Chapter 4. Isolation and Characterisation Of Vascular Progenitors 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. (a) (b) (c) 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 124 Chapter 4. Isolation and Characterisation Of Vascular Progenitors 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 (VECadherin) 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. 125 Chapter 4. Isolation and Characterisation Of Vascular Progenitors UCB-EPC FB-EPC CD31 CD144 vWF acLDL 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). 126 Chapter 4. Isolation and Characterisation Of Vascular Progenitors 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. Sample FB-EPC UCB-EPC Average concentration (ng/ul) 220 646 FBEPC A260 A280 260/280 260/230 5.623 2.674 2.1 1.24 16.151 7.584 2.13 1.48 UCBEPC 28S 18S 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. 127 Chapter 4. Isolation and Characterisation Of Vascular Progenitors 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 FBEPC. Six genes were found to be common to these four processes: Kinase insert domain receptor, Jagged 1, Placental Growth Factor, Interleukin 8, angiopoietic-like 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. 128 Chapter 4. Isolation and Characterisation Of Vascular Progenitors UCB FB 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. 129 Chapter 4. Isolation and Characterisation Of Vascular Progenitors Category GO:7275: development GO:9653: morphogenesis GO:30154: cell differentiation GO:30216: keratinocyte differentiation GO:1525: angiogenesis GO:48514: blood vessel morphogenesis GO:1944: vasculature development GO:1568: blood vessel development GO:1709: cell fate determination GO:45446: endothelial cell differentiation GO:45165: cell fate commitment GO:48637: skeletal muscle development GO:48741: skeletal muscle fiber development GO:48747: muscle fiber development GO:45445: myoblast differentiation GO:42692: muscle cell differentiation GO:9887: organ morphogenesis GO:7519: striated muscle development GO:8360: regulation of cell shape GO:45596: negative regulation of cell differentiation GO:45671: negative regulation of osteoclast differentiation GO:45670: regulation of osteoclast differentiation GO:51093: negative regulation of development GO:48534: hemopoietic or lymphoid organ development GO:30097: hemopoiesis GO:30316: osteoclast differentiation GO:45656: negative regulation of monocyte differentiation GO:45638: negative regulation of myeloid cell differentiation Genes in Category 4849 1562 1285 26 183 190 190 190 45 23 58 96 96 96 60 68 530 149 100 27 35 217 217 12 12 13 % of Genes in Category 16.29 5.246 4.316 0.0873 0.615 0.638 0.638 0.638 0.151 0.0772 0.195 0.322 0.322 0.322 0.202 0.228 1.78 0.5 0.336 0.0907 0.0235 0.0269 0.118 0.729 0.729 0.0403 0.0403 0.0437 Genes in List in Category 71 31 25 9 9 5 6 5 12 2 6 2 % of Genes in List in Category 38.59 16.85 13.59 2.717 4.891 4.891 4.891 4.891 2.717 2.174 2.717 3.261 3.261 3.261 2.717 2.717 6.522 3.261 2.717 1.63 1.087 1.087 1.63 3.261 3.261 1.087 1.087 1.087 p-Value 2.82E-13 8.52E-09 4.03E-07 5.05E-07 2.23E-06 3.04E-06 3.04E-06 3.04E-06 8.53E-06 1.14E-05 3.00E-05 3.00E-05 3.00E-05 3.00E-05 3.54E-05 6.49E-05 0.000123 0.000339 0.0004 6.09E-04 0.000782 0.00104 0.00131 0.00237 0.00237 0.00241 0.00241 0.00283 130 Chapter 4. Isolation and Characterisation Of Vascular Progenitors GO:48513: organ development GO:45655: regulation of monocyte differentiation GO:46847: filopodium formation GO:902: cellular morphogenesis 1524 15 19 770 5.118 0.0504 0.0638 2.586 19 2 11 10.33 1.087 1.087 5.978 0.00293 0.00378 0.00606 0.00867 Genes in List in Category 43 16 14 2 7 2 % of Genes in List in Category 33.08 12.31 10.77 1.538 1.538 5.385 5.385 1.538 5.385 1.538 1.538 1.538 p-Value 1.89E-06 0.0013 0.00683 0.000392 0.00413 0.00307 0.00307 0.00531 0.000502 0.00143 0.00143 0.00121 Table 4-1: List of ontological processes upregulated in FB-EPC compared to UCB-EPC Category GO:7275: development GO:9653: morphogenesis GO:48513: organ development GO:45666: positive regulation of neuron differentiation GO:45664: regulation of neuron differentiation GO:8361: regulation of cell size GO:16049: cell growth GO:1502: cartilage condensation GO:1558: regulation of cell growth GO:48627: myoblast development GO:48628: myoblast maturation GO:7520: myoblast fusion Genes in Category 4849 1562 1524 22 435 435 25 317 13 13 12 % of Genes in Category 16.29 5.246 5.118 0.0235 0.0739 1.461 1.461 0.084 1.065 0.0437 0.0437 0.0403 Table 4-2: List of ontological processes downregulated in FB-EPC compared to UCB-EPC 131 Chapter 4. Isolation and Characterisation Of Vascular Progenitors 221009_s_at 201005_at 204470_at 221731_x_at 201852_x_at 202157_s_at 201430_s_at 229800_at 203881_s_at 208297_s_at 208394_x_at 203184_at 1555480_a_at 218665_at 227405_s_at 230559_x_at 219901_at 209604_s_at 204689_at 201162_at 211506_s_at 209098_s_at 201596_x_at 203934_at 221841_s_at 204153_s_at 202291_s_at 209086_x_at 218678_at 202149_at 204105_s_at 218902_at 210809_s_at 209652_s_at 1559400_s_at 205128_x_at 235131_at 226028_at 217591_at 202363_at 212558_at 201998_at 206283_s_at 235086_at 232068_s_at 204653_at 209909_s_at angiopoietin-like CD9 antigen (p24) chemokine (C-X-C motif) ligand (melanoma growth stimulating activity, alpha) chondroitin sulfate proteoglycan (versican) collagen, type III, alpha (Ehlers-Danlos syndrome type IV, autosomal dominant) CUG triplet repeat, RNA binding protein dihydropyrimidinase-like Doublecortin and CaM kinase-like dystrophin (muscular dystrophy, Duchenne and Becker types) ecotropic viral integration site endothelial cell-specific molecule fibrillin (congenital contractural arachnodactyly) filamin binding LIM protein frizzled homolog (Drosophila) frizzled homolog (Drosophila) FYVE, RhoGEF and PH domain containing FYVE, RhoGEF and PH domain containing GATA binding protein hematopoietically expressed homeobox insulin-like growth factor binding protein interleukin jagged (Alagille syndrome) keratin 18 kinase insert domain receptor (a type III receptor tyrosine kinase) Kruppel-like factor (gut) manic fringe homolog (Drosophila) matrix Gla protein melanoma cell adhesion molecule nestin neural precursor cell expressed, developmentally down-regulated neuronal cell adhesion molecule Notch homolog 1, translocation-associated (Drosophila) periostin, osteoblast specific factor placental growth factor, vascular endothelial growth factor-related protein pregnancy-associated plasma protein A, pappalysin prostaglandin-endoperoxide synthase (prostaglandin G/H synthase and cyclooxygenase) ras homolog gene family, member J roundabout homolog 4, magic roundabout (Drosophila) SKI-like sparc/osteonectin, cwcv and kazal-like domains proteoglycan (testican) sprouty homolog 1, antagonist of FGF signaling (Drosophila) ST6 beta-galactosamide alpha-2,6-sialyltranferase T-cell acute lymphocytic leukemia Thrombospondin toll-like receptor transcription factor AP-2 alpha (activating enhancer binding protein alpha) transforming growth factor, beta Table 4-3: List of genes upregulated in FB-EPC compared to UCB-EPC 132 Chapter 4. Isolation and Characterisation Of Vascular Progenitors 226777_at 205609_at 214297_at 202436_s_at 226281_at 227646_at 206115_at 225079_at 210310_s_at 204451_at 210220_at 218469_at 235521_at 228904_at 201185_at 201508_at 203851_at 226534_at 210302_s_at 228425_at 238852_at 205111_s_at 225975_at 232231_at 202037_s_at 206056_x_at 228347_at 202935_s_at 222557_at 225665_at 203372_s_at 232528_at 205990_s_at ADAM metallopeptidase domain 12 (meltrin alpha) angiopoietin Chondroitin sulfate proteoglycan (melanoma-associated) cytochrome P450, family 1, subfamily B, polypeptide delta-notch-like EGF repeat-containing transmembrane Early B-cell factor early growth response epithelial membrane protein fibroblast growth factor frizzled homolog (Drosophila) frizzled homolog (Drosophila) gremlin 1, cysteine knot superfamily, homolog (Xenopus laevis) homeo box A3 Homeo box B3 HtrA serine peptidase insulin-like growth factor binding protein insulin-like growth factor binding protein KIT ligand mab-21-like (C. elegans) Paired box gene Paired related homeobox phospholipase C, epsilon protocadherin 18 runt-related transcription factor secreted frizzled-related protein sialophorin (gpL115, leukosialin, CD43) Sine oculis homeobox homolog (Drosophila) SRY (sex determining region Y)-box (campomelic dysplasia, autosomal sex-reversal) stathmin-like sterile alpha motif and leucine zipper containing kinase AZK suppressor of cytokine signaling Ubiquitin-conjugating enzyme E2E (UBC4/5 homolog, yeast) wingless-type MMTV integration site family, member 5A Table 4-4: List of genes downregulated in FB-EPC compared to UCB-EPC 133 Chapter 4. Isolation and Characterisation Of Vascular Progenitors 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. . 143 Chapter 4. Isolation and Characterisation Of Vascular Progenitors 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 144 Chapter 4. Isolation and Characterisation Of Vascular Progenitors 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. 145 Chapter 4. Isolation and Characterisation Of Vascular Progenitors 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 CFU-F from every 106 mononuclear cells seeded to CFU-F per 106 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 146 Chapter 4. Isolation and Characterisation Of Vascular Progenitors 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. 147 Chapter 4. Isolation and Characterisation Of Vascular Progenitors 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 106 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 (1.3/106 cells) and cord blood from term deliveries (0.35/106 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/106 cells during early second trimester (24 – 28 weeks gestational age), and diminishing frequencies with increased 148 Chapter 4. Isolation and Characterisation Of Vascular Progenitors gestational age(Javed 2008). Intriguingly, this gradual reduction in MSC frequency was accompanied with an increase in EPC frequency, indicating a temporal change in the proportion of circulating stem / progenitor populations. Alternatively, the apparent reduced proportion of EPC in second trimester fetal blood may be explained by a greater plasticity and potential for transdifferentiation of FB-EPC. Evidence to support this line of though is borne out out by my results demonstrating that a large proportion of FB-EPC continues to demonstrate an ability to take up acLDL at passage three (92.3%). 4.3.2.2.2 Increased vasculogenic potential of FB EPC Matrigel tube formation assays are commonly used as an in vitro assay of endothelial function (Arnaoutova 2009). Compared to UCB EPC, FB EPC appeared to have more robust tube forming capability, with the establishment of macro-networks visible to the naked eye and evidence of secondary angiogenic sprouting. Two possible mechanisms may account for this increased vasculogenecity. First, I postulated that the heterogeneous population comprising endothelial lineage and MSC interact synergistically towards the formation of blood vessel networks. MSC populations have recently been shown to stabilise vascular networks in vivo (Au 2008). In response to EC, MSC upregulate myocardin and migrate towards them, stabilising the newly formed blood vessel. MSC have also been shown to secrete the angiogenic factors VEGF and bFGF, and are capable of increasing angiogenesis in vivo (Han 2006). In a recent study, Cyr61, an angiogenic factor, was found to be secreted by MSC and contribute greatly to capillary formation (Estrada 2009). 149 Chapter 4. Isolation and Characterisation Of Vascular Progenitors An alternative explanation lies in the primitive nature of the FB EPC. Tissues in the developing foetus are regions of intense vasculogenic activity and remodelling. It thus follows that FB EPC may have greater vasculogenic capacity. The results from my microarray experiments appear to support this, with six upregulated genes involved in angiogenesis, blood vessel development and vasculogenesis. Of these, Kinase Domain Receptor (KDR) stands out, as it is one of the markers of endothelial progenitor cells (the other two being CD34 and CD133) (Peichev 2000). Jagged-1 (Jag-1), Placental Growth Factor (PlGF) and Interleukin (IL-8) have also been found to be involved in various aspects of endothelial progenitor cell biology, including homing, migration and cell fate determination. Jag-1 has been directly implicated in EPC mobilisation and commitment, and Kwon et al demonstrated that Jag-1 knockout mice had drastically reduced EPC numbers and functionality. In vitro, they demonstrated that lack of functionality could be reversed by co-culture with cells overexpressing Jag-1. Similarly, PlGF is a well-established vasculogenic ligand for VEGFR1, is known to have profound effects on EPC biology (Autiero 2003), and knockout animals demonstrated impaired vascularisation. IL-8, on the other hand, appears to be involved primarily in EPC recruitment and chemotaxis (He 2005). The last two genes, angiopoietin-like (ANGPTL4) and roundabout homolog (roho4), have not directly been implicated in EPC biology. However, the angiopoietins have well described roles in late stages of embryonic vasculogenesis, suggesting a possible role in fetal EPC biology as well (Pham 2001). In adults, ANGPTL4 is secreted mainly by adipocytes in response to fasting (Gealekman 2008), or from EC in response to hypoxia (Le Jan 2003). While the data from these two groups on the mechanism of action appears to be conflicting, it is clear that 150 Chapter 4. Isolation and Characterisation Of Vascular Progenitors ANGPTL4 is involved in the stabilisation of blood vessels, contributing to increased vasculogenic activity observed in Matrigel and chicken chorioallantoic membrane assay. Similarly, Robo4 has recently been implicated in embryonic blood vessel development, Jones et al found it to be exclusively expressed in vascular beds of the developing mouse (Jones 2008). In vivo, Robo4 was found to have a role in reducing vessel permeability and stabilising neovascular networks. Taken together, these genes appear to have an important role in developmental vasculogenesis. 4.3.2.2.3 Potential application in vascular tissue engineering My results confirm the presence of EPC in fetal blood, and show that they are phenotypically different from UCB-EPC. FB-EPC appear to have a greater role in development and vasculogenesis, lending support to the possible application in biomimetic tissue engineering that better recapitulates the vascular development process. However, due to the lack of lineage commitment, and a tendency of the cell to de-differentiate in culture, further work remains to better define a suitable milieu to expand the cells. 4.3.2.3 Fetal liver endothelial progenitor cells (FL-EPC) 4.3.2.3.1 Immunoselection of EPC Selection by adherent culture resulted in the isolation of fibroblsatic cell types, despite the use of endothelial permissive medium. This was unsurprising, however, as the medium used contained several mitogenic factors favouring the expansion of fibroblasts and MSC, which exist in high frequencies in the fetal liver, likely surpassing that of EPC (Campagnoli 2001; Dan 2006). Thus, despite the use of 151 Chapter 4. Isolation and Characterisation Of Vascular Progenitors endothelial permissive medium, the adhered fibroblasts and MSC quickly outgrew the EPC population. To verify the presence of haematopoietic and endothelial lineage cells in the liver, I studied the expression profile of the fetal liver cells by flow cytometry. My results demonstrated a significant proportion of cells (2.14 ± 1.26 %) expressing CD133, a haematopoietic stem cell marker reportedly expressed on haemangioblastic cells (Loges 2004). I also verified the expression of endothelial markers, including VEGFR2 (also known as KDR, which is similarly upregulated in FB-EPC), which was expressed on 0.35 ± 0.27% of the liver MNC. Nava et al previously reported 16% of fetal liver cells expressing CD133 and 8% expressing VEGR2 during the 18th week of gestation (Nava 2005), further suggesting the presence of EPC in the liver. The discrepancies in numbers can be explained by difference in isolation techniques, as well as the use of different antibodies. 4.3.2.3.2 In vitro culture of fetal liver CD133+ cells My results from the MACS isolation of fetal liver CD133+ (FL-133) resulted in varying yields, probably due to inter-sample variability. FL-133 were highly enriched, and grew largely as suspension cultures. Similarly, Peichev et al described the isolation and culture of CD34+ cells from fetal liver, and demonstrated that they were maintained in culture as non-adherent cells. Following culture for two weeks, the CD34+ cells gave rise to adherent EC-like populations. However, I failed to observe this in my experiments, but found instead that the cells continued to expand in suspension. This may have been due to the fact that CD133+ cells are more primitive 152 Chapter 4. Isolation and Characterisation Of Vascular Progenitors and may comprise a large subpopulation of cells that requires further differentiation.(Yin 1997). Wu et al demonstrated that the CD34+ subpopulation of CD133+ cell were capable of generating endothelial progeny, and taken together with my results, appear to suggest that the CD34 and CD133 are co-expressed on the endothelial-lineage committed fraction. Separately, Gehling et al described the use of CD133 to select for haemangioblastic cells from leukapheretic products (Gehling 2006). Following selection, the cells could be expanded in culture using a cytokine-rich medium. These cells continued to demonstrate haematopoietic and angioblastic properties. My results demonstrated that a similar haemangioblastic population could be derived from fetal liver. Although immunocytochemistry revealed that an endothelial-like population could be generated, the cells were difficult to maintain, and diminished in numbers after extended culture. Due to lack of time, I was unable to continue studying this cell source, and decided that UCB- currently presents the most viable option for vascular tissue engineering. 153 Chapter 5. Functionalisation of µXPCL Surfaces Using Radio Frequency Glow Discharge (RFGD) Plasma Functionalisation of µXPCL Surfaces Using Radio Frequency Glow Discharge (RFGD) Plasma 154 Chapter 5. Functionalisation of µXPCL Surfaces Using Radio Frequency Glow Discharge (RFGD) Plasma 5.1 Introduction The field of biomaterials has evolved from the use of permanent materials with the aim of reduced toxic responses to the new generation of bioresorbable materials, which are eventually replaced by host tissue (Hench 2002). Accordingly, the current paradigm focussed on modulating, rather than totally avoid tissue response. This epitomises the concept of biocompatibility, where the aim is to elicit an appropriate host response in an adequate fashion (Williams 1999). Polymeric materials used in implant applications often perform a purely structural role, and the material is thus typically selected on the basis of mechanical properties and degradation kinetics. Host responses, however, are resolved at the surface, as biological interactions are limited to the tissue-material interface. Modulation of the tissue responses can thus be achieved by adequate modification of the material surface, while preserving the bulk properties. Thus, my work in this chapter focuses on surface engineering of µXPCL films. Surface engineering can be achieved by simply altering the surface free energy. Synthetic materials lack cell-recognisable sites, and tissue responses are instead mediated by the recognition of the adsorbed protein layer, which is determined in turn by surface energetics (Vogler 1998). Thus, surface wettability of biomaterials have been altered to engineer favourable responses ranging from blood clotting (Hoffman 1983) to cell adhesion and tissue integration (Lee 1998). 155 Chapter 5. Functionalisation of µXPCL Surfaces Using Radio Frequency Glow Discharge (RFGD) Plasma Surface modification of wettability can be achieved by a variety of techniques, as described in Chapter 1. In particular, Radio Frequency Glow Discharge (RFGD) plasma-based methods stand out because of the shallow depth of penetration (Kato 2003). This results in true surface modification, without alteration of bulk properties. Recently, Park et al demonstrated the use of plasma techniques to induce copolymerisation of acrylic acid monomers onto polyester nanofibres (Park 2007). Using a different approach, Nitschke et al described the immobilisation of nano-thin hydrogel layers, including polyacrylic acid, onto organic substrates by plasmainduced cross-linking (Nitschke 2004). The modified materials had altered hydrophilicity, and Park showed improved cell attachment and proliferation on the modified surfaces. It is apparent, however, that this strategy is dependent on random, non-specific protein interactions and may lead to a plethora of unrelated and undesired tissue responses. To elicit more specific and directed responses, functional biomolecules may be introduced to surface. For example, where increased cell adhesion is required, extracellular matrix proteins such as collagen are engrafted onto biomaterial surfaces (Okada 1996). Alternatively, biologically functional molecules such as heparin have been immobilised to mediate specific host responses (Hoffman 1983). More recently, “capture antibodies” have been studied to anchor cells or proteins to the substrate for very specific applications (McFarland 1998; Avci-Adali 2008). This chapter covers two parts. First, I functionalised the µXPCL surface by plasma immobilisation of hydrogel layers, to achieve a suitably wettable surface for cell adhesion. I then proceeded to study the immobilisation of biomolecules onto the 156 Chapter 5. Functionalisation of µXPCL Surfaces Using Radio Frequency Glow Discharge (RFGD) Plasma modified film surfaces. This has important implications in developing surfaces for cell culture and for optimising film surfaces for specific applications. 5.2 Plasma Immoblisation of Hydrogels 5.2.1 Plasma Immoblisation of Polyacrylic Acid (PAAc) Here, I investigated the immobilisation of polyacrylic acid onto µXPCL films. Polyacrylic acid grafted surfaces have previously been used for tissue engineering applications (Gupta 2002; Cheng 2004). Collectively, these previous works have shown that PAAc engrafted surfaces acquire improved wettability for improved cell adhesion, as well as the potential for subsequent engraftment of amine-containing biomolecules through the use of carbodiimide chemistry. 5.2.1.1 Toluidine BlueO staining To visualise the coating and distribution of polyacrylic acid, I stained the films with Toluidine BlueO (TBO). TBO is a metachromatic dye capable of complexing with carboxyl groups. PAAc was successfully engrafted on the surface, as evidenced by he blue stain (Figure 5-1). However, I also observed that the staining on was not uniform, and prone to streaking as a result of the coating process. Thus, I studied the possibility of repeating the grafting process, and demonstrated that improved distribution was achieved, together with a higher density of carboxyl groups, as evidenced by the darker colouration. 157 Chapter 5. Functionalisation of µXPCL Surfaces Using Radio Frequency Glow Discharge (RFGD) Plasma (a) ~ ~ Plasma preacativation Air exposure for 10 mins Spin coat PAAc Plasma induced crosslinking Repeat grafting (b) Pristine 1x 3x Figure 5-1: Polyacrylic acid immobilisation process (a) Schema of polyacrylic acid (PAAc) immobilisation. µXPCL films were pretreated with plasma to increase hydrophilicity. PAAc-methanol solution was spin-coated on the surface and immobilised. The process was repeated to increase amount of PAAc grafted. (b) Photomicrographs (10x magnification) of TBO stained films. Carboxyl groups stain blue after complexing with TBO. Staining shows increased density and uniformity after repeat grafting 5.2.1.2 Water contact angle measurements PAAc is highly anionic, and when engrafted onto a substrate, confers hydrophilicity to the modified surface. Here, I found a significant drop in water contact angle on µXPCL films following PAAc engraftment from 70.9 ± 0.3° to 59.6 ± 0.3° (p[...]... 96 hours 137 Chapter 4 Isolation and Characterisation Of Vascular Progenitors 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 FL 133 + cells generated... 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 145 Chapter 4 Isolation and Characterisation Of Vascular Progenitors 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. .. subpopulation of cells that requires further differentiation.(Yin 1997) Wu et al demonstrated that the CD34+ subpopulation of CD 133 + cell were capable of generating endothelial progeny, and taken together with my results, appear to suggest that the CD34 and CD 133 are co-expressed on the endothelial-lineage committed fraction Separately, Gehling et al described the use of CD 133 to select for haemangioblastic cells... use of different antibodies 4 .3. 2 .3. 2 In vitro culture of fetal liver CD 133 + cells My results from the MACS isolation of fetal liver CD 133 + (FL- 133 ) resulted in varying yields, probably due to inter-sample variability FL- 133 were highly enriched, and grew largely as suspension cultures Similarly, Peichev et al described the isolation and culture of CD34+ cells from fetal liver, and demonstrated that they... to the lack of lineage commitment, and a tendency of the cell to de-differentiate in culture, further work remains to better define a suitable milieu to expand the cells 4 .3. 2 .3 Fetal liver endothelial progenitor cells (FL-EPC) 4 .3. 2 .3. 1 Immunoselection of EPC Selection by adherent culture resulted in the isolation of fibroblsatic cell types, despite the use of endothelial permissive medium This was... 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 CFUHills, are associated with angioblasts and the adult haemangioblast (Hill 20 03; Loges 2004) However, the cells were unable to undergo further proliferation and gradually diminished under... population To verify the presence of haematopoietic and endothelial lineage cells in the liver, I studied the expression profile of the fetal liver cells by flow cytometry My results demonstrated a significant proportion of cells (2.14 ± 1.26 %) expressing CD 133 , a haematopoietic stem cell marker reportedly expressed on haemangioblastic cells (Loges 2004) I also verified the expression of endothelial markers,... 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... 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 CD 133 , a stem/progenitor cell marker, was chosen because of the reported ability to distinguish EPC from mature EC (Peichev 2000) Furthermore, CD 133 + cells isolated from cord blood has been shown to capable of differentiation towards endothelial lineage, and... 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 . Colony forming assays of expanded FL 133 + 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. 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. 5 .38 5 0.000502 GO:48627: myoblast development 13 0.0 437 2 1. 538 0.001 43 GO:48628: myoblast maturation 13 0.0 437 2 1. 538 0.001 43 GO:7520: myoblast fusion 12 0.04 03