Genetic Engineering of Human Stem Cells for Enhanced Angiogenesis
Using Biodegradable Polymeric Nanoparticles
Rohit Mohan & Liju J.Kumar
(Sixth semester Biotechnology & Biochemical Engineering
Mohandas College of Engineering &Technology)

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Abstract
Stem cells hold great potential as cell-based therapies to promote vascularization and tissue
regeneration.Vascular endothelial growth factor (VEGF) high-expressing, transiently modified stem cells
promote angiogenesis. Nonviral, biodegradable polymeric nanoparticles are developed to deliver hVEGF
gene to human mesenchymal stem cells (hMSCs) and human embryonic stem cell-derived cells (hESdCs).
Treated stem cells demonstrated markedly enhanced hVEGF production, cell viability, and engraftment
into target tissues. S.c. implantation of scaffolds seeded with VEGF- expressing stem cells (hMSCs and
hESdCs) led to 2- to 4-fold-higher vessel densities 2 weeks after implantation, compared with control cells
or cells transfected with VEGF by using Lipofectamine 2000, a leading commercial reagent. Four weeks
after intramuscular injection into mouse ischemic hindlimbs, genetically modified hMSCs substantially
enhanced angiogenesis and limb salvage while reducing muscle degeneration and tissue fibrosis. These
results indicate that stem cells engineered with biodegradable polymer nanoparticles may be therapeutic
tools for vascularizing tissue constructs and treating ischemic disease.
Genetic modification of stem cells to express angiogenic factors enhance the efficacy of stem cells for
therapeutic angiogenesis. However, previous studies have largely relied on viral vectors to deliver the
therapeutic genes to stem cells, which are associated with safety concerns. Nonviral delivery systems, such
as polyethylenimine and Lipofectamine are often associated with toxicity and provide significantly lower
transfection efficiency than a viral-based approach.
Key Words: VEGF, hMSC, hESdC, angioenic, DNA nanoparticles.

Introduction
Angiogenesis is the physiological process
involving the growth of new blood vessels from
pre-existing vessels.It is an important process
occurring in the body to heal the wounds & to
restore blood flow to the tissues after injury.
However, some injuries or genetic modifications
may demand enhanced angiogenesis. Human
stem cells modified using optimized poly(β-
amino esters)-DNA nanoparticles to express an
angiogenic gene encoding VEGF can enhance
angiogenesis.


Angiogenesis
The process of angiogenesis occurs as an orderly
series of events:
1. Diseased or injured tissues produce and
release angiogenic growth factors that
diffuse into the nearby tissues.
2. The angiogenic growth factors bind to
specific receptors located on the
endothelial cells (EC) of nearby
preexisting blood vessels.
3. Once growth factors bind to their
receptors, the endothelial cells become
activated. Signals are sent from the
cell's surface to the nucleus.
4. The endothelial cell's machinery begins
to produce new molecules including
enzymes. These enzymes dissolve tiny
holes in the sheath-like covering basement membrane) surrounding all
existing blood vessels.
5. The endothelial cells begin to divide
(proliferate) and migrate out through the
dissolved holes of the existing vessel
towards the diseased tissue.
6. Specialized molecules called adhesion
molecules called integrins serve as
grappling hooks to help pull the
sprouting new blood vessel sprout
forward.
7. Additional enzymes are produced to
dissolve the tissue in front of the
sprouting vessel tip in order to
accommodate it. As the vessel extends,
the tissue is remolded around the vessel.
8. Sprouting endothelial cells roll up to
form a blood vessel tube.
9. Individual blood vessel tubes connect to
form blood vessel loops that can
circulate blood.
10. Finally, newly formed blood vessel
tubes are stabilized by specialized
muscle cells that provide structural
support. Blood flow then begins.

Polymer Synthesis
Poly(β-amino esters) (PBAE) were synthesized
after a two-step procedure, in which C32-Ac was
first prepared by polymerization by using excess
diacrylate over amine monomer and C32-Ac was
then reacted with various amine reagents to
generate amine-capped polymer chains (Fig.
1B). Here, we chose three leading end-modified
C32 polymers (C32-103, C32-117, and C32-
122), which demonstrated high transfection
efficiency in stem cells.

Materials and Methods
Transfection
Bone marrow-derived hMSCs and hESdCs were
obtained and cultured as previously described.
Cells were transfected with VEGF plasmid or
control plasmid (EGFP or luciferase) by using
optimized poly(β-amino esters) transfection
conditions. Lipofectamine 2000 (Invitrogen), a
commercially available transfection reagent, was
used for control transfection
S.C. Implantation Of Stem Cell- Seeded
Scaffolds
All procedures for surgery were approved by the
Committee on Animal Care of Massachusetts
Institute of Technology. All constructs were
implanted into s.c. space in the dorsal region of
athymic mice. Three experimental groups were
studied for hMSCs transfected with: (i) C32-
103/VEGF, (ii) C32-117/VEGF, (iii) C32-
122/VEGF. Three control groups include (i)
hMSC-C32-103/Luc, (ii) hMSC-Lipo/VEGF,
and (iii) acellular scaffold alone. For hESdCs,
cells were transfected by using either C32-
117/VEGF or C32–117/Luc, and the acellular
scaffold group was examined as blank control.
All tissue constructs were harvested at 2 or 3
weeks after implantation for analyses
Transplantation Of Stem Cells Into
Ischemic Mouse Hindlimb
Hindlimb ischemia was induced in a mouse
model as previously described (6). Immedi-ately
after arterial dissection, cells were suspended in
100 μL of hMSC growth medium and injected
intramuscularly into two sites of the gracilis
muscle in the medial thigh. Five experimental
groups were examined as following: (i) PBS, (ii)
no transfection, (iii) hMSC-C32-122/EGFP, (iv)
hMSC-Lipo/VEGF, and (v) hMSC-C32-
122/VEGF.
Results
In Vitro VEGF Production
VEGF production by transfected stem cells was
examined by measuring the VEGF concentration
in the supernatant of transfected cells by using
ELISA. Four days after transfection, VEGF
secretion from PBAE-transfected hMSCs or
hESdCs was ≈1- to 3-fold higher than their
respective untransfected controls, and ≈1- to 2-
fold higher compared with Lipofectamine 2000
secretion from day 4 to day 9 slightly decreased
and was still significantly higher in PBAE-
transfected groups than the control groups. Cell
viability after the PBAE-mediated transfection
was 80–90% in both stem cell types.

Enhancement Of Angiogenesis In The
S.C.Space
Angiogenesis in s.c. space was examined 2 or 3
weeks after implantation. Compared with
acellular scaffold controls, scaffolds seeded with
VEGF-transfected hMSCs using three poly(β-
amino esters) (PBAE) led to markedly increased
blood vessel migration into the constructs from
adjacent tissues (Fig. 2A), whereas the control
groups (hMSCs transfected with C32-103/Luc or
Lipo/VEGF) did not appear to be much different
from the acellular control. H&E and mouse
endothelial cell antigen (MECA) staining of the
harvested tissue sections demonstrated 3- to 4-
fold-higher vessel density in the hMSC-
PBAE/VEGF groups compared with the controls
(Fig. 2 A and B), and a similar trend was
observed with hESdC groups.

Enhanced VEGF Production And
Homing Factor Expressions
VEGF production by the transplanted stem cells
in vivo was examined by hVEGF ELISA. Two
days after transplantation, C32-122/VEGF-
transfected hMSCs produced 6-fold-higher
VEGF than did untransfected cells or cells

Enhanced VEGF Production And
Homing Factor Expressions
VEGF production by the transplanted stem cells
in vivo was examined by hVEGF ELISA. Two
days after transplantation, C32-122/VEGF-
transfected hMSCs produced 6-fold-higher
VEGF than did untransfected cells or cells