Practical Analysis of Stem Cells by Flow Cytometry · PDF filePractical Analysis of Stem Cells...

Practical Analysis of Stem Cells by Flow Cytometry

Established Methods and Emerging Technologies

Bill Telford

Experimental Transplantation and Immunology BranchFlow Cytometry Laboratory

National Cancer InstituteNational Institutes of Health

Flow Cytometry in Stem Cell Biology

Flow cytometry and cell sorting are absolutely indispensable techniquesfor both the identification and isolation of embryonic and adult stem cells

in both bone marrow and other tissues.

• Fluorescent immunophenotyping for stem cell markers

• Aldehyde dehydrogenase activity detection usingfluorogenic substrates

• G0/G1 discrimination using Hoechst 33342/pyronin Y

• Hoechst side population analysis

Quiescent SCCD34-c-Kit+CD34-c-Kit-

Stem cell subpopulations and their identificationHematopoetic stem cells (HSCs)

Sel

f-ren

ewal

Turnover time: 30-60 days

Activated SC CD34+ c-Kit -

Sel

f-ren

ewal

More frequent turnover

Circulating CD34+ Stem Cell

CD34+ c-Kit+Progenitor Cell

Immature differentiating cells

From DS Donnely DS and Krause DS, Leukemia and Lymphoma, 40, 221-234 (2001)

Isolation of Stem Cells by Flow Cytometry

As with most things in biology, there is no single characteristicthat adequately identifies stem cells by itself.

We need to look at multiple phenotypic characteristics includingcell surface markers and biochemical / physiological characteristics.

And, if possible, we need to look at these multiple characteristicsin a single multiparametric assay.

And of course, flow cytometry is an ideal way to do this!

R1

R2

R3

R4

R5

FSC

SS

C

Bone Marrow Cell Differentiation by Scatter Measurement

RBCs(Ter119)

granulocytes/monocytes(Gr-1/Mac-1)

stem cells / progenitors

mature lymphocytes

(CD3/B220)

Phenotypic markers for mammalian hematopoetic stem cells

• Human stem cells

• Lineage depletion • CD34• HLA-DR• AC133 (CD133)• CD7

• Rodent stem cells

• Lineage depletion • CD34 • Sca-1• c-kit (CD117)• MAC-1 (+/-)

Phenotypic markers for mammalian stem cells

CD34

• A single chain transmembrane glycoprotein expressed on HSCs, vascular endothelium, embryonic fibroblasts and some cells in fetal and adult nervous tissue

• Promotes adhesive interactions between stem cells and stromalelements in the bone marrow

• Probably regulates association between stems cells and the “niche”microenvironment via indirect regulation of other adhesion factors

• The dominant marker for clinical identification and separation ofstem cells


Sca-1

• Stem cell antigen, a GPI-linked surface protein expressed on 30% of mouse bone marrow including pluripotent HSC and committed lymphoid and myeloid progenitors, also was found in osteoblastskidney apithelia(Ly-6A/E).

• HSCs from Sca-1 KO mice have impaired repopulation potential.

• Lower engraftment of secondary transplants in Sca-1 deficient animals also suggests a defect in HSC self-renewal.

• In addition to HSC deficiencies in Sca-1-deficient mice, specific cell lineages and progenitor subpopulations are also affected. (Ito CY, Li CY, Bernstein A, Dick JE, Stanford WL. Blood 101, 517-23, 2002).


c-kit (steel factor)

• Transmembrane glycoprotein, PTK-R in HSC, was found in melanocytes and primordial germ cells

• Mice lacking the receptor tyrosine kinase c-Kit (c-KitW/W) have hematopoietic defects causing perinatal death

• A viable c-KitW/W mouse shows an age-dependent, progressive decline of pro-T and pro-B cells accompanied by loss of common lymphoid progenitors in the bone marrow in adult mice lacking c-kit.

• Adult c-KitW/W hematopoietic stem cells can engraft in host bone marrow but fail to radioprotect, form spleen colonies, or establish sustained lymphopoiesis (Waskow C, Paul S, Haller C, GassmannM, Rodewald H., Immunity 3, 277-88, 2003)

Benchtop analyzers

• BD FACScan – one laser, three colors

• BD FACScalibur – two lasers, four colors

• Beckman Coulter EPICS XL – one laserfour colors

PE-Cy5-Sca-1

AP

C-C

-kit

Sca-1only

c-kit only

Sca-1 and c-kit expression in mouse bone marrowThree color analysis

APC-c-kit

FITC

-line

age

Lineage depletion with biotinylatedantibodies against B220, CD3, Ly6C+G,GR-1, Ter119, CD5 and NK1.1

FITC “dump” channel

APC-anti-c-kitPE-Cy5-anti-Sca-1FITC-Lineage

side scatter

PE-C

y7-L

inea

ge

FITC-c-kit

PE

-Cy5

.5-S

ca-1

PE-CD34

side

sca

tter

side

sca

tter

forward scatter forward scatter

PE-Cy7 labeling for lineage panel(CD3, B220, Ly6C + G, CD11b, Ter119, NK1.1, CD5)

FITC-c-kitPE-CD34PE-Cy5-Sca-1PE-Cy7-Lineage

Sca-1, c-kit and CD34 expression in mouse bone marrowFour color analysis

Discrimination of the Hoechst SP on the flow cytometer

Samples from Drs. Atsushi Terasumi and John Vogel, DB, NCI, NIH

Hoechst 33342 Red(675/20 nm)

Hoe

chst

333

42 B

lue

(440

/10)

Murine bone marrowunseparated(150,000 events)

0.04%

• When loaded with the fluorescent DNA dye Hoechst 33342, murinebone marrow stem cells preferentially pump out the dye via the ABCG2 membrane transporter.

• These “side population” (SP) cells can be distinguished onthe basis of their reduced Hoechst dye fluorescence(Goodell et al., JEM 183, 1797 (1996)).

• Both DNA-bound (Hoechst blue fluorescence) and unbound(Hoechst red fluorescence) used to distinguish side population cells.

• SP cells are enriched for Sca-1 expression and show nolineage marker expression. SP cells can reconstitute irradiated mice with both lymphoid and myeloid lineages.

Mouse L929Hoechst 33342 2 :g/ml

BD FACSVantage SE

Discrimination of the Hoechst SP on the flow cytometer

krypton-ion UV (tertiary)laser path

split mirror

argon-ion 488 nm(primary)laser path

675/20

440/

10HOblue

HOred

Mouse L929Hoechst 33342 2 :g/ml

Verapamil blocks the accumulation of Hoechst SP cells

Samples from Drs. Atsushi Terasumi and John Vogel, DB, NCI, NIH

Hoe

chst

333

42 B

lue

(440

nm

)

Hoechst 33342 Red (675 nm)

verapamil 10 :M30 m preincubationno priorenrichment

0.03%0.11%

no verapamilno priorenrichment

Negative selection of lineage-positive cells by magnetic bead depletion

Label bone marrow with haptenor PE-conjugated antibodiesagainst lineage markers

CD3B220Ly6C + GGr-1Ter119NK1.1CD5 PE

Discrimination of Hoechst SP on the flow cytometer

Murine bone marrowLineage+ antibodyretained fraction

Murine bone marrowLineage+ antibody columnflow through cell fraction

Samples from Drs. Paul Love and Ella Frolova, NICHD, NIH

Hoe

chst

333

42 B

lue

(440

nm

)


Lineage panel includes CD3, B220,CD11b, Ly6G, Ter-119


560

SP

575/

26

split mirror

Sca-1


Lin530/30

675/20

440/

10HOblue

HOred

Discrimination of Hoechst SP on the flow cytometer

Lineage “dump” channel(CD3, B220, Ly6C + G, CD11b, Ter119, NK1.1, CD5)

It is very possible (and highly desirable) to combine Hoechst SPanalysis and cell surface immunophenotyping.

Lineage marker expression in Hoechst SP cells

Lin expression

Lin+ fraction

Cells with increasing SP phenotypeshow decreased Lin expression

Hoe

chst

333

42 B

lue

(440

nm

)



all cells

lineagenegative

Lineage exclusion and Hoechst SPanalysis in mouse bone marrow

side scatter

PE

-Cy7

-Lin

eage


Lineage exclusion enriches for stem cells, but is insufficient alone for good isolation.

4.2%

25.3%

63.9%

6.5%

Sca-1 expression in SP subpopulation cells

Sca-1 expression

Cells with increasing SP phenotypeare enriched for Sca-1 expressing cells

Hoe

chst

333

42 B

lue

(440

nm

)



Lineage exclusion, Sca-1 and c-kit immunophenotyping in mouse bonemarrow Hoechst SP analysis

PE-Cy5-Sca-1

AP

C-C

-kit

Sca-1only

c-kit only

all cells

lineagenegative

lineagenegative

Sca-1+c-kit+


Sorting Hoechst SP cellsH

oech

st 3

3342

Blu

e (4

40 n

m)


Murine bone marrowLineage+ antibody columnflow through cell fractionsorted

Murine bone marrowLineage+ antibody columnflow through cell fractionunsorted

Lineage panel includes CD3, B220,CD11b, Ly6G, Ter-119

Tissue and species distribution of the Hoechst SP phenotype

Mouse skeletal-muscle cells

Mouse ES

Monkey BM

Mouse BM

A wide variety of stem cell types(hematopoetic and mesenchymal,

embryonic and adult) from bothnon-primate and primate tissues

exhibit some degree of ABCdependent SP activity.

HeNe

argon-ion

krypton-ion

Laser sources on the FACSVantage SE

Equipment required for analysis of Hoechst side population…

• Large scale cell sorter (i.e. FACSVantage DiVa,Beckman-Coulter Altra or Cytomation MoFlo

• High power argon-ion or krypton-ion laser (US$ 30,000)

• Total equipment cost = US$ 400,000

The equipment for analyzing Hoechst SP is prohibitivelyexpensive for most institutions.

Blue-green 488 nm laserdetectorconfiguration

• BD LSR II, Beckman-Coulter FC500,Cytomation CyAn

• Polychromatic cell sorting using a variety of laser sources

• Up to 10 colors simultaneously using up to four lasers

Polychromatic flow cytometers

Diode-pumped solid state488 nm (blue-green)

HeNe 633 nm (red)

VLD 408 nm (violet)

Near-UV laser diode 374 nm

BD Biosciences LSR IILaser sources

Novel laser sources for flow cytometry

• Laser diodes

• Near-infrared and red• Blue• Violet• Near-ultraviolet

• Diode-pumped solid state

• Diode pumping of a solid state laser medium (such as yttrium aluminum garnet (YAG), or neodymium-YAG

• Frequency doubling or tripling of can generate interestinglaser lines for flow cytometry

• DPSS green 532 nm• DPSS blue-green 460 – 490 nm

• Mode-locked Nd-YAG frequency-tripled 355 nm UV laser (quasi-CW)

Lightwave mode-locked Nd-YAG 355 nm laser

NdYag 355 nm 22 mWBD LSR II

Hoechst red fluorescence (650 LP)

Hoe

chst

blu

e flu

ores

cenc

e (4

50/5

0)

Cost is still high (about US$ 30,000)

Laser diodes

42062083012402500

InGaN/Al2O3

InGaP/GaAs

blue/violet/near-UV diodes

red/near-IR diodes

“orange gap”

AlGaAs/GaAs

InGaAs/GaAs

InGaAsP/InP

InGaAsSb/Ga/Sb

visible rangeLaser diodes currently cover the infraredto the near-ultraviolet, with a few critical gaps.

Wavelength (nm)

Single Transverse Mode Violet Laser Diode

• can emit from 397 to 408 nm• may provide a useful near UV laser

source for both flow and laserscanning cytometry

Violet lasers on benchtop flow cytometers

BD FACSVantage SEBD LSR II

Violet laser diode 405 nm 18 mW• Solid-state violet laser diodes (VLDs) are nowstandard equipment on a wide variety of flowcytometers

• BD LSR II and FACSAria• DakoCytomation CyAn• Compucyte LSC2 and iCys

• These small, reliable laser sources havebroadened the use of violet-excitedfluorochromes such as DAPI, Cascade Blueand Pacific Blue.

Hoechst SP Analysis using a Violet Laser DiodePower Technology 408 nm 15 mW

Hoe

chst

333

42 B

lue

(440

nm

)

Hoechst 33342 Red (675 nm) Murine bone marrowNo purification

Kr MLUV 100 mWFACSVantage DiVa

Violet laser diode 408 nmBD LSR II

Violet laser diodes allow detection of the SP population,but with very low resolution.

350 400 450 500 600 650300 550Wavelength (nm)

Rel

ativ

e flu

ores

cenc

e

450/50

Hoechst 33342

Spectral Properties of Hoechst 33342

N

NH+

NCH3+NH

N

NH+

O

Cl

H

Hoechst 33342

The spectra of Hoechst33342 suggests that it

would be poorly excited by violet laser light.

SP = 1.58% SP = 0.58%

SP = 0.28%SP = 5.72%

0.31%

Hoechst redH

oech

st b

lue

FITC-Sca-1A

PC

-c-k

it

2.14%

no inhibitor fumitremorgin CSca-1 (+) c-kit (+)

gated cells

Hoe

chst

blu

e

SP = 76.5%

SP = 65.8%

Hoechst red

Violet diode 408 nm 25 mW

mouse BMno separation

mouse BMlineage depletion(SC-enriched)

Hoechst SP Analysis using a Violet Laser Diode

Violet laser diodes allow detection of the SP population,but with very low resolution.

Is good Hoechst SP resolution necessary?

Yes, it is!

Hoechst 33342 labeledbone marrow oftenproduces more thanone hypodlploid population.

We aren’t sure what theselow-blue populations are,but they are NOT stem cells.

This is especially true ofprimate bone marrow andnon-hematopoetictissues.

Suboptimal excitation ofthe Hoechst-labeled cellsoften fails to distinguishtrue SP cells from othernon-stem SP populations.

Lineage (+)Sca-1 (-)c-kit (-)CD34 (-)

Lineage (+)c-kit (+)

Hoe

chst

333

42 B

lue

(440

nm

)


Hoe

chst

333

42 B

lue

(440

nm

)

Hoechst 33342 Red (675 nm) Murine bone marrowNo purification

Kr MLUV 100 mWFACSVantage DiVa

Violet laser diode 401 nmBD LSR II

Even short-wavelength violet diodes do not significantly improve SP resolution

Hoechst SP Analysis using a Violet Laser DiodePower Technology 401 nm 15 mW

Near-UV laser diodes (NUVLDs)on benchtop flow cytometers

NUVLD sources can be mounted on some benchtopinstruments like the LSR II and used for previously

troublesome UV- dependent applications

Near-UV laser diodes (NUVLDs) for Hoechst 33342 side population analysis

NUVLD 374 nm 8 mWBD LSR II

Krypton-ion MLUV 100 mWBD FACSVantage DiVa

Hoechst red fluorescence (650 LP)

Hoe

chst

blu

e flu

ores

cenc

e (4

50/5

0)

NUVLDs on cuvette instruments give better SP resolution than gas lasers on stream-in-air instruments.

all three

Sca-1c-kit

CD34c-kit

c-kit onlySca-1 only CD34 only

CD34Sca-1

lineage-negative

all

Six color bone marrow stem cell analysisRequirements for simultaneous Hoechst SP analysis

forward scatter

side

sca

tter

PE

Cy5

.5-S

ca-1

PE-C

D34

FITC-c-kit

PE

-Cy7

Lin

eage

FITC-c-kit PE-Cy5.5-Sca-1

forward scatter

side

sca

tter


FITC-c-kitPE-CD34PE-Cy5.5-Sca-1PE-Cy7-Lineage

Hoechst red

Hoe

chst

blu

e

Cost of producing a UV laser line for Hoechst SP analysis…

Mode-locked Nd-YAG 355 nm laser

= US$ 30,000

Near-UV laser diode 375 nm laser= US$ 7,000

Krypton-ion multiline UV 361 - 365 nm laser

= US$ 30,000

Cost of instruments capable of doing Hoechst SP analysis…

BD LSR II withNUVLD or Nd-YAG laser

or

Cytomation CyAnwith Enterprise II laser

= US$ 250,000

FACSVantage withAr or Kr ion UV laser

= US$ 400,000

Still pricey!

NPE Analyzer

Next-generation flow cytometry technologystill in the advanced development stage.

Utilizes a unique Coulter sizing transducerincorporated into a flow cytometry flowcell to allow both highly accurate electroniccell sizing and fluorescence analysis.

stepper motors

for X-Y lampalignment

mercury arc lamp

Coulter transducer/flow cell

cell inlet

photodiodedetector

stepper motors

for X-Y lampalignment

NPE AnalyzerTransducer flow cell assembly

chargereservoir

mercury arclamp

chargeplate

photodiode

flow cell

mirror

injectionnozzle

chargeplate

Can we use the NPE Analyzer to measure Hoechst SP?Hg arc lamp derived UV excitation

366

405

546

313

578

435

Wavelength

Hg arc lamp excitation

405 nm violet

435 nm blue

546 nm green

We can “extract” most of the high-output linesfrom the Hg arc lamp for excitation of as varietyof fluorochromes.

The Hg lamp emits a strong UV line at 365 nm.

mirror

450/

20650 LP

linearphotodiode

mirror

450/

20

High-sensitivityPMT

Hig

h-se

nsiti

vity

PM

T

• Photodiodes have severalunique advantages as opticaldetectors, are notas sensitive as traditionalphotomultiplier tubes

• NPE can be equipped with high-sensitivity photomultipliertubes sensitive to as few asa few hundred photons per total detector area

• NPE set up a custom opticalbench for us with two detectorpositions, both with PMTs.

Can we use the NPE Analyzer to measure Hoechst SP?Optical layout

(-)

(-)(-)

(-)

(+)

(+)

(-)(-)

(-)

(-)(-)

(-)

(+)

(+)

(+)(+)

(+)

Coulter sizing provides a far more accurate measurement of particle size thantraditional forward scatter measurement by flow cytometry

The NPE Analyzer works on the same principle as the Coulter Counter, via the electrical resistance generated across an orifice by the occlusion of a particle.

Can we use the NPE Analyzer to measure Hoechst SP?Electronic cell volume

Can we use the NPE Analyzer to measure Hoechst SP?Electronic volume measurement

2.0 µm3.2 µm

4.5 µm5.6 µm

5.6 µm

6.0 µm 6.4 µm

The NPE Analyzer can Measure electronic cell volume with a highdegree of precision.

Measurement of stemcell electronic volumemight provide a valuablenew phenotypic markerfor stem cell-ness (likescatter does).

forward scatter

side

sca

tter

electronic cell volumeH

O33

342

blue

mirror

450/

20HOred

HOred

675/20

610 SP


mirror


675/2044

0/10HO

blue

HOred

610 SP

Hoe

chst

333

42 B

lue

(440

/10)

SP population= 0.16%


Hoechst 33342 Red (675/20 nm)


NPE Analyzer

FACSVantage SE

NPE Analyzer

FACSVantage SE

With assistance from Drs. Paul Love and Ella Frolova, NICHD

Can we use the NPE Analyzer to measure Hoechst SP?

Can we use the NPE Analyzer to measure Hoechst SP?

Yes, we can.

But…the same problem as with the violet diode, namely suboptimalexcitation. Lower precision … and poorer “junk” separation … than morepowerful UV sources.

The Hg arc lamp is a thoeretical point source – the actual power levelof laser light reaching the flow cell is probably less than 1 mW, makingit marginal for reproducible SP analysis (we have the same problem withlow-power NUVLDs on cuvette flow cytometers)

This is plenty of UV light for DAPI cell cycle…

…but less than optimal for applications requiring stronger UV excitation.

mercury arclamp

injectionnozzle

flow cell

NUVLD

dichroicmirror

Mounting a NUVLD laser on the NPE Analyzer

• A NUVLD can be mounted inthe NPE Analyzer and thebeam steered to the flow cell

mirror

450/

10HOblue

HOred

675/20

610 SP

Mounting a NUVLD laser on the NPE Analyzer

dichroic mirrornear-UV diode laser

Mounting a NUVLD laser on the NPE AnalyzerMounting a NUVLD laser on the NPE Analyzer

dichroic mirror

near-UV diode laser

Hoechst SP on the NPE Analyzer

NPE AnalyzerHg arc lamp 365 nm80 WHO blue = 450/20HO red = 675/20

NPE AnalyzerNUVLD 374 nm7 mWHO blue = 450/20HO red = 675/20

NUVLDs not only give better Hoechst SP resolution, they give greater contrast between the SP population and other non-stem cell hypodiploid populations

Hoechst red

Hoe

chst

blu

e

Today’s Stem Cell Wet Workshop…

We will label whole mouse bone marrow cells and A549 cells (an ABCG2 overexpressing lung carcinoma cell line) with Hoechst 33342 for Hoechst SP analysis.

We will then analyze these cells on the NPE Analyzer, first with themercury arc lamp UV source, then with the NUVLD.

This is a very new and continually evolving technique … any resultswe obtain today may have real experimental value.

Acknowledgements

Atsushi TeranumiJohn VogelMark Udey

Dermatology Branch, NCI

Veena Kapoor

NCI ETIB Flow Lab

Michael BustinElla Frolova

Molecular Biology Branch, NCI

NPE Systems

Richard ThomasErnie ThomasRaquel CabanaMichael Brochu, Sr. Michael Brochu, Jr.

Larry DuckettJoe Trotter

BD Biosciences

James Jackson

Power Technology

Awtar Krishan

The University of MiamiMedical School

Susan BatesRobert Robey

Developmental TherapeuticsBranch, NCI

Visit our WWW site at…

http://home.ncifcrf.gov/ccr/flowcore/index.htm

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