Isolation and Analysis of Pluripotent, Neural, and

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


Stem Cell Background

Schematic adapted from Wobus AM. and Boheler, KR., Physiol. Rev.2005; 85:635-678.

Embryonic

Adult

iPS

Oct3/4Sox2Klf4

c-MycNanogLIN28


Stem Cell Background

Schematic adapted from http://stemcells.nih.gov/index.asp


Challenges in Stem Cell Research

• Identify and isolate cells of interest from a heterogeneous pool

• Analyze cells for quality and purity

• Analyze cell function


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


BD Biosciences

First to commercialize tools for isolating and analyzing hematopoietic stem cells, now bringing this expertise to the

broader stem cell field


Portfolio of High Quality Antibodies

For 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


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


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


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%


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


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 .


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


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


Day 0 17 20 357



• 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



• 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




-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


Cell Surface Marker Screening with BD Lyoplate™ Human CD Marker Panel

CD Marker Tuj1 Merge

CD marker screening in 96-well format by imaging






Day 0 17 20 357




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


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






Day 0 17 20 357




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


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


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


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


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.


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)


Sorting Based on Markers for Pluripotency and Differentiation

TRA-1-81 Alexa Fluor® 647SSEA-3 PE

SSEA

-3 P

E

SSEA

-1 F

ITC


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


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


Sox17 Hoechst

EndodermEctoderm

H9 P43, P7 sort

Mesoderm

EBs

Sorted H9 hESCs Retain Differentiation Potential


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.


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


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


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



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)



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)



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+



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



Cobblestone cells

Mitomycin-C treated M2-10B4 stromal cells

Unfractionated Bone Marrow KLS CD34hi KLS CD34lo/-

Cobblestone cells



Cobblestone cells

KSL CD34hi

Colony forming assay

CFU



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)


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


AcknowledgmentsStem Cell ResearchBob BalderasJurg RohrerRosanto ParambanJason VidalJulia EmberJeanne EliaNil Emre

UCSDLarry Goldstein

TASSue Reynolds

R&D Cytometry LabDennis SasakiAndrea Nguyen


Questions?

• If you have further questions:• Contact your US Reagent Sales Rep• or e-mail:

[email protected]• 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.

Class I (1) laser product.

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.

BD, BD Logo and all other trademarks are property of Becton, Dickinson and Company. © 2009 BD

Documents

Isolation and Analysis of Pluripotent, Neural, and