RESUMO
The conditioned medium (CM) obtained from mesenchymal stromal cell (MSC) culture has excellent cell growth-promoting activity and is used for cosmetics and healthcare products. Unlike pharmaceuticals, strict efficacy verification is not legally required for these products. However, their efficacy must be substantiated as commercial products. We attempted to simplify CM production and to standardize the evaluation of the growth-promoting activity of CM. CM was obtained through the culturing of two lines of commercially available human adipose tissue-derived MSCs using MEMα with or without 10% fetal bovine serum (FBS) for 24 h. Non-CM control media were produced by the same protocol without MSCs. Growth-promoting activities of the CM were estimated by [3H]-thymidine pulse. CM were subjected to molecular weight fractionation with ultrafiltration using 10 k-, 30 k-, 50 k-, and 100 k-membranes. The FBS-free CMs showed 1.34- to 1.85-fold increases and FBS-containing CMs showed 1.45- to 1.67-fold increases in proliferation-promoting activity compared with non-CM controls, regardless of the source of the cell. The thymidine incorporation levels were approximately three times higher in FBS-containing CMs. Aged cells also showed 1.67- to 2.48-fold increases in the activity due to FBS-containing CM, but not to FBS-free CM. The CM activities were sustained even after 1 year at 4 °C. Molecular weight fractionation showed that the activity was recovered in the fraction above 100 k. Clear and stable cell-growth-promoting activity was confirmed with CMs of commercially available adipose tissue MSCs. The activity was detected in the fraction over 100 k. We propose here the importance of standardizing the production and evaluation of CMs to indicate their specific action.
Assuntos
Tecido Adiposo , Proliferação de Células , Células-Tronco Mesenquimais , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Tecido Adiposo/citologia , Tecido Adiposo/metabolismo , Meios de Cultivo Condicionados/farmacologia , Células Cultivadas , Técnicas de Cultura de Células/métodos , Técnicas de Cultura de Células/normasRESUMO
Ornithine transcarbamylase deficiency (OTCD) is a metabolic and genetic disease caused by dysfunction of the hepatocytic urea cycle. To develop new drugs or therapies for OTCD, it is ideal to use models that are more closely related to human metabolism and pathology. Primary human hepatocytes (HHs) isolated from two patients (a 6-month-old boy and a 5-year-old girl) and a healthy donor were transplanted into host mice (hemi-, hetero-OTCD mice, and control mice, respectively). HHs were isolated from these mice and used for serial transplantation into the next host mouse or for in vitro experiments. Histological, biochemical, and enzyme activity analyses were performed. Cultured HHs were treated with ammonium chloride or therapeutic drugs. Replacement rates exceeded 80% after serial transplantation in both OTCD mice. These highly humanized OTCD mice showed characteristics similar to OTCD patients that included increased blood ammonia levels and urine orotic acid levels enhanced by allopurinol. Hemi-OTCD mice showed defects in OTC expression and significantly low enzymatic activities, while hetero-OTCD mice showed residual OTC expression and activities. A reduction in ammonium metabolism was observed in cultured HHs from OTCD mice, and treatment with the therapeutic drug reduced the ammonia levels in the culture medium. In conclusion, we established in vivo OTC mouse models with hemi- and hetero-patient HHs. HHs isolated from the mice were useful as an in vitro model of OTCD. These OTC models could be a source of valuable patient-derived hepatocytes that would enable large scale and reproducible experiments using the same donor.
Assuntos
Hepatócitos/transplante , Doença da Deficiência de Ornitina Carbomoiltransferase/terapia , Ornitina Carbamoiltransferase/genética , Amônia/sangue , Animais , Pré-Escolar , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Hepatócitos/química , Hepatócitos/citologia , Humanos , Lactente , Masculino , Camundongos , Doença da Deficiência de Ornitina Carbomoiltransferase/genética , Ácido Orótico/urinaRESUMO
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.
Assuntos
Técnicas de Cultura Embrionária/métodos , Doenças Genéticas Ligadas ao Cromossomo X/genética , Reprodução/genética , Animais , Animais Geneticamente Modificados/genética , Cruzamento , Quimera , Clonagem de Organismos/métodos , Modelos Animais de Doenças , Fertilidade , Técnicas de Inativação de Genes/métodos , Engenharia Genética/métodos , Masculino , Técnicas de Transferência Nuclear , Suínos/genéticaRESUMO
Improvement of machine perfusion (MP) technologies is required to enhance organ quality for donor after cardiac death (DCD) grafts. Installing a dialyzer or a filter into the perfusion circuit to maintain the perfusate condition has some advantages. However, the consequences of purification perfusate during subnormothermic machine perfusion (SNMP) remain unexplained. In this study, the effects of initial purification perfusate with simple method of replacing the first 0.5-L perfusate during SNMP were investigated to consider installation effect of the filter or the dialyzer. Porcine liver grafts, which have 60-min warm ischemia time, were procured to imitate the DCD graft condition. Purified SNMP (PSNMP) results were compared with simple cold storage and conventional SNMP. In PSNMP, initial perfusate of 0.5 L was removed to substitute for purification. After preservation process, the preserved grafts were reperfused with diluted autologous blood for 2 h under normothermic machine perfusion condition to evaluate the liver function using an isolated reperfusion model. The vascular pressures, enzyme release rates and the metabolic indexes during reperfusion were analyzed. The pressures in the hepatic artery after reperfusion 60 min were significantly lower in PSNMP group compared with cold storage (CS) and SNMP groups. In addition, lactate dehydrogenase and alkaline phosphatase were significantly lower after PSNMP than after the CS or SNMP. Also, the metabolic indexes of hyaluronic acid and lactate were significantly decreased by purifying the perfusate in MP preservation than in CS or SNMP. The effectiveness of initial purification perfusate during SNMP was investigated.
Assuntos
Transplante de Fígado/métodos , Preservação de Órgãos/métodos , Animais , Morte Súbita Cardíaca , Perfusão/métodos , Suínos , Isquemia QuenteRESUMO
To improve the therapeutic potential of hepatocyte transplantation, the effects of the mitogen-activated protein kinase kinase 4 (MKK4) inhibitor, myricetin (3,3',4',5,5',7-hexahydroxylflavone) were examined using porcine and human hepatocytes in vitro and in vivo. Hepatocytes were cultured, showing the typical morphology of hepatic parenchymal cell under 1-10 µmol/L of myricetin, keeping hepatocyte specific gene expression, and ammonia removal activity. After injecting the hepatocytes into neonatal Severe combined immunodeficiency (SCID) mouse livers, cell colony formation was found at 10-15 weeks after transplantation. The human albumin levels in the sera of engrafted mice were significantly higher in the recipients of myricetin-treated cells than non-treated cells, corresponding to the size of the colonies. In terms of therapeutic efficacy, the injection of myricetin-treated hepatocytes significantly prolonged the survival of ornithine transcarbamylase-deficient SCID mice from 32 days (non-transplant control) to 54 days. Biochemically, the phosphorylation of MKK4 was inhibited in the myricetin-treated hepatocytes. These findings suggest that myricetin has a potentially therapeutic benefit that regulates hepatocyte function and survival, thereby treating liver failure.
Assuntos
Flavonoides/farmacologia , Sobrevivência de Enxerto/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Fígado/metabolismo , Amônia/metabolismo , Animais , Criopreservação , Hepatócitos/citologia , Hepatócitos/transplante , MAP Quinase Quinase 4/metabolismo , Camundongos , Camundongos SCID , Ornitina Carbamoiltransferase/genética , Ornitina Carbamoiltransferase/metabolismo , Fosforilação/efeitos dos fármacos , Albumina Sérica/metabolismo , Suínos , Transplante HeterólogoRESUMO
The use of grafts donated after cardiac death (DCD) would greatly contribute to the expansion of the donor organ pool. Machine perfusion (MP) is a promising technology to improve DCD liver grafts. Several perfusion technologies under various temperature and oxygenation conditions have been suggested and are still debated. It is important to confirm the relationship between oxygen consumption and organ conditions during MP. In this study, we analyzed oxygen consumption during oxygenated MP of porcine DCD liver grafts under different temperature conditions: hypothermic and subnormothermic. Grafts exposed to 60 min of warm ischemia were perfused for 4 h with a modified UW-gluconate perfusate under hypothermic (HMP) and subnormothermic conditions. Oxygen consumption, pressures of the portal vein and hepatic artery, and effluent enzymes were analyzed. Oxygen consumption was strongly related to the graft temperature during MP. Effluent enzyme level of the LDH were lower in the high oxygen consumption group than in the low oxygen consumption group during MP. In summary, we found that high oxygen consumption under subnormothermic temperature conditions has several advantages over HMP for DCD liver graft preservation.
Assuntos
Hipotermia Induzida/métodos , Transplante de Fígado/métodos , Preservação de Órgãos/métodos , Consumo de Oxigênio , Oxigênio/metabolismo , Perfusão/métodos , Animais , Morte , Modelos Animais , SuínosRESUMO
PURPOSE: In Japan, there have been no national surveys on the incidence of de novo malignancy after solid organ transplantation, which is one of the leading causes of death in transplant recipients. METHODS: A questionnaire was distributed to institutions that perform solid organ transplantation in Japan, and clinical information was collected from patients who underwent transplantation between 2001 and 2010 and who exhibited de novo malignancies. RESULTS: Nine thousand two hundred ten solid organ transplants (kidney, 49.9%; liver, 45.9%; heart, 0.9%; lung, 1.2%; pancreas, 1.9%; small intestine, 0.2%) were performed. Four hundred seventy-nine (5.2%) cases of de novo malignancy were identified. The transplanted organs of the patients included the kidney (n = 479, 54.8%), liver (n = 186, 38.8%), heart (n = 5, 0.1%), lung (n = 18, 3.8%), pancreas (n = 9, 1.9%), and small intestine (n = 1, 0.02%). The most common malignancies were post-transplant lymphoproliferative disorder (n = 87) and cancers of the kidney (n = 43), stomach (n = 41), large intestine (n = 41), and lung (n = 36). CONCLUSIONS: This is the first national survey of the incidence of de novo malignancy in Japan. Further study is required to identify the risk of de novo malignancy in organ transplant recipients in comparison to the general population, namely the standardized incidence ratio.
Assuntos
Neoplasias/epidemiologia , Transplante de Órgãos , Complicações Pós-Operatórias/epidemiologia , Causas de Morte , Feminino , Humanos , Imunossupressores/efeitos adversos , Incidência , Neoplasias Intestinais/epidemiologia , Japão/epidemiologia , Neoplasias Renais/epidemiologia , Neoplasias Pulmonares/epidemiologia , Transtornos Linfoproliferativos/epidemiologia , Masculino , Transplante de Órgãos/mortalidade , Transplante de Órgãos/estatística & dados numéricos , Risco , Neoplasias Gástricas/epidemiologia , Inquéritos e Questionários , Fatores de TempoRESUMO
INTRODUCTION: Research has made progress in organ fabrication using an extracellular matrix, cell sheets, or organoids. Human liver tissue has been constructed using a 3-dimensional (3D) bioprinter and showed evidence that an in vitro generated liver bud was reformed in a rodent liver model. This study describes the stages of development of rat fetal organs and liver structure and reviews recent progress in liver organoid transplantation. METHODS: The authors developed the procedures for creating a transected plane for use in experimental microsurgery in rats. A liver lobe was fixed vertically with gauze and it was ligated with 6-0 silk suture in the cut line; the parenchyma was cut, and major vessels were ligated to create the transected plane. The ligated tissue was carefully resected. Hemostasis was not required and hepatic components remained on the transected plane. The plane was covered by omentum. RESULTS: Using this model, we transplanted fetal liver or a 3D bioprinted liver organoid. This microsurgical method enabled creation of an intact liver parenchyma plane. No bleeding was observed. The transplanted liver components successfully engrafted on the liver. CONCLUSION: This method may provide an essential environment for growing liver using portal and arterial blood flow.
Assuntos
Transplante de Fígado , Fígado/cirurgia , Microcirurgia/métodos , Engenharia Tecidual/métodos , Animais , Hepatectomia , RatosRESUMO
PURPOSE: Tracheal cartilage reconstruction is an essential approach for the treatment of tracheal congenital abnormalities or injury. Here, we evaluated the use of allogeneic decellularized tracheas as novel support scaffolds. METHODS: Six weaned pigs (4-week-old domestic males) were transplanted with allogeneic tracheal graft patches (three decellularized and three fresh tracheal scaffolds) onto artificial defects (approximately 15 × 15 mm). After 11 weeks, the tracheas were evaluated by bronchoscopy and histological studies. RESULTS: No pigs displayed airway symptoms during the observation period. Tracheal lumen restored by fresh graft patches showed more advanced narrowing than that treated with decellularized grafts by bronchoscopy. Histologically, fresh grafts induced typical cellular rejection; this was decreased with decellularized grafts. In addition, immunohistochemistry demonstrated regenerating foci of recipient cartilage along the adjacent surface of decellularized tracheal grafts. CONCLUSION: Decellularized allogeneic tracheal scaffolds could be effective materials for restoring impaired trachea.
Assuntos
Aloenxertos/cirurgia , Traqueia/cirurgia , Animais , Masculino , Modelos Animais , Suínos , Engenharia Tecidual/métodos , Alicerces TeciduaisRESUMO
The purpose of this study was to propose a novel evaluation index for the effects of shear stress level and exposure time on hepatocyte damage. Suspensions of rat hepatocytes (0.5 mL) were subjected to shear stress from 1.2 to 3.1 Pa for 10 min (n = 3) using a rheoscope. We counted living and dead cells in photographs taken at 1-min intervals using a digital camera attached to the microscope. Living and dead cells were distinguished using a Trypan blue exclusion test. Under each level of shear stress, at each 1-min time interval, we measured the viability [living-cell number (t)/countable cell number (t)] and the ratio of living cells [RLC: living-cell number (t)/countable cell number in the initial condition]. The effects of shear stress and exposure time on viability and RLC were assessed by multiple regression analysis. As expected, we observed an increase in the number of dead cells and little change in the number of living cells when shear stress was increased. The coefficient of determination (R (2)) to predict the effectiveness of viability and RLC indicated a low to moderate correlation. Viability correlated with shear stress and exposure time (p < 0.001); however, RLC only correlated with exposure time of shear stress (p < 0.001). In this test condition, viability was strongly related not to living-cell damage but to dead-cell damage. Therefore, we propose RLC as a novel and effective index for investigating the effect of shear stress on living hepatocytes.
Assuntos
Hepatócitos/patologia , Animais , Contagem de Células , Técnicas de Cultura de Células , Sobrevivência Celular , Ratos , Reologia , Resistência ao Cisalhamento , Estresse Mecânico , Fatores de Tempo , Azul TripanoRESUMO
Glycogen storage disease type Ib (GSDIb) is caused by a deficiency in the glucose-6-phosphate transporter (G6PT), which leads to neutrophil dysfunction. However, the underlying causes of these dysfunctions and their relationship with glucose homeostasis are unclear. Induced pluripotent stem cells (iPSCs) hold a great promise for advances in developmental biology, cell-based therapy and modeling of human disease. Here, we examined the use of iPSCs as a model for GSDIb. In this study, one 2-year-old patient was genetically screened and diagnosed with GSDIb. We established iPSCs and differentiated these cells into hepatocytes and neutrophils, which comprise the main pathological components of GSDIb. Cells that differentiated into hepatocytes exhibited characteristic albumin secretion and indocyanine green uptake. Moreover, iPSC-derived cells generated from patients with GSDIb metabolic abnormalities recapitulated key pathological features of the diseases affecting the patients from whom they were derived, such as glycogen, lactate, pyruvate and lipid accumulation. Cells that were differentiated into neutrophils also showed the GSDIb pathology. In addition to the expression of neutrophil markers, we showed increased superoxide anion production, increased annexin V binding and activation of caspase-3 and caspase-9, consistent with the GSDIb patient's neutrophils. These results indicate valuable tools for the analysis of this pathology and the development of future treatments.
Assuntos
Doença de Depósito de Glicogênio Tipo I/patologia , Células-Tronco Pluripotentes Induzidas/patologia , Diferenciação Celular , Células Cultivadas , Pré-Escolar , Doença de Depósito de Glicogênio Tipo I/metabolismo , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Neutrófilos/metabolismo , Neutrófilos/patologia , Estresse OxidativoRESUMO
Fireflies produce light through luciferase-catalyzed reactions involving luciferin, oxygen, and adenosine triphosphate, distinct from other luminescent organisms. This unique feature has revolutionized molecular biology and physiology, serving as a valuable tool for cellular research. Luciferase-based bioluminescent imaging enabled the creation of transgenic animals, such as Firefly Rats. Firefly Rats, created in 2006, ubiquitously express luciferase and have become a critical asset in scientific investigations. These rats have significantly contributed to transplantation and tissue engineering studies. Their low immunogenicity reduces graft rejection risk, making them ideal for long-term tracking of organ/tissue/cellular engraftments. Importantly, in the islet transplantation setting, the ubiquitous luciferase expression in these rats does not alter islet morphology or function, ensuring accurate assessments of engrafted islets. Firefly Rats have illuminated the path of transplantation research worldwide for over a decade and continue accelerating scientific advancements in many fields.
Assuntos
Vaga-Lumes , Transplante das Ilhotas Pancreáticas , Animais , Ratos , Vaga-Lumes/metabolismo , Luciferases , Animais Geneticamente Modificados , Diagnóstico por Imagem , Luciferases de Vaga-Lume/genética , Luciferases de Vaga-Lume/metabolismo , Medições LuminescentesRESUMO
Human pluripotent stem cells have been employed in generating organoids, yet their immaturity compared to fetal organs and the limited induction of all constituent cell types remain challenges. Porcine fetal progenitor cells have emerged as promising candidates for co-culturing with human progenitor cells in regeneration and xenotransplantation research. This study focused on identifying proper preservation methods for porcine fetal kidneys, hearts, and livers, aiming to optimize their potential as cell sources. Extracted from fetal microminiature pigs, these organs were dissociated before and after cryopreservation-thawing, with subsequent cell quality evaluations. Kidney cells, dissociated and aggregated after vitrification in a whole-organ form, were successfully differentiated into glomeruli and tubules in vivo. In contrast, freezing hearts and livers before dissociation yielded suboptimal results. Heart cells, frozen after dissociation, exhibited pulsating heart muscle cells similar to non-frozen hearts. As for liver cells, we developed a direct tissue perfusion technique and successfully obtained highly viable liver parenchymal cells. Freezing dissociated liver cells, although inferior to their non-frozen counterparts, maintained the ability for colony formation. The findings of this study provide valuable insights into suitable preservation methods for porcine fetal cells from kidneys, hearts, and livers, contributing to the advancement of regeneration and xenotransplantation research.
Assuntos
Células-Tronco Pluripotentes , Medicina Regenerativa , Animais , Humanos , Suínos , Criopreservação/métodos , Congelamento , VitrificaçãoRESUMO
Hepatocyte-like cells (HLCs) generated from human pluripotent stem cells (PSCs) exhibit hepatocytic properties in vitro; however, their engraftment and functionality in vivo remain unsatisfactory. Despite optimization of differentiation protocols, HLCs did not engraft in a mouse model of liver injury. In contrast, organ-derived hepatocytes reproducibly formed colonies in the liver injury mouse model. As an extension of the phenomenon observed in hematopoietic stem cells giving rise to colonies within the spleen, commonly referred to as "colony-forming units in spleen (CFU-s)", we hypothesize that "colony-forming units in liver (CFU-L)" serves as a reliable indicator of stemness, engraftment, and functionality of hepatocytes. The uniform expression of the randomly inactivated gene in a single colony, as reported by Sugahara et al. 2022, suggests that the colonies generated by isolated hepatocytes likely originate from a single cell. We, therefore, propose that CFU-L can be used to quantify the number of "hepatocytes that engraft and proliferate in vivo" as a quantitative assay for stem cells that utilize colony-forming ability, similar to that observed in hematopoietic stem cells.
Assuntos
Células-Tronco Hematopoéticas , Células-Tronco Pluripotentes , Animais , Camundongos , Humanos , Fígado , Bioensaio , Diferenciação Celular , Modelos Animais de DoençasRESUMO
Stem cell therapy is a current world-wide topic in medical science. Various therapies have been approved based on their effectiveness and put into practical use. In Japan, research and development-related stem cell therapy, generally referred to as regenerative medicine, has been led by the government. The national scheme started in 2002, and support for the transition to clinical trials has been accelerating since 2011. Of the initial 18 projects that were accepted in the budget for preclinical research, 15 projects have begun clinical trials so far. These include the transplantation of retinal, cardiac, and dopamine-producing cells differentiated from human induced pluripotent stem (iPS) cells and hepatocyte-like cells differentiated from human embryonic stem (ES) cells. The distinctive feature of the stem cell research in Japan is the use of iPS cells. A national framework was also been set-up to attain the final goal: health insurance coverage. Now, insurance covers cell transplantation therapies for the repair and recovery of damaged skin, articular cartilage, and stroke as well as therapies introduced from abroad, such as allogeneic mesenchymal stem cells for graft-versus-host disease and chimeric antigen receptor-T (CAR-T) cell therapy. To prepare this review, original information was sought from Japanese authentic websites, which are reliable but a little hard to access due to the fact of multiple less-organized databases and the language barrier. Then, each fact was corroborated by citing its English version or publication in international journals as much as possible. This review provides a summary of progress over the past decade under the national program and a state-of-the-art factual view of research activities, government policy, and regulation in Japan for the realization of stem cell therapy.
RESUMO
To develop novel medical technologies, pig disease models are invaluable especially in the final stages of translational research. Recently, we established a genetically engineered ornithine transcarbamylase-deficient (OTCD) pig strain. Here, we report its characterization and treatment responsiveness. OTCD pigs were obtained by mating an OTCD carrier female (OTC-Xc.186_190delXWT) with a wild-type male. Due to the X-linked recessive mode of inheritance, the disease phenotype emerged only in males. Medication with nitrogen-scavenging agents was based on a clinical protocol. OTCD pigs were born smaller than their wild-type and carrier littermates, showing anemia and faltering. Biochemically, high levels of urinary orotic acid and loss of OTC activity were observed. The natural life course of OTCD pigs was characterized by a decrease in arterial percentage saturation of oxygen and body temperature, as well as an increase in blood ammonia levels; the pigs died in 24.0 ± 5.0 h (mean ± SD, n = 6). The established standard medication composed with nitrogen-scavenging agents and transfusion nearly doubled the survival time to 42.4 ± 13.7 h (n = 6). Our OTCD pig model appropriately mimicked the human pathology. Along with established protocols in handling and medication, this is a first step in developing a large animal disease model that is useful for translational research into novel medical technologies, such as cell transplantation and gene therapy, as well as in relation to urea cycle disorder.
RESUMO
The efficiency of in vitro platelet production is considerably low compared with physiological activity due to the lack of pivotal factors that are essential in vivo. We developed an ex vivo platelet production system, introducing human megakaryocytes into an isolated porcine thighbone and culturing in closed circuit. The efficiency of the ex vivo platelet production system was compared to those in vivo and in vitro. CD61+ platelet-like cells were counted by immunostaining and flow cytometry. Results showed that 4.41 ± 0.27 × 103 CD61+ platelet-like cells were produced by 1 × 103 megakaryocytes in the ex vivo system, while 3.80 ± 0.87 × 103 and 0.12 ± 0.02 × 103 were produced in the in vivo and in vitro systems, respectively. Notably, ex vivo and in vitro production systems generated cells that responded well to thrombin stimulation and expressed functional molecules, such as CD62P. Overall, our ex vivo production system was comparable to in vivo production system and produced platelet-like cells that were functionally superior to those produced in vitro. In future, the present ex vivo production system implementing xenogeneic bone marrow would offer a promising alternative for industrial-scale production of platelet-like cells.
Assuntos
Plaquetas/metabolismo , Células da Medula Óssea/citologia , Animais , Antígenos CD34/metabolismo , Plaquetas/citologia , Diferenciação Celular/efeitos dos fármacos , Humanos , Integrina beta3/metabolismo , Megacariócitos/citologia , Megacariócitos/metabolismo , Suínos , Trombina/farmacologiaRESUMO
INTRODUCTION: Decellularized tissue exhibits cell matrix-like properties, along with reduced antigenicity. We explored the potential of decellularized allogeneic trachea to restore the upper respiratory tract, focusing on pediatric application. This study specifically aimed at long-term observation of tissue regeneration using a micro-miniature pig model. METHODS: Artificial defects (15 × 15 mm) in the subglottis and trachea of micro-miniature pigs were repaired by transplantation of either allogeneic decellularized or fresh (control) tracheal patches. Pigs were evaluated in situ, by bronchoscopy, every three months, and sacrificed for histological examination at six and twelve months after transplantation. RESULTS: No airway symptom was observed in any pig during the observation period. Bronchoscopy revealed the tracheal lumen to be restored by fresh grafts, showing an irregular surface with remarkable longitudinal compression; these changes were mild after restoration with decellularized grafts. Histologically, while fresh graft patches were denatured and replaced by calcified tissue, decellularized patches remained unchanged throughout the observation period. There were regeneration foci of cartilage adjacent to the grafts, and some foci joined the decellularized graft uniformly, suggesting the induction of tracheal reconstitution. CONCLUSION: Allogeneic decellularized tracheal tissue could serve as a promising biomaterial for tracheal restoration, especially for pediatric patients at the growing stage.
RESUMO
Transplantation of liver organoids has been investigated as a treatment alternative to liver transplantation for chronic liver disease. Transportal approach can be considered as a method of delivering organoids to the liver. It is important to set the allowable organoid amount and verify translocation by intraportal transplantation. We first examined the transplantation tolerance and translocation of porcine fetal liver-derived allogeneic organoids using piglets. Fetal liver-derived organoids generated from the Kusabira Orange-transduced pig were transplanted to the 10-day-old piglet liver through the left branch of the portal vein. All recipients survived without any observable adverse events. In contrast, both local and main portal pressures increased transiently during transplantation. In necropsy samples, Kusabira Orange-positive donor cells were detected primarily in the target lobe of the liver and partly in other areas, including the lungs and brain. As we confirmed the transplantation allowance by porcine fetal liver-derived organoids, we performed intraportal transplantation of human-induced pluripotent stem cell (iPSC)-derived liver organoid, which we plan to use in clinical trials, and portal pressure and translocation were investigated. Human iPSC-derived liver organoids were transplanted into the same 10-day-old piglet. Portal hypertension and translocation of human iPSC-derived liver organoids to the lungs were observed in one of two transplanted animals. Translocation occurred in the piglet in which patent ductus venosus (PDV) was observed. Therefore, a 28-day-old piglet capable of surgically ligating PDV was used, and after the PDV was ligated, human iPSC-derived liver organoids with the amount of which is scheduled in clinical trials were transplanted. This procedure inhibited the translocation of human iPSC-derived liver organoids to extrahepatic sites without no portal hypertension. In conclusion, human iPSC-derived liver organoids can be safely transplanted through the portal vein. Ligation of the ductus venosus prior to transplantation was effective in inhibiting extrahepatic translocation in newborns and infants.
Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Organoides/citologia , Animais , Veia Porta/metabolismo , Transplante de Células-Tronco/métodos , SuínosRESUMO
Hepatocytes are an important tool for in vitro toxicology testing. In addition to primary cultures, a limited number of immortalized cell lines have been developed. We here describe a new cell line, designated as HepaMN, which has been established from a liver associated with biliary atresia. Hepatocytes were isolated from a liver of 4-year-old girl with biliary atresia and immortalized by inoculation with CSII-CMV-TERT, CSII-CMV-Tet-Off, CSII-TRE-Tight-cyclin D1 and CSII-TRE-Tight-CDK4R24C (mutant CDK4: an INK4a-resistant form of CDK4) lentiviruses at the multiplicity of infection of 3 to 10. HepaMN cells exhibited morphological homogeneity, displaying hepatocyte-like phenotypes. Phenotypic studies in vivo and in vitro revealed that HepaMN cells showed polarized and functional hepatocyte features along with a canalicular cell phenotype under defined conditions, and constitutively expressed albumin and carbamoyl phosphate synthetase I in addition to epithelial markers. Since HepaMN cells are immortal and subcloned, kinetics and expression profiles were independent of population doublings. HepaMN cells showed increased CYP3A4 expression after exposure to rifampicin, implying that their close resemblance to normal human hepatocytes makes them suitable for research applications including drug metabolism studies.