bluuebunny/crossref_metadata_2025 huggingface dataset:
PuraMatrix™ Facilitates Bone Regeneration in Bone Defects of Calvaria in Mice
Authors: Misawa Haruo , Kobayashi Naoya , Soto-Gutierrez Alejandro , Chen Yong , Yoshida Aki , Rivas-Carrillo Jorge David , Navarro-Alvarez Nalu , Tanaka Kimiaki , Miki Atsushi , Takei Jiro , Ueda Tadayoshi , Tanaka Masato , Endo Hirosuke , Tanaka Noriaki , Ozaki Toshifumi
Published: April 2008

Artificial bones have often used for bone regeneration due to their strength, but they cannot provide an adequate environment for cell penetration and settlement. We therefore attempted to explore various materials that may allow the cells to penetrate and engraft in bone defects. PuraMatrix™ is a self-assembling peptide scaffold that produces a nanoscale environment allowing both cellular penetration and engraftment. The objective of this study was to investigate the effect of PuraMatrix™ on bone regeneration in a mouse bone defect model of the calvaria. Matrigel™ was used as a control. The expression of bone-related genes (alkaline phosphatase, Runx2, and Osterix) in the PuraMatrix™-injected bone defects was stronger than that in the Matrigel™-injected defects. Soft X-ray radiographs revealed that bony bridges were clearly observed in the defects treated with PuraMatrix™, but not in the Matrigel™-treated defects. Notably, PuraMatrix™ treatment induced mature bone tissue while showing cortical bone medullary cavities. The area of newly formed bones at the site of the bone defects was 1.38-fold larger for PuraMatrix™ than Matrigel™. The strength of the regenerated bone was 1.72-fold higher for PuraMatrix™ (146.0 g) than for Matrigel™ (84.7 g). The present study demonstrated that PuraMatrix™ injection favorably induced functional bone regeneration.
Enhanced Functional Maturation of Fetal Porcine Hepatocytes in Three-Dimensional Poly-L-lactic Acid Scaffolds: A Culture Condition Suitable for Engineered Liver Tissues in Large-Scale Animal Studies
Authors: Huang Hongyun , Hanada Sanshiro , Kojima Nobuhiko , Sakai Yasuyuki
Published: April 2008

To engineer liver tissues with a clinically significant size, in vivo evaluation of performance using large-scale animal studies are necessary before proceeding to human clinical trials. As pigs are the most suitable candidates, the development of culture conditions suitable for porcine hepatocyte progenitors is very important to engineer pig liver tissue equivalents. We therefore investigated the efficacy of poly-L-lactic acid (PLLA) three-dimensional (3D) scaffolds on the functional maturation of fetal porcine hepatocytes in the presence of various combinations of biofactors. Cells were isolated from pig fetuses obtained from a local slaughterhouse, and cultured for 15 days both in monolayer and PLLA scaffolds. Although 15 days of culture resulted in almost the same ratio of proliferation (about fivefold) in both monolayer and 3D PLLA culture, the PLLA culture with hepatocyte growth factor (HGF, 10 ng/ml) and sodium butylate (Sb, 1 mM) remarkably enhanced various liver-specific functions of fetal porcine hepatocytes. The final attained functions based on the numbers of immobilized cells on day 1 compared with those of day 1 monolayers; 191fold increase in albumin secretion, 70.5-fold increase in cytochrome P450 IA1/2 capacity, 20.9-fold increase in ammonia removal, and 18.0-fold increase in urea synthesis were obtained. These functions were 2.0–3.3-fold higher than those obtained by the same period of monolayer culture. In addition, final attained unit cell-based functions on day 15 were almost comparable to the levels reported for cultures of adult porcine hepatocytes in both monolayer and 3D spheroid cultures. These results demonstrate that the use of a biodegradable polymer-based 3D culture with an appropriate combination of biofactors is a promising approach to maximize functional maturation of hepatocyte progenitors from large animals. In addition, the established culture conditions are worth using to engineer large liver tissue equivalents for pigs in large-animal-based preclinical studies.
Transplantation of Murine Bone Marrow Stromal Cells under the Kidney Capsule to Secrete Coagulation Factor VIII
Authors: Oh Taekeun , Peister Alexandra , Ohashi Kazuo , Park Frank
Published: April 2008

Ectopic cell transplantation has been studied as an alternative to whole organ transplantation or as a method to produce secretable proteins for genetic disorders. In this study, bone marrow stromal cells isolated from C57Bl/6 mice were genetically modified to express either lacZ- or B-domain-deleted human factor VIII. In vitro modification of the isolated bone marrow stromal cells was initially performed by transducing increased doses of VSV-G pseudotyped lentiviral vectors expressing lacZ. At a MOI of 25, all of the bone marrow stromal cells were X-gal positive, which maintained their ability to expand and differentiate prior to transplantation into mice. Extremely poor engraftment was observed in the liver, but transplantation of the bone marrow stromal cells expressing lacZ under the kidney capsule resulted in long-term viable X-gal-positive cells for at least 8 weeks (length of study). In vitro expression of human factor VIII was detected in a dose-dependent manner following bone marrow stromal cell with a factor VIII-expressing lentiviral vector. Transplantation of the factor VIII-expressing bone marrow stromal cells under the kidney capsule led to long-term therapeutic expression in the mouse plasma (1–3 ng/ml; n = 4–5 mice/group) for 8 weeks. This study demonstrated that ectopic transplantation of bone marrow stromal cells under the kidney capsule can be effective as a method to express secretable proteins in vivo.
Transplanted Human Neural Precursor Cells Migrate Widely but Show no Lesion-Specific Tropism in the 6-Hydroxydopamine Rat Model of Parkinson's Disease
Authors: Jain M. , Armstrong R. J. E. , Elneil S. , Barker R. A.
Published: April 2008

Parkinson's disease (PD), while primarily associated with degeneration of nigrostriatal dopamine neurons, is now increasingly recognized to have more widespread cell loss and so the most effective cell replacement therapy should target all these neuronal losses. Neural precursor cells might be ideal in this regard as in certain circumstances they have been shown to migrate widely following transplantation into the CNS. The aim of this study was to investigate whether transplanted human expanded neural precursor cells (hENPs) could migrate to sites of established or evolving pathology in the adult brain using the 6-hydroxydopamine (6-OHDA) rat model of PD. hENPs were grafted into the striatum prior to, at the same time as, or after the animals received a 6-OHDA lesion to the medial forebrain bundle. The presence of donor cells was then assessed in a distant site of cell loss (substantia nigra) or sites where cell death would not be expected (frontal cortex and globus pallidus). Donor cells were found distant from the site of implantation but the migration of these hENPs was not significantly greater in the 6-OHDA-lesioned brain and the cells did not specifically target the site of cell loss in the substantia nigra. The temporal relationship of grafting relative to the lesion, and therefore dopaminergic cell death, did not affect the migration of hENPs nor their differentiation. We conclude that while transplanted hENPs are capable of migration away from the site of implantation, they show no specific tropism for sites of ongoing or established nigral dopaminergic cell loss in this lesion model. Therefore, the use of such cells to replace the range of neurons lost in PD is likely to require a deeper understanding of the migratory cues in the damaged adult brain and some manipulation of these cells prior to transplantation.
Directed Three-Dimensional Growth of Microvascular Cells and Isolated Microvessel Fragments
Authors: Chang Carlos C. , Hoying James B.
Published: April 2008

Tissue engineering has promise as a means for repairing diseased and damaged tissues. A significant challenge in tissue construction relates to the constraints placed on tissue geometries resulting from diffusion limitations. An ability to incorporate a premade vasculature would overcome these difficulties and promote construct viability once implanted. Most in vitro microvascular fabrication strategies rely on surface-associated cell growth, manipulated cell monolayers, or random arrangement of cells within matrix materials. In contrast, we successfully suspended microvascular cells and isolated microvessel fragments within collagen and then microfluidically drove the mixtures into microfabricated network topologies. Developing within the 3D collagen matrix, patterned cells progressed into cord-like morphologies. These geometries were directed by the surrounding elastomer mold. With similar techniques, suspended fragments formed endothelial sprouts. By avoiding the addition of exogenous growth factors, we allowed constituent cells and fragments to autonomously develop within the constructs, providing a more physiologically relevant system for in vitro microvascular development. In addition, we present the first examples of directed endothelial cell sprouting from parent microvessel fragments. We believe this system may serve as a foundation for future in vivo fabrication of microvascular networks for tissue engineering applications.
Inhibition of p38 MAP Kinase in the Early Posttransplantation Phase Redistributes Blood Vessels from the Surrounding Stroma into the Transplanted Endocrine Tissue
Authors: Johansson Åsa , Sandvik Daniel , Carlsson Per-Ola
Published: April 2008

Transplanted pancreatic islets attain a chronically decreased vascular density following transplantation, despite the increased concentrations of vascular endothelial growth factor (VEGF) secreted from beta-cells in response to hypoxia during culture and in the immediate posttransplantation phase. VEGF, however, exerts dual effects on endothelial cells, and in islet endothelial cells of the adult, the vascular permeability-inducing effects of VEGF seem normally more pronounced than those to induce angiogenesis. p38 MAP kinase activity has recently been shown to serve as a switch to separate these properties of VEGF; inhibition of p38 MAP kinase activity enhances VEGF-induced angiogenesis and, at the same time, abrogates VEGF-induced vascular permeability. We hypothesized that the revascularization of transplanted islets may be hampered by a predisposition of adult islet endothelial cells to react to VEGF by forming fenestrae rather than migrating and proliferating. We therefore administered the p38 MAP kinase inhibitor SB203580 by daily IP injections for the first 14 days following transplantation, and then studied the influence of this treatment on the oxygen tension, blood perfusion, and vascular density of the islet grafts 1 month posttransplantation. SB203580 treatment redistributed islet graft blood vessels from the stroma into the endocrine tissue, and this redistribution of blood vessels into the endocrine tissue was accompanied by an increased oxygenation of the islet cells. However, the total number of blood vessels in the tissue was not affected. The blood perfusion of the islet grafts was also similar in control and SB203580-treated animals. Our results suggest that effects of VEGF to preferentially induce vascular permeability may partially contribute to, but is not the main cause of, low revascularization of transplanted islets.
Isolated Pancreatic Islets in Three-Dimensional Matrices are Responsive to Stimulators and Inhibitors of Angiogenesis
Authors: Kidszun André , Schneider Darius , Erb Doris , Hertl Gundula , Schmidt Veronika , Eckhard Michael , Preissner Klaus T. , Breier Georg , Bretzel Reinhard G. , Linn Thomas
Published: April 2008

The formation of a new microvasculature is essential for the long-term survival and function of the islet graft. In this study we examined endothelium of isolated pancreatic islets by stimulation with growth factors, different culture conditions, and genetic modification. We also inspected the effect of immunosuppressives used in human transplantation on angiogenesis. Isolated islets were embedded in a three-dimensional fibrin or Matrigel matrix. The effect of hyperglycemia, hypoxia, and the addition of VEGF and bFGF was investigated. We exposed islets from transgenic mice expressing the VEGF gene (RIP1VEGF-A) to high glucose (16.7 mmol/L) medium and tested the immunosuppressive agents rapamycin (100 ng/ml) and FK506 (100 ng/ml). To quantify angiogenesis the percentage of sprouting islets was determined. New endothelial capillary-like structures protruded from isolated pancreatic islets. Addition of VEGF to the islets and transgenic RIP-VEGF islets showed a two- to threefold increase of sprouting islets compared to control. Hypoxic culture conditions stimulated angiogenesis, resulting in a twofold increase of capillary sprouting. Rapamycin and FK506 proved to be potent inhibitors of angiogenesis in this system, because a decrease of sprouting islets of more than 20% by both agents was observed. Isolated pancreatic islets are capable of forming new capillary structures and are susceptible to pro- and antiangiogenic stimuli.
Fate of Embryonic Stem Cells Transplanted into the Deafened Mammalian Cochlea
Authors: Coleman B. , Hardman J. , Coco A. , Epp S. , De Silva M. , Crook J. , Shepherd R.
Published: April 2008

Spiral ganglion neurons (SGNs), the primary afferent neurons of the cochlea, degenerate following a sensorineural hearing loss (SNHL) due to lack of trophic support normally received from hair cells. Cell transplantation is emerging as a potential strategy for inner ear rehabilitation, as injected cells may be able to replace damaged SGNs in the deafened cochlea. An increase in the number of surviving SGNs may result in improved efficacy of cochlear implants (CIs). We examined the survival of partially differentiated mouse embryonic stem cells (MESCs), following xenograft transplantation into the deafened guinea pig cochlea (n = 15). Cells were delivered directly into the left scala tympani via microinjection through the round window. Small numbers of MESCs were detected in the scala tympani for up to 4 weeks following transplantation and a proportion of these cells retained expression of neurofilament protein 68 kDa in vivo. While this delivery method requires refinement for effective long-term replacement of damaged SGNs, small numbers of MESCs were capable of survival in the deafened mammalian cochlea for up to 4 weeks, without causing an inflammatory tissue response.
A Novel Bioreactor Microcarrier Cell Culture System for High Yields of Proliferating Autologous Human Keratinocytes
Authors: Liu Jin Yu , Hafner Jürg , Dragieva Galya , Burg Günter
Published: April 2008

Rapid and efficient resurfacing of various skin defects by autologous keratinocyte transplantation is significant in skin wound healing. We developed a novel bioreactor microcarrier cell culture system (Bio-MCCS) to produce autologous human keratinocytes on a large scale. In this Bio-MCCS we used porcine gelatin microbeads as microcarriers for autolgous keratinocytes and spinning bottles as fermentation tanks. First, the microbeads were modified by culturing them with autologous dermal fibroblasts that were subsequently killed when they proliferated to confluence on the microbeads. We then performed the Bio-MCCS by expanding ketatinocytes on the microbeads in spinning bottles at 37°C, 5% CO2. Our results showed that keratinocytes rapidly attached to and actively proliferated on the modified microbeads in the Bio-MCCS, achieving high cell densities on the modified microbeads (MTT assay and PI staining). Keratinocytes cultured on the modified microbeads in the Bio-MCCS remained proliferating potentials as shown by positive PCNA staining and BrdU labeling. In contrast, keratinocytes cultured on nonmodified microbeads in the Bio-MCCS proliferated slowly, rapidly ceased to proliferate, and finally dislodged from the microbeads. When removed from the Bio-MCCS and cultured under static conditions, keratinocytes were able to leave the modified microbeads and formed a multilayered epidermal equivalent on the culture surfaces. While stored at room temperature, keratinocytes remained at higher viabilities on the modified microbeads when compared to those on nonmodified microbeads. The achievement of high yields of proliferating autologous keratinocytes by this Bio-MCCS offers a practical potential of resurfacing various skin defects by direct administration of autologous keratinocyte microbeads on various skin defects.
Porcine Fetal Ventral Mesencephalic Cells are Targets for Primed Xenoreactive Human T Cells
Authors: Koopmans Jan , De Haan Aalzen , Bruin Elinda , van der Gun Ieneke , van Dijk Henk , Rozing Jan , de Leij Lou , Staal Michiel
Published: April 2008

Xenotransplantation of porcine fetal ventral mesencephalic (pfVM) cells to overcome the dopamine shortage in the striatum of patients with Parkinson's disease seems a viable alternative to allotransplantion of human fetal donor tissue, especially because the latter is complicated by both practical and ethical issues. There is, however, little known about the xenospecific immune responses involved in such an intracerebral xenotransplantation. The aim of our study was to investigate whether 1) naive human peripheral blood mononuclear cells (PMBC) display cytotoxicity against pfVM cells of E28 pig fetuses, and 2) priming of human PBMC by xenogeneic antigen presenting cells (APC) modulates pfVM-directed cellular cytotoxicity. For this purpose fresh PMBC from nine individual donors were primed by incubation with either irradiated pfVM cells or porcine spleen cells (PSC) as APC in the presence of IL-2 for 1 week before assessing cytotoxicity in a 51Cr release assay. Also, direct NK reactivity and antibody-dependent cellular cytotoxicity (ADCC) of fresh PMBC against pfVM cells was assessed. No direct cytotoxicity of naive cells (either NK reactivity or ADCC) against pfVM cells could be determined. Only PMBC primed with PSC were capable of lysing pfVM cells. PBMC primed with pfVM cells did not show cytolytic capacity towards pfVM. Interestingly, large differences in xenospecific T-cell responses exist between individual donor PBMC. Thus, human T cells are capable of killing pfVM cells in a xenoreactive response, but only after priming by donor APC. The large interindividual differences between human donors in their xenoreactive response may influence patient selection for xenotransplantation and chances of graft survival for individual patients.
Neural Transplantation in Huntington's Disease: The NEST-UK Donor Tissue Microbiological Screening Program and Review of the Literature
Authors: Farrington M. , Wreghitt T. G. , Lever A. M. L. , Dunnett S. B. , Rosser A. E. , Barker R. A.
Published: April 2008

Neural transplantation of human fetal tissue for Huntington's disease (HD) is now entering the clinical arena. The safety of the procedure has now been demonstrated in a number of studies, although the efficacy of such an approach is still being investigated. Stringent but practicable screening of the donor tissue for potential pathogens is an essential prerequisite for successful implementation of any novel transplant program that uses human fetal tissue. In this article we summarize the UK-NEST protocol for the screening of human fetal tissue being grafted to patients with mild to moderate HD. We describe the results of microbiological screening of 87 potential tissue donors in a pilot study, and of the first four donor–recipient patients included in the UK-NEST series. The rationale for the adoption and interpretation of the various tests is described and our methodology is compared with those previously used by other centers. This article therefore presents a comprehensive, logical yet pragmatic screening program that could be employed in any clinical studies that use human fetal tissue for neurotransplantation.
Chemokines and Their Receptors in Islet Allograft Rejection and as Targets for Tolerance Induction
Authors: Merani Shaheed , Truong Wayne W. , Hancock Wayne , Anderson Colin C. , Shapiro A. M. James
Published: April 2008

Graft rejection is a major barrier to successful outcome of transplantation surgery. Islet transplantation introduces insulin secreting tissue into type 1 diabetes mellitus recipients, relieving patients from exogenous insulin injection. However, insulitis of grafted tissue and allograft rejection prevent long-term insulin independence. Leukocyte trafficking is necessary for the launch of successful immune responses to pathogen or allograft. Chemokines, small chemotactic cytokines, direct the migration of leukocytes through their interaction with chemokine receptors found on cell surfaces of immune cells. Unique receptor expression of leukocytes, and the specificity of chemokine secretion during various states of immune response, suggest that the extracellular chemokine milieu specifically homes certain leukocyte subsets. Thus, only those leukocytes required for the current immune task are attracted to the inflammatory site. Chemokine blockade, using antagonists and monoclonal antibodies directed against chemokine receptors, is an emerging and specific immunosuppressive strategy. Importantly, chemokine blockade may potentiate tolerance induction regimens to be used following transplantation surgery, and prevent the need for life-long immunosuppression of islet transplant recipients. Here, the role for chemokine blockade in islet transplant rejection and tolerance is reviewed.
CNS Gene Therapy and a Nexus of Complexity: Systems and Biology at a Crossroads
Authors: Tyler Carolyn M. , Federoff Howard J.
Published: April 2008

Gene therapy is a potentially promising new treatment for neurodegenerative disorders such as Alzheimer's disease (AD), which has been difficult to treat with conventional therapeutics. Viral vector-mediated somatic gene therapy is a rapidly developing methodology for providing never before achieved capability to deliver specific genes to the CNS in a highly localized and controlled manner. With the advent and refinements of this technology one focus is directed to which genes are the most appropriate to select for specific disease indications. Nerve growth factor (NGF), a potent survival factor for critical cell populations that degenerate in AD, has been chosen already for clinical gene therapy trials in human AD patients. Much knowledge about the pathophysiological underpinnings of AD is still lacking to make clear which patients may benefit from a gene therapy approach. Moreover, a detailed understanding of sustained NGF action in the normal and diseased CNS needs to be resolved before conclusions can be drawn regarding the utility of NGF gene therapy. Systematic efforts to acquire this new knowledge should compel clinically and biologically sophisticated efforts to advance gene therapy for neurodegenerative diseases.
Neural Repair Strategies for Parkinson's Disease: Insights from Primate Models
Authors: Soderstrom Katherine , O'malley Jennifer , Steece-Collier Kathy , Kordower Jeffrey H.
Published: April 2008

Nonhuman primate models of Parkinson's disease (PD) have been invaluable to our understanding of the human disease and in the advancement of novel therapies for its treatment. In this review, we attempt to give a brief overview of the animal models of PD currently used, with a more comprehensive focus on the advantages and disadvantages presented by their use in the nonhuman primate. In particular, discussion addresses the 6-hydroxydopamine (6-OHDA), 1-methyl-1,2,3,6-tetrahydopyridine (MPTP), rotenone, paraquat, and maneb parkinsonian models. Additionally, the role of primate PD models in the development of novel therapies, such as trophic factor delivery, grafting, and deep brain stimulation, are described. Finally, the contribution of primate PD models to our understanding of the etiology and pathology of human PD is discussed.
Ameliorating Injury during Preservation and Isolation of Human Islets Using the Two-Layer Method with Perfluorocarbon and UW Solution
Authors: Salehi Payam , Mirbolooki Mohammadreza , Kin Tatsuya , Tsujimura Toshiaki , Shapiro A. M. James , Churchill Thomas A. , Lakey Jonathan R. T.
Published: April 2008

This study assessed the effects of a two-layer method (TLM), using perfluorocarbon and UW solution, on the quality of human pancreata following storage and islet yield/function after isolation. In part A, TLM was applied immediately after procurement and the energetic profile was compared to a group treated with UW solution only (control) throughout 24-h storage. In part B, cadaveric human pancreata were procured and subjected to a TLM after cold storage in UW solution (TLM group) or UW solution (control group). Energetics, lipid peroxidation, and islet recovery/function were assessed after preservation at 4°C. In part A, after 9-h storage, the energetic profile (ATP, ATP/ADP, energy charge) for the TLM group was superior to controls. In part B, TLM treatment resulted in consistently greater ATP, ATP/ADP, and energy charge values than with storage in UW solution alone (p < 0.05). UW treatment resulted in 40% greater peroxidative damage than in the TLM group (p < 0.05). Islet recovery and functional viability were 30–40% higher following TLM treatment (p < 0.05). These data support the hypothesis that islet viability and yields can be significantly improved using a brief period of TLM treatment following conventional UW storage; reduced energetic and oxidative stress are implicated as potential mechanisms.
Subnormothermic Preservation Maintains Viability and Function in a Porcine Hepatocyte Culture Model Simulating Bioreactor Transport
Authors: Van De Kerkhove M.-P. , Hoekstra R. , Van Nooijen F. C. , Spoelstra F. O. B. , Doorschodt B. M. , Van Wijk A. C. W. A. , Poyck P. P. C. , Chamuleau R. A. F. M. , Van Gulik T. M.
Published: April 2008

Bioartificial liver (BAL) systems have been developed to bridge patients with acute liver failure (ALF) to liver transplantation or liver regeneration. Clinical application of BAL systems is dependent on the supportive quality of cells used and direct availability of the whole system. Reliable transport of BAL systems from the laboratory to remote treatment centers is therefore inevitable. Subsequently, preservation conditions play a crucial role during transport of a BAL, with temperature being one of the most determining factors. In this study, we assessed the effect of subnormothermic preservation on freshly isolated porcine hepatocytes cultured in monolayer under oxygenation. Additionally, the effect of the University of Wisconsin (UW) preservation solution was compared with Williams' E (WE) culture medium at 4°C. The control group was cultured for 3 days at 37°C, whereas the transport groups were cultured at 4°C, 15°C, 21°C, or 28°C for 24 h at day 2. All groups were tested each day for cell damage and hepatic functions. Subnormothermic culture (i.e., 15°C to 28°C) for a period of 24 h did not reduce any hepatic function and did not increase cellular damage. In contrast, culture of hepatocytes in WE medium and preservation in UW solution at 4°C significantly reduced hepatic function. In conclusion, freshly isolated porcine hepatocytes can be preserved for 24 h at subnormothermic temperatures as low as 15°C. Future research will focus on the implementation of the AMC-BAL in an oxygenated culture medium perfusion system for transport between the laboratory and the hospital.
Engrafted Neural Progenitor Cells Express a Tissue-Restricted Reporter Gene Associated with Differentiated Retinal Photoreceptor Cells
Authors: Sam Thien N. , Xiao Jing , Roehrich Heidi , Low Walter C. , Gregerson Dale S.
Published: April 2008

Neural progenitor cells (NPCs) have shown ability to repair injured CNS, and might provide precursors to retinal neurons. NPCs were isolated from the brains of 14 day murine embryos of transgenic mice that express β-galactosidase (β-gal) on the arrestin promoter, which specifically directs expression to retinal photoreceptor cells. NPCs were transferred to adult, syngeneic mice via inoculation into the anterior chamber of the eye, the peritoneal cavity, or the brain. At 14 weeks postgrafting, tissues were collected and examined to determine if differentiated NPC progeny were present in retina based on histochemical detection of β-gal. Four of six anterior chamber-inoculated recipients showed Bluo-gal-stained cells in retina, indicating the presence of transferred NPCs or their progeny. Because the progenitor cells do not express β-gal, positive staining indicates differentiation leading to activation of the arrestin promoter. Two recipients inoculated by the intraperitoneal route also exhibited Bluo-gal staining in retina. The NPCs did not express β-gal if inoculated into brain, but survived and dispersed. Most recipients, regardless of inoculation route, were PCR positive for β-gal DNA in extraocular tissues, but no Bluo-gal staining was found outside of the retina. Injury to the retina promoted, but was not required, for progenitor cell engraftment. β-Gal-positive cells were concentrated in the outer layers of the retina. In summary, a reporter gene specifically expressed in differentiated retinal photoreceptor cells due to the activity of the arrestin promoter was expressed in recipient mouse retina following transfer of NPCs prepared from the β-gal transgenic mice. The presence of β-gal DNA, but not Bluo-gal staining, in spleen and other tissues revealed that the cells also migrated elsewhere and took up residence in other organs, but did not undergo differentiation that led to β-gal expression.
Viability and Functionality of Bovine Chromaffin Cells Encapsulated into Alginate-PLL Microcapsules with a Liquefied Inner Core
Authors: Moustafa T. , Girod S. , Tortosa F. , Li R. , Sol J. C. , Rodriguez F. , Bastide R. , Lazorthes Y. , Sallerin B.
Published: April 2008

Implantation of adrenal medullary bovine chromaffin cells (BCC), which synthesize and secrete a combination of pain-reducing neuroactive compounds including catecholamines and opioid peptides, has been proposed for the treatment of intractable cancer pain. Macro- or microencapsulation of such cells within semi-permeable membranes is expected to protect the transplant from the host's immune system. In the present study, we report the viability and functionality of BCC encapsulated into microcapsules of alginate-poly-L-lysine (PLL) with a liquefied inner core. The experiment was carried out during 44 days. Empty microcapsules were characterized in terms of morphology, permeability, and mechanical resistance. At the same time, the viability and functionality of both encapsulated and nonencapsulated BCC were evaluated in vitro. We obtained viable BCC with excellent functionality: immunocytochemical analysis revealed robust survival of chromaffin cells 30 days after isolation and microencapsulation. HPLC assay showed that encapsulated BCC released catecholamines basally during the time course study. Taken together, these results demonstrate that viable BCC can be successfully encapsulated into alginate-PLL microcapsules with a liquefied inner core.
Comparison of Cooling Systems during Islet Purification
Authors: Swift Sue , Kin Tatsuya , Mirbolooki Mohammadreza , Wilson Richard , Lakey Jonathan R. T.
Published: April 2008

Islet isolation is a complex procedure that includes digestion and purification of pancreatic tissue. As we move towards clinical regulatory control and standardization, understanding of the detailed stages of the procedure have become increasingly important. Purification on a COBE 2991 density gradient allows human islets to be separated from a large volume of acinar tissue. Cooling the gradient and tissue is thought to be important to reduce metabolic activity but cooling systems for the gradient are expensive, with limited availability. In this study, the efficiency of cooling methods for the COBE 2991 cell separator has been investigated. The two cooling systems were: a) COBE 2991 modified internally to allow coolant (polyethylene glycol) from a chiller to circulate either side of the spindle and around the bowl (original system), and b) an air-cooled system using an air conditioner to blow cold air into the bowl from above (air cooler system). Cooling required 20 min for the original system and temperature was stabilized within 4–7°C. The air system cooled rapidly but was not stable. There was an increase in the temperature of the medium with using both systems during centrifugation because of heat generated by the COBE machine; however, the temperature of the medium after centrifugation with the air system was significantly higher than that with the original system (13.3 ± 0.2°C vs. 8.7 ± 0.7°C, p < 0.05). The original cooler system was found to be more efficient at reducing heat generated by the COBE machine than the air system. Further investigation of the importance of the recorded temperatures is required.
Hepatocyte Transplantation in the Long Evans Cinnamon Rat Model of Wilson's Disease
Authors: Park Seon Mee , Vo Kim , Lallier Michel , Cloutier Alexis-Simon , Brochu Pierre , Alvarez Fernando , Martin Steven R.
Published: April 2008

Wilson's disease (WD), caused by a mutation in the P-type copper transporting ATPase (Atp7b) gene, results in excessive accumulation of copper in the liver. Long Evans Cinnamon rats (LEC) bear a mutation in the atp7b gene and share clinical characteristics of human WD. To explore hepatocyte transplantation (HT) as therapy for metabolic liver diseases, 8-week-old LEC rats (n = 12) were transplanted by intrasplenic injection of hepatocytes from donor Long Evans (LE) rats. Immunosuppression was maintained with intraperitoneal tacrolimus. The success of HT was monitored at 24 weeks of life. Serum aminotransferases and bilirubin peaked at 14–21 weeks in both HT rats and nontransplanted controls, but at 24 weeks, survival was 97% in LEC-HT versus 63% in controls. All transplanted rats showed restored biliary copper excretion and reduced liver iron concentration associated with increased ceruloplasmin oxidase activity. Liver tissue expressed atp7b mRNA (11.9 ± 13.6%) indicative of engraftment of normal cells in 7 of 12 HT rats, associated with a reduced liver copper concentration compared to untreated LEC rats. Periportal islets of normal appearing hepatocytes, recognized by atp7b antibody, were observed in transplanted livers while lobular host cells showed persistent pleomorphic changes and inflammatory infiltrates. In conclusion, transplantation of normal hepatocytes prevented fulminant hepatitis, reduces chronic inflammation, and improved 6-month survival in LEC rats. Engraftment of transplanted cells, which express atp7b mRNA, repopulated the recipient liver with normal functional capacity.
Sertoli Cells Induce Systemic Donor-Specific Tolerance in Xenogenic Transplantation Model
Authors: Shamekh R. , El-Badri N. S. , Saporta S. , Pascual C. , Sanberg P. R. , Cameron D. F.
Published: April 2008

Cell therapy is a potentially powerful tool in the treatment of many grave disorders including leukemia, immune deficiencies, autoimmune diseases, and diabetes. However, finding matched donors is challenging and recipients may suffer from the severe complications of systemic immune suppression. Sertoli cells, when cotransplanted with both allo- and xenograft tissues, promote graft acceptance in the absence of systemic immunosuppression. How Sertoli cells do this is not, as yet, clearly defined. We have examined the ability of Sertoli cells to produce systemic immune tolerance. For this purpose, Sertoli cells were injected into an otherwise normal C57/BL6 mouse host via the lateral tail vein. No other immunosuppressive protocols were applied. Six to 8 weeks posttransplantation, blood was collected for analysis of cytokine levels. Tolerance to donor cells was determined by mixed lymphocytic culture, and production of T-cell-dependent antibody was determined by an in vitro anti-sheep red blood cell plaque-forming assay. Results showed a marked modulation of immune cytokines in the transplanted mouse host and donor-specific transplantation tolerance was achieved. Tolerant mouse lymphocytes maintained a competent humoral antibody response. Additionally, C57/BL6 mice transplanted with rat Sertoli cells tolerated rat skin grafts significantly longer than control non-Sertoli cell transplanted mice. We conclude that systemic administration of rat Sertoli cells across xenogenic barrier induces transplantation tolerance without altering systemic immune competence. These data suggest that Sertoli cells may be used as a novel and potentially powerful tool in cell transplantation therapy.
Cryopreserved Fetal Liver Cell Transplants Support the Chronic Failing Liver in Rats with CCl4-Induced Cirrhosis
Authors: Ochenashko Olga V. , Volkova Nataliya A. , Mazur Svetlana P. , Somov Alexander Yu. , Fuller Barry J. , Petrenko Alexander Yu.
Published: April 2008

Hepatocyte transplantation is a promising method for supporting hepatic function in a broad spectrum of liver diseases. The aim of this work was to test the efficacy of human fetal liver cells to support the chronic failing liver in an experimental model of carbon tetrachloride (CCl4)-induced cirrhosis in rats. Liver cirrhosis was induced by intraperitoneal administration of CCl4 at a dose of 0.2 ml (50% v/v solution)/100 g body weight, twice a week for 3 months in rats. Ten days after stopping CCl4 administration (experimental day 0), rats received intrasplenic injection of cryopreserved fetal liver cells (FLC, 1 × 107 cells in 0.3 ml medium). As a cirrhotic control group, CCl4-induced cirrhotic rats were used with intrasplenic injection of an equal volume of medium alone. Animals were sacrificed on experimental day 15. Human fetal liver cell transplantation almost completely prevented the death of cirrhotic animals during the 2 weeks after treatment, while high ongoing mortality was seen in the cirrhotic control group. Cell transplantation into the spleen normalized total bilirubin and TBARSs levels and increased albumin levels in blood serum, as well as restoring mitochondrial function and liver detoxification function (assessed by cytochrome P450 contents and activity) compared with the activities seen in the cirrhosis control group. In parallel with this restoration of biochemical and functional liver indices, morphological patterns of liver recovery or regeneration after liver cell transplantation were demonstrated in day 15 samples by light microscopy. These were absent in the group that had received only medium alone.
End-Stage Organ Failure: Will Regenerative Medicine Keep its Promise?
Authors: Triolo Fabio , Gridelli Bruno
Published: April 2008

End-stage organ failure is a major cause of death worldwide that can occur in patients of all ages and transplantation is the current standard of care for chronic end-stage disease of many organs. Despite the success of organ transplantation, it is becoming clear that there will never be enough organs made available through donation to meet the increasing demand. The past decade's rapid advancement in stem cell biology and tissue engineering generated an explosive outburst of reports that gave rise to regenerative medicine, a new field that promises to “fix” damaged organs through regeneration provided by transplanted cells, stimulation of endogenous repair mechanisms, or implantation of bioengineered tissue. Whether, and if so when, regenerative medicine will keep its promise is uncertain. As we continue to strive to find new effective solutions, alternative approaches based on the development of targeted, preventive interventions aimed at maintaining normal organ function, instead of repairing organ damage, should also be pursued.
The Regenerative Potential of Stem Cells in Acute Renal Failure
Authors: Morigi Marina , Benigni Ariela , Remuzzi Giuseppe , Imberti Barbara
Published: April 2008

Adult stem cells have been characterized in several tissues as a subpopulation of cells able to maintain, generate, and replace terminally differentiated cells in response to physiological cell turnover or tissue injury. Little is known regarding the presence of stem cells in the adult kidney but it is documented that under certain conditions, such as the recovery from acute injury, the kidney can regenerate itself by increasing the proliferation of some resident cells. The origin of these cells is largely undefined; they are often considered to derive from resident renal stem or progenitor cells. Whether these immature cells are a subpopulation preserved from the early stage of nephrogenesis is still a matter of investigation and represents an attractive possibility. Moreover, the contribution of bone marrow-derived stem cells to renal cell turnover and regeneration has been suggested. In mice and humans, there is evidence that extrarenal cells of bone marrow origin take part in tubular epithelium regeneration. Injury to a target organ can be sensed by bone marrow stem cells that migrate to the site of damage, undergo differentiation, and promote structural and functional repair. Recent studies have demonstrated that hematopoietic stem cells were mobilized following ischemia/reperfusion and engrafted the kidney to differentiate into tubular epithelium in the areas of damage. The evidence that mesenchymal stem cells, by virtue of their renoprotective property, restore renal tubular structure and also ameliorate renal function during experimental acute renal failure provides opportunities for therapeutic intervention.
Serum-Free Medium Provides a Clinically Relevant Method to Increase Olfactory Ensheathing Cell Numbers in Olfactory Mucosa Cell Culture
Authors: Ito Daisuke , Fujita Naoki , Ibanez Chrystelle , Sasaki Nobuo , Franklin Robin J. M. , Jeffery Nick D.
Published: April 2008

There is much evidence to suggest that transplanted olfactory ensheathing cells may ameliorate the functional deficits associated with injuries to the nervous system, especially the spinal cord. For clinical implementation of this strategy it will be necessary to derive large numbers of these cells from an accessible and, preferably, autologous source, implying that olfactory mucosa would be ideal. Although olfactory ensheathing cells can be derived from olfactory mucosa, in routine culture conditions the proportion of these cells is unacceptably low for clinical purposes. This study compared the effect of culturing dissociated olfactory bulb and olfactory mucosa in two different media: one containing serum and one serum free. The results indicate that olfactory ensheathing cell proportion, and absolute cell numbers, is greatly increased in serum-free conditions. Further analysis suggests that serum-free medium has a differential effect on contaminating fibronectin-positive and p75-positive cells from olfactory bulb and olfactory mucosa. This study demonstrates that serum-free culture conditions provide a simple and useful means of deriving a sufficient number of olfactory ensheathing cells for transplantation and reveals a difference in biological behavior of the cells contained within olfactory bulb and olfactory mucosa.
Concentration of Bone Marrow Total Nucleated Cells by a Point-of-Care Device Provides a High Yield and Preserves Their Functional Activity
Authors: Hermann Patrick C. , Huber Stephan L. , Herrler Tanja , Von Hesler Christoph , Andrassy Joachim , Kevy Sherwin V. , Jacobson May S. , Heeschen Christopher
Published: April 2008

Stem and progenitor cell therapy is a novel strategy to enhance cardiovascular regeneration. Cell isolation procedures are crucial for the functional activity of the administered cellular product. Therefore, new isolation techniques have to be evaluated in comparison to the Ficoll isolation procedure as the current gold standard. Here we prospectively evaluated a novel point-of-care device (Harvest BMAC System) for the concentration of bone marrow total nucleated cells (TNC) in comparison to the Ficoll isolation procedure for bone marrow mononucleated cells (MNC). The yield in total numbers of TNC was 2.4-fold higher for Harvest compared to Ficoll. Despite significant differences in their cellular compositions, the colony-forming capacity was similar for both products. Intriguingly, the migratory capacity was significantly higher for the Harvest TNC (164 ± 66%; p = 0.007). In a mouse model of hind limb ischemia, the increase in blood flow recovery was similar between Harvest BM-TNC and Ficoll BM-MNC (0.53 ± 0.20 vs. 0.46 ± 0.15; p = 0.88). However, adjustment of the injected cell number based on the higher yield of Harvest TNC resulted in a significant better recovery (0.64 ± 0.16 vs. 0.46 ± 0.15; p = 0.003). Cells concentrated by the Harvest point-of-care device show similar or greater functional activity compared to Ficoll isolation. However, the greater yield of cells and the wider range of cell types for the Harvest device may translate into an even greater therapeutic effect.
Recombinant AAV Viral Vectors Serotype 1, 2, and 5 Mediate Differential Gene Transfer Efficiency in Rat Striatal Fetal Grafts
Authors: Lubansu Alphonse , Abeloos Laurence , Bockstael Olivier , Lehtonen Enni , Blum David , Brotchi Jacques , Levivier Marc , Tenenbaum Liliane
Published: April 2008

Intrastriatal grafts of fetal ganglionic eminences (GE) can reverse symptoms of striatal lesions in animal models of Huntington's disease. On the other hand, neurotrophic factors have been shown to protect host striatal neurons from ongoing degeneration. Neurotrophic gene transfer into GE prior to grafting could combine the benefits of striatal neuron replacement and in situ delivery of neurotrophic factors. Here we evaluate the potency of recombinant adeno-associated viruses (rAAV) as vectors for gene delivery into rat embryonic (E15) GE using the eGFP reporter gene under the control of the strong cytomegalovirus (CMV) promoter. We observed a very efficient expression of the eGFP reporter gene in organotypic cultures of GE infected with rAAV serotype 1 from 4 days until at least 4 weeks postinfection. In contrast, transduction was low and absent when using serotype 2 and serotype 5 rAAV, respectively. Two months after transplantation of rAAV2/1-infected embryonic GE in adult rat striatum, more than 20% of grafted cells expressed eGFP. The majority of transduced cells in the graft were neurons as indicated by colabeling of GFP-immunoreactive cells with the NeuN marker. Our study suggests that GE transduced by rAAV-serotype 1 vectors could be an interesting tool to mediate efficient expression of a gene coding a neurotrophic factor in Huntington's disease.
The TIM Family of Cosignaling Receptors: Emerging Targets for the Regulation of Autoimmune Disease and Transplantation Tolerance
Authors: Truong Wayne , Shapiro A. M. James
Published: April 2008

Currently, lifelong immune suppression regimens are required for solid organ and cellular transplantation and carry significant increased risk of infection, malignancy, and toxicity. For non-life-saving procedures such as islet transplantation, the risk/benefit ratio of lifelong immunosuppression versus benefit from transplantation requires even more careful balance. The search for specific agents to modulate the immune system without chronic immunosuppression is important for the broad application of islet transplantation. The T-cell immunoglobulin mucin (TIM) family is a distinct group of coreceptors that are differentially expressed on TH1 and TH2 cells, and have the potential to regulate both cytotoxic and humoral immune responses. Completed murine studies demonstrate Tim pathways may be important in the regulation of tolerance to self (auto), harmless (allergic), and transplant (allo) antigen; however, the potential impact of targeting Tim coreceptors has yet to be fully explored in transplantation tolerance induction or autoimmune disease. The current review examines the impact of Tim coreceptor targeting as an emerging therapeutic option for regulating autoimmune diseases and prevention of allograft rejection.