bluuebunny/crossref_metadata_2025 huggingface dataset:
Assessment of the Reproducibility of Alginate Encapsulation of Pancreatic Islets Using the MTT Colorimetric Assay
Authors: Heald K.A. , Jay T.R. , Downing R.
Published: June 2017

Radioisotope diffusion experiments demonstrate that alginate/polyaminoacid encapsulation can prevent antibody and cytotoxic cell contact in vitro. The unpredictable outcome of xenotransplantation of encapsulated islets may reflect incomplete encapsulation. We have assessed a cytotoxic/MTT (tetrazolium) assay to test antibody permeability of capsules.

Samples of free porcine islet tissue, and islet tissue encapsulated in alginate/poly-l-lysine/alginate microspheres were incubated with fresh autologous pig serum or normal human serum overnight. Cell metabolism was assessed by the MTT assay.

Data from eight experiments (10 replicates/experiment) were analyzed using the Mann-Whitney U-test. Values were deemed significant when p < 0.05. Free islets cultured in human serum showed a significant reduction in metabolism when compared with islets cultured in pig serum: percentage reduction 52 ± 23% (mean ± SD). The differences in formazan production were significant in all experiments (p < 0.05). Alginate encapsulation of islets or removal of xeno-reactive antibodies in human serum by adsorption was shown to prevent the effects of complement-mediated lysis. However, in one of the eight experiments, there was a significant reduction in islet metabolism after exposure of encapsulated porcine islets to human serum.

In conclusion, it has been shown that alginate encapsulation can prevent complement-mediated lysis. However, the encapsulation process employed was imperfect and did not prevent complement-mediated lysis of porcine islets in all experiments. The cytotoxicity/MTT assay allows investigation of the permeability of capsules to serum antibodies and could be performed to determine the viability of the islets and the integrity of microcapsules prior to transplantation.
In Vivo Cell Transformation: Neogenesis of Beta Cells from Pancreatic Ductal Cells
Authors: Rosenberg Lawrence
Published: June 2017

During embryogenesis, islet cells differentiate from primitive duct-like cells. This process leads to the formation of islets in the mesenchyme adjacent to the ducts. In the postnatal period, any further expansion of the pancreatic endocrine cell mass will manifest itself either by a limited proliferation of the existing islet cells, or by a reiteration of ontogenetic development. It is the latter, cell transformation by a process of differentiation from a multipotential cell, that will be referred to in this review as islet neogenesis. To better appreciate the mechanisms underlying islet cell neogenesis, some of the basic concepts of developmental biology will be reviewed. Considerable discussion is devoted to the subject of transdifferentiation, a change in a cell or in its progeny from one differentiated phenotype to another, where the change includes both morphological and functional phenotypic markers. While in vitro studies with fetal and neonatal pancreata strongly suggest that new islet tissue is derived from ductal epithelium, what is not established is whether the primary cell is a committed endocrine cell or duct-like cell capable of transdifferentiation. Next, research in the field of β-cell neogenesis is surveyed, in preparation for the examination of whether there is a physiological means of inducing islet cell regeneration, and whether the new islet mass will function in a regulated manner to reverse or stabilize a diabetic state? Our belief is that the pancreas retains the ability to regenerate a functioning islet cell mass in the postnatal period, and that the process of cell transformation leading to islet neogenesis is mediated by growth factors that are intrinsic to the gland. Furthermore, it is our contention that these factors act directly or indirectly on a multipotential cell, probably associated with the ductular epithelium, to induce endocrine cell differentiation. In other words, new islet formation in the postnatal period reiterates the normal ontogeny of islet cell development. These ideas will be fully developed in a discussion of the Partial Duct Obstruction (PDO) Model.
Amelioration of the Behavioral Phenotype in Genetically Ataxic Mice through Bilateral Intracerebellar Grafting of Fetal Purkinje Cells
Authors: Triarhou Lazaros C. , Zhang Zei , Le Wei-Hua
Published: June 2017

We have previously applied neural grafting to “Purkinje cell degeneration” mutant mice (gene symbol pcd, mouse chromosome 13), a model of recessively inherited cerebello-olivary atrophy, to create appropriate interactions between wild-type and mutant cells in elucidating gene effects on the involved neuron populations and to address issues of the structural integration of donor Purkinje cells into the disrupted cerebellar loop. Behaviorally, pcd homozygotes manifest ataxic signs beginning at 3-4 wk of age. The functional effects of cerebellar transplants on motor performance have long remained an open question. The aim of the present study was to determine the recovery of motor responses in pcd mutants in a battery of behavioral tasks after bilateral transplantation of cerebellar cell suspensions (prepared from wild-type mice) into the parenchyma of the deep cerebellar nuclei of the hosts, according to a protocol that emphasizes the reconstruction of the missing inhibitory cortico-nuclear projection. With this approach, the denervated deep nuclei of the host receive a new Purkinje axonal innervation; further, most transplanted Purkinje cells end up occupying cortical localities anyway and display a correct dendritic tree orientation toward the pia. Motor coordination and fatigue resistance were assessed in a rotarod treadmill apparatus, a behavioral paradigm useful in studying various brain abiotrophies and treatments, including developmental perturbations of the cerebellar cytoarchitecture. Locomotor activity was quantified by the number of squares mice crossed as they moved about in an open-field matrix. Grafted pcd mice performed significantly better than sham-operated mutants in both of these tasks. Moreover, graft-recipient mice were able to sustain their abdomen above the floor on their limbs during movement, contrasting to the typical lowered, widened stance of sham-operated pcd mutants. These findings clearly demonstrate that bilateral transplants of fetal Purkinje cells have functional effects on motor performance in the pcd model of hereditary cerebellar ataxia.
Clinical Study of Fetal Mesencephalic Intracerebral Transplants for the Treatment of Parkinson's Disease
Authors: Kopyov Oleg V. , Jacques Deane “Skip” , Lieberman Abraham , Duma Christopher M. , Rogers Robert L.
Published: June 2017

This study reports our findings from 22 patients (ages ranging from 42 to 73 yr; mean = 55.2) with recalcitrant idiopathic Parkinson's disease (PD) who received implants of fetal ventral mesencephalic tissue using an MRI-guided stereotactic procedure and who have been followed for at least 6 mo postoperatively, employing the guidelines established by the Core Assessment Program for Intracerebral Transplantations. Evaluations were videotaped and were performed both on and off levodopa medications. To date, we have seven patients with 24 mo, three with 18 mo, three with 12 mo, and nine with 6 mo of postsurgical assessments. Comparing surgical outcomes to levels prior to fetal transplants we found: 1) mean levodopa levels were reduced 46% at 6 mo, 12% at 12 mo, 20% at 18 mo, and 54% at 24 mo; 2) Unified Parkinson's Disease Rating Scale (UPDRS) scores with patients on levodopa were improved by an average of 38% (6 mo), 50.2% (12 mo), 69.3% (18 mo), and 73.9% (24 mo), while off medication scores showed reductions ranging from 24.7% at 6 mo to 55.1% at 24 mo. Other measures, including Hoehn-Yahr staging, Activities of Daily Living, and dyskinesia rating scales, were also significantly improved following fetal transplants. Timed motor tasks (finger dexterity, supination-pronation, foot tapping, and Stand-Walk-Sit) performance also demonstrated highly significant improvements. Patient's self-rating scores indicated that the patients typically perceived substantial improvements in their condition. However, substantial variability in the improvements following surgery still persists and range from nominal improvements in performance to significant changes that can be classified as altering the overall lifestyle of the patients. To date, 4 of the 22 subjects were considered by the physicians to be nonresponders; that is, there were no clinically relevant improvements in these patients' conditions.
Long-Term Expression of a Fluorescent Reporter Gene via Direct Injection of Plasmid Vector into Mouse Skeletal Muscle: Comparison of Human Creatine Kinase and Cmv Promoter Expression Levels in Vivo
Authors: Bartlett R.J. , Secore S.L. , Singer J.T. , Bodo M. , Sharma K. , Ricordi C.
Published: June 2017

Expression of a fluorescent reporter gene has been studied using two alternate promoters to transcribe the green fluorescent protein (gfp) from Aequorea victoria. The human cytomegalovirus (CMV) enhancer/promoter or the human muscle-specific creatine kinase promoter (CKM) were inserted along with the gfp cDNA into a plasmid expression vector based on a modified adeno-associated virus genome. Naked plasmid DNA was injected into the hamstring muscle of mdx mice and gfp gene expression determined from frozen muscle sections taken at 4, 14, and 42 days postinjection. Fluorescence patterns obtained by photomicroscopy and quantitative fluorescence measurements indicated a near-linear increase in the accumulation of the gfp in skeletal muscle during the length of the study, with gfp expression at 42 days being roughly four times the values obtained at 4 days. The levels of expression of gfp from the CKM construct were consistantly higher than for the CMV construct. The CKM promoter/expression vector combination demonstrates significant potential for simple, direct delivery and long-term, high-level expression of genes in skeletal muscle.
Production and Characterization of Fetal Sheep Pancreatic Islet-Like Cell Clusters
Authors: Tuch Bernard E. , Madrid Jocelyn C. , Summers Ellie , Smith Murray S.R.
Published: June 2017

Explants of fetal sheep pancreas transplanted into diabetic athymic mice survive for many months but there is only partial differentiation of the endocrine cells. As an alternative form of graft we examined the possibility of creating islet-like cell clusters (ICCs) by collagenase digestion of the fetal sheep pancreas, as has been described for human and porcine fetal pancreas. Such ICCs did form at the rate of 6-23 per 10 mg pancreas; their size varied between 65 and 474 μm (median 232 μm) and their insulin content was 1.6 ± 0.2 mU per 20 ICCs. Laser scanning confocal analysis showed that 4.6 ± 0.7% of the cells contained insulin. Insulin was secreted from ICCs maintained in culture at the daily rate of 2.5 mU per 30 ICCs. Arginine but not glucose or theophylline enhanced acute insulin secretion in vitro. Transplantation of up to 1000 ICCs into athymic and scid mice resulted in sparse growth of the epithelial-like cells in the graft and only partial differentiation of the endocrine cells. Hyperglycaemia in diabetic recipients was not normalized. Thus, while functioning ICCs can be created from fetal sheep pancreas, they do not appear to be appropriate for transplantation to reverse diabetes in mice.
Short-Term Hypothermic Preservation of Porcine Hepatocyte Spheroids using uw Solution
Authors: Sakai Yasuyuki , Naruse Katsutoshi , Nagashima Ikuo , Muto Tetsuichiro , Suzuki Motoyuki
Published: June 2017

The feasibility of University of Wisconsin (UW) solution in short-term hypothermic preservation of porcine hepatocyte spheroids was investigated, because they have great potential in bioartificial liver (BAL) systems. Porcine hepatocyte spheroids preserved for 3 days expressed almost comparable levels of albumin secretion as those without preservation, during 8 subsequent days of recultivation in continuous rotational culture, whereas isolated single cells did not reorganize into spheroids and completely lost their function in recultivation. Although for 3-day–preserved spheroids, the albumin secretion was lowered immediately after recultivation (Days 0–2), it was completely restored to that of nonpreserved ones. The function was completely lost in recultivation for 7-day–preserved ones. These results demonstrate that reorganization into spheroids is effective in preventing the functional loss of porcine hepatocytes occurring in hypothermic preservation, and that spheroid formation should precede the preservation as long as spheroid culture is finally used in BAL systems. Also, porcine hepatocyte spheroids are shown to be satisfactory stored in UW solution up to 3 days without significant cellular or functional loss.
Growth Properties of Neural Cell Lines Immortalized with the Tsa58 Allele of Sv40 Large T Antigen
Authors: Truckenmiller M.E. , Dillon-Carter Ora , Tornatore Carlo , Kulaga Henrietta , Takashima Hidetoshi , Freed William J.
Published: June 2017

In vitro growth properties of three CNS-derived cell lines were compared under a variety of culture conditions. The M213-20 and J30a cell lines were each derived from embryonic CNS culture with the temperature-sensitive (ts) allele of SV40 large T antigen, tsA58, while the A7 cell line was immortalized using wild-type SV40 large T antigen. Cells immortalized with tsA58 SV40 large T proliferate at the permissive temperature, 33° C, while growth is expected to be suppressed at the nonpermissive temperature, 39.5°C. Both the M213-20 and J30a cell lines were capable of proliferating at 39.5°C continuously for up to 6 mo. All three cell lines showed no appreciable differences in growth rates related to temperature over a 7-day period in either serum-containing or defined serum-free media. The percentage of cells in S-phase of the cell cycle did not decrease or was elevated at 39.5°C for all three cell lines. After 3 wk at 39.5°C, the three cell lines also showed positive immunostaining using two monoclonal antibodies reacting with different epitopes of SV40 large T antigen. Double strand DNA sequence analyses of a 300 base pair (bp) fragment of the large T gene from each cell line, which included the ts locus, revealed mutations in both the J30a and M213-20 cell lines. The J30a cell line ts mutation had reverted to wild type, and two additional loci with bp substitutions with predicted amino acid changes were also found. While the ts mutation of the M213-20 cells was retained, an additional bp substitution with a predicted amino acid change was found. The A7 cell line sequence was identical to the reference wild-type sequence. These findings suggest that (a) nucleic acid sequences in the temperature-sensitive region of the tsA58 allele of SV40 large T are not necessarily stable, and (b) temperature sensitivity of cell lines immortalized with tsA58 is not necessarily retained.
Cerebellar Grafts Partially Reverse Amino Acid Receptor Changes Observed in the Cerebellum of Mice with Hereditary Ataxia: Quantitative Autoradiographic Studies
Authors: Stasi Kalliope , Mitsacos Adamantia , Triarhou Lazaros C. , Kouvelas Elias D.
Published: June 2017

We used quantitative autoradiography of [3H]CNQX (200 nM), [3H]muscimol (13 nM), and [3H]flunitrazepam (10 nM) binding to study the distribution of non-NMDA and GABAAreceptors in the cerebellum of pcd mutant mice with unilateral cerebellar grafts. Nonspecific binding was determined by incubation with 1 mM Glu, 200 μM GABA, or 1 μM clonazepam, respectively. Saturation parameters were defined in wild-type and mutant cerebella. In mutants, non-NMDA receptors were reduced by 38% in the molecular layer and by 47% in the granule cell layer. The reduction of non-NMDA receptors in the pcd cerebellar cortex supports their localization on Purkinje cells. [3H] CNQX binding sites were visualized at higher density in grafts that had migrated to the cerebellar cortex of the hosts (4.1 and 11.0 pmol/mg protein, respectively, at 23 and 37 days after grafting) than in grafts arrested intraparen-chymally (2.6 and 6.2 pmol/mg protein, respectively, at 23 and 37 days after grafting). The pattern of expression of non-NMDA receptors in cortical vs. parenchymal grafts suggests a possible regulation of their levels by transacting elements from host parallel fibers. GABAAbinding levels in the grafts for both ligands used were similar to normal molecular layer. Binding was increased in the deep cerebellar nuclei of pcd mutants: the increase in [3H]muscimol binding over normal was 215% and the increase in [3H]flunitrazepam binding was 89%. Such increases in the pcd deep cerebellar nuclei may reflect a denervation-induced supersensitivity subsequent to the loss of Purkinje axon terminal innervation. In the deep nuclei of pcd mutants with unilateral cerebellar grafts, [3H]muscimol binding was 31% lower in the grafted side than in the contralateral nongrafted side at 37 days after transplantation; [3H]fluni-trazepam binding was also lower in the grafted side by 15% compared to the nongrafted side. Such changes in GABAAreceptors suggest a significant, albeit partial, normalizing trend of cerebellar grafts on the state of postsynaptic supersensitive receptors in the host cerebellar nuclei.
Use of Allogeneic Bone Marrow Labeled with Neomycin Resistance Gene to Examine Bone Marrow-Derived Chimerism in Experimental Organ Transplantation
Authors: Smith J. P. , Kasten-Jolly J. , Rebellato L. , Haisch Carl E. , Thomas Judith M.
Published: June 2017

Posttransplant infusion of viable donor bone marrow cells (DBMC) has been shown in our previous studies to promote acceptance of incompatible kidney allografts in rhesus monkeys after treatment with polyclonal antithymocyte globulin to deplete peripheral T-lymphocytes. In this nonhuman primate model, the infusion of the DBMC is requisite for the induction of functional graft tolerance and specific MLR and CTLp unresponsiveness, although the relevant role and fate of bone marrow-derived chimeric cells is uncertain. Standard immunological and molecular techniques applied to this monkey model are unable to differentiate between chimeric cells derived from the infused DBMC and those derived from allograft-borne passenger leukocyte emigrants. To distinguish chimerism due to infused DBMC, we transduced DBMC with a functional neomycin resistance gene (Neor) using the retroviral vector pHSG-Neo. Neor-Mransduced BMC were infused into recipients approximately 2 wk after kidney transplantation and treatment with rabbit antithymocyte globulin. No maintenance immunosuppressive drugs were given. Genomic DNA isolated from peripheral blood leukocytes was used to monitor the presence of Neor-positive cells. Tissue samples obtained at necropsy also were assessed for Neor-positive chimeric cells. The presence of DBMC-derived chimerism was assessed by polymerase chain reaction using Neor sequence-specific primers (PCR-SSP). Chimerism was detectable in recipient tissues at various times for up to 6 mo after DBMC infusion. These studies using gene transduction methodology indicate that a stable genetic marker can provide capability to examine DBMC-derived chimerism for prolonged periods in a nonhuman primate model. This approach should facilitate future studies in preclinical models to study the role and type of chimeric cell lineages in relation to functional allograft tolerance.
Towards the Development of a Bioartificial Pancreas: Effects of Poly-l-Lysine on Alginate Beads with BTC3 Cells
Authors: Benson J.P. , Papas K.K. , Constantinidis I. , Sambanis A.
Published: June 2017

A bioartificial tissue construct that consists of insulin-secreting cells entrapped in an alginate/poly-l-lysine (PLL) matrix offers a promising approach for the treatment of type I diabetes. Use of transformed cells has been proposed as a solution to the cell availability problem posed by islets. The growth characteristics of transformed cells in their sequestered environment and the effects of PLL on their metabolic and secretory activities have not yet been characterized. Our data demonstrate that mouse insulinoma βTC3 cells proliferate while they are entrapped in both PLL-free and PLL-coated alginate beads. During this process, cell aggregates develop in the bead periphery, which increase in number and size with time. PLL is crucial for the long-term in vitro structural stability of beads, and it does not appear to affect the metabolic and secretory activities of entrapped βTC3 cells. The implications of these findings in the development of a bioartificial pancreatic construct based on transformed cells are discussed.
An Internet Multicast Symposium Concerning the Microcirculation of the Islets of Langerhans
Authors: Brunicardi F. Charles , Moldovan Stefan , Gilman Stewart , Livingston Edward , Passaro Edward
Published: June 2017

The Internet Globally-linked Computer System was used to conduct an international scientific symposium. The symposium was held at the VAMC-Long Beach and consisted of prepared lectures that were multicast over the Internet. The basic unit of hardware used for the Internet Multicast was the Silicon Graphics Indy Unix Workstation, which was equipped with a color video camera. The multicast required four additional pieces of software from the file transfer protocol. The multicast backbone protocol allowed for simultaneous audio and video signals (the presenter, the slides, and the videotape images of islet microcirculation studies) to be transmitted over the computer network. The faculty included 12 experts in microcirculation, who gave 15-min lectures that included a question and answer period. All lectures were received at 14 computer stations in six countries. Eleven of the faculty gave their lectures at the VAMC-Long Beach, and one gave her lecture at the Massachusetts Institute of Technology in Boston, MA. The presenter from Boston was able to receive and answer questions from the faculty at the VAMC-Long Beach. An estimated $12,000 was saved in travel, hotel, and food costs and an estimated 180 travel hours were saved by viewers who did not have to travel to the symposium. We have demonstrated that a scientific symposium can be conducted using the Internet. We propose that many of our future meetings will be organized over the computer network. This format of multiimage projections allows us to effectively communicate in a personal way with a reduction in expensive and time-consuming travel.
Immunological Responses to Injury and Grafting in the Central Nervous System of Nonhuman Primates
Authors: Bakay Roy A.E. , Boyer Kevin L. , Freed Curt R. , Ansari Aftab A.
Published: June 2017

Allogeneic transplantation for the therapy of human Parkinson's disease is being considered as a viable approach at several clinical centers worldwide. As an attempt to understand the basic biology of central nervous system (CNS) transplantation, our laboratory has developed an experimental nonhuman primate model for human Parkinson's disease and carried out preliminary studies directed at evaluating the potential pathology at the graft site. In addition, studies have been conducted to examine whether such transplantation procedures lead to specific and/or nonspecific immunologic sensitization of the host or results in generalized immunosuppression. Groups of rhesus macaques (Macaca mulatta) were either controls operated (n = 6), autografted with adrenal medullary and peripheral nerve tissue (n = 3), or allografted with fetal mesencephalic tissue (n = 6). Immunohistological studies demonstrated the presence of mononuclear cell infiltrates as early as 1 wk and up to 1 yr postoperatively, although the frequency of the infiltrating cells declined with time. The infiltrates consisted of variable numbers of cells which express CD2+, CD3+, CD4+, CD8+, CD19+, CD22+, CD25+, and CD68+. There appeared to be no difference in the frequency, kinetics, or phenotype of the infiltrating cells in operative controls compared with recipients of auto- or allografts. Tissue sections obtained postoperatively showed low levels of major histocompatibility complex (MHC) Class I antigens and no detectable level of MHC-Class II antigens in neural tissue. A small aliquot of tissue from the operative site was placed in vitro with media containing interleukin-2 (IL-2), which led to the exudation and growth of mononuclear cells that were predominantly CD4+ cells. Phenotypic studies of peripheral blood mononuclear cells (PBMC) from operative controls, auto- and allograft recipient monkeys performed at varying time periods postoperatively failed to show differences in the frequencies of subsets of T-cells, B-cells, NK-cells, or monocytes. Studies on aliquots of the same PBMC failed to show major functional differences in NK-cells, LAK cells, or response to polyclonal mitogens. Finally, recipients of allogeneic mesencephalic grafts failed to show evidence of donor-specific humoral or cellular sensitization. These data indicate that transplantation of autograft adrenal or allograft fetal mesencephalic tissues in the CNS of nonhuman primate did not induce detectable donor-specific sensitization nor nonspecific immunosuppression.
Trophic Effect of Porcine Sertoli Cells on Rat and Human Ventral Mesencephalic Cells and Hnt Neurons in Vitro
Authors: Othberg Agneta I. , Willing Alison E. , Cameron Don F. , Anton Alex , Saporta Samuel , Freeman Thomas B. , Sanberg Paul R.
Published: June 2017

The poor survival of embryonic dopaminergic (DA) neurons transplanted into patients with Parkinson's disease (PD) has encouraged researchers to search for new methods to affect the short- as well as long-term survival of these neurons after transplantation. In several previous rodent studies Sertoli cells increased survival of islet cells and chromaffin cells when cotransplanted in vivo. The aims of this study were to investigate whether porcine Sertoli cells had a positive effect on the survival and maturation of rat and human DA neurons, and whether the Sertoli cells had an effect on differentiation of neurons derived from a human teratocarcinoma cell line (hNT neurons). A significant increase of tyrosine hydroxylase (TH)-positive neurons of both rat and human ventral mesencephalic tissue was found when cocultured with Sertoli cells. Furthermore, there was a significantly increased soma size and neurite outgrowth of neurons in the coculture treated group. The Sertoli cell and hNT coculture also revealed an increased number of TH-positive cells. These results demonstrate that the wide variety of proteins and factors secreted by porcine Sertoli cells benefit the survival and maturation of embryonic DA neurons and suggest that cotransplantation of Sertoli cells and embryonic DA neurons may be useful for a cell transplantation therapy in PD.
Cellular Delivery of Cntf but not Nt-4/5 Prevents Degeneration of Striatal Neurons in a Rodent Model of Huntington's Disease
Authors: Emerich Dwaine F. , Bruhn Susan , Chu Yaping , Kordower Jeffrey H.
Published: June 2017

The delivery of neurotrophic factors to the central nervous system (CNS) has gained considerable attention as a potential treatment strategy for neurodegenerative disorders such as Huntington's disease (HD). In the present study, we directly compared the ability of two neurotrophic factors, ciliary neurotrophic factor (CNTF), and neurotrophin-4/5 (NT-4/5), to prevent the degeneration of striatal neurons following intrastriatal injections of quinolinic acid (QA). Expression vectors containing either the human CNTF or NT-4/5 gene were transfected into a baby hamster kidney fibroblast cell line (BHK). Using a polymeric device, encapsulated BHK-control cells and those secreting either CNTF (BHK-CNTF) or NT-4/5 (BHK-NT-4/5) were transplanted unilaterally into the rat lateral ventricle. Seven days later, the same animals received unilateral injections of QA (225 nmol) into the ipsilateral striatum. Nissl-stained sections demonstrated that the BHK-CNTF cells significantly reduced the volume of striatal damage produced by QA. Quantitative analysis of striatal neurons further demonstrated that both choline acetyltransferase (ChAT)- and glutamic acid decarboxylase (GAD)-immunoreactive neurons were protected by CNTF implants. In contrast, the volume of striatal damage and loss of striatal ChAT and GAD-positive neurons in animals receiving BHK-NT-4/5 implants did not differ from control-implanted animals. These results help better define the scope of neuronal protection that can be afforded following cellular delivery of various neurotrophic factors. Moreover, these data further support the concept that implants of polymer-encapsulated CNTF-releasing cells can be used to protect striatal neurons from excitotoxic damage, and that this strategy may ultimately prove relevant for the treatment of HD.
In Vivo Identification, Survival, and Functional Efficacy of Transplanted Hepatocytes in Acute Liver Failure Mice Model by Fish Using Y-Chromosome Probe
Authors: Vanaja Donkena Krishna , Sivakumar Bhattiprolu , Jesudasan Rachel A. , Singh Lalji , Janardanasarma Marasanapalle Kalle , Habibullah Chittoor Mohammed
Published: June 2017

Hepatocyte transplantation has excited much interest in lending temporary metabolic support to a failing liver following acute liver injury. The exact site from which they act and the clinical, biochemical, and histological changes in the recipient body following hepatocyte transplantation is yet to be worked out. The present study is an attempt to delineate location and function of transplanted hepatocytes and also the overall survival of these cells with a fluorescent in situ hybridization (FISH) technique using a Y-chromosome–specific probe in a carbon tetrachloride (CCl4)-induced mice model of fulminant hepatic failure. Fifty-five syngenic adult Swiss female mice of approximately the same age and body weight were divided into three groups. Group-1 (n = 15), which received mineral oil, served as a negative control. Group-II (n = 15) received CCl4 (3 mL/kg) 40% vol/vol in mineral oil, by gavage served as positive control for hepatic failure. Group-III (n = 25) received intrasplenic transplantation of syngenic single cell suspension of hepatocytes in Hanks medium, after 30 h of CCl4 administration. Male Swiss adult mice (n = 15) served as donors of hepatocytes. The overall survival of animals in groups I to III was 100, 0, and 70%, respectively, by 2 wk of the study period. Transplanted hepatocytes were identified by Periodic Acid Schiff (PAS) staining and confirmed with a FISH technique using the Y-chromosome probe. The majority of exogenously transplanted hepatocytes were found in the liver and spleen sections even after 1 wk of hepatocyte transplantation. Transplanted cells were mostly found to be translocated into the sinusoids of the liver. Transplanted hepatocytes were found to be beneficial as a temporary liver support in a failing liver, significantly improving the survival of the animals. In the present study, the FISH technique was used to unequivocally distinguish the transplanted cells from the host, and thus describes a model for studying the distribution and survival of the transplanted cells.
Human SeruM Albumin Microspheres Approximate Initial Organ-Specific Biodistributions of Transplanted Hepatocytes and Are Effective Cell Surrogates for Safety Studies
Authors: Rajvanshi Pankaj , Bhargava Kuldeep K. , Afriyie Menes , Camaya Maria V. , Gagandeep S. , Vasa Srinivasa Rao G. , Palestro Christopher J. , Gupta Sanjeev
Published: June 2017

Liver repopulation with transplanted hepatocytes will generate novel cell-based therapies, although translocation of transplanted cells into lungs through portasystemic shunts has the potential for embolic complications. To facilitate safety analysis of hepatocyte transplantation, we wished to obtain effective cell surrogates and analyzed biodistributions of similarly sized99mTc-labeled human serum albumin microspheres and rat hepatocytes. Image analysis with dual99mTc and111In labels indicated that cells and microspheres were similarly distributed in the liver when injected into normal rats via the spleen. Also, their distributions were similar when injected via a femoral vein or the superior mesenteric vein with cells and microspheres localizing in lungs or liver, respectively. Upon intraportal injection in rats with portal hypertension, microspheres localized in both liver and lungs, consistent with portasystemic shunting. These data demonstrate that human serum albumin microspheres are effective cell surrogates for approximating the safety of hepatocyte transplantation and should be clinically useful.
Islet Allograft Rejection in Rats: a Time Course Study Characterizing Adhesion Molecule Expression, Mhc Expression, and Infiltrate Immunophenotypes
Authors: Coddington Douglas A. , Yang Hua , Rowden Geoffrey , Colp Pat , Issekutz Thomas B. , James R. Wright
Published: June 2017

Wistar Furth (RT1u) islets transplanted under the renal capsules of streptozotocin-diabetic Lewis (RT1l) rats reject after 5–6 days of normoglycemia. Hand-picked WF islets (1500–2000) were transplanted under the kidney capsules of diabetic Lew or WF rats. Rats bearing iso- or allografts were killed on posttransplant days 2, 4, and 6. Serial frozen sections of grafts and controls were stained by immunoperoxidase for rat MAC-1, class II MHC, CD2, CD4, CD8, B-cells, VLA-4, LFA-1, L-selectin, ICAM-1, and VCAM-1. Infiltrating cells, parenchymal cells, and endothelial cells in five distinct compartments (i.e., peritoneal reflection, subcapsular perivascular space, islet grafts, graft–kidney interface, and kidney) were evaluated for expression of the various markers at each interval. Significant infiltrates arrived in three distinct waves in both iso- and allografts. First, macrophages blanketed the peritoneal capsular reflection and infiltrated by day 2. Second, the first wave of lymphocytes arrived in the edematous subcapsular soft tissue via capsular vessels by day 2 (allo > iso). Third, the second wave of lymphocytes arrived from the renal parenchyma to form a dense band at the graft–kidney interface and around grafts by days 4 and 6 (allo >>> iso); CD4+ cells vastly outnumbered CD8+ cells, with CD4+ cells being mobilized first and from interstitial vessels throughout the entire kidney. CD8+ cells emigrated only from renal interstitial vessels adjacent to the graft. Large numbers of L-selectin+, VLA-4+, and LFA-1+ cells were seen in the infiltrates with the most intensely staining cells being intravascular. B-cells composed a very small proportion of infiltrating cells in both allo- and isografts. Endothelial staining for ICAM-1 and VCAM-1 was prominent throughout. Both class II MHC and ICAM-1 expression were induced on renal tubular epithelial cells, but neither was found on islet parenchymal cells. In conclusion, this study shows that islet allograft rejection is more complex than previously realized.
TILAPIA—A Source of Hypoxia-resistant Islet Cells for Encapsulation
Authors: James R. Wright , Yang Hua , Dooley Kent C.
Published: June 2017

Encapsulation of pancreatic islets prevents graft revascularization after transplantation, resulting in graft hypoxia and attrition. Hypoxia-resistant islets would be ideal for encapsulation. Tilapia, a tropical teleost fish, have large, anatomically discrete islets that can be easily harvested without expensive, fickle islet isolation procedures and that provide mammalian-like glucose tolerance profiles when transplanted into diabetic recipients. Because tilapia can live in stagnant water, we speculated that tilapia islets might tolerate lower oxygen tensions than mammalian islets. Tilapia and rat islets (n = 30) were placed in paired 60-mm Petri dishes containing 10 mL of deoxygenated CMRL-1066 media and cultured together in sealed chambers gassed with 95% N2/5% CO2. Islet viability was determined by fluorscein diacetate/ethidium bromide staining at intervals varying from 2.5 h to 7 days; blood gas measurements were obtained on media samples at the end of selected incubation intervals. Rat islets underwent near-total necrosis and fragmentation in <24 h; occasional viable single cells could be identified until about 72 h. On the other hand, the fish islets showed no loss of viability until about 72 h when some showed mild central necrosis. Even at 7 days, all fish islets appeared roughly 50% viable. Fish islets cultured under hypoxic conditions for 72 h (media, pO2 = 27.8 mmHg) and then transplanted into streptozotocin-diabetic athymic nude mice were viable (6/6) but showed some diminished function (3/6) over a 25-day follow-up period. Our results suggest that tilapia islets will survive and function at lower oxygen tensions than mammalian islets.
Reconstruction of the Hematopoietic System after Stem Cell Transplantation
Authors: Gordon M.Y. , Blackett N.M.
Published: June 2017

The practice of hematopoietic stem cell transplantation to rescue patients from the myeloablative effects of chemo- or radiotherapy, or to replace defective hematopoiesis, is based on the assumption that hematopoietic stem cells in the graft have sufficient proliferative potential to supply mature blood cells for the remainder of the recipient's lifespan. However, the mechanism(s) whereby this is achieved are not well understood. Here we address the reconstruction of the hematopoietic system by considering the effects of stem cell and progenitor cell renewal and differentiation. We conclude that stem cell self-renewal is necessary for hematological recovery and that infused committed progenitor cells (CFU-GM) may contribute to the neutrophil count in the early posttransplant period.
Effect of a New Immunosuppressive Agent, Fty720, on Survival of Islet Allografts
Authors: Yamasaki Toru , Inoue Kazutomo , Hayashi Hiroyuki , Gu Yuanjun , Setoyama Hiroshi , Ida Jun , Cui Wanxing , Kawakami Yoshiyuki , Kogire Masafumi , Imamura Masayuki
Published: June 2017

A newly developed immunosuppressant, FTY720, has a unique mechanism that is quite different from those of conventional immunosuppressants, and is presumed to be mediated through decreases in the number of peripheral lymphocytes, especially helper T cells. This study was performed to ascertain whether this innovative drug could prolong islet allograft survival. The donors were inbred Lewis rats and the recipients were ACI rats rendered hyperglycemic with intravenous streptozotocin. In the study group, FTY720 dissolved in distilled water was orally administered at a dose of 5 mg/kg to the recipient ACI rats 1 day before and on the day of grafting. In the control group, only distilled water was orally administered to the recipient ACI rats on the day before and the day of grafting. Two thousand islets were transplanted into the portal vein of the recipient rats in the study and control groups immediately after isolation. The graft survival time in the study group was significantly longer than that in the control group, indicating that FTY720 retains a potent effect on the prolongation of islet allograft survival. FTY720 could become a useful immunosuppressant for future clinical islet allotransplantation.
Complement Permeability in Bio-Artificial Endocrine Pancreas Using a Diffusion Chamber
Authors: Hirotani Sachiko , Ohgawara Hisako
Published: June 2017

Immunoisolation is a potentially important approach to transplanting islets without any immunosuppressive therapy. This study was designed to investigate whether complement penetrates into the diffusion chamber that we developed for a bioartificial endocrine pancreas (Bio-AEP). The results showed that within 12–24 h, when a Nuclepore membrane (Nuclepore Corp., California) was the sole barrier, complement permeated 100%. However, the Nuclepore membrane used together with a mixed matrix prevented complement penetration. Also, the complement become inactivated during penetration through both the mixed matrix and the Nuclepore membrane with a pore size of 0.1 μm. In conclusion, immunoisolation of cells by a mixed matrix and a Nuclepore membrane of 0.1 μm pore size is a particularly effective method for transplanting xenogeneic cells. Thus, the clinical application of our Bio-AEP shows promise for long-term xenotransplantation without an immunosuppressant.
Xenogeneic Cell Therapy: Current Progress and Future Developments in Porcine Cell Transplantation
Authors: Edge Albert S.B. , Gosse Marilyn E. , Dinsmore Jonathan
Published: June 2017

The multitude of distinct cell types present in mature and developing tissues display unique physiologic characteristics. Cellular therapy is a novel technology with the promise of utilizing this diversity to treat a wide range of human degenerative diseases. Intractable diseases, disorders, and injuries are characterized by cell death or aberrant cellular function. Cell transplantation can replace diseased or lost tissue to provide restorative therapy for these conditions. The limited use of cell transplants as a basis for current therapy can, in part, be attributed to the lack of available human cells suitable for transplantation. This has prevented further realization of the promise of cell transplantation as a platform technology. Accordingly, cell-based therapies such as blood transfusions, for which the cells are readily available, are a standard part of current medical practice. Despite numerous attempts to expand primary human cells in tissue culture, current technological limitations of this approach in regard to proliferative capacity and maintenance of the differentiated phenotype has prevented their use for transplantation. Further, use of human stem cells for the derivation of specific cell types for transplantation is an area of future application with great potential, but hurdles remain in regard to deriving and sufficiently expanding these multi-potential cells. Thus, it appears that primary cells are at present a superior source for transplantation. This review focuses on pigs as a source of a variety of primary cells to advance cell therapy to the clinic and implement achievement of its full potential. We outline the advantages and disadvantages of xenogeneic cell therapy while underscoring the utility of transplantable porcine cells for the treatment of human disease. © 1998 Elsevier Science Inc.
The hNT Human Neuronal Cell Line Survives and Migrates into Rat Retina
Authors: Konobu Toshifuml , Sessler Francis , Luo Lai-Yi , Lehmann John
Published: June 2017

The present studies were undertaken to determine if hNT cells can survive in the vitreous of the eye and migrate into the retina. The hNT neuronal cell line represents a uniform source of human tissue that may be of use in retinal grafts. hNT cells stored in liquid nitrogen were thawed and labeled with the fluorescent dye 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine Perchlorate (DiI). Thirty thousand cells in 1 μL were injected epiretinally in rat. At survival times of 3, 14, 28, or 56 days, retinal sections were examined quantitatively by epifluorescence to reveal DiI-labeled cells. hNT cells survived in the vitreous at all time points without evidence of vascularization. At 3 days, essentially no hNT cells were found in deep retina, and only very few were attached to retina. At days 14, 28, and 56, hNT cells were found to cluster on the vitreal/retinal interface, and in deeper layers. The clusters of hNT cells took on the shape of a funnel at 14 days, and inverted funnel at 28 days, and by 56 days, populated the photoreceptor layer as a stratum. It is possible that hNT cells took on the morphology and function of photoreceptors. These results suggest that hNT cells injected epiretinally survive in the vitreous at least 56 days, migrate to the retinal/vitreous interface, and may migrate through the retina. This system permits the independent and quantitative evaluation of survival and migratory trophic responses. © 1998 Elsevier Science Inc.
Characterization and Evaluation of Detoxification Functions of a Nontumorigenic Immortalized Porcine Hepatocyte Cell Line (HepLiu)
Authors: Liu Jin , Pan Jing , Naik Sharda , Santangini Henry , Trenkler Donna , Thompson Nancy , Rifai Abdalla , Chowdhury Jayanta Roy , Jauregui Hugo O.
Published: June 2017

Primary porcine hepatocytes (PPH) are currently used in research and therapeutic applications as the biological component of extracorporeal liver assist devices to overcome the shortage of human hepatocytes. However, their finite life span and typically rapid loss of functions limit their utility. An immortalized, nontumorigenic, highly differentiated porcine hepatocyte cell line was developed in our laboratory to resolve these disadvantages. PPH were transfected with simian virus 40 (SV40) T antigen under the control of the SV40 early promoter. From the established 69 clones, 23 clones displaying hepatocyte-like morphology were screened for diazepam metabolism. One clone, HepLiu D63, has been maintained in culture for > 2 years, through more than 60 passages and 240 divisions. Albumin protein, present in early passages, was lost at later passages, but albumin transcript still was detectable in later passages. Carbamoyl phosphate synthetase, a gateway enzyme of the urea cycle, was consistently detectable in HepLiu cells. Cytokeratin 18, a characteristic marker of primary hepatocytes, was detected by both immunofluorescent staining and Western blot in HepLiu cells. Furthermore, maintenance of P450 functions in HepLiu cells was evidenced by diazepam and 7-ethoxycoumarin metabolites measured by HPLC. Phase II conjugative function was measured as acetaminophen glucuronidation. P450 dealkylase was demonstrated microscopically by the conversion of a nonfluorescent substrate to a fluorescent product. Both Northern blot analysis and immunofluorescent staining showed SV40 T antigen expression in the nuclei of HepLiu cells. No tumor formation occurred when HepLiu cells were injected into severe combined immunodeficient (SCID) mice nor was the TA1 (a tumor marker) mRNA expressed, even in later passages. This immortalized, nontumorigenic, highly functional cell line may provide a valuable tool for drug/toxicological studies, liver biologic regulation studies, and artificial liver support systems.
Characterization of Iris Pigment Epithelial Cell for Auto Cell Transplantation
Authors: Abe Toshiaki , Tomita Hiroshi , Ohashi Toshifumi , Yamada Katsura , Takeda Yoshiyuki , Akaishi Keiko , Yoshida Madoka , Sato Masami , Tamai Makoto
Published: June 2017

To establish auto iris pigment epithelial (IPE) transplantation, we characterized the properties of IPE cells and the method of culture using auto serum. Monkey and human IPE cells were obtained and cultured in several conditions, using auto, mouse, rabbit, bovine, or human serum. Immunocytochemical study was performed to confirm that the cells were epithelial in origin. The proliferation rate of the IPE was also calculated from fresh human IPE cells, which were obtained during filtering glaucoma surgery. Proliferation rate was also compared to that of retinal pigment epithelial (RPE) cells. Reverse-transcriptase and polymerase chain reaction for melanogenesis was performed, and the amount of pigment in the IPE cells was also calculated. Mouse and rabbit sera were not effective for the monkey IPE cell culture. Conversely, the cells grew well in the medium with auto, bovine, or human serum. Human IPE cells grew exponentially by the described methods and reached to 60,000 cells after about 4–5 weeks. When we compared them by proliferation rate, IPE cells were less proliferative than RPE cells. The gene expression for melanogenesis and the amount of pigment in the IPE gradually decreased through successive passages. Transplantation has been tried for the treatment of age-related macular degeneration using RPE from fetus or from eye bank eyes. However, focal rejection may play an important role in the clinical results. The establishment of auto IPE cell transplantation may improve the problem of rejection. In the present study, we established auto IPE cell culture using auto serum. The cultured IPE cell showed pigment epithelial cell properties until around five passages in both human and monkey.