The complexity of organogenesis hinders in vitro generation of organs derived from a patient's pluripotent stem cells (PSCs), an ultimate goal of regenerative medicine. Mouse wild-type PSCs injected into Pdx1 −/− (pancreatogenesis-disabled) mouse blastocysts developmentally compensated vacancy of the pancreatic “developmental niche,” generating almost entirely PSC-derived pancreas. To examine the potential for xenogenic approaches in blastocyst complementation, we injected mouse or rat PSCs into rat or mouse blastocysts, respectively, generating interspecific chimeras and thus confirming that PSCs can contribute to xenogenic development between mouse and rat. The development of these mouse/rat chimeras was primarily influenced by host blastocyst and/or foster mother, evident by body size and species-specific organogenesis. We further injected rat wild-type PSCs into Pdx1 −/− mouse blastocysts, generating normally functioning rat pancreas in Pdx1 −/− mice. These data constitute proof of principle for interspecific blastocyst complementation and for generation in vivo of organs derived from donor PSCs using a xenogenic environment.

Finally, by combining the principle of blastocyst complementation with the production of interspecific chimeras, we succeeded in generating rat pancreas in Pdx1 −/− mice. These sets of experiments provide proof-of-principle data for donor iPSC-derived organ generation in a xenogenic environment.

Generation of interspecific chimeras in livestock animals using preimplantation embryos of each species is described; the “geep,” or chimera between goat and sheep, is famous as an interspecific live chimera (). In rodents, however, methods like those producing the geep succeeded only between mouse subspecies, such as Mus musculus and Mus caroli, that are closely related but not capable of interbreeding (). Many groups have sought to generate interspecific chimeras between mouse and rat, successful with chimeric preimplantation embryos in vitro but failing with live chimeric animals (). Extraembryonic lineage cells like trophectoderm or primitive endoderm, derived from xenogenic embryos, might suffer inhibition of implantation on exposure to host uterus; inhibition of further intrauterine development also is possible (). Only cells of pre-blastocyst origin can contribute to extraembryonic lineage cells, and mESCs/iPSCs do not thus contribute (). Therefore, we rechallenged this old issue with new technology, using iPSCs or ESCs that are not capable of contributing to extraembryonic tissues. Using recently established culture conditions with a combination of signaling inhibitors (), we generated rat-ESCs and -iPSCs (rESCs, riPSCs). Our work has now confirmed the existence of interspecific chimeras generated with PSCs, using injection not only of mouse PSCs into rat embryos but also of rat PSCs into mouse embryos.

Although our results verified our initial hypothesis, this system cannot be applied to generate human organs. A xenogenic, but not allogenic, blastocyst complementation system must therefore be established. However, little is known about the nature of the xenogenic barrier, how organogenesis by donor PSCs is influenced, or how intrinsic developmental programs can be regulated in xenogenic environments. To address these issues, we attempted to generate interspecific chimeras between mouse and rat using a blastocyst injection technique with mouse and rat PSCs.

We hypothesized that with blastocysts derived from a mutant mouse strain in which the gene necessary to form a particular organ is deficient, the same principle might apply. To test this hypothesis, we used, in this study, blastocyst complementation to generate functional pancreas from donor PSCs. The pancreas, consisting of endocrine and exocrine glands, is formed by early embryonic interactions of mesenchyme and epithelium (). Pdx1 (pancreatic and duodenal homeobox1) is a Hox-type transcription factor that plays a critical role in pancreatic development and β cell maturation. Homozygous deficiency of Pdx1 in the mouse results in death soon after birth due to pancreatic insufficiency (). Targeted disruption of Pdx1 thus should empty a pancreatic “developmental niche” in embryos derived from Pdx1blastocysts. Therefore, injection of mouse PSCs (mPSCs) into Pdx1blastocysts should result in generation of pancreata almost entirely derived from injected mPSCs.

Blastocyst complementation was first reported by Chen et al. They demonstrated that deficiency of T and B lymphocyte lineages in Rag2-deficient (Rag2) mice was complemented by injecting normal mouse embryonic stem cells (mESCs) into Rag2mouse-derived blastocysts (). Because Rag2 is an indispensable enzyme for rearrangement of immunoglobulin and T cell receptor genes, the T and B cells generated in the complemented animals were mESC derived; there were no host T or B lymphocytes. We assumed that this complementation was possible because the Rag2host, incapable of generating mature T and B cells, provided a “developmental niche” for ESC-derived T and B cells.

Current stem cell therapy mainly targets diseases that can be treated by cell replacement, such as Parkinson's disease or diabetes mellitus. One of the ultimate goals of regenerative medicine, however, is to grow organs using the patient's own stem cells and to transplant those organs into the patient. With the development of induced pluripotent stem cell (iPSC) technology, we are now able to obtain patient-derived PSCs (), although actual developmental potentials remain to be defined, as do risks associated with somatic cell reprogramming. The real challenge is to create a reproductive system for generation of PSC-derived organs. The interactions among cells and tissues during development and organogenesis are so complex that the recapitulation of these interactions to generate organs in vitro is essentially impractical. We have challenged this goal using the biology of blastocyst complementation.

As in wild-type experiments, successful maturation into adulthood (8 weeks) was uncommon in Pdx1mice complemented with riPSCs; however, adult mice with riPSC-derived pancreas ( Figure 7 C; n = 2) had intact pancreas expressing EGFP ( Figures 7 E and 7F). Quantitative analysis of the sections by image J software revealed percentages of EGFP-positive cells to be 81.9% ± 3.4%. Additionally, on GTT in adulthood, insulin was secreted in response to glucose loading and normal serum glucose levels were maintained ( Figure 7 D). These results indicate that generation of a xenogenic iPSC-derived organ is possible via interspecific blastocyst complementation.

The contribution of EGFP-marked riPSC-derived cells in the pancreas of neonatal Pdx1interspecific chimeras was small relative to that of host-derived cells, as seen in whole-body chimerism (Pdx1+ riPSCs in Figure 7 A bottom panel; n = 5). In contrast, the pancreatic epithelia in Pdx1interspecific chimeras were entirely composed of EGFP-marked riPSC-derived cells (Pdx1+ riPSCs in Figure 7 A top panel; n = 10). Each genotype was confirmed by PCR using genomic DNA extracted from FACS-sorted mouse CD45 (mCD45)-positive splenocytes ( Figure S7 A ). Neonates with entirely EGFP-positive pancreata thus were clearly identified as of Pdx1genotype ( Figure S7 B). The existence of interspecific chimeras between mouse and rat was also confirmed by FACS patterning, which demonstrated distinct populations of mCD45- and rat CD45 (rCD45)-positive cells, with only rCD45-positive cells expressing EGFP after riPSC injection ( Figure S7 A). FACS-sorted mCD45- and rCD45-positive cells were also confirmed as, respectively, mouse or rat in origin by genomic PCR testing using Oct3/4 locus primers ( Figure S7 C), which clearly identified cell origin. On immunostaining, riPSC-derived pancreas expressed EGFP almost universally ( Figure 7 B) and also expressed α-amylase (an exocrine tissue marker) and insulin, glucagon, and somatostatin (endocrine tissue markers; Figure 7 B).

(F) Histological studies of sections obtained from riPSC-derived adult pancreas revealed clear differences in the distributions of riPSC-derived cells after staining by anti-EGFP antibody with DAPI nuclear counterstaining (right panel). Sections were also stained with HE.

(B) Immunohistological studies of sections obtained from riPSC-derived pancreas. Serial frozen sections were immunostained for EGFP, α-amylase, insulin, glucagon, and somatostatin with DAPI nuclear counterstaining. See also Figure S7 for genotyping to identify Pdx1 status and origin of each species in interspecific chimera.

(A) riPSC-derived pancreas in neonatal Pdx1 −/− mouse. In Pdx1 −/− mice complemented with riPSCs, almost all pancreata expressed EGFP, indicating riPSC origin (top panel). On the other hand, in Pdx1 +/− mice complemented with riPSCs, pancreata only partially expressed EGFP, indicating a composite of both riPSC- and mouse-derived cells (bottom panel).

Our last goal was to generate xenogenic rat pancreas in Pdx1mice by interspecific blastocyst complementation. For efficient production of Pdx1mice, embryos were generated by intercross of Pdx1founder male mice (in which pancreas arose principally from exogenous miPSCs) with Pdx1female mice. With this founder system, half the mice born were Pdx1(in contrast to intercross of Pdx1heterozygotes in which only 25% of offspring were Pdx1). For donor riPSCs, we used the riPSC#3 line ( Figure S4 A). This line was selected among 11 established riPSC clones for embryonic developmental rate and degree of chimerism after injection into mouse embryos (data not shown). After injection, 139 embryos were transferred into uteri of pseudopregnant mice and 34 mice were born. They were analyzed at neonatal and adult stages.

To see the xenogenic contribution to germ cells, testis and developing gonad of the interspecific chimeras were examined. Cells were not found that coexpressed EGFP or Venus and the germ cell marker mouse vasa homolog (MVH) ( Figure 5 F). As the injected miPSCs or rESCs were both confirmed as germline-competent pluripotent stem cells in intraspecific settings (data not shown), germ cell development may be impaired or more limited in the xenogenic environment.

It is known that rats do not have gallbladders, whereas mice do. To date, when mPSCs were injected into r-blastocysts, the interspecific chimeras produced (generally the size of rats) have not had gallbladders (n = 8). In contrast, interspecific chimeras generated from m-blastocysts complemented with rPSCs (more like mice in size) have had gallbladders (n = 4).

We determined the distribution of mouse- or rat-iPSC-derived EGFP-positive cells in neonatal interspecific chimeras. With miPSC injection into r-blastocyst ( Figure S6 A ) and with riPSC injection into m-blastocyst, almost all organs contained EGFP-positive cells ( Figure S6 B). In sections immunostained with anti-EGFP antibody, representative images demonstrated that EGFP-positive cells become various types of tissues ( Figure 5 D). Of particular note was a pancreatic islet generated by injection of miPSCs into r-blastocyst. Cells in this islet marked immunohistochemically for insulin; the islet was a composite of EGFP-positive and -negative cells, indicating that the islet consisted of host rat cells and exogenous donor mouse cells ( Figure 5 E). Islet polyclonality also was seen with intraspecific chimeras ( Figure 2 E). The expression of other functional molecules in xenogenic cells (i.e., hepatocytes or cholangiocytes in the liver or leukocytes in the peripheral blood) was also detected ( Figure 5 E and Figure S6 C).

(C) FACS analysis, using species-specific antibodies against mouse and rat CD45, of peripheral blood (PB) of inter-specific chimera generated by miPSCs injection into r-blastocyst. Almost all cells expressing CD45 also express EGFP. Staining: Anti-mCD45 with anti-mouse Gr1 and -Mac1 cocktails and anti-mouse B220 or with anti-mouse CD4 and anti-mouse CD8. Both mCD45- and EGFP-positive cells are gated and shown.

Adult rats typically are ten times bigger than adult mice, whereas newborn rats are three times bigger than newborn mice. Because mouse and rat gestations are of similar length (19 and 21 days, respectively), organogenesis requires more cell proliferation and differentiation during rat development than during mouse development. What determines the size of interspecific chimeras is an intriguing biological question. Interestingly, body size and weight of interspecific chimeras born from rat foster mothers were similar to those of normal newborn rats, whereas for those born from mouse foster mothers they were similar to those of newborn mice ( Figures 5 A and 5C). However, high contributions by xenogenic cells may also affect interspecific chimera size. One chimera obtained after injection of miPSCs into r-blastocysts showed body weight and size equivalent to those of a newborn mouse, although its birth mother was a rat ( Figure 5 B). This particular chimera's donor miPSC-derived cell contribution was extremely high ( Figure 5 B, inlet). However, chimeras with high contributions of xenogenic iPSC-derived cells generally were not identified, suggesting an association with embryonic lethality. In most chimeras, the size of newborns seemed to conform with that in the species from which the blastocyst originated. A correlation between body weight and contribution of donor iPSC-derived cells as estimated from hair color and peripheral blood cells is shown in Figure 6 . The origins of placenta and uterus may have a key role in size determination. Injected xenogenic PSCs never developed into placenta, which was always of host blastocyst origin ( Figure 4 A, Figure S5 A). Therefore it is difficult to determine whether it is placenta or uterine environment that influences the size of embryos.

To investigate the developmental potential of generated chimeras and to assess the functionality of the cells, tissues, or organs derived from injected cells, we analyzed interspecific chimeras at neonatal and adult stages. Mouse- or rat-iPSC-injected interspecific chimeras survived after birth and expressed EGFP ubiquitously (as did intraspecific chimeras; Figure 5 A , r-blastocyst + miPSCs: n = 5, m-blastocyst + riPSCs: n = 10). As these chimeras developed into adulthood, chimerism could be judged by coat color because miPSCs (C57BL/6, black coat) were injected into r-blastocyst (Wistar, white coat) or riPSCs (Wistar) were injected into m-blastocyst (BDF11 × C57BL/6, black coat) ( Figure 5 C, r-blastocyst + miPSCs: n = 8, m-blastocyst + riPSCs: n = 4). The full-term development rate of interspecific chimeras, either with miPSCs into r-blastocyst or with riPSCs into m-blastocyst, was ∼20%. In both settings it was lower than that for intraspecific chimeras (∼50%).

Sections in (D) were observed under fluorescence microscopy and in (E) and (F) were observed under confocal laser scanning microscopy. See also Figure S6 for macroscopic images of neonates and results of peripheral blood analysis of adults. Scale bars in (A) and (B), 10 mm; in (D), 100 μm; (E) and (F), 50 μm.

(E) Neonatal pancreas and adult liver of interspecific chimera generated from r-blastocyst injected with miPSC, immunostained for EGFP and insulin or for EGFP and albumin or CK19 with DAPI nuclear counterstaining. In pancreas, an islet-like cell cluster contains cells producing insulin composed of both miPSC-derived cells that express EGFP and r-blastocyst-derived cells that do not. In liver, albumin-positive hepatocytes and CK19-positive cholangiocytes (arrowhead) also express EGFP, indicating miPSC origin.

(D) Analysis of chimerism in neonates by organ or tissue. Interspecific chimeras generated by miPSC injection into r-blastocyst (left panels) and by riPSC injection into m-blastocyst (right panels) are shown. Sections of representative organs (heart, liver, pancreas, and kidney) after immunostaining for EGFP antibody with DAPI nuclear counterstaining are shown.

(B) Neonatal body weights of interspecific chimeras were measured and plotted. One chimera obtained after injection of miPSCs into r-blastocyst showed a high contribution of mouse cells as shown in the insert, with body weight and size equivalent to those of newborn mouse (right panel).

To investigate the influence of iPSC contribution to xenogenic development at the fetal stage, we assessed embryonic development rate of interspecific chimeras, and the extent of chimerism, by FACS analysis using established embryonic fibroblasts. Both embryonic development rate and degree of chimerism were lower in interspecific chimeras than in intraspecific chimeras ( Figures 4 F and 4G). In addition, high contributions by xenogenic cells appeared to be associated with morphological abnormalities and embryonic lethality (data not shown). To exclude the possibility that these abnormalities were caused by donor iPSCs, we also attempted to generate interspecific chimeras using mouse or rat ESCs. DsRed-marked EB3DR mESCs could also generate interspecific rat chimeras (top panels in Figure S5 A ), with embryonic development rate and degree of chimerism similar to those generated by miPSC injection ( Figure S5 B). The Venus-marked WIv3i-1 and -5 rESC lines, with high contribution to rat embryo development and germline competency (), could also generate interspecific chimeras after injection into m-blastocyst (middle and bottom panels in Figure S5 A), but embryonic development rate and degree of chimerism were lower than reported for intraspecific chimeras ( Figure S5 C). These results suggest that generation of interspecific chimeras between mouse and rat is less efficient than generation of intraspecific chimeras.

To further confirm interspecific chimerism, genomic DNA extracted from FACS-sorted cells expressing CD45 was PCR-amplified using primers common to the mouse and rat Oct3/4 loci ( Figure 4 C). PCR products of different lengths, indicating origin in each species, were clearly present ( Figure 4 D). These results strongly indicated that the animals harboring these cells were mouse/rat interspecific chimeras.

Next, we tried to quantitate contribution of mouse or rat iPSCs to these interspecific chimeras. Chimerism in interspecific embryos appeared to vary individual-to-individual and organ-to-organ. Since quantitation of PSC-derived cells was difficult in organs, we analyzed embryonic fibroblasts and hematopoietic cells. FACS analysis of embryonic fibroblasts revealed that donor-derived EGFPcell percentages of about 28.0% and 26.5%, respectively, were detected in mouse and rat interspecific chimeras (representative FACS data shown in Figure 4 B). We also examined chimerism in hematopoietic cells by staining cells from livers of interspecific chimera fetuses with antibodies specific for mouse and rat CD45 antigens. Cells that expressed mouse or rat CD45 represented distinct populations in interspecific chimeras, with only cells derived from injected iPSCs expressing EGFP ( Figure 4 B). Whereas a high proportion (28.3%) of mouse blood cells was detected in r-blastocyst-derived chimeric fetal liver, rat blood cells were only rarely present (less than 3.3%) in m-blastocyst-derived chimeric fetal liver ( Figure 4 B). This tendency was specific to interspecific chimeras and not observed in intraspecific chimeras ( Figure 4 E). It is not clear why the difference in contribution of iPSCs to hematopoietic cells between mouse and rat interspecific chimeras was so marked.

We injected EGFP-marked GT3.2 miPSCs into rat blastocysts (r-blastocysts) or EGFP-marked riPSCs ( Figures S4 A and S4B ) into mouse blastocysts (m-blastocysts). Because post-implantation development reportedly is severely hampered after intrauterine transfer of xenogenic blastocysts (), injected r-blastocysts or m-blastocysts were transferred, respectively, into the uteri of pseudopregnant rats or mice. After intrauterine transfer of injected r-blastocysts or m-blastocysts with development to the fetal stage, we evaluated EGFP expression by fluorescence microscopy for each transferred embryo. EGFP-expressing cells were found in the body of each injected conceptus, but never in placenta ( Figure 4 A ). This finding indicates that injected mouse or rat iPSCs can contribute to xenogenic development, with generation of interspecific chimeras.

(G) Chimerism analysis of embryonic fibroblasts from chimeras generated by injection of miPSCs or riPSCs into mouse or rat blastocysts. Fibroblasts were obtained from chimeras and analyzed for EGFP intensity by FACS. Plotted dots show degrees of chimerism for individual embryos. See also Figure S5 for interspecific chimeras using ESCs.

(E) Correlation of chimerism between embryonic fibroblasts and CD45 + hematopoietic cells in fetal liver. Cells were prepared from interspecific chimeras generated by injection of riPSCs into m-blastocysts or from intraspecific chimeras generated by injection of riPSCs into r-blastocysts.

(C) Schema of mouse and rat Oct3/4 loci (mOct3/4 and rOct3/4). Of note is that in the rat Oct3/4 locus the 2 introns flanking exon 3 are longer than in the mouse Oct3/4 locus. This difference in length can distinguish origins of otherwise similar cells. Arrowheads indicate common primers of each species for PCR. PCR product sizes for both species are shown below.

(B) Representative data of FACS analysis on cells from fetal liver and on embryonic fibroblasts derived from interspecific chimeras. Right panels show cells from fetal liver immunostained with anti-mCD45 and -rCD45 antibodies. Note that anti-mouse or -rat monoclonal antibodies (mAb) against CD45 can distinguish CD45-expressing hematopoietic cells in a species-specific manner. Almost all CD45-expressing cells derived from injected cells express EGFP, indicating iPSC origin.

(A) Interspecific chimera fetuses generated by injection of miPSCs into r-blastocyst (rBL: left panels) and by injection of riPSCs into m-blastocyst (mBL: right panels). With rat embryo manipulation, fetuses were analyzed 12 days after embryo transfer into uteri of 3.5 dpc pseudopregnant rats (embryonic day (E) 15.5). With mouse embryo manipulation, fetuses were analyzed 11 days after embryo transfer into uteri of 2.5 dpc pseudopregnant mice (E13.5). See also Figure S4 for characteristics of riPSCs and interspecific embryos after injection of riPSCs into mouse 8-cell/morula stage embryos.

Our second goal was to generate interspecific chimeras between mouse and rat. To achieve this goal, we generated not only miPSCs but also riPSCs and rESCs using an established protocol (). These mouse and rat PSCs enabled us bidirectionally to generate interspecific chimeras.

To prevent nonspecific loss of islets due to inflammation, anti-inflammatory cytokine monoclonal antibody (mAb) cocktails were given at transplantation and 2 and 4 days thereafter (arrows, Figure 3 E), as described (). Two months after transplant, EGFP-expressing miPSC-derived islets were still detectable at the graft site ( Figures 3 B and 3C). Production of insulin by the transplanted islets was confirmed immunohistologically ( Figure 3 D). The induced-diabetic recipients no longer exhibited hyperglycemia; they maintained normal blood glucose levels and responded normally to GTT ( Figures 3 E and 3F). This is in contrast to the therapeutic effect conferred by islets, composites of blastocyst- and miPSC-derived cells, obtained from pancreas of Pdx1chimeric mice (C57BL/6 × DBA2 or C57BL/6 × BDF1 F1 origin). This effect lasted only for a short time, presumably due to immune rejection by the host C57BL/6 diabetic mice ( Figure 3 E). These data strongly indicate that the miPSC-derived pancreas, with islets, formed in an allogenic host is functional and that the “autologous” islets thus formed can be used to treat diabetes, without rejection.

To assess the functionality of miPSC-derived pancreas, islets from miPSC-derived pancreas were transplanted into mice of the original strain (C57BL/6) in which STZ administration had induced diabetes. As a control, islets from pancreas in Pdx1chimeric mice injected with miPSCs were transplanted. Blastocysts were obtained by an intercross of Pdx1mice (C57BL/6 × DBA2 or C57BL/6 × BDF1 F1 strains) that were semi-allogenic to miPSCs used in this study. EGFP-expressing miPSC-derived islets ( Figure 3 A ) were isolated conventionally based on a method developed previously () and were transplanted beneath the renal capsule of recipient mice. Nonfasting blood glucose levels were then monitored. As these islets were of donor origin (i.e., C57BL/6 strain), an immunosuppressive regimen was not used.

Sections in (C) were observed under fluorescence microscopy and in (D) were observed under confocal laser scanning microscopy. Scale bar in (D), 50 μm. Error bars in (E) and (F) indicate ± SD.

(E) Transplantation of miPSC-derived islets into mice with STZ-induced diabetes. Each mouse received 150 islets. Arrows indicate time points at which an mAb cocktail (anti-INF-γ, anti-TNF-α, anti-IL-1β) was administered. Nonfasting blood glucose levels were measured weekly for 2 months after transplantation. Glucose levels are shown for STZ-induced diabetic mice transplanted with miPSC-derived islets (green; n = 3), with islets derived from Pdx1 +/− chimeric mice (orange; n = 2), with islets derived from syngenic strain of host strain (C57BL/6) as positive control (blue; n = 2), and with sham transplantation control mice as negative control (purple; n = 3).

To test whether miPSC-derived pancreata are functional in Pdx1chimeric mice, we performed glucose tolerance testing (GTT). Whereas streptozotocin (STZ)-induced diabetic mice failed to respond to GTT, Pdx1chimeric mice responded well, indicating that Pdx1mice with miPSC-derived pancreas secreted insulin in response to glucose loading and maintained normal serum glucose levels ( Figure 2 E). Histology and function of exogenously derived pancreas were essentially the same for mice derived from miPSC- and from mESC-complemented blastocysts ( Figure S3 ). These data demonstrate that the vacant “pancreatic niche” provided in Pdx1mice can be occupied, with developmental compensation, by miPSC- or mESC-derived cells after intraspecific blastocyst complementation, generating functionally intact pancreas.

In (C) sections were observed by fluorescence microscopy; in (D) and (E) they were observed by confocal laser scanning microscopy. Scale bars in (C), 200 μm; in (D) and (E), 100 μm.

(C) Immunohistological studies of sections obtained from pancreas revealed clear differences in the distributions of mESC-derived cells. Sections were stained with HE or immunostained for EGFP with DAPI nuclear counterstaining.

As expected, in both Pdx1and Pdx1mice injected with EGFP-miPSC, miPSC-derived cells contributed to all the nonpancreatic tissues of the body, including lung, heart, liver, muscle, testis, and brain (data not shown). The extent of contribution varied from tissue to tissue and also varied depending on individual chimera. However, the pancreas in adult Pdx1mice was entirely derived from donor miPSCs (Pdx1in Figure 2 B). Detailed analysis of EGFP distributions in adult miPSC-derived pancreas demonstrated that pancreatic islets, exocrine tissues, and duct epithelia were entirely derived from donor miPSCs, as already shown in neonates (Pdx1in Figures 2 C and 2D). In expected contrast, pancreas from Pdx1mice was a composite of host and donor derivatives (Pdx1in Figures 2 B and 2C). Quantitative analysis of these sections by image J software revealed percentages of EGFP-positive cells to be 27.4% ± 27.8% in the miPSC + Pdx1setting, 95.6% ± 4.6% in the miPSC + Pdx1setting. Of note is that most individual islets were composed of both host-derived and miPSC-derived cells (Pdx1in Figure 2 D), indicating, as previously reported (), nonclonal origin of pancreatic islets.

We next addressed whether mPSCs can rescue Pdx1lethality via blastocyst complementation. As we predicted based on neonatal analysis ( Figure 1 ), both mESCs and miPSCs contributed to pancreatic organogenesis; Pdx1chimeric mice survived to adulthood (mESCs injection: n = 4, miPSCs injection: n = 15 in Figure 2 A ). They even served as Pdx1founders, transmitting their genotype to the next generation. Mating Pdx1founder male mice with Pdx1female mice increased to 50% the proportion of pups of homozygously disrupted genotype ( Figure 2 A). These data indicate that blastocyst complementation can be used to generate a functional organ and to yield homozygous founder mice even when uncomplemented homozygosity is fatal. The method described here thus can be useful for efficient production of gene-targeted mice that are embryonic lethal without introducing a conditional gene targeting system.

Sections in (C) were observed under fluorescence microscopy and in (D) were observed under confocal laser scanning microscopy. Scale bars in (C), 100 μm; in (D), 50 μm. Error bars in (E) indicate ± SD.

(E) Results of GTT in Pdx1 −/− (■) and Pdx1 +/− (▴) mice (n = 6 each) complemented with miPSCs. Mice with STZ-induced diabetes (• and ♦) served as controls. Fasting after 20 hr, blood was sampled via tail vein at intraperitoneal glucose administration (1 g/kg; 0 min) and 15, 30, 60, and 120 min thereafter.

(D) Immunohistological analysis of a generated islet, showing the distribution of miPSC-derived cells. Sections were stained for EGFP, with anti-insulin antibodies, and with DAPI nuclear counterstaining. Yellow lines show borders of endocrine and exocrine tissues. See also Figure S3 for analysis of mESC-derived pancreas in adult mice.

(C) Immunohistological studies of sections obtained from pancreas revealed clear differences in the distributions of miPSC-derived cells. Sections were stained with HE or for EGFP with DAPI nuclear counterstaining.

In all postnatal chimeric mice derived from blastocysts injected with either miPSCs or mESCs, pancreas was present regardless of host genotype (including Pdx1). As expected, the pancreas in Pdx1or Pdx1chimeric mice was a composite of host-derived cells and EGFP-miPSC- or mESC-derived cells, as in whole-body chimerism (Pdx1+ miPSCs or mESCs in Figure 1 A ). In contrast, the pancreatic epithelium in Pdx1chimeric mice was almost entirely composed of EGFP-marked miPSC- or mESC-derived cells (Pdx1+ miPSCs or mESCs in Figure 1 A). The pancreas of these mice was grossly and histologically normal. We examined these pancreatic tissues further for contributions of donor mPSCs to different pancreatic lineages. Both miPSCs and mESCs supplied all pancreatic cell lineages (exocrine and endocrine tissues, including ductal epithelia), but pancreatic stromal elements—vessels, nerves, and fibrocytes—were composites of host- and mPSC-derived cells in all mice ( Figure 1 B).

(B) Histological analysis of the distribution of mESC-derived cells in neonatal mouse pancreas. Sections of pancreas generated in a Pdx1 −/− mouse complemented with mESCs were immunostained for EGFP; for pancreatic endocrine markers (insulin, glucagon, and somatostatin); for the pancreatic exocrine marker α-amylase; for the ductal marker rhodamine-conjugated DBA-lectin; and for blood vessels with PECAM-1, with DAPI nuclear counterstaining.

(A) mPSC-derived pancreas at neonatal stage. In Pdx1mice complemented with mPSCs, the pancreas was almost entirely positive for EGFP, indicating mPSC origin (left panels in +miPSCs and +mESCs). On the other hand, in Pdx1mice complemented with mPSCs, the pancreas was partially positive for EGFP, indicating a composite of both mPSC- and host-derived cells (right panels in +miPSCs and +mESCs). Right panel shows absence of pancreas in a nonchimeric Pdx1mouse at the same developmental stage. See also Figure S1 for detailed characteristics of miPSCs and Figure S2 for genotyping of Pdx1 status.

For complementation donor cells, we used GT3.2 mouse iPSCs (miPSCs) that had been generated from tail tip fibroblasts (TTFs) of an adult EGFP-transgenic (TG) C57BL/6 mouse () using three factors (Oct3/4, Sox2, Klf4) in retroviral vectors ( Figure S1 available online). These GT3.2 miPSCs were injected into blastocysts obtained by an intercross of heterozygote Pdx1-LacZ knockin mice (Pdx1), offspring of C57BL/6(Pdx1) × DBA(Pdx1) or C57BL/6(Pdx1) × BDF1(Pdx) mice. G4.2 mESCs were also injected for comparison. Neonates were assessed macroscopically and histologically for pancreatic development and were genotyped for Pdx1. These neonates were highly chimeric: Due to silencing of EGFP and to contamination by donor cells, accurate determination of the genotypes required analysis at the single-cell level. To identify the genotypes of these neonates accurately, EGFP-negative, c-Kit-positive, and Sca1-positive, lineage marker-negative (EGFPKSL) bone marrow cells were clone-sorted by fluorescence-activated cell sorting (FACS) to obtain single-cell-derived hematopoietic colonies ( Figure S2 A ). After culture for 2 weeks, cells collected from each colony were subjected to PCR analysis ( Figure S2 B). This “colony PCR” method unambiguously determined the host embryo genotype and proved that pancreas had been formed in Pdx1mice by blastocyst complementation ( Figure S2 C).

(A) FACS analysis of bone marrow cells. Gating and staining profiles of bone marrow cells after staining with anti-c-Kit, -Sca1, and -lineage marker antibodies are shown. Cells in the EGFP-negative hematopoietic stem/progenitor cell population (EGFP - Kit + Sca1 + Lin - cells) were clone-sorted and cultured for genotyping by genomic PCR.

(E and F) Chimeric mice derived from GT3.2 miPSCs were analyzed at E13.5 (E) or neonatal (F) stages. BDF1 × C57BL/6-derived blastocysts injected with miPSCs were transplanted into the uteri of foster mothers. The yellow dashed line in (F) demarcates an EGFP - nonchimeric neonate.

(D) Teratoma formation by GT3.2 miPSCs. Approximately 5 × 10 6 cells were transplanted under the kidney capsule. Four weeks later, the resultant tumor mass was excised. HE-stained histologic sections were evaluated by microscopy. Shown are representative derivatives of the 3 germ layers, with gut-like structures (upper panel), epidermal structures (middle panel), and muscle-like structures (bottom panel).

(B) Genomic DNA extracted from GT3.2 or GT3.3 miPSCs was analyzed by PCR to detect integrated viral-vector-derived DNA. As a positive control for four factor-derived miPSCs, Tg mice were used that contained the Nanog-GFP-IRES-Puroreporter construct-derived miPSC line (NgiPSCs), kindly provided by S. Yamanaka, Kyoto University (). The E14.1 mESC line served as a negative control and myogenin (Myog) served as a loading control.

Our first goal was to generate pancreas from mPSCs. We performed a blastocyst complementation experiment using Pdx1blastocysts that would provide a niche for pancreatic organogenesis. These mice exhibit pancreatic agenesis due to the absence of Pdx1-specified interactions between prepancreatic epithelium and mesenchyme, a key step in pancreatic development. Homozygote Pdx1-LacZ knockin mice (Pdx1) are born alive but die within 1 week after birth, presumably due to pancreatic insufficiency ().

Discussion

We report three innovative observations, using proof-of-principle approaches. (1) If an empty developmental niche for an organ is provided (as with the Pdx1−/− mouse and the pancreatic niche), PSC-derived cellular progeny can occupy that niche and developmentally compensate for the missing contents of the niche, forming an organ almost entirely composed of cells derived from donor PSCs. (2) Generation of interspecific chimeras between mouse and rat is possible with injection of mouse or rat PSCs into embryos from the other species; injected PSC-derived cells are distributed throughout the body and appear to function normally. (3) The combination of (1) and (2) successfully generates rat pancreas in mouse with injection of riPSCs into Pdx1−/− mouse embryos, a technique that we term “interspecific blastocyst complementation.”

−/− mice derived from blastocysts complemented with mPSCs were born with functional pancreas almost entirely derived from donor PSCs and grew into adulthood without showing any evidence of pancreatic insufficiency. Several groups have used the same technique to study the development of thymic epithelium ( Muller et al., 2005 Muller S.M.

Terszowski G.

Blum C.

Haller C.

Anquez V.

Kuschert S.

Carmeliet P.

Augustin H.G.

Rodewald H.R. Gene targeting of VEGF-A in thymus epithelium disrupts thymus blood vessel architecture. Fraidenraich et al., 2004 Fraidenraich D.

Stillwell E.

Romero E.

Wilkes D.

Manova K.

Basson C.T.

Benezra R. Rescue of cardiac defects in id knockout embryos by injection of embryonic stem cells. Ueno et al., 2009 Ueno H.

Turnbull B.B.

Weissman I.L. Two-step oligoclonal development of male germ cells. Ueno and Weissman, 2006 Ueno H.

Weissman I.L. Clonal analysis of mouse development reveals a polyclonal origin for yolk sac blood islands. Stanger et al., 2007 Stanger B.Z.

Tanaka A.J.

Melton D.A. Organ size is limited by the number of embryonic progenitor cells in the pancreas but not the liver. We demonstrated that the Pdx1mice derived from blastocysts complemented with mPSCs were born with functional pancreas almost entirely derived from donor PSCs and grew into adulthood without showing any evidence of pancreatic insufficiency. Several groups have used the same technique to study the development of thymic epithelium (), to compensate for cardiac defects (), or to determine if yolk sac hematopoiesis and germ cell development are of clonal or nonclonal origin (). Although Stanger et al. tested development of pancreas and liver in embryos to define organ size determinants (), no study has exploited this technique to produce donor-derived functional organs and rescued a lethal phenotype to adulthood.

Kroon et al., 2008 Kroon E.

Martinson L.A.

Kadoya K.

Bang A.G.

Kelly O.G.

Eliazer S.

Young H.

Richardson M.

Smart N.G.

Cunningham J.

et al. Pancreatic endoderm derived from human embryonic stem cells generates glucose-responsive insulin-secreting cells in vivo. Miura et al., 2009 Miura K.

Okada Y.

Aoi T.

Okada A.

Takahashi K.

Okita K.

Nakagawa M.

Koyanagi M.

Tanabe K.

Ohnuki M.

et al. Variation in the safety of induced pluripotent stem cell lines. Nakagawa et al., 2008 Nakagawa M.

Koyanagi M.

Tanabe K.

Takahashi K.

Ichisaka T.

Aoi T.

Okita K.

Mochiduki Y.

Takizawa N.

Yamanaka S. Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Okita et al., 2008 Okita K.

Nakagawa M.

Hyenjong H.

Ichisaka T.

Yamanaka S. Generation of mouse induced pluripotent stem cells without viral vectors. Direct in vitro differentiation of insulin-producing cells from PSCs has been a major focus of stem cell therapy, as recently demonstrated by a sophisticated protocol to generate pancreatic endoderm efficiently via stepwise endodermal differentiation (). However, the in vitro generation of insulin-producing cells still needs further improvement in differentiation efficiency, in insulin production levels, or in speed of insulin response to glucose changes. In addition, the risk of tumor development due to contamination with undifferentiated PSCs must be rigorously assessed before clinical use. Compared with those generated in vitro, insulin-producing cells obtained from pancreas that is formed in vivo by blastocyst complementation must have gone through near-normal differentiation processes with proper epigenetic changes. The tissues obtained, such as insulin-producing cells, thus are presumed to be fully functional and the risk of teratoma development due to contamination of undifferentiated PSCs to be negligible. Nonetheless, the oncogenicity of iPSC-derived cells due to reactivation of introduced genes () or to genome abnormality due to long-term culture remains to be assessed. To establish iPSCs without genomic integration of a retroviral sequence should further reduce the risk associated with the use of iPSCs ().

We assumed that aggregation of early embryos would lead to the presence in trophectoderm of xenogenic cells that are reported to be harmful to embryonic development after uterine implantation. We therefore injected mESCs/iPSCs, but not blastomere cells, into r-blastocysts. As predicted, EGFP-positive PSC-derived cells were not detected in placentas ( Figure 4 A and Figure S5 A), and we succeeded in generating interspecific chimeras. To confirm this further, we then attempted to generate interspecific chimeras by injecting rESCs/iPSCs into m-blastocysts. After injection into 8-cell/morula stage mouse embryos, the riPSCs were eventually enclosed within the inner cell mass of the m-blastocyst and were never detected in the m-blastocyst trophectoderm ( Figures S4 C and S4D). Our study, consistent with others, indicates that the presence of xenogenic cells among extraembryonic lineage cells, with exposure of xenogenic cells to the uterine environment, is inhibitory to implantation and/or to further intrauterine development of interspecific chimeras.

It is of prime interest that body size and weight of interspecific chimeras conformed with those of the species of the foster mother ( Figures 5 A–5C). What xenogenic components contribute to the phenotypic determination of interspecific chimeras? It seems that placenta and/or uterine environment are responsible for size determination of adult interspecific chimeras both as embryos and as adults. Given that placenta must be of the same origin as the foster mother for successful generation of interspecific chimeras, it is not clear which is primarily responsible for this determination. Degree of chimerism may also influence the phenotype. As observed in intraspecific chimeras, contribution and distribution of xenogenic cells in interspecific chimeras vary organ-to-organ. There is a negative correlation between contribution of donor (mouse) PSC-derived cells and body weight ( Figure 6 ). Although we could obtain interspecific chimeras consistently ( Figure 4 and Figure 5 ), embryonic lethality was high and postnatal development was poor. Some interactions between mouse- and rat-derived cells indispensable for organism survival may not work across species, resulting in death during embryonic development or in retarded postnatal development.

Another example is the formation of gallbladders in interspecific chimeras. Those derived from m-blastocysts have gallbladders but those from r-blastocysts do not. It is conceivable that the temporo-spatial development of donor PSC-derived cells is regulated by the xenogenic host microenvironment, which governs morphogenesis and organogenesis. However, the data also indicate that the intrinsic developmental program imprinted in PSCs may create competition between host blastocyst-derived and donor PSC-derived cells to form chimeric organs. The balance between host and donor cells at certain critical points during embryonic development thus may be important. Xenogenic developmental systems may be useful to elucidate these and other developmental questions and to help in generation of chimeras between species evolutionarily more distant from one another than are mouse and rat.

−/− mice via interspecific blastocyst complementation. In all interspecific neonates derived from Pdx1−/− blastocysts injected with riPSCs, pancreas was present. Although full maturation into adulthood was not common, once the mice matured into adulthood, the generated riPSC-derived pancreas was morphologically and histologically normal and was not associated with any sign of diabetes or other abnormalities; GTT results strongly indicated normal function. Generation of functional cells (sperm, hepatocytes) in xenogenic environments has been reported ( Mercer et al., 2001 Mercer D.F.

Schiller D.E.

Elliott J.F.

Douglas D.N.

Hao C.

Rinfret A.

Addison W.R.

Fischer K.P.

Churchill T.A.

Lakey J.R.

et al. Hepatitis C virus replication in mice with chimeric human livers. Shinohara et al., 2006 Shinohara T.

Kato M.

Takehashi M.

Lee J.

Chuma S.

Nakatsuji N.

Kanatsu-Shinohara M.

Hirabayashi M. Rats produced by interspecies spermatogonial transplantation in mice and in vitro microinsemination. Kamel-Reid and Dick, 1988 Kamel-Reid S.

Dick J.E. Engraftment of immune-deficient mice with human hematopoietic stem cells. McCune et al., 1988 McCune J.M.

Namikawa R.

Kaneshima H.

Shultz L.D.

Lieberman M.

Weissman I.L. The SCID-hu mouse: murine model for the analysis of human hematolymphoid differentiation and function. Most importantly, we succeeded in generating functional rat pancreas in Pdx1mice via interspecific blastocyst complementation. In all interspecific neonates derived from Pdx1blastocysts injected with riPSCs, pancreas was present. Although full maturation into adulthood was not common, once the mice matured into adulthood, the generated riPSC-derived pancreas was morphologically and histologically normal and was not associated with any sign of diabetes or other abnormalities; GTT results strongly indicated normal function. Generation of functional cells (sperm, hepatocytes) in xenogenic environments has been reported (). In addition, hematopoietic xeno-chimeras have been used commonly as a method to study functionality of hematopoietic stem cells (). No study, however, has demonstrated generation in a xenogenic environment of a PSC-derived functional organ that can rescue embryonic lethality to adulthood.

The organ generation system described may be applied to treat organ failure in humans if pigs or other large animals are used. There are, however, several issues that need to be addressed to bring this principle into the clinic. For example, though we were able to generate interspecific chimeras between mouse and rat, their embryonic lethality is high and maturation into adulthood is uncommon. The nature of this xenogenic barrier is not clear, but it is evident that the evolutionary distance accounts for this, as we do not see these problems in intraspecific chimeras. Livestock animals such as pigs or sheep may be too distant evolutionarily for successful complementation. In addition, as described in the allogenic system, vessels, nerves, and some interstitial elements that are not under the influence of Pdx1 expression were composites of host- and miPSC- or mESC-derived cells. Although we showed in this study that islets prepared from miPSC-derived pancreas generated in allogenic hosts indeed were successfully transplanted into “autologous” mice with STZ-induced diabetes without rejection, whether the same principle applies to transplantation of islets obtained from pancreas generated in xenogenic animals remains to be seen. Transplantation of islets from rat pancreas generated in Pdx1−/− mice into diabetic rats should answer this question. However, due to the size difference between mouse and rat, and to high embryonic lethality and poor postnatal maturation of interspecific chimeras, it is not possible to obtain sufficient numbers of islets to treat diabetic rats. Generation of mouse pancreas in Pdx1−/− rats will be necessary to do such experiments.

Lai et al., 2002 Lai L.

Kolber-Simonds D.

Park K.W.

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Greenstein J.L.

Im G.S.

Samuel M.

Bonk A.

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et al. Production of alpha-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning. Nagashima et al., 2004 Nagashima H.

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Nottle M.B. Sex differentiation and germ cell production in chimeric pigs produced by inner cell mass injection into blastocysts. Nichols and Smith, 2009 Nichols J.

Smith A. Naive and primed pluripotent states. Goldstein et al., 2002 Goldstein R.S.

Drukker M.

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Benvenisty N. Integration and differentiation of human embryonic stem cells transplanted to the chick embryo. James et al., 2006 James D.

Noggle S.A.

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Brivanlou A.H. Contribution of human embryonic stem cells to mouse blastocysts. Production of organ-deficient livestock animals and generation of chimeras is another issue, but nuclear transfer technology available for livestock animals may permit establishment of organ-deficient pig lines, for example (). The successful generation of pig chimeras using blastocyst injection has also been reported (). The major difficulty seems to be that primate and rodent PSCs differ (); limits of primate PSCs in contributing to embryo development have been suspected. Poor contribution of human ESCs to embryo development after injection into mouse blastocysts or chick embryo has been demonstrated (). Generation of interspecific chimera technology may prove a tool useful in assessment of pluripotent stem cell potential, thereby addressing this issue.

Another issue of concern is the fact that PSC-derived cells are found not only in pancreas but in all organs and tissues, including brain and gonads. Therefore, without proper control of the differentiation potential of PSCs, generation of human organs in livestock animals will face an ethical issue. There are several approaches to address this. One is use of committed stem or progenitor cells in place of PSCs. If they are introduced into an appropriate microenvironment at an appropriate developmental time point, to restrict differentiation toward a particular organ may be possible. An alternative is to use genetically modified PSCs whose differentiation potential is restricted to certain tissues or organs.

In conclusion, the approach described here will be of use not only for better understanding of the mechanism of organogenesis but also as an initial step toward the ultimate regenerative medicine of the future.