platelets - university of birmingham · megakaryocytes/platelets and models for human disorders to...
TRANSCRIPT
This article was downloaded by:[University of Birmingham]On: 29 August 2007Access Details: [subscription number 768418694]Publisher: Informa HealthcareInforma Ltd Registered in England and Wales Registered Number: 1072954Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
PlateletsPublication details, including instructions for authors and subscription information:http://www.informaworld.com/smpp/title~content=t713442010
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
PLEASE SCROLL DOWN FOR ARTICLE
Full terms and conditions of use: http://www.informaworld.com/terms-and-conditions-of-access.pdf
This article maybe used for research, teaching and private study purposes. Any substantial or systematic reproduction,re-distribution, re-selling, loan or sub-licensing, systematic supply or distribution in any form to anyone is expresslyforbidden.
The publisher does not give any warranty express or implied or make any representation that the contents will becomplete or accurate or up to date. The accuracy of any instructions, formulae and drug doses should beindependently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings,demand or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with orarising out of the use of this material.
© Taylor and Francis 2007
Dow
nloa
ded
By:
[Uni
vers
ity o
f Birm
ingh
am] A
t: 09
:59
29 A
ugus
t 200
7
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: [email protected]
ISSN 0953–7104 print/ISSN 1369–1635 online � 2007 Informa UK Ltd.
DOI: 10.1080/09537100701288012
Dow
nloa
ded
By:
[Uni
vers
ity o
f Birm
ingh
am] A
t: 09
:59
29 A
ugus
t 200
7
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.
Dow
nloa
ded
By:
[Uni
vers
ity o
f Birm
ingh
am] A
t: 09
:59
29 A
ugus
t 200
7
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
Dow
nloa
ded
By:
[Uni
vers
ity o
f Birm
ingh
am] A
t: 09
:59
29 A
ugus
t 200
7
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.
Dow
nloa
ded
By:
[Uni
vers
ity o
f Birm
ingh
am] A
t: 09
:59
29 A
ugus
t 200
7
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
Dow
nloa
ded
By:
[Uni
vers
ity o
f Birm
ingh
am] A
t: 09
:59
29 A
ugus
t 200
7
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.
Dow
nloa
ded
By:
[Uni
vers
ity o
f Birm
ingh
am] A
t: 09
:59
29 A
ugus
t 200
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
Dow
nloa
ded
By:
[Uni
vers
ity o
f Birm
ingh
am] A
t: 09
:59
29 A
ugus
t 200
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.
Dow
nloa
ded
By:
[Uni
vers
ity o
f Birm
ingh
am] A
t: 09
:59
29 A
ugus
t 200
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
Methods for genetic modification of megakaryocytes and platelets 401
Dow
nloa
ded
By:
[Uni
vers
ity o
f Birm
ingh
am] A
t: 09
:59
29 A
ugus
t 200
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
402 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 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.
Methods for genetic modification of megakaryocytes and platelets 403
Dow
nloa
ded
By:
[Uni
vers
ity o
f Birm
ingh
am] A
t: 09
:59
29 A
ugus
t 200
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].
404 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 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
Dow
nloa
ded
By:
[Uni
vers
ity o
f Birm
ingh
am] A
t: 09
:59
29 A
ugus
t 200
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.