Incorporation of endothelial progenitor cells into the neovasculature of malignant glioma xenograft

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SHORT REPORT SHORT REPORT

*Correspondence to: Claire F. Jessup; Email: [email protected]: 12/03/10; Revised: 03/07/11; Accepted: 03/07/11DOI:

Pancreatic islets come under a myriad of cellular assaults dur-ing isolation including ischemia, enzymatic damage and physi-cal stress. Following transplantation, the instant blood-mediated inflammatory reaction (IBMIR), proinflammatory cytokines, alloimmunity and existing autoimmunity further assail the frag-ile islets.1,2 These factors combine to cause apoptosis in up to 70% of transplanted β-cells within 48 h of transplantation3-5 and dis-ruption of the extracellular matrix surrounding islets can further predispose these cells to immune cell infiltration.6 Furthermore, reestablishment of the dense and specialized intraislet endothe-lium is slow (taking up to two weeks7) and incomplete, resulting in lower vascular density and oxygen tension.8

Endothelial progenitor cells (EPC) are vasculogenic, bone-marrow derived cells9 that have the potential to improve the engraftment of transplanted cells such as pancreatic islets. EPC secrete multiple humoral factors10 including proangiogenic and remodelling factors11 that may improve β-cell survival. In addi-tion, EPC stimulate the formation and repair of vasculature either by recruiting and differentiating other cell types, or by incorporating into vessels themselves.12 Thus codelivered EPC may improve both the survival and the ultimate function of transplanted pancreatic islets.

The optimal mode of delivery of EPC is unknown. When delivered as a direct transplant, EPC improve myocardial infarc-tion.10 Due to the expression of CXCR4,13 EPC effectively home

Pancreatic islet transplantation is limited by extensive apoptosis and suboptimal function of the implanted islets in the longer term. Endothelial progenitor cells (EPC) may be ideal for enhancing both the survival and function of transplanted islets. Here, we describe for the first time the in vitro formation of rat mosaic pseudoislets comprised of pancreatic β-cells with interspersed vasculogenic EPC. Bone marrow-derived EPC displayed a similar phenotype to non-adherent EPC, recently described in the human and mouse. Mosaic pseudoislet formation was enhanced by the use of an embryoid body forming medium (BPEL) and a spin protocol. Mosaic pseudoislets maintained function in vitro and may represent an enhanced cell therapy delivery approach to enhance the survival and revascularization of transplanted islets.

Incorporation of endothelial progenitor cells into mosaic pseudoislets

Daniella Penko,1,2 Daisy Mohanasundaram,1 Shaundeep Sen,1,3 Christopher Drogemuller,1 Clare Mee,1 Claudine S. Bonder,2-4 P. Toby H. Coates1,2,4 and Claire F. Jessup1,2,4,*

1Islet Transplantation and Dendritic Cell Biology Laboratory; Central Northern Adelaide Renal and Transplantation Service; Royal Adelaide Hospital; 2School of Medicine; 4Centre for Stem Cell Research; The Robinson Institute; University of Adelaide; 3Vascular Biology and Cell Trafficking Laboratory; Centre for Cancer Biology; Adelaide, Australia

Key words: endothelial progenitor cells, pancreatic islets, transplantation, revascularization, pseudoislet

Abbreviations: EPC, endothelial progenitor cells; EC, mature endothelial cells; IEQ, islet equivalents; BSA, bovine serum albumin; BPEL, BSA-polyvinylalcohol essential lipids containing medium; FCS, foetal calf serum; AcLDL, acetylated low density

lipoprotein; TMRE, tetramethylrhodamine ethyl ester; 7AAD, 7-aminoactinomycin D; ROCK, Roh-associated kinase; DAPI, 4',6-diamidino-2-phenylindole

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to sites of wound repair when delivered systemically, and improve hindlimb ischemia9,12 and kidney ischemic reperfusion injury14 in animal models. The difficulty with islet transplantation is that an effective cellular therapy requires access to the intraislet envi-ronment—mature endothelial cells (EC), for example, cannot penetrate into the dense central islet region.15 EPC, on the other hand, exhibit the plasticity of immature vascular cells required during new vessel formation following transplantation.

In this study, the in vitro delivery of EPC throughout the inte-rior of the islet structure, via a reaggregation technique was inves-tigated. We used an embryoid body-forming medium, combined with a low-speed spin protocol, to enhance the reaggregation of dispersed rat islet cells and rat EPC to form mosaic pseudoislets in culture.

The ability of BPEL, a medium originally devised to enhance the aggregation of human embryonic stem cells,16 to enhance the formation of pseudoislets from dispersed rat islet cells was investigated. ROCK inhibitor was added to assist cell survival.17 Rat serum has previously been described to assist pseudoislet for-mation.18 However, preliminary experiments showed that RPMI with 10% rat serum was inferior to BPEL for islet cell reaggre-gation. Thus, BPEL was selected as the preferred reaggregation medium for further experiments.

The number of islet equivalents (IEQ) required to form pseu-doislets was investigated. Pseudoislets of increasing size formed


2 Islets Volume 3 Issue 3

Figure 1. Formation of pseudoislets of various sizes by alteration of input islet equivalents (IEQ). Dispersed rat islets were plated at various IEQ per well in BPEL medium with or without FCS. Whole islets (70–80% purity) were used as a control. (A) Cells plated at day 0. (B) Pseudoislets formed by day 3 in culture. Images captured at the inverted light microcope. Scale bars represent 100 μm.



culture. Cultures expressed low levels of CD45 and CD14 (data not shown).

To form mosaic pseudoislets, dispersed islets were mixed (2:1) with day 4–7 EPC. Three-dimensional clusters formed by day 3 in culture, with an average diameter of 320 ± 11 μm (Fig. 3A), and were not significantly larger than pseudoislets formed by islet cells alone. Whole islets were used as a control, and formed larger clumped structures by day 3 with and without added EPC measuring 376 ± 15 μm (n = 22) and 433 ± 17 μm (n = 22) respectively. The viability of the mosaic pseudoislets was assessed by flow cytometry at 7 days post-cluster formation. The viabil-ity of β-cells (defined as Newport green (NG)-positive/7AAD-negative) within intact islets, reformed islets and mosaic pseudoislets was 78, 68 and 74% respectively. Interestingly, the intensity of NG staining, which indicates the high level of zinc within β-cells, was markedly higher in the mosaic pseudoislets (mean fluorescence intensity (MFI) = 89) compared to intact and reformed islets (MFI = 42 and 36 respectively), possibly indicat-ing an enhancement of islet function by EPC coculture.

In order to observe the distribution of EPC throughout the pseudoislet, EPC were prestained with DiI-labeled AcLDL prior to incorporation into mosaic pseudoislets. To monitor the fate of incorporated EPC, pseudoislet clusters were dissociated at

by day 3 in BPEL with increasing input of islet cells (Fig. 1). The inclusion of 10% FCS did not affect the appearance of the reformed pseudoislets and was included in subsequent experi-ments. Ten IEQ of dispersed islets formed pseudoislets with a diameter of 300 ± 11 μm (n = 22) and was used as the islet cell input per well for subsequent experiments.

Bone marrow-derived rat endothelial progenitor cells (EPC) were isolated by enrichment culture on fibronectin with endo-thelial growth factors as previously optimized for murine EPC.14 Characteristic endothelial outgrowth colonies developed over the first week in culture (Fig. 2A) and repassaged EPC demon-strated characteristic endothelial-like morphology (Sup. Fig. 1). EPC were further characterized by uptake of acetylated low den-sity lipoprotein (AcLDL) (Fig. 2B) and tube formation in vitro (Fig. 2C). By flow cytometric analysis, EPC expressed moder-ate levels of endothelial markers VEGFR-2 and CD31 (Fig. 2D). Upon maturation, the mature endothelial cell marker VCAM-1 was markedly upregulated and by day 14 most cells expressed VCAM-1 (Sup. Fig. 1), indicating the level of enrichment for the endothelial lineage using this culture method. Flow cytometric analysis of day 4 EPC showed that most cells took up high lev-els of DiI-labeled AcLDL (Sup. Fig. 2) which is characteristic of EPC, indicating the high prevalence of EPC at this early stage of

Figure 2. Characterization of rat bone marrow-derived endothelial progenitor cells. (A) EPC colony at day 6 on fibronectin by light microscopy. (B) Uptake of DiI-labeled Ac-LDL (red) by fluorescence microscopy. Scale bar = 50 um. (C) Tube formation at day 7 (light microscopy after 7 h plated on MatrigelTM). (D) Surface phenotype of EPC at days 7 and 14 in culture by flow cytometry (n = 2; ±SEM).



approach for EPC delivery, where vasculogenic cells are interspersed with β-cells in a three-dimensional struc-ture in vitro.

Islet cells have been artificially reaggregated for vari-ous applications including gene transfer19 and size stan-dardization.20 Dispersed rat islets reaggregated via the hanging drop technique took 5–6 days to form,20 com-pared to 1–2 days with the current BPEL spin aggrega-tion described here.

In the absence of a definitive marker for EPC, we are currently unable to unambiguously identify these cells from others, such as mature ECs and haemato-poietic progenitors.21 Furthermore, there are at least three described populations of EPC including 1) col-ony forming units, which probably comprise circulat-ing EC, 2) late outgrowth EPC and 3) a more recently described non-adherent EPC population22 (Brice and Bonder, manuscript in preparation). The rat EPC described here are most like the latter: non-adherent cells at day 2, that upregulate endothelial markers and have angiogenic potential upon maturation. Compared to the wealth of data on human and murine EPC, there is limited literature on rat bone-marrow derived EPC. However, rat EPC share many characteristics including

uptake of Ac-LDL and tube formation in matrigel,23-25 as was also shown in this study.

The distribution of EPC throughout the mosaic pseudois-let was unexepected. In previous studies when insulinoma and primary rat β-cells were combined, the insulinoma cells were excluded to the periphery of the reformed structures.26 In the native pancreas, caherins are responsible for the clustering of islet cells.27 Islets express VEGF,28 and EPC are mobilized by VEGF;29 thus, the relatively ischemic centres of the reforming structures may be responsible for the resultant EPC distribution.

Swift revascularization of transplanted pancreatic islets is vital for their health and ultimate function, and may involve vascular endothelial cells from a number of sources. Following transplantation, islets are slowly revascularized with both recipi-ent (recruited endothelial and bone-marrow derived) and donor (intraislet endothelial) cells incorporating into the new vascu-lature.30 The overexpression of proangiogenic factors31-34 may enhance this process, but the production of angiostatic media-tors35 by islets is a likely barrier to efficacy. Mature endothelial cells (EC),15,36 and mesenchymal stem cells (MSC)37 have been used to coat islets in animal transplantation models, but their lack of plasticity and risk of tumorgenicity, respectively, remain as significant hurdles. EPC are ideal candidates for enhanc-ing islet revascularization with the ability to home to ischemic sites (via chemotactic factors), position themselves within the microenvironment (via adhesion molecules), express appropri-ate growth factors (such as vascular endothelial growth factor) to stimulate local cells and/or incorporate into growing vasculature themselves. Here, we have described the novel construction of a mosaic pseudoislet structure where EPC may be perfectly placed to perform their dual role of supporting early islet cell survival while enhancing intraislet revascularization.

days 3 and 7 in culture and analysed for DiI-labeled cells by flow cytometry. Initially, EPC made up 41% of the mosaic pseudo-islet clusters (day 3) (Sup. Fig. 3), slightly more than expected from a 2:1 islet:EPC cell input ratio. This may result from the proliferation of EPC and/or death of islet cells. The latter is not likely to be a cause, since the viability and function of the mosaic clusters appears to be unaffected as examined by 7AAD stain-ing and insulin release (see below). The EPC fraction reduced to 21% by day 7, indicating possible loss of EPC over this extended culture period or expansion of other cells. Confocal microscopy demonstrated that at day 3 post-cluster formation EPC were distributed throughout the structure (Fig. 4 and Sup. Fig. 4). Interestingly, in some areas incorporated EPC appeared to cluster and stretch towards neighbouring EPC (Fig. 4A), forming tube-like structures.

Functionally, the mosaic pseudoislets were compared to reformed islet clusters and intact cultured islets in a static glucose stimulated insulin secretion (GSIS) assay (Fig. 3B). Reformed clusters containing islet cells alone had deregulated responses to high and low glucose (stimulation index (SI) = 0.9), while EPC-containing mosaic pseudoislets produced significantly more insulin in response to high glucose compared to low glucose (SI = 1.7). In addition, mosaic pseudoislets had superior release of insulin in response to high glucose (11,347 ± 1,134 pg/ml) compared to reformed islets without EPC (6,719 ± 246 pg/ml). The equivalent number of intact cultured islets produced 8,584 ± 1,818 pg/ml insulin in response to high glucose, with an SI of 1.5.

Outcomes for islet transplantation may be improved by two distinct approaches: 1) by providing protective or prosur-vival humoral factors at the transplant site and 2) by improving the speed and extent of intraislet revascularization. EPC have the potential to provide both, and this study describes a novel

Figure 3. Formation and in vitro function of mosaic islet cells/EPC pseudoislets. Dispersed islets and day 7 EPC were mixed (2:1) and replated at 10 IEQ per well in BPEL (10% FCS). Clusters are shown at day 0 (A) and day 3 (B) postaggregation taken at the inverted light microscope. Scale bar represents 100 μm. (C) Glucose-stimulated insulin release. Insulin secretion (y axis) was compared between whole intact islets (solid bars), reformed pseudoislets (white bars) and mosaic pseudois-lets + EPC (hatched bars) in low (2.8 mM) or high (25 mM) glucose. Bars represent mean ± SEM. (n = 3; 10 clusters of 10 IEQ per test; *p = 0.01 by Students t-test, **p < 0.05 by one-way ANOVA).



and phase-contrast microscopy on an inverted microscope and images were recorded using a digital camera (CKX41; Olympus Corporation). For labeling, DiI-conjugated acetylated low- density lipoprotein (DiI-Ac-LDL; 10 μg/ml; Biomedical Technology Inc., #BT-902) was incubated with cells at 37°C for 4 h. Following PBS rinse, cells were resuspended in complete M199. Labeling was confirmed at the flow cytometer (FACScan; BD Biosciences).

Rat pancreatic islet isolation. Rat pancreatic islets were isolated by collagenase (Sigma, #C7657) digestion method as previously described in reference 38. Briefly, after Laparotomy, pancreatic duct was identified, cold HBSS was injected into the pancreatic duct to inflate the pancreas. After full infla-tion, the pancreas was removed, minced and then digested with Collagenase (type XI, Sigma) for 16 min. The digestion was stopped by adding cold HBSS to the digest. After washing twice, islets were purified by density separation (Histopaque; Sigma, #H8889) and hand-picked using a Pasteur pipette. Islet equivalents (IEQ) count was performed as previously described in reference 39, with addition of the zinc-binding dye Dithizone (Sigma, #D5130).

Materials and Methods

Animals. Albino Wistar rats and NOD-SCID mice were housed at the Animal Care Facility (IMVS, Adelaide, Australia) and used between 6–12 weeks of age. All experimental procedures were approved by the Animal Ethics Committee of the IMVS/CNAHS Animal Ethics Committee and conform to the guide-lines established by the “Australian Code of Practice for the Care and Use of Animals for Scientific Purposes.”

Endothelial progenitor cell isolation, culture and label-ing. Bone marrow cells were obtained by flushing femurs and tibiae of 6–12 week old Albino Wistar rats with M199 medium (Sigma, #M0393). Cells were cultured on fibronectin (Fn; 50 μl/ml; Roche, #10838039001) in 20% fetal calf serum. Unless otherwise stated, all medium was supplemented with endothelial cell growth supplement and heparin (both at 15 μg/ml; BD Biosciences, #356006). Non-adherent cells (termed ‘EPC’) were removed from early outgrowth ‘ECmonos’ and plated onto fresh fibronectin-coated flasks at 48 h. Cells were harvested by trypsin-EDTA (10 min at 37°C) when required. Cells were visualized using 4x/0.13 NA and 10x/0.3 NA objectives

Figure 4. Cellular distribution within reformed pseudoislets. Confocal images of reformed clusters containing islet cells alone (A) or mosaic pseudois-lets containing EPC (B and C). Images depict Newport green-stained β-cells (green), DiI-labeled EPC (red) and DAPI-stained nuclei (blue). (A) A central optical slice of a reformed islet cluster (islet cells only). (B) A central of a reformed mosaic pseudoislet cluster, showing EPC distributed throughout. (C) An optical slice of a reformed pseudoislet shows the formation of a tube-like structure, bounded by elongated and interacting EPC (enlarged in box). For clarity, green NG staining has been removed from (B and C) and the outer edges of islet structure is shown by solid green line line. Scale bar represents 50 μm (A and B) or 20 μm (C).



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Flow cytometry. Cells were incubated with anti-rat CD45-FITC (23 ug/ml; #554877), anti-rat VCAM-1-PE (2.5 ug/ml; #559229), biotinylated anti-VEGFR-2 (5 ug/ml; cat #nb100-40753) (BD Biosciences); biotinylated goat anti-rat CD34 (25 ug/ml; R&D systems, #BAF4117); rabbit anti-CXCR4 (50 ug/ml; Abcam, #2074-100) or rabbit anti-CD14 (2 ug/ml; Santa Cruz, #sc-9150) for 30 min at 4°C. For sec-ondary detection, goat anti-rabbit FITC (Jackson Laboratories, #111-095-144) or SAPE (10 ug/ml; BD Biosciences; #554061) was added for 30 min at 4°C. Cells were resuspended in 10 μg/ml 7AAD (to detect non-viable cells; Invitrogen, #A1310) prior to analysis at the flow cytometer (FACSCantoII; BD Biosciences).

Matrigel tubule assay. As previously described in reference 40, MatrigelTM (BD Biosciences, #354234) was added to a pre-cooled 96-well plate (95 μl/well) and incubated at 37°C for 30 min. EPC were seeded (1 x 104/well) in duplicate and moni-tored regularly over 8 h at 37°C using an inverted microscope (CKX41; Olympus Corporation). Human umbilical vein endo-thelial cells were used as a EC control (4 x 104 cells/well).

Formation of pseudoislet clusters. Embryoid body-forming BPEL medium was prepared as described in referene 16, with the addition of 10 μM ROCK inhibitor (Sigma, #Y0503). Purified rat islets were dissociated with Accutase (Sigma, #A6964) for 5–10 min at 37°C. Dispersed cells were washed twice in RPMI (10% FCS) and passed through a 0.45 μM strain cap (Invitrogen, #352235). 1–100 IEQ dispersed islet cells or whole islets were plated in 96-well round bottomed non-adherent plates (Nunc, #174908) in BPEL and centrifuged at 400 g for 2 min at room temperature. Cell clusters were observed at the inverted microscope (CKX41; Olympus Corporation). Round islet-like clusters formed in culture by days 2–5.

Confocal microscopy. Whole islets and pseudoislets were washed with PBS containing 1% Albumex®20 and incubated with 1 mM Newport green (NG) (1/1,000; Invitrogen, #N24191) and DAPI (1 μg/ml; Sigma, #D9564) at 37°C for 1 h. Whole islets/pseudo-islets were washed with PBS before mounting on SuperFrost®Plus microscope slides (Lomb, #5910) using SecureSealTM Imaging Spacers (0.12 mm; Sigma, #S7685) with DAKO mounting medium (Sigma, #S3023). Images were obtained at the confocal microscope (Eclipse TE2000; Nikon Corporation, Tokyo, Japan).

Glucose-stimulated insulin release. Whole islets and pseudois-lets were placed in RPMI (10% FCS) at 37°C overnight to recover, followed by RPMI with low (2.8 mM) or high (25 mM) glucose at 37°C for 2 h. Insulin concentrations in supernatants were quanti-fied by ELISA (High sensitivity rat insulin ELISA; Crystal Chem, #90060). Stimulation indices (SI) were calculated as the mean insulin production at high glucose divided by the mean insulin production at low glucose.

Acknowledgements

D. Penko was supported by a TQEH Research Foundation Scholarship. C. Jessup was funded by the NHMRC C.J. Martin Overseas Training Fellowship (ID 375170) and C. Bonder is a Royal Adelaide Hospital Florey Fellowship holder. The authors thank Mr. Clyde Milner for his editorial input, Professor Ed Stanley (Embryonic Stem Cell Differentiation Laboratory, Monash University) for the provision of the BPEL aggregation medium recipe and Dr. Ghafar Sarvestani (Hanson Institute, Adelaide) for assistance with confocal imaging.

Note

Supplemental materials can be found at:www.landesbioscience.com/journals/islets/article/15392



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Incorporation of endothelial progenitor cells into the neovasculature of malignant glioma xenograft

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