Interspecies organogenesis generates autologous functional islets

Abstract

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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Figure 1: Generation of apancreatic rats by TALEN-mediated mutagenesis.
Figure 2: Generation of rat pancreata by blastocyst complementation.
Figure 3: Generation of mouse pancreata in rat by interspecies blastocyst complementation.
Figure 4: Transplantation of mouseR islets into mice with drug-induced diabetes.

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Acknowledgements

We thank A. Oshima and H. Tsukui for technical support, K. Okada for secretarial support, H. Nagashima and M. Watanabe for advice in preparing the manuscript, A. S. Knisely for reading of the manuscript and Japan Insulin-Dependent Diabetes Mellitus (IDDM) Network for continuous support. This work was supported by grants from Japan Science and Technology Agency, Exploratory Research for Advanced Technology, Leading Advanced Projects for medical innovation, Japan Agency for Medical Research and Development, Japan Society for the Promotion of Science, KAKENHI Grant Number 26460358, research grant for type 1 diabetes, Japan IDDM Network and California Institute for Regenerative Medicine.

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Authors

Contributions

T.Y. and H.S. designed, performed, and analysed all experiments and wrote the manuscript. M.I.-K., T.G., H.H., M.S., T.K., A.Y., and A.U. performed embryo manipulation. N.M. performed data analysis. Y.O. performed histopathological analysis. S.H. performed establishment of iPSCs. H.M. performed data analysis. D.T.R. wrote the manuscript. M.H. performed embryo manipulation and data analysis. H.N. designed the study and wrote the manuscript.

Corresponding author

Correspondence to Hiromitsu Nakauchi.

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Competing interests

H.N. is a co-founder and shareholder of iCELL Inc., ChimaERA Corporation and ReproCELL Inc.

Additional information

Reviewer Information Nature thanks H. Lickert, Q. Zhou and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 The birth rate of Pdx1mu/mu rats and phenotype of Pdx1+/mu rats.

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. Source data

Extended Data Figure 2 Immunofluorescence photomicrographs of rES-cell-derived pancreata generated in Pdx1mu/mu chimaeric rats.

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). Source data

Extended Data Figure 3 Immunofluorescence photomicrographs of miPSC-derived-pancreata generated in Pdx1mu/mu chimaeric rats.

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). Source data

Extended Data Figure 4 Clinical biochemistry and histologic observations in Pdx1mu/mu chimaeric rat with diabetic-like symptoms.

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. Source data

Extended Data Figure 5 Injection studies of pancreatic duct through common bile duct.

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.

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.

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. Source data

Extended Data Table 1 Sequence analysis of predicted off-target sites of Pdx1 TALEN

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Yamaguchi, T., Sato, H., Kato-Itoh, M. et al. Interspecies organogenesis generates autologous functional islets. Nature 542, 191–196 (2017). https://doi.org/10.1038/nature21070

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