Kyung-Mee Park

Systemic Decellularization for Multi-organ Scaffolds in Rats

INTRODUCTION Bioscaffolds derived from animal organs are promising materials for xenotransplantation and regenerative medicine. For effective generation of biological scaffolds from diverse organs, there have been many technical challenges. In this study, we introduced a novel approach to create multiorgan bioscaffolds through systemic decellularization. METHODS AND MATERIALS To obtain acellular bioscaffolds, the healthy adult rats were systemically perfused with ionic detergent through the carotid artery. Additional liver perfusion was set up to prevent potential obstruction from the influx of the decellularized debris via the portal vein. The perfusion system was controlled to maintain a constant physiological cardiac output of approximately 50 mL/min and was designed to minimize air entrapment. After decellularization, every organ designated for bioscaffold was harvested for evaluation of vascular structure and histology. RESULTS The perfusion times were different for each organ. In our histological analysis, the decellularized bioscaffolds harvested from most organs including major solid organs (ie, heart, liver, and kidney) as well as the others (such as stomach, intestines, spleen, etc) represented no evidence of residual cellular materials. Furthermore, the well-preserved collagen materials and intact vascular structures were also confirmed. CONCLUSION The results from this study suggested that this systemic decellularization has the advantages to obtain a variety of bioscaffolds from single donor, and we can even decellularize organs with complex influx vascular structures. This method may also be used to study organ bioengineering for patients who need simultaneous combined organ transplantation.

Biocompatibility evaluation of tissue-engineered decellularized scaffolds for biomedical application.

Biomaterials based on seeding of cells on decellularized scaffolds have gained increasing interest in the last few years and suggested to serve as an alternative approach to bioengineer artificial organs and tissues for transplantation. The reaction of the host toward the decellularized scaffold and transplanted cells depends on the biocompatibility of the construct. Before proceeding to the clinical application step of decellularized scaffolds, it is greatly important to apply a number of biocompatibility tests in vitro and in vivo. This review describes the different methodology involved in cytotoxicity, pathogenicity, immunogenicity and biodegradability testing for evaluating the biocompatibility of various decellularized matrices obtained from human or animals.

Porcine Bioengineered Scaffolds as New Frontiers in Regenerative Medicine

Porcine organs are attractive for xenotransplantation, if severe immunologic concerns can be overcome. Recently, reengineered organs, with heterologous cellular materials removed but preserved organ architecture and vasculature have been created using small rodents in an effort to produce customized bioengineered organs. However, few studies have been performed to generate bioengineered organs from porcine sources. The aim of this work was to produce 3-D bioengineered scaffolds from major porcine organs, preserving the native morphology and vascular structures with complete removal of cellular and nuclear materials. We decellularized porcine heart, liver, and kidney using a peristaltic pump system with 1% sodium dodecyl sulfate. The preservation of major architecture and vasculature was confirmed by gross findings, ultrasonography, and angiography. Hematoxylin and eosin staining revealed no evidence of nuclear or cytoplasmic residues. Quantitative DNA analysis demonstrated a substantial reduction (0%-8%) of porcine DNA in the scaffolds. These results suggested that 3-D bioengineered scaffolds of porcine organs may have tremendous potential to produce non-immunogenic transplantable organs as well as beneficial tools for biomedical studies on organ re-engineering and repair.

Preparation of immunogen-reduced and biocompatible extracellular matrices from porcine liver

Decellularized biologic matrices are plausible biomedical materials for the bioengineering in liver transplantation. However, one of the concerns for safe medical application is the lack of objective assessment of the immunogen within the materials and the in vivo immune responses to the matrices. The purpose of this study was the production of immunogen-reduced and biocompatible matrices from porcine liver. In the present study, 0.1% SDS solution was effective for removing DNA fragments and sequences encoding possible immunogenic and viral antigens within the matrices. The PCR analysis showed that galactose-α-1,3 galactose β-1,4-N-acetylglucosamine (1,3 gal), swine leukocyte antigen (SLA), and porcine endogenous retrovirus (PERV) were completely removed in the matrices. Collagen and glycosaminoglycans (GAGs) were preserved over 63%-71%, respectively, compared to those of native liver. The implanted decellularized tissues showed minimal host responses and naturally degraded within 10 weeks. In this study, we produced immunogen-reduced and biocompatible extracellular matrices from porcine liver. Although future investigations would be required to determine the mechanism of the host reaction, this study could provide useful information of porcine liver-derived biologic matrices for liver researches.

Acute Rejection After Swine Leukocyte Antigen-Matched Kidney Allo-Transplantation in Cloned Miniature Pigs With Different Mitochondrial DNA-Encoded Minor Histocompatibility Antigen

INTRODUCTION Graft rejection remains a major cause of morbidity and mortality following renal transplantation. One of the main determinants of success after renal transplantation is histocompatibility between donor and recipient. Most of the research on this topic has addressed human leukocyte antigen (HLA), but the roles played by minor histocompatibility antigens (mHAgs), such as mitochondrially transmitted antigens, are poorly understood. In this study, we evaluated immune responses induced by minor antigens originating from mitochondrial DNA (mtDNA) in a large animal model. METHODS To characterize whole swine leukocyte antigen (SLA) allele in 8 cloned pigs, we performed SLA genotyping for SLA-1, SLA-2, SLA-3, SLA-DQB1, and SLA-DRB1 as well as the hypervariable region 1 (HV1) of mtDNA. Renal transplantation was performed using SLA-matched pigs with different mtDNA as well as SLA-mismatched cloned animals. Cytokine profiling was performed by incubating peripheral leukocytes with cellular components from SLA-matched different mtDNA and SLA-mismatched cells to evaluate mtDNA-mediated immune response. RESULTS SLA types were confirmed to be identical, but mtDNA sequences of HV1 varied among cloned pigs. Rejection episodes in the SLA-matched group with different mtDNA were similar to those in the SLA-mismatched group; that is, plasma creatinine and BUN levels were increased and mononuclear cell infiltration was observed in perivascular regions in the matched and SLA-mismatched groups. Furthermore, in vitro studies showed interleukin (IL)-1β expression to be elevated in SLA-matched and SLA-mismatched groups. CONCLUSION Cloned pigs are a useful preclinical model to evaluate the immunogenicity of mtDNA encoding minor antigens. The mtDNA originating from nongenomic DNA induced cell-mediated immune rejection after kidney transplantation.

Successful Management of Multidrug-Resistant Pseudomonas aeruginosa Pneumonia after Kidney Transplantation in a Dog

ABSTRACT An 8-year-old male mongrel dog that had undergone renal transplantation was presented 25 days later with an acute cough, anorexia and exercise intolerance. During the investigation, neutrophilic leukocytosis was noted, and thoracic radiographs revealed caudal lung lobe infiltration. While being treated with two broad-spectrum antibiotics, clinical signs worsened. Pneumonia due to infection with multidrug-resistant (MDR) Pseudomonas (P.) aeruginosa, sensitive only to imipenem and amikacin, was confirmed by bacteria isolation. After treatment with imipenem-cilastatin without reducing the immunosuppressant dose, clinical signs completely resolved. During the 2-year follow-up period, no recurrence was observed. To the best of authors’ knowledge, this is the first report of pneumonia caused by MDR P. aeruginosa in a renal recipient dog and successful management of this disease.

Hepatic Differentiation of Porcine Embryonic Stem Cells for Translational Research of Hepatocyte Transplantation

Porcine embryonic stem cells (ES) are considered attractive preclinical research tools for human liver diseases. Although several studies previously reported generation of porcine ES, none of these studies has described hepatic differentiation from porcine ES. The aim of this study was to generate hepatocytes from porcine ES and analyze their characteristics. We optimized conditions for definitive endoderm induction and developed a 4-step hepatic differentiation protocol. A brief serum-free condition with activin A efficiently induced definitive endoderm differentiation from porcine ES. The porcine ES-derived hepatocyte-like cells highly expressed hepatic markers including albumin and α-fetoprotein, and displayed liver characteristics such as glycogen storage, lipid production, and low-density lipoprotein uptake. For the first time, we describe a highly efficient protocol for hepatic differentiation from porcine ES. Our findings provide valuable information for translational liver research using porcine models, including hepatic regeneration and transplant studies, drug screening, and toxicology.

Kidney injury molecule-1 is involved in the chemotactic migration of mesenchymal stem cells

A better understanding of the organ specific factors that regulate the migration of mesenchymal stem cells (MSCs) into the target organ is essential for optimization of strategies to improve the repair after injury. In the present study, we showed that the kidney injury molecule-1 (KIM-1), a well-known kidney-specific biomarker, enhanced the in vitro migration capacity of MSCs as a potent kidney-specific chemo-attractant or an inducer. The in vitro roles were verified by migration assay using KIM1-PK1 cell lines, the mouse proximal tubular epithelial cells (mPTEs) and recombinant human KIM-1 proteins (rhKIM-1). Immunofluorescence staining displayed specific ectodomain binding of KIM-1 on the surface of MSCs. Upregulation of chemokine receptor type 4 (CXCR4) protein when treated with tumor necrosis factor alpha (TNF-α) was shown. The effect of KIM-1 on migration of MSCs was augmented by TNF-α pretreatment in a dose-dependent manner, and reduced by AMD3100, an antagonist of CXCR4. These results suggest that KIM-1 is a potential chemo-ligand of CXCR4 and may play an important role in kidney-specific migration of MSCs via interaction between KIM-1 and CXCR4.

Disparate Hypervariable Region-1 of Mitochondrial DNA Did Not Induce Skin Allograft Rejection in Cloned Porcine Models

INTRODUCTION Alloantigen recognition in skin transplantation is the bane for surgeons. Several studies have mainly focused on the immunogenicity of major histocompatibility (MHC) antigens and H-Y minor histocompatibility antigens. However, the roles of the mitochondrial DNA (mtDNA) encorded miHA have not been identified. Therefore, we sought to address the antigenicity of the hypervariable region 1 (HV-1) of mtDNA in skin transplantation using cloned pig models. METHODS Swine leukocyte antigen and HV-1 of mtDNA were analyzed using sequencing methods. Skin transplantation was performed between MHC-matched, mtDNA-mismatched cloned miniature pigs. Full-thickness skin was grafted between cloned pigs without any immunosuppressants. The grafted tissues were observed for 3 months and evaluated histologically. RESULTS The cloned pigs shared identical MHC but mtDNA mismatched at 9 positions. Skin grafts between the cloned pigs were accepted and hair growth maintained, whereas MHC-mismatched grafts showed acute rejection within 7 days after transplantation and were replaced by hairless scar tissue. CONCLUSIONS HV-1 disparate skin grafts were not recognized as alloantigenic by MHC-matched cloned pigs.

A novel mouse model of diabetes mellitus using unilateral nephrectomy.

Diabetes mellitus (DM) is a major cause of morbidity and mortality worldwide, and its complications are prominent public health issues. Many experimental models of streptozotocin (STZ)-induced and high-fat diet (HF)-induced DM have been used to study this disease. Studies have indicated that unilateral nephrectomy (UN) accelerates the development of diabetic nephropathy. We hypothesized that UN stimulates HF and STZ combination-induced DM in mice. Seventy-two female C57BL/6J mice were divided into four treatment groups: HF; HF + STZ120 (HF and STZ, 120 mg/kg); UN + HF + STZ120 (UN, HF and STZ, 120 mg/kg); and HF + STZ200 (HF and STZ, 200 mg/kg). Onset of DM, survival rate, blood pressure, urine glucose level, and pancreatic histology were investigated. Additionally, renal function was evaluated in the UN + HF + STZ120 group after STZ injection. DM was induced in the UN + HF + STZ120 and HF + STZ200 groups within one week. The UN + HF + STZ120 group had lower mortality than the HF + STZ200 group and greater pancreatic destruction than the HF and HF + STZ120 groups. Two weeks after STZ injection, blood pressure was not significantly different among the groups. Nephrotoxicity associated with the combination of UN and STZ was not observed. In conclusion, the combination of these three techniques – UN, HF and STZ induced DM rapidly and effectively.