Pluripotent stem cell–derived islets (SC-islets) have emerged as a new source for β-cell replacement therapy. The function of human islet transplants is hampered by excessive cell death posttransplantation; contributing factors include inflammatory reactions, insufficient revascularization, and islet amyloid formation. However, there is a gap in knowledge of the engraftment process of SC-islets. In this experimental study, we investigated the engraftment capability of SC-islets at 3 months posttransplantation and observed that cell apoptosis rates were lower but vascular density was similar in SC-islets compared with human islets. Whereas the human islet transplant vascular structures were a mixture of remnant donor endothelium and ingrowing blood vessels, the SC-islets contained ingrowing blood vessels only. Oxygenation in the SC-islet grafts was twice as high as that in the corresponding grafts of human islets, suggesting better vascular functionality. Similar to the blood vessel ingrowth, reinnervation of the SC-islets was four- to fivefold higher than that of the human islets. Both SC-islets and human islets contained amyloid at 1 and 3 months posttransplantation. We conclude that the vascular and neural engraftment of SC-islets are superior to those of human islets, but grafts of both origins develop amyloid, with potential long-term consequences.
There is a gap in knowledge of the engraftment process of stem cell–derived islets (SC-islets).
We investigated the engraftment capability of SC-islets compared with that of human islets after transplantation.
Vascular density was similar in SC-islets and human islets, but neural density was four- to fivefold higher in SC-islets than in human islets.
Vascular and neural engraftment of SC-islets are superior to those of human islets.
Introduction
β-Cell replacement therapies for type 1 diabetes have over the years been restricted to either transplantation of the whole pancreas or isolated pancreatic islets, both obtained from brain-dead cadaveric donors. Clinical use of such diabetes reversal protocols has so far been limited, mainly because of the shortage of organ donors, but also because of the need for chronic immunosuppressive treatment in recipients. Islet transplantation is a much simpler and safer procedure than whole-pancreas transplantation, but long-term results have been inferior to those of whole-gland transplantation. Despite the progressive improvements in islet transplantation during the last decades (1), many insulin-producing cells are lost posttransplantation (2). Important contributing factors to this excessive cell death include instant blood-mediated inflammatory reaction when the islet tissue becomes exposed to portal blood (3), gluco- and lipotoxicity (4), and insufficient vascular engraftment (5). The decreased blood perfusion and oxygenation of transplanted islets may also contribute, by impaired drainage of synthesized and secreted islet amyloid polypeptide (IAPP), to the progressive β-cell toxic amyloid formation observed in human islet grafts (6).
The limited availability of tissue for β-cell replacement may be solved by using embryonic or induced pluripotent stem cells, generating large numbers of insulin-producing cells. There are also emerging strategies to manipulate such immature cells for induced hypoimmunity and tolerance with regard to rejection and recurrence of autoimmune disease after their differentiation (as reported by Deuse et al. [7], for example). Still, even if the generated cells have a similar potency for insulin secretion as human β-cells (8–11), implantation of a sufficient number of insulin-producing cells to obtain sustained reversal of hyperglycemia will pose a problem if the conditions for their long-term survival and function are not optimal posttransplantation. Like primary islets, stem cell–derived islets (SC-islets) depend on the ingrowth of blood vessels from surrounding tissue for their oxygen supply. Blood vessels are also central for glucose sensing, dispersal of secreted hormones, and paracrine functional support (12). The specific importance of blood vessels for the function and capacity of human islets and SC pancreatic progenitors in diabetes reversal was recently reported (13). Moreover, proper innervation is essential to regain full and coordinated β-cell function (14). To address the gap in knowledge of the engraftment capability of SC-islets, we investigated the capability of transplanted SC-islets to become vascularized and innervated by comparing them with primary human islets.
Research Design and Methods
Animals
NOD.Cg-PrkdcscidI12rgtm1Sug mice (Taconic M&B, Ejby, Denmark; female and male mice, 23.7 ± 1.0 g, age 7–20 weeks) were used as recipients for the transplantation of SC-islets and human islets. All experiments were approved by the Animal Ethical Committee in Uppsala, Sweden.
Derivation of SC-Islets
Human Islets
The regional ethics board in Uppsala, Sweden, approved the use of human pancreatic tissue for this study. Pancreata from adult heart-beating brain-dead donors were provided by the Nordic Network for Clinical Islet Transplantation and used for the isolation of human islets at the human islet isolation facility, Rudbeck Laboratory, Uppsala University Hospital. The average age of the organ donors was 51.5 ± 4.1 years (range 19–74), and no donor had diabetes (HbA1c 35.8 ± 0.9 mmol/mol). Available information on islet donors and islet characteristics is listed in Table 1.
Human islet donor information
Donor no. . | Age, years . | Sex . | BMI, kg/m2 . | HbA1c, mmol/mol . | Cold ischemia time, h . | Dynamic index* . |
---|---|---|---|---|---|---|
1 | 56 | Female | 24.0 | 33.0 | 07:01 | 0.9 |
2 | 56 | Female | 22.2 | 34.0 | 07:40 | 19.6 |
3 | 66 | Male | 26.9 | 38.0 | 06:49 | 3.6 |
4 | 49 | Male | 30.9 | 39.0 | 17:57 | NA |
5 | 43 | Male | 29.1 | 30.0 | 04:37 | 24.7 |
6 | 41 | Female | 27.4 | 38.0 | 17:25 | 11.1 |
7 | 54 | Male | 42.6 | 37.0 | 15:29 | 1.5 |
8 | 62 | Female | 25.7 | 33.0 | 10:57 | 4.0 |
9 | 53 | Female | 28.7 | 33.0 | 15:32 | 2.6 |
10 | 26 | Female | 34.9 | 35.0 | 21:21 | 1.6 |
11 | 19 | Female | 26.7 | 35.0 | 19:07 | NA |
12 | 74 | Female | 22.7 | 34.0 | 07:03 | 3.6 |
13 | 70 | Female | 40.8 | 42.0 | 04:39 | 1.6 |
14 | 39 | Male | 26.6 | 34.0 | 14:34 | 18.0 |
15 | 65 | Male | 30.1 | 42.0 | 09:46 | 5.3 |
Donor no. . | Age, years . | Sex . | BMI, kg/m2 . | HbA1c, mmol/mol . | Cold ischemia time, h . | Dynamic index* . |
---|---|---|---|---|---|---|
1 | 56 | Female | 24.0 | 33.0 | 07:01 | 0.9 |
2 | 56 | Female | 22.2 | 34.0 | 07:40 | 19.6 |
3 | 66 | Male | 26.9 | 38.0 | 06:49 | 3.6 |
4 | 49 | Male | 30.9 | 39.0 | 17:57 | NA |
5 | 43 | Male | 29.1 | 30.0 | 04:37 | 24.7 |
6 | 41 | Female | 27.4 | 38.0 | 17:25 | 11.1 |
7 | 54 | Male | 42.6 | 37.0 | 15:29 | 1.5 |
8 | 62 | Female | 25.7 | 33.0 | 10:57 | 4.0 |
9 | 53 | Female | 28.7 | 33.0 | 15:32 | 2.6 |
10 | 26 | Female | 34.9 | 35.0 | 21:21 | 1.6 |
11 | 19 | Female | 26.7 | 35.0 | 19:07 | NA |
12 | 74 | Female | 22.7 | 34.0 | 07:03 | 3.6 |
13 | 70 | Female | 40.8 | 42.0 | 04:39 | 1.6 |
14 | 39 | Male | 26.6 | 34.0 | 14:34 | 18.0 |
15 | 65 | Male | 30.1 | 42.0 | 09:46 | 5.3 |
NA, not available.
Dynamic index was calculated as ratio of insulin secretion response to rise in glucose concentration in islet perifusion medium from 1.67 to 20 mmol/L.
Cell Transplantation
For implantation of human islets or SC-islets (fully differentiated at stage 7 week 3–4), 700–800 islet equivalents were packed in PE-50 tubing. Mice were anesthetized with isoflurane, and the kidney was exposed. A small opening in the kidney capsule was made, and the capsule locally gently separated from the kidney parenchyma with a glass rod. The tubing was inserted into the opening, and the human islets/SC-islets were implanted using a Hamilton syringe. Subcutaneous carprofen (5 mg/kg; Norocarp) was used for analgesia.
Human C-Peptide Levels
One and 3 months posttransplantation, stimulated (i.e., 10 min postinjection of glucose intravenously at 2 g/kg bodyweight) human C-peptide levels were measured in blood plasma using ultrasensitive human C-peptide ELISA (Mercodia, Uppsala, Sweden).
Immunohistochemistry
One or 3 months posttransplantation, grafts were fixed in 4% paraformaldehyde at 4°C overnight, followed by incubation in 15% sucrose-PBS solution for 2–3 h at room temperature and then in 30% sucrose-PBS solution at 4°C overnight and frozen. Human islets and SC-islets were fixed in 4% paraformaldehyde for 20 min at room temperature, washed in PBS, and frozen. Cryosections of the tissues were prepared, with a thickness of 8 μm. Sections were incubated with primary antibody overnight at +4°C, followed by incubation with secondary antibody (antibodies listed in Supplementary Table 4). The nuclei were stained with DAPI.
Amyloid Detection
To quantify the amount of amyloid in the transplanted human islets and SC-islets, the grafts were stained for amyloid using the oligothiophene probe pentameric formylthiophene acetic acid with cyano (16) and diluted 30 μmol/L in 0.1 mmol/L phosphate buffer, in combination with immunolabeling for mouse CD31 to evaluate the distance between amyloid and nearest blood vessels and for chromogranin A to delineate the endocrine cells.
Confocal Imaging
All images were obtained using laser scanning confocal microscope Zeiss LSM 780 (Carl Zeiss AG, Oberkochen, Germany) with Plan-Apochromat 10×/0.45 M27 and 20×/0.8 M27 objectives.
Image Analysis
Three levels separated by 250 μm were included in each biological replicate for the image analysis. Exceptions were made for some analysis of Lycopersicon esculentum agglutinin (LEA) staining, where only one to two levels were analyzed because of limited material. For the image analysis of the composition of cells before and after transplantation, Zeiss ZEN software (blue edition) was used.
The ingrowth of blood vessels and neurons in the grafts was calculated in tissue sections after immunolabeling. Chromogranin A was used to identify the area of endocrine cells, and mouse blood vessels and human blood vessels were identified with species-specific anti-CD31 antibodies, pericytes with NG2, and neurons with anti–NF-L. Vascular and neuronal density were reported as percentages of the endocrine area. Percentages of insulin-, glucagon-, and somatostatin-positive cells are reported as percentages of the sum of these cells. Amyloid is reported as a percentage of the endocrine area, and β-cell apoptosis is reported as the percentage of insulin-positive cells also staining positive for caspase-3.
Quantifications of vascular density, nerve density, and amyloid formation were performed using Imaris software (Bitplane). Analyses of β-cell apoptosis and additional nerve markers (i.e., tyrosine hydroxylase and vesicular acetylcholine transporter), pericytes, and LEA were performed using Fiji software (Image J 1.53t; National Institutes of Health) (17).
Blood Flow and Oxygen Tension Measurements
Animals undergoing transplantation were anesthetized with intraperitoneal administration of 0.02 mL/g bodyweight Avertin (2.5% v/v solution of 10 g 97% v/v 2,2,2-tribromo-ethanol [Sigma-Aldrich] in 10 mL 2-methyl-2-butanol [BDH Merck, Ltd., Poole, U.K.]). The animals were placed on an operating table maintained at 37°C and tracheostomized. Blood flow in the graft and kidney cortex in the immediate vicinity of the graft (reference organ) was measured using laser Doppler flowmetry (probe diameter 1.2 mm; Transonic BLF21 Series; Transonic, Ithaca, NY). Blood flow values were recorded as arbitrary tissue perfusion units because the equipment is not easily calibrated in physical units of blood flow.
Oxygen tension in the graft and kidney cortex in the immediate vicinity of the graft (reference organ) was measured using custom-made Clark microelectrodes (tip diameter 2–5 μm; Unisense, Aarhus, Denmark). The mean of all these measurements (five or more per location) in each animal was calculated and considered to be one experiment. All measurements were performed according to previously established protocols (18).
Gene Expression
Total RNA was extracted using the RNeasy Plus Micro Kit (Qiagen) according to the manufacturer’s instructions. RNA purity and concentrations were determined using a NanoDrop ND-1000 (NanoDrop). Five batches of SC-islets and human islets from seven donors were sent for RNA sequencing by Novogene Co., Ltd. (Beijing, China). Preparation of the RNA library and transcriptome sequencing was conducted by Novogene. Bulk RNA sequencing analysis was performed, as described in the Supplementary Material. Results from the gene expression analysis together with the raw sequences were deposited into the Gene Expression Omnibus under accession GSE221383.
For quantitative RT-PCR, 500 ng total RNA was converted to cDNA using the SuperScript IV Cells Direct cDNA Synthesis Kit (Thermo Fisher Scientific) at a volume of 20 μL. RT-PCR was performed using the PowerUp SYBR Green Master Mix (Applied Biosystems) on the CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA). Relative expression levels were normalized to the internal control, GAPDH. All primers used are listed in Supplementary Table 1.
Statistical Analysis
Values are expressed as mean ± SEM. In these studies, one biological replicate corresponded to one mouse undergoing transplantation with SC-islets from one differentiation or islets from one human donor, but exceptions were made for the blood flow and oxygen tension measurements, where two mice received human islet transplants from the same donor, and four mice received SC-islet transplants from two differentiations (i.e., two mice per differentiation). For analyses of endocrine cell composition and vascular density, one-way ANOVA and the Šídák multiple comparisons test was applied. For analyses of amyloid, neural density, blood flow, and oxygen tension measurements, the Student unpaired t test was used, whereas Mann-Whitney test was used for β-cell apoptosis. P < 0.05 was considered statistically significant. All statistical analyses were carried out using GraphPad Prism 9.0 software (GraphPad, San Diego, CA).
Data and Resource Availability
All RNA sequencing raw data generated for this study are publicly available at Gene Expression Omnibus accession GSE221383. All data are available from the corresponding author upon reasonable request.
Results
The stimulated C-peptide levels at 10 min postinjection of glucose were 440.6 ± 53.22 pmol/L (n = 6) for human islet grafts and 592.7 ± 157.6 pmol/L (n = 6) for SC-islet grafts 1 month after transplantation. Three months posttransplantation, the stimulated C-peptide levels were 564.9 ± 35.8 pmol/L (n = 6) for human islet grafts and 1,054.0 ± 402.0 pmol/L (n = 5) for SC-islet grafts (Supplementary Fig. 1). Before transplantation, human islets and SC-islets had similar cellular compositions, with ∼50% insulin-positive cells, 40% glucagon-positive cells, and 5–10% somatostatin-positive cells (Fig. 1). However, SC-islets changed in cellular composition after transplantation. In SC-islets retrieved after 3 months of implantation, a decrease in insulin-producing cells and a concomitant increase in glucagon-positive cells were observed (Fig. 1). Both human islets and SC-islets expressed the maturation markers MAFA, NEUROD1, NKX6.1, NKX2.2, and PDX1 pre- and posttransplantation (Fig. 2 and Supplementary Fig. 2). In both human islets and SC-islets, the cellular proliferation rate was low before and after transplantation, as evaluated by the expression of PCNA (Fig. 2). The extent of amyloid in transplanted human islets was similar at 1 and 3 months posttransplantation (Fig. 3). There was minimal amyloid deposition in SC-islet grafts 1 month posttransplantation. However, the extent of amyloid increased to a similar magnitude as human islet grafts 3 months posttransplantation.
Endocrine composition of human islets and SC-islets before and after transplantation. A: Images of human islets and SC-islets immunostained for insulin (ins; yellow), glucagon (glu; green), somatostatin (som; red), and nuclei (blue) before and after transplantation. B: Comparison of percentages of endocrine cells in human islets and SC-islets before and after transplantation. All values are given as mean ± SEM (n = 4–10). One biological replicate corresponded to one mouse receiving a transplant of SC-islets from one differentiation or islets from one human donor. Scale bar, 50 μm. ****P < 0.0001 by one-way ANOVA and Šídák multiple comparisons test.
Endocrine composition of human islets and SC-islets before and after transplantation. A: Images of human islets and SC-islets immunostained for insulin (ins; yellow), glucagon (glu; green), somatostatin (som; red), and nuclei (blue) before and after transplantation. B: Comparison of percentages of endocrine cells in human islets and SC-islets before and after transplantation. All values are given as mean ± SEM (n = 4–10). One biological replicate corresponded to one mouse receiving a transplant of SC-islets from one differentiation or islets from one human donor. Scale bar, 50 μm. ****P < 0.0001 by one-way ANOVA and Šídák multiple comparisons test.
Maturation markers of human islets and SC-islets before and after transplantation. Images of human islets and SC-islets immunostained for MAFA, NEUROD1, NKX6.1, NKX2.2, PCNA, and PDX1 (green), insulin (ins; red), and nuclei (blue) before and after transplantation. Scale bar, 50 μm.
Maturation markers of human islets and SC-islets before and after transplantation. Images of human islets and SC-islets immunostained for MAFA, NEUROD1, NKX6.1, NKX2.2, PCNA, and PDX1 (green), insulin (ins; red), and nuclei (blue) before and after transplantation. Scale bar, 50 μm.
Amyloid detected in SC-islets transplanted beneath the renal capsule. A–D: One month posttransplantation, retrieved human islet grafts (A) (C is higher magnification) and SC-islet grafts (B) (D is higher magnification) were immunostained for the endocrine marker chromogranin A (CgA; gray), mouse endothelial marker CD31 (red), and amyloid marker pentameric formylthiophene acetic acid (pFTAA; green). E–H: Three months posttransplantation, human islet grafts (E) (G is higher magnification) and SC-islet grafts (F) (H is higher magnification) were also immunostained for amyloid. I: Quantification of amyloid was performed in both human islet grafts and SC-islet grafts. J: Measurement of the distance from amyloid to blood vessels. All values are given as mean ± SEM (n = 4–6). One biological replicate corresponded to one mouse receiving a transplant of SC-islets from one differentiation or islets from one human donor. Scale bar, 100 μm. *P < 0.05 by two-tailed Student t test.
Amyloid detected in SC-islets transplanted beneath the renal capsule. A–D: One month posttransplantation, retrieved human islet grafts (A) (C is higher magnification) and SC-islet grafts (B) (D is higher magnification) were immunostained for the endocrine marker chromogranin A (CgA; gray), mouse endothelial marker CD31 (red), and amyloid marker pentameric formylthiophene acetic acid (pFTAA; green). E–H: Three months posttransplantation, human islet grafts (E) (G is higher magnification) and SC-islet grafts (F) (H is higher magnification) were also immunostained for amyloid. I: Quantification of amyloid was performed in both human islet grafts and SC-islet grafts. J: Measurement of the distance from amyloid to blood vessels. All values are given as mean ± SEM (n = 4–6). One biological replicate corresponded to one mouse receiving a transplant of SC-islets from one differentiation or islets from one human donor. Scale bar, 100 μm. *P < 0.05 by two-tailed Student t test.
Three months posttransplantation, vascular density was similar in the human islets and SC-islets (Fig. 4). However, whereas the vascular network in the human islets was a mixture of ingrowing recipient blood vessels (positive for mouse CD31) and donor-derived human endothelial cells (positive for human CD31), there were only recipient blood vessels and no human endothelial cells in the SC-islets. These findings were confirmed with an additional vascular marker, LEA (Supplementary Fig. 3). The number of pericytes, as evaluated by NG2 staining, was similar in the human islet and SC-islet grafts (Supplementary Fig. 4). To assess the functionality of the blood vessels, especially when considering their partly different origin and that some vascular structures of human origin may be remnant and not perfused, we complemented these studies with blood flow and oxygen tension measurements of the different grafts. Although blood perfusion was just slightly higher in the SC-islets (2.2 ± 0.4 [n = 9] vs. 1.3 ± 0.2 mmHg [n = 8]; P = 0.067), the oxygenation of these cells was doubled when compared with corresponding grafts of human islets (23.1 ± 3.1 [n = 8] vs. 11.6 ± 1.9 mmHg [n = 7]; P < 0.01) (Fig. 4).
Characterization of blood vessels in transplanted human islets or SC-islets. A: Three months posttransplantation, retrieved human islet grafts and SC-islet grafts were immunostained for the endocrine marker chromogranin A (gray), human endothelial marker CD31 (green), mouse endothelial marker CD31 (red), and nuclei (blue). Bottom row shows grafts above in higher magnification. Vascular density in these grafts was evaluated. All values are given as mean ± SEM (n = 6). B: Tendency toward increased blood flow in SC-islet grafts compared with human islet grafts (P = 0.067 by two-tailed Student t test; n = 8–9). Oxygen tension was superior in SC-islet grafts compared with human islet grafts (n = 7–8). All values are given as mean ± SEM. Measurements of blood flow and oxygen tension in the superficial kidney cortex (1–2 mm) in the immediate vicinity of the graft is included for reference. One biological replicate corresponded to one mouse receiving a transplant of SC-islets from one differentiation or islets from one human donor, but exceptions were made for blood flow and oxygen tension measurements where two mice received transplants with human islets from same donor and four mice received transplants with SC-islets from two differentiations (i.e., two mice per differentiation). Scale bar, 50 μm. *P < 0.05, **P < 0.01 by one-way ANOVA and Šídák multiple comparisons test (A) or two-tailed Student t test (B).
Characterization of blood vessels in transplanted human islets or SC-islets. A: Three months posttransplantation, retrieved human islet grafts and SC-islet grafts were immunostained for the endocrine marker chromogranin A (gray), human endothelial marker CD31 (green), mouse endothelial marker CD31 (red), and nuclei (blue). Bottom row shows grafts above in higher magnification. Vascular density in these grafts was evaluated. All values are given as mean ± SEM (n = 6). B: Tendency toward increased blood flow in SC-islet grafts compared with human islet grafts (P = 0.067 by two-tailed Student t test; n = 8–9). Oxygen tension was superior in SC-islet grafts compared with human islet grafts (n = 7–8). All values are given as mean ± SEM. Measurements of blood flow and oxygen tension in the superficial kidney cortex (1–2 mm) in the immediate vicinity of the graft is included for reference. One biological replicate corresponded to one mouse receiving a transplant of SC-islets from one differentiation or islets from one human donor, but exceptions were made for blood flow and oxygen tension measurements where two mice received transplants with human islets from same donor and four mice received transplants with SC-islets from two differentiations (i.e., two mice per differentiation). Scale bar, 50 μm. *P < 0.05, **P < 0.01 by one-way ANOVA and Šídák multiple comparisons test (A) or two-tailed Student t test (B).
Potential differences in the innervation of the grafts were also examined, and because many of the cues for angiogenesis and neurogenesis in tissues are shared, we evaluated neural density in parallel with vascular density measurements. Costaining of mouse CD31 (vascular marker) and NF-L (neural marker) showed an ingrowth of nerves and blood vessels in close vicinity of each other (Fig. 5). Similar to the ingrowth of recipient blood vessels (positive for mouse CD31), the ingrowth of nerves in the SC-islets was four to five times higher than in the human islets when evaluated 3 months posttransplantation (Fig. 5). To further delineate the nature of ingrowing nerves, additional nerve markers were used: tyrosine hydroxylase (sympathetic nerve marker) (Supplementary Fig. 5) and vesicular acetylcholine transporter (parasympathetic nerves) (Supplementary Fig. 6). There were more sympathetic nerves in SC-islet grafts when compared with human islet grafts (Supplementary Fig. 5E), whereas the number of parasympathetic nerves was comparable in SC-islet grafts and human islet grafts (Supplementary Fig. 6E).
Nerve density in transplanted human islets or SC-islets. Three months posttransplantation, retrieved human islet grafts and SC-islet grafts were immunostained for the endocrine marker chromogranin A (gray), nerve marker NF-L (green), mouse endothelial marker CD31 (red), and nuclei (blue). Bottom row shows grafts above in higher magnification. Nerve density in these grafts was evaluated. All values are given as mean ± SEM (n = 6). One biological replicate corresponded to one mouse transplanted with SC-islets from one differentiation or islets from one human donor. Scale bar, 50 μm. ***P < 0.001 by two-tailed Student t test.
Nerve density in transplanted human islets or SC-islets. Three months posttransplantation, retrieved human islet grafts and SC-islet grafts were immunostained for the endocrine marker chromogranin A (gray), nerve marker NF-L (green), mouse endothelial marker CD31 (red), and nuclei (blue). Bottom row shows grafts above in higher magnification. Nerve density in these grafts was evaluated. All values are given as mean ± SEM (n = 6). One biological replicate corresponded to one mouse transplanted with SC-islets from one differentiation or islets from one human donor. Scale bar, 50 μm. ***P < 0.001 by two-tailed Student t test.
To elucidate the importance of improved vascularization (including improved oxygenation) and innervation on graft survival, we compared apoptotic rates in the implanted SC-islets and human islets. Three months posttransplantation, there was a fourfold higher level of apoptosis of insulin-positive cells in human islets when compared with SC-islets (Fig. 6).
β-Cell apoptosis of human islets and SC-islets 3 months after transplantation. A–D: Three months posttransplantation, retrieved human islet grafts (A) (C is higher magnification) and SC-islet grafts (B) (D is higher magnification) were immunostained for insulin (gray), apoptosis marker, active cleaved caspase-3 (green), and nuclei (blue). E: Caspase-3 reactivity was evaluated in both human islet grafts and SC-islet grafts. All values are given as mean ± SEM (n = 6). One biological replicate corresponded to one mouse receiving a transplant of SC-islets from one differentiation or islets from one human donor. Scale bar, 100 μm. *P < 0.05 by Mann-Whitney test.
β-Cell apoptosis of human islets and SC-islets 3 months after transplantation. A–D: Three months posttransplantation, retrieved human islet grafts (A) (C is higher magnification) and SC-islet grafts (B) (D is higher magnification) were immunostained for insulin (gray), apoptosis marker, active cleaved caspase-3 (green), and nuclei (blue). E: Caspase-3 reactivity was evaluated in both human islet grafts and SC-islet grafts. All values are given as mean ± SEM (n = 6). One biological replicate corresponded to one mouse receiving a transplant of SC-islets from one differentiation or islets from one human donor. Scale bar, 100 μm. *P < 0.05 by Mann-Whitney test.
RNA sequencing was performed to identify differences in factors known to affect angiogenesis and neurogenesis between human islets and SC-islets before their transplantation (complete list of significant differentially expressed genes [DEGs] is provided in Supplementary Table 2). Principal component analysis showed a clear distinction between the SC-islets and human islets, with PC1 (accounting for 48.4% of total variation) separating the two groups (Fig. 7A). The differential expression analysis showed 5,109 DEGs (false discovery rate [FDR] <0.01) (Supplementary Table 2). Approximately half of the DEGs were expressed at a higher level in the human islets (2,657), and the other half were expressed higher in the SC-islets (2,452) (Fig. 7B). Enrichment analysis showed that the DEGs were enriched for a multitude of pathways (Supplementary Fig. 7 and Supplementary Table 3). However, of particular interest concerning the phenotypic findings were the enrichments for positive regulation of angiogenesis (GO:0045766; 51 of 183 genes in this category had significantly higher expression in human islets compared with SC-islets; FDR 6.83e−10) (Supplementary Fig. 8) and regulation of neurogenesis (GO:0050767; 59 of 361 genes in this category had significantly higher expression in SC-islets compared with human islets; FDR 2.46e−3 (Supplementary Fig. 9). Some of the angiogenesis and neurogenesis genes are highlighted in Fig. 7C.
RNA sequencing (RNA seq) of human islets and SC-islets before transplantation. A: Principal component analysis. Samples group together by sample type (human islets vs. SC-islets). Majority of the expression variation among samples (PC1) is explained by sample type. B: Volcano plot showing expression differences between human islets and SC-islets. Human islets are treated as reference for fold change (FC). DE genes belonging to gene ontology (GO) term positive regulation of angiogenesis (GO:0045766) are highlighted; colors correspond to sample types in which the expression is higher. C: Heat map of selected genes with known functions in angiogenesis and neurogenesis. D: Gene expression analysis of human islets or SC-islets before transplantation. Log2 transformation of FC of the relative mRNA expression levels in the 11 DEGs (genes involved in angiogenesis and neurogenesis) selected for validation with quantitative RT-PCR (qRT-PCR; black bars) parallels the direction of change in RNA seq (blue bars).
RNA sequencing (RNA seq) of human islets and SC-islets before transplantation. A: Principal component analysis. Samples group together by sample type (human islets vs. SC-islets). Majority of the expression variation among samples (PC1) is explained by sample type. B: Volcano plot showing expression differences between human islets and SC-islets. Human islets are treated as reference for fold change (FC). DE genes belonging to gene ontology (GO) term positive regulation of angiogenesis (GO:0045766) are highlighted; colors correspond to sample types in which the expression is higher. C: Heat map of selected genes with known functions in angiogenesis and neurogenesis. D: Gene expression analysis of human islets or SC-islets before transplantation. Log2 transformation of FC of the relative mRNA expression levels in the 11 DEGs (genes involved in angiogenesis and neurogenesis) selected for validation with quantitative RT-PCR (qRT-PCR; black bars) parallels the direction of change in RNA seq (blue bars).
Differences in gene expression of particular interest were confirmed by quantitative RT-PCR. Most of the genes that differed in expression between human islets and SC-islets were less expressed in the SC tissue, with one exception (NRXN2) (Fig. 7D). Several factors with reduced expression in the SC-islets have angiostatic (antiangiogenic) and/or neurostatic characteristics (e.g., ADAMTS1, THBS1, and THBS2, which are all part of the thrombospondin family, as well as tissue inhibitors of metalloproteinases TIMP1, TIMP2, and TIMP3).
Discussion
Promising clinical phase 1/2 trials of SC-islets in a perforated macrocapsule implanted subcutaneously (19,20) or SC-islets transplanted into the liver (21) have demonstrated safety and tolerability and, in the latter study, also improved glycemic control at 1 year posttransplantation. However, although clinical islet transplantation is characterized by a progressive deterioration of islet graft function, the long-term outcome of β-cell replacement with SC-islets is still unknown. This experimental study investigated factors potentially involved in the chronic failure of human islet grafts, the vascular and neural engraftment, and the tendency toward amyloid formation in transplanted SC-islets versus primary human islets. A remarkable difference was observed in the capacity of SC-islets to become revascularized and reinnervated. At 3 months posttransplantation, the ingrowth of blood vessels was more than twofold higher and that of nerves was four- to fivefold higher in the SC-islets compared with in the human islets. However, both the SC-islets and human islets had detectable amyloid in the grafts, which poses a threat to long-term cell survival (22,23).
In contrast to the vascular network in transplanted human islets, which is a mosaic of remnant donor endothelium and ingrowing recipient blood vessels (24–26), a substantially higher number of ingrowing blood vessels (stained positively for mouse CD31) were observed in SC-islet grafts, but human CD31-positive cells were absent in SC-islet grafts. To assess the consequences of this difference for vascular function, both the blood flow and the oxygen tension of the different grafts were measured. Although blood flow tended to be higher in the SC-islet grafts, the oxygenation of these grafts was double that of the grafts of human islets. A probable explanation for the increased vascular functionality in the SC-islet transplants is the presence of unperfused vascular structures composed of remnant donor endothelial structures in the human islet transplants. Improved oxygenation may also reflect less oxygen consumption (i.e., function of the tissue). However, in our recent study of the functionality of SC-islets when compared with human islets, we observed both similar functional activity and similar oxygen consumption at normoglycemia (10). We then compared apoptotic rates in the implanted SC-islets and human islets to elucidate the importance of the improved vascular functionality and innervation on graft survival. Three months posttransplantation, the apoptotic rate was four times higher in the human islets, suggesting that long-term β-cell survival may be better in SC-islet grafts.
Angiogenesis does not normally occur in pancreatic islets, but it can be activated by either increased expression of proangiogenic factors or a decrease in angiostatic factors in the islets. Increased vascular density in transplanted islets has been shown after, for example, overexpression of VEGF-A (27–30), increased activity of matrix metalloproteinase-9 (MMP-9) (31–33), or depletion of the angiostatic factor thrombospondin-1 (THBS1) in the tissue (34). As also observed in the current study, ingrowing blood vessels and nerves were closely aligned, which suggests similar cues for their attraction (35). That vascular signals, specifically in islets, are important to coordinate innervation via vascular scaffolding has also previously been reported (36). RNA sequencing and RT-PCR experiments were performed to elucidate mechanisms for the substantial ingrowth of blood vessels and nerves in the SC-islets when compared with the human islets. A common observation was that most factors previously described to be involved in angiogenesis or neurogenesis were less expressed in the SC-islets, except for NRXN2, which is highly expressed in enterochromaffin cells. It should be noted that enterochromaffin cells are not found in human islets. Noteworthy, several potent angiostatic factors were among these, including ADAMTS1, THBS1, and THBS2 of the thrombospondin family. This could therefore be an explanation for the better vascular and neural ingrowth in SC-islets. Thrombospondins are normally bound in the extracellular matrix (37), and THBS1 (thrombospondin-1), for example, induces apoptosis selectively in proliferating endothelial cells (i.e., those that are forming new blood vessels) (38). THBS1 also inhibits the mobilization of VEGF-A and MMP-9 and blocks their binding to coreceptors on the endothelial surface (39). Also, tissue inhibitors of metalloproteinases (TIMP1, TIMP2, and TIMP3) were less expressed in the SC-islets, which allows, for example, the activity of MMP-9, previously shown to be essential for physiological β-cell function and islet vascularization (40).
Amyloid depositions have been suggested to contribute to the failure of β-cells in type 2 diabetes (41). Amyloid is also known to develop in human islets during culture, as well as after experimental and clinical transplantation (6,42), and experimental studies have indicated that such deposits limit the viability and result in the functional failure of islet grafts (22,23). The exact mechanism for amyloid formation remains to be solved, but active β-cells are a prerequisite for the synthesis of IAPP in transplanted islets (43). Inadequate numbers of blood vessels, providing for the efficient export of secretory products, might also contribute to the IAPP accumulation in transplanted islets. In fact, considerable amounts of amyloid form in the nonvascularized setting of experimentally transplanted encapsulated islets (44), as well as in clinically transplanted encapsulated islets (43,45). Device-encapsulated SC β-like cells also developed amyloid after experimental transplantation (43). In the current study, we show that amyloid also forms in freely transplanted SC-islets. Of note, there was minimal amyloid deposition in SC-islet grafts 1 month posttransplantation, but the extent of amyloid increased to a similar magnitude as that of human islet grafts at 3 months. Amyloid formation is a nucleation-dependent phenomenon, meaning that any sequester of amyloid triggers further formation of amyloid, and the mass grows over time (46). The long-term consequences of this in a clinical setting remain to be determined, but if progressive, such accumulation of amyloid may result in deteriorating function and lead to graft failure (22,23).
Some plasticity was observed in the cellular composition of the grafts posttransplantation. In the SC-islets, the fraction of β-cells decreased while the fraction of glucagon positive cells increased. In our previous characterization of SC-islets, >95% of the insulin-positive cells were monohormonal at stage 7, week 6, before transplantation, and only ∼0.5% were Ki67 positive (10). The generated SC-islets also responded to glucose stimuli, for example, and had the capacity to cure diabetic recipient mice. In the current study, we observed very low expression of the cell proliferation marker PCNA and expression of maturation markers MAFA, NEUROD1, NKX6.1, NKX2.2, and PDX1 both before and 3 months after transplantation. Future studies are required to address whether transdifferentiation still may have occurred or if the finding can be explained by differentiation of the small fraction of remaining progenitor-like cells into glucagon positive cells or a selective expansion of glucagon-positive cells resulting from a higher proliferation rate.
There were some limitations to our study, one being that only normoglycemic recipients were used, considering that a chronic diabetic condition may cause both angiopathy and neuropathy. However, with regard to revascularization and reinnervation, we and others have shown that neither process is affected by a diabetic condition (47,48). We also limited the functional testing to vascular function, excluding neural function, considering previous reports of the variability in neural function between different implantation sites for islets. Sympathetic nerve innervation can influence insulin secretion, as previously elegantly described by the Caicedo research group for islets implanted in the anterior chamber of the eye, via the constricting effects of pericytes surrounding blood vessels (49). In our study, however, there was a tendency toward increased blood perfusion in the SC-islet grafts when compared with the human islet grafts, which suggests that at least the normal sympathetic activity does not substantially influence blood perfusion and secondarily insulin release in SC-islets. Furthermore, oxygen tension was higher in the SC-islet grafts than in the human islet grafts, indicating no severe vasoconstriction in the former affecting β-cell function. Moreover, there was no difference in the number of pericytes surrounding blood vessels between the human islet grafts and SC-islet grafts, despite the higher number of murine blood vessels in the SC-islet grafts. Therefore, the fractional pericyte numbers were lower in the SC-islets compared with the human islet grafts. For islets implanted beneath the renal capsule, there is also less nerve functionality compared with other investigated sites (50). Finally, testing of graft functionality was limited to glucose-stimulated human C-peptide and did not include experiments with reversal of diabetes. Such studies are instead included in our other publication (10).
We conclude that SC-islets have superior capacity compared with human islets for vascular and neural engraftment, but there may still be concerns regarding their long-term function posttransplantation, because amyloid formation occurs. If the amyloid formation proves to be a critical problem, genetic modifications of human pluripotent stem cells to inhibit IAPP expression in derived β-like cells may be desirable to prevent their functional decline and death.
This article contains supplementary material online at https://doi.org/10.2337/figshare.25571928.
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Acknowledgments. The authors thank Zhanchun Li, Lisbeth Ahlqvist, and My Quach for their skilled technical assistance.
Funding. This work was supported by the Swedish Research Council (55X-15043, 2017-01343), the Swedish Child Diabetes Fund, the Swedish Diabetes Foundation, Diabetes Wellness Sverige, the Novo Nordisk Foundation, the Erling Persson Family Foundation, the Ernfors Family Foundation, and national strategic research programs Excellence of Diabetes Research in Sweden (Exodiab) and StemTherapy. The work in Helsinki was supported by the Academy of Finland Center of Excellence Metastem (312437), the Sigrid Jusélius Foundation, the Novo Nordisk Foundation, and the Wellcome Collaborative Award (224600/Z/21/Z).
Duality of Interest. No potential conflicts of interest relevant to this article were reported.
Author Contributions. All authors conducted the study and approved the final version of the manuscript. P.-O.C. and J.L. designed the study and drafted the manuscript, and the other authors revised it critically for intellectual content. J.L. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.