Islet transplantation is an established therapy for diabetes. We have previously shown that rat pancreata can be created from rat pluripotent stem cells (PSCs) in mice through interspecies blastocyst complementation. Although they were functional and composed of rat-derived cells, the resulting pancreata were of mouse size, rendering them insufficient for isolating the numbers of islets required to treat diabetes in a rat model. Here, by performing the reverse experiment, injecting mouse PSCs into Pdx-1-deficient rat blastocysts, we generated rat-sized pancreata composed of mouse-PSC-derived cells. Islets subsequently prepared from these mouse–rat chimaeric pancreata were transplanted into mice with streptozotocin-induced diabetes. The transplanted islets successfully normalized and maintained host blood glucose levels for over 370 days in the absence of immunosuppression (excluding the first 5 days after transplant). These data provide proof-of-principle evidence for the therapeutic potential of PSC-derived islets generated by blastocyst complementation in a xenogeneic host.
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Extended data figures and tables
Extended Data Figures
- Extended Data Figure 1: The birth rate of Pdx1mu/mu rats and phenotype of Pdx1+/mu rats. (324 KB)
a, Results of mating Pdx1+/muA with Pdx1+/muB. Wild-type, Pdx1+/mu and Pdx1mu/mu rats were born in the mendelian ratio (Χ2 = 2, P = 0.37 by chi-squared test). b, Results of GTTs in Pdx1+/mu. MODY-like hyper-glycaemia were observed in Pdx1+/muA (2 of 6 rats) and Pdx1+/muB (3 of 6 rats). c, Amino acid sequences of Pdx1muA, Pdx1muB and wild-type Pdx1, which are predicted from full-length cDNA derived from mRNA in duodenum of Pdx1muA/muB or Pdx1+/+ rats.
- Extended Data Figure 2: Immunofluorescence photomicrographs of rES-cell-derived pancreata generated in Pdx1mu/mu chimaeric rats. (823 KB)
a, Pancreata sections were stained with antibodies against rat glucagon, rat insulin, rat somatostatin, rat CK19 and rat α-amylase. Scale bars, 100 μm. b, Quantitative analysis of sections of pancreata. Percentages of EGFP-expressing areas in areas that were positive for insulin, glucagon, somatostatin, CK19 or α-amylase were analysed by image J software (n = 3). The mean values ± s.d. were obtained from 3 biological replicates. c, The area under glucose curve (AUC glucose), calculated from GTT data in Fig. 2e. The mean values ± s.d. were obtained from 2 (Pdx1mu/mu + rES cells, WT chimaera), 3 (WT), or 4 (Pdx+/mu + rES cells) independent experiments (P = 0.24 Pdx1+/mu + rESCs versus Pdx1mu/mu + rESCs; P = 0.88 Pdx1mu/mu + rESCs versus WT; P = 0.08 WT chimaera versus WT; P = 0.19 WT chimaera versus Pdx1+/mu + rESCs; Student’s t-test).
- Extended Data Figure 3: Immunofluorescence photomicrographs of miPSC-derived-pancreata generated in Pdx1mu/mu chimaeric rats. (800 KB)
a, Pancreata sections were stained with antibodies against mouse glucagon, mouse insulin, mouse somatostatin, mouse CK19 and mouse α-amylase. Scale bars, 100 μm. b, Quantitative analysis of sections of pancreata. Percentages of EGFP-expressing areas in areas that were positive for insulin, glucagon, somatostatin, CK19 or α-amylase were analysed by Image J software (n = 3). The mean values ± s.d. were obtained from 3 biological replicates. c, AUC glucose, calculated from GTT data in Fig. 3e. The mean values ± s.d. were obtained from 3 (Pdx+/mu + mPSCs; WT), or 6 (Pdxmu/mu + mPSCs) independent experiments (P = 0.20 Pdx1+/mu + mPSCs versus Pdx1mu/mu + mPSCs; P = 0.14 Pdx1mu/mu + mPSCs versus WT; Student’s t-test).
- Extended Data Figure 4: Clinical biochemistry and histologic observations in Pdx1mu/mu chimaeric rat with diabetic-like symptoms. (730 KB)
a, GTTs results, 6 weeks and 10 weeks after birth, in Pdx1mu/mu chimaeric rats that possess mPSC-derived pancreata (chimaeras 183 and 186). Blood sampling at the same time points as in Fig. 3e. Chimaera 186 showed diabetic-like symptoms. b, Photomicrographs of pancreata of Pdx1mu/mu chimaeric rat (chimaera 186) (left) and C57BL/6N mouse pancreata (right). IL, islet of Langerhans; PD, pancreatic duct.
- Extended Data Figure 5: Injection studies of pancreatic duct through common bile duct. (306 KB)
PBS-containing trypan blue was injected from the duodenum into the common bile duct (see diagram, bottom) of a wild-type Wistar rat (upper left, before injection; lower left, after injection), Pdx1+/mu chimaeric rat (upper middle, before injection; lower middle, after injection), and a Pdx1mu/mu chimaeric rat (upper right, before injection; lower right, after injection). PBS diffused throughout the pancreata in wild-type rat and Pdx1+/mu chimaeric rat, whereas, in the Pdx1mu/mu chimaeric rat, the PBS was retained in common bile duct and flowed backward to duodenum.
- Extended Data Figure 6: Analysis of transplanted islets. (842 KB)
a, Bright-field (top) and fluorescent (bottom) images of hemi-nephrectomized kidney with (right) or without (left) transplanted islets. b, Transplanted islets, kidney capsule; anti-CD3 and –CD11b, hematoxylin counterstain. Islets lie inside the dotted line (Scale bars: 100 nm). c, Immunohistological analysis of engrafted islets under kidney capsule. Sections were stained with antibody against EGFP, insulin, glucagon and somatostatin and with DAPI. Scale bars, 100 nm. d, Quantitative analysis of sections of engrafted islets. Ratio of insulin-, glucagon- and somatostatin-positive cells were analysed by Image J software (n = 3). The mean values ± s.d. were obtained from 3 biological replicates (P = 0.93 insulin+ area in mouseR islets versus in WT mouse islets; P = 0.89 glucagon+ area in mouseR islets versus in WT mouse islets; P = 0.77 somatostatin+ area in mouseR islets versus in WT mouse islets; Student’s t-test). e, Top, FACS diagrams, dispersed small samples of WT rat kidney (left), WT mouse kidney (middle) and mouse kidney with transplanted islets (right). Cells were stained with fluorophore-tagged antibodies against mouse and rat CD31 (mCD31 and rCD31, respectively). Bottom, FACS diagram, EGFP expression by dispersed cells of mouse kidney with transplanted islets (right) (n = 3).
- Extended Data Figure 7: Analysis of mouseR islets transplanted mice. (173 KB)
a, AUC glucose, calculated from GTT data in Fig. 4d. The mean values ± s.d. were obtained from 3 independent experiments (P < 0.01 A, B or C versus D, E or F, Student’s t-test). b, Nonfasting mouse c-peptide level (pmol l−1) in serum from Pdx1muA/muB + mPSCs chimaera, mouse transplanted with mouseR islets, and C57BL/6 mouse. The lowest value of the x axis represent the lowest limit of detection. The mean values ± s.d. were obtained from 3 biological replicates except for Pdx1mu/mu + mPSCs that was from 2 biological replicates (P < 0.01 Pdx1mu/mu + mPSCs chimaera and mouseR islets transplanted mouse versus STZ treated mouse; P < 0.01 STZ treated mouse versus WT mouse; Student’s t-test). c, Nonfasting rat c-peptide level (pmol l−1) in serum of Pdx1muA/muB + miPSCs chimaera, mouse transplanted with mouseR islets, C57BL/6 mouse and Wistar rat. Values are mean ± s.d. N.D., not detected. The lowest value of the x axis represent the lowest limit of detection. The mean values ± s.d. were obtained from 3 biological replicates, except for Pdx1mu/mu + mPSCs, for which they were obtained from 2 biological replicates.