Kazuhiro Umeyama

Generating Porcine Chimeras Using Inner Cell Mass Cells and Parthenogenetic Preimplantation Embryos

Background The development and validation of stem cell therapies using induced pluripotent stem (iPS) cells can be optimized through translational research using pigs as large animal models, because pigs have the closest characteristics to humans among non-primate animals. As the recent investigations have been heading for establishment of the human iPS cells with naïve type characteristics, it is an indispensable challenge to develop naïve type porcine iPS cells. The pluripotency of the porcine iPS cells can be evaluated using their abilities to form chimeras. Here, we describe a simple aggregation method using parthenogenetic host embryos that offers a reliable and effective means of determining the chimera formation ability of pluripotent porcine cells. Methodology/Significant Principal Findings In this study, we show that a high yield of chimeric blastocysts can be achieved by aggregating the inner cell mass (ICM) from porcine blastocysts with parthenogenetic porcine embryos. ICMs cultured with morulae or 4–8 cell-stage parthenogenetic embryos derived from in vitro-matured (IVM) oocytes can aggregate to form chimeric blastocysts that can develop into chimeric fetuses after transfer. The rate of production of chimeric blastocysts after aggregation with host morulae (20/24, 83.3%) was similar to that after the injection of ICMs into morulae (24/29, 82.8%). We also found that 4–8 cell-stage embryos could be used; chimeric blastocysts were produced with a similar efficiency (17/26, 65.4%). After transfer into recipients, these blastocysts yielded chimeric fetuses at frequencies of 36.0% and 13.6%, respectively. Conclusion/Significance Our findings indicate that the aggregation method using parthenogenetic morulae or 4–8 cell-stage embryos offers a highly reproducible approach for producing chimeric fetuses from porcine pluripotent cells. This method provides a practical and highly accurate system for evaluating pluripotency of undifferentiated cells, such as iPS cells, based on their ability to form chimeras.

Advancing Pig Cloning Technologies Towards Application in Regenerative Medicine

Regenerative medicine is expected to make a significant contribution by development of novel therapeutic treatments for intractable diseases and for improving the quality of life of patients. Many advances in regenerative medicine, including basic and translational research, have been developed and tested in experimental animals; pigs have played an important role in various aspects of this work. The value of pigs as a model species is being enhanced by the generation of specially designed animals through cloning and genetic modifications, enabling more sophisticated research to be performed and thus accelerating the clinical application of regenerative medicine. This article reviews the significant aspects of the creation and application of cloned and genetically modified pigs in regenerative medicine research and considers the possible future directions of the technology. We also discuss the importance of reproductive biology as an interface between basic science and clinical medicine.

Diffuse glomerular nodular lesions in diabetic pigs carrying a dominant-negative mutant hepatocyte nuclear factor 1-alpha, an inheritant diabetic gene in humans.

Glomerular nodular lesions, known as Kimmelstiel-Wilson nodules, are a pathological hallmark of progressive human diabetic nephropathy. We have induced severe diabetes in pigs carrying a dominant-negative mutant hepatocyte nuclear factor 1-alpha (HNF1α) P291fsinsC, a maturity-onset diabetes of the young type-3 (MODY3) gene in humans. In this model, glomerular pathology revealed that formation of diffuse glomerular nodules commenced as young as 1 month of age and increased in size and incidence until the age of 10 months, the end of the study period. Immunohistochemistry showed that the nodules consisted of various collagen types (I, III, IV, V and VI) with advanced glycation end-product (AGE) and N ε-carboxymethyl-lysine (CML) deposition, similar to those in human diabetic nodules, except for collagen type I. Transforming growth factor-beta (TGF-β) was also expressed exclusively in the nodules. The ultrastructure of the nodules comprised predominant interstitial-type collagen deposition arising from the mesangial matrices. Curiously, these nodules were found predominantly in the deep cortex. However, diabetic pigs failed to show any of the features characteristic of human diabetic nephropathy; e.g., proteinuria, glomerular basement membrane thickening, exudative lesions, mesangiolysis, tubular atrophy, interstitial fibrosis, and vascular hyalinosis. The pigs showed only Armanni-Ebstein lesions, a characteristic tubular manifestation in human diabetes. RT-PCR analysis showed that glomeruli in wild-type pigs did not express endogenous HNF1α and HNF1β, indicating that mutant HNF1α did not directly contribute to glomerular nodular formation in diabetic pigs. In conclusion, pigs harboring the dominant-negative mutant human MODY3 gene showed reproducible and distinct glomerular nodules, possibly due to AGE- and CML-based collagen accumulation. Although the pathology differed in several respects from that of human glomerular nodular lesions, the somewhat acute and constitutive formation of nodules in this mammalian model might provide information facilitating identification of the principal mechanism underlying diabetic nodular sclerosis.

Transgenic Pig Expressing the Red Fluorescent Protein Kusabira-Orange as a Novel Tool for Preclinical Studies on Hepatocyte Transplantation

INTRODUCTIONnResearch on hepatocyte transplantation as an alternative or supplementary treatment for liver transplantation is progressing. However, to advance to clinical trials, confidence in the technique must be established and its safety must be validated by conducting experiments using animals of comparable sizes to humans, such as pigs. We used transgenic pigs expressing red fluorescence protein for investigating the distribution and survival of transplanted cells.nnnMATERIALS AND METHODSnDonor hepatocytes were isolated from transgenic Kusabira-Orange (KO)-expressing pigs (age, 41 days; weight, 10 kg) created by in vitro fertilization using sperm from a transgenic-cloned KO pig by Matsunari et al. and ova from a domestic pig. The hepatocyte transplant recipients were the nontransgenic, KO-negative littermates. In these recipient pigs, double lumen cannulae were inserted into the supramesenteric veins to access the hepatic portal region. KO-positive donor hepatocytes from the transgenic male pig were isolated using collagenase perfusion. Hepatocytes (1 × 10(9) cells) were transplanted through the cannula. For estimating allogeneic immunogenicity, full-thickness skin (3 × 3 cm) from the same donor was grafted orthotopically on the neck region of the recipients. Immunosuppressive treatment was not implemented. The recipient pigs were humanely killed at 7 and 39 days after transplantation, and the organs were harvested, including the lungs, heart, liver, pancreas, and kidneys.nnnRESULTSnStrong red fluorescence was detected in both the parenchymal and nonparenchymal hepatocytes of the transgenic male donor pig by fluorescent microscopy. Transplanted cells were detected in the liver and lung of the recipient pigs at 7 days after perfusion. Hepatocytes remained in the liver and lung of recipients on day 39, with lower numbers than that on day 7.nnnCONCLUSIONnTransgenic pigs expressing the fluorescent protein KO serve as a useful model of cell transplantation in preclinical studies.

Modeling lethal X-linked genetic disorders in pigs with ensured fertility

Significance The development of therapies for rare and intractable genetic disorders represents a significant unmet medical need. Disease model pigs characterized by physiological, anatomical, and pathogenetic similarities to humans allow translational studies to be performed, yielding valuable data that can be extrapolated to patients. The establishment of an efficient reproduction system is a key element in the practical application of disease model pigs, which often suffer from reproductive inability due to severe symptoms. Here, we showed that the valuable trait of genetically modified disease model pigs can be maximized by generating unique chimeric boars composed of mutant and normal cells. Genetically engineered pigs play an indispensable role in the study of rare monogenic diseases. Pigs harboring a gene responsible for a specific disease can be efficiently generated via somatic cell cloning. The generation of somatic cell-cloned pigs from male cells with mutation(s) in an X chromosomal gene is a reliable and straightforward method for reproducing X-linked genetic diseases (XLGDs) in pigs. However, the severe symptoms of XLGDs are often accompanied by impaired growth and reproductive disorders, which hinder the reproduction of these valuable model animals. Here, we generated unique chimeric boars composed of mutant cells harboring a lethal XLGD and normal cells. The chimeric boars exhibited the cured phenotype with fertility while carrying and transmitting the genotype of the XLGD. This unique reproduction system permits routine production of XLGD model pigs through the male-based breeding, thereby opening an avenue for translational research using disease model pigs.

Temporal Changes in Cellular Repopulation and Collagen Fibril Remodeling and Regeneration After Allograft Anterior Cruciate Ligament Reconstruction: An Experimental Study Using Kusabira-Orange Transgenic Pigs.

Background: Distinguishing recipient cells from donor ligament cells is difficult in the early graft-healing phase after anterior cruciate ligament (ACL) reconstruction. The ability to track the distribution and differentiation of recipient cells using genetically engineered transgenic (Tg) animals would have significant clinical and research effects on graft healing after ACL reconstruction. Hypothesis: Kusabira-Orange Tg pigs may allow the tracking of recipient cells infiltrating the graft after ACL reconstruction. The repopulation of recipient cells within the graft would be apparent even in the early graft-healing phase when necrotic donor cells are still present. Study Design: Descriptive laboratory study. Methods: In 17 genetically engineered Tg pigs, which carried the red fluorescent protein Kusabira-Orange, ACL reconstruction was performed on the right knee using a digital flexor tendon harvested from wild-type pigs. Tissue samples harvested at different time points were subjected to histological, immunohistochemical, and electron microscopic analyses. Results: At 3 weeks postoperatively, recipient cells expressing red fluorescence embraced the graft and were infiltrating the central part of the graft. These cells with oval nuclei gradually infiltrated the gap of collagen fibers, losing their regular orientation. At 6 weeks, cellularity within the graft had doubled to match that of the native ACL, while acellular necrotic regions still existed centrally. Ubiquitous cellular distributions resembling the native ACL were observed at 24 weeks. Electron microscopic analysis showed that the mean collagen fibril diameter and density gradually decreased over 24 weeks. Conclusion: Genetically engineered pigs carrying the Kusabira-Orange gene were useful animal models for analyzing intrinsic and extrinsic cellular dynamics during the course of graft healing after ACL reconstruction. Cellular repopulation by recipient cells occurred in the very early stage, and the cellular distribution within the graft resembled that in the native ACL by 24 weeks, but the reconstructed graft had not restored the ultrastructure of the native ACL by that stage. Clinical Relevance: In allograft ACL reconstruction in a pig model, cellular repopulation was completed by 24 weeks after surgery, but the collagen matrix had not resumed the ultrastructure of the native ACL. Surgeons should be aware that risks may remain with returning to sports activities at 24 weeks after surgery.