bluuebunny/crossref_metadata_2025 huggingface dataset:
Efficacy of a Larger Version of the Hybrid Artificial Liver Support System Using a Polyurethane Foam/Spheroid Packed-Bed Module in a Warm Ischemic Liver Failure Pig Model for Preclinical Experiments
Authors: Yamashita Yo-Ichi , Shimada Mitsuo , Tsujita Eiji , Shirabe Ken , Ijima Hiroyuki , Nakazawa Kohji , Sakiyama Ryoichi , Fukuda Junji , Funatsu Kazumori , Sugimachi Keizo
Published: September 2014

We have reported the usefulness of a polyurethane foam packed-bed culture system of hepatocyte spheroids as a hybrid artificial liver support system (PUF-HALSS). The aim of this study was to evaluate in detail the efficacy in serum parameters regarding the liver function of a larger version of the PUF-HALSS containing 2 × 1010 porcine hepatocytes for clinical use in warm ischemic liver failure pigs. Warm ischemic liver failure pigs weighing 25 kg were divided into two groups: (1) a control group (n = 3), in which each pig was attached to a PUF-HALSS without hepatocytes, and (2) a HALSS group (n = 3), in which each pig was attached to a PUF-HALSS. In the HALSS group, the increase of blood ammonia was completely suppressed and blood lactate levels were significantly suppressed. The Fisher's ratio was better maintained, and the increase of total bile acid, glycochenodeoxycholic acid, and taurochenodeoxycholic acid was significantly suppressed in the HALSS group. Serum creatinine levels were significantly lower, and blood glucose levels were significantly higher in the HALSS group. Serum levels of tumor necrosis factor- a were not elevated in either group. In conclusion, the larger version of the PUF-HALSS demonstrated many advantages as a liver support system in warm ischemic liver failure pigs.
Digital Imaging as a Possible Approach in Evaluation of Islet Yield
Authors: Girman Peter , Kříž Jan , Friedmanský Jozef , Saudek FrantišEk
Published: September 2014

Digital image analysis (DIA) is a new method in assessment of islet amount, which is expected to provide reliable and consistent results. We compared this method with conventional counting of small numbers of rat islets. Islets were isolated from 8 pancreases and counted in 24 samples in duplicate, first routinely by sizing according to estimated diameters under a calibrated reticule and then by processing of islets pictures taken by camera. As presumed, no significant difference was found in absolute numbers of islets per sample between DIA and conventional assessment. Volumes of islets per sample measured by DIA were on average more than 10% higher than amounts evaluated conventionally, which was statistically significant. DIA has been shown to be an important method to remove operator bias and provide consistent results. Evaluation of only two dimensions of three-dimensional objects still represents a certain limitation of this technique. With lowering of computer prices the system could become easily available for islet laboratories.
Fetal Lateral Ganglionic Eminence Attracts One of Two Morphologically Different Types of Tyrosine Hydroxylase-Positive Nerve Fibers Formed by Cultured Ventral Mesencephalon
Authors: Johansson Saga , Strömberg Ingrid
Published: September 2014

The purpose of this study was to investigate the influence of fetal lateral ganglionic eminence (LGE) on nerve fiber outgrowth formed by fetal ventral mesencephalon (VM). Organotypic tissue cultures of fetal VM and LGE plated as single or cocultures were employed. Survival time was 3–21 days in vitro. Nerve fiber outgrowth and migration of astrocytes were analyzed using immunohistochemistry for tyrosine hydroxylase (TH) and S100. In addition, cultures were labeled with the TUNEL technique and with antibodies directed against neurofilament (NF) in order to study apoptosis and retraction of nerve fibers, respectively. The results revealed two morphologically different types of TH-positive outgrowth growing into the substrate. The initially formed TH-positive outgrowth radiated continuously without changing direction, while a second wave of TH-positive outgrowth became obvious when the initial growth already had reached a distance of approximately 1000 μm. The second wave of TH-positive outgrowth radiated from the tissue, but at a certain distance changed direction and formed a network surrounding the culture. The initially formed TH-positive growth was not associated with the presence of S100-positive astrocytes and avoided to grow into the LGE. At longer time points the first wave of TH-positive nerve fibers appeared dotted, with disrupted NF-immunoreactive fibers and in most cultures these long distance growing fibers had disappeared at 21 days in vitro. The second wave of TH-positive nerve fibers was growing onto a layer of glia and never reached the distance of the first wave. LGE became innervated by TH-positive fibers at the time point for when the second wave of TH-positive growth had been initiated, and the innervation appeared in TH-dense patches that also showed a high density of S100-positive astrocytes. Significantly increased TUNEL activity within LGE portion of cocultures was observed when TH-positive fibers entered the LGE and formed patches. In conclusion, two morphologically different types of TH-positive outgrowth were found and the initially formed fibers neither targeted the LGE nor were they guided by glial cells, but their potential to grow for long distances was high.
De Novo Liver Tissue Formation in Rats Using a Novel Collagen-Polypropylene Scaffold
Authors: Takimoto Yukinobu , Dixit Vivek , Arthur Marika , Gitnick Gary
Published: September 2014

In experimental and clinical settings hepatocyte transplantation has provided limited benefit to patients with chronic liver disease because the transplanted hepatocytes were short-lived and were merely maintained for a brief period within the body. Except for whole-liver transplantation, creation of de novo liver tissue is necessary to treat this condition on a long-term basis. The aim of this study was to facilitate the formation of new tissue by actual self-regeneration, rather than by compensatory hypertrophy, or scar formation, with our collagen-polypropylene composite scaffold. Collagen-polypropylene composite scaffolds, not containing hepatocytes, were implanted into the median liver lobe and the dynamics of new liver tissue formation was analyzed immunohistochemically over a 6-month period. Control scaffolds consisted of polypropylene scaffolds without collagen matrix. The control scaffold implants remained hollow throughout the study period and became encapsulated with a hard connective tissue capsule 1 week after implantation. In contrast, the collagen-polypropylene composite scaffold was filled with regenerating tissue structures 3 weeks after implantation. At this time, the predominant cell type within the scaffold was sesmin-positive stellate cells. A week earlier, oval cells were identified using monoclonal antibody staining (OV-6). Subsequently, these cells differentiated into α-fetoprotein-positive immature hepatocytes. After 6 months, mature liver tissue, juxtaposed with bile ducts and blood vessels, was seen within the polypropylene scaffolds. We report the first evidence of de novo formation of liver tissue within a polypropylene scaffold, following implantation in the liver. This scaffold may play a role in treating chronic liver diseases requiring organ replacement therapy.
Photoconstructs of Nerve Guidance Prosthesis Using Photoreactive Gelatin as a Scaffold
Authors: Gámez E. , Ikezaki K. , Fukui M. , Matsuda T.
Published: September 2014

We devised a novel nerve prosthesis composed of an elastomeric gelatinous tube and multifilament gelatinous fibers, both of which were prepared from styrene-derivatized gelatin, which allows in situ formation of a bioactive substance-incorporated gel. An in vitro study showed that the axonal regeneration potential of a photocured gelatin layer impregnated with laminin, fibronectin, and NGF was almost comparable with that of coated Matrigel. A nerve conduit and fibers prepared from photoreactive gelatin was subjected to visible-light irradiation with rotation in the presence of camphorquinone as a photoinitiator using a custom-designed apparatus. A sample of transparent gelatinous conduit with an inner diameter of 1.2 mm and a wall thickness of 0.6 mm and gelatin fibers ranging from 10 to 100 μm in diameter were produced. The photocured elastomeric gelatinous tube was flexible and had structural integrity that allowed mechanical handling without breaking. A novel nerve guidance prosthesis composed of tubes packed with fibers was assembled. This photofabrication technology may enable the design of a tailor-made shape and rapid morphogenesis and functional recovery of damaged nerve tissue.
Transplanted Endothelial Progenitor Cells Augment the Survival Areas of Rat Dorsal Flaps
Authors: Kubota Yoshiaki , Kishi Kazuo , Satoh Hiroko , Tanaka Takara , Nakajima Hideo , Nakajima Tatsuo
Published: September 2014

Endothelial progenitor cells (EPCs) have been identified in peripheral blood, and have been reported to be incorporated into ischemic regions such as the ischemic hindlimb. In this study, we examined whether or not transplantation of EPCs is useful for salvaging surgical flaps in vivo. At the same time, we quantitatively compared the neovascularization ability of transplanted EPCs and that of mature endothelial cells (ECs). ECs obtained from the aorta of rats by explantation and passaged several times were used in the present study. EPCs were obtained from the blood of rat hearts. The blood samples were separated by density gradient centrifugation. Light-density mononuclear cells (MNCs) were collected and cultured on plastic plates coated with rat plasma vitronectin. Cells attached at day 7 of culture were deemed to be EPCs. Then PBS (control), ECs, or EPCs (3.0 × 105 suspended in 1.0 ml PBS) were injected at the middle of a flap. Seven days after surgery, the survival lengths of the flaps were evaluated. EPC-transplanted groups revealed statistically significant augmentation of survival length compared with the other two groups (p < 0.003). EPC-transplanted groups had significantly more angiographically detectable blood vessels (p < 0.003) and significantly higher capillary density (p < 0.03) than the other two groups. Confocal microscopy revealed that EPCs were incorporated into enhanced neovascularization. These results suggest that transplantation of EPCs may be useful for salvaging surgical flaps, and EPCs are superior to ECs in neovascularization ability.
Human Cord Blood-Derived Mesenchymal Stem Cells Home and Survive in the Marrow of Immunodeficient Mice after Systemic Infusion
Authors: Erices Alejandro A. , Allers Carolina I. , Conget Paulette A. , Rojas Cecilia V. , Minguell José J.
Published: September 2014

Bone marrow is the residence site of mesenchymal stem cells (MSC), which upon commitment and maturation develop into several mesenchymal phenotypes. Recently, we have described the presence of MSC in human cord blood (cbMSC) and informed that their properties are the same as those for MSC obtained from adult bone marrow. In this study we have investigated the capability of transplanted cbMSC to home and survive in the marrow of unconditioned nude mice. cbMSC utilized for transplantation studies were characterized by morphology, differentiation potential, and immunophenotype. After transplantation by systemic infusion, human DNA (as detected by PCR amplification of human-specific β-globin gene) was detected in the marrow of recipients as well as in ex vivo-expanded stromal cells prepared from the marrow of transplanted animals. These results demonstrate homing and survival of cbMSC into the recipient marrow and also suggest a mesenchymal-orientated fate of engrafted cells, because human DNA was also detected in cells of other recipient tissues, like cardiac muscle, teeth, and spleen.
Bridging a Patient with Acute Liver Failure to Liver Transplantation by the AMC-Bioartificial Liver
Authors: Van De Kerkhove Maarten-Paul , Di Florio Ernesto , Scuderi Vincenzo , Mancini Antonio , Belli Antonello , Bracco Adele , Scala Daniela , Scala Simona , Zeuli Laura , Di Nicuolo Giuseppe , Amoroso Pietro , Calise Fulvio , Chamuleau Robert A. F. M.
Published: September 2014

Recently a phase I clinical trial has been started in Italy to bridge patients with acute liver failure (ALF) to orthotopic liver transplantation (OLT) by the AMC-bioartificial liver (AMC-BAL). The AMC-BAL is charged with 10 × 109 viable primary porcine hepatocytes isolated from a specified pathogen-free (SPF) pig. Here we report a patient with ALF due to acute HBV infection. This patient was treated for 35 h by two AMC-BAL treatments and was bridged to OLT. There was improvement of biochemical and clinical parameters during the treatment. No severe adverse events were observed during treatment and follow-up of 15 months after hospital discharge. Possible porcine endogenous retrovirus (PERV) activity could not be detected in the patient's blood or blood cells up to 12 months after treatment.
Hypothermic Storage and Cryopreservation of Hepatocytes: The Protective Effect of Alginate Gel against Cell Damages
Authors: Mahler Stephan , Desille Mireille , Frémond Benjamin , Chesné Christophe , Guillouzo André , Campion Jean-Pierre , Clément Bruno
Published: September 2014

Hepatocyte-based therapy has been proposed as an alternative to organ transplantation in the treatment of liver disorders. In the clinical context, a major issue is the constant supply of quality assurance-controlled hepatocytes, thereby requiring their cold storage in good conditions. We have analyzed the protective effects of alginate entrapment of rat hepatocytes after either 24 or 48 h of hypothermic storage or cryopreservation on the cell viability, cell yield, both mitochondrial and other cytoplasmic functional activities, and apoptosis. Decrease in viability, as evaluated by the MTT inclusion test, was 4% and 13% (24 h at 4°C), 15% and 33% (48 h at 4°C), and 9% and 19% (liquid nitrogen) for entrapped and free suspended hepatocytes, respectively. Viable cell yields were 86 ± 8% and 51 ± 6% for cryopreserved entrapped and free suspended hepatocytes, respectively. The mitochondrial (MTS assay), 7-ethoxyresorufin O-deethylase (EROD), and glutathione-S-transferase (GST) activities were better preserved in entrapped than in free suspended hepatocytes. Both hypothermic storage and cryopreservation were found to induce early caspase-3-like activities, being always much lower in entrapped hepatocytes, particularly after cryopreservation (98.4 ± 42.4 vs. 6.4 ± 4.0 fluorescence arbitrary units/hours/μg protein). Thus, cold-induced apoptosis in hepatocytes can be significantly reduced following their entrapment within alginate gel beads and this is associated with an improvement of both their viability and function.
Factors Influencing Insulin Secretion from Encapsulated Islets
Authors: De Haan Bart J. , Faas Marijke M. , De Vos Paul
Published: September 2014

Adequate regulation of glucose levels by a microencapsulated pancreatic islet graft requires a minute-to-minute regulation of blood glucose. To design such a transplant, it is mandatory to have sufficient insight in factors influencing the kinetics of insulin secretion by encapsulated islets. The present study investigates factors influencing the glucose-induced insulin response of encapsulated islets in vitro. We applied static incubations and did the following observations. (i) Small islets (90–120 μm) showed a similar instead of a lower glucose-induced insulin response, suggesting that inclusion of only small islets, which are associated with lower protrusion and failing rates, has no consequences for the functional performance of the graft. (ii) A capsule diameter of 800 μm showed identical rather than lower glucose-induced insulin responses as smaller, 500-μm capsules. (iii) Capsule membranes constructed with a conventional permeability interfered with diffusion of insulin, as illustrated by a lower response of islets in capsules with a 10-min poly-L-lysine (PLL) membrane than islets in capsules with a 5-min PLL membrane. (iv) Irrespective of the tested porosity, the capsules provided sufficient immunoprotection because the 10-min PLL membranes did block diffusion of the cytokines IL-1β (17 kDa) and TNF-α (70 kDa) while the 5-min PLL membranes interfered with the diffusion of the vast majority of the cytokines. We conclude that capsules containing small islets (90–120 μm) and a membrane with a lower permeability than routinely applied is preferred in order to obtain a graft with adequate glucose-induced insulin responses.
Polymers for Induction of Revascularization in the Rat Fascial Flap: Application of Vascular Endothelial Growth Factor and Pancreatic Islet Cells
Authors: Linn Thomas , Erb Doris , Schneider Darius , Kidszun Andre , Elçin A. Eser , Bretzel Reinhard G. , Elçin Y. Murat
Published: September 2014

One of the major obstacles in transplanting avascular tissue or metabolically active cells for ischemic diseases is the loss of transplanted cells due to lack of oxygen and nutrients in the early posttransplantation period. Biodegradable polymeric tissue engineering scaffolds and hydrogels have a potential to incorporate cells or cellular organoids such as islets of Langerhans and growth factors. In this study, we tested the efficiency of two types of polymeric materials to carry recombinant human vascular endothelial growth factor (rhVEGF) or pancreatic tumor cell lines, namely Ins-1 and AR42J, for the induction of new vessels. Chitosan hydrogel fibers with micropores were prepared and molded into a cylinder construct (5 mm φ 8 mm height). Macroporous PLGA scaffolds with a pore size of 250–400 μm were prepared and cut into cylinders (6 mm φ 3 mm height). Both chitosan and PLGA constructs were loaded with rhVEGF (3 μg) or seeded with the cell lines (5 × 105 cells and 3 × 105 cells/construct, respectively, for AR42J and INS-1 cells), and transplanted into the fascial flaps of Wistar rats. At distinct time points up to 4 weeks postimplantation, polymers were explanted, fixed, and vessel density was counted on sections stained with anti-Factor-VIII antibody. Additionally, the kinetics of rhVEGF release from PLGA microspheres (φ of 50–80 μm) was determined using VEGF Elisa. Endogenous VEGF release from pancreatic rat cell lines was also determined. Light microscopy study was performed on H&E-stained paraffin sections of the islet-polymer samples. The vascular density of rhVEGF-loaded chitosan constructs was increased fourfold 2 weeks after subcutaneous transplantation compared with rhVEGF-unloaded controls (465 ± 144 vs. 104 ± 80 vessels per mm2, p < 0.05). Protein leakage occurred, but was not observed after 2 weeks. Higher insulin content was detected in rat islet grafts transplanted following VEGF application. More than 50% of total rhVEGF was released on the first day of in vitro culture of PLGA microspheres. rhVEGF secretion had another, but smaller, peak on the third day followed by a constant release. By comparison, endogeneous VEGF secretion of pancreatic tumor cells was measured within a 3-day culture period. Biodegradable polymer scaffolds and hydrogels may have potential use as solid supports to induce angiogenesis for pancreatic cell transplantation.
The Effects of Poly(Ethyleneimine) (PEI) Molecular Weight on Reinforcement of Alginate Hydrogels
Authors: Kong Hyun Joon , Mooney David J.
Published: September 2014

Alginate hydrogels are widely used for cell encapsulation and transplantation, and they are frequently surface reinforced with secondary polymers to enhance their mechanical rigidity and stability. We hypothesized that the molecular weight (MW) of the polymer utilized to reinforce alginate would be an important factor in their stability, particularly when the gel network was homogeneously reinforced with the polymer. This hypothesis was investigated with alginate hydrogels cross-linked with Ca2+, and reinforced throughout the bulk of the gel with poly(ethyleneimine) (PEI) having different MWs. Interactions between the two polymers became significant following gelation, leading to higher elastic moduli (E) than gels with no PEI. The decrease in E of gels incubated in isotonic salt solutions over time, utilized as an indication of gel break down, was ameliorated with an increase in the MW of the PEI. In addition, the dependencies of the moduli and visco-elasticity on the temperature also became smaller with the use of high MW PEI. This is likely due to the limited mobility of high MW PEI, leading to a higher energy for dissociation. The stable interactions between the alginate and PEI prevented alterations of the pore structure in the gels, and slowed the deterioration of gel properties even under continuous agitation in a bioreactor. The results of this study will likely be useful in designing alginate encapsulation strategies for various applications.
Staging and Preparation of Human Fetal Striatal Tissue for Neural Transplantation in Huntington's Disease
Authors: Rosser A. E. , Barker R. A. , Armstrong R. J. E. , Elneil S. , Jain M. , Hurelbrink C. B. , Prentice A. , Carne C. , Thornton S. , Hutchinson H. , Dunnett S. B.
Published: September 2014

Transplantation of human fetal central nervous system tissue has been shown to be of benefit in Parkinson's disease, and is currently being explored as a therapeutic option in Huntington's disease. The success of a neural transplant is dependent on a number of factors, including the requirement that donor cells are harvested within a given developmental window and that the cell preparation protocols take account of the biological parameters identified in animal models. Although many of the criteria necessary for a successful neural transplant have been defined in animal models, ultimately they must be validated in human studies, and some issues can only ever be addressed in human studies. Furthermore, because neural transplantation of human fetal tissue is limited to small numbers of patients in any one surgical center, largely due to practical constraints, it is crucial that tissue preparation protocols are clearly defined and reproducible, so that (i) multicenter trials are possible and are based on consistent tissue preparation parameters, and (ii) results between centers can be meaningfully analyzed. Here we describe the preparation of human fetal striatum for neural transplantation in Huntington's disease, and report on the validation of a method for estimating the developmental stage of the fetus based on direct morphometric measurements of the embryonic tissue.
In Vitro Expansion of Human Hepatocytes is Restricted by Telomere-Dependent Replicative Aging
Authors: Wege Henning , Chui Michael S. , Le Hai T. , Strom Stephen C. , Zern Mark A.
Published: September 2014

Currently, different techniques to expand human hepatocytes in vitro are being investigated to generate enough cells for liver-directed cell therapies. However, based on observations in fibroblasts and other cell types, telomere attrition limits the proliferative capacity of normal somatic cells. Therefore, we explored whether telomere-dependent replicative aging restricts the in vitro proliferation of human hepatocytes. Subpopulations of cells isolated from a neonatal liver and characterized as hepatocyte derived by RT-PCR and flow cytometry started to proliferate 5–7 days after plating and were termed proliferating human hepatocytes (PHH). Following retroviral-mediated transduction of the catalytic telomerase subunit, telomerase reverse transcriptase (hTERT), telomerase activity increased from almost undetectable levels to levels as high as in HepG2 and other telomerase-positive cell lines. As expected, untransduced PHH progressively lost telomeric repeats and arrested after 30–35 cell divisions with telomeres of less than 5 kilo bases. In comparison, telomerase-reconstituted PHH maintained elongated telomeres and continued to proliferate as shown by colorimetric assays and cell counts. In this study, telomere stabilization extended the proliferative capacity of in vitro proliferating human neonatal hepatocytes. Therefore, telomere attrition needs to be addressed when developing techniques to expand human hepatocytes.
Neural Precursor Cells as Carriers for a Gene Therapeutical Approach in Tumor Therapy
Authors: Arnhold S. , Hilgers M. , Lenartz D. , Semkova I. , Kochanek S. , Voges J. , Andressen C. , Addicks K.
Published: September 2014

Conventional therapeutical approaches such as surgery, radiotherapy, or chemotherapy have been shown to be rather unsuccessful in the treatment of infiltrative growing tumors such as the malignant glioblastoma multiforme. Thus, new therapeutical strategies have to be developed that are suitable for inducing cell death also in migrating tumor cells. These new therapeutical stategies include cell and/or gene therapeutical approaches. We demonstrate that glial-restricted progenitor cells as well as embryonic stem cell-derived neural stem cells belong to cell populations applicable to such therapeutical concepts. Both cell types can be efficiently transduced using a third-generation high-capacity “gutless” adenoviral vector, and show a tropism for the F98 glioma cells by migrating towards a spheroid of F98 glioma cells with a tendency to form a barrier around the tumor spheroid in an in vitro tumor confrontation model. Moreover, in a migration assay, secretion products of glial-restricted precursor cells have shown a potency to inhibit the migratory activity of glioma cells in vitro. In vivo, F98 glioma cell-derived tumor formation in the right striatum resulted in migration of glial as well as neural precursor cells towards the tumor area when cotransplanted in the corpus callosum of the contralateral hemisphere. After arrival, both cell types surround the tumor mass and even invade the experimentally induced tumor. These data indicate that glial-restricted as well as embryonic stem cell-derived neural precursor cells are good candidates as carriers for an ex vivo gene therapeutical approach in tumor therapy.
Insulin-Induced Normoglycemia Reduces Islet Number Needed to Achieve Normoglycemia after Allogeneic Islet Transplantation in Diabetic Mice
Authors: Ferrer-Garcia Juan C. , Merino-Torres Juan F. , Bermejo Gema Pérez , Herrera-Vela Carmen , Ponce-Marco Jose L. , Piñon-Selles Francisco
Published: September 2014

The Edmonton protocol established that insulin independence could be reached with the transplantation of an appropriate number of islet cells. However, to effect a cure, islets from two or three pancreases are needed. The aim of this study was to examine whether normoglycemia, with insulin treatment before and after transplantation, reduces the islet number needed to achieve normoglycemia in allogeneic islet transplantation. Swiss mice were used as donors and recipients. Diabetes was induced by IP administration of streptozotocin (180 mg/kg BW). Diabetic mice were transplanted with 300 (n = 16), 400 (n = 16), or 500 (n = 16) islets under the left kidney capsule. For every group, half the animals were kept normoglycemic with insulin treatment from day 4 before transplantation to day 10 after transplantation. At the end of the study, all normoglycemic mice were given an IP glucose tolerance test (IPGTT). For statistical analysis, paired or unpaired Student's t-test or ANOVA was used. Only insulin-treated mice achieved normoglycemia by the end of the study (37.5% of animals transplanted with 400 islets and 50% transplanted with 300 or 500 islets). At the end of the study, normoglycemic mice transplanted with 300 allogeneic islets showed better glycosylated hemoglobin (HbA1C) than did normoglycemic mice transplanted with 500 islets (300 islets: 2.7 ± 0.2%; 500 islets: 3.6 ± 0.2%; p < 0.05). After the IPGTT, insulin-treated mice transplanted with 500 islets showed abnormal glucose tolerance; however, insulin-treated mice transplanted with 300 or 400 islets showed normal glucose tolerance. Insulin treatment reduced the islet number needed to achieve normoglycemia in allogeneic islet transplantation. The HbA1C and IPGTT results suggest that transplanting smaller numbers of allogeneic islets improves β-cell function; some studies suggest that this may be due to lower immunogenicity, hypoxia, and inflammation.
Transgenic Sertoli Cells as a Vehicle for Gene Therapy
Authors: Dufour Jannette M. , Gores Paul , Hemendinger Richelle , Emerich Dwaine F. , Halberstadt Craig R.
Published: September 2014

Gene therapy involves the manipulation of genetic material to replace defective or deficient proteins to restore function in disease states. These genes are introduced into cells by mechanical, chemical, and biological approaches. To date, cell-based gene therapy has been hampered by the lack of an abundant, safe, and immunologically acceptable source of tissue. As an alternative, transgenic animals designed to produce therapeutic proteins could overcome some of the issues facing gene therapy but the problem of immune rejection of the tissue remains. This article reports on recently published work indicating the potential to use transgenic Sertoli cells surviving in an allogeneic host by virtue of their ability to create a locally immunoprivileged environment, thereby providing for the continued delivery of a therapeutic protein to the systemic circulation.
Use of D-Stat™ to Prevent Bleeding following Percutaneous Transhepatic Intraportal Islet Transplantation
Authors: Froud Tatiana , Yrizarry Jose M. , Alejandro Rodolfo , Ricordi Camillo
Published: September 2014

An infrequent but nevertheless concerning complication associated with percutaneous transhepatic islet transplantation is bleeding. Historically in 61 procedures at this institution, we experienced four bleeding complications in three patients (6.6%), two requiring blood transfusion (3.3%) and two asymptomatic intraperitoneal bleeds detected sonographically at 24 h postprocedure (3.3%). It is suggested that the source of the majority of these bleeds is the liver parenchymal tract following removal of the infusion catheter combined with a significant dose of heparin administered to prevent portal vein thrombosis. Various techniques have been used to reduce the risk of tract bleeding, including gelfoam, intravascular coils, and cautery. In our experience gelfoam alone has been used to plug the catheter tract (n = 47); however, in the aforementioned three patients, this technique failed, either due to dislodgement of, or bleeding peripheral to, the plug. This article describes the use of D-Stat™, a collagen/thrombin paste that is injected into the peripheral tract. In five consecutive cases performed using D-Stat™, there has been no bleeding or thromboses detected. D-Stat™ combined with a single gelfoam plug offers a quick, easy, efficacious way of sealing the entire catheter tract without leaving any permanent hardware in the liver. This new method may simplify tract closure and reduce bleeding complications in islet transplantation.
Short-Term Storage of the Ischemically Damaged Human Pancreas by the Two-Layer Method Prior to Islet Isolation
Authors: Tsujimura Toshiaki , Kuroda Yoshikazu , Churchill Thomas A. , Avila Jose G. , Kin Tatsuya , Shapiro A. M. James , Lakey Jonathan R. T.
Published: September 2014

A two-layer cold storage method (TLM) allows sufficient oxygen delivery to pancreata during preservation and resuscitates the viability of ischemically damaged pancreata in the canine pancreas transplant model. In this study, we applied a short-term preservation of the TLM to human pancreata after prolonged cold ischemia prior to islet isolation, and investigated the mechanisms of resuscitation of the ischemically damaged human pancreas by the TLM. Human pancreata were procured from cadaveric donors and preserved by the TLM for 3.2 ± 0.5 h after 11.1 ± 0.9 h of cold storage in UW (TLM group), or by cold UW alone for 11.0 ± 0.3 h (UW group). Islet isolations of all pancreata were performed using the Edmonton protocol. Islet recovery and in vitro functional viability of isolated islets were significantly increased in the TLM group compared with the UW group. According to the criteria of the Edmonton protocol, 10/14 cases (71%) in the TLM group were transplanted to patients with type I diabetes mellitus compared with only 5/21 cases (24%) in the UW group. In the metabolic assessment of human pancreata, levels of energetic parameters (ATP, total adenylates, and energy charge) were significantly increased, and malondialdehyde (MDA) levels were significantly decreased after the TLM preservation. There was no observable change in the incidence or degree of mitochondrial injury after the TLM preservation. Additional short-term storage by the TLM resuscitates the ischemically damaged human pancreas by regenerating the energetic status and prevents further damage by oxidative stress, ultimately leading to improvements of islet recovery and in vitro function. Use of the TLM following prolonged storage in UW provides an excellent adjunctive protocol for treating human pancreata for the rigors of the islet isolation process.
Clonal Population of Adult Stem Cells: Life Span and Differentiation Potential
Authors: Seruya Mitchel , Shah Anup , Pedrotty Dawn , Du Laney Tracey , Melgiri Ryan , Mckee J. Andrew , Young Henry E. , Niklason Laura E.
Published: September 2014

Adult stem cells derived from bone marrow, connective tissue, and solid organs can exhibit a range of differentiation potentials. Some controversy exists regarding the classification of mesenchymal stem cells as bona fide stem cells, which is in part derived from the limited ability to propagate true clonal populations of precursor cells. We isolated putative mesenchymal stem cells from the connective tissue of an adult rat (rMSC), and generated clonal populations via three rounds of dilutional cloning. The replicative potential of the clonal rMSC line far exceeded Hayflick's limit of 50–70 population doublings. The high capacity for self-renewal in vitro correlated with telomerase activity, as demonstrated by telomerase repeat amplification protocol (TRAP) assay. Exposure to nonspecific differentiation culture medium revealed multilineage differentiation potential of rMSC clones. Immunostaining confirmed the appearance of mesodermal phenotypes, including adipocytes possessing lipid-rich vacuoles, chondrocytes depositing pericellular type II collagen, and skeletal myoblasts expressing MyoD1. Importantly, the spectrum of differentiation capability was sustained through repeated passaging. Furthermore, serum-free conditions that led to high-efficiency smooth muscle differentiation were identified. rMSCs plated on collagen IV-coated surfaces and exposed to transforming growth factor-β1 (TGF-β1) differentiated into a homogeneous population expressing α-actin and calponin. Hence, clonogenic analysis confirmed the presence of a putative MSC population derived from the connective tissue of rat skeletal muscle. The ability to differentiate into a smooth muscle cell (SMC) phenotype, combined with a high proliferative capacity, make such a connective tissue-derived MSC population ideal for applications in vascular tissue construction.
Intraspinal Transplantation of CD34+ Human Umbilical Cord Blood Cells after Spinal Cord Hemisection Injury Improves Functional Recovery in Adult Rats
Authors: Zhao Zong Mao , Li Hong Jun , Liu Hai Ying , Lu Shi Hong , Yang Ren Chi , Zhang Qing Jun , Han Zhong Chao
Published: September 2014

The present study was designed to compare the functional outcome of the intraspinal transplantation of CD34+ human umbilical cord blood (CB) cells with that of human bone marrow stromal (BMS) cells in adult rats with spinal cord injury. Sixty adult Wistar rats were subjected to left spinal cord hemisection, and then divided into three groups randomly. The control group received an injection of PBS without cells, while the two other groups of rats received a transplantation of 5 × 105 CD34+ CB or BMS cells, respectively. Functional outcome was measured using the modified Tarlov score at days 1, 7, 14, 21, and 28 after transplantation. A statistically significant improvement in functional outcome and survival rate in the experimental groups of rats was observed compared with the control group. Rats that received CD34+ CB cells achieved a better improvement in functional score than those that received BMS cells at days 7 and 14 after transplantation. Histological evaluation revealed that bromodeoxyuridine (BrdU)-labeled CD34+ CB and BMS cells survived and migrated into the injured area. Some of these cells expressed glial fibriliary acidic protein (GFAP) or neuronal nuclear antigen (NeuN). Our data demonstrate for the first time that intraspinal transplantation of human CD34+ CB cells provides benefit in function recovery after spinal cord hemisection in rats and suggest that CD34+ CB cells may be an excellent choice of cells as routine starting material of allogenic and autologous transplantations for the treatment of spinal cord injury.
Transplanted hNT Cells (“LBS Neurons”) in a Rat Model of Huntington's Disease: Good Survival, Incomplete Differentiation, and Limited Functional Recovery
Authors: Fricker-Gates Rosemary A. , Muir Janice A. , Dunnett Stephen B.
Published: September 2014

A variety of immortalized cell lines have been proposed to exhibit sufficient phenotypic plasticity to allow them to replace primary embryonic neurons for restorative cell transplantation. In the present experiments we evaluate the functional viability of one particular cell line, the hNT cells developed by Layton Bioscience, to replace lost neurons and alleviate asymmetrical motor deficits in a unilateral excitotoxic lesion model of Huntington's disease. Because the grafts involved implantation of human-derived cells into a rat host environment, all animals were immunosuppressed. Cyclosporin A and FK-506 were similar in providing effective immunoprotection of the hNT xenografts, and whereas the lesions induced a marked inflammatory response in the host brain, this was not exacerbated by the presence of xenograft cells. The presence of grafted cells was determined with the human-specific antigen HuNu, and good graft survival was demonstrated in almost all animals up to the longest survival examined, 16 weeks posttransplantation. Although the cells exhibited progressively greater maturation and differentiation at 10-day, 4- and 16-week time points, staining for the mature neuronal marker NeuN was at best very weak, and we were unable to detect unequivocal staining with any markers of mature striatal phenotype, including DARPP-32, calbindin, parvalbumin, choline acetyl transferase, or NADPH diaphorase (with in all cases positive control provided by good staining on the intact contralateral side of the brain). Nor were we able to detect any differences between rats with lesions alone and rats with grafts in the contralateral motor deficits exhibited in a test of skilled paw reaching or cylinder placing. These results suggest that further and more extensive studies should be undertaken to assess whether hNT neurons can show more extensive and appropriate maturation and be associated with recovery in appropriate behavioral models, before they may be considered a suitable replacement for primary embryonic cells for clinical application in Huntington's disease.
Natural Antibodies Prevent in Vivo Transmission of Porcine Islet-Derived Endogenous Retrovirus to Human Cells
Authors: Mckane Brice W. , Ramachandran Sabarinathan , Xu Xiao-Chun , Olack Barbara J. , Chapman William C. , Mohanakumar T.
Published: September 2014

The discovery of porcine endogenous retroviruses (PERV) has raised concerns regarding the safety of porcine xenotransplantation. However, transmission of PERV had not been observed in humans exposed to porcine tissue. We examined whether PERV derived from porcine pancreatic islet cells could infect human cells in vivo and the role of natural antibodies in inhibiting PERV infection. In vivo infective potential of PERV was studied in SCID mice reconstituted with human peripheral blood leucocytes. Porcine islets were transplanted under the kidney capsule. PERV infection was determined by analyzing PERV gene expression in graft infiltrating lymphocytes (GIL) harvested 21 days posttransplantation. Mice were administered normal human serum prior to and 2 days posttransplantation to study their role in protection of human cells against PERV infection. PERV genes were expressed in all porcine tissues examined, including purified porcine islets. PERV expression was detected in GILs from three of five human-SCID mice. Administration of human serum blocked PERV infection in GILs in five of five human-SCID mice. These results indicate that PERV from porcine islets can infect human cells in vivo. Normal human serum blocks transmission of retrovirus in vivo, suggesting that natural xenoreactive antibodies can prevent PERV infection.
Fate of a Chimeric Joint Construct in an Ectopic Site in SCID Mice
Authors: Warden Scott , Zaleske David J. , Glowacki Julie
Published: September 2014

This study examines the use of a devitalized biological knee as a scaffold for repopulation with chondrocytes and tests the hypothesis that the devitalized scaffold would become repopulated with the foreign chondrocytes when placed in a suitable environment. Chimeric knee constructs were engineered in vitro and their ectopic in vivo fate was examined in SCID mice. The constructs were made by applying porous collagen sponges that contained viable bovine articular chondrocytes to shaved articular surfaces of devitalized embryonic chick knees. The chimeric joints were cultured for 1 week and were subsequently transplanted into dorsal subcutaneous pouches of 5-week-old mice. Specimens were prepared for histological analysis at 1, 3, 6, or 8 weeks after transplantation. Controls included empty collagen sponges, collagen sponges seeded with viable bovine chondrocytes, and devitalized chick knees without collagen sponge inserts. One week after in vitro incubation of the constructs, the porous collagen sponges with viable bovine chondrocytes were adherent to the shaved articular surfaces of the devitalized chick joints. There was abundant metachromatic neomatrix around the chondrocytes in the collagen sponges. During maintenance of the constructs in vivo, the chimeric joints exhibited dramatic changes. Bovine chondrocytes proliferated in the collagen sponges and formed abundant new matrix. Bovine chondrocytes migrated into preexisting chick cartilage canals at 1 week. Subsequently, bovine chondrocytes invaded the matrix of the devitalized chick knees. Bovine neocartilage obliterated the interface between the collagen sponge and the devitalized chick cartilage. With time in vivo, the bovine neocartilage expanded and replaced the chick matrix. The devitalized cartilage appears to provide a framework for supporting chondrogenesis in a chimeric joint.
Plasma as a Scaffold for Regeneration of Neural Precursor Cells after Transplantation into Rats with Spinal Cord Injury
Authors: Takenaga Mitsuko , Ohta Yuki , Tokura Yukie , Hamaguchi Akemi , Suzuki Noboru , Nakamura Masaya , Okano Hideyuki , Igarashi Rie
Published: September 2014

The present study investigated whether plasma could be useful as a scaffold for cell transplantation in rats with spinal cord injury (SCI). Transplantation of cells with plasma promoted the recovery of SCI-induced motor dysfunction. Immunohistochemical analysis revealed that the grafted cells had differentiated into the neural lineage. When dissociated neural precursor cells were cultured with plasma, extensive neurite outgrowth was observed along with increased expression of p35 and NF68. Neural markers were also expressed by the cultured cells. Culture with plasma reduced thymidine incorporation, but promoted cell growth and increased the RNA contents. These findings suggest that the cells underwent differentiation into neurons in the presence of plasma. In conclusion, plasma could be a promising scaffold for cell transplantation therapy.
Induction of Neurotrophin Expression via Human Adult Mesenchymal Stem Cells: Implication for Cell Therapy in Neurodegenerative Diseases
Authors: Pisati Federica , Bossolasco Patrizia , Meregalli Mirella , Cova Lidia , Belicchi Marzia , Gavina Manuela , Marchesi Chiara , Calzarossa Cinzia , Soligo Davide , Lambertenghi-Deliliers Giorgio , Bresolin Nereo , Silani Vincenzo , Torrente Yvan , Polli Elio
Published: September 2014

In animal models of neurological disorders for cerebral ischemia, Parkinson's disease, and spinal cord lesions, transplantation of mesenchymal stem cells (MSCs) has been reported to improve functional outcome. Three mechanisms have been suggested for the effects of the MSCs: transdifferentiation of the grafted cells with replacement of degenerating neural cells, cell fusion, and neuroprotection of the dying cells. Here we demonstrate that a restricted number of cells with differentiated astroglial features can be obtained from human adult MSCs (hMSCs) both in vitro using different induction protocols and in vivo after transplantation into the developing mouse brain. We then examined the in vitro differentiation capacity of the hMSCs in coculture with slices of neonatal brain cortex. In this condition the hMSCs did not show any neuronal transdifferentiation but expressed neurotrophin low-affinity (NGFRp75) and high-affinity (trkC) receptors and released nerve growth factor (NGF) and neurotrophin-3 (NT-3). The same neurotrophin's expression was demonstrated 45 days after the intracerebral transplantation of hMSCs into nude mice with surviving astroglial cells. These data further confirm the limited capability of adult hMSC to differentiate into neurons whereas they differentiated in astroglial cells. Moreover, the secretion of neurotrophic factors combined with activation of the specific receptors of transplanted hMSCs demonstrated an alternative mechanism for neuroprotection of degenerating neurons. hMSCs are further defined in their transplantation potential for treating neurological disorders.
Tea Polyphenol Inhibits Allostimulation in Mixed Lymphocyte Culture
Authors: Kim Jong-Yoon , Kina Tatsuo , Iwanaga Yasuhiro , Noguchi Hirofumi , Matsumura Kazuaki , Hyon Suong-Hyu
Published: September 2014

Green tea polyphenols are known to protect allogenic donor tissues from acute rejection by their recipients. This immunosuppressive effect may be generated by a unique chemical property of the major component, epigallocatechin-o-gallate (EGCG), which can block specific cell surface molecules of the donor tissues. To test this hypothesis, we examined the effects of EGCG on the murine mixed lymphocyte reactions. EGCG treatment of stimulator cells significantly attenuated the proliferation of responder T cells. The proliferation did not recover upon the secondary stimulations by fresh untreated cells or exogenous IL-2. Flow cytometric analyses showed that EGCG treatment decreased the staining intensities of various cell surface molecules including MHC II, which plays a major role in antigen presentation, and B7.1, B7.2, and their ligand, CD28, which are required for costimulatory signals in T-cell activation. These results suggest that an anergic state of alloreactive T cells may be induced by either weakening of antigen signaling or blockage of costimulatory signals with EGCG. Other possible mechanisms behind the immunosuppressive effect and a potential use of EGCG treatment of donor tissues in transplantation medicine are discussed.