The members of the VANGUARD Consoritum are listed at the end of the paper
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Bio-engineering of pre-vascularized islet organoids for the treatment of type 1 diabetes.
Lack of rapid revascularization and inflammatory attacks at the site of transplantation contribute to impaired islet engraftment and suboptimal metabolic control after clinical islet transplantation. In order to overcome these limitations and enhance engraftment and revascularization, we have generated and transplanted pre-vascularized insulin-secreting organoids composed of rat islet cells, human amniotic epithelial cells (hAECs), and human umbilical vein endothelial cells (HUVECs). Our study demonstrates that pre-vascularized islet organoids exhibit enhanced
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 (
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 (
Despite the fact that islets represent only 1–2% of pancreatic tissue volume, they receive 10–15% of the total pancreatic blood flow (
Islet isolation and culture lead to the disruption of the islet capillary system, with significant loss of ECs due to de-differentiation or necrosis (
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 size-controlled spheroids leads to improvement of function and viability, thanks to heterotypic cell–cell interactions and reproduction of the complex natural morphology of the islet (
All reagents and antibodies used in this study are listed in
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‐Rag1tm1Mom/J (abbreviated NOD–
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.
Human umbilical vein endothelial cells (HUVECs) were isolated using a method adapted from a previously published protocol (
hAECs were isolated, cultured and characterized as described previously (
Medium was changed every 48 h. Confluent cells were recovered by mild trypsinization and were cryopreserved for later utilization.
Rat islets were isolated by enzymatic digestion (collagenase V) and purified using a discontinuous Ficoll gradient (
HUVECs and hAECs were analyzed for expression of previously reported endothelial cell surface markers or specific amniotic epithelial cell surface markers by flow cytometry.
For analysis, cells (2.5 × 105) were stained by incubation for 30 min with primary or isotype control antibody in 100 µl PBS with 0.2% BSA, washed twice with PBS, and analyzed. Antibodies used for HUVECs were: AlexaFluor 657-conjugated anti-CD144 (1:40 dilution), PE-conjugated anti-CD31 and PerCP-Cy 5.5-conjugated anti-CD45 (1:25 dilution). Antibodies used for hAECs were: FITC-conjugated anti-human CD105 (clone 266), BV421-conjugated anti-human CD326 (clone EBA-1), PerCP-Cy5.5 conjugated anti-SSEA4 (clone MC813-70) (1:50 dilution), PE-Cy7 conjugated anti-human CD90 (clone 5E10; 1:100 dilution), PE-conjugated anti-human HLA-E (clone 3D12) and APC-conjugated anti-human HLA-G (clone 87G; 1:20 dilution).
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).
The tube formation assay was performed according to manufacturer’s protocols of Corning® Matrigel® Matrix. 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 24-well plates. After 1 h of incubation at 37°C, cells (8 × 104) were seeded onto the Matrigel and tube formation of HUVECs was observed and photographed using an inverted phase-contrast microscope during 6 h.
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.
Pre-vascularized islet organoids (PIO) were generated on AggreWell™400 24-well plates by seeding mixture of ICs, HUVECs and hAECs at a ratio of 5:4:1 (800 cells/organoid). Undissociated native islets (NI), ICs spheroids (400 ICs/spheroid), hereafter referred to as pseudo-islets (PI), and IC:HUVEC spheroids (ratio 1:1, 800 cells/spheroid), hereafter referred to as IC + HUVEC served as controls. PIO, PI and IC + HUVEC were cultured for 4 days to allow cell aggregation at 37°C, 21% O2 and 5% CO2.
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).
PIO, PI and NI were collected fixed in formalin and embedded in paraffin. Serial sections of 5 μm were cut and processed for immunofluorescent staining. Slides were stained with the following primary antibodies: guinea pig anti-insulin (1:100), chicken anti-GFP (1:500), and rabbit anti-CK-7 (1:100). The following secondary antibodies were then applied: donkey anti–guinea pig Alexa 555 Fluor-conjugated (1:300), donkey anti–guinea pig FITC‐conjugated (1:200), donkey anti-mouse AMCA-conjugated (1:50), goat anti-chicken Alexa Fluor 488 (1:500).
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% CO2, and 100% humidity. As control, one hundred IC + HUVEC spheroids and PI were cultured in the same way in the hydrogel.
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.
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 (
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 capacity to clear glucose
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 left ventricle, followed by 1 ml of 4% PFA. The graft bearing EFPs were collected and fixed overnight in 4% PFA at 4°C. They were then maintained in 30% sucrose at 4°C until used for histology.
Grafts were recovered, fixed in formalin and embedded in paraffin. Serial sections of 5 μm were cut and processed for immunofluorescent staining. Tissue samples were permeabilized with 0.5% Triton X-100/PBS for 30 min, followed by 1-h incubation in 0.5% BSA/PBS at room temperature to block unspecific sites. Slides were then incubated with the following primary antibodies: guinea pig anti-insulin (1:100), rabbit anti-CD34 (1:2,000), chicken anti-GFP (1:500), and rabbit anti-VEGF (1:100). The following secondary antibodies were then applied: donkey anti–guinea pig Alexa 555 Fluor-conjugated (1:300), donkey anti–guinea pig FITC‐conjugated (1:200), donkey anti‐rabbit Alexa 555 Fluor-conjugated (1:300) and goat anti-chicken Alexa Fluor 488 (1:500). Both primary and secondary antibodies were diluted in PBS-0.5% BSA. Finally, slides were mounted with aqueous mounting medium containing DAPI for nuclear staining. Slides were processed on a Zeiss Axioscan.Z1 slide scanner and a Zeiss Axiocam. To analyse vascularization, six pictures per condition were taken and the number of CD34+ cells were counted and normalized by the graft area.
Morphometric analysis was performed using Zen 2.3 Blue Edition software.
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
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 log‐rank (Mantel‐Cox) test. A
HUVECs reached 80% confluence within 5 days with initial seeding density of 6,000 cells/cm2. Morphologically, cells displayed typical elliptic shape (
HUVEC characterization and
When cultured on Matrigel, HUVECs formed well-shaped vascular-like structures over a period of 6 h (
To track HUVECs within organoids both
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
Generation of PIO and PI is described in
Organoids generation.
To investigate the angiogenic potential of the PIO, collagen-based sprouting assays were performed. Our results demonstrated that PIO showed more extensive sprouting in surrounding matrix compared to IC + HUVEC (
To assess whether incorporation of hAECs and HUVECs into the islet organoids could promote engraftment and function
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,
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/cm2) than in the NI samples (7.6 ± 0.9,
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 (
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
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.
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 post-transplantation 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 (
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 (
HUVECs are the most commonly used, robust source of human endothelial cells in regenerative medicine and tissue engineering (
Another important finding is the existence of a cross-talk between the islet, endothelial and amniotic epithelial cells associated within one organoid (summarized in
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)
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 (
In this study, we demonstrate a novel approach to generate pre-vascularized 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, size-controlled 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 (
Moreover, the SP5D can be automatized, and robotic-mediated 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.
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
The School of Medicine of the Università del Piemonte Orientale “Amedeo Avogadro”: Chiara Borsotti, Simone Merlin. IRCCS Ospedale San Raffaele: Lorenzo Piemonti, Antonio Citro, Silvia Pellegrini. Ludwig-Maximilians-Universität München: Jochen Seissler, Lelia Wolf-van Buerck, Mohsen Honarpisheh. Lyon Claude Bernard University: Olivier Thaunat. Erasmus University Medical Center Rotterdam: Emma Massey, Antonia Cronin, Eline Bunnik, Dide de Jongh. European Society for Organ Transplantation: Luca Segantini, Giovanna Rossi. Kugelmeiers AG: Patrick Kugelmeier, Petra Wolint. Accelopment Switzerland Ltd. : Marco Cavallaro, Julia Götz, Jeanette Müller.
The original contributions presented in the study are included in the article/
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.
C-HW: Performing experiments, data analysis and interpretation, manuscript writing. FL, DC-D, and KB: Performing experiments. MC, TB, CO, AF, and DB: Manuscript editing. LP: Technical support. AF, CO, VO-G and BT: Provision of study material. EB: Conception and design, supervision of project, financial support, administrative support, manuscript writing, final approval of manuscript. All other authors edited and approved the manuscript.
This work is supported by grants from the European Commission (Horizon 2020 Framework Program; VANGUARD grant 874700), the European Foundation for the Study of Diabetes (EFSD), the Juvenile Diabetes Research Foundation (JDRF; grant 3-SRA-2020-926-S-B), the Shota Rustaveli National Science Foundation (grant FR-19-19760) and the Swiss National Science Foundation (grant 310030_173138).
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The Supplementary Material for this article can be found online at:
Aminomethylcoumarin Acetate
area under the curve
basic fibroblast growth factor
Bovine Serum Albumine
Commission Cantonale d’Ethique de la Recherche
Cytokeratin 7
4′,6-diamidino-2-phénylindole
Dulbecco’s Modified Eagle Medium
endothelial cell
epididymal fat pad
epidermal growth factor
fetal bovine serum
green fluorescent protein
Glucoagon-like peptide 1 receptor
human amniotic epithelial cell
heparin binding epithelial growth factor
Hanks’ balanced salt solution
Hepatocyte growth factor
Hypoxia-inducible factor 1-alpha
human umbilical vein endothelial cell
islet cell
islet equivalent
insulin-like growth factor-1
intraperitoneal glucose tolerance test
lentiviral
Minimum essential medium non-essential amino acids
multiplicity of infection
native islet
non-diabetic control
Dubbelco’s Phosphate buffer saline
Proprotein Convertase Subtilisin/Kexin Type 1
Proprotein Convertase Subtilisin/Kexin Type 2
pancreatic and duodenal homeobox 1
Paraformaldehyde
pseudo-islet
prevascularized islet organoid
ribosomal protein lateral stalk subunit P1
reverse transcriptase polymerase chain reaction
stimulation index
streptozotocin
vascular endothelial cadherin
Vascular endothelial growth factor A