Development of cell sheet constructs for layer by layer tissue engineering using the blood vessel as an experimental model 3

Chapter 4. Isolation and Characterisation Of Vascular Progenitors
124

4.2.2.2 Matrigel tube formation assay
I then employed the Matrigel tube formation assay to compare the vasculogenic
capacity of fetal blood and cord blood derived EPC. When the cells were trypsinised
and transferred to a Matrigel, substrate, established networks were formed from both
groups within 16 hours. However, it was noted that FB-RPC was capable of more
robust tube formation, resulting in networks visible to the naked eye. Microscopic
examination of the networks revealed structures indicative of secondary angiogenic
sprouting.

Figure 4-5: Matrigel vascularisation assay
Tube formation in Matrigel by (a) UCB-EPC and (b) FB-EPC. Greatly increased
network formation was found in FB-EPC, with secondary angiogenic sprouting
(indicated with red arrow heads). (c) Networks were visible to the un-aided naked eye

(a)
(b) (c)
Chapter 4. Isolation and Characterisation Of Vascular Progenitors
125

4.2.2.3 Immunocytochemistry
Characterisation of the cells was conducted through immunocytochemical staining at
passage four. Endothelial-like cells were derived from both UCB-EPC and FB-EPC,
as evidenced by the expression of endothelial markers CD31 (PECAM), CD144 (VE-
Cadherin) and vWF (von Willebrand Factor).

It was noted, however, that the staining pattern was different across both groups.
While the markers were consistently expressed in UCB-EPC, CD31 and CD144 were
only expressed on some clusters of FB-EPC. The stained cells were also noted to

adopt a more fibroblastic orphology. Similarly, vWF was sporadically expressed on
the cells.

A functional assay of acLDL uptake was performed to confirm the endothelial nature
of the cells. In contrast to the immunostaining results, acLDL was taken up by more
than 90% of all cells in both groups, suggesting their functional endothelial identity.

Chapter 4. Isolation and Characterisation Of Vascular Progenitors
126


Figure 4-6: Expression of endothelial markers
Immunocytochemical assays demonstrate the expression of surface endothelial
markers CD31 (PECAM) and CD144 (VE-Cadherin). Cells also express von
Willebrand factor in well defined Weider-Palade bodies, albeit at reduced levels in
FB-EPC. All cells took up acetylated low density lipoproteins (acLDL), seen as
perivascular vesicles in the cytosol, an indication of endothelial functionality. Cell
nuclei were counterstained (represented in red).
UCB-EPC FB-EPC
CD31
CD144
vWF
acLDL
Chapter 4. Isolation and Characterisation Of Vascular Progenitors
127


4.2.2.4 Microarray analysis of blood outgrowth cells
To further elucidate differences between UCB-EPC and FB-EPC, I proceeded to
compare the differences in expression of the two populations (n=1) through a whole

genome expression array approach. As the cell population appeared to be
contaminated with fibroblast-like cells, I decided to carry out MAC Sorting to enrich
the population for CD31 (an endothelial marker) expressing cells prior to RNA
extraction for microarray. The selected cells were plated overnight to reduce changes
in expression level due to the sorting process. The quality of extracted RNA was
assessed using spectrophotometry and formaldehyde gel electrophoresis prior to
labelling for microarray.

Figure 4-6: Assessment of RNA quality.
Quantity of RNA was assessed by spectrophotometry prior to labelling for microarray.
Sufficient, good quality RNA was obtained, as determined by denaturing gel
electrophoresis.

Sample
Average concentration
(ng/ul) A260 A280

260/280

260/230

FB-EPC 220 5.623 2.674

2.1 1.24
UCB-EPC 646 16.151

7.584

2.13 1.48
FB-

EPC
UCB-
EPC
28S
18S
Chapter 4. Isolation and Characterisation Of Vascular Progenitors
128

Analysis of the microarray data shows 27,530 out of 55,000 genes to be appreciably
expressed in either line. The profiles of both groups were largely similar, and thus, of
these genes, 434 were found to be differentially expressed by five-fold, with 251
upregulated and 183 downregulated in fetal blood. To identify genes of interest, I then
proceeded to use the Gene Ontology Browser to identify processes associate with
these genes. In particular, I chose to look for overlaps with ontologies associated with
the development process which may be significant. Thus, I identified 33
developmental processes that were upregulated in FB (summarized in Table 4-1)
comprising 47 genes (Table 4-3) and 13 processes that were upregulated in UCB-EPC
(Table 4-2), comprising 33 genes (Table 4-4).

Studying the processes which have been significantly upregulated in FB-EPC, a
correlation with processes involved in blood vessel formation was found, including
angiogenesis, blood vessel morphogenesis, vasculature development and blood vessel
development, and thus supporting a possible greater vasculogenic potential of FB-
EPC. Six genes were found to be common to these four processes: Kinase insert
domain receptor, Jagged 1, Placental Growth Factor, Interleukin 8, angiopoietic-like 4
and roundabout homolog 4.

In contrast, UCB-EPC are largely involved directing cell differentiation, as well as
processes involving regulation of cell growth and size. The common genes involved
in regulation of cell growth and size are HtrA serine peptidase 1, insulin-like growth

factor binding protein 4, suppressor of cytokine signalling 2, insulin-like growth
factor binding protein 6, phospholipase C an dubiquitin conjugating enzyme E2E 3.
Chapter 4. Isolation and Characterisation Of Vascular Progenitors
129


Figure 4-7: Comparative gene expression of UCB-EPC versus FB-EPC
(a) Microarray analysis shows differentially regulated genes in UCB-EPC compared
with FB-EPC (b) 434 genes demonstrate five-fold differences in gene expression.
UCB

FB

Chapter 4. Isolation and Characterisation Of Vascular Progenitors
130

Category
Genes in
Category

% of
Genes in
Category
Genes in
List in
Category

% of
Genes in
List in

Category

p-Value
GO:7275: development 4849

16.29

71

38.59

2.82E-13

GO:9653: morphogenesis 1562

5.246

31

16.85

8.52E-09

GO:30154: cell differentiation 1285

4.316

25

13.59

4.03E-07

GO:30216: keratinocyte differentiation 26

0.0873

5

2.717

5.05E-07

GO:1525: angiogenesis 183

0.615

9

4.891

2.23E-06

GO:48514: blood vessel morphogenesis 190

0.638

9

4.891

3.04E-06

GO:1944: vasculature development 190

0.638

9

4.891

3.04E-06

GO:1568: blood vessel development 190

0.638

9

4.891

3.04E-06

GO:1709: cell fate determination 45

0.151

5

2.717

8.53E-06

GO:45446: endothelial cell differentiation 23

0.0772

4

2.174

1.14E-05

GO:45165: cell fate commitment 58

0.195

5

2.717

3.00E-05

GO:48637: skeletal muscle development 96

0.322

6

3.261

3.00E-05

GO:48741: skeletal muscle fiber development 96

0.322

6

3.261

3.00E-05

GO:48747: muscle fiber development 96

0.322

6

3.261

3.00E-05

GO:45445: myoblast differentiation 60

0.202

5

2.717

3.54E-05

GO:42692: muscle cell differentiation 68

0.228

5

2.717

6.49E-05

GO:9887: organ morphogenesis 530

1.78

12

6.522

0.000123

GO:7519: striated muscle development 149

0.5

6

3.261

0.000339

GO:8360: regulation of cell shape 100

0.336

5

2.717

0.0004

GO:45596: negative regulation of cell differentiation 27

0.0907

3

1.63

6.09E-04

GO:45671: negative regulation of osteoclast differentiation 7

0.0235

2

1.087

0.000782

GO:45670: regulation of osteoclast differentiation 8

0.0269

2

1.087

0.00104

GO:51093: negative regulation of development 35

0.118

3

1.63

0.00131

GO:48534: hemopoietic or lymphoid organ development 217

0.729

6

3.261

0.00237

GO:30097: hemopoiesis 217

0.729

6

3.261

0.00237

GO:30316: osteoclast differentiation 12

0.0403

2

1.087

0.00241

GO:45656: negative regulation of monocyte differentiation 12

0.0403

2

1.087

0.00241

GO:45638: negative regulation of myeloid cell differentiation 13

0.0437

2

1.087

0.00283

Chapter 4. Isolation and Characterisation Of Vascular Progenitors
131

GO:48513: organ development 1524

5.118

19

10.33

0.00293

GO:45655: regulation of monocyte differentiation 15

0.0504

2

1.087

0.00378

GO:46847: filopodium formation 19

0.0638

2

1.087

0.00606

GO:902: cellular morphogenesis 770

2.586

11

5.978

0.00867


Table 4-1: List of ontological processes upregulated in FB-EPC compared to UCB-EPC

Category

Genes in
Category

% of
Genes in
Category
Genes in
List in
Category

% of
Genes in
List in
Category

p-Value
GO:7275: development 4849

16.29

43

33.08

1.89E-06

GO:9653: morphogenesis 1562

5.246

16

12.31

0.0013

GO:48513: organ development 1524

5.118

14

10.77

0.00683

GO:45666: positive regulation of neuron differentiation 7

0.0235

2

1.538

0.000392

GO:45664: regulation of neuron differentiation 22

0.0739

2

1.538

0.00413

GO:8361: regulation of cell size 435

1.461

7

5.385

0.00307

GO:16049: cell growth 435

1.461

7

5.385

0.00307

GO:1502: cartilage condensation 25

0.084

2

1.538

0.00531

GO:1558: regulation of cell growth 317

1.065

7

5.385

0.000502

GO:48627: myoblast development 13

0.0437

2

1.538

0.00143

GO:48628: myoblast maturation 13

0.0437

2

1.538

0.00143

GO:7520: myoblast fusion 12

0.0403

2

1.538

0.00121


Table 4-2: List of ontological processes downregulated in FB-EPC compared to UCB-EPC
Chapter 4. Isolation and Characterisation Of Vascular Progenitors


132


221009_s_at angiopoietin-like 4
201005_at CD9 antigen (p24)
204470_at chemokine (C-X-C motif) ligand 1 (melanoma growth stimulating activity, alpha)
221731_x_at chondroitin sulfate proteoglycan 2 (versican)
201852_x_at collagen, type III, alpha 1 (Ehlers-Danlos syndrome type IV, autosomal dominant)
202157_s_at CUG triplet repeat, RNA binding protein 2

201430_s_at dihydropyrimidinase-like 3
229800_at Doublecortin and CaM kinase-like 1
203881_s_at dystrophin (muscular dystrophy, Duchenne and Becker types)
208297_s_at ecotropic viral integration site 5
208394_x_at endothelial cell-specific molecule 1
203184_at fibrillin 2 (congenital contractural arachnodactyly)
1555480_a_at filamin binding LIM protein 1
218665_at frizzled homolog 4 (Drosophila)
227405_s_at frizzled homolog 8 (Drosophila)
230559_x_at FYVE, RhoGEF and PH domain containing 4
219901_at FYVE, RhoGEF and PH domain containing 6
209604_s_at GATA binding protein 3
204689_at hematopoietically expressed homeobox
201162_at insulin-like growth factor binding protein 7
211506_s_at interleukin 8
209098_s_at jagged 1 (Alagille syndrome)
201596_x_at keratin 18
203934_at kinase insert domain receptor (a type III receptor tyrosine kinase)
221841_s_at Kruppel-like factor 4 (gut)
204153_s_at manic fringe homolog (Drosophila)
202291_s_at matrix Gla protein
209086_x_at melanoma cell adhesion molecule
218678_at nestin
202149_at neural precursor cell expressed, developmentally down-regulated 9
204105_s_at neuronal cell adhesion molecule
218902_at Notch homolog 1, translocation-associated (Drosophila)
210809_s_at periostin, osteoblast specific factor
209652_s_at placental growth factor, vascular endothelial growth factor-related protein
1559400_s_at pregnancy-associated plasma protein A, pappalysin 1
205128_x_at

prostaglandin-endoperoxide synthase 1 (prostaglandin G/H synthase and
cyclooxygenase)
235131_at ras homolog gene family, member J
226028_at roundabout homolog 4, magic roundabout (Drosophila)
217591_at SKI-like
202363_at sparc/osteonectin, cwcv and kazal-like domains proteoglycan (testican)
212558_at sprouty homolog 1, antagonist of FGF signaling (Drosophila)
201998_at ST6 beta-galactosamide alpha-2,6-sialyltranferase 1
206283_s_at T-cell acute lymphocytic leukemia 1
235086_at Thrombospondin 1
232068_s_at toll-like receptor 4
204653_at transcription factor AP-2 alpha (activating enhancer binding protein 2 alpha)
209909_s_at transforming growth factor, beta 2

Table 4-3: List of genes upregulated in FB-EPC compared to UCB-EPC
Chapter 4. Isolation and Characterisation Of Vascular Progenitors


133


226777_at ADAM metallopeptidase domain 12 (meltrin alpha)
205609_at angiopoietin 1
214297_at Chondroitin sulfate proteoglycan 4 (melanoma-associated)
202436_s_at

cytochrome P450, family 1, subfamily B, polypeptide 1
226281_at delta-notch-like EGF repeat-containing transmembrane
227646_at Early B-cell factor
206115_at early growth response 3

225079_at epithelial membrane protein 2
210310_s_at

fibroblast growth factor 5
204451_at frizzled homolog 1 (Drosophila)
210220_at frizzled homolog 2 (Drosophila)
218469_at gremlin 1, cysteine knot superfamily, homolog (Xenopus laevis)
235521_at homeo box A3
228904_at Homeo box B3
201185_at HtrA serine peptidase 1
201508_at insulin-like growth factor binding protein 4
203851_at insulin-like growth factor binding protein 6
226534_at KIT ligand
210302_s_at

mab-21-like 2 (C. elegans)
228425_at Paired box gene 8
238852_at Paired related homeobox 1
205111_s_at

phospholipase C, epsilon 1
225975_at protocadherin 18
232231_at runt-related transcription factor 2
202037_s_at

secreted frizzled-related protein 1
206056_x_at

sialophorin (gpL115, leukosialin, CD43)
228347_at Sine oculis homeobox homolog 1 (Drosophila)

202935_s_at

SRY (sex determining region Y)-box 9 (campomelic dysplasia, autosomal sex-reversal)
222557_at stathmin-like 3
225665_at sterile alpha motif and leucine zipper containing kinase AZK
203372_s_at

suppressor of cytokine signaling 2
232528_at Ubiquitin-conjugating enzyme E2E 3 (UBC4/5 homolog, yeast)
205990_s_at

wingless-type MMTV integration site family, member 5A

Table 4-4: List of genes downregulated in FB-EPC compared to UCB-EPC
Chapter 4. Isolation and Characterisation Of Vascular Progenitors


134


4.2.2.5 Flow cytometry Analysis of fetal liver cells
As the adhesion culture failed to generate endothelial progeny from fetal liver cells, I
evaluated the liver mononuclear cell population using flow cytometry for markers
associated with EPC. The profile suggests that liver MNCs contains a significant
population of cells expressing endothelial markers (VEGFR2: 0.35%, CD31: 7.64%,
CD105: 6.34%). Significantly, a large proportion of cells were found to express
haematopoietic stem cell marker, including CD34 (10.7%) and CD133 (2.14%).
Similar to cord blood, the pan-haematopoietic marker CD45 was also highly
expressed (88.8%). These results suggested that EPC, which stem from
haematopoietic sources, do exist in fetal liver, and that enrichment had to be

employed prior to culture to provide a selective advantage over highly proliferative
fibroblasts.
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Endothelial markers
VEGFR2 0.35 ± 0.27
CD31 7.74 ± 7.26
CD105 6.34 ± 2.04
Stem cell markers
CD34 10.70 ± 3.26
CD133 2.14 ± 1.26
CD117 15.52 ± 10.84
Haematopoietic markers
CD45 88.77 ± 5.93

Figure 4-8: Flow cytometric analysis of surface expression markers on fetal liver
MNCs.
Fetal liver MNC express stem cell markers at a higher frequency than umbilical cord
blood counterparts. Of particular interest is the CD133+ population, where frequency
is 5.5 fold higher than that found in cord blood, as reported in literature (McGuckin
2004). These results suggest the presence of EPC in liver.


CD31

VEGFR

CD45

CD117

CD34

CD133

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136

4.2.2.6 Fetal liver selection and expansion
In light of the evidence to suggest the presence of EPC in the fetal liver, I then
proceeded to study the possible selection of EPC using immunoselection. CD133, a
stem/progenitor cell marker, was chosen because of the reported ability to distinguish
EPC from mature EC (Peichev 2000). Furthermore, CD133+ cells isolated from cord
blood has been shown to capable of differentiation towards endothelial lineage, and
for the construction of microvascular networks (Wu 2004).

Using enrichment by Magnetic Affinity Cell Sorting (MACS), purity was consistently
above 80% as determined by flow cytometry. Recovery rate, however, was highly
variable, and the CD133+ cell yield was 1.7±2.2% of total mononuclear cells (n=7).
Expansion in a cytokine-rich cocktail (detailed in 2.1.10.1) resulted in expansion of
the putative stem cell population, with a doubling time of 96 hours.
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137


Figure 4-9: Fetal liver CD133+ cells
Following immunoselection by Magnetic Affinity Cell Sorting (MACS), >80%
CD133+ enriched populations can be derived (Red: Isotype control, Blue: Liver MNC,
Black CD133 enriched population). These cells expand in culture with a doubling
time of about 96 hours.
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138


4.2.2.7 Haematopoietic assays
Following expansion for one week, I transferred a portion of the cells to semi-solid
cultures for haematopoietic colony forming assays as an assessment of the retention of
haematopoietic character following expansion. As seen in Figure 4-10, Colony
forming assays of expanded FL133+ cells generated CFU-E (Colony Forming Unit
Erythrocyte, 1.33/10
3
cells), BFU-E (Burst Forming Unit Erythrocyte, 3.00/10
3
cells),
CFU-M (Colony Forming Unit Macrophage, 11.7/10
3
cells), CFU-G (Colony
Forming Unit Granulocyte, 7.7/10
3

cells) and CFU-GEMM (Colony Forming Unit
granulocyte, erythrocyte, macrophage, megakaryocyte, 6.67/10
3
cells). My results
demonstrate that a range of haematopoietic colonies could be generated, suggesting
that the cells continued to retain HSC character. Of interest, cobblestone area forming
colonies (CAFC) were formed, which is taken as an indicator of haematopoietic stem
property (van Os 2008).

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139



CFU
Count
(colonies per 10
3
cells)

CFU-E 1.33±1.15
BFU-E 3.00 ± 1.00
CFU-M 11.7 ± 4.6
CFU-G 7.67 ± 2.51
CFU-GM 6.67 ± 1.15

CAFC 4.33 ± 2.51

Figure 4-10: Haemopoietic assays on FL-EPC
Colony forming assays of expanded FL133+ cells generated haemopoietic colonies
including (a) CFU-E (Colony Forming Unit Erythrocyte) (b) BFU-E (Burst Forming
Unit Erythrocyte) (c) CFU-M (Colony Forming Unit Macrophage) and (d) CFU-
GEMM (Colony Forming Unit granulocyte, erythrocyte, macrophage,
megakaryocyte). (e) Cobblestone Area Forming Colonies (CAFC), associated with
long term culture initiating colonies, were also found.
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140


4.2.2.8 Endothelial Differentiation
In parallel with the haematopoietic differentiation assay, I subjected a separate portion
of expanded CD133 cells to endothelial differentiation by removing Flt-3 ligand.
Lightly adherent colonies surrounding loose clusters of cells, reminiscent of blood
islands, could be observed over a week. Live staining with antibodies against CD31
suggests that the adherent cells are endothelial in nature. This was further supported
by the ability of the cells to take up acLDL. Such colonies, previously termed CFU-
Hills, are associated with angioblasts and the adult haemangioblast (Hill 2003; Loges
2004). However, the cells were unable to undergo further proliferation and gradually
diminished under extended culture.
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141


Figure 4-11: Endothelial differentiation of FL-EPC

Following transfer to endothelial differentiation medium, (a,b) tightly adherent
colonies surrounding loose clusters of cells (CFU-Hill) emerged after a week.
Adherent cells express (c) CD31 (green) and (d,e) take up acLDL (red)
(a)

(b)

(c)

(d)

(e)

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142


4.2.2.9 Immunocytochemistry
To confirm the capability of the putative liver-derived EPC (FL-EPC) to generate
endothelial progeny, I assayed for the expression of various endothelial markers,
namely CD31, CD144, VEGFR2, as well as vWF. The staining profile was compared
with FB-EPC, as well as HUVE cells (Figure 4-12).

As seen in Figure 4-12, terminally differentiated EC such as HUVEC exhibit
hexagonal morphology, and uniform staining of CD31 and CD144, localised at the
gap junctions. FB-EPC appeared elongated, and although CD31 and CD144 were
similarly found to be expressed at gap junctions, the positive cells appeared in tight
clusters, interspersed with negative cells. Consistent with previous studies,

endogenous VEGFR2 could be found in all three cell types distributed across the cell
cytotosol, possibly on endosomes, as well as on the plasma membranes (Ewan 2006).
Finally, vWF is found to be highly localised in granular ultrastructures corresponding
to Weibel-Palade bodies. In the case of FB-EPC, although similar granular structures
could be found, only a fraction of cells tested positive for vWF.

In comparison, FL-EPC are rounder and smaller, with similarly well-defined staining
of gap junction proteins (CD31 and CD144) restricted to cell periphery. I noted that
VEGFR2 expression was largely localised at the cell membrane. vWF staining pattern
was also found to be different, appearing only in some cells, and lacked organisation
within the cytosol.
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143



Figure 4-12: Immunocytochemical profile of fetal EPC
Fetal EPC are capable of generating endothelial progeny. Despite the obvious
differences in morphology, expression of endothelial markers are similar. An
exception is the expression of von Willebrand factor (vWF). Although vWF is
expressed in both FL-EPC and FBEOC, it is confined to well defined Weider-Pallade
bodies within FBEOC. vWF expression is lower in both FL-EPC and FBEOC as
compared to HUVEC, possibly due to the more primitive nature of the EPC.
.
Chapter 4. Isolation and Characterisation Of Vascular Progenitors


144

4.3 Discussion
4.3.1 Summary of Results
In this chapter, I detailed my efforts to identify good cellular sources for vascular
tissue engineering. While several studies have already demonstrated potential cells
sources, these are typically limited by the lack of capacity for proliferation and / or
functional differentiation. I have described the advantages of perinatal stem cell
sources for transplant therapies. In particular, previous work carried out in Professor
Nicholas Fisk’s laboratory has demonstrated that perinatal mesenchymal stem cell
sources exhibit an advantage over adult counterparts in terms of proliferation rate and
capacity (Guillot 2007), with four-fold more doublings achieved in culture over 50
days, 1.5 fold longer telomeres and up to 5.7 fold greater telomerase activity. Based
on these findings, I proceeded to study the use of perinatal vascular progenitor cell
sources for vascular tissue engineering.

UCPVC were found to demonstrate proliferation and clonogenic capability. Besides
displaying a profile suggesting mesenchymal stem cell character, UCPVC were found
to express smooth muscle cell differentiation markers actin and calponin, but not
myosin heavy chain (MHC). Serum starvation has previously been found to induce
differentiation of synthetic SMC cell types towards a contractile phenotype (Lavender
2005). Similarly, I found that serum starvation resulted in upregulation of actin and
calponin, and was accompanied by expression of MHC. This likely involves serum
response factor binding to CArG motifs and subsequent upregulation of smooth
muscle markers, (Han 2006). Indeed, increased expression of calponin was similarly
found when fetal MSC were similarly subjected to serum starvation. The capacity for
Chapter 4. Isolation and Characterisation Of Vascular Progenitors


145

expansion and differentiation support the use of UCPVC for vascular tissue
engineering.

I then proceeded to study perinatal sources of EPC. EPC derived from cord blood
have been well-reported (Ingram 2004), and I derived a similar population of cells by
adhesion selection, which were capable of generating endothelial progeny. Using the
same technique, I was able to derive a heterogeneous population of cells that
contained EPC. Endothelial progeny could be generated from this population, which
demonstrated visibly increased in vitro vasculogenic capability. Continued culture of
outgrowth cells eventually results in completely fibroblastic colonies with decreased
expression of endothelial markers. Microarray comparison of UCB and FB derived
EC demonstrate marked differences in expression profiles, including an increased
expression of genes associated with developmental vasculogenesis in FB EPC.
Although such primitive nature may be favourable for biomimetic tissue engineering
(Ingber 2006), the small numbers isolated from the limited blood sampling possible
(Chan 2008), the heterogeneity of the population, and difficulties associated in
enrichment and maintenance of phenotype makes it impracticable for vascular tissue
engineering currently. In my studies with fetal liver as an alternative haematopoietic
source of EPC, I found that adhesion selection was unfeasible. MACs enrichment
followed by expansion resulted in the generation of a haemangioblast-like population.
However, these cells were incapable of further expansion. In the light of these
findings, UCB currently represent the best source of EPC for vascular tissue
engineering.

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146

4.3.2 Critical Assessment
4.3.2.1 Umbilical Cord Perivascular Cells (UCPVC)
4.3.2.1.1 Isolation of MSC-like cells from perivascular regions of the umbilical
cord
Stem cell populations can be derived from umbilical cord tissue, including Wharton’s
Jelly (WJ), umbilical cord blood and the perivascular regions. In particular, WJ MSC
can be isolated in large starting numbers, and could be further expanded up to 80
population doublings (Mitchell 2003). WJ MSC are reported to differentiate along
bone, cartilage, adipose, as well as cardiomyogenic lineages (Wang 2004;
Karahuseyinoglu 2007). Aside from the Wharton’s Jelly, MSC-like cells have
reportedly been isolated from umbilical cord blood (UCB) (Erices 2000). Like WJ
MSC, UCB MSC have been shown to be capable of multilineage differentiation (Jang
2006). However, UCB MSC are notoriously difficult to isolate, with a high degree of
operator dependency. Consequently, conflicting data on the frequency of these cells
range from 1 CFU-F from every 10
6
mononuclear cells seeded to 3 CFU-F per 10
6

cells (Goodwin 2001; Bieback 2004). Even with optimised harvesting procedures, the
highest reported efficiencies at 63% of 27 samples capable of generating MSC
colonies (Bieback 2004), which has not been reproduced by other laboratories.

More recently, the perivascular region has been shown to be a source of MSC-like
pericytes, referred to as human umbilical cord perivascular cells (referred to here as
UCPVC) (Sarugaser 2005). This is in line with recent evidence provided by Crisan et
al suggesting a perivascular origin for all MSC (Crisan 2008). Indeed, UCPVC have
been found to express the panel of mesenchymal stem cell markers, and are similarly
Chapter 4. Isolation and Characterisation Of Vascular Progenitors

147

capable of multilineage differentiation. In addition, compared to adult bone marrow
derived MSC, UCPVC were found to proliferate twice as fast, and exhibit 1.2 times
greater clonogenecity (Zhang 2009).

4.3.2.1.2 Characterisation of smooth muscle cell character
Following optimisation of the harvesting protocol, I was able to successfully retrieve
UCPVC from all umbilical cord artery samples (n=5). Besides mesenchymal stem cell
markers, I also found that some UCPVC express smooth muscle actin and calponin,
suggesting a predisposition towards smooth muscle lineage. This is in agreement with
evidence showing a dependence of mesenchymal stem cell character on anatomical
origins (in 't Anker 2003; Koga 2008; Zhang 2009). Although smooth muscle Myosin
Heavy Chain (smMHC) was initially not expressed, culture in serum-free conditions
resulted in increased expression, accompanied with upregulation of actin and calponin.
My results are consistent with previous studies on smooth muscle differentiation,
demonstrating the gradual increase of late differentiation markers (Galmiche 1993). In
a separate study, Han et al reported on the harvest of a similar population of cells,
which they termed umbilical vascular smooth muscle cells. Following serum
starvation, they report a similar increased expression of smooth muscle markers,
including calponin and MHC (Han 2006). Tamama et al describe the induction of
bone marrow MSC towards smooth muscle cell lineage by serum starvation, with the
upregulation of early- and mid- smooth muscle cell differentiation markers (Tamama
2008). However, MEK inhibition was further required to induce expression of myosin
heavy chain. Similarly, I found that fMSC could be induced to upregulate calponin by
serum starvation, but failed to express myosin heavy chain.

Chapter 4. Isolation and Characterisation Of Vascular Progenitors

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4.3.2.2 Fetal blood endothelial progenitor cells (FB-EPC)
4.3.2.2.1 Adhesion selection of EPC
Endothelial and haematopoietic cells are believed to originate from a common
haemangioblastic precursor. Hence, EPC were first shown to be present in adult
peripheral blood, and later, in umbilical cord blood (Asahara 1997; Murohara 2001).
Adhesion plating has been shown to be capable of isolating a hierarchy of endothelial
progenitor cells (Ingram 2004), and similarly, I have demonstrated here the presence
of EPC in fetal blood, with cells expressing endothelial markers. However, in
comparing UCB-EPC with FB-EPC, I found that FB-EPC represent a more
heterogeneous population. In the CFU generation assays, aside from cobblestone
morphologies, many colonies could be found with spindle shaped morphologies.
These cell populations are reminiscent of fetal MSC (fMSC). Similarly, the
immunocytochemistry profile suggests a heterogeneous population, with non-uniform
expression of endothelial markers. Like EPC, MSC are known to be closely
associated with haematopoietic stem cells. fMSC have previously been shown to exist
in fetal circulation, and can be isolated from first trimester fetal blood by adhesion
selection in FBS-supplemented DMEM, at a frequency of 8.2 colonies per 10
6
cells
seeded (Campagnoli 2001). These fetal blood MSC (FB MSC) are capable of at least
twenty passages, demonstrate rapid cycling times, and in vitro differentiation towards
bone, cartilage and fat could be achieved. In the same paper, Campagnoli reported
reduced fMSC frequency in second trimester blood (1.3/10
6
cells) and cord blood
from term deliveries (0.35/10
6

cells). In a separate study, Jawed et al showed the
generation of a similar population of cells by culture in EGM-2 on collagen coated
surfaces, appearing at a frequency of 0.15 cells/10
6
cells during early second trimester
(24 – 28 weeks gestational age), and diminishing frequencies with increased