Bio-Engineering of Pre-Vascularized Islet Organoids for the Treatment of Type 1 Diabetes

Graphical Abstract Bio-engineering of pre-vascularized islet organoids for the treatment of type 1 diabetes.


INTRODUCTION
Allogenic transplantation of pancreatic islets is a cell therapy option that holds great promise in the treatment of type 1 diabetes. The development of the Edmonton protocol has drastically increased the success rate of islet transplantation, and has proven to be able to achieve insulin independence in patients with type 1 diabetes (1). Most importantly, pancreatic islet transplantation confers a significant improvement in glycemic control and prevents life-threatening severe hypoglycaemia (2). Despite its efficacy, clinical islet transplantation is facing a number of challenges that limit achievement of steady functional success comparable to whole organ transplantation (3). One of the major challenges is the suboptimal long term graft function caused by the loss of the large portion of intraportally transplanted islets due to the IBMIR reaction, pro-inflammatory microenvironment, low oxygen tension in the liver, impaired vascularization and immunosuppressive drug toxicity (3). Therefore, the search for a suitable alternative transplantation site is a major focus of research in the field. Other limiting factors hampering the widespread application of islet transplantation are shortage of donor organs and need for lifelong immunosuppression (4). Xenogenic islets and stem cell-derived beta cells are the two major potentially unlimited sources of insulin-producing tissue (5).
In recent years, substantial progress has been made in generating and characterizing functional stem cell-derived beta cells, which will undoubtedly change the way we will treat type 1 diabetes (6). The first attempts of clinical application of microencapsulated porcine islets or stem cell-derived endocrine tissue incorporated into macrodevices have already taken place (7,8) and re-enforce the need to identify a site as functional as portal vein infusion but allowing easy graft removal-a site that to date this remains clinically elusive.
Despite the fact that islets represent only 1-2% of pancreatic tissue volume, they receive 10-15% of the total pancreatic blood flow (9). Each islet possesses 1 to 3 pre-arterioles (10), depending on islet size, that rapidly branch out into a multitude of fenestrated capillaries and form an important intra-islet microcirculation that is five time denser than in the exocrine tissue (11). The cross-talk between endocrine and endothelial cells is vital for proper islet development, configuration and vascularization. Islet cells secrete vascular endothelial growth factor-A (VEGF-A) and angiopoietin-1 in order to recruit endothelial cells (ECs) that are necessary for islet development, survival and function. On the other hand, ECs are involved in cell differentiation, insulin gene expression and cell segregation during embryogenesis (12,13). In addition, they secrete components of the intra-islet basement membrane that are crucial for proper endocrine function (11).
Islet isolation and culture lead to the disruption of the islet capillary system, with significant loss of ECs due to dedifferentiation or necrosis (14). In addition, islets vary in size, ranging from 50 to 400 μm in diameter. In the immediate posttransplantation period, avascular islets are supplied with oxygen and nutrients solely by diffusion until re-establishment of the blood flow, a process that can take about 2 weeks (9). Because of that, larger islets fail to engraft due to insufficient vascularization and subsequent necrosis (15). Significant efforts have been made to develop new strategies to minimize hypoxia-induced β cell death.
Several scientific groups, including our own, have demonstrated that re-aggregation of islet cells in combination with other cell types into homogeneous, round shaped and sizecontrolled spheroids leads to improvement of function and viability, thanks to heterotypic cell-cell interactions and reproduction of the complex natural morphology of the islet (16)(17)(18)(19)(20). In our previous studies, we have shown that incorporation of human amniotic epithelial cells (hAECs) into insulin-secreting organoids protected islet cells from oxidative stress in vitro, subsequently improving ß cell viability, function and engraftment (17,20). Here, we propose an improved approach, in which we engineer pre-vascularized organoids that provide both control over their size and composition, and prompt re-establishment of the cross-talk between ECs and islet cells, thereby facilitating graft revascularization after transplantation.

Reagents and Antibodies
All reagents and antibodies used in this study are listed in Supplementary Tables S1-S3.

Animals
Animal experiments were performed in accordance with the Geneva veterinary authorities and approved by the Institutional Animal Care and Use Committee of the University of Geneva. Ten-week-old, pregnant female, Lewis rats were purchased from Janvier Laboratory (Le Genest St-Isle, France) and bred in our animal facility at the Geneva University. Fifteen-to 21-week-old male rats were used for pancreatic islet isolation. Six-to 9-week old male B6.129S7-Rag1 tm1Mom /J (abbreviated NOD-Rag1 null bred at Charles River Laboratories, Saint-Germain-Nuelles, France) mice were used as transplantation recipients. All animals were kept under conventional housing conditions with free access to water and food.

Human Tissues
Studies involving human tissues were approved by the Commission Cantonale d'Ethique de la Recherche (CCER; protocol PB_2017-00101), in compliance with the Swiss Human Research Act (810.30).
Placentas were obtained from women undergoing elective caesarean section of uncomplicated, term pregnancies. Informed, written consent was obtained from each donor prior to tissue collection.

Isolation and Culture of Human Umbilical Vein Endothelial Cells and Human Amniotic Epithelial Cells
Human umbilical vein endothelial cells (HUVECs) were isolated using a method adapted from a previously published protocol (21). Briefly, the umbilical vein was rinsed, then distended with Collagenase A solution (2 mg/ml) and incubated at 37°C for 12 min. Released cells were then collected by flushing the vein with cold HBSS supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 mg/ml streptomycin and 0.25 mg/ml amphotericin B. Isolated HUVECs were plated in a 75 cm 2 flasks and cultured at 37°C, 21% O 2 and 5% CO 2 in M199 medium supplemented with 20% FBS, 100 U/ml Penicillin and 0.1 mg/ml Streptomycin (1% of a L-Glutamin-Penicillin-Streptomycin stock solution), Fungin 0.1%, 30 μg/ml endothelial cell growth supplement and 100 μg/ml heparin. HUVECs from passage 2 to 7 were used in this study.
Medium was changed every 48 h. Confluent cells were recovered by mild trypsinization and were cryopreserved for later utilization.

Characterization of Human Umbilical Vein Endothelial Cells and Human Amniotic Epithelial Cells
HUVECs and hAECs were analyzed for expression of previously reported endothelial cell surface markers or specific amniotic epithelial cell surface markers by flow cytometry.
Flow cytometry analysis was performed on a Gallios cytometer using the Kaluza Analysis software.
HUVECs were further characterized by immunostaining. Immunofluorescent assessment was performed on the cells cultured on gelatine-coated glass coverslips. Fixed cells were washed, permeabilized and stained with the following primary antibodies: mouse anti-CD31 (1:50 dilution), rabbit anti-von Willebrand factor (1:100 dilution) and mouse anti-vimentin (1:50 dilution). Cells were then incubated with corresponding Alexa Fluor and FITC-conjugated secondary antibodies. For nuclear counterstaining samples were mounted with aqueous solution containing 4,6 diamidino-2-phenylindole (DAPI). Briefly, Matrigel thawed overnight at 4°C was mixed with VEGF (200 ng/ml) and 250 μl of matrix was added to each well osf 24well plates. After 1 h of incubation at 37°C, cells (8 × 10 4 ) were seeded onto the Matrigel and tube formation of HUVECs was observed and photographed using an inverted phase-contrast microscope during 6 h.

Lentiviral Transduction
Lentiviral vector carrying the green fluorescent protein (GFP) under the control of an endothelial specific promoter Vascular endothelial cadherin (VEC/Cdh5) (LV-VEC.GFP) was provided by Prof. A. Follenzi (Università del Piemonte Orientale). HUVECs were transduced with LV-VEC.GFP at passage 3 using a multiplicity of infection (MOI) of 10 (MOI 10). Transduction efficiency was assessed by fluorescent microscopy and flow cytometry and considered successful when at least 80% of cells showed expression of GFP.
Culture medium for PIO was prepared by mixing equal volumes of complete DMEM, DMEM/F12 and M199 medium, hereafter referred to as organoid medium. IC + HUVEC were cultured in the mixture of complete DMEM and M199 medium at the ratio 1:1. Finally, PI and NI were cultured in complete DMEM medium. Culture medium was changed every other day. Mean diameter of NI, PIO and PI were calculated on the images taken on light microscope using ImageJ software.
In order to observe PIO composition and cell distribution during culture, fluorescent carbocyanine dyes CM-DiL (red) prelabeled hAECs and GFP transduced HUVECs were used. Pictures were taken using an epifluorescent microscope (DMi8 manual microscope).

Organoids Sprouting Assay
One hundred PIO were resuspended in a collagen solution, transferred into prewarmed 24-well plates and allowed to gelify for 30 min. Next, 0.1 ml organoid medium supplemented with VEGF-A at the concentration of 200 ng/ml was pipetted on top of each hydrogel containing PIO. The hydrogels were cultured for 24 h at 37°C, 5% CO 2 , and 100% humidity. As control, one hundred IC + HUVEC spheroids and PI were cultured in the same way in the hydrogel.

In Vitro Functional Assessment
To assess functional capacity, 300 NI and an equivalent number of PIO and PI, were incubated in duplicates for 1 h at 37°C in Krebs-Ringer solution containing low glucose (2.8 mmol/L) in order to equilibrate the samples. After a change of medium, islets and aggregates were incubated at 37°C for another hour in Krebs-Ringer solution containing low glucose (2.8 mmol/L), followed by 1 h at high glucose (16.7 mmol/L). Supernatants were collected and stored at −20°C. Insulin concentration in supernatants was measured using a rat insulin ELISA kit and normalized to the total insulin content. Results are expressed as the ratio between insulin secreted in high glucose to low glucose, referred to as stimulation index (SI). In addition, total insulin content per IC was measured by dividing the total insulin content by the number of ICs present in the NI, PI and PIO.

Diabetes Induction and Xenogeneic Transplantation
Three days before transplantation mice were subjected to intraperitoneal injection of STZ (180 mg/kg). Non-fasting blood glucose levels were then checked daily using a portable glucometer. Only mice with blood glucose levels over 18 mmol/L for 3 consecutive days were used in this study. Glycemia readings over 28 mmol/L, indicated as "high" on glucometer, were recorded as 30 mmol/L.
A marginal mass of 300 islet equivalents (IEQ) for NI and 1200 PIO, PI and IC + HUVEC were transplanted. Number of organoids was based on the average number of islet cells per IEQ, previously estimated as 1,560 ICs/IEQ (25).
At the day of transplantation, NI and engineered constructs were recovered from culture, packed in PE50 tubing and transplanted into the epididymal fat pad (EFP) of diabetic mice. Non-fasting glucose was assessed daily during the first week and 3 times per week thereafter. Normoglycemia was defined as two consecutive blood glucose levels under 11.1 mmol/L.

Graft Metabolic Function Assessment
Graft capacity to clear glucose in vivo was assessed dynamically by intraperitoneal glucose tolerance test (IPGTT) at 30 days after transplantation. Mice were fasted for 6 h and intraperitoneally injected with 2 g of glucose/kg. Blood glucose measurements were taken at 0, 15, 30, 45, 60 and 120 min.

Lectin Injection
Functional graft vasculature was assessed by infusing DyLight 594-conjugated Lycopersicon Esculentum (Tomato) lectin into the beating left ventricle of mice hearts. Mice were injected with 100 μl of undiluted lectin. Lectin was allowed to circulate for 1 min. Then, the right ventricle was cut to allow blood flow decompression and a volume of 3 ml of PBS was injected into the

Real-Time Quantitative PCR
Graft bearing EFPs recovered at 3 and 30 days after transplantation were processed for PCR analysis. RNA was extracted using the RNeasy minikit and reverse transcribed with a High Capacity cDNA Reverse transcription kit. Gene amplification was performed by RT-PCR using TaqMan Fast Advance Master Mix. Primers used for amplification are listed in Supplementary Table S4. RPLP1 was used as a housekeeping gene to normalize gene expression values. Data were calculated using the comparative cycle threshold Ct method (2 −ΔCt method) and are expressed in arbitrary units.

Statistical Analysis
Continuous variables are expressed as mean ± SEM. Multiple comparisons were analyzed using one-way ANOVA followed by Dunnett multiple comparisons test while two-way comparisons were analyzed using the Student's t-test. Cumulative number of animals reaching normoglycemia was compared using the logrank (Mantel-Cox) test. A p value ≤0.05 was considered statistically significant. All statistical analyses were performed with the Prism software 8.0.

Human Umbilical Vein Endothelial Cell Characterization and Transduction
HUVECs reached 80% confluence within 5 days with initial seeding density of 6,000 cells/cm 2 . Morphologically, cells displayed typical elliptic shape ( Figure 1A) and were positive for von Willebrand factor and CD31 ( Figure 1B). Endothelial origin of the cells was additionally confirmed by flow cytometry. Cells were positive for CD31 and CD144 (97.8% ± 0.7 and 98.1% ± 0.6, respectively) and negative for CD45 (95.8%) ( Figure 1C).
When cultured on Matrigel, HUVECs formed well-shaped vascular-like structures over a period of 6 h ( Figure 1D).
To track HUVECs within organoids both in vitro and in vivo, cells were transduced with LVs carrying green fluorescent protein (GFP) gene under the control of the VEC promotor. HUVEC positivity for GFP was observed during culture and confirmed by flow cytometry 3 days after transduction with 86.6% of GFP+ cells ( Figure 1E right and left panel, respectively).

Human Amniotic Epithelial Cells Characterization
hAECs used in this study were isolated from six different placentas. Flow cytometry analysis demonstrated strong positivity of hAECs for the embryonic cell surface marker SSEA-4 (88.4 ± 5.0%) and the epithelial cell adhesion molecule (CD326; 95.9 ± 1.3%). HLA-E and HLA-G were expressed in 16.9 ± 4.7% and 48.6 ± 12.3% of the cells, respectively. Finally, expression of CD105 and CD90 by hAECs were 17.6 ± 5.6%, 50.1 ± 7.1, respectively. The results of each hAEC preparation are described in Supplementary Figure S1.

Cellular Composition, Endocrine Function and Angiogenic Activity of Pre-Vascularized Islet Organoids
Generation of PIO and PI is described in Figure 2A. Aggregation and incorporation of the different cell types occurred within 4 days (Figures 2B,C). Mean diameter of NI, PI and PIO was 144.4 ± 6.6, 105.8 ± 1.2 and 134.3 ± 2.3 μm, respectively ( Figure 2D). NI showed the biggest heterogeneity in size. PI exhibited a significantly smaller mean diameter in comparison with PIO (p < 0.0001), due to fewer cellular content. Cellular composition observed by fluorescent microscopy showed that all 3 cell types were present in the PIO ( Figure 2E). The functional capacity of the constructs was evaluated by glucose-stimulated insulin secretion (GSIS) assay. PI and PIO demonstrated significantly improved insulin secretion in response of glucose stimulation (SI 7.8 ± 1.5 and 7.7 ± 1.2), compared to NI (SI 2.0 ± 0.5, p 0.013 and p 0.014, respectively). No significant difference was observed between PI and PIO ( Figure 2F). In addition, total insulin content/IC was measured and compared between the three groups. PI and PIO demonstrated an increased insulin content/IC (0.01 ± 0.003 and 0.008 ± 0.002 pmol/L, respectively) in comparison with NI (0.002 ± 0.0004 pmol/L). These dramatic enhancement of static GSIS secretion in our constructs compared to unmodified native islets indicate that better oxygen and nutrient access, and improved transport of glucose and insulin, enhanced survival and function of PI and PIO. Our findings are consistent with previous reports on better in vitro performance of smaller pseudoislets (26,27). To investigate the angiogenic potential of the PIO, collagenbased sprouting assays were performed. Our results demonstrated that PIO showed more extensive sprouting in surrounding matrix compared to IC + HUVEC (Supplementary Figure S2). In contrast, no sprouting was observed from PI (data not shown). Furthermore, immunofluorescence revealed GFP positive cells confirming their endothelial nature.

Pre-Vascularized Islet Organoids Improve Glycaemic Control in Immunodeficient Diabetic Mice
To assess whether incorporation of hAECs and HUVECs into the islet organoids could promote engraftment and function in vivo, diabetic NOD-Rag1 null mice were transplanted with a marginal mass of PIO (n 14), NI (n 13) and PI (n 9). Mice transplanted with PIO demonstrated significant improvement of glycaemic control compared to both controls. Average blood glucose levels were significantly lower in the PIO group compared to NI and PI ( Figure 3A). Normoglycemia was reached in 78.6% of animals (11/14) in the PIO group, in comparison with 55.6% (5/9) and 46.2% (6/13) for the PI and NI groups, respectively ( Figure 3B). Median time to achieve normoglycemia was 6 days in the PIO group, 21 days in the PI group and >30 days in the NI group. To investigate secretory function of the graft, IPGTT was performed at 30 days post-transplantation. Mice transplanted with PIO and nondiabetic controls (NDC) showed lower blood glucose levels  when compared to animals transplanted with PI and NI ( Figure 3C). This is illustrated by the increasing area under the curve (AUC) of the different groups, with PIO (966.8 ± 113.7), PI (1783 ± 351.1, p 0.05 vs. PIO) and NI (1856 ± 294.5, p 0.014 vs. PIO; Figure 3D). We further investigated whether the improved glycemic control in the PIO group was associated with insulin production from the transplanted β cells. Remarkable upregulation of rat insulin mRNA levels in the graft was found in the PIO group in comparison to controls (PIO vs. PI, p 0.013, PIO vs. NI, p 0.013; Figure 3E). These results were supported by insulin measurements in the serum taken from the same mice ( Figure 3F). Although a statistical significance wasn't achieved, a ten-fold increase in insulin levels was detected in the PIO group (1,259 ± 521 pmol/L), in comparison to both controls (NI: 140.6 ± 22.1 pmol/L, PI: 159.8 ± 14.4 pmol/L, p ns).
Glp-1r, pdx1 are known to be critical for promoting insulin secretion (28)(29)(30)(31). Therefore, we investigated whether these genes were involved in the improved secretory outcomes of PIO. Gene expression analyses revealed upregulation of genes involved in β-cell function (pdx1, pcsk1, pcsk2 and glp-1r) in PIO at 30 days post-transplantation, compared to controls (pdx1: PIO vs. PI, p 0.0009, PIO vs. native islet, p 0.0009; glp-1r: PIO vs. PI, p 0.002, PIO vs. native islet, p 0.002; pcsk1: PIO vs. PI, p 0.02, PIO vs native islet p 0.021 and pcsk2: PIO vs. PI, p 0.0005, PIO vs. native islet, p 0.0006; Figure 3G). Interestingly, at an earlier time points (3 days), a similar increase in gene expression was observed in PI and PIO in comparison with NI group, although without reaching statistical differences (Supplementary Figure S3). These data indicate that incorporation of accessory cells into the organoids supports long term secretory function of β cells.

Transplantation of Pre-Vascularized Islet Organoids Accelerates Graft Revascularization
To evaluate engraftment and revascularization, graft-bearing EFPs were removed at 30 days post-transplantation and processed for histology. Immunohistochemical staining for CD34, a marker for endothelial cells, showed that vessel density was significantly higher in the PIO samples (22.6 ± 3.5 CD34 + cells/cm 2 ) than in the NI samples (7.6 ± 0.9, p 0.002; Figures 4A,B). Furthermore, in the PIO group, vessels were observed not only around graft, but mainly within β-cell positive area.
To investigate whether the blood vessels formed within the engrafted tissue constructs become functional and contribute to graft perfusion, we used intravascular injection of fluorescently labeled Lectin. Histological assessment of the Lectin-perfused grafts demonstrated the presence of functional Lectin positive vascular network within the PIO, in contrast only few vessels were present within NI ( Figure 4C).
Next, we examined the mechanisms by which supportive cells (HUVECs and hAECs) contributed to rapid neovascularization of the graft. To this end, we investigated whether these cells might induce the production of angiogenic factors, such as vegf-a ( Figure 4D). We observed, that rat vegf-a mRNA expression was significantly higher in PIO group (0.365 ± 0.033 AU) compared to NI (0.038 ± 0.005 AU; p 0.0006) group. This finding was further confirmed by immunohistochemical staining for vegf-a of recovered samples, demonstrating higher fluorescent intensity in the PIO compared to NI ( Figure 4E). These data indicate that incorporation of HUVEC and hAEC into PIO contribute to graft revascularization.

Human Amniotic Epithelial Cells Incorporation Into Organoids Improves Function and HUVEC-Derived Revascularization
Finally, we evaluated whether incorporation of hAECs into the organoids was essential for the engraftment and vascularization of the PIO. To this end, we added an additional group of mice transplanted with spheroids composed of IC: HUVEC (1:1 ratio) to the three existing groups. Figure 5 summarizes the results obtained with this group. Blood glucose control was significantly lower in the IC + HUVEC group in comparison to the PIO group ( Figure 5A). The IPGTT performed at 30 days post-transplantation demonstrated a poor glucose clearance in the IC + HUVEC group ( Figure 5B). Response to increased blood glucose levels was significantly lower than for the PIO group as demonstrated by the AUC (2044 ± 578.1 vs. 966.8 ± 113.7, p 0.008, respectively; Figure 5C).
After demonstrating that incorporation of supportive cells into the PIO improved graft revascularization, we investigated the degree to which these cells were contributing to new vessel development in the graft. To easily identify donor-derived new vessels, GFP-transduced HUVECs were incorporated into the PIO. Graft-bearing EFPs were recovered at 30 days posttransplantation and processed for immunohistological analysis. Interestingly, GFP positive cells were found inside the graft in the PIO group, while none was found in the IC + HUVEC group ( Figure 5D). Both human and mouse vessels were positively stained by anti-CD34 confirming the establishment of anastomoses between donor derived HUVECs and mouse blood vessels. Furthermore, GFP/CD34 double positive endothelial cells were found at the graft periphery, inside capillaries containing erythrocytes, indicating that HUVECs were able to migrate and merge with a murine vascular system, forming functionally perfused blood vessels, as shown in Figure 5E. These data indicate that hAECs support HUVECs inside the organoids and thus contribute to accelerated revascularization.

DISCUSSION
Impaired and delayed revascularization of the graft is a major issue in islet transplantation and represents a main limitation to the search for extrahepatic sites for islet transplantation. Common vascularization strategies focus either on the combination of accessory cells with islets (32) or | Crosstalk between the hAEC, the endothelial cell (EC) and the islet β cell (IC) within the PIO. hAEC enhances revascularization of the PIO in a direct manner by secreting 1) angiogenic factors and 2) vegf that improve EC viability, function, proliferation and blood vessel formation, and 3) by producing ECM-degrading proteases (MMP-1) that facilitate EC migration and sprouting. Additionally, hAECs secrete EGF that 4) upregulates IC pdx1 expression, leading to higher IC survival and proliferation, as well as 5) glp1-r expression, leading to an up-regulation of glycolytic genes and vegf-a through the mTOR/HIF-1a pathway, resulting in 6) an improved insulin secretion and 7) a better revascularization of the PIO. incorporation of endothelial cells into islet-like constructs generated from embryonic stem cell-derived ß cells (30) or ß cell lines (31), and are mainly based on in vitro testing. In this study, we successfully generated functional pre-vascularized islet organoids using multiple cell types. The major finding of this study is that incorporation of hAECs and HUVECs into insulin-producing organoids hastens the rate of graft revascularization, and subsequently results in better engraftment of the β-cell mass.
HUVECs are the most commonly used, robust source of human endothelial cells in regenerative medicine and tissue engineering (33). However, limited proliferative potential of these cells hinders their clinical application. hAECs isolated from the amniotic membrane of discarded placenta is considered a non-controversial stem cell source (34). These cells demonstrated profound anti-fibrotic, anti-inflammatory, non-tumorigenic and low antigenic properties (35,36). Furthermore, hAECs possess pluripotent stem cells characteristics, can be isolated in large quantities and are thus considered as an evolving therapeutic tool for the development of various clinical applications (35). Previously, we have shown that the generation of insulin-secreting organoids from primary IC in combination with hAECs improved islet engraftment and vascularization primarily by stimulating VEGF-A production from the graft via HIF1-α signaling pathway (17,20). Therefore, in this study, we evaluated whether hAECs could accelerate the angiogenic potential of mature endothelial cells (HUVECs). Our results show that chimeric, prevascularized insulin secreting organoids are capable of establishing new vascular networks in vitro and in vivo when co-cultured with hAECs and HUVECs. The enhancement of the angiogenic potential of HUVECs by hAECs can be explained by three possible mechanisms: 1) via the secretion of ECM-degrading proteases facilitating EC migration and sprouting (37), 2) by upregulating VEGF expression in endothelial and islet cells (38), and 3) by the reduction or suppression of inflammatory responses (39,40). Our in vivo experiments have demonstrated the superiority of pre-vascularized islet organoids for insulin secretion and revascularization.
Another important finding is the existence of a cross-talk between the islet, endothelial and amniotic epithelial cells associated within one organoid (summarized in Figure 6), and that this communication can be successfully employed for improving outcomes of islet transplantation. In terms of revascularization, we observe that both blood vessel density and number of functional vessels were significantly higher in the grafts explanted from mice transplanted with PIO in comparison to control groups. VEGF-A is a proangiogenic factor that recruits endothelial cells and circulating endothelial progenitors (11). Our results demonstrated significant upregulation of VEGF-A gene expression in the grafts explanted from mice transplanted with pre-vascularized organoids. Immunohistochemical analysis of the explanted grafts confirmed that the major producers of VEGF-A were islet cells. This finding was in agreement with our previous studies, demonstrating that hAECs markedly increase production of VEGF-A in islet cells via paracrine signalling (17). In addition, hAECs themselves are known to secrete VEGF-A (41), which on the other hand could also enhance performance of HUVECs within the organoids. To verify this hypothesis, we used GFP-HUVECs and tracked transplanted cells inside the graft. We found GFP-HUVECs both inside and in the vicinity of the graft. At the same time, GFP-HUVECs were also detected to be integrated into the peri-islet functional blood vessels containing red blood cells. This indicates that the donor derived endothelial cells anastomosed with the murine vascular system and formed functionally perfused blood vessels. Interestingly, the same was not observed in mice transplanted with IC + HUVECs, in which no GFP-HUVECs were found in the recovered grafts. In addition, almost no blood circulation was observed inside the graft area. This indicates that hAECs contribute to the process of endothelial cell remodelling and stabilization finally leading to mature vessel formation. Our findings are in agreement with previously reported data, demonstrating that hAECs enhance EC viability, function, proliferation, migration and blood vessel formation in vitro and in vivo (41). Furthermore, amniotic cells secrete additional factors that are critical for angiogenesis, such as EGF, HB-EGF, bFGF, HGF, IGF-1 (42). Taken together, these data suggest that hAECs promote revascularization both directly by secreting angiogenic factors and indirectly by stimulating VEGF-A secretion by islet cells.
Accelerated revascularization can also provide important survival cues to the islet cells. Another important challenge to islet transplantation is to achieve stable, long-term insulin independence, preferably with single donor islet transplantation. In this study, improved revascularization was accompanied by prompt return of severely diabetic mice to a normoglycaemic state after transplantation of minimal mass of prevascularized islet organoids. Mice transplanted with PIO showed significantly improved insulin secretion and better glucose clearance compared to mice transplanted with PI, NI and IC + HUVECs. Investigations of underlying mechanisms showed that superior function of β-cells in PIOs was mediated by the GLP-1R signalling pathway. GLP-1R has been found to regulate homeostasis of β-cell mass by inducing β-cell proliferation and protecting against apoptosis. On the other hand, activation of the GLP-1R leads to the activation of multiple downstream pathways, including EGF receptor signalling (43), which in turn stimulates proliferation of β cells (44). EGF has been shown to enhance glucose-dependent insulin secretion and upregulate PDX1 expression (20). Although the precise mechanisms underlying this pattern of increased gene expression in the PIOs are not fully understood, we speculate that growth factor expression profile of hAECs, mainly EGF, could stimulate upregulation of the expression of genes involved in β-cell function (GLP-1R, PDX-1).

CONCLUSION
In this study, we demonstrate a novel approach to generate prevascularized islet organoids by combining primary ICs with two additional supportive cell types, HUVECs and hAECs, and address some of the challenges of clinical islet transplantation such as donor supply scarcity, impaired islet engraftment and revascularization. Furthermore, our data demonstrate that hAECs not only promote cell viability and engraftment, but most importantly, play a primordial supporting role in the development of HUVEC-derived neo-vessels within the transplanted tissue. However, to generate large numbers of uniform, sizecontrolled and functional prevascularized islet organoids, a scalable platform technology is a prerequisite to ensure standardization and reproducibility for new and innovative beta cell replacement strategies.
Addressing this challenge, recently, we showed that several spheroid generating methods are suitable to assemble uniform, size-controlled and functional islet-like clusters (45). The compared techniques included native islets as controls (IEQs), a self-aggregation technique, the hanging drop technique, the agarose 3D microwell technique and the Sphericalplate SP5D. We demonstrated that up to 9000 islet organoids can be easily generated per plate.
Moreover, the SP5D can be automatized, and roboticmediated spheroid generation can further reduce variability and therefore improve standardization and reproducibility.
Taken together, these findings could be a basis for the design of novel extra-hepatic, extra-vascular islet transplantation sites.

CAPSULE SENTENCE SUMMARY
The pre-vascularized islet organoids were generated from dissociated islet cells, human amniotic epithelial cells (hAECs), and human umbilical vein endothelial cells (HUVECs). Our study demonstrates that pre-vascularized islet organoids exhibit enhanced in vitro function and most importantly, improved engraftment and accelerated vascularization in vivo in a murine model.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by the Commission Cantonale d'Ethique de la Recherche (CCER), in compliance with the Swiss Human Research Act (810.30). The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by the Institutional Animal Care and Use Committee of the University of Geneva.