Cardiovascular tissue engineering and repair. Composite scaffold provides a cell delivery platform for cardiovascular repair.

1. Amandine F. G. Godier-Furnémonta, Timothy P. Martensa, b, Michael S. Koeckerta, Leo Wana, Jonathan Parksa,
Kotaro Araic, Geping Zhangb, Barry Hudsonb, Shunichi Hommac, and Gordana
aDepartment of Biomedical Engineering, Columbia University, New York, NY 10032; and Departments of bSurgery and
cMedicine, Columbia University Medical Center, New York, NY 10032
Procedures of the National Academy of Sciences
PNAS May 10, 2011 vol. 108 no. 19 7974-7979
(published online on April 20, 2011)
Raul Soto
Biol504
Composite scaffold provides
a cell delivery platform for
cardiovascular repair

2. Myocardial Infarction
http://nursingcrib.com/nursing-notes-reviewer/myocardial-infarction/
One or more regions
of the heart muscle
experience a severe
and prolonged
decrease in oxygen
supply because of
insufficient coronary
blood flow.
The affected muscle
tissue subsequently
becomes necrotic.

3. Demographics
• 425,000 deaths in the United States in 2006 (about one of every six
deaths)
• 1,255,000 new and recurrent attacks per year.
• About 34 percent of people who experience a coronary attack in a
given year die from it.
• 17,600,000 victims of angina (chest pain due to coronary heart
disease), heart attack and other forms of coronary heart disease are
still living.

4. “The Story”
Decellularized a section of human
heart muscle
Isolated MPC cells from human
bone marrow
Applied human mesenchymal
progenitor cells (MPCs) to
decellularized scaffold
After 4 weeks:
Heart explant & analysis
In Vitro: MPCs characterization
In Vivo 1: Cell Delivery via
composite scaffolds into acute
and chronic MI models
Implanted composite scaffold in
rat models for acute and chronic
myocardial infarction
In Vivo 2: functional effects of
MPC implant
This left the ECM scaffold with
intact architecture and mechanical
properties
Figure 1
Figure 2
Figure 3
Figure 4

5. “The Story”
Figure 1
• Complete decellularization of human
myocardium leaves intact most
elements of ECM and underlying
mechanical properties
• Stem cell - matrix composite scaffold
was built by applying fibrin hydrogel
with suspended cells (MPCs) to
decellularized sheets of human
myocardium
• This composite scaffold was implanted
in rat models of cardiac infaction
Figure 2
• Characterized (in vitro) myogenic and
vasculogenic potential of human
MPCs
• Showed that in vitro conditioning of
MPCs with TGF-β promotes
arteriogenesis
Figure 3
• Implanted composite scaffold
enhances the formation of vascular
network by:
• (1) secretion of paracrine factors like
SDF-1
• (2) migration of MPCs into ischemic
myocardium (but not normal
myocardium)
Figure 4
• Baseline levels of left ventricle systolic
dimensions and contractility
recovered after 4 weeks of composite
scaffold implantation

6. Methods and Assays
• Figure 1
o Myocardial Tissue Sectioning (A, B)
o Myocardial Tissue Decellularization
(A)
o Immunoselection (A)
o FACS / Flow Cytometry (A)
o SEM (C-F, K)
o Spectrophotometry (S1 D)
o Immunostaining (G-J)
o Uniaxial Tensile Test (L)
• Figure 2
o Q-PCR (A-I)
o RNA microarray (J)
• Figure 3
o Light microscopy (A-Q)
o ELISA (R)
o Western blotting (S)
o Matrigel Assay (T)
• Figure 4
o Echocardiography

7. Figure 1A
Composite scaffolds containing STRO-3 MPCs and cell-free scaffolds were
studied in nude rat models of acute and chronic cardiac infarction
Decellularize human myocardium:
2hr lysis in Tris + EDTA buffer;
6hr solubilization in SDS
Washed in PBS
incubated in PBS w/Rnase & Dnase
Washed in HBSS with mixing
Human STRO-1+ MPCs isolated from
bone marrow aspirate to >99% purity,
using human STRO-1 mouse IgM
primary AB, goat anti-mouse IgM
secondary AB, and streptavidin
microbeads
Verified purity of samples of STRO-1+
and STRO-1- with flow cytometry,
labeled with PE-conjugated streptavidin
TissueSectioningand
Decellularization
MPCsCellIsolation
TGF-
β

8. Figure 1B
• Architecture of the
decellularized matrix
depends on direction of
sectioning
• Circumferential sections
were used to create
scaffolds
• Large interconnected pores,
smooth channels enable
rapid infiltration of cells
and fluid
Tissue Sectioning

9. Figure 1CDEF
Method: SEM
Dehydrate tissue samples
through ethanol gradient
Dry tissue samples using
BalTec Critical Point Dryer
Sputter-coat with 15nm of
Gold-Palladium
Imaging with Hitachi 4700
SEM

10. Figure S1 D
• DNA quantification of decellularized tissue after several
variations of lysis/detergent/DNase treatment compared
with native tissue.
• Control: native tissue (negative)
• Critique: Methods section in paper supplement does not
provide details about this assay
Residual DNA in decellularized
tissue sample was quantified to
determine extent of
decellularization
Digested tissue samples with
Quant-it Picogreen Assay Kit
and BioTek spectrophotometer
Method:
DNA Quantification

11. Figure 1 GHIJ
Stained sections with
monoclonal antibodies (mAbs)
against rat factor VIII
Staining with mAb against SMA
differentiated arterioles from
large capillaries
Human cells identified using
mAb A21325, specific for
human mitochondria
Staining done using:
• Immunoperoxidase
• avidin-bioting blocking kit
• rat-absorbed biotinylated
anti-mouse IgG
• Peroxidase conjugate
Analyzed rat histological
sections at 40x magnification
by image processing to
determine
•# of MPCs
• Factor VIII-positive vessels
• SMA-positive vessels
Method:
Immunostaining
Tissue Characterization
Staining of ECM components of circumferential sections
Compared native myocardial tissue v decellularized tissue
Collagen IV, Laminin, Vitronectin maintain their presence.
Fibronectin is lost from the ECM (shown by patchy immunostaining)
Method: light microscopy, immunostaining
Critique: no discussion of possible effects of lack of fibronectin in
decellularized tissue

12. Figure 1K
Tissue Characterization
SEM images were used to measure average pore diameter
in decellularized scaffold
Controls : none
Critique:
• histogram should include raw data table
• should have measured pore diameters in native tissue as
a negative control
Method: SEM
Dehydrate tissue samples
through ethanol gradient
Dry tissue samples using
BalTec Critical Point Dryer
Sputter-coat with 15nm of
Gold-Palladium
Imaging with Hitachi 4700
SEM

13. Incubate radial and
cylindrical sections of fresh
and decellularized tissue
in cardioplegic solution
Used Instron 5848 Micro
tester to perform pull test
Stretched samples
uniaxially by applying
force in the apex-to-base
direction, at 0.1% per
second, up to 20% strain
Calculated tangential
modulus from stress –
strain chart
Uniaxial Tensile Strength (stress as a function of strain)
Native vs Decellularized tissue comparison
Decellularized tissue behavior under uniaxial stress is similar
to native tissue.
Controls: native tissue (circumferential, radial)
Critique: testing remained in elastic region of stress-strain
curve, should have continued until ultimate stress/ fracture
point. Also, should include the raw data table.
Figure 1L
Method: Uniaxial
Tensile Testing

14. Figure 2 A-IqPCR
Passage 2
Passage 5
Fig 2 ABC
Cell Marker Expression
Passage 2 vs 5 MPCs
Fig 2 D-I
Passage 5 MPCs, effect
of TGF-β conditioning
at various concentrations
0.1 ng/mL
1.0 ng/mL 10 ng/mL
Fig 2 J
Vascular profile of MPCs
• Encapsulated / not
• TGF-β / not
TGF-β,
encap
No TGF-β
encap No TGF-β
No encap
TGF-β,
no encap
Whole genome
array

15. Figure 2 A-I
Obtained MPCs from bone
marrow donors
Expanded MPCs in
DMEM up to passage 5
and cultured for 7d:
• with and without TGF-β
(neg control)
• in monolayers and on
composite scaffolds (neg
control)
RNA isolated from fresh cells
using RNAqueous Isolation Kit
DNA contamination removed
enzymatically
Built cDNA library using 1ug
RNA; Reaction Ready cDNA
Synthesis Kit and Biorad
MyCycler
PCR arrays run on Stratagene
MC-3005 cycler
PCR arrays analyzed with
MxPro 3.0 software
FC expressed using DCt
method, raw data normalized
to RPL32 housekeeper QuantitativePCR
Method: qPCR

16. Figure 2 ABC
MPC Phenotype Characterization
Compares the expression of developmental, cardiac,
and vascular cell markers between passage 2 and
passage 5 MPCs
At all passages, cells demonstrated high levels of mRNA
for several developmentally relevant receptors
Y-axis: increase or decrease of expression between
passage 5 and passage 2
C: Significant up-regulation for some vascular markers
observed in passage 5.
Passage 5 MPCs were used for all further experiments
due to their enhanced vascular profile
Critiques:
• No controls used
• Use of mRNA instead of measuring protein
expression directly
• Figures only shows data from passages 2
and 5, should show data from all passages
• Y-axis shows difference in expression
between passage 5 and passage 2, it should
show the actual measured values instead
• No explanation for why expression of
vascular markers is higher in passage 5
MPCs versus previous passages

17. Figure 2 DEFGHI
MPC Phenotype Characterization
Shows how conditioning MPCs with various
concentrations of TGF-β affects the expression of various
markers in passage 5 cells
TGF-β is implicated in vascular morphogenesis
Four treatments:
• No TGF-β concentration (0.0 ng/mL)
• Low TGF-β concentration (0.1 ng/mL)
• Medium TGF-β concentrarion (1.0 ng/mL)
• High TGF-β concentrarion (10.0 ng/mL)
Graphs show how expression of 4 osteochondral markers
(D-G) and 2 vasculogenic markers (H-I) changed with
various concentrations of TGF-β
Controls:
Negative control (TGF-β = 0.0 baseline for comparison)
Critiques:
• Use of mRNA instead of measuring protein expression directly
• Should test other growth factors involved in angiogenesis such as AC133, Ang1, Ang2
• Y-axis shows difference in expression, should show the actual measured values
• Should keep increasing concentration of TGF-β until a plateau is reached
• Significance of up-regulation or down-regulation of these markers in passage 5 MPCs is not addressed.

18. Figure 2 J
Obtained MPCs from bone
marrow donors
Expanded MPCs in
DMEM up to passage 5
and cultured for 7d:
• with and without TGF-β
(negative control)
• with and without fibrin
encapsulation (negative
control)
Encapsulated in Fibrin
NOT Encapsulated in Fibrin
TGF-β
TGF-β
no
TGF-β
no
TGF-β
STRO-1 –selected MPCs expanded
to passage 5
Cultured with OR without
(negative control) TFG-β for 5d
Encapsulated in fibrin OR kept in
monolayer culture ( negative
control ); with OR without TFG-β
supplementation, for 2 d
Samples flash-frozen in LN2 and
shipped on dry ice
RNA isolated, samples profiled
with Ilumina Human HT-12 v4
bead chips
Method:
Whole Genome RNA Array

19. Shows how gene expression of vascular markers
in MPCs was affected by fibrin encapsulation and
by TGF-β conditioning.
Independent confirmation of vascular profile of
MPCs
Some markers were up-regulated or down-
regulated significantly
Controls:
• negative controls: no TGF-β, no fibrin
encapsulation
Critiques:
• x-axis is supposed to show MPCs (TFGβ +
fibrin encapsulation) vs MPCs (no TFGβ, no
fibrin encapsulation), but it not clear which is
which
• y-axis shows difference in expression, should
show the actual measured values
• Significance of up-regulation or down-
regulation of these markers in passage 5 MPCs
is not addressed.
• RNA quantity not always a good predictor of
actual protein expression
Figure 2 J

20. Figure 2 J
Critiques:
This would be a better way to present the
results for Figure 2:
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
SDF-1 EDN-1 VEGFA VEGFB
MPCs + TGFβ + fibrin
MPCs + TGFβ
MPCs + fibrin
MPCs only
vascular markers
Expression(pg/mL)

21. Figure 3
1 – 3: MPCs conditioned with TGF-β, seeded into
scaffold
1: acute MI 2: chronic MI 3: normal mice
4 – 6: MPCs with no TGF-β, seeded into scaffold
4: acute MI 5: chronic MI 6: normal mice
7: scaffold without MPCs, without TGF-β
7: acute MI
Passage-5 MPCs cultured with TGF-
β for 7d
BuildCompositeScaffold
106 conditioned MPCs loaded into
decellularized scaffold,
polymerized for 3 min, added 0.1
ng/mL TGF-β to experimentals, not
to controls
• Scaffolds with MPCs conditioned
with TGF-β
• scaffolds with nonconditioned
MPCs (neg control)
• scaffolds with no cells (neg
control)
Anesthesize rats, exposed heart
through left thoracotomy
Attached engineered patch with
sutures to the infarct bed in the
myocardium
Implant/Explant
Explant hearts after 4 weeks &
analysis
1 2 3
4 5 6
7
TGF-β
No TGF-β
No TGF-β, no MPCs
Acute MI Chronic MI normal

22. Figure 3 A-K
After 4 weeks of implantation, acute MI mice with SMT showed enhanced angiogenesis and
arteriogenesis on infarct bed
• A-D: Factor VIII – positive vessels per view field (indicator of angiogenesis)
• Number of vessels in SMT > SM > S
• E-H: SMA-positive vessels per view field (smooth muscle actin)
• Number of vessels in SMT > SM > S
• I : TGF-β increased # of MPCs in infarct bed
• J-K: MPCs found close to endogenous vessels
• Method : light microscopy
SMT = scaffold +
MPCs + TGF-β
SM = scaffold +
MPCs
S = scaffold only

23. • Controls:
o Scaffold with MPCs but no TGF-β (negative)
o Scaffold without MPCs or TGF-β (negative)
o Normal mice vs acute MI vs chronic MI (negative)
• Critique:
o Negative controls should include normal (healthy), acute MI and
chronic MI mice without any treatment
o Negative controls should include scaffold without MPCs or TGF-β for
chronic MI mice, not only acute MI mice
o Should include photos of tissues from mice without any treatment, to
compare against B-D, F-H, J-K. How do we know if tissue was ok and
the scaffold without MPCs or TGF-β damaged it?
o In 3J-K, they should have included photos of healthy hearts, and
untreated diseased hearts for comparison.
Figure 3 A-K

24. Figure 3 L-P
Presence of TGF-β conditioned MPCs on
scaffolds in acute MI, chronic MI, and no MI
models
• [L-O] Greater cell migration into infarct bed for
SMT than for SM
• Yellow arrows = cell clusters
• No migration of MPCs into healthy myocardium
• Regions of inflammation stimulated MPC
migration, for example wall adhesions (3N),
microinfarction caused by sutures (3O).
• Controls:
o normal tissue / no infarct
• Critique:
o should include photos of untreated normal
tissue and untreated infact tissue (acute and
chronic) for comparison

25. Figure 3 Q
• Shows how conditioning passage 5
MPCs with various concentrations of
TGF-β affected the expression of SDF-1,
four weeks after implantation of the
scaffold
• Passage 5 MPCs show greater expression
of SDF-1 when supplemented with TGF-
β
• Method: Q-PCR
Controls:
• no TGF-β (negative control, not shown)
Critique
• Y-axis shows difference in expression,
should show the actual measured values
• the baseline (MPCs not supplemented
with TGF-β, negative control) is not
shown
• Should increase TGF-β concentration
until graph reaches a plateau.

26. Figure 3R
• ELISA of supernatants from MPCs after culture with or without TGF-
β and with or without hypoxia.
• Expression of SDF-1 increases as TGF-β concentration increases
• Culture under hypoxic conditions showed the highest expression
level of SDF-1, means cells can recover their capability to release
SDF-1 upon migration into the ischemic infarct bed
Controls:
• no TGF-β (negative)
• Hypoxia induced / not induced (negative)
Critique
• Only MPCs without TGF-β were grown under hypoxic conditions.
Experiment should have included MPCs grown under hypoxic
conditions, conditioned with various concentrations of TGF-β .
106 MPCs cultured for 7d in
DMEM supplemented with
TGF-β. Media changed every 3d
SDF-1 ELISA normalized to cell
number to exclude proliferative
effects of TGF-β
Method:
Hypoxic Cell Culture
and SDF-1 ELISA
For hypoxia, cells were
cultured in environmental
chamber for 24h under 93%
nitrogen, 5% CO2, and 2% O2,
with O2 monitoring
5 groups: MPCs conditioned
with 0, 0.1, 1.0, 10.0 ng/mL of
TGF-β; MPCs grown under
hypoxic conditions

27. • Western blot demonstrates SDF-1 protein expression.
• Shows that SDF-1 expression increases as the
concentration of TGF-β increases.
Controls:
• GAPDH (loading control)
• no TGF-β (negative)
Critique:
• Blot photo does not show a clear difference between
TGF-β = 0, 0.1, or 1.0 ng/mL
• Western Blot does not show a line for hypoxia, probably
because the test is not sensitive enough to detect/display a
difference
• Method used for Western Blot not described in paper
Figure 3S
MPCs were treated with 0, 0.1,
1.0, 10.0 ng.mL of TGF-β
Lyse cells with detergent
Method:
Western Blot
Centrifuge cells, wash pellets
with lysis buffer 3 times
Immunoprecipitates dissolved
Stain with primary antibody,
wash, incubate with secondary
antibody
Analyze with Western blotting

28. • Matrigel assay of coronary
endothelium with (Upper) and
without (Lower) SDF-1
supplementation.
• Supplementation of exogenous
SDF-1 to coronary endothelium
enhances and stabilizes
endothelial network formation
• As time increases, EC + SDF-1
shows more and thicker lines
Control:
• With / without SDF-1
supplementation (negative control)
Critique:
• Needs to show photos for time = 0
hours for proper comparison
• Method for Matrigel Assay not
included in paper
Thaw Matrigel at 4°C overnight
on ice . Chill pipettes, plates
and tips at -20°C .
Wash cells 3 times with DMEM
containing 1% FBS
Figure 3T
Method: Matrigel
Assay
(typically used to study metastatic
potential of tumor cells)
Resuspend 10^6 cells/mL in
media with 1% FBS
Wash gelled matrigel with
serum free-culture media, and
put 100 uL of cell suspension in
Matrigel
Incubate at 37°C for 20h – 40h
(depends on cell type)
Stain with DiffQuick solution,
scrape off non-invaded cells
View / count invaded cells
under light microscope

29. Figure 4
Changes in ventricular function after composite scaffold implant,
after 4 weeks
• Baseline : no myocardial infarctium (healthy) (negative
control)
• Control: induced MI, no treatment (negative control)
• MPC: MPCs with TGF-b delivered by intramyocardial
injection (neg control for scaffold)
• Patch: MPCs with TGF-b delivered by scaffold
• MPC and Patch show the greatest improvement
• Patch performance is superior to injected MPCs
• Method : Echocardiogram
Rats scanned in parasternal
short-axis view using anesthesia
2D and M-mode imaging using
Sonos 5500 System and
broadband high frequency
Calculated left ventricular FS
and FAC
Measured LVDA and LVSA
Measured maximum and
minimum values in cardiac
cycle by tracing endocardial LV
border
Method: Echocardiography

30. Suggested Experiment
• Quantify expression of paracrine factors other than SDF-1 (ANGPT-1,
EDN-1, EDNRA, FLT1, VCAM, CX43, SMA, SM-MHC, TIE1, TIE2,
Myocardin) to better characterize MPC’s response to TGF-β, using a
protein microarray.
0.0 (negative
control)
0.1 ng/mL 1.0 ng.mL
10.0 ng/mL
10^6 MPCs, cultured for 7 days
in DMEM, supplemented with
TGF-β in various concentrations
Lyse the cells. Arrays are
incubated with lysate
Arrays are treated with
secondary fluorescence-labeled
antibody (Cy3, Cy5) for
visualizing interactions of
array antibodies with vascular
factors of interest
Slides are scanned with high
resolution convocal scanner,
fluorescent signals are digitized
Run computer algorithms for
data normalization,
multivariable statistics
1
1 2
2
3 4
3 4

31. Suggested Experiment
-30
-20
-10
0
10
20
30
40
Up/downregulation
(Xfold)
Vascular factors
0.1 ng/mL TGFβ
1.0 ng/mL TGFβ
10 ng/mL TGFβ

32. Summary
What they Did
• Developed a cell therapy
approach to treat myocardial
infarction (tissue damage after
heart attack)
• Combined the use of:
o Decellularized scaffold to
deliver cells to damaged
heart
o Stem cells (MPCs)
conditioned to maximize
ability to revascularize and
improve blood flow to the
damaged tissue
Results
• Composite scaffold promoted
growth of new blood vessels
• Composite scaffold released
signaling molecules that
stimulated native tissue to
repair itself

34. Glossary
Term Definition
aSMA Smooth muscle actin
Cells Passage
Passage is the # of times a cell line has been re-plated and allowed to grow back to confluency. Each time you
trypsinize an adherent cell line to replate it, add +1 to the passage number. The higher the passage #, the farther away
you are from the primary explant the cell line was derived from. Keep track of the passage number, to make sure you
don't lose properties you want to study from that cell line
Chelating Combine metal ion with a chemical compound to form a ring, remove heavy metal from bloodstream
confluency
confluency is commonly used as a measure of the number of the cells in a cell culture dish or a flask and refers to the
coverage of the dish or the flask by the cells. For example, 100 percent confluency means the dish is completely
covered by the cells and there is no more room left for the cells to grow whereas 50 percent confluency means
roughly half of the dish is covered and there is room for cells to grow.
DMEM Dulbecco Modified Eagle Medium, cell culture medium (w/ aminoacids, salts, glucose, vitamins) for mammalian cells
Dnase Catalyzes hydrolysis of DNA
EDTA Crystalline acid, strong chelating agent
Endothelium Thin layer of cells that lines interior surface of blood vessels
expand Allow cells to multiply
Factor VIII Blood clotting factor, essential
Fibronectin
Fibronectin is a high-molecular weight (~440kDa) glycoprotein of the extracellular matrix that binds to membrane-
spanning receptor proteins called integrins. In addition to integrins, fibronectin also binds extracellular matrix
components such as collagen, fibrin and heparan sulfate proteoglycans (e.g. syndecans).
HBSS Hank's balanced salt solution
IS buffer Used to buffer solutions from drastic pH changes, maintain them at ph 7 - 9
ischemic Restriction in blood supply
Matrigel Substrate for cell culture, stimulates complex cell behavior
MSC, MPC
Mesenchymal stem cells, these are multipotent stem cells that can differentiate into osteoblasts (bone), chondrocytes
(cartilage), adipocytes (fat cells).

35. Glossary
Term Definition
PBS Phosphate buffered saline solution
Rnase Catalyzes hydrolysis of RNA
Scaffold
Fully decellularized human myocardium,with preserved composition, structure, mechanics of ECM & fibrin hydrogel
containing human mesenchymal stem cells conditioned in vitro, in nude rat implant model, using molecular,
structural, and functional assays.
SDS Detergent that dissolves lipids
SDF-1 Stromal cell-derived factors 1-alpha and 1-beta are small cytokines that belong to the intercrine family
STRO-1
Cell surface marker protein, STRO-1 + cells are generally more homogeneous, have higher rates of adherence and
higher rates of proliferation. It's not clear what are the differences between STRO-1+ cells and MSC
STRO-3 monoclonal antibody used as a tag, reacts with STRO-1 cells
TGF β
Transforming growth factor beta, controls proliferation, cell differentiation, other functions. Antiproliferative factor in
normal epithelial cells
Trypsinization
Trypsinization is the process of using trypsin, a proteolytic enzyme which breaks down proteins, to dissociate
adherent cells from the vessel in which they are being cultured. When added to a cell culture, trypsin breaks down
the proteins which enable the cells to adhere to the vessel. Trypsinization is often used to passage cells to a new
vessel.
Vitronectin
Vitronectin also known as VTN is a protein which in humans is encoded by the VTN gene. The protein encoded by this
gene is a member of the pexin family. Vitronectin is an abundant glycoprotein found in serum and the extracellular
matrix and promotes cell adhesion and spreading, inhibits the membrane-damaging effect of the terminal cytolytic
complement pathway, and binds to several serpin serine protease inhibitors