis kits contain fix and perm buffers
Human Pluripotent Stem Cell Sorting and Analysis Kit Hu
SSEA-3 PETra-1-81 AlexaFluor® 647SSEA-1 FITC
- -
Human and Mouse Pluripotency Analysis Kit
HuMs
Oct3/4 PerCP-Cy5.5SSEA-4 AlexaFluor® 647SSEA-1 PE
-
Human Pluripotency Analysis Kit-TF Hu
Oct3/4 PerCP-Cy5.5hNanog PESox2 AlexaFluor® 647
- -
Mouse Pluripotency Analysis Kit-TF Ms
Oct3/4 PerCP-Cy5.5mNanog PESox2 AlexaFluor® 647
- -
Mouse HSC Isolation Kit Ms
c-Kit PESca-1 PE-CyTM7CD34 FITCLineage Cocktail APC
- -
Sorti
ng
Drop
-ins
GFPKit Sp Antibodies IC A
naly
sis
CS A
naly
sis
50For Research Use Only. Not for use in diagnostic or therapeutic procedures.
AcknowledgmentsStem Cell ResearchBob BalderasJurg RohrerRosanto ParambanJason VidalJulia EmberJeanne EliaNil Emre
UCSDLarry Goldstein
TASSue Reynolds
R&D Cytometry LabDennis SasakiAndrea Nguyen
51For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Questions?
• If you have further questions:• Contact your US Reagent Sales Rep• or e-mail:
ResearchApplications@bd.com• Please visit our BD Stem Cell Source page:
• bdbiosciences.com/stemcellsource
Alexa Fluor® is a registered trademark of Molecular Probes, Inc.
mTeSR™ is trademark of WiCell Research Institute.
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