ABSTRACT
BACKGROUND: Organ donor scarcity remains a restricting factor for pancreatic islet transplantation. To date, limited information is available on the impact of long-distance transportation on transplantable pancreatic islets. The objective of this study was to assess the effects of transportation on the viability and function of murine pancreatic islet cells. METHODS: The isolated murine pancreatic islets were transported from Japan to Korea with the use of commercial modes of transportation: subway and commercial airplane. After transportation, the islets were assessed by performing a viability assay and by evaluating the islets' insulin secretion in response to glucose stimulation. A comparative study was performed for evaluating the insulin secretory responses of transported and control islets (not transported). RESULTS: There was no evidence of contamination in the transported pancreatic islets. No significant differences were observed in the viability and functionality of the transported and control islet cells. CONCLUSIONS: These findings show the feasibility of pancreatic islet transportation from Japan to Korea. Our data could be used not only for the inter-Asian but also for global advancement of animal and human islet transportation methods and transplantation research.
Subject(s)
Islets of Langerhans Transplantation , Islets of Langerhans/physiology , Tissue and Organ Procurement/methods , Transportation , Animals , Feasibility Studies , Humans , International Cooperation , Japan , Mice , Mice, Inbred C57BL , Republic of KoreaABSTRACT
Laboratory behavioural observation and computational fluid dynamics (CFD) analysis were conducted to examine whether the movement of the elongated dorsal and pelvic spines changed the hydrodynamic drag in white-streaked grouper Epinephelus ongus larvae. The behavioural observation in the tank revealed that the larvae extended the dorsal and pelvic spines during passive transport and retracted during swimming; the angles of the dorsal and pelvic spines in relation to the anteroposterior axis were larger during the passive transport (mean ± S.D. = 28.84 ± 14.27 and 20.35 ± 15.05°) than those during the swimming (mean ± S.D. = 2.59 ± 5.55 and 0.32 ± 6.49°). The CFD analysis indicated that the relative hydrodynamic drag acting on the larvae was approximately 1.25 times higher when the spines were extended (passive transport) than when the spines were retracted (swimming), suggesting that the E. ongus larvae have an ability to adjust their hydrodynamic drag depending on the behavioural context.
Subject(s)
Perciformes/anatomy & histology , Spine/anatomy & histology , Swimming , Animals , Hydrodynamics , Larva/anatomy & histology , PostureABSTRACT
Autologous chondrocyte transplantation involves isolating chondrocytes from a patient's cartilage tissue. Storage conditions such as storage time and temperature are important for the quality of the isolated cells. However, few studies have focused on variables for optimum tissue preservation, and there is neither an established method for storing cartilage nor reliable reports on how different conditions affect the isolated chondrocytes. Therefore, in the present study, we stored cartilage in various preservation solutions, under different temperatures, and for varying durations and determined their effects on the characteristics and viability of isolated chondrocytes. We assessed chondrocyte viability with the use of a cell proliferation assay and determined their chondrogenic potential with the use of reverse-transcription polymerase chain reaction, enzyme-linked immunosorbent assay, and glycosaminoglycan assays. Our results demonstrated that cartilage tissue stored in a preservation medium composed of dimethyl sulfoxide, fetal bovine serum, and Dulbecco Modified Eagle Medium at a ratio of 1:2:7 (v/v) or stored with a commercially available preservation solution generated greater numbers of chondrocytes when the storage temperature was -80°C than when it was 4°C. The viability of isolated cells decreased with time at 4°C, whereas these values remained constant for tissues stored at -80°C. Our data suggest that an optimal method for preserving cartilage tissue is required to ensure the quality of cells used for transplantation.
Subject(s)
Cartilage, Articular/drug effects , Chondrocytes/drug effects , Cold Temperature , Cryopreservation/methods , Organ Preservation Solutions/pharmacology , Animals , Autografts , Cartilage, Articular/metabolism , Cartilage, Articular/pathology , Cartilage, Articular/surgery , Cell Proliferation/drug effects , Cell Survival/drug effects , Cells, Cultured , Chondrocytes/metabolism , Chondrocytes/pathology , Chondrocytes/transplantation , Chondrogenesis/drug effects , Chondrogenesis/genetics , Culture Media/pharmacology , Dimethyl Sulfoxide/pharmacology , Gene Expression Regulation , Glycosaminoglycans/metabolism , Male , RNA, Messenger/metabolism , Rabbits , Serum , Time FactorsABSTRACT
Autologous chondrocyte transplantation (ACT) is an effective and safe therapy for repairing articular cartilage defects and requires cell preservation and subculture before transplantation. We compared the effects of cryopreservation and passaging on cell viability, proliferation, and maintenance of the function of chondrocytes and synovium-derived mesenchymal stem cells (MSCs) used as sources for ACT. These cells were isolated from the knee joints of rabbits and were cultured, passaged serially, and divided into 2 groups that were either cryopreserved or not. The morphology, viability, gene expression, and differentiation potential of the 2 groups were compared. Maintenance of the potential to undergo chondrogenic differentiation was determined with the use of a 3-dimensional culture method. Passaging and cryopreservation significantly affected the ability of chondrocytes to maintain their morphology, express chondrogenic genes, and differentiate. In contrast, synovium-derived cells were not affected by passaging and cryopreservation. Our results may serve as the foundation for the application of passaged and cryopreserved chondrocyte or other source cells of MSCs in ACT.
Subject(s)
Cell Proliferation , Chondrocytes/pathology , Cryopreservation , Mesenchymal Stem Cells/pathology , Synovial Membrane/pathology , Animals , Autografts , Cell Differentiation/genetics , Cell Shape , Cell Survival , Cells, Cultured , Chondrocytes/metabolism , Chondrocytes/transplantation , Chondrogenesis/genetics , Gene Expression Regulation , Glycosaminoglycans/metabolism , Male , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/metabolism , RNA, Messenger/metabolism , Rabbits , Synovial Membrane/metabolism , Synovial Membrane/transplantationABSTRACT
Pancreatic islets have been the focus of recent studies exploring the pathologic mechanisms of diabetes mellitus as well as more effective and radical treatments for this disease. Islet transplantation is a promising therapeutic strategy; however, isolation of pancreatic islets for this purpose has been challenging, because the technique is time consuming and technically difficult, and tissue handling can be variable. Pseudo-islets can be used as an alternative to naïve islets, but require cellular sources or artificial materials. In this study, pancreas-derived cells were used to generate pseudo-islets. Because the pancreas is composed of a variety of cell types, namely α cells, ß cells, δ cells, and other pancreatic cells that perform different functions, we used 3 different cell lines-NIT-1 (a ß-cell line), α TC1 clone 6 (an α-cell line), and TGP52 (a pancreatic epithelial-like cell line)-which we cocultured in nonadhesive culture plates to produce hybrid cellular spheroids. These pseudo-islets had an oval shape and were morphologically similar to naïve islets; additionally, they expressed and secreted the pancreatic hormones insulin, glucagon, and somatostatin, as confirmed by reverse-transcription polymerase chain reaction and enzyme-linked immunosorbent assay. The results demonstrate that pseudo-islets that mimic naïve islets can be successfully generated by a coculture method. These artificial islets can potentially be used for in vitro tests related to diabetes mellitus, specifically, in drug discovery or for investigating pathology. Moreover, they can be useful for examining basic questions pertaining to cell-cell interactions and tissue development.
Subject(s)
Bioartificial Organs , Islets of Langerhans/cytology , Tissue Engineering/methods , Animals , Cell Communication , Cell Line, Tumor , Cell Shape , Coculture Techniques , Gene Expression Regulation , Glucagon/genetics , Glucagon/metabolism , Insulin/genetics , Insulin/metabolism , Insulin Secretion , Islets of Langerhans/metabolism , Islets of Langerhans Transplantation , Mice , RNA, Messenger/metabolism , Somatostatin/genetics , Somatostatin/metabolism , Spheroids, Cellular , Time FactorsABSTRACT
Intravital imaging techniques will be a valuable tool to monitor the post-transplantation dynamics of the cells/tissues in regenerative medicine research. Among the conventional live imaging techniques, the cranial window model has various advantages regarding resolution, longevity, and easy manipulability. We describe the use of the cranial window model to visualize the post-transplantation processes of primary pancreatic islets in the living mouse. Macroscopic or microscopic analyses were performed to evaluate the post-transplantation dynamics of primary murine islets, including the revascularization process inside the cranium. Consistent with earlier literature on clinical outcomes of islet transplantation, marked loss of transplanted islets was observed within 7 days. Intravital confocal microscope analysis revealed that functional revascularization seldom occurred in the central regions of the transplants. Our results suggest that the cranial window model offers an ideal platform for understanding cellular dynamics, through the possibility of long-term imaging studies over time scales. This platform is possibly applied not only for transplant studies of pancreatic islets, but also for other endodermal cell/tissue types in vivo.
Subject(s)
Graft Survival , Islets of Langerhans Transplantation , Islets of Langerhans/surgery , Microscopy, Confocal , Skull/surgery , Animals , Green Fluorescent Proteins/biosynthesis , Green Fluorescent Proteins/genetics , Islets of Langerhans/blood supply , Islets of Langerhans/metabolism , Islets of Langerhans/pathology , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, SCID , Mice, Transgenic , Models, Animal , Neovascularization, Physiologic , Skull/blood supply , Skull/pathology , Time Factors , Tissue SurvivalABSTRACT
BACKGROUND: The animal model with humanized liver is useful for testing drug metabolism and toxicity in preclinical studies. A mouse model has been reported in which the liver was repopulated more than 90% with human hepatocytes; however, in the rat, the target is far from being reached. In this study, we attempt to develop a humanized liver model with an immunodeficient rat. METHODS: Rag1 knockout rats were treated with neonatal thymectomy. At 3 and 4 weeks of age, they were injected with hepatotoxin retrorsine; 2 weeks after, the animals were subjected to 70% partial hepatectomy and transplanted with immature human hepatocytes via portal vein. The recipients were also treated with anti-asialo GM1 antibody weekly from the day before transplantation and were injected with FK506 every 3 days after transplantation. RESULTS: In Rag1 knockout rats, B lymphocytes were deleted almost completely in peripheral blood. However, T and natural killer (NK) lymphocytes were kept present. When they were treated additionally with neonatal thymectomy for T-lymphocyte deletion and suppressed neutralized NK lymphocytes with anti-asialo GM1, B, T, and NK cells in lymphocytes were reduced to very low levels of 0.75%, 1.58%, and 0.26%, respectively. After transplanting human donor hepatocytes into retrorsine-treated recipient livers, at week 3 the human cell-derived hepatic colonies were expanded in the recipient liver and the liver repopulation rate with human hepatocytes reached approximately 17%. The human hepatocyte-specific genes, albumin, CYP3A4, CYP2C18, and CYP2C9, also could be detected in the recipient rat. CONCLUSION: It is possible to generate a chimera animal with humanized liver in a novel severely immunodeficient rat model.
Subject(s)
Genes, RAG-1 , Hepatocytes/transplantation , Liver Transplantation/methods , Liver/surgery , Animals , Antibodies/pharmacology , Aryl Hydrocarbon Hydroxylases/genetics , Aryl Hydrocarbon Hydroxylases/metabolism , B-Lymphocytes/immunology , Cell Proliferation , Cytochrome P-450 CYP2C9/genetics , Cytochrome P-450 CYP2C9/metabolism , Cytochrome P-450 CYP3A/genetics , Cytochrome P-450 CYP3A/metabolism , G(M1) Ganglioside/immunology , Genetic Markers , Hepatectomy , Hepatocytes/drug effects , Hepatocytes/immunology , Hepatocytes/metabolism , Humans , Immunocompromised Host , Immunosuppressive Agents/pharmacology , Liver/drug effects , Liver/immunology , Liver/metabolism , Liver Regeneration , Models, Animal , Pyrrolizidine Alkaloids/pharmacology , RNA, Messenger/metabolism , Rats, Sprague-Dawley , Rats, Transgenic , Serum Albumin/genetics , Serum Albumin/metabolism , Serum Albumin, Human , Species Specificity , T-Lymphocytes/immunology , Tacrolimus/pharmacology , Thymectomy , Time Factors , Transplantation ChimeraABSTRACT
Mass-scale production of hepatocytes from human induced pluripotent stem cells (iPSCs) with functional properties of primary hepatocytes is of great value in clinical transplantation for liver failure as well as in facilitating drug development by predicting humanized drug metabolism profiles. In this report, we generated human hepatocyte-like cells from human iPSCs with the use of a stepwise protocol. Aiming at future clinical and industrial application, it is important to determine the suitable stage of iPSC-derived hepatic cells that possess high proliferative capacity to intensively expand the hepatic cells. Ki67 immunostaining showed that human iPSC-derived hepatic endoderm cells contained Ki67(+) cells at the highest level in the middle stage of hepatic differentiation, suggesting that the abundance of proliferating hepatic progenitor cells exists in this stage. Extensive expansion and differentiation of human iPSC-derived hepatic progenitors will provide future perspectives in transplantation therapy and drug development.
Subject(s)
Cell Differentiation , Cell Proliferation , Hepatocytes/physiology , Induced Pluripotent Stem Cells/physiology , Biomarkers/metabolism , Cell Differentiation/genetics , Cell Proliferation/genetics , Cells, Cultured , Gene Expression Regulation, Developmental , Hepatocytes/metabolism , Humans , Induced Pluripotent Stem Cells/metabolism , Ki-67 Antigen/metabolism , Time Factors , TransfectionABSTRACT
BACKGROUND: The visualization of induced pluripotent stem (iPS) cells with the use of fluorescent techniques is useful for the in vivo evaluation of iPS-derived functional cells following differentiation and distribution of the transplanted cells. The data obtained from the fluorescently labeled iPS cells would lead to amelioration and validation of protocols directing the differentiation of iPS cells into functional cells. In this study, we established enhanced green fluorescent protein (EGFP)-labeled iPS cells to enable their easy visualization. METHODS: Human iPS cells were transfected with (a) 2 transcription activator-like effector nuclease (TALEN) vectors targeted to the adeno-associated virus integration site 1 (AAVS1) locus and (b) the targeting vector carrying the homology arms, EGFP gene, and a drug-selection marker. RESULTS: Several puromycin-resistant clones were obtained after transfection of the targeting vector and corresponding TALEN-expressing vectors. EGFP expression in these clones was observed with the use of a fluorescent microscope. Clones were examined for specific recombination, which revealed precise targeting at the AAVS1 locus. Furthermore, EGFP protein expression was sustained after directed differentiation into a hepatic lineage. CONCLUSIONS: We were successful in evaluating the behavior of iPS-derived hepatic cells. The data suggest that genomic knock-in at the AAVS1 locus is suitable for long-term observation of iPS-derived cells. Manipulation of the iPS genome can also be applied for the purification of hepatic cells during iPS cell differentiation by introducing the fluorescent protein under the regulation of a hepatic cell-specific promoter. Another application involves gene correction of iPS cells from patients with hepatic disease for regenerative medicine.
Subject(s)
Cell Tracking/methods , Deoxyribonucleases/metabolism , Dependovirus/genetics , Genes, Reporter , Genetic Vectors , Green Fluorescent Proteins/biosynthesis , Hepatocytes/metabolism , Induced Pluripotent Stem Cells/metabolism , Microscopy, Fluorescence , Transfection/methods , Biomarkers/metabolism , Cell Differentiation , Cell Lineage , Cells, Cultured , Deoxyribonucleases/genetics , Gene Expression Regulation , Gene Knock-In Techniques , Green Fluorescent Proteins/genetics , HumansABSTRACT
Current surgical intervention of craniofacial defects caused by injuries or abnormalities uses reconstructive materials, such as autologous cartilage grafts. Transplantation of autologous tissues, however, places a significant invasiveness on patients, and many efforts have been made for establishing an alternative graft. Recently, we and others have shown the potential use of reconstructed elastic cartilage from ear-derived chondrocytes or progenitors with the unique elastic properties. Here, we examined the differentiation potential of canine joint cartilage-derived chondrocytes into elastic cartilage for expanding the cell sources, such as hyaline cartilage. Articular chondrocytes are isolated from canine joint, cultivated, and compared regarding characteristic differences with auricular chondrocytes, including proliferation rates, gene expression, extracellular matrix production, and cartilage reconstruction capability after transplantation. Canine articular chondrocytes proliferated less robustly than auricular chondrocytes, but there was no significant difference in the amount of sulfated glycosaminoglycan produced from redifferentiated chondrocytes. Furthermore, in vitro expanded and redifferentiated articular chondrocytes have been shown to reconstruct elastic cartilage on transplantation that has histologic characteristics distinct from hyaline cartilage. Taken together, cultured hyaline cartilage-derived chondrocytes are a possible cell source for elastic cartilage reconstruction.
Subject(s)
Chondrocytes/transplantation , Chondrogenesis , Elastic Cartilage/metabolism , Hyaline Cartilage/transplantation , Regeneration , Tissue Engineering/methods , Animals , Autografts , Cell Differentiation , Cell Proliferation , Cells, Cultured , Chondrocytes/metabolism , Chondrogenesis/genetics , Dogs , Gene Expression Regulation , Glycosaminoglycans/metabolism , Hyaline Cartilage/cytology , Hyaline Cartilage/metabolism , Male , Regeneration/genetics , Time FactorsABSTRACT
Induction and promotion of angiogenesis play a role in a diverse range of physiologic and pathophysiologic processes that are especially relevant to the field of regenerative medicine. For assessing vasculogenesis and neo-angiogenesis, identifying angiogenic factors, angiocrine factors, and vascular niche, facilitating tissue-repair and tumor growth, efficiently generating induced pluripotent stem cells, and coculturing with organ-specific stem cells, isolation and characterization of the subpopulation of human umbilical vein endothelial cells (HUVECs) and their endothelial progenitor cells (EPCs) are needed. In this study, primary HUVECs were collected from fresh umbilical cords and fractionated and characterized with the use of flow cytometry. Clonal colony assay showed that endothelial colony-forming units in culture frequently existed in fresh HUVECs. Antigenic profiling demonstrated that undifferentiated EPCs in HUVECs had normal endothelial marker CD31 with a subpopulation of cells positive for hematopoietic stem cell marker CD34 and c-Kit. With continuing passages, EPC markers CD34 and vascular endothelial growth factor receptor 2 expression decreased dramatically. Moreover, a distinct subpopulation with different proliferative capability and angiogenesis from the early-passage HUVECs was shown. In conclusion, it is possible to isolate accurately and to enrich EPCs or hematoangioblast-like cells from a heterogeneous population of HUVECs, and to explore the differential process with flow cytometry for further investigations.
Subject(s)
Human Umbilical Vein Endothelial Cells/physiology , Neovascularization, Physiologic , Stem Cells/physiology , Antigens, CD34/metabolism , Biomarkers/metabolism , Cell Differentiation , Cell Proliferation , Cell Separation/methods , Cells, Cultured , Flow Cytometry , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Phenotype , Platelet Endothelial Cell Adhesion Molecule-1/metabolism , Proto-Oncogene Proteins c-kit/metabolism , Stem Cells/metabolism , Vascular Endothelial Growth Factor Receptor-2/metabolismABSTRACT
BACKGROUND: Recent study has demonstrated the important role of endothelial-mesenchymal interactions in 3-dimensional self-organization of immature progenitor populations with the use of mimicking of organogenesis. Here, we show that the same principle can be applicable to adult mature cells, ie, human adult hepatocytes (hAHs). METHODS: We cultivated hAHs with fluorescence-labeled human mesenchymal cells (hMSCs) and human umbilical vein endothelial cells (HUVECs) in micro-well culture plates and observed them for 9 days. Fluorescence microscopy imaging analyses were performed to evaluate the internal structures of generated 3-dimensional tissues. Maintenance of in vitro protein production capacity was examined with the use of enzyme-linked immunosorbent assay (ELISA). RESULTS: hAHs started to self-organize into 3-dimensional tissue with the use of coculturing with hMSCs and HUVECs. Live imaging analyses showed that endothelial cells started sprouting inside the generated tissues after 2 days of culture. ELISA showed that human albumin production capacity was improved with the use of coculture compared with hAHs-only culture after 9 days. CONCLUSIONS: We demonstrated that 3-dimensional vascularized hepatic tissue could be generated from hAHs by reconstituting endothelial-mesenchymal interactions. Future studies are needed to evaluate the therapeutic potential of vascularized hepatic tissue transplantation, and this may pave a new way to establish a new transplantation modality as an alternative to hepatocyte transplantation.
Subject(s)
Bioartificial Organs , Cell Communication , Hepatocytes/physiology , Human Umbilical Vein Endothelial Cells/physiology , Liver/blood supply , Liver/cytology , Neovascularization, Physiologic , Tissue Engineering/methods , Adult , Cell Culture Techniques , Cells, Cultured , Hepatocytes/metabolism , Hepatocytes/transplantation , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Liver/metabolism , Liver Transplantation/methods , Luminescent Proteins/biosynthesis , Luminescent Proteins/genetics , Mesoderm/cytology , Microscopy, Fluorescence , Serum Albumin/metabolism , Serum Albumin, Human , Time Factors , Transfection , Red Fluorescent ProteinABSTRACT
Autologous chondrocyte transplantation (ACT) has been established to contribute cartilage regeneration over the past years; however, many obstacles need to be overcome. Recently, newer ACT technique involves cotransplantation of chondrocytes and biomaterial. Although various proposed intelligent biomaterials exist, many of them remain insufficient and controversial. In this study, we aimed to examine the effects of natural extracellular matrix (ECM) to the proliferation rate and differentiation on the chondrocytes. We first derived a natural ECM sheet from 10-µm-thick frozen sections of porcine knee cartilages. We then cultured the chondrocytes derived from a rabbit's knee on a dish precoated with the natural ECM. Then we assessed differentiation and chondrogenic potential of the cells compared with those grown in untreated culture dishes. We characterized the gene expression of chondrogenic markers, such as collagen type II, SOX-9, and aggrecan, as well as the level of ECM protein with the use of reverse-transcription polymerase chain reaction analysis. The cells cultured with the ECM sheet showed highest chondrogenic potential and differentiation. Therefore, we can induce good chondrogenesis by with the use of a natural ECM sheet on the culture dish. The readily available and easy-to-handle thin ECM sheets create an environment that promotes efficient cartilage regeneration. Our data suggest that this natural ECM scaffold improved the chondrogenic differentiation of the cells in vitro by providing a favorable microenvironment.
Subject(s)
Cartilage/cytology , Cartilage/metabolism , Cellular Microenvironment , Chondrocytes/metabolism , Chondrogenesis , Extracellular Matrix/metabolism , Tissue Engineering/methods , Animals , Cartilage/transplantation , Cell Differentiation , Cell Proliferation , Cells, Cultured , Chondrocytes/transplantation , Chondrogenesis/genetics , Gene Expression Regulation , Genetic Markers , Male , Rabbits , Regeneration , SwineABSTRACT
In vitro organogenesis is now becoming a realistic goal of stem cell biology, as one can obtain an unlimited number of pluripotent stem cells through reprogramming technology. One practical challenge is to develop a four-dimensional (4-D) stem cell culture system whereby multiple progenitors communicate in a spatiotemporal manner, as observed during in vivo organogenesis. This perspective summarizes the potential for emerging culture platforms in the future application of induced pluripotent stem cell (iPSC)-derived miniature organs by recapitulating early embryogenesis.
Subject(s)
Drug Discovery/methods , Induced Pluripotent Stem Cells/drug effects , Induced Pluripotent Stem Cells/metabolism , Tissue Engineering , Toxicity Tests/methods , Cell Culture Techniques , Cells, Cultured , Humans , OrganogenesisABSTRACT
BACKGROUND: Careful orchestration among endodermal epithelial, endothelial, and mesenchymal cells initiate liver organogenesis prior to vascular function. Nonparenchymal endothelial or mesenchymal cells not only form passive conduits, but also establish an organogenic stimulus. Herein, we have evaluated the potential roles of primitive endothelial and mesenchymal cells toward hepatic organization in vitro. METHODS: To track the cellular movements and localization, we retrovirally transduced enhanced green fluorescence protein and kusabira orange into human fetal liver cells (GFP-hFLCs) and human umbilical vein endothelial cells (KO-HUVECs), respectively. GFP-hFLCs were cocultivated with KO-HUVECs and human mesenchymal stem cells (hMSCs) under conventional two-dimensional (2D) conditions. RESULTS: Even under 2D culture, fetal liver, endothelial, and mesenchymal cells self-organized into a macroscopically visible three-dimensional (3D) organoid. Time-lapse confocal imaging showed dynamic cellular organizations of GFP-hFLCs and KO-HUVECs. Endothelial cells organized into patterned clusters wrapping fetal liver cells, forming vessel-like lumens inside. Mesenchymal cells supported the generated organoid from outside. CONCLUSION: Generation of whole organ architecture remains a great challenge so far. Our preliminary results showed that recapitulation of primitive cellular interactions during organogenesis elicit the intrinsic self-organizing capacity to form hepatic organoids. Future studies to define precise conditions mimicking organogenesis may ultimately lead to the generation of a functional liver for transplantation and for other applications such as drug development.
Subject(s)
Cell Communication , Human Umbilical Vein Endothelial Cells/physiology , Liver/physiology , Mesenchymal Stem Cells/physiology , Cells, Cultured , Coculture Techniques , Green Fluorescent Proteins/biosynthesis , Green Fluorescent Proteins/genetics , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Liver/embryology , Liver/metabolism , Luminescent Proteins/biosynthesis , Luminescent Proteins/genetics , Mesenchymal Stem Cells/metabolism , Microscopy, Confocal , Organogenesis , Organoids , Time Factors , Time-Lapse Imaging , Transduction, Genetic , Transfection , Red Fluorescent ProteinABSTRACT
Islet transplantation is considered to be one of the most promising treatment for type I diabetes mellitus (TID). Development of the Edmonton protocol opened the possibility of insulin independence for the patients with TID. However, there is the problem of the donor shortage. Herein we have discussed recent approaches to overcome the problem. It is neccessary to develop a new cellular source for donor islets and to achieve a high engraftment rate. One advantage in TID therapy is that allogeneic islet transplantation is allowed to avoid autoimmunity. That opens broad candidates for the beta-cell source. To achieve a high engraftment rate, is several attempts have sought to develop an appropriate site for transplantation and to modify beta-cells for long-term survival. It is also important to achieve early onset of blood perfusion after transplantation by prevascularization of the islets in vitro. These multiple approaches will bring a milestone in diabetes therapy.
Subject(s)
Cell Transplantation , Diabetes Mellitus, Type 1/surgery , Islets of Langerhans Transplantation , Islets of Langerhans/surgery , Regeneration , Regenerative Medicine/methods , Tissue Donors/supply & distribution , Animals , Cell Differentiation , Cell Proliferation , Cell Survival , Cell Transplantation/adverse effects , Diabetes Mellitus, Type 1/pathology , Diabetes Mellitus, Type 1/physiopathology , Humans , Islets of Langerhans/pathology , Islets of Langerhans/physiopathology , Islets of Langerhans Transplantation/adverse effects , Treatment OutcomeABSTRACT
BACKGROUND: Although hepatocytes can be an option for liver transplantation, the shortage of donor organs continues to worsen. Since the development of induced pluripotent stem (iPS) cell technology, it is eagerly anticipated to produce functional elements from pluripotent stem cells. These functional cells differentiated from iPS cells could be used for transplantation, drug screening, and in vitro toxicology. METHODS: Human iPS cells are maintained on Mitomycin C-treated mouse embryonic fibroblast layers in DMEM-Ham F12-based medium supplemented with Knockout Serum Replacement, nonessential amino acids, 2-mercaptoethanol, and Glutamax. Differentiation of human iPS cells into a definitive endodermal lineage was induced with PRMI 1640 medium supplemented with B27 and 100 ng/mL human activin A. Two B27 supplements were examined with and without insulin. Furthermore, the PI3 kinase inhibitor LY294002 was used to examine the effect of inhibiting insulin signaling. RESULTS AND DISCUSSION: We established efficient induction of definitive endodermal differentiation from iPS cells. Quantitative analysis revealed efficient (93.03 ± 2.74%) differentiation of human iPS cells into definitive endoderm cells using B27 minus insulin. This protocol may contribute as a fundamental technique to promote human iPS studies to develop cellular sources for transplantation.
Subject(s)
Cell Differentiation , Cell Lineage , Endoderm/cytology , Induced Pluripotent Stem Cells/physiology , Activins/metabolism , Animals , Cell Differentiation/drug effects , Cell Lineage/drug effects , Cells, Cultured , Coculture Techniques , Culture Media/chemistry , Culture Media/metabolism , Endoderm/metabolism , Endoderm/transplantation , Humans , Induced Pluripotent Stem Cells/drug effects , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/transplantation , Insulin/metabolism , Mice , Phosphatidylinositol 3-Kinase/metabolism , Phosphoinositide-3 Kinase Inhibitors , Protein Kinase Inhibitors/pharmacology , Signal TransductionABSTRACT
BACKGROUND: One of the major obstacles in regenerating thick, complex tissues such as the liver is their need for vascularization, which is essential to maintain cell viability during tissue growth and to induce structural organization. Herein, we have described a method to engineer a functional human vascular network. METHODS: Enhanced green fluorescence protein-labeled human umbilical vein endothelial cells (GFP-HUVECs) were cocultivated with kusabira orange-labeled human mesenchymal stem cells (KO-hMSCs) inside a collagen/fibronectin matrix. Premature vascular network formation was visualized by fluorescence microscopy imaging. Furthermore, constructs prevascularized in vitro were implanted into a transparency window in immunodeficient mice. RESULTS: Following several days of cultivation, GFP-HUVECs formed vessel-like structures that were stabilized by pericytes differentiated from KO-hMSCs. After implantation in vivo, the patency of human vascular structures was proved by rhodamine dextran infusion. These functional vascular structures remained for over 2 months. DISCUSSION: Vascularization is the key challenge to organ generation. We successfully generated human vascular networks inside a matrix. Integration of parenchymal cells using our engineering technique should facilitate future efforts to reconstitute vascularized human organ systems in vitro.
Subject(s)
Blood Vessel Prosthesis , Blood Vessels/physiology , Cell Communication , Human Umbilical Vein Endothelial Cells/physiology , Mesenchymal Stem Cells/physiology , Neovascularization, Physiologic , Tissue Engineering , Animals , Blood Vessel Prosthesis Implantation , Blood Vessels/metabolism , Cell Differentiation , Cells, Cultured , Coculture Techniques , Collagen Type I/metabolism , Fibronectins/metabolism , Green Fluorescent Proteins/biosynthesis , Green Fluorescent Proteins/genetics , Human Umbilical Vein Endothelial Cells/metabolism , Human Umbilical Vein Endothelial Cells/transplantation , Humans , Luminescent Proteins/biosynthesis , Luminescent Proteins/genetics , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/metabolism , Mice , Mice, Inbred NOD , Mice, SCID , Microscopy, Fluorescence , Pericytes/physiology , Time Factors , Tissue Engineering/methods , Transfection , Vascular Patency , Red Fluorescent ProteinABSTRACT
Transplantation of bioengineered elastic cartilage is considered to be a promising approach for patients with craniofacial defects. We have previously shown that human ear perichondrium harbors a population of cartilage progenitor cells (CPCs). The aim of this study was to examine the use of a rotating wall vessel (RWV) bioreactor for CPCs to engineer 3-D elastic cartilage in vitro. Human CPCs isolated from ear perichondrium were expanded and differentiated into chondrocytes under 2-D culture conditions. Fully differentiated CPCs were seeded into recently developed pC-HAp/ChS (porous material consisted of collagen, hydroxyapatite, and chondroitinsulfate) scaffolds and 3-D cultivated utilizing a RWV bioreactor. 3-D engineered constructs appeared shiny with a yellowish, cartilage-like morphology. The shape of the molded scaffold was maintained after RWV cultivation. Hematoxylin and eosin staining showed engraftment of CPCs inside pC-HAp/ChS. Alcian blue and Elastica Van Gieson staining showed of proteoglycan and elastic fibers, which are unique extracellular matrices of elastic cartilage. Thus, human CPCs formed elastic cartilage-like tissue after 3-D cultivation in a RWV bioreactor. These techniques may assist future efforts to reconstruct complicate structures composed of elastic cartilage in vitro.
Subject(s)
Bioreactors , Cell Differentiation , Chondrocytes/metabolism , Chondrogenesis , Elastic Cartilage/metabolism , Stem Cells/metabolism , Tissue Engineering/instrumentation , Biomarkers/metabolism , Cell Culture Techniques , Cells, Cultured , Chondrocytes/pathology , Chondroitin Sulfates/metabolism , Collagen/metabolism , Congenital Abnormalities/pathology , Congenital Microtia , Durapatite/metabolism , Ear/abnormalities , Ear/pathology , Elastic Cartilage/pathology , Humans , Rotation , Stem Cells/pathology , Time Factors , Tissue Engineering/methods , Tissue ScaffoldsABSTRACT
Induction of hepatocyte growth factor/scatter factor (HGF/SF) may be one of the critical steps in organ regeneration, wound healing, and embryogenesis. We previously reported the production of HGF/SF from various human leukemia cell lines and a high level of the growth factor in blood and bone marrow plasma from patients with various types of leukemia. We determined here the effects of hematopoietic cytokines on HGF/SF production in human leukemia cell lines, KG-1, a myeloid cell line, and RPMI-8226, a B cell line. Interferon (IFN)-gamma remarkably stimulated HGF/SF production in both cell lines at concentrations of more than 0.1 or 1 IU/ml. IFN-alpha and IFN-beta were as effective as IFN-gamma in RPMI-8226 cells, but less than IFN-gamma in KG-1 cells. HGF/SF gene expression in KG-1 cells was also up-regulated by IFN-gamma. Granulocyte colony-stimulating factor (G-CSF), granulocyte/macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-5 and IL-6 had no effect on HGF/SF production in the 2 leukemia cell lines. We also determined the effects of HGF/SF inducers known for human fibroblasts on the growth factor production in leukemia cells. Out of phorbol 12-myristate 13-acetate (PMA), cholera toxin, IL-1 beta, and tumor necrosis factor (TNF)-alpha, the former three were as effective as IFN-gamma in KG-1 cells, but only TNF-alpha stimulated HGF/SF production in RPMI-8226 cells, whose effect was less than those of IFN-alpha, IFN-beta, and IFN-gamma. The effect of IFN-gamma in KG-1 cells was synergistic with that of PMA. In contrast with the effect in leukemia cells, HGF/SF induction by IFN-gamma in human skin fibroblasts was much less than that by PMA or cholera toxin. These results indicated that IFN-gamma is a potent inducer of HGF/SF in human leukemia cells. This finding suggests the presence of a homeostatic control mechanism in liver regeneration and repair: hepatic injury, DNA synthesis inhibition, or apoptosis caused by IFN-gamma is subsequently overcome by cytokine-induced HGF/SF, a potent promoter of liver DNA synthesis.