Xenogenic Engraftment of Human-Induced Pluripotent Stem Cell-Derived Pancreatic Islet Cells in an Immunosuppressive Diabetic Göttingen Mini-Pig Model

Abstract

In the development of cell therapy products, immunocompromised animal models closer in size to humans are valuable for enhancing the translatability of in vivo findings to clinical trials. In the present study, we generated immunocompromised type 1 diabetic Göttingen mini-pig models and demonstrated the engraftment of human-induced pluripotent stem cell-derived pancreatic islet cells (iPICs). We induced hyperglycemia with a concomitant reduction in endogenous C-peptide levels in pigs that underwent thymectomy and splenectomy. After estimating the effective in vivo dose of immunosuppressants (ISs) via in vitro testing, we conducted exploratory implantation of iPICs using various implantation methods under IS treatments in one pig. Five weeks after implantation, histological analysis of the implanted iPICs embedded in fibrin gel revealed numerous islet-like structures with insulin-positive cells. Moreover, the area of the insulin-positive cells in the pre-peritoneally implanted grafts was greater than in the subcutaneously implanted grafts. Immunohistochemical analyses further revealed that these iPIC grafts contained cells positive for glucagon, somatostatin, and pancreatic polypeptides, similar to naturally occurring islets. The engraftment of iPICs was successfully reproduced. These data support the observation that the iPICs engrafted well, particularly in the pre-peritoneal space of the newly generated immunocompromised diabetic mini-pigs, forming islet-like endocrine clusters. Future evaluation of human cells in this immunocompromised pig model could accelerate and development of cell therapy products.

Keywords: cell therapy; iPS cell; immunosuppressant; islet; type 1 diabetes.

Graphical Abstract

Figure 1.

Schematic representation of the intervention in pigs. To investigate iPIC engraftment within an immunosuppressive diabetic mini-pig model, ID 1 to 3 pigs underwent the following procedures. Initially, the pigs underwent a thymectomy, followed 3 to 4 weeks later by a splenectomy and gastrostomy. A central venous (CV) catheter was placed either simultaneously or 4 weeks later. Three to 4 weeks later, streptozotocin (STZ) was administered to induce diabetes. Post-diabetes induction, continuous insulin administration was initiated. The iPICs were implanted 4 weeks after the STZ treatment. Immunosuppressants (ISs) were administered 1 week prior to the transplantation and continued throughout the experiment. An intravenous glucose tolerance test (ivGTT) was conducted before and after STZ administration (white boxes).

Figure 2.

Generation of the thymectomized and splenectomized T1DM mini-pig model. (A, B) Blood glucose levels (A) and plasma porcine C-peptide levels (B) during the early period after administration of streptozotocin (200 mg/kg) to the mini-pigs. Glucose was administered as needed to avoid severe hypoglycemia during the period from 10 to 30 h after STZ injection. (C–F) ivGTTs performed before (open circle) and 3 weeks after STZ injection (closed circle). Blood glucose levels (C) and porcine plasma C-peptide levels (D) were measured after administration of glucose (0.5 mg/kg). Paired t-test was used to compare groups by time point (*P < 0.05). Multiplicity was adjusted using the Bonferroni correction method (n = 5, mean ± standard deviation [SD]). AUC for blood glucose levels (E) and porcine plasma C-peptide levels (F). Paired t-test was used to compare groups (n = 5, mean ± SD; **P < 0.01, *P < 0.05). (G, H) Fasting blood glucose levels (G) and body weight changes (H) were monitored throughout the study period.

Figure 3.

Determination of the target blood concentrations of MPA and CsA in mini-pig. MPA and CsA exist in both protein-bound and unbound forms in the blood. To determine the total effective concentrations in vivo, the unbound effective concentrations in vitro, determined using the assay media, were used. This was based on the assumption that the effective unbound concentration in vitro was equivalent to the pharmacologically effective unbound concentration in mini-pig blood in vivo. (A, B) Dose-dependent inhibition of PHA-induced PBMC proliferation by MPA (A) and CsA (B) in the assay medium in vitro. (C, D) Schematic representations of the calculation steps. The target concentration in the plasma was calculated using the in vitro-to-plasma ratio of the unbound form concentrations of MPA (C) and CsA (D). The target blood concentration of CsA was calculated using the blood-to-plasma concentration ratio. Data in (A) and (B) represent the mean ± SD of four experiments.

Figure 4.

iPIC engraftment at a pre-peritoneal site and subcutaneous sites in a diabetic mini-pig. iPICs were implanted at multiple sites in the ID 1 pig using various methods. Details of these implants are described in Table 1. (A) Plasma human C-peptide levels before and after iPIC implantation. Blood samples were collected pre-feeding (open circles) and post-feeding (closed circles). (B) Representative HE-stained images of the retrieved grafts 5 weeks after iPIC implantation. “Non-treatment” indicates intact SC tissue. White arrowhead: iPICs; black arrowhead: multinucleated giant cells; S: suture; Poly-p: polypropylene meshes; P: polyester meshes; Mem: polycarbonate membrane; Alg: alginate gel. Scale bars: 500 μm. (C) Representative images of PP and SC-1-1 stained with an anti-insulin antibody and visualized with diaminobenzidine (DAB). Note: The appearance of sites SC-1-2, SC-2-1, and SC-2-2 is similar to that of SC-1-1. Scale bars: 250 μm. (D) Quantification of DAB substrate-detected human INS-positive cell counts at each implantation site, normalized to the number of clusters initially implanted. Analysis included ten sites per graft. Data are expressed as mean ± SD. The Welch–Aspin test was used for statistical comparison of positive areas at each implantation site (***P < 0.001). (E–K) Representative immunofluorescence images from the pre-peritoneal site PP (E left, F–H) and a subcutaneous site SC-1-1 (E right, I–K). Scale bars: 500 μm in E left and E right, 100 μm in F–K. INS: insulin; GCG: glucagon; SST: somatostatin; PPY: pancreatic polypeptide.

Figure 5.

Changes in blood C-peptide levels after the implantation of a single graft into one pig. (A) iPICs were implanted into the PP site of diabetic ID 2 pig. (B) iPICs were implanted into the SC site of the diabetic ID 3 pig. Left panels indicate the plasma human C-peptide levels. Blood samples were collected before feeding (open circles) and after feeding (closed circles). Right panels show representative images of the retrieved grafts at 5 to 6 weeks after iPIC implantation. Tissue sections were stained with an anti-insulin antibody and visualized with DAB. Details of the implants are described in Table 2. Note: Plasma human C-peptide levels and images of engrafted iPICs in ID 2 pig were not obtained possibly due to an accidental infection at the implantation site approximately 1 month post-iPIC implantation.