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Methods for genetic modification of megakaryocytesand platelets

Online Publication Date: 01 September 2007To cite this Article: Pendaries, Caroline, Watson, Stephen P. and Spalton, JenniferC. (2007) 'Methods for genetic modification of megakaryocytes and platelets',Platelets, 18:6, 393 - 408To link to this article: DOI: 10.1080/09537100701288012URL: http://dx.doi.org/10.1080/09537100701288012

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Platelets, September 2007; 18(6): 393–408

REVIEW

Methods for genetic modification of megakaryocytes and platelets

CAROLINE PENDARIES, STEPHEN P. WATSON, & JENNIFER C. SPALTON

Centre for Cardiovascular Sciences, Institute for Biomedical Research, Wolfson Drive,

The Medical School, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK

(Received 14 February 2007; accepted 19 February 2007)

AbstractDuring recent decades there have been major advances in the fields of thrombosis and haemostasis, in part throughdevelopment of powerful molecular and genetic technologies. Nevertheless, genetic modification of megakaryocytes andgeneration of mutant platelets in vitro remains a highly specialized area of research. Developments are hampered by the lowfrequency of megakaryocytes and their progenitors, a poor efficiency of transfection and a lack of understanding with regardto the mechanism by which megakaryocytes release platelets. Current methods used in the generation of geneticallymodified megakaryocytes and platelets include mutant mouse models, cell line studies and use of viruses to transformprimary megakaryocytes or haematopoietic precursor cells. This review summarizes the advantages, limitations andtechnical challenges of such methods, with a particular focus on recent successes and advances in this rapidly progressingfield including the potential for use in gene therapy for treatment of patients with platelet disorders.

Keywords: Megakaryocytes, genetic modification, platelets

Abbreviations: ALV-A, Subgroup A avian leukosis virus; BFU-MK, Burst-forming unit-megakaryocyte; BMEC,Bone marrow endothelial cell; CFU-MK, Colony-forming unit-megakaryocyte; GM-CSF, Granulocyte-macrophagecolony-stimulating factor; HPP-CFC, High proliferative potential colony-forming cell; HSC, Haematopoietic stem cells;RISC, RNA-induced silencing complex; VSV-G, Vesicular stomatitis virus G

Introduction

Platelets provide a first line of defence following

injury, forming thrombi that patch-up

damaged tissue, thereby playing an indispensable

role in haemostasis. Vessel wall damage exposes

subendothelial proteins that trigger platelet adhesion,

platelet aggregation, granule secretion and exposure

of a procoagulant surface, leading to formation of a

vascular plug. Platelet activation is reinforced by the

secondary agonists, ADP and thromboxane

A2, and by generation of thrombin through the

coagulation cascade. The critical role of platelets in

haemostasis is illustrated by the profound

bleeding exhibited by patients deficient in the major

platelet glycoprotein receptors, GPIb-IX-V and

GPIIbIIIa.

Platelets represent an important clinical target for

prevention of pathological thrombosis in diseased

blood vessels. Clinical trials have demonstrated the

effectiveness of anti-platelet agents in the secondary

prevention of acute coronary syndromes and ischae-

mic stroke, hence confirming the pivotal role of

platelets in arterial thromboembolism. Nevertheless,

antiplatelet agents carry the risk of excessive and

life-threatening bleeding. While this may be an

inevitable problem of antiplatelet therapy, the further

understanding of platelet biology may identify new

ways to minimize this risk.

The anucleate nature of the platelet limits their

study by classical molecular biology approaches. As

a consequence, platelets have been primarily char-

acterized using biochemical and pharmacological

techniques, although such studies are limited by the

Correspondence: Jennifer C. Spalton, Centre for Cardiovascular Sciences, Institute for Biomedical Research, Wolfson Drive, The Medical School,

University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK. E-mail: j.c.spalton@bham.ac.uk

ISSN 0953–7104 print/ISSN 1369–1635 online � 2007 Informa UK Ltd.

DOI: 10.1080/09537100701288012

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availability and specificity of inhibitors. In this

regard, the development of mouse models of

haemostasis and thrombosis and application of

gene targeting strategies to the mouse genome has

enabled considerable advances to be made in our

understanding of specific proteins in platelet func-

tion. Nevertheless, there is an urgent need for

development of techniques that will provide access

to an increased number and variety of mutant

platelets for both in vivo and in vitro studies. This

has led to a focus on the manipulation of haemato-

poietic stem cells and megakaryocytes as a source of

genetically-modified platelets. In this review we will

discuss the various technologies for genetic-targeting

of megakaryocytes and generation of mutant plate-

lets. This will include a discussion of the advantages,

technical challenges and limitations associated with

such approaches, and will define outstanding

questions and future directions in this rapidly

progressing field.

Megakaryocytopoiesis

To perform genetic modifications on megakaryocytes

requires an understanding of the processes that

influence megakaryocyte maturation, proplatelet

formation and platelet release. These steps are

summarized in Figure 1. Megakaryopoiesis is the

process that gives rise to mature megakaryocytes,

generated by the passage of pluripotent

haematopoietic stem cells (HSCs) through stages of

proliferation, differentiation and maturation.

Nuclear maturation in megakaryocytes, a process

known as endoreplication, proceeds in concert with

cytoplasmic maturation and expression of platelet

surface markers. Clusters of differentiation markers

are used to analyse megakaryocyte maturation. The

expression of CD34 (stem cell marker), CD38

(ADP-ribosyl cyclase) and CD41 (GPIIb or �IIb)are typically used to analyse early megakaryopoiesis,

while CD42, CD42a (GPIb and GPIX respectively)

and GPVI are markers of the later stages of

differentiation (Figure 1).

Terminally differentiated megakaryocytes are large

cells (50–100 mm diameter) with high polyploidy and

a unique set of organelles, including �-granules anddense bodies [1–5]. Megakaryocytes also have

an extensive system of internal membranes that

contribute to the demarcation membrane system

[6], which are believed to be the source of proplatelet

membranes [7]. The process of platelet generation

and shedding from megakaryocytes is not completely

understood, due in part to the difficulty of studying

this dynamic process in vivo. However, it is now

recognized that as megakaryocytes mature and

differentiate, they migrate to sinusoidal endothelial

cells in the bone marrow where they form trans-

endothelial projections that fragment into 1000–5000

platelets for release into the intravascular space

[1–5]. During the release phase, the megakaryocyte

cytoplasm converts into long branched protrusions

called proplatelets and disc-shaped platelets are

assembled de novo within these extensions [8].

Microtubule and actin cytoskeleton participate

actively in this process [9].

A key step in thrombopoiesis is migration of

maturing megakaryocytes from the proliferative

osteoblastic niche within the bone marrow

microenvironment, where HSCs reside, to the

capillary rich vascular niche, where proplatelets are

formed [10]. This process is regulated by a variety of

chemokines and cytokines (Figure 1), as well as by

adhesive interactions with interstitial cells and

extracellular matrix proteins [11]. The chemokine

thrompoboietin (TPO) plays a major role in the

humoral regulation of thrombopoiesis, both at

Figure 1. Maturation steps leading, from haematopoietic stem cell, to mature MK (megakaryopoiesis) and platelet (thrombopoiesis)

generation. Cell types are abbreviated as follows: HPP-CFC, high proliferative potential colony-forming cell; HPP-CFU-MK, high

proliferative potential colony-forming unit-megakaryocyte; BFU-MK, burst-forming unit-megakaryocyte; CFU-MK, colony-forming

unit-megakaryocyte. Growth factors and cytokines involved in MK growth and maturation are mentioned between each cell type.

Associated surface markers are shown below.

394 C. Pendaries et al.

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the level of HSCs and terminal megakaryocyte

differentiation. TPO is produced constitutively in

the liver and by bone marrow stromal cells and its

circulating levels are regulated by binding to the

receptor, c-Mpl, on platelets [2].

Megakaryocytes are estimated to constitute

approximately 0.4% of the total nucleated cells

within bone marrow [12]. Thus, due to their relative

scarcity, it is easier to study their differentiation

in vitro in the presence of TPO, with the advantage

that this allows selection and synchronisation of

distinct phases of megakaryocyte formation.

Megakaryocytes can be cultured from precursors

obtained from bone marrow, neonatal cord blood or

adult peripheral blood. However, purification of

megakaryocytes and their precursors is not straight-

forward because of their low number and by binding

of activated platelets to CD34þ cells, thus giving rise

to an artefactual phenotype [13]. For these

reasons, the majority of studies on megakaryocyte

differentiation and platelet production are performed

on megakaryocyte cell lines or on megakaryocytes

derived from in vitro differentiation of bone marrow

HSC. The techniques of primary megakaryocyte

isolation, culture of megakaryocyte precursors and

proplatelet formation are thoroughly explained in

two recent publications [14, 15].

The above discussion raises the question of

whether genetic modification of megakaryocytes

should be performed on stem cells, immature

progenitors or at later stages of differentiation.

For example, if the protein of interest plays a role

in cell cycle, cytoskeleton regulation or membrane

trafficking, and the aim is to study the role of this

targeted protein in platelet function, it will be

necessary to work at later stages using inducible

systems or transduction/transfection of mature

megakaryocytes in order to avoid an effect on

proliferation, differentiation and maturation.

Current approaches in genetic modification

of megakaryocyte progenitors

Several methods of genetic modification of

haematopoietic cells and their progenitors have

been developed. The following section is an overview

of available methods, with specific examples of

genetic modification of haematopoietic precursors,

primary megakaryocytes and megakaryocytic cell

lines as summarized in Figure 2 and Table I.

Mutant mice as a source of genetically-modified

megakaryocytes/platelets and models for

human disorders

To date the most successful means of obtaining

genetically-modified platelets is through the use of

mutant mice. These provide a rich source of

genetically-modified platelets and have enabled

major advances in understanding the mechanisms

of platelet regulation and functional roles.

The generation of mutant mice, however, is a

costly and time-consuming process and problems

can arise if the elimination of the target gene

produces lethality during embryonic development

or shortly after birth. The generation of conditional

knockouts or knockins can overcome this problem,

but this involves further expense and time.

A major advantage of using mutant mice is that

mutant megakaryocytes and platelets can be studied

both in vitro and in the whole animal. There are

many examples of mutant mouse lines that have

provided a useful resource for platelet research,

including those deficient in GPIb-IX-V and

GPIIb-IIIa receptors; the collagen receptors, GPVI

and GPIaIIa; the protease activated receptor

(PAR-4); and the purinergic receptors, P2Y1 and

P2Y12 [16]. Different clinical syndromes, combined

with studies in mouse and in vitro models, have

revealed the importance of specific genes for normal

haematopoiesis. This includes defects in genes

that give rise to inherited thrombocytopenia dis-

orders, including Bernard-Soulier, Paris-Trousseau/

Jacobsen, and Wiskott-Aldrich syndromes.

Characterization of mutations in these disorders

has contributed greatly to our understanding of

megakaryocyte and platelet development. For

example, the mouse model of Bernard-Soulier

syndrome, was generated by a targeted disruption

of the gene encoding the glycoprotein GPIb� subunit

Figure 2. Overview of the different approaches developed to generate genetically modified platelet precursors, with examples of published

data discussed in this review. 1Bernard-Soulier syndrome: GPIb� KO [18], GPIb� KO [17]; Glanzmann thrombasthenia, GPIIIa integrin

KO [19, 96]; Hermansky-Pudlak syndrome [20]. 2Embryonic stem cell line [60]; K562 cell line [97]; 3Human embryonic stem cell line

[98]; 4UT7 cell line [99]; 5Dami [36], K562 [39, 40] cell lines; 6[7, 100]; 7[63, 64, 66]; 8[45]; 9[101]; 10[102]; 11[85, 86, 89–91, 93–95].

Methods for genetic modification of megakaryocytes and platelets 395

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Tab

leI.

Megakaryo

cyte

and

plateletgen

etic

modification

evolution:from

themouse

mutantmodelsto

therecentgen

etherap

yap

proaches

using

viraltran

sduction

ofhem

atopoieticstem

cells.

Thistable

presentstheevolutionofresearch

ongen

eticmodificationsin

megakaryo

cytes.Rep

resentative

exam

pleswithefficien

cy,ad

vantages

anddrawbacksofeach

approachareshown.BM

,bone-marrow;

CM

V,cytomegalovirus;

ESC,em

bryonic

stem

cell;HSC,haematopoieticstem

cell;KSL,c-kitþ,Sca1þ,lineage-;KO,Knock-O

ut;KI,Knock-In;M

K,M

egakaryo

cyte;M

uLV,murineleukaemia

virus;

PGK,phosphoglycerate

kinase;

PTS,Paris-T

rousseausyndrome;

SIN

,Self-inactivate

vector;

SIV

,Sim

ianIm

munodeficiency

lentivirusvector.

Models

Approaches

Exam

ples:

Reagen

tor

Virus/celltype/Transfected

ortran

sducedco

nstructs[ref]

Transfection/T

ransduction

efficien

cyAdvantages

Drawbacks

Mutantmice

Knock

out

Knock

in

GPIb�KO

[18],

GPIb�KO

[17],

GPIIIa

integrinKO

[19,96].

Invivogen

erationofplatelets

and

studyofmegakaryo

poiesis/

thrombopoiesisprocesses.

Modelsforhuman

diseases.

Tim

eco

nsuming,unexpected

phen

otype.

Megakaryo

cytic

celllines

Lipid-based

tran

s-

fectionreagen

ts

Lipofectam

ine,

Fugen

e/Dam

i

cells/factorVIIIcD

NA/G

PIIb

orCM

Vpromoter[36,37].

Bestefficien

cywithlineagespecific

promoters.

Quick,easy

tocarryout.

Transform

edcellsfunctionally

far

from

norm

al.

Lipofectam

ine/K562cells/siRNA

[38,39].

Electroporation

UT7-G

Mcells/RUNX1cD

NA,

siRNA

[99].

Highefficien

cyoftran

sfection

compareto

primarycells.

Noproductionofplatelets.

Viral

tran

sduction

Len

tivirus/Dam

icells/GPIb�

cDNA/G

PIIbpromoter[61].

97.3%

Stable

tran

sfectionpossible.

Len

tivirusHIV

derived

vector/M

O7ecells/RGS16

shRNA

[50].

2–3fold

#in

protein

level.

BetterexpressionwithGPIIb,

plateletfactor4an

dGPIb�

lineagespecific

promoters

than

CM

V.

SIV

lentivirus/UT7-T

PO

cells/

hfactorVIIIcD

NA/G

PIb�,

GPIIb,GPVIpromoter[66].

Bestefficien

cywithGPIb�

promoter.

GPIb�promoteristhestrongestin

celllines.

Primarymature

MKs

Lipid

based

tran

sfection

DOTAPreagen

t/PKC�-ribozyme

[49].

50%

Effectonproplateletform

ingan

d

platelet-releasingM

Ks.

Uncyclingcells�requires

large

no.ofcells.

Poortran

sfection

efficien

cy.Nostab

le

tran

sfection.

Mirustran

sfectionreagen

t/primary

MKsfrom

mouse

BM

/murine

CIB

1siRNA

[51].

40–60%

#ofprotein

level.

Transfectionofmature

MKs

avoidsim

pactonM

K

maturation.

Viral

tran

sduction

Len

tivirusHIV

derived

vector/pri-

maryM

Ksfrom

CD34þ

human

cord

blood/RGS16

RNAi[50].

2fold

#in

protein

level.

Len

tivirusesareab

leto

tran

sduce

non-cyclingcellslikemature

MKs.

Sinbis

virus/primaryM

Ksfrom

mouse

BM

/CIB

1cD

NA

[51].

10–13fold

"in

protein

level.

Hem

atopoietican

d

embryonic

stem

cells(H

SC,ESC)

Viral

tran

sduction

Retrovirus/human

cord

blood

CD34HSC/stable

expression

(G418Rco

lony)

[52].

67-83%

forHPP-C

FC,25–82%

forCFU-G

EM

M

HSC

both

self-ren

ewan

ddiffer-

entiateinto

allbloodlineages.

PurificationofHSC.

396 C. Pendaries et al.

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Retrovirus/M

plreceptor/BM

reco

nstitutionwithretroviral

vector-tran

sducedmouse

BM

cells[53].

50%

prior,

90%

afterG418selec-

tion.10.5

weekspost-trans-

plantation,98%

ofpositive

colonies.

HSC

representlong-term

marrow

repopulatingcellsforgen

e

therap

y.

DifferentiationofHSC

before

exvivogen

erationofM

Ks

orplatelets.

Aden

ovirus/CD34þ/C

D9[54].

90%

inM

Ksan

dproplatelets.

TransducedHSC

areab

leto

differentiatein

MK

lineagean

d

exvivoM

Ksarefunctional.

Safetyprocedure

required

with

virus.

Lackofspecificitywith

non-lineagespecific

promoters.

Aden

ovirus/human

CD34þHSC/

GFPcD

NA/C

MV

promoter

[55].

45%

Possible

stab

leexpressionofthe

tran

sducedgen

einto

single

purified

stem

/progen

itorcells.

Applicable

forfuture

clinical

studies.

MuLV

retrovirus/Pl(A2)alloan

ti-

gen

form

ofGPIIIa/G

PIIbpro-

moter[56].

20%

DifferenciationdowntheM

K

lineage.

Lineagespecific

expression,

"Biosafety.

MuLV

vs.HIV

based

retrovirus

vectors/G

FPcD

NA/EF1�,

CM

V,PGK

promoters

[58].

20–30%

oftran

sduction:

HIV

4M

ulV.

Expression:EF1�

prom4

CM

V,PGK.

"tran

sductionefficien

cywith

HIV

-1vector.Highestlevelof

expressionwithEF1�

promoter.

HIV

-SIN

vector"Biosafety

but

#expression.

VSV-G

pseudotyped

HIV

-1SIN

retrovirus/human

bloodCD34/

hGPIIbpromoter[57].

"length

ofexpressiontime.

Lineagespecific

expression.

"Biosafety

withSIN

vector.

Len

tivirus/co

rdbloodHSC/G

FP

cDNA

[59].

Atweek10,40%

ofGFPexpres-

sionin

CFU-derived

colony.

Long-term

culture

expan

sion:

expan

ded

1000fold

inlong-

term

culturesþgrowth

factors,

FLT-3

ligan

d).

Len

tivirus/ESC/C

alDEG-G

EFI

DNA/O

P9feed

ercells,

TPO,

IL6,IL

7co

-culture

[60].

10–50%

tran

sfectionefficien

cy.

Largepolyploid

mature

MKsan

d

producingproplatelets.

Importan

ceofsupplemen

tary

factors

forthedifferentiation.

Viral

tran

sduction

andgen

e

therap

y

Bernard

Soulier

Syndrome

50%

oftheM

Kderived

from

the

stem

cellsexpress

the

tran

sgen

e.

GPIb-IX-V

functionrestored.�IIb

promoterdirects

high-level

expressionan

disactive

earlyin

megakaryo

cytopoiesis.

‘‘Tolerance

challenge’’:

Individualscanberefractory

to

infuseddonorplatelets

dueto

productionofan

tibodies

against

mismatch

edalloan

ti-

gen

icdeterminan

tsofthe

tran

sgen

eresultingin

clearance

ofthetran

sfusedplatelets.

(continued)

Methods for genetic modification of megakaryocytes and platelets 397

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Tab

leI.

Continued

.

Models

Approaches

Exam

ples:

Reagen

tor

Virus/celltype/Transfected

ortran

sducedco

nstructs[ref]

Transfection/T

ransduction

efficien

cyAdvantages

Drawbacks

Len

tivirus/CD34þ

HSC

from

human

blood/G

PIb�cD

NA/

GPIIbpromoter[61].

Paris-Trousseau/Jacobsen

Thrombopenia

Patient1:FLI1

expression�9.5.

Patient2:FL1expression�1.5.

RestorationofM

Kphen

otypein

2

PTSpatients

afterFLI1

cDNA

tran

sfer

invitro."totalnumber

ofmature

MKs(�

3to

8),

proplateletproducingM

Ks

(�5).

HIV

-derived

Len

tivalsystem

/

CD34þ

cellsfrom

peripheral

bloodofPTSpatients/FLI1

cDNA/PGK

promoter[63].

Hem

ophilia

AExpressionofthetran

sgen

ein

mouse

platelets:7–11%

for

CM

V,16–27%

forGPIb

�

promoter.

GPIb�most

potentplatelet-

specific

promoterforin

vivo

experim

ents.Detectable

tran

-

scripts

inBM

andspleen

forat

least90days.

SIV

vectors:

safety

advantageforclinical

applications.

Partially

corrected

hem

ophilia

Aphen

otype.

SIV

/human

CD34þ

derived

MK

KSLcells,murineHSC/human

factorVIIIcD

NA/C

MV,

GPIb�,GPIIb,GPVIpromo-

ters

[66].

Glanzmannthrombasthenia

–19%

ofpatientM

Kareex

vivo

tran

sduced[65].

Exvivophen

otypic

correctionof

MK

from

GT

patients

[65].

–M

uLV/bloodCD34(þ

)cells

from

GT

patients/hGPIIIa

cDNA/G

PIIbpromoter[65].

–Human

GPIIIa

expression"�3

to6onmouse

platelets

[64].

Hyb

ridemurineGPIIb-human

GPIIIa

integrinexpression,

improvedbleed

ingtimein

mice

andtolerance

bytheim

mune

system

ofthehost

[64].

–HIV

-1SIN

vector/mouse

stem

cellsGPIIIa

deficient/hGPIIIa

cDNA/G

PIIbpromoter[64].

398 C. Pendaries et al.

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7 [17] or, more recently, the � subunit [18] of the

GPIb-IX-V complex. Both models are associated

with a macrothrombocytopenia and a severe bleeding

phenotype. Another example is the depletion of the

GPIIIa subunit in mouse that mimics the human

bleeding disorder Glanzmann thrombasthenia [19].

The GPIIIa-null mice show all the cardinal features

of the disorder including defects in platelet aggrega-

tion and clot retraction, prolonged bleeding times,

and cutaneous and gastrointestinal bleeding.

Another model is the ‘Sandy’ (Sdy) mouse line, a

model for the human disorder Hermansky-Pudlak

syndrome type 7, which manifests in the form of

platelet dysfunction resulting from defective dense

granular storage and release [20, 21].

The development of mutant mice will continue to

remain a popular and powerful technique due to the

vast amount of information that can be gained from a

single knockout line, despite the labour-intensive

and time-consuming nature of the work. Moreover,

there have been a number of recent developments

in knockout technology resulting in the birth

of techniques such as recombineering [22]

and gene trapping [23], making knockouts more

widely available to the research community and

removing some of the more difficult aspects of the

process.

Genetic modification of megakaryocytic cell lines

Cell lines are widely used in research due to their

availability, ease of culture and optimization of

methods for their genetic modification, although

the extent to which they mimic cells in vivo will

always raise concern. Cell lines are immortalized in

order for their growth to continue beyond the cell’s

natural lifespan. Further, they are cultured in

an artificial environment away from neighbouring

cells, growth factors and stimuli. The difficulties

encountered with harvesting and culturing primary

megakaryocytes led to the use of fibroblast cell lines

transfected with DNA encoding platelet proteins as

a model for the study of megakaryocyte biology.

This approach made use of a range of transfection

methods and has furthered the knowledge of platelet

surface proteins including von Willebrand factor

[24], GPIb-IX [25], and GPIIb-IIIa [26–28], and is

still widely used today.

Megakaryocytic cell lines have provided us

with a unique opportunity to study proliferation,

differentiation and maturation of megakaryocytes.

More than 20 human or animal cell lines that

express megakaryocytic features have been described

[29, 30]. The human megakaryocyte cell lines Dami,

MEG-01, K562, HEL, MO7e, MEGA2, UT-7,

CMK, ELF-153, T33 and CHRF-288-1 cell lines

were established from blood or bone marrow of

patients with megakaryoblastic leukaemia. All of

these lines can be genetically modified to varying

extents through overexpression of a gene of interest

or by reducing expression using RNA interference

(RNAi). The generation of ‘knockout’ megakaryo-

cyte cell lines by homologous recombination is,

however, a very involved procedure, which does not

justify the effort and costs involved.

All data using megakaryocyte-like cell lines

should be interpreted with caution. For example,

investigations on TPO/Mpl signalling have revealed

cell line-specific responses that differ to those in

primary cells [31]. However, whilst the use of cell

lines may not be completely representative of their

in vivo counterparts, they are often appropriate as a

base to gain information about the protein of interest.

Nevertheless, primary cells are preferable

because of their increased physiological relevance.

Consequently, more and more studies are now

focusing on primary bone marrow cells and intact

animal models rather than transformed cell lines.

Transfection methods for cell lines are well

established, the earliest being the use of the

cationic polymer DEAE-dextran in 1965 [32],

which associates with DNA to form a complex,

facilitating its association with the plasma membrane

and uptake into the cell by endocytosis. This method

has been used successfully for transient transfections,

for example in studies on the effects of TPO on the

HEL cell line [33], and the characterization of the

thromboxane A2 receptor, as expressed in COS7

cells [34], but cannot be used for generation of stable

transfectants. Following on from this, the relatively

simple technique of calcium phosphate precipitation

was devised by Graham and van der Eb [35]. DNA is

mixed with calcium chloride and added to a buffered

phosphate solution that generates a precipitate as the

calcium ions coat the DNA, facilitating its passage

across the plasma membrane by endocytosis or

phagocytosis. Although this method is widely used,

it is not effective for all cell lines, which has driven

efforts towards the development of alternative

transfection reagents.

The main disadvantage of the DEAE-dextran and

calcium phosphate chemical methods is that they

cannot be used for in vivo transfer of DNA and so

this led to the development of artificial liposomes

that function in a similar way but are effective on a

wider range of cells. The general principle is based

on the cationic lipid forming a complex with

negatively charged DNA that enables it to interact

with the cell membrane and be taken up by

endocytosis without requiring any further stimuli.

Such reagents are widely available commercially and

easy to use but tend to be expensive and

often produce low transfection efficiencies, lack of

reproducibility and cytotoxic side-effects. They are

also not suitable for all cell types and indeed most

primary cells are recalcitrant to transfection by such

reagents.

Methods for genetic modification of megakaryocytes and platelets 399

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7 Transfection using lipid-based reagents is by far the

most widely used method for transfection of immor-

talised megakaryocyte cell lines. Shi et al. [36]

developed a system in which Dami cells were

transfected with a Factor VIII expression cassette

under the control of the megakaryocyte/platelet-

specific GPIIb promoter, using the lipofectamine

reagent (Invitrogen, Paisley, UK). This study was

followed up by Rodriguez et al. [37] who used the

FuGENE transfection reagent (Roche Applied

Science, West Sussex, UK) and also modified the

construct to improve the levels of expression.

Subsequent studies confirmed that expressed Factor

VIII was biologically active. Potentially, such an

approach could be used to treat haemophilia, with

the advantage that Factor VIII is liberated at the site of

injury. Additionally, these reagents have been used to

transfect megakaryocyte cell lines with siRNA

duplexes, including K562, MEG-01 and HEL lines

[38–40].

The transfer of DNA into cells can be facilitated by

mechanical means, using electrical impulses

to induce temporary permeability of the plasma

membrane, a technique known as electroporation.

This approach is quick and easy to carry out, but the

mechanical nature of the procedure means that

the level of cell damage can be high if conditions

are not optimized correctly [41]. The use of

this technique is therefore a compromise between

transfection efficiency and cell survival.

Electroporation tends to be more effective on

primary cells than the lipid-based reagents, but the

low levels of viability are a major disadvantage

considering that the starting number of cells is

likely to be low. This is of particular concern when

working with primary megakaryocytes as the yield of

these cells is low so cell loss during manipulation

should be minimized.

An early example of the use of electroporation in

megakaryocyte research is the study of Block et al.

[42]. They used a rat marrow expression system to

study the terminal differentiation of primary cells into

megakaryocytes, with a focus on the GPIIb gene as a

model megakaryocyte-specific gene. Electroporation

has also been used successfully on both human and

mouse HSC. Wu and colleagues presented work in

2001 on the optimization of electroporation in the

transfection of CD34þ HSC selected from human

umbilical cord blood, achieving an efficiency of

approximately 30% [43, 44]. The number of viable

cells at 48 hours post-transfection was high (�77%), a

result that was attributed to the addition of plasma to

the ex vivo cell cultures, which aided transgene

expression and survival. Oliveira and Goodell were

able to achieve transfection efficiencies of approxi-

mately 80% with mouse HSC when electroporation

conditions were optimized [45].

Additional mechanical methods include

microinjection of DNA into the nucleus.

However, this is not appropriate for experiments

that require large numbers of cells due to the labour-

intensive nature of the technique. The more recently

developed strategy of nucleofection, devised by

the company Amaxa (and which is similar to

electroporation) enables DNA to be transported

directly into the nucleus. Nucleofector technology

is designed with primary cells in mind and has

recently been used successfully on a range of cell

types including human bone marrow-derived

mesenchymal stem cells [46], haematopoietic stem

cells [47], and the K562 cell line [48].

Primary megakaryocytes

Human primary megakaryocytes can only be

obtained in reasonable yield through invasive

surgery, such as bone marrow extraction or during

hip replacement operations. They can also be

obtained from peripheral blood, although the

number of cells is extremely low and so this source

is not routinely used. Primary megakaryocytes can

also be isolated from foetal mouse liver or adult bone

marrow aspirates, although their rarity means that

samples from several mice are usually pooled

to generate a reasonable number of cells for

experimentation. Such cells are fragile when isolated

and must be handled with extreme care. Because of

their fragile nature they are difficult to transfect

and manipulate by the standard method of

chemical-based transfection.

There are very few examples of genetic modifica-

tion of primary mature megakaryocytes. One of the

earliest examples of primary murine megakaryocyte

manipulation was the use of the lipid-based transfec-

tion reagent DOTAP to transfect a FITC-conjugated

ribozyme to assess the effect of protein kinase C

isoform expression on proplatelet formation [49].

They achieved a transfection efficiency of approxi-

mately 50% at 24 hours post-transfection, and

PKC�-specific ribozymes were found to reduce the

number of proplatelet-forming megakaryocytes

by 38–50%.

Greater success with regard to genetic manipula-

tion has been achieved with the use of viral methods.

Viral transduction is a powerful technique but tends

to be used only when other methods have been

exhausted due to the time-consuming nature of viral

preparation and the specialist facilities and safety

procedures that are required. Health and safety is of

the utmost importance when starting viral work

and particular consideration must be given to the

assessment of the recombinant virus as particular

gene families such as those involved in cell cycle and

transcriptional regulation can make the virus

particularly dangerous.

Recent advances have enabled the development

of second generation, replication defective,

self-inactivating (SIN) retroviruses, which has greatly

400 C. Pendaries et al.

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7 increased their safety and enabled the majority of

work to be carried out at safety level 2. Retroviral

systems have the advantage that they can be used

both in vitro and in vivo, although current systems

tend to be complex, lack specificity, and show

poor levels of induction. It is hoped that future

developments may include conditional systems such

as drug-inducible expression so that transgene

expression can be more rigorously controlled and

more robust levels of induction achieved.

In 2005 Berthebaud and colleagues used

lentiviral-mediated RNAi to knockdown expression

of RGS16, a negative regulator of G-protein-coupled

receptor signalling, in both the MO7e cell line and

primary megakaryocytes [50]. CD34þ cells obtained

from human cord blood were stimulated with TPO

and stem cell factor and the resulting megakaryocytes

transduced with HIV-derived lentiviral vectors

carrying siRNA for RGS16. This method enabled a

50% reduction in expression of RGS16, and

implicated this protein in the negative regulation of

signalling through the SDF-1 chemokine receptor

CXCR4. Similarly, Yuan et al. [51] obtained mature

megakaryocytes by differentiation of murine bone

marrow and transduced these with constructs for

wild type and mutant calcium and integrin

binding protein-1 (CIB-1) using Sindbis virus. This

research implicated CIB-1 as a negative regulator of

agonist-induced GPIIbIIIa activation in murine

megakaryocytes through a direct interaction with

the GPIIb integrin subunit tail.

Overall, whilst there has been limited success in

the use of viral approaches to express exogenous

proteins in primary megakaryocytes, the difficulty in

obtaining such cells, combined with their fragile

nature and subsequent resistance to transfection

means that it is desirable to find an alternative cell

type that can be modified and used as a source of

genetically modified platelets.

Haematopoietic stem cells as a source of

megakaryocytes and platelets

The difficulties encountered with transfecting

primary megakaryocytes have led to a greater focus

on the manipulation of their precursor, HSC.

This has the wider benefit that the stem cells can

be modified prior to the induction of differentiation

down a specific haematopoietic lineage.

Viral transduction is a very powerful approach as

HSCs can be selected based on expression of

the surface antigen CD34, transduced with viral

particles, and differentiated down the megakaryocy-

tic lineage. One of the earliest attempts at modifying

cord blood stem cells was by Lu et al. in 1993 [52].

They attribute their success to the use of a cocktail of

cytokines to stimulate the HSCs prior to

retroviral transduction. The cocktail consisted of

erythropoietin (EPO), steel factor, interleukin-3 and

granulocyte-macrophage colony stimulating factor

(GM-CSF). Their findings were amongst the first to

suggest that this technique has great potential for use

in gene therapy to correct inherited disorders.

Yan et al. [53] later had success with retroviral

transduction of mouse bone marrow cells, achieving

greater than 90% transduction of HSC after

antibiotic selection. Their focus was on the TPO

receptor Mpl, and their system serves as a powerful

example of how retroviral gene transfer can be used

to over-express a cell surface receptor in the

haematopoietic lineage, thereby allowing its

biological function to be investigated. CD34þ cells

have also been successfully transduced with the CD9

surface receptor by adenoviral methods [54] with

expression confirmed in both megakaryocyte and

proplatelets obtained after stimulation with SCF and

TPO. Further studies by Faraday et al. [55] provided

the critical finding that megakaryocytes obtained by

stimulating differentiation of transduced HSC down

the megakaryocytic lineage in vitro, termed ex vivo

megakaryocytopoiesis, are not only able to express

the transgene but also remain functional despite viral

infection, as assessed by agonist-induced GPIIbIIIa

activation.

Whilst these early efforts were successful, the one

common draw back of these approaches is the lack of

specificity of transgene expression. Consequently,

efforts have focussed on designing a system

that results in high transduction efficiencies

with expression restricted to cells of the

megakaryocyte lineage. The first study showing

megakaryocyte-specific gene expression was that of

Wilcox et al. [56], who made use of a fragment from

the GPIIb gene to drive expression of the transgene

in order to induce early and specific transgene

expression during megakaryopoiesis. CD34þ HSC

were transduced with a murine leukaemia retrovirus

(MuLV) and lineage–specific expression after

differentiation down the megakaryocyte lineage was

confirmed. The efficiency of transduction achieved

was only 20%, but a comparison of the promoters

used demonstrated 67% lineage-specific expression

in cells transfected with the GPIIb promoter

construct, compared with only 32% megakaryocytic

expression where the CMV promoter was used.

Yasui et al. [57] used the initial findings of Wilcox

and colleagues as a starting point for the further

development of this system. They constructed a

VSV-G pseudotyped HIV-1 self-inactivating (SIN)

vector that expressed green fluorescent protein under

the control of the human GPIIb promoter in order to

restrict expression to the MK lineage. Use of the HIV

vector was preferable to MuLV due to greater

efficiency of transduction, improved length of

expression time, and increased biosafety. Salmon

et al. [58] further developed the lentiviral vector

system, making modifications to improve the

degree of transduction of human HSC. They ran

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7 a side-by-side comparison between MuLV- and

HIV-based vectors and also tested different promo-

ters in order to assess which system gave the best

transduction efficiency and expression levels.

The HIV vector was found to have an improved

transduction efficiency and the EF1� promoter was

found to result in the highest level of expression

relative to CMV and PGK promoters. Indeed the low

expression levels achieved with the PGK promoter

were further decreased when used with the HIV

vector, suggesting that the self-inactivating features

have a deleterious effect where expression levels

are already quite low. They concluded that it is

preferable to use HIV-derived SIN vectors in

combination with the EF1� promoter in order to

achieve high efficiency transduction and expression

of transgenes in human HSC and their derivatives.

The genetic modification resulting from lentiviral

transduction of HSC has also been shown to be

maintained during long-term culture expansion, for

example, transgene expression in HSC was seen in

cultures supplemented with Flt-3 ligand and

expanded 1000-fold [59]. Once again, this highlights

the potential of using lentiviral vectors in the gene

therapy of haematopoietic disorders.

A common theme in the optimization of viral

transduction of primary HSC is the importance of

the supplementary factors that are added to the

culture. These stimulate differentiation down the

correct lineage, but also impact on the transduction

efficiency and survival of transduced cells in culture.

Eto et al. [60] found that optimum survival of

transduced megakaryocytes derived from embryonic

stem cells (ESC) was obtained when the modified

ESC were co-cultured with OP9 feeder cells in the

presence of TPO, IL-6, and IL-7. These conditions

enabled them to obtain large polyploid megakaryo-

cytes that expressed megakaryocytic markers, did

not express HSC markers, were able to produce

proplatelets and showed normal bidirectional

signalling with respect to integrin GPIIbIIIa. Their

studies went on to look at the over-expression of the

Rap1 exchange factor CalDEG-GEFI, which was

found to enhance agonist-induced activation of

GPIIbIIIa. Using this approach they were able to

achieve between 10–50% transduction efficiency.

A disadvantage of this approach, however, is the

use of feeder cells. Although they help to supply the

HSC with growth factors that aid their survival and

differentiation down the MK lineage, it also makes it

harder to collect the modified cells as they must be

separated out from the feeder cells.

Several groups researching the genetic modifica-

tion of HSC have a common goal of devising a

system to be used in gene therapy. Although such

approaches are still in the early stages, many patients

with platelet disorders could benefit from this.

One such disorder is Bernard-Soulier syndrome, a

severe congenital disorder in which patients lack the

platelet membrane glycoprotein complex

GPIb-IX-V. Shi et al. [61] used a lentiviral vector

to transduce CD34þ cells from human peripheral

blood, and achieved megakaryocyte/platelet specifi-

city of GPIb� expression through the GPIIb

promoter. Around 50% of megakaryocytes derived

from the modified stem cells were shown to express

the transgene, with expression confirmed as cell-type

specific and GPIb-IX-V function restored.

Megakaryocyte differentiation was stimulated by

supplying the cells with recombinant human IL-3,

IL-6, IL-11, SCF, Flt-3 ligand and TPO. These

findings confirm that this approach could be used to

treat GPIb�-deficient forms of Bernard-Soulier

syndrome as the attenuated lentiviral system

serves as a good delivery vector for transduction of

multipotent stem cells without compromising

the self-renewing capacity of these cells after

transplanting them back in vivo.

Paris-Trousseau syndrome, also known as

Jacobsen’s syndrome, is also seen as a good

target for viral-based gene therapy approaches.

This disorder results in patients having two distinct

populations of megakaryocytes as a consequence of

dysmegakaryopoiesis, caused by a deficiency of the

FLI-1 transcription factor [62]. Raslova et al. [63]

expressed FLI-1 in CD34þ cells taken from three

Paris-Trousseau syndrome patients using a lentiviral

vector; two showed a significant increase in the total

number of cells surviving and a corresponding

increase in the percentage of megakaryocytes, whilst

in the third patient they observed a decrease in the

amount of initial cell death, an improvement in

megakaryocyte maturation, and an increase in the

number of proplatelet-producing megakaryocytes.

There is clearly considerable potential for the use

of genetically modified megakaryocytes in gene

therapy applications, although there are aspects of

this approach that require further investigation and

validation before it can be used clinically. Fang et al.

[64] investigated some of these issues in their study

on a murine model for Glanzmann thrombasthenia.

They aimed to restore platelet function in

GPIIIa-deficient mice by supplying stem cells with

the transgene so that the megakaryocyte progeny of

these modified stem cells would then be able to

synthesize GPIIIa and thus make the GPIIbIIIa

complex necessary for platelet function. In addition,

they also set out to investigate if the expression of the

transgene could be maintained at a therapeutic

level for a reasonable length of time and, more

importantly, if this gene product could be tolerated

by the immune system of the host. They used an

HIV-1 self-inactivating vector, in this instance using

the GPIIb promoter, in order to achieve

MK-specificity. The expression of the transgene

was able to restore platelet function, and although

immune responses were triggered by the presence of

this exogenous protein they were able to overcome

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7 the immune clearance of genetically modified

platelets by administering intravenous immunoglo-

bulin, findings that emphasize the potential of this

approach.

Gene therapy for Glanzmann thrombasthenia was

also the target of a study by Wilcox et al. [65].

They used an MuLV-derived vector to transduce the

wild type GPIIIa gene into peripheral blood CD34þ

cells, with expression driven by the GPIIb promoter.

They observed the expression of the GPIIbIIIa

complex in transduced megakaryocytes, which was

able to form an activated comformation and

bind fibrinogen as a result of stimulation with

agonists such as adrenaline and the thrombin

receptor-activating peptide (TRAP). Again, these

findings support the use of CD34þ cells as a target

for gene therapy of platelet disorders.

Ohmori et al. [66] presented a study showing the

phenotypic correction of the coagulation abnormality

haemophilia A, using a simian immunodeficiency

virus. This vector presents a safety advantage for

clinical applications of gene therapy as it is derived

from SIVagmTYO1 and it is nonpathogenic to its

natural host and to infected asian macaques. In this

study, the GPIb� promoter was shown to be the

most potent platelet-specific promoter for in vivo

experiments as it showed strongest activity in

differentiated megakaryocytes, and in particular

during the later phases of megakaryopoiesis.

The transduction of mouse haematopoietic stem

cells resulted in the expression of the transgene

in 20% of platelets after bone-marrow transplanta-

tion, with a phenotypic correction of haemophilia

A mice.

RNA interference

Lentiviral vectors can be used not only to provide

cells with the means to express an exogenous

product, but also to introduce RNAi constructs to

reduce expression of a specific protein in the cell type

of interest. RNAi involves use of fragments of

double-stranded RNA that interfere with expression

of the target gene. A major breakthrough came when

it was discovered that effective silencing could be

achieved with 21- and 22-nucleotide RNAs [67].

Further research has enabled characterization of the

cellular mechanisms, including formation of the

RNA-induced silencing complex, which processes

the double-stranded RNA into single strands and

cleaves the target mRNA to silence gene expression

[68]. More detailed information can be found in

recent reviews [69–71].

RNAi technology has progressed considerably over

the last decade, but the main drawback of this

technique remains the poor transfection efficiencies

obtained when using conventional lipid-based

transfection reagents. This is a particular problem

in the case of RNAi as it is rare to achieve either

100% knockdown of the target or 100% transfection

efficiency so effects can be masked by untransfected

cells or residual protein in transfected cells.

These problems can be overcome by use of lentiviral

vectors that express an RNAi cassette such as short

hairpin RNA. One example of the successful use of

this system is that by Schomber et al. (2004) who

were able to achieve up to 50% transduction

efficiency of human CD34þ cells and up to a 95%

reduction in expression of the target gene at

the mRNA level, with specific and stable gene

silencing [72].

If this technique can be further modified to

maximize its efficiency whilst minimizing any

detrimental side-effects, it will provide a new

dimension in platelet/megakaryocyte research.

Although there will always be a demand for knockout

mice due to the wealth of information they provide,

RNAi provides a simpler and quicker method of

determining the effects of a specific protein in vitro,

which may provide sufficient information to

determine its role and importance in that cell type.

Moreover, it has an additional advantage that it can

be performed in primary cells that already lack a gene

of interest, thereby generating a dual/multiple

knockout.

Further development of genetic technologies

In 1999 Murphy and Leavitt developed a system that

combined the use of transgenic mice with retroviral

techniques to generate a pure population of geneti-

cally modified primary CD41þmegakaryocytes [73].

They produced a transgenic mouse line that

expresses the subgroup A avian leukosis virus

(ALV-A) receptor, TVA, on the surface of cells,

which confers susceptibility to infection by ALV-A

and retroviruses pseudotyped with the ALV-A

envelope protein. ALV-A expression in this model

was driven by the GPIb� 50 regulatory sequence so

that TVA expression and subsequent infection by

ALV-A pseudotyped viruses was restricted to cells of

the megakaryocyte lineage. Selective infection was

achieved both in vitro and in vivo, enabling a large

pure population of genetically modified primary

megakaryocyte precursors to be obtained. Further

to this, the TVA-expressing mice can also be crossed

with knockout lines providing a null genetic

background in which the effects of a specific

gene product can be assessed. For example,

TVA-expressing mice were crossed with mice

deficient for the TPO receptor Mpl, followed by

transduction of the primary megakaryocytes with

wild type or mutant Mpl, enabling the identification

of key functional regions of the receptor [74].

However, although this method was successful,

there have been no follow up studies from other

groups.

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7 A particularly powerful approach that has been

used by the Koretzky lab in the investigation

of structure-activity relationships in vivo is the

introduction of HSCs that express a mutant gene

into lethally irradiated mice that lack the gene under

investigation. They have used this technique to study

functional relationships in the tyrosine kinase Syk

and the adapter SLP-76. Bone marrow cells were

isolated from Syk and SLP-76 knockout embryos

and modified by transducing cells with either the

wild-type protein or point mutants using the MIGR1

retroviral system. These modified stem cells were

transplanted back into the bone marrow of lethally

irradiated mice and studies carried out on the

neutrophils and platelets derived from these geneti-

cally modified HSC, thus enabling determination

of the structure-activity relationships of the

proteins [75–77].

From genetically modified megakaryocytes

to culture-derived platelets

An important consideration is the extent to which

genetically modified megakaryocytes can be used as a

source of genetically modified platelets. Over the

course of the last 30 years many laboratories

have focused their research on understanding the

processes allowing platelet generation from

megakaryocytes in vitro. The key to success seems

to reside in finding the appropriate combination

of chemokines, extracellular matrix proteins and

co-cultured cells to enable investigation of proplate-

let formation and platelet release. Indeed, the distinct

location of particular extracellular matrix proteins

within the bone marrow [78] supports the notion that

they may play an important mechanistic role in

various stages of megakaryocyte maturation and

proplatelet formation. Several extracellular

matrix components, including basement membrane,

vitronectin, collagen through GPVI, matrigel and

VWF are known to play a role as promoters of

in vitro proplatelet generation. Recently it has been

shown that fibrinogen was also able to regulate

proplatelet formation via a GPIIbIIIa-dependent

mechanism [79]. On the other hand, other bone

marrow components like collagen through GPIaIIa

or stroma cell contact inhibit proplatelet formation

(review [80]).

The interaction of megakaryocytes with sinusoidal

bone marrow endothelial cells appears to be a critical

step in the later stages of megakaryocyte maturation,

proplatelet formation and platelet release.

Indeed, once in contact with endothelial cells,

megakaryocytes form distinct transendothelial pseu-

dopods, send cytoplasmic projections into the

lumen and release platelets directly into the

marrow-intravascular sinusoidal space [11, 81, 82].

The model developed by Hamada et al. (1999) using

a transmigration assay of megakaryocytes through

a confluent monolayer of bone marrow endothelial

cells in response to the chemokine SDF-1 [81], was

an attempt to mimic the physiological process of

platelet release through endothelial cells. This assay

enabled generation of functional platelets in the

lower chamber of the transwell.

Ungerer et al. [83] developed a method in which

they could generate large amounts of culture-derived

platelets from megakaryocyte progenitor cells.

CD34þ cells were isolated from human and mouse

peripheral blood and then treated with a cocktail of

cytokines in order to stimulate differentiation and

platelet shedding. A ten-fold higher yield of platelets

was achieved when the CD34þ progenitors were

cultured with TPO, interleukin-6, interleukin-1� and

stem cell factor, compared with TPO and stem cell

factor alone or TPO and interleukin-1� alone.

These culture-derived platelets showed similar

morphological and functional characteristics when

compared with platelets isolated from peripheral

blood, including expression of cell surface antigens,

the presence and release of �- and dense-granules,

and aggregation. In addition to this, they demon-

strated the key finding that if the progenitor

cells underwent genetic modification by adenoviral

infection, stable transgene expression was carried

over to the culture-derived platelets. Following on

from this initial breakthrough, the same group

focussed on modification of megakaryocyte

precursors prior to generation of platelets, with the

aim of establishing a homogeneous population.

They infected CD34þ cells from human peripheral

blood with adenoviruses or retroviruses and then

used the previously established method to gain

genetically modified platelets. Interestingly, they

found that transduction of megakaryocyte precursors

with adenoviruses resulted in an alteration of

activation patterns of the clusters of differentiation

markers in platelets, such as a reduction in receptor

activation in response to agonists, including TRAP

(thrombin receptor activating peptide). In contrast,

retroviral infection had no impact on expression

profiles and responses of platelet receptors, thereby

enabling use of these platelets in further functional

studies. Antibiotic selection through the presence of

a co-transduced plasmid carrying the neomycin

resistance gene resulted in greater than 90% homo-

geneity in the resulting population and the platelets

showed normal aggregation responses to agonists

including ADP and thrombin. They concluded that

the use of retroviruses was preferable to adenoviruses

due to the higher transduction efficiency achieved

and the reduction in side-effects. In addition,

sufficient platelets were generated to facilitate

injection back into mice, permitting in vitro studies

to be carried out on the modified platelets by

intravital video fluorescence microscopy, such as

adhesion and thrombus formation [84].

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7 Discussion/perspectives

In this review we have outlined the variety of

approaches that have been used to obtain genetically

modified megakaryocytes and platelets. These tech-

nologies are essential in the further understanding of

megakaryopoiesis and thrombopoiesis. A recent

interesting study from Schulze et al. [7] using a

combination of strategies illustrates how informative

these technologies are in furthering understanding of

the physiological and molecular mechanisms

of platelet production. They used (i) primary

megakaryocytes from a mouse strain in which the

GPIIb locus was replaced by cDNA encoding

enhanced yellow fluorescent protein modified by a

C-terminal farnesylation site which allowed it to be

incorporated in cellular membranes; (ii) retroviral

expression of EGFP-tagged PIP4K� and

EGFP-tagged PH domain of PLC�1 in primary

megakaryocytes (in order to follow respectively the

PIP4K� enzyme and its product, PI(4,5)P2, to show

that PI(4,5)P2 accumulates in internal membranes of

the demarcation membrane system; and (iii) PIP4K�short hairpin RNA retroviral infection to show a

specific requirement for this enzyme in terminal

maturation of megakaryocytes, and the expansion in

size and organization of the demarcation membrane

system. They were able to demonstrate that

PI(4,5)P2 in the demarcation membrane system

activates the WASp-Arp2/3 complex and thereby

nucleates F-actin in preparation for platelet release.

This study represents a considerable advance in our

understanding of how megakaryocyte membranes

and cytoskeleton are coordinated for platelet assem-

bly and release. Thus, we now know that each stage

of megakaryocyte maturation represents a potential

target for interference and that, in time, it may

be possible to consider thrombopoietic disorders

targeted to a specific stage in megakaryocyte.

The main limit for in vitro megakaryopoiesis/

thrombopoiesis studies or genetic therapy

approaches is that we do not have a full under-

standing of the mechanism by which megakaryocytes

release platelets. The majority of previous studies

have shown very low amounts of culture-derived

platelets, and these are not sufficient in number to

perform classical physiological assays to assess

platelet functions, including aggregation, secretion,

calcium mobilization, spreading, clot retraction,

and protein expression by western blotting.

The complexity of this technique means that most

studies focus on megakaryocytes as being represen-

tative of platelets, but this is an area requiring further

development and understanding.

Importantly, new animal models derived from all

these technologies are now being developed for the

validation of therapeutic targets. The model of

syngenic transplantation of genetically modified

murine bone marrow or HSC into irradiated mice

provides a means to follow in vivo thrombopoiesis

[85, 86]. This is the most straightforward way to

analyse the reconstitution of platelet content by

haematopoietic cells. This model can be used in

immunology, angiogenesis, and cancer research, in

addition to the field of thrombosis, as haematopoietic

stem cells give rise to all the haematopoietic lineages.

An interesting model was established by Wilcox

et al. [87] in their study on the expression of a Factor

VIII transgene in megakaryocytes. Following the

demonstration of functional Factor VIII expression

in both CD34þ cells from human peripheral

blood and cells from murine bone marrow, factor

VIII-transduced human peripheral blood cells were

xeno-transplanted into NOD-SCID mice. Platelets

isolated post-transplantation were demonstrated to

express factor VIII, which retained its association

with VWF, suggesting that it will be possible to

generate an inducible pool of factor VIII as a form of

treatment for haemophilia A sufferers. Indeed,

further research has generated promising results

with regard to the long-term expression of the

transgene in a lineage-specific manner [88].

Factor VIII-null mice were transplanted with

bone marrow transduced by a lentiviral construct

expressing Factor VIII from the platelet-specific

GPIIb promoter. Analysis of platelets from the

modified mice confirmed expression of functional

factor VIII and correction of the haemophilia

A phenotype. The most encouraging finding from

this study was that the phenotype was also corrected

in secondary recipients of the transduced bone

marrow cells, demonstrating that the gene

transfer occurred within long-term repopulating

haematopoietic stem cells.

An additional interesting model for the future is

the humanised murine model where irradiated

immunodeficient mice are xenotransplanted with

human cells. One major problem for in vivo studies

of compounds optimized for clinical use is that of

species differences. For example, the protease-

activated receptor 1 (PAR1) targeted by thrombin

in human platelets is not present on mouse platelets

and so it is not possible to test compounds against

PAR1 in rodent thrombosis models. For this reason

the humanised murine models will be very useful

to improve understanding of thrombopoiesis

mechanisms and to evaluate the in vivo effectiveness

of anti-human therapy. In the humanised model,

mice are used as recipients for human megakaryocy-

tic cell engraftment resulting in a model that allows

the study of human megakaryopoiesis and thrombo-

poiesis. Recent data have already shown the

feasibility of this approach using the xenotransplan-

tation of either human peripheral blood or cord

blood CD34þ cells or even directly human platelets

[89–95].

In conclusion, this review sheds light on the

considerable advances in the haemostasis and

Methods for genetic modification of megakaryocytes and platelets 405

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7 thrombosis fields. Although the use of

genetic modification by retro/lentiviral transduction

of haematopoietic stem cells for treatment of patients

with haemostasis disorders remains a long-term goal,

it is clear that we are on the way to solving the many

challenges that lie ahead.

Acknowledgements

Work in the authors’ laboratory is funded by the

British Heart Foundation and Wellcome Trust, SPW

holds a BHF chair.

References

1. Hartwig J, Italiano Jr J. The birth of the platelet. J Thromb

Haemost 2003;1:1580–1586.

2. Kaushansky K. The molecular mechanisms that control

thrombopoiesis. J Clin Invest 2005;115:3339–3347.

3. Pang L, Weiss MJ, Poncz M. Megakaryocyte biology and

related disorders. J Clin Invest 2005;115:3332–3338.

4. Schulze H, Shivdasani RA. Mechanisms of thrombopoiesis.

J Thromb Haemost 2005;3:1717–1724.

5. Szalai G, Larue AC, Watson DK. Molecular mechanisms of

megakaryopoiesis. Cell Mol Life Sci 2006;63:2460–2476.

6. Behnke O. An electron microscope study of the

megacaryocyte of the rat bone marrow. I. The development

of the demarcation membrane system and the platelet surface

coat. J Ultrastruct Res 1968;24:412–433.

7. Schulze H, Korpal M, Hurov J, Kim SW, Zhang J,

Cantley LC. Characterizationof the megakaryocyte demarca-

tion membrane system and its role in thrombopoiesis. Blood

2006;107:3868–3875.

8. Italiano Jr JE, Lecine P, Shivdasani RA, Hartwig JH. Blood

platelets are assembled principally at the ends of proplatelet

processes produced by differentiated megakaryocytes. J Cell

Biol 1999;147:1299–1312.

9. Tablin FM, Castro M, Leven RM. Blood platelet formation

in vitro. The role of the cytoskeleton in megakaryocyte

fragmentation. J Cell Sci 1990;97:59–70.

10. Wilson A, Trumpp A. Bone-marrow haematopoietic-

stem-cell niches. Nat Rev Immunol 2006;6:93–106.

11. Avecilla ST, Hattori K, Heissig B, Tejada R, Liao F, Shido K.

Chemokine-mediated interaction of hematopoietic progeni-

tors with the bone marrow vascular niche is required for

thrombopoiesis. Nat Med 2004;10:64–71.

12. Levine RF. Isolation and characterization of normal human

megakaryocytes. Br J Haematol 1980;45:487–497.

13. Dercksen MW, Weimar IS, Richel DJ, Breton-Gorius J,

Vainchenker W, Slaper-Cortenbach CM. The value of flow

cytometric analysis of platelet glycoprotein expression of

CD34þ cells measured under conditions that prevent

P-selectin-mediated binding of platelets. Blood 1995;

86:3771–3782.

14. Debili NF, Louache F, Vainchenker W. Isolation and culture

of megakaryocyte precursors. Methods Mol Biol 2004;

272:293–308.

15. Leven RM. Isolation of primary megakaryocytes and studies

of proplatelet formation. Methods Mol Biol 2004;

272:281–291.

16. Ware J. Dysfunctional platelet membrane receptors:

From humans to mice. Thromb Haemost 2004;92:478–485.

17. Ware J, Russell S, Ruggeri ZM. Generation and rescue of a

murine model of platelet dysfunction: The Bernard-Soulier

syndrome. Proc Natl Acad Sci USA 2000;97:2803–2808.

18. Kato K, Martinez C, Russell S, Nurden P, Nurden A,

Fiering S. Genetic deletion of mouse platelet glycopro-

tein Ibbeta produces a Bernard-Soulier phenotype

with increased alpha-granule size. Blood 2004;

104:2339–2344.

19. Hodivala-Dilke KM, McHugh KP, Tsakiris DA, Rayburn H,

Crowley D, Ullman-Cullere M. Beta3-integrin-deficient

mice are a model for Glanzmann thrombasthenia showing

placental defects and reduced survival. J Clin Invest

1999;103:229–238.

20. Li W, Zhang Q, Oiso N, Novak EK, Gautam R, O’Brien EP.

Hermansky-Pudlak syndrome type 7 (HPS-7) results from

mutant dysbindin, a member of the biogenesis of lysosome-

related organelles complex 1 (BLOC-1). Nat Genet

2003;35:84–89.

21. Swank RT, Reddington M, Novak EK. Inherited prolonged

bleeding time and platelet storage pool deficiency in the subtle

gray (sut) mouse. Lab Anim Sci 1996;46:56–60.

22. Copeland NG, Jenkins NA, Court DL. Recombineering:

A powerful new tool for mouse functional genomics.

Nat Rev Genet 2001;2:769–779.

23. Evans MJ, Carlton MB, Russ AP. Gene trapping and

functional genomics. Trends Genet 1997;13:370–374.

24. Bonthron DT, Handin RI, Kaufman RJ, Wasley LC, Orr EC,

Mitsock LM. Structure of pre-pro-von Willebrand factor

and its expression in heterologous cells. Nature 1986;

324:270–273.

25. Lopez JA, Leung B, Reynolds CC, Li CQ, Fox JE. Efficient

plasma membrane expression of a functional platelet glyco-

protein Ib-IX complex requires the presence of its three

subunits. J Biol Chem 1992;267:12851–12859.

26. Bajt ML, Ginsberg MH, Frelinger AL 3rd, Berndt MC,

Loftus JC. A spontaneous mutation of integrin alpha IIb beta

3 (platelet glycoprotein IIb-IIIa) helps define a ligand binding

site. J Biol Chem 1992;267:3789–3794.

27. O’Toole TE, Loftus JC, Plow EF, Glass AA, Harper JR,

Ginsberg MH. Efficient surface expression of platelet GPIIb-

IIIa requires both subunits. Blood 1989;74:14–18.

28. Kashiwagi H, Schwartz MA, Eigenthaler M, Davis KA,

Ginsberg MH, Shattil SJ. Affinity modulation of platelet

integrin alphaIIbbeta3 by beta3-endonexin, a selective bind-

ing partner of the beta3 integrin cytoplasmic tail. J Cell Biol

1997;137:1433–1443.

29. Baatout S. Cell lines with potential for megakaryocytic

differentiation: A review. Haematologia (Budap) 1996;27:

161–183.

30. Saito H. Megakaryocytic cell lines. Baillieres Clin Haematol

1997;10:47–63.

31. Drachman JG, Rojnuckarin P, Kaushansky K.

Thrombopoietin signal transduction: Studies from cell lines

and primary cells. Methods 1999;17:238–249.

32. Vaheri A, Pagano JS. Infectious poliovirus RNA: A sensitive

method of assay. Virology 1965;27:434–436.

33. Zauli G, Gibellini D, Vitale M, Secchiero P, Celeghini C,

Bassini A. The induction of megakaryocyte differentiation is

accompanied by selective Ser133 phosphorylation of the

transcription factor CREB in both HEL cell line and primary

CD34þ cells. Blood 1998;92:472–480.

34. Pawate S, Schey KL, Meier GP, Ullian ME, Mais DE,

Halushka PV. Expression, characterization, and

purification of C-terminally hexahistidine-tagged thrombox-

ane A2 receptors. J Biol Chem 1998;273:22753–22760.

35. Graham FL, van der Eb AJ. Transformation of rat

cells by DNA of human adenovirus 5. Virology 1973;54:

536–539.

36. Shi Q, Wilcox DA, Fahs SA, Kroner PA, Montgomery RR.

Expression of human factor VIII under control of the platelet-

specific alphaIIb promoter in megakaryocytic cell line as well

as storage together with VWF. Mol Genet Metab 2003;

79:25–33.

406 C. Pendaries et al.

Dow

nloa

ded

By:

[Uni

vers

ity o

f Birm

ingh

am] A

t: 09

:59

29 A

ugus

t 200

7 37. Rodriguez MH, Plantier JL, Enjolras N, Rea M, Leboeuf M,

Uzan G. Biosynthesis of FVIII in megakaryocytic cells:

Improved production and biochemical characterization. Br J

Haematol 2004;127:568–575.

38. Eriksson M, Arminen L, Karjalainen-Lindsberg ML,

Leppa S. AP-1 regulates alpha2beta1 integrin expression by

ERK-dependent signals during megakaryocytic differentiation

of K562 cells. Exp Cell Res 2005;304:175–186.

39. Huang CL, Cheng JC, Liao CH, Stern A, Hsieh JT,

Wang CH. Disabled-2 is a negative regulator of integrin

alpha(IIb)beta(3)-mediated fibrinogen adhesion and cell

signaling. J Biol Chem 2004;279:42279–42289.

40. Tseng CP, Huang CL, Huang CH, Cheng JC, Stern A,

Tseng CH. Disabled-2 small interfering RNA modulates

cellular adhesive function and MAPK activity during

megakaryocytic differentiation of K562 cells. FEBS Lett

2003;541:21–27.

41. Gehl J. Electroporation: Theory and methods, perspectives

for drug delivery, gene therapy and research. Acta Physiol

Scand 2003;177:437–447.

42. Block KL, Ravid K, Phung QH, Poncz M. Characterization of

regulatory elements in the 5’-flanking region of the rat GPIIb

gene by studies in a primary rat marrow culture system. Blood

1994;84:3385–3393.

43. Wu MH, Smith SL, Danet GH, Lin AM, Williams SF,

Liebowitz DN. Optimization of culture conditions to enhance

transfection of human CD34þ cells by electroporation. Bone

Marrow Transplant 2001;27:1201–1209.

44. Wu MH, Smith SL, Dolan ME. High efficiency electropora-

tion of human umbilical cord blood CD34þ hematopoietic

precursor cells. Stem Cells 2001;19:492–499.

45. Oliveira DM, Goodell MA. Transient RNA interference in

hematopoietic progenitors with functional consequences.

Genesis 2003;36:203–208.

46. Aluigi M, Fogli M, Curti A, Isidori A, Gruppioni E,

Chiodoni C. Nucleofection is an efficient nonviral transfec-

tion technique for human bone marrow-derived mesenchymal

stem cells. Stem Cells 2006;24:454–461.

47. von Levetzow G, Spanholtz J, Beckmann J, Fischer J,

Kogler G, Wernet P. Nucleofection, an efficient

nonviral method to transfer genes into human

hematopoietic stem and progenitor cells. Stem Cells Dev

2006;15:278–285.

48. Schakowski F, Buttgereit P, Mazur M, Marten A,

Schottker B, Gorschluter M. Novel non-viral method for

transfection of primary leukemia cells and cell lines. Genet

Vaccines Ther 2004;2:1.

49. Rojnuckarin P, Kaushansky K. Actin reorganization and

proplatelet formation in murine megakaryocytes: The role of

protein kinase calpha. Blood 2001;97:154–161.

50. Berthebaud M, Riviere C, Jarrier P, Foudi A, Zhang Y,

Compagno D. RGS16 is a negative regulator of SDF-1-

CXCR4 signaling in megakaryocytes. Blood 2005;106:

2962–2968.

51. Yuan W, Leisner TM, McFadden AW, Wang Z, Larson MK,

Clark S. CIB1 is an endogenous inhibitor of agonist-induced

integrin alphaIIbbeta3 activation. J Cell Biol 2006;172:

169–175.

52. Lu L, Xiao M, Clapp DW, Li ZH, Broxmeyer HE. High

efficiency retroviral mediated gene transduction into single

isolated immature and replatable CD34(3þ) hematopoietic

stem/progenitor cells from human umbilical cord blood.

J Exp Med 1993;178:2089–2096.

53. Yan XQ, Lacey DL, Saris C, Mu S, Hill D, Hawley RG.

Ectopic overexpression of c-mpl by retroviral-mediated gene

transfer suppressed megakaryopoiesis but enhanced erythro-

poiesis in mice. Exp Hematol 1999;27:1409–1417.

54. Burstein SA, Dubart A, Norol F, Debili N, Friese P,

Downs T. Expression of a foreign protein in human

megakaryocytes and platelets by retrovirally mediated gene

transfer. Exp Hematol 1999;27:110–116.

55. Faraday N, Rade JJ, Johns DC, Khetawat G, Noga SJ,

DiPersio JF. Ex vivo cultured megakaryocytes express

functional glycoprotein IIb-IIIa receptors and are capable of

adenovirus-mediated transgene expression. Blood 1999;94:

4084–4092.

56. Wilcox DA, Olsen JC, Ishizawa L, Griffith M, White GC 2nd.

Integrin alphaIIb promoter-targeted expression of gene

products in megakaryocytes derived from retrovirus-trans-

duced human hematopoietic cells. Proc Natl Acad Sci USA

1999;96:9654–9659.

57. Yasui K, Furuta RA, Matsumoto K, Tani Y, Fujisawa J.

HIV-1-derived self-inactivating lentivirus vector induces

megakaryocyte lineage-specific gene expression. Microbes

Infect 2005;7:240–247.

58. Salmon P, Kindler V, Ducrey O, Chapuis B, Zubler RH,

Trono D. High-level transgene expression in human hema-

topoietic progenitors and differentiated blood lineages after

transduction with improved lentiviral vectors. Blood

2000;96:3392–3398.

59. Luther-Wyrsch A, Costello E, Thali M, Buetti E, Nissen C,

Surbek D. Stable transduction with lentiviral vectors and

amplification of immature hematopoietic progenitors from

cord blood of preterm human fetuses. Hum Gene Ther

2001;12:377–389.

60. Eto K, Murphy R, Kerrigan SW, Bertoni A, Stuhlmann H,

Nakano T. Megakaryocytes derived from embryonic stem

cells implicate CalDAG-GEFI in integrin signaling. Proc Natl

Acad Sci USA 2002;99:12819–12824.

61. Shi Q, Wilcox DA, Morateck PA, Fahs SA, Kenny D,

Montgomery RR. Targeting platelet GPIbalpha transgene

expression to human megakaryocytes and forming a complete

complex with endogenous GPIbbeta and GPIX. J Thromb

Haemost 2004;2:1989–1997.

62. Hart A, Melet F, Grossfeld P, Chien K, Jones C,

Tunnacliffe A. Fli-1 is required for murine vascular and

megakaryocytic development and is hemizygously deleted in

patients with thrombocytopenia. Immunity 2000;13:167–177.

63. Raslova H, Komura E, Le Couedic JP, Larbret F, Debili N,

Feunteun J. FLI1 monoallelic expression combined with its

hemizygous loss underlies Paris-Trousseau/Jacobsen throm-

bopenia. J Clin Invest 2004;114:77–84.

64. Fang J, Hodivala-Dilke K, Johnson BD, Du LM, Hynes RO,

White GC 2nd. Therapeutic expression of the platelet-specific

integrin, alphaIIbbeta3, in a murine model for Glanzmann

thrombasthenia. Blood 2005;106:2671–2679.

65. Wilcox DA, Olsen JC, Ishizawa L, Bray PF, French DL,

Steeber DA. Megakaryocyte-targeted synthesis of the integrin

beta(3)-subunit results in the phenotypic correction of

Glanzmann thrombasthenia. Blood 2000;95:3645–3651.

66. Ohmori T, Mimuro J, Takano K, Madoiwa S,

Kashiwakura Y, Ishiwata A. Efficient expression of a

transgene in platelets using simian immunodeficiency virus-

based vector harboring glycoprotein Ibalpha promoter: In vivo

model for platelet-targeting gene therapy. Faseb J 2006;

20:1522–1524.

67. Elbashir SM, Lendeckel W, Tuschl T. RNA interference is

mediated by 21- and 22-nucleotide RNAs. Genes Dev

2001;15:188–200.

68. Hammond SM, Bernstein E, Beach D, Hannon GJ. An RNA-

directed nuclease mediates post-transcriptional gene silencing

in Drosophila cells. Nature 2000;404:293–296.

69. Bantounas I, Phylactou LA, Uney JB. RNA interference

and the use of small interfering RNA to study gene

function in mammalian systems. J Mol Endocrinol 2004;

33:545–557.

70. Downward J. RNA interference. BMJ 2004;328:1245–1248.

71. Tuschl T. RNA interference and small interfering RNAs.

Chembiochem 2001;2:239–245.

Methods for genetic modification of megakaryocytes and platelets 407

Dow

nloa

ded

By:

[Uni

vers

ity o

f Birm

ingh

am] A

t: 09

:59

29 A

ugus

t 200

7 72. Schomber T, Kalberer CP, Wodnar-Filipowicz A, Skoda RC.

Gene silencing by lentivirus-mediated delivery of siRNA in

human CD34þ cells. Blood 2004;103:4511–4513.

73. Murphy GJ, Leavitt AD. A model for studying megakaryocyte

development and biology. Proc Natl Acad Sci USA

1999;96:3065–3070.

74. Gaur M, Murphy GJ, deSauvage FJ, Leavcitt AD.

Characterization of Mpl mutants using primary megakaryo-

cyte-lineage cells from mpl(�/�) mice: A new system for Mpl

structure-function studies. Blood 2001;97:1653–1661.

75. Abtahian F, Bezman N, Clemens R, Sebzda E, Cheng L,

Shattil SJ. Evidence for the requirement of ITAM domains

but not SLP-76/Gads interaction for integrin signaling in

hematopoietic cells. Mol Cell Biol 2006;26:6936–6949.

76. Abtahian F, Guerriero A, Sebzda E, Lu MM, Zhou R,

Mocsai A. Regulation of blood and lymphatic vascular

separation by signaling proteins SLP-76 and Syk. Science

2003;299:247–251.

77. Sebzda E, Hibbard C, Sweeney S, Abtahian F, Bezman N,

Clemens G. Syk and Slp-76 mutant mice reveal a cell-auto-

nomous hematopoietic cell contribution to vascular develop-

ment. Dev Cell 2006;11:349–361.

78. Nilsson SK, Debatis ME, Dooner MS, Madri JA,

Quesenberry PJ, Becker PS. Immunofluorescence character-

ization of key extracellular matrix proteins in murine bone

marrow in situ. J Histochem Cytochem 1998;46:371–377.

79. Larson MK, Watson SP. Regulation of proplatelet formation

and platelet release by integrin alpha IIb beta3. Blood

2006;108:1509–1514.

80. Larson MK, Watson SP. A product of their environment:

Do megakaryocytes rely on extracellular cues for proplatelet

formation? Platelets 2006;17:435–440.

81. Hamada T, Mohle R, Hesselgesser J, Hoxie J, Nachman RL,

Moore MA. Transendothelial migration of megakaryocytes in

response to stromal cell-derived factor 1 (SDF-1) enhances

platelet formation. J Exp Med 1998;188:539–548.

82. Tavassoli M, Aoki M. Localization of megakaryocytes in the

bone marrow. Blood Cells 1989;15:3–14.

83. Ungerer M, Peluso M, Gillitzer A, Massberg S,

Heinzmann U, Schulz C. Generation of functional culture-

derived platelets from CD34þ progenitor cells to study

transgenes in the platelet environment. Circ Res 2004;

95:e36–e44.

84. Gillitzer A, Peluso M, Laugwitz KL, Munch G, Massberg S,

Konrad I. Retroviral infection and selection of culture-derived

platelets allows study of the effect of transgenes on platelet

physiology ex vivo and on thrombus formation in vivo.

Arterioscler Thromb Vasc Biol 2005;25:1750–1755.

85. Fibbe WE, Heemskerk DP, Laterveer L, Pruijt JF, Foster D,

Kaushansky K. Accelerated reconstitution of platelets

and erythrocytes after syngeneic transplantation of bone

marrow cells derived from thrombopoietin pretreated donor

mice. Blood 1995;86:3308–3313.

86. Jin H, Aiyer A, Su J, Borgstrom P, Stupack D, Friedlander M.

A homing mechanism for bone marrow-derived progenitor

cell recruitment to the neovasculature. J Clin Invest

2006;116:652–662.

87. Wilcox DA, Shi Q, Nurden P, Haberichter SL, Rosenberg JB,

Johnson BD. Induction of megakaryocytes to synthesize and

store a releasable pool of human factor VIII. J Thromb

Haemost 2003;1:2477–2489.

88. Shi Q, Wilcox DA, Fahs SA, Fang J, Johnson BD, Du LM.

Lentivirus-mediated platelet-derived factor VIII gene therapy

in murine haemophilia A. J Thromb Haemost 2007;5:

352–361.

89. Boylan B, Berndt MC, Kahn ML, Newman PJ.

Activation-independent, antibody-mediated removal of

GPVI from circulating human platelets: Development of a

novel NOD/SCID mouse model to evaluate the in vivo

effectiveness of anti-human platelet agents. Blood

2006;108:908–914.

90. Bruno S, Gunetti M, Gammaitoni L, Dane A, Cavalloni G,

Sanavio F. In vitro and in vivo megakaryocyte differentiation

of fresh and ex-vivo expanded cord blood cells: Rapid and

transient megakaryocyte reconstitution. Haematologica

2003;88:379–387.

91. Bruno S, Gunetti M, Gammaitoni L, Perissinotto E,

Caione L, Sanavio F. Fast but durable megakaryocyte

repopulation and platelet production in NOD/SCID mice

transplanted with ex-vivo expanded human cord blood

CD34þ cells. Stem Cells 2004;22:135–143.

92. Fibbe WE, Noort WA, Schipper F, Willemzei R. Ex vivo

expansion and engraftment potential of cord blood-derived

CD34þ cells in NOD/SCID mice. Ann N Y Acad Sci

2001;938:9–17.

93. Macchiarini F, Manz MG, Palucka AK, Shultz LD.

Humanized mice: Are we there yet? J Exp Med

2005;202:1307–1311.

94. Miyoshi H, Smith KA, Mosier DE, Verma IM, Torbett BE.

Transduction of human CD34þ cells that mediate long-

term engraftment of NOD/SCID mice by HIV vectors.

Science 1999;283:682–686.

95. Perez LE, Rinder HM, Wang C, Tracey JB, Maun N,

Krause DS. Xenotransplantation of immunodeficient

mice with mobilized human blood CD34þ cells provides

an in vivo model for human megakaryocytopoiesis and

platelet production. Blood 2001;97:1635–1643.

96. Emambokus NR, Frampton J. The glycoprotein IIb mole-

cule is expressed on early murine hematopoietic progenitors

and regulates their numbers in sites of hematopoiesis.

Immunity 2003;19:33–45.

97. Goldfarb AN, Delehanty LL, Wang D, Racke FK,

Hussaini IM. Stromal inhibition of megakaryocytic differ-

entiation correlates with blockade of signaling by protein

kinase C-epsilon and ERK/MAPK. J Biol Chem

2001;276:29526–29530.

98. Gaur M, Kamata T, Wang S, Moran B, Shattil SJ,

Leavitt AD. Megakaryocytes derived from human embryonic

stem cells: A genetically tractable system to study mega-

karyocytopoiesis and integrin function. J Thromb Haemost

2006;4:436–442.

99. Nagai R, Matsuura E, Hoshika Y, Nakata E, Nagura H,

Watanabe A. RUNX1 suppression induces megakaryocytic

differentiation of UT-7/GM cells. Biochem Biophys Res

Commun 2006;345:78–84.

100. Murphy GJ, Gottgens B, Vegiopoulos A, Sanchez MJ,

Leavitt AD, Watson SP. Manipulation of mouse hemato-

poietic progenitors by specific retroviral infection. J Biol

Chem 2003;278:43556–43563.

101. Tadokoro S, Shattil SJ, Eto K, Tai V, Liddington RC,

de Pereda JM. Talin binding to integrin beta tails:

A final common step in integrin activation. Science

2003;302:103–106.

102. Zou GM, Chen JJ, Yoder MC, Wu W, Rowley JD.

Knockdown of Pu.1 by small interfering RNA in CD34þ

embryoid body cells derived from mouse ES cells turns cell

fate determination to pro-B cells. Proc Natl Acad Sci USA

2005;102:13236–13241.

408 C. Pendaries et al.