bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of Trophic Factor Expression by Innervating Target Regions in Intraocular Double Transplants
Authors: Willis L. , Quintero E. M. , Nelson M. , Granholm A.-Ch.
Published: April 2008

Trophic factors have been found to play a significant role both in long-term survival processes and in more rapid and dynamic processes in the brain and spinal cord. However, little is known regarding the regulation of expression of growth factors, and how these proteins interact on a cell-to-cell basis. We have studied protein levels of one growth factor known to affect the noradrenergic innervation of the hippocampal formation, namely brain-derived neurotrophic factor (BDNF). The purpose of the present study was to determine if appropriate innervation or contact between the LC noradrenergic neurons and their target, the hippocampus, affects expression of this growth factor in either brain region. Fetal brain stem tissue, containing the LC, and hippocampal formation were dissected from embryonic day 17 rat fetuses and transplanted together or alone into the anterior chamber of the eye of adult Fisher 344 rats. The tissue was grown together for 6 weeks, after which the animals were sacrificed and ELISAs for BDNF were undertaken. Transplantation to the anterior chamber of the eye increased the expression of BDNF in the hippocampal but not the brain stem tissue, compared with levels observed in fetal and adult rats in vivo. In addition, double grafting with hippocampal tissue more than tripled BDNF levels in brain stem grafts and doubled BDNF levels in the hippocampal portion of double grafts compared with hippocampal single grafts. Triple grafts containing basal forebrain, hippocampus, and brain stem LC tissue increased brain stem and hippocampal BDNF levels even further. Colchicine treatment of LC-hippocampal double grafts gave rise to a significant decrease in hippocampal BDNF levels to levels seen in single hippocampal grafts, while only a partial reduction of BDNF levels was seen in the brain stem portion of the same double grafts treated with colchicine. The findings suggest that an appropriate hippocampal innervation or contact with its target tissues is essential for regulation of BDNF expression in the brain stem, and that retrograde transport of BDNF can occur between double grafted fetal tissues in oculo.
Autologous Bone Marrow Transplantation in Patients with Subacute and Chronic Spinal Cord Injury
Authors: Syková Eva , Homola Aleš , Mazanec Radim , Lachmann Hynek , Konrádová ŠImona Langkramer , Kobylka Petr , Pádr Radek , Neuwirth Jiří , Komrska Vladimír , Vávra Vladimír , Štulík Jan , Bojar Martin
Published: April 2008

Stem cell transplants into spinal cord lesions may help to improve regeneration and spinal cord function. Clinical studies are necessary for transferring preclinical findings from animal experiments to humans. We investigated the transplantation of unmanipulated autologous bone marrow in patients with transversal spinal cord injury (SCI) with respect to safety, therapeutic time window, implantation strategy, method of administration, and functional improvement. We report data from 20 patients with complete SCI who received transplants 10 to 467 days postinjury. The follow-up examinations were done at 3, 6, and 12 months after implantation by two independent neurologists using standard neurological classification of SCI, including the ASIA protocol, the Frankel score, the recording of motor and somatosensory evoked potentials, and MRI evaluation of lesion size. We compared intra-arterial (via catheterization of a. vertebralis) versus intravenous administration of all mononuclear cells in groups of acute (10–30 days post-SCI, n = 7) and chronic patients (2–17 months postinjury, n = 13). Improvement in motor and/or sensory functions was observed within 3 months in 5 of 6 patients with intra-arterial application, in 5 of 7 acute, and in 1 of 13 chronic patients. Our case study shows that the implantation of autologous bone marrow cells appears to be safe, as there have been no complications following implantation to date (11 patients followed up for more than 2 years), but longer follow-ups are required to determine that implantation is definitively safe. Also, we cannot yet confirm that the observed beneficial effects were due to the cell therapy. However, the outcomes following transplantation in acute patients, and in one chronic patient who was in stable condition for several months prior to cell implantation, are promising. It is evident that transplantation within a therapeutic window of 3–4 weeks following injury will play an important role in any type of stem cell SCI treatment. Trials involving a larger population of patients and different cell types are needed before further conclusions can be drawn.
Adenovirus-Mediated Expression of the Anticoagulant Hirudin in Human Islets: A Tool to Make the Islets Biocompatible to Blood
Authors: Cabric Sanja , Elgue Graciela , Nilsson Bo , Korsgren Olle , Schmidt Peter
Published: April 2008

Human islets induce an injurious clotting reaction at the time of transplantation. A potential strategy to counteract this reaction would be to allow the islets to express hirudin, a protein with direct anticoagulative activity. Human islets were transduced with an adenoviral vector encoding hirudin, an empty corresponding vector, or left untreated. Islet culture supernatants were analyzed for hirudin using an ELISA, a chromogenic substrate assay based on the thrombin-binding properties of hirudin and in a whole blood viscosimetry assay. Immunohistochemical evaluation and determination of hirudin content revealed an abundant expression of hirudin after transduction. Hirudin content in transduced islets was in the range of the insulin content levels. A delay in human whole blood clotting time could be observed after addition of supernatants taken from islet cultures expressing hirudin. However, transduced islets showed an impaired glucose-stimulated insulin release, but could readily be retrieved 6 weeks after transplantation to athymic mice. A marked expression and secretion of hirudin with functional capacity can be induced in human islets using an adenoviral vector. The impairment in glucose-stimulated insulin release in hirudin-secreting islets, compared to controls, indicates that the additional protein synthesis affects the functional capacity of the islets.
Human Bone Marrow Mesenchymal Stem Cells Seeded on Modified Collagen Improved Dermal Regeneration In Vivo
Authors: Markowicz Marta , Koellensperger Eva , Neuss Sabine , Koenigschulte Sarah , Bindler Christine , Pallua Norbert
Published: April 2008

In the correction of functional and aesthetic impairments, loss of soft connective tissue creates the need for adequate implant material. The reconstruction of defects resulting from radical excisions, trauma, or hereditary diseases has seen the use of combined grafts and flaps. With the aim of minimizing donor site morbidity, new methods have been evaluated. Because of a low rate of vascularization, with artificial dermal templates the take has only been poor. As shown in previous studies, improved angiogenetic potency and epidermal formation has been obtained in modified, cell-seeded collagen matrices. We have now investigated the suitability of adult bone marrow mesenchymal stem cells (hMSC) for soft tissue engineering. In this study, hMSC were isolated and expanded. Cells (106) were seeded onto EDC cross-linked collagen sponges and implanted in 30 immunodeficient mice. Collagen sponges without cells were used as controls. The grafts were evaluated after 2 and 6 weeks. After explantation, macroscopic appearance, weights, and histology (scaffold degradation, cellularity, and invasion depth of the seeded cells) were all assessed. After 2 and 6 weeks in vivo, new vessels were found macroscopically on all cell-seeded collagen grafts. The control grafts appeared to be degraded with a lower rate of vessel ingrowth. In the experimental group, weight gain was significant after 2 and 6 weeks in vivo compared to the same grafts after 72 h in vitro, while weight increased only slightly in the control group. Histologically, populated scaffolds showed a high density of vascularization under a capsule. The control sponges showed single capillaries and a thicker capsule. Compared to the controls, cellularity (cells/field) was greater in cell-containing collagen grafts after 2 and 6 weeks. The results obtained demonstrate that in vitro cultured human mesenchymal stem cells seeded on modified collagen sponges may be able to act as a replacement for soft tissue.
Increased Sensitivities of Peripheral Blood Mononuclear Cells to Immunosuppressive Drugs in Cirrhosis Patients Awaiting Liver Transplantation
Authors: Mijiti Abuduxhukuer , Matsuno Naoto , Iwahori Tohru , Takeuchi Hironori , Nagao Takeshi , Oka Kitaro , Hirano Toshihiko
Published: April 2008

Successful immunosuppressive therapy is critical for liver transplantation. However, a considerable number of patients show clinical resistance to the therapy and experience rejection episodes, or alternatively exhibits serious adverse effects of drugs. We examined the in vitro response of peripheral blood mononuclear cells (PBMCs) to immunosuppressive drugs in cirrhosis patients awaiting liver transplantation. We evaluated the suppressive efficacy of prednisolone, methylprednisolone, cyclosporine, and tacrolimus on the in vitro blastogenesis of PBMCs obtained from 22 cirrhosis patients and 31 healthy subjects. In vitro drug concentrations giving 50% inhibition of PBMC blastogenesis (IC50s) were calculated. Two out of these 22 patients received liver transplantation from living donors, and their clinical courses were surveyed until 5 weeks after operation. The median IC50 values for prednisolone, cyclosporine, and tacrolimus against blastogenesis of PBMCs from cirrhosis patients were significantly lower than those of PBMCs from healthy subjects (p < 0.01). However, large individual differences were observed in the IC50 values of the immunosuppressive drugs examined, especially in the cirrhosis patients. One recipient exhibiting high PBMC sensitivity to tacrolimus (IC50 = 0.001 ng/ml) showed good clinical course without rejection until 5 weeks after liver transplantation. The other recipient exhibiting relatively low PBMC sensitivity to taclolimus (IC50 = 0.30) showed allograft rejection at 1 week after operation. We concluded from these observations that PBMCs of cirrhosis patients are vulnerable to the immunosuppressive effects of prednisolone and calcineurin inhibitors. However, large individual variations in the IC50 values suggest that patients exhibiting relatively lower sensitivity to these drugs may have risks of rejection, whereas highly sensitive patients are possibly able to reduce the dose of immunosuppressive drugs to avoid serious drug-adverse effects, after liver transplantation.
Maintenance of Neovascularization at the Implantation Site of an Artificial Device by bFGF and Endothelial Cell Transplant
Authors: Miki Atsushi , Rivas-Carrillo Jorge D. , Navarro-Alvarez Nalu , Soto-Gutierrez Alejandro , Chen Yong , Tanaka Kimiaki , Narushima Michiki , Tabata Yasuhiko , Okitsu Teru , Noguchi Hirofumi , Matsumoto Shinichi , Tanaka Noriaki , Kobayashi Naoya
Published: April 2008

Development of a subcutaneously implantable bioartificial pancreas (BAP) with immunoisolatory function could have a great impact on the treatment of diabetes mellitus. We have developed an implantable BAP device with an ethylene vinyl alcohol (EVAL) membrane. In the present study, we used basic fibroblast growth factors (bFGF), which was incorporated in a carrier for sustained release, in order to induce neovascularization when the device was implanted subcutaneously. To maintain the vasculature thus formed, a cell infusion port was attached to the BAP device, through which the device was filled with human liver vascular endothelial cell line TMNK-1, and the vasculature could be adequately maintained. Mice were divided into the following three groups. In group 1, a bFGF-free BAP device was implanted subcutaneously. In group 2, a sustained-release bFGF-impregnated BAP device was implanted. In group 3, a sustained-release bFGF-impregnated BAP device was implanted, and 3 × 106 TMNK-1 cells were infused into the implanted device every week. Neovascularization induced in the subcutaneous tissue around the implanted BAP device was macroscopically examined and histologically evaluated. In addition, the tissue blood flow was measured using a laser blood flow meter. In mice in group 3, neovascularization was significantly induced and maintained until week 8 postimplantation. It was confirmed by scanning electron microscopy that infused TMNK-1 cells adhered to the inner polyethylene surface of the device. It was demonstrated that the use of bFGF and vascular endothelial TMNK-1 cells induced and maintained adequate vasculature and tissue blood flow surrounding the implantable bag-type BAP device. We believe that the present study will contribute to BAP development for the treatment of diabetes.
Self-Assembling Peptide Nanofiber as a Novel Culture System for Isolated Porcine Hepatocytes
Authors: Navarro-Alvarez Nalu , Soto-Gutierrez Alejandro , Rivas-Carrillo Jorge D. , Chen Yong , Yamamoto Tsuyoshi , Yuasa Takeshi , Misawa Haruo , Takei Jiro , Tanaka Noriaki , Kobayashi Naoya
Published: April 2008

Freshly isolated porcine hepatocytes are a very attractive cell source in the cell-based therapies to treat liver failure because of unlimited availability. However, due to the loss of hepatocyte functions in vitro, there is a need to develop a functional culture system to keep the cells metabolically active. Here we compared the effect of a self-assembling peptide nanofiber (SAPNF) as an extracellular matrix (ECM) with collagen type I on hepatocyte metabolic and secretion activities following hepatocyte isolation. Isolated porcine hepatocytes were cultured in SAPNF and collagen type I. Morphological assessment at different time points was performed by using SEM and phase contrast microscope. Metabolic and secretion activities were comparatively performed in the groups, by means of ammonia, lidocaine, and diazepam as well as albumin. Hepatocytes cultured on SAPNF revealed a three-dimensional spheroidal formation, thus maintaining cell differentiation status during 2 weeks of culture. On the other hand, hepatocytes in collagen revealed a spread shape, and by day 14 no hepatocyte-like cells were observed, but cells with long shape were present, thus revealing a degree of dedifferentiation in collagen culture. Hepatocytes in SAPNF were capable of drug-metabolizing activities and albumin secretion in higher ratio than those cultured on collagen. The present work clearly demonstrates the usefulness of SAPNF for maintaining differentiated functions of porcine hepatocytes in culture.
Improvement of Hepatocyte Viability after Cryopreservation by Supplementation of Long-Chain Oligosaccharide in the Freezing Medium in Rats and Humans
Authors: Miyamoto Yoshitaka , Suzuki Satoshi , Nomura Kou , Enosawa Shin
Published: April 2008

Factors affecting cell viability, plating efficiency, and survival of hepatocytes after cryopreservation have been investigated. We focused especially on the effect of including trehalose and related oligosaccharides in the cryopreservation fluid. This was supplemented with either glucose, trehalose, maltotriose, or other sugars, in addition to dimethyl sulfoxide (10%) and first tested with primary rat hepatocytes cooled in a controlled rate freezer. After thawing, viability by trypan blue exclusion of cells frozen in oligosaccharide-supplemented medium was significantly higher than for those cryopreserved without oligosaccharides. Use of oligosaccharides with higher molecular weights resulted in greatest improvement in viability. Moreover, attachment and survival rates in plastic dishes were approximately 1.2–1.8-fold greater after freezing in the presence of di-, tri-, and tetrasaccharides. Human hepatocytes isolated from untransplantable liver showed the same tendency regarding viability, but cell adherence was not similarly improved by the addition of oligosaccharides. Possible reasons for these differences may be prior cell damage during extended cold ischemia of the human liver, donor age, or cell degradation caused by progression of fatty liver in humans, and/or species differences.
Preservation of Rat Aortic Tissue Transplant with Green Tea Polyphenols
Authors: Hyon S.-H. , Kim D.-H. , Cui W. , Matsumura K. , Kim J.-Y. , Tsutsumi S.
Published: April 2008

Green tea polyphenols have recently attracted medical attention as bioactive agents with anticancer, antimicrobial, and antiviral effects. We discovered their new usage as preservative agents for tissue transplants. We preserved rat aortas in a DMEM solution containing polyphenols extracted from green tea leaves. The preserved aortas retained original structures and mechanical strength, and were devoid of any undesirable cell secretions for over a month under physiological conditions. In addition, aortas from Lewis rats preserved for a month and transplanted to allogenic ACI rats completely avoided rejection by the host, suggesting that the polyphenols have immunosuppressive actions on the aortic tissues. From these results, we conclude that polyphenol treatment of aortic tissue transplant can maintain its viability for extended periods of time either before or after transplantation, and the method can be applicable to other transplantation situations.
The Use of the BD Oxygen Biosensor System to Assess Isolated Human Islets of Langerhans: Oxygen Consumption as a Potential Measure of Islet Potency
Authors: Fraker Chris , Timmins Mark R. , Guarino Richard D. , Haaland Perry D. , Ichii Hirohito , Molano Damaris , Pileggi Antonello , Poggioli Raffaella , Presnell Sharon C. , Inverardi Luca , Zehtab Mitra , Ricordi Camillo
Published: April 2008

The measurement of cellular oxygen consumption rate (OCR) is a potential tool for the assessment of metabolic potency of isolated islets of Langerhans prior to clinical transplantation. We used a commercially available 96-well plate fluoroprobe, the BD Oxygen Biosensor System (OBS), to estimate OCR in 27 human islet preparations, and compared these results to those of concurrent mouse transplantations. OCR was estimated both from the dO2 at steady state and from the transient rate of change of dO2 during the initial culture period immediately after seeding (“dO2 slope”). To demonstrate the validity of the OBS-derived values, it was shown that they scaled linearly with islet equivalent number/DNA concentration and with each other. These measurements were obtained for each preparation of islets incubated in media supplemented with either low (2.2 mM) or high (22 mM) glucose. Concurrently, one to three athymic nude mice were transplanted with 2,000 IEQs under the kidney capsule. The OCR Index, defined as the ratio of the DNA-normalized “dO2 slope” in high glucose to that in low glucose, proved highly predictive of mouse transplant results. Of the 69 mice transplanted, those receiving islets where the OCR Index exceeded 1.27 were 90% likely to reverse within 3 days, whereas those receiving islets with an OCR Index below 1.27 took significantly longer, often failing to reverse at all over a 35-day time period. These results suggest that the OBS could be a useful tool for the pretransplant assessment of islet cell potency.
Murine Brain Progenitor Cells have the Ability to Differentiate into Functional Neurons and Integrate into the CNS
Authors: Ding Shinghua , Messam Conrad A. , Li Peiying , Selzer Michael E. , Dichter Marc A. , Haydon Philip G.
Published: April 2008

Although neural stem and progenitor cells have been shown to differentiate into neurons, few studies have examined the physiological properties of the differentiated neurons derived from stem cells. Here we show that mouse brain progenitor cells (mBPCs) differentiated in culture by removal of mitogenic factors or addition of BDNF or GDNF express neuronal-specific proteins including MAP-2 and synaptobrevin II. However, these cells demonstrate small voltage-gated Na+ currents and are not able to generate action potentials. When the mBPCs are cocultured with developing rat hippocampal neurons, the stem cells differentiate into neurons expressing MAP-2, develop large voltage-gated Na+ currents, and are able to generate action potentials. To investigate the influence of a mature CNS environment on survival, differentiation, migration, and morphological integration, mBPCs were transplanted into the spinal cord of adult mice. Undifferentiated cells transplanted into the spinal cord exhibited limited migration and expressed NG2, but did not differentiate to express MAP-2. Predifferentiated cells migrated to both gray and white matter with about 23% cells developing MAP-2 immunoreactivity after 8 weeks. These results suggest that both the environment and state of differentiation may dictate migration and the differentiation pathway of stem cells after transplantation.
Microencapsulation of Engineered Cells to Deliver Sustained High Circulating Levels of Interleukin-6 to Study Hepatocellular Carcinoma Progression
Authors: Moran Diarmuid M. , Koniaris Leonidas G. , Jablonski Elizabeth M. , Cahill Paul A. , Halberstadt Craig R. , Mckillop Iain H.
Published: April 2008

Interlukin-6 (IL-6) is a pleitropic cytokine that plays a central role in normal and abnormal hepatic function and response. The aims of the current study were to determine the viability of using cell encapsulation technology to introduce a genetically modified xenogeneic (CHO) cell population to elevate circulating levels of rhIL-6 in a rat model and determine the effects of sustained high rhIL-6 levels on hepatocellular carcinoma (HCC) progression in vivo. An alginate matrix was combined with transfected CHO cells, selected for their ability to synthesize rhIL-6, and used to generate uniform alginate–cell beads. Once encapsulated transfected cells continued to undergo replication, formed colonies within the bead, and synthesized/released large quantities of rhIL-6 into culture medium in vitro. Intraperitoneal implantation of beads into rats resulted in significantly increased circulating and intrahepatic levels of rhIL-6 up to 4 days postimplantation. Prolonged implantation led to the escape of CHO cells from the bead, resulting in a host response and CHO cell death within the bead. Subsequently CHO-IL-6 encapsulated cells were implanted into rats previously inoculated intrahepatically with the H4IIE HCC cell line. These studies demonstrated the maintenance of high circulating/intrahepatic rhIL-6 levels in this model. Despite significantly increased rhIL-6, this technique did not significantly alter the rate of net tumor progression. However, Stat3 activity was significantly increased in both normal liver and HCC tissue resected from animals implanted with CHO-IL-6 cells. Collectively these data demonstrate the short-term viability of using cell encapsulation technology to generate high levels of active circulating and intrahepatic cytokines and raise the possibility of modifying specific signal transduction cascades identified to be important during tumor progression.
Immunologic Consequences of Multiple, High-Dose Administration of Allogeneic Mesenchymal Stem Cells to Baboons
Authors: Beggs Kirstin J. , Lyubimov Alex , Borneman Jade N. , Bartholomew Amelia , Moseley Annemarie , Dodds Robert , Archambault Michael P. , Smith Alan K. , Mcintosh Kevin R.
Published: April 2008

Mesenchymal stem cells (MSCs) express low immunogenicity and demonstrate immunomodulatory properties in vitro that may safely allow their transplantation into unrelated immunocompetent recipients without the use of pharmacologic immunosuppression. To test this hypothesis, three groups of baboons (three animals per group) were injected as follows: group 1 animals were injected with vehicle; group 2 animals were injected IV with DiI-labeled MSCs (5 × 106 MSCs/kg body weight) followed 6 weeks later by IM injections of DiO-labeled MSCs (5 × 106 MSCs/kg) from the same donor; and group 3 animals were treated similarly as group 2 except that MSCs were derived from two different donors. Muscle biopsies, performed 4 weeks after the second injection of MSCs, showed persistence of DiO-labeled MSCs in 50% of the recipients. Blood was drawn at intervals for evaluation of basic immune parameters (Con A mitogen responsiveness, PBMC phenotyping, immunoglobulin levels), and to determine T-cell and alloantibody responses to donor alloantigens. Host T-cell responses to donor alloantigens were decreased in the majority of recipients without suppressing the overall T-cell response to Con A, or affecting basic parameters of the immune system. All recipient baboons produced alloantibodies that reacted with donor PBMCs. Two of six animals produced alloantibodies that reacted with MSCs. We conclude that multiple administrations of high doses of allogeneic MSCs affected alloreactive immune responses without compromising the overall immune system of recipient baboons. The induction of host T-cell hyporesponsiveness to donor alloantigens may facilitate MSC survival.
Establishment of Cocultures of Osteoblasts, Schwann Cells, and Neurons towards a Tissue-Engineered Approach for Orofacial Reconstruction
Authors: Haastert Kirsten , Semmler Nicole , Wesemann Maike , Rücker Martin , Gellrich Nils-Claudius , Grothe Claudia
Published: April 2008

In orofacial reconstruction not only the osseous structures themselves but also neighboring cranial nerves need to be regenerated. To replace autologous bone implants, biocompatible tissue-engineered scaffolds are under investigation at least for bone replacement but until now these studies have not focused on parallel reconstruction of injured cranial nerves. The present study contributes to the development of optimized tissue-engineered products that will enable regeneration of both bone and nervous tissue. For the first time, cocultures of primary osteoblasts (rat or human) and primary Schwann cells (rat or human) were established. The suitability of monocultures of osteoblasts and cocultures of osteoblasts plus Schwann cells as substrate for sensory neurons as well as motoneurons was tested here. The results suggest that whereas osteoblasts provide a good substrate for sensory neurons, motoneurons depend on the presence of Schwann cells for survival and neurite outgrowth. For prolonged availability of regeneration-promoting growth factors at the site of the graft, those proteins should be delivered by the transplanted cells themselves. To enable this, we established electroporation-based nonviral transfection of osteoblasts as well as Schwann cells. Our new cell culture system will enable investigations of the effect of graft-derived growth factors on osteoblasts and Schwann cells as well as on neurite outgrowth from cocultured neurons of the sensory and motor system.
Induction of Tolerance across Fully Mismatched Barriers by a Nonmyeloablative Treatment excluding Antibodies or Irradiation Use
Authors: Stephan Lionel , Pichavant Christophe , Bouchentouf Manaf , Mills Philippe , Camirand Geoffrey , Tagmouti Saloua , Rothstein David , Tremblay Jacques P.
Published: April 2008

A mixed-chimerism approach is a major goal to circumvent sustained immunosuppression, but most of the proposed protocols need antibody treatment or host irradiation. Another promising experience involves busulfan combined with cyclophosphamide treatment. Additionally, recent publications demonstrated that, differing from busulfan, treosulfan administration does not present severe organ or hemato toxicities. Currently, Duchenne muscular dystrophy (DMD) patients are treated with chronic immunosuppression for muscle precursor cell transplantation (MT). We have developed a safe tolerance approach within this cellular allotransplantation therapy background. Thus, we have conditioned, prior to a donor BALB/c MT, the dystrophic mouse model C57Bl10J mdx/mdx, with our treatment based on a donor-specific transfusion, then a treosulfan treatment combined with single cyclophosphamide dose, and finally a donor bone marrow transplantation (TTCB). A first MT was performed in all mixed chimeric mice resulting from the TTCB treatment in the left tibialis anterior (TA) muscles. A second MT from the same donor strain was performed 100 days later in the right TA without any additional therapy. Results show that all treated mice developed permanent mixed chimerism. Long-lasting donor-positive fibers were present in both TAs of the mice, which received MT after the TTCB treatment. Only a basal level of infiltration was observed around donor fibers and mixed chimeric mice rejected third-party haplotype skin grafts. Thus, mixed chimerism development with this TTCB conditioning regimen promotes donor-specific stable tolerance, avoiding costimulatory blockade antibodies or irradiation use and side effects of sustained immunosuppressive treatments. This protocol could be eventually applied for MT to DMD patients or others tissue transplantations.
The Effects of Immunosuppressive Agents on the Function of Human Hepatocytes In Vitro
Authors: Serrano Trinidad , Mitry Ragai R. , Terry Claire , Lehec Sharon C. , Dhawan Anil , Hughes Robin D.
Published: April 2008

Calcineurin inhibitors (tacrolimus) and steroids continue to be an important component of hepatocyte transplantation protocols, despite reports of hepatotoxicity and inhibitory effects of steroids on cell proliferation. The aim of the study was to investigate whether isolated human hepatocytes were more vulnerable to the toxicity of these agents and also to investigate their effects on hepatocyte VEGF secretion, a vascular permeability factor suggested to be involved in the cell engraftment process. Human hepatocytes were isolated from donor livers/segments rejected or unused for orthotopic liver transplantation using a collagenase perfusion technique. Hepatocytes were plated for cell function tests and to determine VEGF production. Tacrolimus (0–50 ng/ml) and methylprednisolone (0-500 ng/ml) were added to the culture media and cells incubated for 24 h. Cell metabolic activity was assessed using the MTT assay, cell number using the SRB assay, and cell attachment from hepatocyte total protein content and protein synthesis using [14C]leucine incorporation. VEGF in culture supernatants was measured by ELISA. Tacrolimus and methylprednisolone had no statistically significant inhibitory effects on metabolic activity or protein synthesis compared to controls at all concentrations of the agents tested when added after plating. There were also no significant effects on cell attachment when tacrolimus or methylprednisolone was added at the time of cell plating. There were no differences in the responses obtained when either fresh or cryopreserved hepatocytes were used. The amount of VEGF secreted by untreated hepatocytes was highly variable (0–1400 pg/106 cells/24 h). VEGF levels in the culture supernatant from hepatocytes isolated from ≤20-year-old donors (687 + 59 pg/106 cells/24 h) was significantly greater than from older donors (61 +7 pg/106 cells/24 h; p = 0.003). Tacrolimus and methylprednisolone did not significantly affect VEGF secretion by hepatocytes. Tacrolimus and methylprednisolone did not have detrimental effects on the metabolic function of human hepatocytes, cell attachment, or VEGF secretion after cell isolation.
Bone Marrow Stromal Cells for Repair of the Spinal Cord: Towards Clinical Application
Authors: Nandoe Rishi D. S. , Hurtado Andres , Levi Allan D. O. , Grotenhuis Andre , Oudega Martin
Published: April 2008

Stem cells have been recognized and intensively studied for their potential use in restorative approaches for degenerative diseases and traumatic injuries. In the central nervous system (CNS), stem cell-based strategies have been proposed to replace lost neurons in degenerative diseases such as Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis (Lou Gehrig's disease), or to replace lost oligodendrocytes in demyelinating diseases such as multiple sclerosis. Stem cells have also been implicated in repair of the adult spinal cord. An impact to the spinal cord results in immediate damage to tissue including blood vessels, causing loss of neurons, astrocytes, and oligodendrocytes. In time, more tissue nearby or away from the injury site is lost due to secondary injury. In case of relatively minor damage to the cord some return of function can be observed, but in most cases the neurological loss is permanent. This review will focus on in vitro and in vivo studies on the use of bone marrow stromal cells (BMSCs), a heterogeneous cell population that includes mesenchymal stem cells, for repair of the spinal cord in experimental injury models and their potential for human application. To optimally benefit from BMSCs for repair of the spinal cord it is imperative to develop in vitro techniques that will generate the desired cell type and/or a large enough number for in vivo transplantation approaches. We will also assess the potential and possible pitfalls for use of BMSCs in humans and ongoing clinical trials.
Resolution of Neurotoxicity and β-Cell Toxicity in an Islet Transplant Recipient following Substitution of Tacrolimus with MMF
Authors: Froud Tatiana , Baidal David A. , Ponte Gaston , Ferreira Jacqueline V. , Ricordi Camillo , Alejandro Rodolfo
Published: April 2008

Calcineurin inhibitors such as tacrolimus have well-recognized efficacy in organ transplantation but side effects of nephrotoxicity, neurotoxicity, and β-cell toxicity that can be particularly detrimental in islet transplantation. Neuro- and nephrotoxicity have been demonstrated in multiple islet transplant recipients despite the relatively low serum maintenance levels typically used (3–5 ng/ml). We describe a single patient in whom symptoms and signs of neurotoxicity necessitated substitution of tacrolimus with mycophenolate mofetil (MMF), which resulted in complete symptom resolution over the subsequent 9 months. Concomitantly noted were an almost immediate improvement in glycemic control and an improved response to stimulation testing, suggesting remission of tacrolimus-induced β-cell toxicity and insulin resistance. At 18 months post-“switch,” 30 months posttransplant, the patient remains insulin independent with good glycemic control. The goal to remove calcineurin inhibitors from regimens of islet transplantation is a worthy one.
In Vitro Culture Duration does Not Impact the Ability of Encapsulated Choroid Plexus Transplants to Prevent Neurological Deficits in an Excitotoxin-Lesioned Rat Model of Huntington's Disease
Authors: Emerich Dwaine F. , Thanos Christopher G.
Published: April 2008

Delivery of neurotrophic molecules to the CNS is a potential treatment strategy for preventing the neuronal loss accompanying many neurological disorders. Choroid plexus (CP) epithelial cells secrete a cocktail of neurotrophic factors, and encapsulated CP transplants are neuroprotective in animal models of stroke and Huntington's disease (HD). Prior to clinical use, it is essential to identify and optimize parameters such as the length of time that transplant products such as encapsulated CP can be maintained. In the present study, neonatal porcine CP was encapsulated within alginate microcapsules and maintained in vitro for 1, 2, or 7 months. The encapsulated cells remained viable (>80%) at all time points and were transplanted unilaterally into the rat striatum. Seven days later, the same animals received unilateral injections of quinolinic acid (QA; 225 nmol) adjacent to the implant site. Separate groups of animals served as controls and received QA alone. After surgery, animals were periodically evaluated for weight loss and were tested for motor function 14 days post-QA. In controls, QA lesions produced a significant loss of body weight and impaired function of the contralateral forelimb. In contrast, implants of CP were potently neuroprotective as rats receiving CP transplants did not lose body weight and were not significantly impaired when tested for motor function. These benefits were independent of the length of time that the cells were held in vitro and demonstrate that the potential potency of alginate encapsulated CP cells can be retained for extremely long periods of time in vitro.
Tetracycline-Regulated Expression of VEGF-A in Beta Cells Induces Angiogenesis: Improvement of Engraftment following Transplantation
Authors: Mathe Zoltan , Dupraz Philippe , Rinsch Chris , Thorens Bernard , Bosco Domenico , Zbinden Marie , Morel Philippe , Berney Thierry , Pepper Michael S.
Published: April 2008

Early revascularization of pancreatic islet cells after transplantation is crucial for engraftment, and it has been suggested that vascular endothelial growth factor-A (VEGF-A) plays a significant role in this process. Although VEGF gene therapy can improve angiogenesis, uncontrolled VEGF secretion can lead to vascular tumor formation. Here we have explored the role of temporal VEGF expression, controlled by a tetracycline (TC)-regulated promoter, on revascularization and engraftment of genetically modified beta cells following transplantation. To this end, we modified the CDM3D beta cell line using a lentiviral vector to promote secretion of VEGF-A either in a TC-regulated (TET cells) or a constitutive (PGK cells) manner. VEGF secretion, angiogenesis, cell proliferation, and stimulated insulin secretion were assessed in vitro. VEGF secretion was increased in TET and PGK cells, and VEGF delivery resulted in angiogenesis, whereas addition of TC inhibited these processes. Insulin secretion by the three cell types was similar. We used a syngeneic mouse model of transplantation to assess the effects of this controlled VEGF expression in vivo. Time to normoglycemia, intraperitoneal glucose tolerance test, graft vascular density, and cellular mass were evaluated. Increased expression of VEGF resulted in significantly better revascularization and engraftment after transplantation when compared to control cells. In vivo, there was a significant increase in vascular density in grafted TET and PGK cells versus control cells. Moreover, the time for diabetic mice to return to normoglycemia and the stimulated plasma glucose clearance were also significantly accelerated in mice transplanted with TET and PGK cells when compared to control cells. VEGF was only needed during the first 2–3 weeks after transplantation; when removed, normoglycemia and graft vascularization were maintained. TC-treated mice grafted with TC-treated cells failed to restore normoglycemia. This approach allowed us to switch off VEGF secretion when the desired effects had been achieved. TC-regulated temporal expression of VEGF using a gene therapy approach presents a novel way to improve early revascularization and engraftment after islet cell transplantation.
Application of a New Subretinal Injection Device in the Dog
Authors: Komáromy András M. , Varner Signe E. , De Juan Eugene , Acland Gregory M. , Aguirre Gustavo D.
Published: April 2008

The use of a new subretinal injection device (RetinaJect™ Subretinal Cannula, SurModics, Inc., Eden Prairie, MN) to access the subretinal space in the canine model was evaluated. Subretinal injections were performed in 33 mongrel dogs between 2 and 52 months of age (median = 9 months). In 5 normal dogs the injection of 150 μl saline or India ink occurred by using a conventional subretinal injection device (CSID) with a 30-gauge anterior chamber irrigating cannula. The sclera had to be surgically exposed and penetrated before the subretinal injection with the CSID could occur. After removing the CSID, the conjunctiva over the sclerotomy site had to be closed. In a second group of 28 dogs [16 normals, 10 RPE65 mutants, and 2 with progressive rod cone degeneration (prcd)], the 25-gauge needle of the RetinaJect™ was used to penetrate the conjunctiva and the sclera. Once the tip of the needle was close to the retinal surface, a 39-gauge polyimide cannula was extended and brought into apposition with the retina for the subsequent subretinal injection of 150 μl saline, India ink, or adeno-associated virus (AAV). No closure of the conjunctiva was required. The animals were clinically monitored between 1 and 59 weeks after surgery. From this second group 25 eyes were harvested for routine histological analysis either immediately after surgery or after a clinical observation time of between 1 and 40 weeks. Both devices provided equally successful access to the subretinal space. The main advantage of the RetinaJect™ was that no surgical dissection was required; this led to a shorter procedure time and milder postoperative conjunctival swelling. In summary, the use of the RetinaJect™ can be recommended as an alternative to the CSID for subretinal injections in dogs.
Myoblast Therapy: From Bench to Bedside
Authors: Liu Zhongmin , Wu Yanling , Chen Bing-Guan
Published: April 2008

Myoblasts are defined as stem cells containing skeletal muscle cell precursors. A decade of experimental work has revealed many properties of myoblasts, including the stability of resulting hybrid myofibers without immune suppression, the persistence of transgene expression, and the lack of tumorigenicity. Early phase clinical trials also showed that myoblast-based therapy is a promising approach for many intractable clinical conditions, including both muscle-related and non-muscle-related diseases. The potential application of myoblast therapy may be in the treatment of genetic muscle diseases, cardiomyocyte damaged heart diseases, and urinary incontinence. This review will provide an overview of myoblast biology, along with discussion of the potential application in clinical medicine. In addition, problems in current myoblast therapy and possible future improvements will be addressed.
Endothelial Cell Preservation at 10°C Minimizes Catalytic Iron, Oxidative Stress, and Cold-Induced Injury
Authors: Zieger Michael A. J. , Gupta Mahesh P.
Published: April 2008

There is growing evidence that oxidative stress plays an important role in mediating the injury induced by hypothermia during the preservation of cells and tissues for clinical or research use. In cardiovascular allografts, endothelial cell loss or injury may lead to impaired control of vascular permeability and tone, thrombosis, and inflammation. We hypothesized that hypothermia-induced damage to the endothelium is linked to increases in intracellular catalytic iron pools and oxidative stress. In this study, bovine aortic endothelial cells and cell culture methods were used to model the response of the endothelium of cardiovascular tissues to hypothermia. Confluent cells were stored at 0°C to 25°C and cell damage was measured by lipid peroxidation (LPO) and lactate dehydrogenase release. Varying the bleomycin-detectible iron (BDI) in cells modulated cold-induced LPO and cell injury. In untreated cells, injury was highest at 0°C and a minimum at 10°C. A similar temperature-dependent trend was found in BDI levels and cell plating efficiencies. Arrhenius plots of cell killing and iron accumulation rates showed biphasic temperature dependence, with minima at 10°C and matching activation energies above and below 10°C. These findings imply that the mechanisms underlying the hypothermic increase in catalytic iron, oxidative stress, and cell killing are the same and that preservation of the endothelium may be optimized at temperatures above those routinely used.
Periosteal Cell Pellet Culture System: A New Technique for Bone Engineering
Authors: Akiyama Mari , Nonomura Hidehiko , Kamil Syed H. , Ignotz Ronald A.
Published: April 2008

To treat bone loss that is induced by disease or wounds, bone grafts are commonly used. In dentistry, guided tissue regeneration is effective in the treatment of periodontal diseases. However, bone resorption after implantation is a major problem with the bone graft and guided tissue regeneration technique. This study examines a cell pellet culture system without exogenous scaffolds for bone regeneration. First, we examined the effect of ascorbic acid on cells. Transmission electron microscopic observation revealed that cells formed a three-dimensional structure of multiple cell layers after 5 weeks of culturing in medium containing 50 μg/ml ascorbic acid with the medium changed every 7 days. A single cell pellet was produced by centrifuging cells that were gathered from 10 tissue culture dishes. Van Gieson staining and collagen type I immunostaining showed that the pellet contained collagen fibers and cells that adhered to the collagen fibers. Several of these cell pellets were implanted subcutaneously on the backs of nude mice for 6 weeks. Histology and immunohistochemistry results indicated new bone formation, vascular invasion, and insular areas of calcification. Bone tissue was surrounded by osteoblasts. The appearance of new bone formation is similar to that seen in intramembranous ossification. The present pellet system is reliable and might solve problems of bone resorption after implantation.
Bridging Nigrostriatal Pathway with Fibroblast Growth Factor-Primed Peripheral Nerves and Fetal Ventral Mesencephalon Transplant Recuperates from Deficits in Parkinsonian Rats
Authors: Chiang Yung-Hsiao , Lin Shinn-Zong , Zhou Feng C.
Published: April 2008

Previous studies have indicated that the nigrostriatal dopaminergic (DA) pathway can be reconstructed in hemiparkinsonian rats with a bridge transplantation technique involving fetal ventral mesencephalic transplants and glial cell line-derived neurotrophic factor. In this study, we examined if the nigrostriatal pathway can be restored by combining peripheral nervous tissue with the fetal ventral mesencephalon transplants. Adult rats were injected with 6-hydroxydopamine into left median forebrain bundle. Those with marked rotational behavior, which has been previously shown to indicate complete DA dennervtion, were used for transplant treatments. One month after the lesion, fetal ventral mesencephalic cells were transplanted into the nigral region followed by nigral-striatal grafting of peripheral nerves as a bridge. The bridging nerves (sciatic or intercostals) were pretreated with basic fibrous growth factor (nerve+bFGF+) or Hank's saline (nerve+bFGF-). We found that (a) animals receiving transplants of VM and bFGF+ nerve had a reduction in rotational behavior; (b) animals receiving bFGF- nerve bridge only had a partial improvement in rotation. Reinnervation of tyrosine hydroxylase (TH)-immunoreactive (ir) fibers into the striatum was found in both of the above groups with more innervation in the former than in the latter. No TH-ir fibers in lesioned striatum or reduction in rotational behavior were found in animals receiving VM only, or VM plus bFGF. Taken together, our data indicate that peripheral nerve, with the aid of bFGF, greatly facilitates the reconstitution of the TH pathway from nigra to striatum and improves motor function in hemiparkinsonian rats.