Endothelial progenitor cells

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Endothelial progenitor cells

• Mehrdad Abedi, MD
• Journal Club Presentation

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Blood vessel formation

• Two categories
• a.) vasculogenesis
• de novo blood vessel generation from
  vascular progenitor cells
• b.) angiogenesis
• formation of new blood vessels via
  extension or remodeling of existing blood
  vessels

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Blood vessel formation

• Vasculogenesis
• a.) during embryonic development
• b.) during adulthood associated with
  circulating progenitor cells
• Angiogenesis
• a.) embryonic development
• b.) adulthood wound healing, menstrual
  cycle, tumour-angiogenesis

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Vasculogenesis

• The vascular system is one of the earliest organ
  system that developes during embryogenesis
• One of the first markers of angioblast precursors
  Flk-1 (VEGF-R2)
• Further important early markers are Brachyury
  and C-Kit

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Vasculogenesis

• 1. First phase
• Initiated from the generation of hemangioblasts
  leave the primitive streak in the posterior
  region of the embryo a part of splanchnic
  mesoderm
• 2. Second phase
• Angioblasts proliferate and differentiate into
  endothelial cells
• 3. Third phase
• Endothelial cells form primary capillary plexus

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Vasculogenesis

• Extraembryonic Vasculogenesis
• Intraembryonic Vasculogenesis

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Extraembryonic Vasculogenesis

• First apparent as blood islands in yolk sac
• Blood islands are foci of hemangioblasts
• Differentiate in situ
• a.) loose inner mass of embryonic
  hematopoietic precursors
• b.) outer layer of angioblasts
• by the merge of individual blood islands
  capillary networks are formed
• Yolk sac vasculogenesis communicate with fetal
  circulation via the vitelline vein

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Extraembryonic Vasculogenesis
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Intraembryonic vasculogenesis

• para-aortic mesoderm
• AGM (aorta-gonad-mesonephros)
• First dorsal aorta and
• cardinal veins are built
• Endocardium - vascular plexus is generated
• Development of bilateral embryonic aortae
• Then allantoic vasculature occurs

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Embryonic circulatory system
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• Subsequent vascular development primarly via
  angiogenesis
• Some endoderm derived organs, however, are also
  capable for vasculogenesis

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Developmental angiogenesis

• Majority of vascular development occurs via
  angiogenesis
• Growth of new blood vessels from existing vessels
• Two distinct mechanisms available
• a.) sprouting angiogenesis
• b.) intussusceptive angiogenesis

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Sprouting angiogenesis
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Sprouting angiogenesis

• Sprouting invasion of new capillaries into
  unvascularized tissue from existing mature
  vasculature
• - degradation of matrix proteins
• - detachment and migration of ECs
• - proliferation
• Guided by endothelial tip cells and influenced by
  various attractant and repulsive factors (Ephrin,
  Netrin, Plexin)

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Intussusceptive angiogenesis

• Intussusceptive or non sprouting angiogenesis
• - remodelling of excisting vessels
• - vessel enlarges
• - pinches inward
• - splits into two vessels

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Intussusceptive angiogenesis
Das Endothel ein multifunktonelles Organ
Entdeckung, Funktionen und molekulare
Regulation Stürzl M., et al
Cell Tissue Res (2003) 314107117 DOI
10.1007/s00441-003-0784-3
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Building of blood vessels in adulthood
Endothelial precursors
Angiogenic sprouting
Intussusceptive growth
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Important factors guiding angiogenesis - VEGF
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Important factors guiding angiogenesis

• bFGF proliferation, differentiation, maturation
• TGFb stabilize the mature capillary network by
  strengthen the ECM structures
• PDGF recruits the pericytes to provide the
  mechanical flexibility to the capillary
• MMP inhibitors suppresses angiogenesis
• Endostatin cleaved C-terminal fragment of
  collagen XVII binds to VEGF to interfere the
  binding to VEGFR

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Differentiation of stem cells to vascular cells
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Differentiation of ES cells to vascular cells

• Stem cells cultivated with a defined cocktail mix
  (BMP-4, VEGF, SCF, Tpo, Flt3-ligand) in serum
  free medium to generate embryoid bodies
• EBs dissociated and cultivated in specific medium
  or EBs seeded for outgrowth

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KDRlow/C-Kitneg population gives rise to
cardiomyocytes, SMCs and Ecs common progenitor
Lei Yang et al., Nature Letters, 2008
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Generation of functional hemangioblasts from
embryonic stem cells
LDL red vWF - green
LDL red VE-cad- green
CD31 red vWF - green
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Alternatives to human embryonic stem cells

• Stem cells derived from single blastomeres
• Stem cells through nuclear reprogramming
• Induced pluripotent stem cells (iPS) through
  expression of stem cell specific proteins in
  differentiated cells

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Human embryonic cell lines derived from single
blastomeres without emberyo destruction
ectoderm 3-tubulin
endoderm alpha-fetoprotein
mesoderm SMC
Figure 1. Derivation and Characterization of hESC
Lines from Single Blastomeres without Embryo
Destruction (A) Stages of derivation of hES cells
from single blastomere. (a) Blastomere biopsy,
(b) biopsied blastomere (arrow) and parent embryo
are developing next to each other, (c) initial
outgrowth of single blastomere on MEFs, 6 days,
and (d) colony of single blastomere-derived hES
cells.
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Stem cells through nuclear reprogramming -
overview

• Adult and stem cells are genetically equivalent
• Differential gene expression is a result of
  epigenetic changes during development
• Nuclear reprogramming reversal of the
  differentiation state of a mature cell to one
  that is characteristic of the undifferentiated
  embryonic state
• A. Nuclear transfer
• B. Cellular fusion
• C. Cell extracts
• D. Culture induced reprogramming

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Stem cells through nuclear reprogramming -
overview
Nuclear transfer
Experimental Approach Reproductive
cloning functional test for reprogramming to
totipotency Somatic cell nuclear
transfer efficient derivation of genetically
matched ES cells with normal potency Mechanistic
insights Allows epigenetic changes (reversible)
to be distinguished from genetic changes
(irreversible)
Limitations Reproductive cloning is very
inefficient There are abnormalities at all stages
of development
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Nuclear exchange to generate stem cells
http//www.hhmi.org/biointeractive/stemcells/anima
tions.html
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Cybrid embryos - human chromosomes with animal
eggs
In vitro fertilization
Intracytoplasmic sperm injection
Somatic cell nuclear transfer
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Stem cells through nuclear reprogramming -
overview
Cell fusion
Experimental Approach Nuclear reprogramming of
somatic genome in hybrids generated with
pluripotent cells in most hybrids less
differentiated partner is predominant Mechanistic
insights Allows study of genetics of
reprogramming Question chromosomes of somatic
cells reprogrammed or silenced nucleus or
cytoblast required for molecular
reprogramming Limitations Fusion rate is very
low Tetraploid cells are generated
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Stem cells through nuclear reprogramming -
overview
Cell extract
Experimental Approach Exposure of somatic nuclei
or permeabilized cells to extracts from oocytes
or pluripotent cells Mechanistic
insights Allows biochemical and kinetic analysis
of reprogramming Limitations No functional
reprogramming done
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Stem cells through nuclear reprogramming -
overview
Cell explantation
Experimental Approach Explantation in culture
selects for pluripotent, reprogrammed cells
certain physiological conditions entire cells can
de-differentiate (Teratokarzinoma) Mechanistic
insights Allows study of genetics of
reprogramming Limitations May be limited to
germ line cells
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Stem cells through nuclear reprogramming

• Molecular mediators of reprogramming and
  pluripotency
• Chromatin remodelling factors
• DNA modification
• Histone modification
• Pluripotency maintained by a combination of
  extra- and intracellular signals
• Extracellular signals STAT3, BMP, WNT
• Intracellular signals factors at transcriptional
  level (Oct-4, Nanog, Sox2)

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Induced pluripotent stem cells (iPS) and cellular
alchemy

• introducing of factors in fibroblasts - induced
  pluripotent stem cells
• Able to produce many cell types
• Initially 24 genes selected
• Transduced into mouse embryonic fibroblasts
• Resistance gene for G418 under control of Fbx15
  promoter, which is only active in pluripotent
  cells
• Drug resistant colonies appeared, which resembled
  ES cells
• Expressed transcripts and proteins considered to
  be part of ES cell signature
• Termed induced pluripotent stem cells (iPS)
• Formed all three germ layers in vitro and in vivo
• Best combination Oct-4, Sox2. c-Myc, Klf4

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Adult progenitor and stem cells sources and
transdifferentiation
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Adult stem cells
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Adult progenitor cells
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Mobilization of vascular progenitors

• Tissue ischemia results in expression of
  cytokines like VEGF
• Recruites progenitor cells
• Steady state 0.01 MNCs in blood are CEPs
• Amount of circulating progenitors are increased
  after trauma, infectious injuries or tumour
  growth
• 24 h after injury 12

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Mobilization of vascular progenitors

• Mobilization mediated through metalloproteinases,
  adhesion molecules, VEGF and PLGF (placental
  growth factor)
• Induction of MMP9 causes release of stem-cell
  active soluble kit ligand

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Isolation of adult vascular progenitors
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Prospective therapeutical applications

• Tumour homing Trojan horse principle
• Organ revascularization and regeneration
• Wound healing
• Heart diseases
• Blood diseases

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Tumour homing Trojan horse principle
Home to places of active neoangiogenesis Vascular
progenitor transduced with a therapeutic
gene Vehicle for targeting therapeutic gene
expression to tumour Bystander effect of
advantage Gene directed enzyme prodrug
therapy (CYT-P450-Ifosfamide HSV-TK/Ganciclovir)
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Organ vascularization and regeneration

• After pathological ischemic events in the body
  exogenous introduction of vascular progenitors
  may facilitate restoration
• Bone marrow, rich reservoir of tissue-specific
  stem and progenitor cells
• Possible applications ischemic limbs,
  postmyocardial infarction, endothelialization of
  vasclular grafts, atherosclerosis, retinal and
  lymphoid organ neovascularization

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Potential use of adult stem and progenitor cells
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Peripheral Artery Disease

• Figure 5.?Angiographic analysis of collateral
  vessel formation in patients in group A
  Collateral branches were strikingly increased at
  (A) knee and upper-tibia and (B) lower-tibia,
  ankle, and foot before and 24 weeks after marrow
  implantation. Contrast densities in suprafemoral,
  posterior-tibial, and dorsal pedal arteries
  (arrows) are similar before and after
  implantation.

Tateishi-Yuyuama et al., The Lancet, Aug, 2002
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Impaired wound healing
Figure 4.Limb salvage after marrow implantation
in two patients in group ANon-healing ulcer on
heel (A) and ischaemic necrosis on big toe (B)
showed improvement 8 weeks after implantation.
Tateishi-Yuyuama et al., The Lancet, Aug, 2002
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Circulating EPCs aid in cardiac repair

• CD34, CD133, and VEGF2R
• Circulate in blood stream
• Contribute to repair of vascular or myocardial
  injury and collateral formation

Asahara T et al. Science. 1997275964-7. Takahash
i T et al. Nature Med. 19995434-8.
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EPC in CV events

• Originate in bone marrow
• Circulate in blood stream
• Number and function (proliferation, migration,
  homing) modulated by age, CV risk factors, and
  disease
• Release stimulated by organ and vascular injury
• Participate in vascular repair (collateralization)
  and re-endothelialization, partly by paracrine
  effects
• Circulating numbers ?by exercise and drugs
  (statins and ACE inhibitors)
• Independent predictors of endothelial dysfunction
  and long-term prognosis in patients with CAD

Hill JM et al. N Engl J Med. 2003348593-600.
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EPC number has prognostic importance
N 519 males with CAD, mean age 66 y
1.00
Group 3 (high EPC level)
0.98
0.96
Group 2 (medium EPC level)
Event-free survival
0.94
Group 1 (low EPC level)
0.92
0.90
0
100
200
300
365
0
Days
Werner N et al. N Engl J Med. 2005353999-1007.
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Mobilization of EPCs after myocardial infarction
N 16 patients with AMI, 8 controls
P lt 0.001 P lt 0.001
P lt 0.001 P lt 0.05
300
200
MNCCD34 (/106WBCs)
100
0
1 3 7 14 28
Day
Time after onset
Shintani S et al. Circulation. 20011032776-9.
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VEGF levels correlate with increase in EPCs
450
r 0.35 P 0.01
400
350
300
250
200
MNCCD34 (cells/106 WBCs)
150
100
50
0
0
50
100
150
200
250
300
350
400
450
Plasma VEGF (pg/mL)
Shintani S et al. Circulation. 20011032776-9.
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Common Problems with Using EPCs in Clinical
Settings.

• low frequency of less than one ECFC per one
  million nucleated cells in steady-state
  peripheral blood.
• despite rapid mobilization, EPCs cryopreserved
  after harvest lost functionality after thawing
  and thus a method for large-scale EPC preparation
  is presently lacking.
• The currently established ECFC enrichment methods
  start with density-gradient separation followed
  by subsequent adherence to matrix protein-coated
  tissue culture surfaces and depend on the
  presence of selected lots of fetal bovine serum
  (FBS).
• Current protocols for ECFC propagation are not
  yet suited to comprehensive testing or
  application of ECFCs as an investigational new
  drug (IND).

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• Blood samples (n 20) were obtained after
  written informed consent.
• Steady-state venous peripheral blood (PB) was
  obtained from healthy volunteers (max. 4 x 6 mL
  4 male, 2 female age 26 50 years) and patients
  with cardiovascular disease (CVD 1 x 6 mL, 4
  male, 3 female, age 45 86).
• Umbilical cord blood (UCB 20 45 mL n 7) was
  collected after full term pregnancies.

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• Endothelial growth medium (EGM) was prepared with
  replacing FBS 10 pooled human platelet lysate
  (pHPL) to create
• The pHPL was prepared from pooled platelet-rich
  plasma derived from a minimum of 40 whole blood
  donations as previously reported.

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• A maximum volume of 24 mL peripheral blood per
  volunteer or CVD-patient, respectively was
  collected in 6 mL vacuette tubes preloaded with
  108 IU/6ml of preservative-free sodium heparin.
  Umbilical cord blood of term pregnancies was
  collected immediately after delivery in 50mL
  tubes (Falcon) preloaded with preservative- free
  heparin.
• Endothelial growth medium (EGM) was prepared by
  supplementing endothelial cell basal medium
  (EBM-2) medium with hydrocortisone, human
  epidermal growth factor, vascular endothelial
  cell growth factor, human fibroblast growth
  factor B, R3 insulin-like growth factor-1, and
  ascorbic acid. The EGM was heparinized with 10
  U/mL of preservative-free Heparin (Biochrom AG)
  prior to supplementation with 10 pooled human
  platelet lysate (pHPL) and 2mM L-Glutamine,
  100U/mL penicillin and 100µg/mL Streptomycin.

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• Heparinized blood was diluted without additional
  cell separation in EGM/10 pHPL and seeded
  directly into the culture vessels.
• Nonadherent cells were removed by washing with
  pre-warmed PBS after overnight culture.
• Cultures were maintained until the outgrowth of
  cobblestone-type colonies (for a maximum of four
  weeks).

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• we also succeeded in generating ECFCs under
  animal protein-free conditions from unseparated
  UCB in seven of seven samples
• Large-scale expansions of primary ECFCs (16 33
  colonies) from three CVD patients did not show
  significant differences in final cell number
  compared to healthy volunteers

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In vivo functionality

• 3 x 105 bone marrow derived MSCs were mixed with
  1.2 x 106 ECFCs in Matrigel immediately before a
  subcutaneous injection of 0.2 mL aliquots into
  the right flank of immune-deficient

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Preserved ECFC phenotype, proliferation,
progenitor hierarchy and genomicstability after
large-scale expansion and cryopreservation
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• Despite robust proliferation, the expanded EPCss
  are genomically stable as evidenced by normal
  karyotype and balanced array-comparative genomic
  hybridization (array CGH) profiles.
• They display an endothelial phenotype and can be
  cryopreserved.
• After thawing, ECFCs showed preserved robust
  proliferation and maintained a progenitor
  hierarchy.
• Expanded ECFCs were successfully applied in test
  systems to form vascular networks in vitro and
  perfused human vessels in immune deficient
  experimental animals in vivo.

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