bluuebunny/crossref_metadata_2025 huggingface dataset:
The Neointima Formed in Endothelial Cell Sodded ePTFE Vascular Grafts Results from Both Cellular-Hyperplasia and Extracellular-Hypertrophy
Authors: Kleinert Leigh B. , Hoying James B. , Williams Stuart K.
Published: June 2017

Endothelial cell transplantation onto polymeric vascular grafts results in the formation of a neointima. The formation of this neointima is often suggested to result from a chronic cellular hyperplasia where the terms intimal hyperplasia and intimal thickening are used interchangeably. While the formation of a midgraft neointima in sodded grafts involves a level of cell proliferation, the synthesis and deposition of extracellular matrix proteins is also a ubiquitous observation in these grafts. To assess the composition of midgraft neointima in sodded grafts, a morphometric method was developed to provide a differential quantitation of the cellular-hyperplastic and extracellular-hypertrophic elements of intimal thickening. The formed neointima on microvessel endothelial cell sodded and control (noncell-treated) ePTFE vascular grafts was quantified after 3, 12, and 52 wk of graft implantation in a canine carotid artery model. Midgraft sections of grafts were evaluated for both intimal thickness (IT) and cell density per unit volume and quantified using a PC-based image analysis program. Sodded grafts explanted at 3 wk exhibited an average neointimal cell density (3 × 109 cells/cm3; IT 30 μm) equivalent to cell densities observed in normal arterial media. After 12 wk the mean cell density approached a hyperplastic value (3.7 × 109 cells/cm3; IT 76 μm), while grafts explanted after 52 wk exhibited a mean cell density (2.8 × 109 cells/cm3; IT 30 μm) similar to 3-wk values. Control grafts that received no cells exhibited no midgraft cellular coverage. These results indicate that neointima formation in the midgraft region of sodded grafts occurred via mechanisms involving both a cellular hyperplasia and an extracellular hypertrophy. Differential responses occur presumably due to localized differences in cellular proliferation and cellular biosynthetic activity.
Sertoli Cell-Induced Effects on Functional and Structural Characteristics of Isolated Neonatal Porcine Islets
Authors: Selawry H.P. , Wang X. , Alloush L.
Published: June 2017

A lack of a sufficient number of human donor pancreases has stimulated interest in isolation and cryopreservation techniques for islets from the porcine pancreas. But because of a poorly developed outer membrane porcine islets are particularly susceptible to damage during cryopreservation. The aims of this study were twofold: 1) to develop a method for isolation and storage of islets from neonatal porcine pancreas and, 2) to examine effects of Sertoli cells on islet yield and function in Sertoli cell-islet cell cocultures. A total of 170 neonatal porcine pancreases were processed by means of a short period of digestion with collagenase and culture of the tissues at 32°C for periods up to 7 days following isolation. Results were: The mean ±SEM, number of viable islets, and percentage loss of cells following 7 days of culture were 29,442 ± 1,119 and 22.2 ± 1.2, respectively. Cryopreservation had a marked impact on recovery of viable islets: In absence of Sertoli cells an average of only 64% of islets remained viable; by contrast, when cryopreserved islets were cocultured with Sertoli cells, a mean of 82% was recovered. Glucose at 16.7 mmol/L had the capacity to elicit insulin release from 3-day-old cultured islets. The concentration in absence of Sertoli cells was 57.3 ± 3.8, uU/mL/10 islets; in the presence of Sertoli cells the level increased to a mean ± SEM of 112.8 ± 17.7, uU/mL/10 islets. Similar results were obtained following cryopreservation: glucose at 16.7 mmol/L stimulated a mean ± SEM of 27.9 ± 6.6, uU/mL/10 islets, of insulin in absence of, and 44.9 ± 9.9, uU/mL/10 islets, in presence of, Sertoli cells. Our results show that isolation and cryopreservation of neonatal porcine islets can be successfully accomplished. In addition, coculture with Sertoli cells significantly improves both the yield and functional capacity of islets following cryopreservation.
Constitutive Expression of Glutamic Acid Decarboxylase (GAD) by Striatal Cell Lines Immortalized using the tsA58 Allele of the SV40 Large T Antigen
Authors: Giordano Magda , Takashima Hideotoshi , Poltorak Maciej , Geller Herbert M. , Freed William J.
Published: June 2017

Rodent striatal cells were immortalized using the A58 temperature-sensitive allele of the SV40 large T antigen. Seventy-eight clones and 10 mixed cultures were characterized at the nonpermissive and permissive temperatures. Based on morphology and expression of proteins, cells were classified into three primary types, with types b and c expressing some neuronal characteristics. Type a cells have an epithelial-like morphology with coarse cytoplasmic extensions and occasional fine processes. These cells express vimentin, do not grow well under serum-free conditions and, when confluent, form a uniform monolayer. Type b cells have a polygonal shape and usually extend multiple thin processes. These cells possess large nuclei with multiple nucleoli and do not express vimentin. Type c cells have a fibroblast-like appearance, are unipolar or multipolar, and their soma is smaller than that of type b cells. Type c cells do not express vimentin, and when confluent form a uniform monolayer. Some type b and c clones express NCAM and MAP-2. Several type b and c cell lines were found to consistently express glutamic acid decarboxylase (GAD) immunoreactivity under several tissue culture conditions. Selected cell lines were transplanted into the intact adult rat brain in several locations. Cells survived well for 15 wk and did not form tumors. The proteins expressed in vivo were similar to those expressed in vitro.
Incorporation of β-Galactosidase-Expressing Endothelial Cells into the Skeletal Muscle Microvascular Bed of Mice
Authors: Schwärzler Christoph , Webersinke Gerald , Fässler Reinhard , Lametschwandtner Alois , Bauer Hannelore , Bauer Hans-Christian
Published: June 2017

Cloned murine endothelial cells (cEC) were used as a carrier system for introducing a foreign gene into the microvascular bed of the hind limb of inbred mice. cEC were transfected with a β-galactosidase-neo fusion construct, which enables both selection for DNA uptake in the presence of G 418 and the staining of cells for β-galactosidase activity. Transfected cEC adhered and integrated readily into confluent monolayers of nontransfected cEC (up to 26% of total cell number). Seeding lacZ-transfected cEC on explanted arteries revealed rapid adhesion of the cells (within minutes) to the intact endothelium. After injection of 106transfected EC via the femoral artery into the microvascular bed of the hind limb their presence was documented by β-galactosidase staining after various time periods (1 h to 4 wk). Implanted cEC were detected in numerous elements of the microcirculation both in frozen sections and in squash preparations of the hind limb muscle and in the femoral bone up to 4 wk after the injection. The microvascular bed of skeletal muscle of the mouse as a recipient site for transduced syngeneic endothelial cells is, thus, a suitable experimental model to study various strategies for somatic gene therapy. Copyright © 1997 Elsevier Science Inc.
Isolation and Characterization of a Cell Line from the Epithelial Cells of the Human Fetal Pancreas
Authors: Wang Sijian , Beattie Gillian M. , Mally Martin I. , Cirulli Vincenzo , Itkin-Ansari Pam , Lopez Ana D. , Hayek Alberto , Levine Fred
Published: June 2017

Pancreatic cell lines are useful for basic studies of pancreatic biology and for possible application to cell transplantation therapies for diabetes. A retroviral vector expressing simian virus 40 (SV40) T antigen and H-rasval12 was used to infect a monolayer culture of epithelial cells from an 18-wk human fetal pancreas. Infected cells gave rise to a clonal epithelial cell line, designated TRM-1. This cell line expresses epithelial markers as well as glut2 and small amounts of insulin and glucagon. TRM-1 is the first cell line to be generated from the human fetal pancreas and also the first cell line derived directly from the fetal pancreas of any species. The approach that we have used to develop TRM-1 should be applicable to isolating cell lines from other stages of human pancreatic development. Copyright © 1997 Elsevier Science, Inc.
Culture Expanded Canine Mesenchymal Stem Cells Possess Osteochondrogenic Potential in Vivo and in Vitro
Authors: Kadiyala S. , Young R. G. , Thiede M. A. , Bruder S. P.
Published: June 2017

Mesenchymal Stem Cells (MSCs) possessing the capacity to differentiate into various cell types such as osteoblasts, chondrocytes, myoblasts, and adipocytes have been previously isolated from the marrow and periosteum of human, murine, lapine, and avian species. This study documents the existence of similar multipotential stem cells in canine marrow. The cells were isolated from marrow aspirates using a modification of techniques previously established for human MSCs (hMSCs), and found to possess similar growth and morphological characteristics, as well as osteochondrogenic potential in vivo and in vitro. On the basis of these results, the multipotential cells that were isolated and culture expanded are considered to be canine MSCs (cMSCs). The occurrence of cMSCs in the marrow was determined to be one per 2.5 × 104 nucleated cells. After enrichment of the cMSCs by centrifugation on a Per-coll cushion, the cells were cultivated in selected lots of serum. Like the hMSCs, cMSCs grew as colonies in primary culture and on replating, grew as a monolayer culture with very uniform spindle morphology. The population doubling time for these cMSCs was approximately 2 days. The morphology and the growth kinetics of the cMSCs were retained following repeated passaging. The osteogenic phenotype could be induced in the cMSC cultures by the addition of a synthetic glucocorticoid, dexamethasone. In these osteogenic cultures, alkaline phosphatase activity was elevated up to 10-fold, and mineralized matrix production was evident. When cMSCs were loaded onto porous ceramics and implanted in autologous canine or athymic murine hosts, copious amounts of bone and cartilage were formed in the pores of the implants. The MSC-mediated osteogenesis obtained by the implantation of the various MSC-loaded matrix combinations is the first evidence of osteogenesis in a canine model by implantation of culture expanded autologous stem cells. The identification and isolation of cMSCs now makes it feasible to pursue preclinical models of bone and cartilage regeneration in canine hosts.
Cellular Delivery of Human Cntf Prevents Motor and Cognitive Dysfunction in a Rodent Model of Huntington's Disease
Authors: Emerich Dwaine F. , Cain Chris K. , Greco Corinne , Saydoff Joel A. , Hu Zhong Yi , Liu Hanjiu , Lindner Mark D.
Published: June 2017

The delivery of ciliary neurotrophic factor (CNTF) to the central nervous system has recently been proposed as a potential means of halting or slowing the neural degeneration associated with Huntington's disease (HD). The following set of experiments examined, in detail, the ability of human CNTF (hCNTF) to prevent the onset of behavioral dysfunction in a rodent model of HD. A DHFR-based expression vector containing the hCNTF gene was transfected into a baby hamster kidney fibroblast cell line (BHK). Using a polymeric device, encapsulated BHK-control cells and those secreting hCNTF were transplanted bilaterally into rat lateral ventricles. Eight days later, the same animals received bilateral injections of quinolinic acid (QA, 225 nmol) into the previously implanted striata. A third group received sham surgery (incision only) and served as a normal control group. Bilateral infusions of QA produced a significant loss of body weight and mortality that was prevented by prior implantation with hCNTF-secreting cells. Moreover, QA produced a marked hyperactivity, an inability to use the forelimbs to retrieve food pellets in a staircase test, increased the latency of the rats to remove adhesive stimuli from their paws, and decreased the number of steps taken in a bracing test that assessed motor rigidity. Finally, the QA-infused animals were impaired in tests of cognitive function — the Morris water maze spatial learning task, and the delayed nonmatching-to-position operant test of working memory. Prior implantation with hCNTF-secreting cells prevented the onset of all the above deficits such that implanted animals were nondistinguishable from sham-lesioned controls. At the conclusion of behavioral testing, 19 days following QA, the animals were sacrificed for neurochemical determination of striatal choline acetyltransferase (ChAT) and glutamic acid decarboxylase (GAD) levels. This analysis revealed that QA decreased striatal ChAT levels by 35% and striatal GAD levels by 45%. In contrast, hCNTF-treated animals did not exhibit any decrease in ChAT levels and only a 10% decrease in GAD levels. These results support the concepts that implants of polymer-encapsulated hCNTF-releasing cells can be used to protect striatal neurons from excitotoxic damage, produce extensive behavioral protection as a result of that neuronal sparing, and that this strategy may ultimately prove relevant for the treatment of HD.
Intranigral Grafts of Fetal Ventral Mesencephalic Tissue in Adult 6-Hydroxydopamine-Lesioned Rats can Induce Behavioral Recovery
Authors: Johnston R.E. , Becker Jill B.
Published: June 2017

Intrastriatal grafts of fetal ventral mesencephalon in rats with unilateral 6-hydroxydopamine lesions can reduce and even reverse rotational behavior in response to direct and indirect dopamine agonists. These grafts can ameliorate deficits on simple spontaneous behaviors, but do not improve complex behaviors that require the skilled integration of the use of both paws. We report here that rats with grafts into the DA-depleted substantia nigra, that receive cyclosporine A, can experience recovery on spontaneous behaviors that mimic those observed in Parkinson's disease. Specific cyclosporine A treatment conditions can differentially affect whether intranigral grafts normalize paw use during initiation or termination of a movement sequence. These findings may have important implications for the treatment of Parkinson's disease.
Functional Characterization of Ngf-Secreting Cell Grafts to the Acutely Injured Spinal Cord
Authors: Tuszynski Mark H. , Murai Keith , Blesch Armin , Grill Ray , Miller Ian
Published: June 2017

Previously we reported that grafts of cells genetically modified to produce human nerve growth factor (hNGF) promoted specific and robust sprouting of spinal sensory, motor, and noradrenergic axons. In the present study we extend these investigations to assess NGF effects on corticospinal motor axons and on functional outcomes after spinal cord injury. Fibroblasts from adult rats were transduced to express human NGF; control cells were not genetically modified. Fibroblasts were then grafted to sites of midthoracic spinal cord dorsal hemisection lesions. Three months later, recipients of NGF-secreting grafts showed deficits on conditioned locomotion over a wire mesh that did not differ in extent from control-lesioned animals. On histological examination, NGF-secreting grafts elicited specific sprouting from spinal primary sensory afferent axons, local motor axons, and putative cerulospinal axons as previously reported, but no specific responses from corticospinal axons. Axons responding to NGF robustly penetrated the grafts but did not exit the grafts to extend to normal innervation territories distal to grafts. Grafted cells continued to express NGF protein through the experimental period of the study. These findings indicate that 1) spinal cord axons show directionally sensitive growth responses to neurotrophic factors, 2) growth of axons responding to a neurotrophic factor beyond an injury site and back to their natural target regions will likely require delivery of concentration gradients of neurotrophic factors toward the target, 3) corticospinal axons do not grow toward a cellular source of NGF, and 4) functional impairments are not improved by strictly local sprouting response of nonmotor systems.
Analysis of Hepatocyte Distribution and Survival in Vascular Beds with Cells Marked by 99mTC or Endogenous Dipeptidyl Peptidase IV Activity
Authors: Gupta Sanjeev , Vasa Srinivasa Rao G. , Rajvanshi Pankaj , Zuckier Lionel S. , Palestro Christopher J. , Bhargava Kuldeep K.
Published: June 2017

Knowledge of the kinetics of cell distribution in vascular beds will help optimize engraftment of transplanted hepatocytes. To noninvasively localize transplanted cells in vivo, we developed conditions for labeling rat hepatocytes with 99mTc–pertechnetate. The incorporated 99mTc was bound to intracellular proteins and did not impair cell viability. When 99mTc hepatocytes were intrasplenically injected into normal rats, cells entered liver sinusoids with time–activity curves demonstrating instantaneous cell translocations. 99mTc activity in removed organs was in liver or spleen, and lungs showed little activity. However, when cells were intrasplenically transplanted into rats with portasystemic collaterals, 99mTc appeared in both liver sinusoids and pulmonary alveolar capillaries. To further localize cells, we transplanted DPPIV+ F344 rat hepatocytes into syngeneic DPPIV – recipients. Histochemical staining for DPPIV activity demonstrated engraftment of intrasplenically transplanted cells in liver parenchyma. In contrast, when 99mTc hepatocytes were injected into a peripheral vein, cells were entrapped in pulmonary capillaries but were subsequently broken down with redistribution of 99mTc activity elsewhere. Intact DPPIV+ hepatocytes were identified in lungs, whereas only cell fragments were present in liver, spleen, or kidneys. These findings indicate that although the pulmonary vascular bed offers advantages of easy accessibility and a relatively large capacity, significant early cell destruction is an important limitation.
Alginate Polylysine Microcapsules as Immune Barrier: Permeability of Cytokines and Immunoglobulins over the Capsule Membrane
Authors: Kulseng Bård , Thu Beate , Espevik Terje , Skjåk-Bræk Gudmund
Published: June 2017

Transplantation of pancreatic islets in alginate polylysine microcapsules is a potential useful method for treating type I diabetes. In this study, the permeability for alginate-polylysine microcapsules to cytokines an immunoglobulines has been investigated by a newly developed method. Magnetic monodisperse polymer particles (Dynabeads) coated with antibodies against selected proteins were encapsulated in 0.7 mm alginate polylysine microcapsules. The capsule membrane permeability to IgG (150 kDa). Transferrin (81 kDa), Tumor necrosis factor (TNF, 51 kDa), Interleukin-1β (IL-1β, 17.5 kDa), and insulin (5.8 kDa) was estimated by measuring the binding of 125I-labeled proteins to the encapsulated antibody coated Dynabeads. Capsules with an inhomogeneous solid gel core were made of alginates with high guluronic or high mannuronic acid content and poly-l (PLL)- or poly-d-lysine (PDL) of concentrations varied from 0.05-0.2%. The various capsules examined were all impermeable to IgG. The capsules made with a PLL-, but not PDL-membranes were permeable for transferrin. IL-1β was found to penetrate all of the different capsule types. The high-G capsules, however, could be made impermeable to TNF and still allowed transferrin to pass. The permeability of these capsules to IL-1β, but not to TNF was confirmed in an assay where mouse islets of Langerhans were incubated with TNF and IL-1β, and comparing the IL-6 for encapsulated and non-encapsulated islets.
Selective Intraportal Transplantation of DiI-marked Isolated Rat Hepatocytes
Authors: Markus P. M. , Koenig S. , Krause P. , Becker H.
Published: June 2017

Transplantation of isolated hepatocytes is a promising alternative to orthotopic liver transplantation in experimental animal models with acute hepatic failure and hereditary enzyme defects. Conventional light microscopy identification of hepatocytes within recipient livers has been limited due to the inability to distinguish between donor and recipient liver cells. In this study, we labeled hepatocytes intracellularly with the fluorescent dye DiI-18 prior to selective intraportal or intrasplenic transplantation. Syngeneic LEW rat hepatocytes were isolated and 2 × 107 fluorescence-labeled cells were transplanted by intraportal infusion selectively into 2/3 of the recipient liver lobules to avoid lethal portal hypertension. Rats were sacrificed on postop days 1, 3, 5, 10, 20, and 40. Histological examination was performed using light and fluorescence microscopy counterstained by light green dye. The quantity of transplanted hepatocytes residing within the recipient liver was determined by FACS analysis after enzymatic digestion of the recipient liver lobules. Engrafted hepatocytes were identified in the periportal regions of transplanted liver lobules. The stained hepatocytes were retrieved up to 20 days postop using fluorescent microscopy. Using FACS analysis the number of labeled hepatocytes was found to diminish over time following transplantation from 2.1% on postop day 1 to 0.5% on day 10. Labeled hepatocytes transplanted into the spleen were retrieved in clusters up to 20 days postop (the last day of observation). Furthermore, the migration of labeled hepatocytes from spleen to liver parenchyma was observed following intrasplenic transplantation. However, after selective intraportal transplantation, only fluorescent debris was found in splenic and pulmonary tissue upon examination of various organs. This article describes the method of fluorescent labeling of rat hepatocytes and reports the feasibility and limitations of using DiI-18 as a marker.
Organization and Histochemical Phenotype of Human Fetal Cerebellar Cells following Transplantation into the Cerebellum of Nude Mice
Authors: Pundt L. L. , Jörn E. A. , Conrad J. A. , Low W. C.
Published: June 2017

Previous rodent studies have demonstrated the capacity of cerebellar transplants to organize into trilaminar cell layers typically observed in the normal cerebellum. In Purkinje Cell (PC)-deficient animals, PCs will migrate into the host and form synaptic connections. Recently, fetal cerebellar grafts transplanted into the Purkinje cell degeneration (pcd) mutant mouse were shown to result in an improvement of motor behaviors. These studies indicate the potential therapeutic use of neural transplantation in patients with cerebellar degeneration. In the present study, human fetal cerebellar tissue (8.5 wk postconception) was dissociated and transplanted into the normal cerebellum of nude mice. Six months following transplantation, histological analysis revealed donor cells in recipient mice. Immunostaining for the 28 kDa calcium-binding protein (calbindin) revealed the presence of donor PCs that were organized in discrete cellular layers within the transplant neuropil. In most cases the dendritic processes were oriented in a planar fashion perpendicular to the transplant cell layer. Human neurofilament immunostaining revealed bundles of donor fibers within the core of the transplant and/or at the periphery. These bundles were found to be calbindin positive (PC fibers). Three animals provided evidence of donor PC axon growth ventrally into host white matter, and in one case, this ventral migration reached the deep cerebellar nuclei. Most notable was the development of a pronounced folia-like organization by the implanted cell suspensions. Glial processes within the grafts were aligned perpendicular to the long axis of the transplant folia. These results demonstrate the capacity of human fetal cerebellar cell suspension to reorganize into cell layers typical of the normal cerebellum following transplantation into the rodent cerebellum, and develop an organotypic folia-like organization.
Bone Marrow Mononuclear Cell Count does not Predict Neutrophil and Platelet Recovery following Autologous Bone Marrow Transplant: Value of the Colony-Forming Unit Granulocyte-Macrophage (CFU-GM) Assay
Authors: Al-Fiar Fahad , Prince H. Miles , Imrie Kevin , Stewart A. Keith , Crump Michael , Keating Armand
Published: June 2017

The common use of the marrow autograft mononuclear cell (MNC) count derives from positive correlative studies following allogeneic transplantation and from earlier conflicting data regarding the value of the bone marrow autograft colony-forming unit granulocyte-macrophage (CFU-GM) assay for predicting hematologic recovery after ABMT. We conducted a retrospective analysis at our institution to determine whether autograft CFU-GM levels predict engraftment of neutrophils and platelets after ABMT in heavily pretreated patients with hematologic malignancies. Between 1 January 1993 and 1 March 1995, 58 heavily pretreated patients received only marrow cells as the autograft product. Patients with Hodgkin's disease (n = 25), acute myeloid leukemia (n = 19), and non-Hodgkin's lymphoma (n = 14) underwent intensive therapy with etoposide and melphalan. Unpurged marrow containing a minimum of 1.5 × 108/kg (range: 1.5-4.8) was infused. Median time to an absolute neutrophil count ≥0.5 × 109/L was 21 days (range 10-270) and median time to a platelet count ≥20 × 109/L independent of transfusions was 44 days (range 13-317). There was no correlation between autograft MNC count and neutrophil or platelet engraftment. However, a correlation between autograft CFU-GM and both platelet and neutrophil recovery was demonstrated with a threshold CFU-GM of 3 × 104/kg; delayed neutrophil recovery was observed in 79% of patients below this threshold compared to only 9% in those with an autograft CFU-GM level of more than 3 × 104/kg (p = 0.0001). Similarly, platelet recovery was delayed in 76% of patients below, and 20% of those above this threshold (p = 0.003). We conclude that marrow autograft CFU-GM is predictive of engraftment of both platelets and neutrophils in heavily pretreated patients after ABMT for hematological malignancies.
Implantation of Xenogeneic Transgenic Neural Plate Tissues into Parkinsonian Rat Brain
Authors: Hara Koichi , Uchida Koichi , Fukunaga Atsushi , Toya Shigeo , Kawase Takeshi
Published: June 2017

Xenografting must be considered as a means of establishing neural transplantation therapy and of securing fetal neural tissues as donor material. The early stage (embryonic day 8.5, E8.5) embryonic mesencephalic neural plate (NP) from transgenic mice was examined for possible application in effective xenografting therapy. As recipients, Parkinsonian rats treated with 6-hydroxydopamine were used, and as donors, GT4-2 mice into which a β-galactosidase gene was introduced to allow brain tissue differentiation from the recipients by X-gal staining. Three microscopic pieces of E8.5 GT4-2 mice NP were injected into the striatum of the Parkinsonian rats. Some hosts were given immunosuppressants (cyclophosphamide and FK506) (IS group), others were not (non-IS group). Amphetamine-induced rotation was examined at days 11 and 21 after grafting (D11 and D21, respectively), and morphological investigations were performed using hematoxylin-eosin (H-E), X-gal, and thyrosine hydroxylase (TH) staining. The rotations were counted in 30 of the 38 transplanted rats before and after grafting. Histological data were obtained from 19 of these 30 rats. In 11 of them the grafts survived (survival group) and in the remaining 8, the grafts were unsuccessful (rejection group). In the survival group at D11, the mean number of rotations made by transplanted rats expressed as a percentage of the number before grafting (rotation percentage) decreased to 43.8% (n = 9), which, in comparison with the average of 125.9% (n = 6) in the rejection group, reveals significant behavioral recovery (p < 0.01). The rotation percentage at D21 was 23.8% in the survival group (n = 4) and 84.5% in the rejection group (n = 3). Behavioral recovery was thus seen to improve with time in the survival group. In the IS group (n = 19), the rotation percentages averaged 74.9% (D11, n = 15) and 51.1% (D21, n = 7), while the non-IS group averages were 136.7% (D11, n = 9) and 140.7% (D21, n = 9), indicating a tendency for better behavioral recovery in the IS group than in the non-IS group (p < 0.05). Fifteen IS group rats were studied histologically, 10 (sacrificed on D11, D21) from the survival group and 5 (sacrificed on D11, D21) from the rejection group. In the non-IS group (n = 4), there was a graft in only one rat sacrificed on D11. There were many X-gal positive and TH positive cells in the grafts, suggesting that mouse NP survived, and differentiated into TH positive neurons in the rat brain. Xenografted NP has the potential to cure central nervous system diseases.
Subcutaneous Xenotransplantation of Hybrid Artificial Pancreas Encapsulating Pancreatic B Cell Line (MIN6): Functional and Histological Study
Authors: Kawakami Yoshiyuki , Inoue Kazutomo , Hayashi Hiroyuki , Wang W.j. , Setoyama Hiroshi , Gu Y.J. , Imamura Masayuki , Iwata Hiroo , Ikada Yoshito , Nozawa Masumi , Miyazaki Jun-Ichi
Published: June 2017

The biohybrid artificial pancreas is designed to enclose pancreatic endocrine tissues with a selectively permeable membrane that immunoisolates the graft from the host immune system, allowing those endocrine tissues to survive and control glucose metabolism for an extended period of time. The pancreatic B cell line MIN6 is established from a pancreas B cell tumor occurring in transgenic mice harboring the human insulin promoter gene connected to the SV40 T-antigen hybrid gene. It has been proven that glucose-stimulated insulin secretion in MIN6 cells retains a concentration-dependent response similar to that of normal islets. In this study, we performed the histological and functional examination of three-layer microbeads employing MIN6 cells after subcutaneous xenotransplantation to evaluate this device as bioartificial pancreas. MIN6 cells were microencapsulated in three-layer microbeads formulated with agarose, polystyrene sulfonic acid, polybrene, and carboxymethyl cellulose. Microbeads were xenogenically implanted in the subcutaneous tissue of the back of Lewis rats with streptozotocin-induced diabetes. One week after implantation, microbeads were retrieved and cultured for 24 h before the static incubation. There was no evidence of adhesion to the graft and the fibrosis in the transplantation site as determined by gross visual inspection. Microscopic examination demonstrated that retrieved microbeads maintained normal shape, containing intact MIN6 cells. Histological study showed that these MIN6 cells in the microbeads appeared to be viable without cellular infiltration within or around the microbeads. Immunohistochemical analysis of the microbeads clearly revealed the intense staining of insulin in the cytoplasm of encapsulated MIN6 cells. Insulin productivity of MIN6 cells in the microbeads is strongly suggested to be preserved. In response to 16.7 mM glucose stimulation, static incubation of microbeads 1 wk after implantation caused the 2.3 times increase in insulin secretion seen after 3.3 mM glucose stimulation (84.3 ± 10.0 vs. 37.4 ± 10.7 μU/3 × 106 cells/hr, n = 5 each, p < 0.01). This study demonstrates that three-layer microbeads encapsulating MIN6 cells retain excellent biocompatibility and maintain good insulin secretion even after subcutaneous xenotransplantation, suggesting the possible future clinical application of this unique bioartificial pancreas to subcutaneous xenotransplantation.
Gelatin Density Gradient for Isolation of Islets of Langerhans
Authors: Samejima Tadashi , Yamaguchi Kenji , Iwata Hiroo , Morikawa Noriyuki , Ikada Yoshito
Published: June 2017

Isolation of islets of Langerhans (islets) has been performed by means of collagenase digestion of the pancreatic tissue combined with density gradient separation of islets from unwanted exocrine tissues. An enormous number of islets are necessary for clinical islet transplantation. The density gradient used for isolation of a large number of islets should satisfy several requirements in addition to those for the conventional density gradients, such as high viscosity for creating fine interfaces with a large area, easy sterilization, and low cost. This study is concerned with the development of a new density gradient made of low-molecular-weight gelatin. We isolated islets from the hamster pancreatic tissue using the gelatin density gradients. The yield and purity of islet and its insulin release function were compared with those of islets isolated using Ficoll and Ficoll-Conray density gradients that have been conventionally used. The new gelatin density gradient can separate islets from the unwanted exocrine tissue as effectively as the Ficoll density gradient and more effectively than the Ficoll-Conray density gradients. The islets collected using the gelatin gradient retain ability of insulin release increase in response to glucose stimulation, similar to those isolated by the Ficoll-Conray gradient and more than those collected by the Ficoll gradient. In addition, the gelatin effectively inhibited enzyme activities, that is, collagenase and proteolytic enzymes released from the exocrine tissue, and thus it can inhibit overdigestion of islets during their density gradient isolation. The gelatin gradient satisfies most of the additional requirements for islet isolation from the pancreatic tissue of large animals mentioned above. Although several factors, such as molecular weight of gelatin, osmolality of the gradient, and centrifugal conditions, still remain to be optimized, our results suggest that the gelatin gradient has potentiality to isolate islets from the pancreatic tissue of a large animal.
Number and Volume of Islets Transplanted in Immunobarrier Devices
Authors: Suzuki Kazuhisa , Bonner-Weir Susan , Hollister-Lock Jennifer , Colton Clark K. , Weir Gordon C.
Published: June 2017

Immunobarrier devices may prevent immune destruction of transplanted islets, but there are concerns about survival within such devices. Islets were transplanted in diffusion chambers that employed two laminated polytetrafluoroethylene membranes held together with titanium rings. Five hundred syngeneic mouse islets placed in devices were transplanted into the epididymal fat pads of streptozotocin (STZ) diabetic mice (B6AF1). After 2 wk the devices were removed. Sections were made parallel to the membrane surface. Eight to 13 systematically selected sections of each device were analyzed by planimetry to determine the area of the device space and of the islets within that space. From these data we estimated total volume of the device, volume of islets, and number of islets in a device. The data were segregated into two groups: group I (blood glucose less than 100 mg/dL 2 wk after implantation), and group II (over 150 mg/dL). The volume (mean + SE) of devices implanted for 2 wk was 2.1 + 0.4 μL in group I and 2.2 + 0.2 μL in group II. The islet volume and number within devices were 0.30 + 0.06 and 0.17 + 0.01 μL, or 340 + 50 and 230 + 20 islets in group I and group II, respectively. The volume of fibrous tissue in devices was about 0.50 μL. About 10% of the islet tissue had central necrosis. The beta cell volume in a membrane device needed for cure is comparable to that required with islets under the kidney capsule (0.25-0.80 μL). The mass of islets contained within membrane devices needed to cure diabetes is equivalent to that of a graft in an optimal transplant site such as under the kidney capsule.
Intrastriatal Grafts from Multiple Donors do not Result in a Proportional Increase in Survival of Dopamine Neurons in Nonhuman Primates
Authors: Sladek John R. , Collier Timothy J. , Elsworth John D. , Roth Robert H. , Taylor Jane R. , Redmond D. Eugene
Published: June 2017

We examined the potential for “double grafts,” i.e., grafts from two donors in each recipient, to enhance the total number of ventral mesencephalic dopamine neurons that survive grafting in adult African green monkeys. Because dopamine cell survival in grafts represents a small percentage of the total number of neurons grafted, several human clinical trials recently have employed grafts of tissue from multiple donors (e.g., from two to eight embryos per host recipient) in attempts to increase the total number of dopamine neurons that survive in grafts. Presumably, this is intended to elevate dopamine levels by providing more dopamine neurons to the damaged brain to alleviate the symptoms of parkinsonism. While well-developed grafts with several thousand dopamine neurons were found in most recipient animals, we observed a reduced total number of tyrosine hydroxylase positive neurons in the grafts in spite of the presence of some double grafts that were larger than normal. The overall growth of the grafts was impressive; some grafts were so large that they spanned the full dorsoventral extent of the caudate nucleus, probably reflecting the fact that twice as much tissue was implanted in each drop site in comparison to our standard protocol. However, some animals revealed atypical patterns of neurite outgrowth that appeared limited to the grafted tissue, and at least one monkey revealed “amorphous” grafts generally lacking in cellular structure, which suggests a possible rejection phenomenon. These findings raise questions about the use of multiple donors and suggest that the likelihood of rejection and/or cell death may be enhanced, which is of potential importance in the design of grafting strategies for clinical applications.
Liposome-Mediated Gene Transfer into Established CNS Cell Lines, Primary Glial Cells, and in Vivo
Authors: Kofler Patrick , Wiesenhofer Bettina , Rehrl Christine , Baier Gottfried , Stockhammer Günter , Humpel Christian
Published: June 2017

Sufficient gene transfer into CNS-derived cells is the most crucial step to develop strategies for gene therapy. In this study liposome-mediated gene transfer using a β-galactosidase (β-GAL) reporter gene was performed in vitro (C6 glioma cells, NT2 neuronal precursor cells, 3T3 fibroblasts, primary glial cells) and in vivo. Using Trypan blue exclusion staining, optimal lipid concentration was observed in the range of 10-12 μg/mL. Under optimal conditions (80,000 cells/16 mm well, incubation overnight, lipid/DNA ratio = 1:18) a high transfection rate was achieved (<9% for C6 cells; <1% for NT2 cells). In primary cultures of glial cells a fair amount of positive stained cells (glial cell) was found, but the transfection efficiency was lower (<0.1%). A “boost-lipofection” markedly increased (twice) lipofection efficiency in C6 cells. Expression of β-GAL reached a maximum after 3-5 days. When the liposome–DNA complexes were injected/infused directly into the brains of adult rats, several weakly stained cells could be observed in the brain region adjacent to the injection site. It is concluded that liposome-mediated gene transfer is an efficient method for gene transfer into CNS cells in vitro, but the transfection efficiency into the rat brain in vivo is far too low and therefore not applicable.
Kidney Cografts Enhance Fiber Outgrowth from Ventral Mesencephalic Grafts to the 6-Ohda–Lesioned Striatum, and Improve Behavioral Recovery
Authors: Granholm Ann-Charlotte , Henry Stephanie , Hebert Meleik A. , Eken Servet , Gerhardt Greg A. , Van Horne Craig
Published: June 2017

Recent studies have demonstrated the presence of many different neurotrophic factors in the developing and adult kidney. Due to its production of this mixture of neurotrophic factors, we wanted to investigate whether fetal kidney tissue could be beneficial for neuritic fiber growth and/or cell survival in intracranial transplants of fetal ventral mesencephalic tissue (VM). A retrograde lesion of nigral dopaminergic neurons was performed in adult Fischer 344 male rats by injecting 6-hydroxydopamine into the medial forebain. The animals were monitored for spontaneous locomotor activity in addition to apomorphine-induced rotations once a week. Four weeks following the lesion, animals were anesthetized and embryonic day 14 VM tissue from rat fetuses was implanted stereotaxically into the dorsal striatum. One group of animals received a cograft of kidney tissue from the same embryos in the same needle track. The animals were then monitored behaviorally for an additional 4 months. There was a significant improvement in both spontaneous locomotor activity (distance traveled) and apomorphine-induced rotations with both single VM grafts and VM–kidney cografts, with the VM–kidney double grafts enhancing the motor behaviors to a significantly greater degree. Tyrosine hydroxylase (TH) immunohistochemistry and image analysis revealed a significantly denser innervation of the host striatum from the VM–kidney cografts than from the single VM grafts. TH-positive neurons were also significantly larger in the cografts compared to the single VM grafts. In addition to the dense TH-immunoreactive innervation, the kidney portion of cografts contained a rich cholinergic innervation, as evidenced from antibodies against choline acetyltransferase (ChAT). The striatal cholinergic cell bodies surrounding the VM–kidney cografts were enlarged and had a slightly higher staining density for ChAT. Taken together, these data support the hypothesis that neurotrophic factors secreted from fetal kidney grafts stimulated both TH-positive neurons in the VM cografts and cholinergic neurons in the host striatum. Thus, these factors may be combined for treatment of degenerative diseases involving both dopaminergic and cholinergic neurons.
One-Year Chromaffin Cell Allograft Survival in Cancer Patients with Chronic Pain: Morphological and Functional Evidence
Authors: Bés Jean C. , Tkaczuk Jean , Czech Kimberly A. , Tafani Mathieu , Bastide Raymond , Caratero Claude , Pappas George D. , Lazorthes Yves
Published: June 2017

The control of chronic pain through transplantation of chromaffin cells has been reported over the past few years. Analgesic effects are principally due to the production of opioid peptides and catecholamines by chromaffin cells. Clinical trials have been reported with allografts consisting of whole-tissue fragments implanted into the subarachnoid space of the lumbar spinal cord (14,19,36). In the present study, allogeneic grafts were successfully used to control chronic pain in two patients over a period of 1 yr based on patient reported pain scores, morphine intake, and CSF levels of Met-enkephalin. Macroscopic examination at autopsy located the transplanted tissue fragments in the form of multilobulated nodules at the level of the spinal axis and cauda equina. Immunocytochemical microscopy showed neuroendocrine cells are positive for chromagranin A (CGA), and enzymes tyrosine hydroxylase (TH) and dopamine-β-hydroxylase (DβH). The results suggest that there is a relationship between analgesic effect, Met-enkephalin levels in CSF, and the presence of chromaffin cells surviving in spinal subarachnoid space.
Perfusion Enhances Functions of Bone Marrow Stromal Cells in Three-Dimensional Culture
Authors: Glowacki Julie , Mizuno Shuichi , Greenberger Joel S.
Published: June 2017

Perfusion of medium through three-dimensional (3D) collagen sponges enhanced viability and function of cocultivated marrow stromal and hematopoietic cell lines. Cells of the murine bone marrow stromal cell line GPIa were cultured in novel 3D collagen sponges, made from pepsin-digested bovine skin. Static cultures of sponges were maintained in dishes with media changes every other day. Perfused sponges were contained in a glass column with medium flow set at 1.3 mL/min. In some sponges, the 32D cl3 c-fmsm (CRX-1) hematopoietic progenitor cell line was added 7 days after GPIa cells. At 7 and 16 days, light microscopic evaluation showed poor viability of cells in static sponge cultures. In perfused sponge cultures, there was greater cellularity throughout the sponge and abundant accumulation of metachromatic extracellular matrix surrounding GPIa cells. Chondroitin 6-sulfate and heparan sulfate were identified as components of the matrix by immunohistochemical methods. DNA synthesis was evaluated by 15-h exposure of cultures to bromodeoxyuridine (BrdU), with subsequent immunohistochemical localization with monoclonal anti-BrdU antibody. Cells positive for BrdU were identified at the outer surfaces of both static and perfused sponges; however, positive cells were also seen throughout the internal areas of the sponges that were perfused. These results suggest that better nutrient exchange occurred in perfused sponges. In static cocultures of GPIa and CRX-1 cells, there was no detectable viability of the IL-3–dependent CRX-1 cells; however, under perfused conditions, CRX-1 cells flourished within the sponges as documented by BrdU incorporation. Thus, medium perfusion enhanced GPIa stromal cell line viability and function in 3D collagen sponge cultures, as demonstrated by BrdU incorporation, matrix production, and support of CRX-1 cells. This novel culture system may be useful for examining the interactions of bone marrow stromal cells with extracellular matrix molecules, soluble and matrix-bound factors, and with other cell types.
Overcoming the Histocompatibility Barrier: Transferrins as Carriers and Modulators of Immunogenic Identity
Authors: Pierpaoli Walter
Published: June 2017

Proteic molecules were found in the bone marrow that were later identified as transferrins. When applied to transplantation of genetically incompatible bone marrow in supralethally irradiated recipient mice, the transferrins obtained from plasma of bone marrow donors promoted engraftment, permanent hemopoietic chimerism, and donor-type immune character. A combination of donor-matched transferrins and antigens was needed for induction of xenogeneic (interspecies) “tolerance” or unresponsiveness to donor antigens in chemically immunosuppressed mice treated with human transferrins and donor leucocytes. This novel and unique property of transferrins may explain the genesis and maintenance of immunogenic identity and allow a reshaping of the immune system. © 1998 Elsevier Science Inc.
Development of Approaches to Improve the Healing following Muscle Contusion
Authors: Kasemkijwattana Channarong , Menetrey Jacques , Somogyi George , Moreland Morey S. , Fu Freddie H. , Buranapanitkit Boonsin , Watkins Simon C. , Huard Johnny
Published: June 2017

Muscle injuries are a challenging problem in traumatology, and the most frequent occurrence in sports medicine. Muscle contusions are among the most common muscle injuries. Although this injury is capable of healing, an incomplete functional recovery often occurs, depending on the severity of the blunt trauma. We have developed an animal model of muscle contusion in mice (high energy blunt trauma) and characterized the muscle's ability to heal following this injury using histology and immunohistochemistry to determine the level of muscle regeneration and the development of scar tissue. We have observed a massive muscle regeneration occurring in the first 2 wk postinjury that is subsequently followed by the development of muscle fibrosis. Based on these observations, we propose that the enhancement of muscle growth and regeneration, as well as the prevention of fibrotic development, could be used as approach(es) to improve the healing of muscle injuries. In fact, we have identified three growth factors (bFGF, IGF-1, and NGF) capable of enhancing myoblast proliferation and differentiation in vitro and improving the healing of the injured muscle in vivo. Furthermore, the ability of adenovirus to mediate direct and ex vivo gene transfer of β-galactosidase into the injured site opens possibilities of delivering an efficient and persistent expression of these growth factors in the injured muscle. These studies should help in the development of strategies to promote efficient muscle healing with complete functional recovery following muscle contusion. © 1998 Elsevier Science Inc.