bluuebunny/crossref_metadata_2025 huggingface dataset:
Extensive Axonal and Glial Fiber Growth from Fetal Porcine Cortical Xenografts in the Adult Rat Cortex
Authors: Garcia Antony R. , Deacon Terrence W. , Dinsmore Jonathan , Isacson Ole
Published: June 2017

Axonal growth from cortically placed fetal neural transplants to subcortical targets in adult hosts has been difficult to demonstrate and is assumed to be minimal; however, experiments using xenogeneic neural grafts of either human or porcine fetal tissues into the adult rat striatum, mesencephalon, and spinal cord have demonstrated the capability for long-distance axonal growth. This study reports similar results for porcine cortical xenografts placed in the adult rat cerebral cortex and compares these findings with results from cortical allografts. Adult rats that previously received unilateral cortical lesions by an oblique intracortical stereotaxic injection of quinolinic acid, were implanted with suspensions of either E14 rat or E38 xenogeneic porcine fetal cortical cells. Xenografted rats were immunosuppressed by cyclosporin A. The corpus callosum was intact in all cases and grafts were confined to the overlying cortex. After a 31-34 wk posttransplant survival period, acetylcholinesterase (AChE) staining and tyrosine hydroxylase (TH) immunocytochemistry revealed that both allo- and xenografts received host afferents. Retrograde tracer injections into the ipsilateral striatum and cerebral peduncle in allografted animals failed to show any axonal growth to either subcortical target. Using a porcine-specific axonal marker in xenografted animals, we found graft axons in white matter tracts (corpus callosum, internal capsule, cingulum bundle, and medial forebrain bundle) and within the caudate-putamen and both the ipsilateral and contralateral cerebral cortex. Graft axons were not found in the thalamus, midbrain, or spinal cord. In addition, using an antibody to porcine glial fibers, we observed more extensive graft glial fiber growth into the same host fiber tracts, as far caudally as the cerebral peduncle, but not into gray matter targets outside the cortex. These results demonstrate that porcine cortical xenograft axons and glia can extend from lesioned cerebral cortex to cortical and subcortical targets in the adult rat brain. These findings are relevant for prospects of repairing cortical damage and obtaining functional recovery.
Effective Cryopreservation and Long-Term Storage of Primary Human Hepatocytes with Recovery of Viability, Differentiation, and Replicative Potential
Authors: Adams R. Mark , Wang Mary , Crane Ana Maria , Brown Bridgette , Darlington Gretchen J. , Ledley Fred D.
Published: June 2017

Despite reports of successful cryopreservation of primary human hepatocytes, existing methods do not produce sufficient recovery of viable cells to meet the needs of basic research or clinical trials of hepatocellular transplantation. We now describe a protocol for efficient cryopreservation of primary human hepatocytes using University of Wisconsin (UW) solution, fetal bovine serum, and dimethyl sulfoxide (DMSO). This method provides >90% viability of differentiated, primary human hepatocytes 8 mo after cryopreservation as measured by trypan blue exclusion, preserves hepatocyte morphology, liver-specific gene expression α1 antitrypsin), and replication. The effectiveness of UW solution as a cryopreservative agent suggests that metabolic as well as ultrastructural factors may be important in the effective cryopreservation of primary human hepatocytes. The present method represents an effective protocol for cryopreserving differentiated primary human hepatocytes for research. This method may allow characterization and banking of human hepatocytes for clinical applications, including hepatocellular transplantation and hepatic assist devices.
Phenotypic Reversion of Smooth Muscle Cells in Hybrid Vascular Prostheses
Authors: Kanda Keiichi , Miwa Hiromichi , Matsuda Takehisa
Published: June 2017

Our purpose was to evaluate whether or not and when phenotypic modulation of smooth muscle cells (SMCs) in hybrid vascular prostheses preincorporated with SMCs occurs upon implantation. Two types of hybrid vascular grafts incorporated with vascular cells derived from canine jugular veins were prepared: grafts containing a collagen gel layer covered with an endothelial monolayer at the luminal surface (Model I graft) and those containing an endothelial monolayer and SMC multilayer (Model II graft). They were bilaterally implanted into carotid arteries of the same dogs from which the cells had been harvested for 2 wk (n = 3) and 12 wk (n = 3). The time-dependent changes in populations of three SMC phenotypes (synthetic, intermediate, and contractile) in the neoarterial layers were quantified by morphometric evaluation using a transmission electron microscope in hybrid vascular grafts. Before implantation, all the SMCs were of the synthetic phenotype. In Model II grafts at 2 wk, synthetic and intermediate SMCs were dominant especially in the luminal layer. On the other hand, neoarterial layers at 12 wk were dominated by contractile SMCs, which were evenly distributed throughout the entire neoarterial tissues. A markedly delayed phenotypic reversion was noted for the Model I grafts at 12 wk. In the hybrid grafts, during about 3 mo of implantation, neoarterial SMCs transformed from the synthetic to the contractile phenotypes, which was promoted by SMC incorporation.
Amount and Distribution of Collagen in Pancreatic Tissue of Different Species in the Perspective of Islet Isolation Procedures
Authors: van Suylichem Paul T.R. , van Deijnen Jan-Erik H.M. , Wolters Gerrit H.J. , van Schilfgaarde Reinout
Published: June 2017

Because collagen is the major target in the enzymatic dissociation of the pancreas for islet isolation, we determined the amount of collagen and its distribution in a comparative study comprising normal pancreata of rat, dog, man, young pig, and adult pig. Collagen content was determined using a colorimetric method and its distribution was assessed in tissue sections stained with Sirius red. The collagen content is relatively low in the rat and adult pig pancreas, and the amount of collagen is relatively low in the septa of the rat and dog pancreas. Not the amount of collagen in the septa but collagen in the rest of the pancreas, mainly located between the acini, seems to determine the dissociation of the pancreatic tissue. This can be exemplified by the higher islet yields obtained from the adult vs. the young pig pancreas; the latter contains a higher total amount of collagen but a similar, relatively high, amount of collagen in the septa. A high amount of collagen surrounding the islets seems to be of secondary importance in islet isolations, because yields of the same magnitude are obtained from the canine and human pancreas containing a relatively low vs. high amount of collagen around the islets but a similar total collagen content. The rat pancreas contains both a low total amount of collagen and a high amount of collagen around the islets; therefore, the general experience that islet isolation procedures are effective in rats can be readily understood.
Embryonic Stem Cells Differentiated in Vitro as a Novel Source of Cells for Transplantation
Authors: Dinsmore Jonathan , Ratliff Judson , Deacon Terry , Pakzaba Peyman , Jacoby Douglas , Galpern Wendy , Isacson Ole
Published: June 2017

The controlled differentiation of mouse embryonic stem (ES) cells into near homogeneous populations of both neurons and skeletal muscle cells that can survive and function in vivo after transplantation is reported. We show that treatment of pluripotent ES cells with retinoic acid (RA) and dimethylsulfoxide (DMSO) induce differentiation of these cells into highly enriched populations of γ-aminobutyric acid (GABA) expressing neurons and skeletal myoblasts, respectively. For neuronal differentiation, RA alone is sufficient to induce ES cells to differentiate into neuronal cells that show properties of postmitotic neurons both in vitro and in vivo. In vivo function of RA-induced neuronal cells was demonstrated by transplantation into the quinolinic acid lesioned striatum of rats (a rat model for Huntington's disease), where cells integrated and survived for up to 6 wk. The response of embryonic stem cells to DMSO to form muscle was less dramatic than that observed for RA. DMSO-induced ES cells formed mixed populations of muscle cells composed of cardiac, smooth, and skeletal muscle instead of homogeneous populations of a single muscle cell type. To determine whether the response of ES cells to DMSO induction could be further controlled, ES cells were stably transfected with a gene coding for the muscle-specific regulatory factor, MyoD. When induced with DMSO, ES cells constitutively expressing high levels of MyoD differentiated exclusively into skeletal myoblasts (no cardiac or smooth muscle cells) that fused to form myotubes capable of spontaneous contraction. Thus, the specific muscle cell type formed was controlled by the expression of MyoD. These results provided evidence that the specific cell type formed (whether it be muscle, neuronal, or other cell types) can be controlled in vitro. Further, these results demonstrated that ES cells can provide a source of multiple differentiated cell types that can be used for transplantation.
Migration Patterns of Neonatal Subventricular Zone Progenitor Cells Transplanted into the Neonatal Striatum
Authors: Betarbet Ranjita , Zigova Tanja , Bakay Roy A.E. , Luskin Marla B.
Published: June 2017

Our previous studies have shown that the progeny of the neuronal progenitor cells localized in a discrete region of the anterior part of the neonatal subventricular zone, referred to as the SVZa, migrate tangentially along a stereotypical and extended pathway to the olfactory bulb, and then turn radially into one of the overlying cellular layers. In this study we have examined whether the SVZa cells retain their ability to migrate and disperse when heterotopically transplanted into the striatum. SVZa cells from P0–P2 rat pups were microdissected, dissociated, labeled with the lipophilic, fluorescent dye PKH26 or the cell proliferation marker BrdU, and then transplanted into the neonatal (P0–P2) striatum. Examination of the striatum a few days after transplantation revealed aggregates of heavily labeled BrdU-positive, SVZa cells in the striatum, often situated near blood vessels. Two to four weeks after transplantation, however, the labeled SVZa cells had disseminated from their site of implantation and showed three patterns of distribution. In none of the cases was the implantation site detectable in the striatum, signifying that the cells had become incorporated in the host brain. Of the 12 brains analyzed for cell distribution, transplanted SVZa cells were confined to the striatum in 4 cases. The cells were present as individual cells or in small groups of usually two to four cells. When PKH26 was used, we found that many of the transplanted cells extended processes into the striatum. In 3 out of the 12 animals, the labeled SVZa cells were distributed along the dorsal and lateral aspects of the striatal boundary. In the remaining five animals, labeled SVZa cells appeared in both locations: within the striatum as well as along the striatal boundary. The dispersion of the transplanted cells within the striatum and the presence of the transplanted SVZa cells all along the striatal boundary, a region corresponding to the lateral cortical stream of migration of the developing forebrain, demonstrates that the isochronically transplanted SVZa cells retained their capacity to migrate.
Morphological Differentiation of Astroglial Progenitor Cells from Egf-Responsive Neurospheres in Response to Fetal Calf Serum, Basic Fibroblast Growth Factor, and Retinol
Authors: Chiang Yung H. , Silani Vincenzo , Zhou Feng C.
Published: June 2017

Procurement of multipotential neuroglial stem cells is possible with the addition of epidermal growth factor (EGF). Stem cells will differentiate into neurons and glia upon the removal of EGF from the culture medium. We have previously characterized the neuronal differentiation of stem cells derived from long-term cultured nonpassage neurospheres. In the current study, we (1) characterize the morphological differentiation of the astroglial progenitor cell from 3-mo-old neurospheres, (2) examine whether the astroglial progenitor cells from neurospheres of different brain areas exhibit different differentiation responses to the same exogenous signals, and (3) test the effects of basic fibroblast growth factor (bFGF) and retinol on differentiation. Cerebral cortex, striatum, and mesencephalon cells were obtained from Embryonic Day 14 (E-14) rat fetuses and were dissociated for the procurement of neurospheres in chemically defined medium supplemented with EGF. After 3 mo in culture, the neurospheres, derived from each of the three brain areas, were subcultured into three groups on chamber slides: (1) basal medium, (2) the basal medium plus 20 ng/mL bFGF, and (3) the basal medium plus 10 μM retinol. Phenotypic expression of astroglial cells was examined after 14 days subculture. Our findings indicate that the 3-mo-old cultured nonpassage neurospheres contained numerous multipotential stem cells that stained positive with nestin, and that environmental factors played an important role in influencing the differentiation of astroglial progenitor cells. As detected by glial fibrillary acid protein (GFAP), astroglial progenitor cells turned into protoplasmic astrocytes in the FCS-containing basal medium, fibrous astrocytes in the presence of bFGF, and spindle-shaped astrocytes in the presence of retinol. There were no noticeable differences in differentiation among astroglial progenitor cells of the various brain region-derived neurospheres in any of the three medium conditions. Peculiar varicosity-and growth cone-like structures on the long slender GFAP-positive processes suggest that neuroblasts and glioblast may share common morphologies, features, or common progenitor cells during initial differentiation in vitro.
Cell-Mediated Delivery of Brain-Derived Neurotrophic Factor Enhances Dopamine Levels in an Mpp+ Rat Model of Substantia Nigra Degeneration
Authors: Galpern Wendy R. , Frim David M. , Tatter Stephen B. , Altar C. Anthony , Beal M. Flint , Isacson Ole
Published: June 2017

Brain-derived neurotrophic factor (BDNF) promotes the survival of fetal mesencephalic dopaminergic cells and protects dopaminergic neurons against the toxicity of MPP+ in vitro. Supranigral implantation of fibroblasts genetically engineered to secrete BDNF attenuates the loss of substantia nigra pars compacta (SNc) dopaminergic neurons associated with striatal infusion of MPP+ in the adult rat. Using this MPP+ rat model of nigral degeneration, we evaluated the neurochemical effects of supranigral, cell-mediated delivery of BDNF on substantia nigra (SN) dopamine (DA) content and turnover. Genetically engineered BDNF-secreting fibroblasts (~12 ng BDNF/24 h) were implanted dorsal to the SN 7 days prior to striatal MPP+ administration. The present results demonstrate that BDNF-secreting fibroblasts, as compared to control fibroblasts, enhance SN DA levels ipsilateral as well as contralateral to the graft without altering DA turnover. This augmentation of DA levels suggests that local neurotrophic factor delivery by genetically engineered cells may provide a therapeutic strategy for preventing neuronal death or enhancing neuronal function in neurodegenerative diseases characterized by dopaminergic neuronal dysfunction, such as Parkinson's disease.
Immunomodulation with Intrathymic Grafts or Anti-Lymphocyte Serum Promotes Long-Term Intraspinal Allograft Survival
Authors: Theele Daniel P. , Reier Paul J.
Published: June 2017

In this study, we sought to test whether introduction of fetal cells into the adult rat thymus would promote immunotolerance to subsequent donor-type allografts in the injured spinal cord. To first evaluate intrathymic survival of fetal central nervous system (CNS) tissue, fragments of E14 Sprague–Dawley (SD) fetal spinal cord (FSC,SD) were injected into the thymuses of either adult, outbred SD, or Wistar rats. Histological examination revealed well-differentiated grafts in both the SD (10 out of 13) and Wistar (7 out of 13) recipients. We next examined whether prior intrathymic exposure to FSC graft-derived alloantigens leads to enhanced survival of subsequent allografts into the injured, adult spinal cord. Wistar rats thus first received FSCSD tissue as intrathymic grafts coupled with single-dose, anti-lymphocyte serum (ALS) ablation of the circulating host T-cell population. Ten days later, FCSSD was transplanted into an aspiration lesion of each intrathymic graft recipient's spinal cord. After 60 days, 87% of two-stage graft recipients (n = 15) exhibited viable intraspinal (IS) grafts compared to 38% (3 out of 8) observed in the controls (i.e., not receiving intrathymic grafts). Another group of Wistar rats that had received ALS (only) at the time of the IS FSCSD transplant (n = 8) also had 75% graft survival rates after 60 days. These initial findings show that the intrathymic microenvironment can be a compatible ectopic site for fetal SC graft development and survival. Also, the enhanced survival of intraspinal grafts in animals with previous intrathymic implants or ALS administered at the time of grafting suggests the potential for inducing immunoprotection of some fetal neural allografts in adult recipients.
Fractions from Commercial Collagenase Preparations: Use in Enzymic Isolation of the Islets of Langerhans from Porcine Pancreas
Authors: Klöck Gerd , Kowalski Matthias B. , Hering Bernhard J. , Eiden Martin E. , Weidemann Astrid , Langer Stefan , Zimmermann Ulrich , Federlin Konrad , Bretzel Reinhard G.
Published: June 2017

Transplantation of isolated islets of Langerhans is an intriguing possibility for the treatment of diabetes mellitus. The isolation of islets from pancreata requires the specific dissociation of the tissue. Commercial collagenases from Clostridium histolyticum are widely used for this purpose. Unfortunately, the effectiveness of these commercial enzymes is not predictable and differs considerably between suppliers and even from lot to lot. This is due mainly to differences in their specific collagenase activity and to the presence of other lytic enzymes, as well as to other contaminants. Free flow zone electrophoresis (FFZE) was used to separate the effective protein components from undesired compounds and to prepare a digestive enzyme mixture with controlled composition of lytic activities. Fractionation of crude collagenases by FFZE resulted in partially purified protein fractions that were enriched for collagenase and tryptic activities, and contained only trace amounts of neutral protease. These preparations proved to be highly effective in an in vitro assay for the liberation of viable islets from porcine pancreas. To scale up the production of these collagenases with defined enzyme composition, we fractionated two different lots of a commercial collagenase from C. histolyticum (one lot effective in islet isolation, the other not) by using fast protein liquid chromatography (FPLC) on hydroxyapatite. Again, high efficacy of islet release from pancreatic tissue was correlated to high specific tryptic and collagenase activities and low levels of neutral protease. The chromatographic protocol developed in this study converted a non-effective collagenase lot into a preparation that allowed successful islet isolation.
Continued Presence of Intrastriatal but not Intraventricular Polymer-Encapsulated PC12 Cells is Required for Alleviation of Behavioral Deficits in Parkinsonian Rodents
Authors: Emerich Dwaine F. , Winn Shelley R. , Lindner Mark D.
Published: June 2017

To date, few studies have systematically evaluated the most appropriate location for grafting catechoiaminergic cells as a potential treatment for Parkinson's disease (PD). The following study was conducted to determine 1) if placement of catecholamine-secreting encapsulated PC12 cells into the lateral ventricle of 6-OHDA–treated rats is as effective as intrastriatal implants on reducing apomorphine-induced rotational behavior, and 2) to determine if the survival of encapsulated PC12 cells is differentially affected by the implant site. Polymer-encapsulated PC12 cells were implanted into either the striatum or lateral ventricle of unilateral 6-OHDA–lesioned rats. Animals were tested for apomorphine-induced rotations over a 6-wk period. Only those animals that received intrastriatal implants of encapsulated PC12 cells showed a reduction in rotation behavior. Moreover, removal of the devices from the striatum resulted in a return to preimplant rotation levels. Postexplant neurochemical analyses demonstrated that the potassium-evoked l-dopa device output increased in vivo while the potassium-evoked dopamine output from the devices decreased over time in vivo. The location of the implant significantly affected catecholamine output from the PC12 cell-loaded devices. The increase in potassium-evoked l-dopa output was greatest, as was the decrease in potassium-evoked dopamine output, from those devices implanted in the striatum. Basal output of dopamine and DOPAC was also significantly higher from devices explanted from the lateral ventricle. These results demonstrate that the continued presence of intrastriatal implants of encapsulated PC12 cells is required to maintain the behavioral effects in 6-OHDA–lesioned rats. In addition, the site of implantation appears to affect device output. These results provide additional support for intraparenchymal delivery of l-dopa and dopamine via polymer encapsulation as a possible treatment for PD.
A Comparative Study of Preparation Techniques for Improving the Viability of Striatal Grafts Using Vital Stains, in Vitro Cultures, and in Vivo Grafts
Authors: Fricker Rosemary A. , Barker Roger A. , Fawcett James W. , Dunnett Stephen B.
Published: June 2017

Cell suspension grafts from embryonic striatal primordia placed into the adult rat striatum survive well and are able to alleviate a number of behavioral deficits caused by excitotoxic lesions to this structure. However, neither the anatomical connectivity between the graft and host nor the functional recovery elicited by the grafts is completely restored. One way in which the survival and function of embryonic striatal grafts may be enhanced is by the improvement of techniques for the preparation of the cell suspension prior to implantation, an issue that has been addressed only to a limited extent. We have evaluated a number of parameters during the preparation procedure, looking at the effects on cell survival over the first 24 h from preparation using vital dyes and the numbers of surviving neurons in vitro, after 4 days in culture, in addition to graft survival and function in vivo. Factors influencing cell survival include the type of trypsinization procedure and the age of donor tissues used for suspension preparation. The presence of DNase has no effect on cell viability but aids the dissociation of the tissue to form single cells. These results have important implications for the use of embryonic striatal grafts in animal models of Huntington's disease, and in any future clinical application of this research.
Development of an Automated Computer-Controlled Islet Isolation System
Authors: Lakey Jonathan R.T. , Warnock Garth L. , Brierton Mark , Ao Ziliang , Hering Bernhard J. , London Nick J.M. , Ricordi Camillo , Corbin Frank , Rajotte Ray V.
Published: June 2017

Before clinical islet transplantation can become an effective and reliable treatment for type 1 diabetic patients, there must be significant improvements in the methods employed for the isolation of islets of Langerhans. We have developed an automated cell extraction system (ACES), which allows computer control of the isolation process. As well, it incorporates a novel method of recombining dissociated pancreatic tissue. Following initial system design and testing to determine the optimal system configuration, a series of 12 consecutive canine islet isolations were performed. Pancreases were perfused with collagenase via the duct and dissociated and recombined using either the standard Ricordi-based protocol (group 1, n = 6) or dissociated and recombined using the ACES system (group 2, n = 6). A total of 90.8 ± 21 x 103 islet equivalents (IE) (mean ± SEM) were recovered in group 1 vs. 99 ± 14 x 103 IE in group 2 (p = NS, student unpaired t-test). Following Ficoll purification the recovery was 56.2 ± 14 x 103 IE for group 1 vs. 54.7 ± 11 x 103 IE for group 2 (p = NS). Viability was equivalent with an 8.6-fold increase in insulin secretion for group 1 and an 8.8-fold increase for group 2 when the islets were exposed to high glucose solution supplemented with IBMX (3-isobutyl-1-methylxanthine) during static incubation. In vivo function was equivalent following transplantation of 2000 IE under the kidney capsule of alloxan-induced diabetic nude mice with five of six and five of seven mice surviving long-term (>50 days posttransplant) (groups 1 and 2, respectively). This data shows that an entirely automated pancreatic islet extraction system can result in effective canine islet recovery without compromising islet yields and viability. The ACES system has several ad van tages over the standard isolation protocol. These include: 1) computer control and monitoring over all phases of the isolation, 2) a single-use sterile disposable tubing set, and 3) a novel method of tissue recombination. Copyright © 1997 Elsevier Science Inc.
Immunological Response against Allogeneic Chondrocytes Transplanted into Joint Surface Defects in Rats
Authors: Hyc Anna , Malejczyk Jacek , Osiecka Anna , Moskalewski Stanislaw
Published: June 2017

Rat chondrocytes isolated from the articular–epiphyseal cartilage complex were transplanted into defects prepared in articular cartilage and subchondral bone. Transplants were taken for examination after 3 and 8 wk. Cartilage formed by syngeneic chondrocytes did not evoke formation of infiltrations. Contrary to that, in the vicinity of cartilage produced by allogeneic chondrocytes numerous infiltrating cells were present and cartilage resorption could be observed. Cyclosporine-A (CsA) treatment of recipients of allogeneic chondrocytes only partially suppressed accumulation of infiltrating cells and matrix resorption. Antichondrocyte immune response of chondrocyte graft recipients was studied by evaluation of spleen mononuclear cells (SMC) stimulation in mixed splenocytechondrocyte cultures and by evaluation of antichondrocyte cytotoxic antibodies. No difference in stimulation of SMC from intact rats by syngeneic and allogeneic chondrocytes was observed. Stimulation by allogeneic chondrocytes was slightly but significantly higher in recipients of syngeneic grafts. SMC of allogenic chondrocyte recipients were strongly stimulated by allogeneic chondrocytes. This response was absent in recipients treated with CsA. Spontaneous antichondrocyte cytotoxic antibody activity was detected in intact rats and in recipients of syngeneic grafts. In recipients of allogeneic chondrocytes the antibody response against allogeneic chondrocytes was raised but was statistically not significant owing to the considerable variation in the level of spontaneously occurring antichondrocyte antibodies.
Comparison of Methods Used for the Removal of Dmso following Cryopreservation and the Development of an Automated Protocol
Authors: Lakey Jonathan R.T. , Warnock Garth L. , Rajotte Ray V.
Published: June 2017

Current methods of islet isolation are limited, thus requiring islets to be pooled from multiple donors to provide sufficient islet mass to permit insulin independence following islet transplantation. Low temperature banking is one approach used to pool islet preparations. Recently, we developed a method for bulk cryopreservation of islets in a single freezer bag system that is less labor-intensive and more readily kept sterile. As a further improvement to this bulk cryopreservation protocol we examined islet survival following slow-step dilution or our standard sucrose dilution protocol. Known numbers of canine islets were cryopreserved in DMSO by slow cooling to -40°C, storing at -196°C, and rapid thawing. When islets were frozen and thawed in glass tubes the recovery of islets after 48 h of tissue culture was significantly higher when the DMSO was removed using either a slow step (71.7 + 2.7%) or a modified slow step (75.7 + 3.9%) protocol as compared with the standard sucrose dilution protocol (65.7 + 3.0%) (p < 0.05, unpaired t-test). Insulin secretion in vitro and in vivo graft function was similar between the experimental groups. Similarly, when islets were frozen then thawed in freezer bags, islet recovery following 48 h postcryopreser-vation tissue culture at 37° C was 74.8 + 2.4% for slow-step dilution compared with 66.2 + 2.7% for the standard sucrose dilution group (p < 0.05, unpaired t-test). Islets thawed in the freezer bag using the modified slow-step dilution protocol showed equivalent functional viability during static incubation to nonfrozen controls. Bulk cryopreservation of isolated islets in single blood freezer bags is a practical alternative to cryopreservation in glass tubes. Development of an automated protocol for the slow stepwise removal of the cryoprotectant from islets in freezer bags will facilitate low temperature tissue banking of islets.
Post-Thaw Viability and Functionality of Cryopreserved Rat Fetal Brain Cells Cocultured with Sertoli Cells
Authors: Cameron D.F. , Othberg A.I. , Borlongan C.V. , Rashed S. , Anton A. , Saporta S. , Sanberg P.R.
Published: June 2017

Testis-derived Sertoli cells have been used to create an immune “privileged” site outside of the testis to facilitate cell transplantation protocols for diabetes and neurodegenerative diseases. In addition to secreting immunoprotective factors, Sertoli cells also secrete growth and trophic factors that appear to enhance the posttransplantation viability of isolated cells and, likewise, the postthaw viability of isolated, cryopreserved cells (26). It would be beneficial if Sertoli cells could be cryopreserved with the transplantable cell type without deleterious effects on the cells. This report describes a protocol for the cocryopreservation of rat Sertoli cells with rat ventral mesencephalic neurons, neurons from the lateral and medial ganglionic eminences and the hNT neuron cell line, and reports on the effects of Sertoli cells on the the postthaw viability of these neurons. Results of trypan blue exclusion analysis indicated that the presence of Sertoli cells did not deleteriously effect cryopreserved neurons and may improve their postthaw recoverability and viability in general. Specifically, results of the tyrosine hydroxylase immunostaining showed that Sertoli cells significantly enhance the postthaw viability of ventral mesencephalic dopaminergic cells in vitro.
The Effects of Storage Conditions and Trophic Supplementation on the Survival of Fetal mesencephalic cells
Authors: Thajeb P. , Ling Z.D. , Potter E.D. , Carvey P.M.
Published: June 2017

It is estimated that only 5-10% of dopamine (DA) neurons implanted into the striatum of patients undergoing fetal-nigral transplantation as a treatment for Parkinson's Disease survive. Because it is often necessary to store fetal tissue prior to transplantation, we evaluated various storage parameters that could influence DA neuron viability in rostral mesencephalic tegmentum (RMT) cultures using tyrosine hydroxylase immunoreactive (THir) cell counts as an index of DA neuron survival. A high K+ hibernation media (HM) was used in all studies. We found that RMT cell viability and THir cell counts decreased as storage duration increased (up to 120 h). Storage at 37°C in HM killed all cells, while storage at 10°C yielded higher survival rates than 4° C. In comparison to trypsinization, mechanical dissociation of tissue increased cell viability. Neutral pH and a storage density of at least 1 × 106 cells/mL were found to be optimal, while striatal coculture of RMT cells with striatal feeder layers increased THir viability up to 16-fold in comparison to monocultures. The nurturing effect of striatal coculture may be explained by the release of autotrophic factors, and we tested this hypothesis by supplementing the HM with human placental cord serum (HPCS, 8%), glial-derived neurotrophic factor (GDNF; 10 μg/mL), and brain-derived neurotrophic factor (BDNF; 10 μg/mL). GDNF and HPCS supplements increased RMT cell viability by 10-15%, while GDNF, BDNF, and HPCS increased viability of THir cells by approximately 40% at all time points studied. As Klenow enzyme labeling technique indicated that 33% of stored RMT cells were undergoing apoptosis, we found that GDNF, BDNF, and HPCS reduced apoptosis by 50%. DNA laddering and DAPI nuclear stain confirmed the presence of apoptosis in hibernated RMT cells, leading us to postulate that the high viability counts seen with trypan blue exclusion are misleading.
Map2 Expression in the Developing Human Fetal Spinal Cord and following Xenotransplantation
Authors: Giovanini Mark A. , Reier Paul J. , Eskin Thomas A. , Anderson Douglas K.
Published: June 2017

Human fetal spinal cord (FSC) tissue was obtained from elective abortions at 6-14 wk gestational age (GA). The specimens were then either immediately processed for immunohistochemical analysis or xenotransplantation. In the latter case, donor tissue was prepared as a dissociated cell suspension and then introduced either sub-pially or intraspinally into contusion lesions of the adult rat midthoracic spinal cord. The xenografts were subsequently examined by conventional histological and immunohistochemical methods at 2-3 mo postgrafting. Immunostaining showed that MAP2 was expressed heavily in cells residing in the mantle layer of the human fetal spinal cord in situ as early as 6 wk GA. Subpial and intraparenchymal xenografts also were intensely immunoreactive for MAP2, but no staining of surrounding host neural tissue was detected. We conclude that the differential expression of MAP2 can be used to distinguish human graft tissue from the surrounding rat spinal cord in this xenograft paradigm. Under appropriate staining conditions, MAP2 can thus serve to facilitate analyses of host-graft integration, donor cell migration, and neuritic outgrowth.
Functional Recovery of Porcine Hepatocytes after Hypothermic or Cryogenic Preservation for Liver Support Systems
Authors: Naik Sharda , Santangini Henry A. , Trenkler Donna M. , Mullon Claudy J.P. , Solomon Barry A. , Pan Jing , Jauregui Hugo O.
Published: June 2017

The provision of an immediate supply of isolated porcine hepatocytes for artificial liver support requires preservation techniques that will allow maintenance of cell viability and detoxification functions. By means of a simple and cost-effective cryopreservation system, porcine hepatocytes can be available for both local and distant medical treatment facilities. Additionally, cryopreservation provides an adequate period for quality control testing to be completed prior to use of any specific cell lot. We are reporting a dual approach, namely the preservation of porcine hepatocytes, at 4°C and at −196°C in liquid nitrogen (LN2). Using a combination of cryoprotectant agents with Chee's modified Eagle's culture media (CEM), collagenase isolated hepatocytes stored at 4°C for 24 h maintained 80% of the initial diazepam metabolism measured in freshly isolated cells and nearly 100% of initial function was preserved in hepatocytes stored up to 6 mo at -196°C. University of Wisconsin solution (UW) was also tested and while adequate for 4°C storage, it certainly did not match the performance of the CEM formulations for preservation of metabolic function of cells stored in liquid nitrogen. Based on our results of viability and detoxification function the combination of CEM with DMSO, polyethylene glycol and serum provided optimal protection for LN2 frozen cells. Other findings in these studies underlined the importance of the gradual introduction of DMSO in the prefreezing process, the period of osmotic equilibration, and the rapid postthaw withdrawal of this agent to minimize cytotoxic effects at these critical stages. Our freezing methodology provides the foundation for further technological developments in the cryopreservation of the large numbers of cells (billions) that are necessary for extracorporeal liver assist devices.
Glucoregulation after Canine Islet Transplantation: Contribution of Insulin Secretory Capacity, Insulin Action, and the Entero-Insular Axis
Authors: Van Der Burg Michael P. M. , Van Suylichem Paul T. R. , Guicherit Onno R. , Frölich Marijke , Lemkes Herman H. P. J. , Gooszen Hein G.
Published: June 2017

The physiological glucoregulatory mechanisms after islet transplantation have been incompletely investigated. We studied the insulin secretory capacity (ISC) by intravenous arginine stimulation during 35-mM glucose clamps, insulin action during hyperinsulinemic euglycemic clamps, and mixed-meal stimulation at 6-9 mo after intrasplenic islet autotransplantation in 8 dogs, as compared with 30 controls. The enteroinsular axis in the recipients was examined by infusion of porcine glucose-dependent insulinotropic polypeptide (GIP) and human glucagon-like peptide-1 (GLP-1) (7-36 amide) under 8.5-mM glycemic clamp conditions in order to mimic the postprandial glycemia after transplantation. The grafts comprised 25% of the native islet mass, and the ISC likewise averaged 25% of the control value. The postprandial insulin response, in contrast, had increased to 140% after transplantation—albeit with a concomitant glucose excursion to approximately 8.5 mM. Insulin action declined on average by 45% posttransplant. The ISC correlated both with the postprandial glucose excursion and insulin action in the grafted dogs. Insulin action did not correlate with the postprandial glucose excursion. Infusion of GIP had no effect, but GLP-1 nearly doubled glucose-stimulated insulin. Thus, a hyperglycemia-enhanced insulinotropic effect of GLP-1, and perhaps other gut hormones, may account for the difference in the insulin response to the intravenous and oral challenges. Because the ISC reflects the engrafted islet mass and appears to be the primary determinant of glucose tolerance, transplantation of higher islet doses should allow prolonged near-normal glucoregulation—at least, in the autotransplant setting.
Enzymatic Activity and Expression of Cytochrome P450 LAω within Intrasplenically Transplanted Fetal Hepatocytes in Spontaneously Hypertensive Rats
Authors: Kato Kazuya , Kato Junji , Hodgson W. John B. , Abraham Nader G. , Onodera Kazuhiko , Imai Masato , Kasai Shinichi , Mito Michio
Published: June 2017

We examined the expression and enzymatic activity of the cytochrome P450 LAω within transplanted hepatocytes. Fetal hepatocytes were harvested at day 20 of gestation from spontaneously hypertensive rats (SHRs) and transplanted into recipient adult SHR spleens. Microscopic examination of the recipient spleens 4 and 10 wk after transplantation revealed masses of hepatocytes with cordlike structures in the red pulp. Immunochemical studies detected cytochrome (cyto) P450 LAω in the fetal hepatocytes before transplantation without prior induction. Although the cyto P450 LAω was not detected by the second week after transplantation, by the 6th and 10th wk after transplantation, it was. Cyto P450-arachidonic acid ω/4oM-1 hydroxylase activity (formation of 20- and 19-hydroxyeico-satetraenoic acid) was detected at 10 wk after transplantation, but not 2 or 6 wk after transplantation. These results demonstrated that fetal hepatocytes can be transplanted successfully into recipient spleens and then grow in the spleens, as in the case of the adult hepatocyte response.
Evaluation of an Immunoisolation Membrane Formed by Incorporating a Polyvinyl Alcohol Hydrogel within a Microporous Filter Support
Authors: Baker Andrew R. , Fournier Ronald L. , Sarver Jeffrey G. , Long Jennifer L. , Goldblatt Peter J. , Horner James M. , Selman Steven H.
Published: June 2017

An immunoisolation membrane formed by incorporating a high water content polyvinyl alcohol (PVA) hydrogel into a microporous polyether sulfone (PES) filter has been investigated in this study. The PVA hydrogel is formed in situ within the filter pores via glutaraldehyde (GA) crosslinking under acidic conditions. The tortuous nature of the microporous filter pores securely anchors the embedded hydrogel to provide excellent structural integrity. The high void fraction of the PES filter support (>80%) and high water content of the PVA hydrogel (>85% water by weight) allow excellent solute transport rates, while an appropriate level of glutaraldehyde crosslinking supplies the required molecular size selectivity. In vitro permeability measurements made with solutes covering a wide range of molecular sizes demonstrate high transport rates for small nutrient molecules with rapidly diminishing permeabilities above a molecular weight of approximately 1,000 Dalton. Implantation experiments show that the membrane properties are not deleteriously affected by prolonged in vivo exposure or common sterilization techniques. Thus, this hybrid hydrogel/filter membrane system offers a promising approach to the immunoisolation of implanted cells.
Proliferative Response of Human T Lymphocytes to Porcine Fetal Brain Cells
Authors: Brevig Thomas , Pedersen Erik B. , Kristensen Tom , Zimmer Jens
Published: June 2017

Intracerebral grafting of porcine fetal brain cells is a potential treatment of neurodegenerative disorders in humans. Although it is well known that the survival of fetal pig brain cells in the rat brain requires immunosuppression, the response of human T lymphocytes to fetal pig brain cells is unknown. Here we report on the proliferative response of human T lymphocytes to freshly isolated cells and 14-18 days cultured cells from 28- or 35-day-old porcine fetal brains. After 5 days of mixed lymphocyte-brain cell culture, we observed no or only minor T cell responses to the freshly isolated brain cells, while both CD4 cells and CD8 cells proliferated in response to the cultured brain cells. Pretreatment of the cultured brain cells with heat-inactivated human serum significantly reduced the proliferative T cell response. The data suggest that the porcine fetal brain contains cells that can stimulate the human cellular immune system, and that this stimulation may be reduced by pretreatment of the fetal pig brain cells with human serum.
Involvement of Male-Specific Minor Histocompatibility Antigen H-Y in Epidermal Equivalent Allograft Rejection
Authors: Beaulieu Marie-Josée , Li Hui , Bergeron Julie , Ross Geneviève , Auger François A. , Rouabhia Mahmoud
Published: June 2017

This study describes the involvement of male-specific minor histocompatibility antigen H-Y in vitro cultured epidermal equivalent (EE) rejection. Male and female Balb/c or C3H/HeN keratinocytes were isolated and cultured separately. Male EE were grafted onto adult male (isografts) and adult female (H-Y allografts) mice. As controls, Balb/c EE were grafted onto adult C3H/HeN (complete allografts) mice. Fourteen, 21, and 30 days postgrafting, histological studies showed well-organized cutaneous tissues with complete basement membranes (laminin and type IV collagen deposition) in H-Y allografts compared to the isografts. This cutaneous organization was altered 150 days postgrafting, which is a sign of the H-Y EE allograft rejection. Complete allografts were totally rejected 21 days postgrafting. Immunological studies revealed leucocyte infiltration of H-Y allografts. Significant infiltration was detected even 150 days postgrafting. Leucocyte phenotyping revealed the presence of Mac-1+, CD8+ and CD4+ cells in the H-Y allografts. Humoral immune analysis revealed the presence of circulating anti-H-Y allogeneic keratinocyte cytotoxic antibodies in female recipient sera. Our data suggest that male-specific minor histocompatibility antigen H-Y induces cellular and humoral activation of the recipient immune system even after grafting EE free of cutaneous active immune cells such as T lymphocytes and Langerhans cells.
Blockade of Cd40-Cd154 Interferes with Human T cell Engraftment in Scid Mice
Authors: Foy Teresa M. , Mcilraith Melissa , Masters Sally R. , Dunn Jonathan J. , Rossini Aldo A. , Shultz Leonard D. , Hesselton Ruth Ann M. , Wagar Eric J. , Lipsky Peter E. , Noelle Randolph J. , Greiner Dale L.
Published: June 2017

Antibodies to the ligand for CD40 (CD154) have been shown to exert profound effects on the development of cell-mediated immune responses in mice. The present study shows that an antibody to human CD154 (hCD40L) inhibits in vivo Tetanus toxoid (TT) specific secondary antibody responses in hu-PBL-scid mice, as well as the expansion of xenoreactive human T cells in the scid mice. A possible cause for the reduced expansion of xenoreactive, human T cells, was the decreased expression of murine B7.1 and B7.2 caused by the administration of anti-hCD40L. Therefore, it may be that defective maturation of murine antigen-presenting cells impeded the priming and expansion of human xenoreactive T cells.
Morphological and Functional Evidence for Enhanced Growth and Potassium-Evoked Dopamine Release in Striatal Grafts Innervated with a Patchy Growth Pattern. an in Oculo Nigrostriatal Cograft Study
Authors: Vidal Nina , Björklund Lars , Strömberg Ingrid
Published: June 2017

During development of the nigrostriatal dopamine system, a patchy and a diffuse type of striatal innervation pattern can be seen. It has been suggested that when fetal dopaminergic neurons, obtained from the ventral mesencephalon (VM), are grafted adjacent to mature striatal tissue, only the diffuse growth is induced. Intraocular grafting studies have indicated that the dopaminergic growth pattern might be influenced by the age of the target area, the lateral ganglionic eminence (LGE). In this study VM grafts were allowed to innervate LGE grafts of different ages. Fetal VM was implanted next to 2-wk-old or 26-day-old striatal in oculo grafts, and the resulting dopaminergic innervation of the striatal grafts was studied using tyrosine hydroxylase (TH) immunohistochemistry. In striatal grafts receiving innervation at the age of 2 wk in oculo, a patchy TH-immunoreactive growth pattern was found, while in striatal grafts innervated at the age of 26 days mainly the diffuse growth pattern was seen. This implies that grafted striatum reached maturity at approximately 1 mo of age. The age of the dopaminergic neurons at dissection and grafting was also studied concerning the ability to induce patchy growth into mature striatum. Thus, VM dissected from 13- and 18-mm fetuses was implanted to either 4-mo-old LGE (grafted in sequence) or to LGE from the same fetus (grafted simultaneously) as controls. TH-positive innervation of striatal tissue, evaluated 4 wk after implantation of VM, revealed a patchy growth pattern in LGE grafted simultaneously with 13- and 18-mm VM. However, when the striatum was mature at the time of innervation, diffuse growth was observed in striatum innervated by VM dissected from 13-mm fetuses. Interestingly, patchy growth was noted in striatal areas close to VM grafts when the dopaminergic neurons were derived from older fetuses (CRL 18 mm). Furthermore, potassium-induced dopamine release was greater in striatal grafts exhibiting the patchy growth than those showing the diffuse pattern of innervation. In conclusion, patchy dopaminergic growth can be induced in mature striatal tissue by grafting VM from older fetuses. Functionally, potassium-evoked dopamine release is enhanced in dopaminergic patches. These results have implications in terms of finding ways to induce patchy growth when grafting to the mature striatum of patients suffering from Parkinson's disease.