bluuebunny/crossref_metadata_2025 huggingface dataset:
Recombinant Sendai Virus-Mediated Gene Transfer to Mouse Pancreatic Stem Cells
Authors: Oishi Koichi , Noguchi Hirofumi , Yukawa Hiroshi , Inoue Makoto , Takagi Soichi , Iwata Hisashi , Hasegawa Mamoru , Hayashi Shuji
Published: June 2017

Efficient gene transfer into stem cells is essential for the basic research and for therapeutic applications in gene-modified regenerative medicine. Adenovirus (AdV) vectors, one of the most commonly used types of vectors, can mediate high, albeit transient, levels of expression of the transgene in pancreatic stem/progenitor cells. However, high multiplicity of infection (MOI) with AdV vectors can result in cellular toxicity. Therefore, AdV vectors have been of limited usefulness in clinical applications. In this study, we investigated the in vitro gene transfer efficiency of Sendai virus (SeV) vectors, a paramyxovirus vector that can efficiently introduce foreign genes without toxicity into several cell types, including pancreatic stem cells. The dose-dependent GFP expression of pancreatic stem cells transfected with SeV vectors after 48 h of culture at 37°C was observed. The transfection of pancreatic stem cells with SeV vectors and AdV vectors results in equal expression of the transgene (GFP expression) in the cells after 48 h of culture at 37°C. Although the transfection of pancreatic stem cells with AdV vectors at high MOIs was cytotoxic, transfection with SeV vectors at high MOIs was rarely cytotoxic. In addition, pancreatic stem cells transfected with SeV maintained their differentiation ability. These data suggest that SeV could provide advantages with respect to safety issues in gene-modified regenerative medicine.
Osteogenesis in Marrow-Derived Mesenchymal Cell Porous Ceramic Composites Transplanted Subcutaneously: Effect of Fibronectin and Laminin on Cell Retention and Rate of Osteogenic Expression
Authors: Dennis James E. , Haynesworth Stephen E. , Young Randell G. , Caplan Arnold I.
Published: June 2017

Cultured-expanded rat marrow-derived mesenchymal cells differentiate into osteoblasts when combined with a porous calcium phosphate delivery vehicle and subsequently implanted in vivo. In this study, the effects of ceramic pretreatment with the cell-binding proteins fibronectin and laminin on the osteogenic expression of marrow-derived mesenchymal cells were assessed by scanning electron microscopy, [3H]-thymidine-labeled cell quantitation, and histological evaluation of bone formation. Scanning electron microscopic observations showed that marrow-derived mesenchymal cells rapidly spread and attach to both fibronectin- or laminin-adsorbed ceramic surfaces but retain a rounded morphology on untreated ceramic surfaces. Quantitation of [3H]-thymidine labeled cells demonstrated that laminin and fibronectin preadsorbed ceramics retain approximately double the number of marrow-derived mesenchymal cells than do untreated ceramics harvested 1 wk postimplantation. Histological observations indicate that the amount of time required to first detect osteogenesis was shortened significantly by pretreatment of the ceramic with either fibronectin or laminin. Fibronectin- and laminin-coated ceramic composite samples were observed to contain bone within 2 wk postimplantation, while in untreated ceramic the earliest observation of bone was at 4 wk postimplantation.

A comparison was made of the initial cell-loading, in vivo cell retention characteristics, and rate of osteogenesis initiation of marrow-derived mesenchymal cells on two types of ceramic with different pore structure and chemical composition, with and without preadsorption with fibronectin or laminin. “Biphasic” ceramics contain randomly distributed pores 200-400 μm in diameter, and “coral-based” ceramics have continuous pores of approximately 200 μm in diameter. Laminin or fibronectin preadsorption significantly increases the number of cells retained in all ceramic test groups by day 7 postimplantation. In addition, by day 7 postimplantation, the biphasic ceramics retain a significantly greater number of cells for all test groups than do coral-based ceramics. The biphasic ceramics consistently have more specimens positive for bone with the identical cell-loading conditions used throughout this study. These results indicate that the retention of cells within the ceramic is an important factor for optimization of marrow mesenchymal cell initiated bone formation. The retention of cells within ceramics is augmented by the adsorption of the cell-binding proteins laminin and fibronectin, but this effect varies depending on ceramic pore structure and/or chemical composition.
Influence of Different Substrates in Detoxification Activity of Adult Rat Hepatocytes in Long-Term Culture: Implications for Transplantation
Authors: Naik Sharda , Santangini Henry , Gann Kathryn , Jauregui Hugo
Published: June 2017

Substrates used to immobilize hepatocytes for transplantation govern attachment and long-term metabolic activity of these cells. The choice of these substrates is based on the familiarity with proteinaceous materials that are constituents of the extracellular matrix. The use of substrates that recognize carbohydrates on the cell surface may provide an alternative method to attach adult mammalian hepatocytes.

In this study, immobilized lectins on tissue culture plasticware were used to support hepatocyte attachment. Long-term cultures with these substrates were compared with control cultures seeded on a mixture of collagen types I and III (Vitrogen).

To evaluate the attachment efficiency and long-term maintenance of diazepam metabolic activity of hepatocytes seeded on different commercially available plasticware, four different types of polymers (supplied as 60-mm dishes) were tested. Diazepam, a benzodiazepine metabolized by the P450 intracytoplasmic pathway, is associated with a synaptic receptor (GABA-benzodiazepine receptor) which plays an important role in hepatic coma.

Polymethylpentene, a derivative of polypropylene treated by plasma discharge, was the best polymer to maintain P450 phenotypic expression, although other polymers provided similar cell attachment efficiencies. The amounts of adsorbed concanavalin A, Arachis hypogaea (peanut), Lens culinaris, and Pisum sativum agglutinin correlate with the percentage values of hepatocyte attachment. Cell attachment to wheat germ agglutinin increased with increased lectin concentrations in spite of constant amounts of adsorbed lectin, whereas hepatocyte attachment to Bandieraea simplicifolia agglutinin was lower and did not change at different lectin concentrations. Long-term cultures of hepatocytes seeded on Vitrogen, concanavalin A, or wheat germ agglutinin showed similar diazepam metabolic activities up to the 10th day, but by day 25, cells seeded on Vitrogen metabolized diazepam at higher values.

This study showed that a polymer used for the manufacture of tissue culture plasticware, which permits a better exchange of gases, contributes to the long-term expression of P450 activity. Lectins proved to be nontoxic for hepatocyte survival, maintained hepatocyte viability, and can be used as an alternative substrate to immobilize hepatocytes to be transplanted in animal models of acute or chronic liver failure.
A Critical Reappraisal of Gastrointestinal Complications of Allogeneic Bone Marrow Transplantation
Authors: Schiller Gary J. , Gale Robert Peter
Published: June 2017

Gastrointestinal toxicity is a common early complication of allogeneic bone marrow transplants. Etiologies include mucosal damage from pretransplant conditioning, opportunistic infection, and graft-versus-host disease. Because the clinical, laboratory, radiographic, and histological findings of acute graft-versus-host disease are nonspecific, accurate diagnosis is difficult or impossible. We review the differential diagnosis of gastrointestinal complications of bone marrow transplants and implications for therapy.
Repeated Transplantation of Microencapsulated Hepatocytes for Sustained Correction of Hyperbilirubinemia in Gunn Rats
Authors: Dixit Vivek , Arthur Marika , Gitnick Gary
Published: June 2017

In previous studies we demonstrated that transplantation of microencapsulated hepatocytes could correct congenital hyperbilirubinemia in Gunn rats for 4 to 6 wks. Reduction in hyperbilirubinemia followed a single transplantation of isolated encapsulated hepatocytes (IEH). After 4 to 6 wks of transplantation IEH gradually lose their functionality. To sustain long-term supplementation of liver function we have investigated the efficacy of monthly IEH transplantations for 6 mo. Hepatocytes, isolated from young Wistar rats, were microencapsulated with a collagen matrix within an alginate-poly L-lysine composite membrane. We transplanted IEH intraperitoneally into homozygous Gunn rats at monthly (4-wk) intervals for 6 mo. Control Gunn rats received intraperitoneal transplantations of empty microcapsules. Total serum bilirubin was measured in the IEH-transplanted and control Gunn rats at weekly intervals for the duration of the 6-month study. A significant (p < 0.01) and sustained decrease (by nearly 50%) in total serum bilirubin levels was observed following monthly IEH transplantations in Gunn rats for the duration of the study. No such decrease in total serum bilirubin levels was seen in the controls. The Gunn rats exhibited good tolerance for the multiple IEH transplantations. Thus, repeated IEH transplantations may be one strategy for providing long-term supplementation of liver function in congenital metabolic liver disease.
The Immunobiology of Cell Transplantation
Authors: Morris Peter J.
Published: June 2017

The potential for cell transplantation is enormous, not only in replacement therapy in conditions such as diabetes but also in approaches to gene therapy and the induction of tolerance to organ transplants. Immunobiological aspects of cell transplantation include: 1) isolation and purification of cells for transplantation, 2) preservation of cells, 3) technical problems of transplantation, 4) the immune response to cell transplants, 5) prevention of the immune response to cell allografts, 6) delivery and regulation of the product of the cell transplant, 7) xenotransplantation, 8) gene therapy. These aspects are briefly discussed.
In Vitro Labeling Strategies for Identifying Primary Neural Tissue and a Neuronal Cell Line after Transplantation in the Cns
Authors: Onifer Stephen M. , White Linda A. , Whittemore Scott R. , Holets Vicky R.
Published: June 2017

Potential labels for identifying embryonic raphe neurons and a clonal, neuronally differentiating, raphe-derived cell line, RN33B, in CNS transplantation studies were tested by first characterizing the labels in vitro. The labels that were tested included 4,6-diamidino-2-phenylindole hydrochloride, 1,1′-dioctadecyl-3,3,3′-tetramethylindocarbocyanine perchlorate, the Escherichia coli lacZ gene, Fast Blue, Fluoro-Gold, fluorescein-conjugated latex microspheres, fluorescein isothiocyanate-conjugated or nonconjugated Phaseolus vulgaris leucoagglutinin, methyl o-(6-amino-3′-imino-3H-xanthen-9-yl) benzoate monohydrochloride, or tetanus toxin C fragment. Subsequently, the optimal in vitro labels for embryonic raphe neurons and for RN33B cells were characterized in vivo after CNS transplantation. In vitro, 1,1-dioctadecyl-3,3,3′-tetramethylindocarbocyanine perchlorate (DiI) optimally labeled embryonic neurons. The Escherichia coli lacZ gene optimally labeled RN33B cells. Most labels were rapidly diluted in cultures of embryonic astrocytes and proliferating RN33B cells. Some labels were toxic and were often retained in cellular debris. In vivo, DiI was visualized in transplanted, DiI-labeled raphe neurons, but not in astrocytes up to 1 mo posttransplant. DiI-labeled host cells were seen after transplantation of lysed, DiI-labeled cells. β-Galactosidase was visualized in transplanted, Escherichia coli lacZ gene-labeled RN33B cells after 15 days in vivo. No β-galactosidase was seen in host cells after transplantation of lysed, lacZ-labeled RN33B cells. The results demonstrate that labels for use in CNS transplantation studies should be optimized for the specific population of donor cells under study, with the initial step being characterization in vitro followed by in vivo analysis. Appropriate controls for false-positive labeling of host cells should always be assessed.
Two PC 12 Pheochromocytoma Lines Sealed in Hollow Fiber-Based Capsules Tonically Release L-Dopa In Vitro
Authors: Lavoie Mark P. , Palmatier Meg , Gentile Frank T. , Kaplan Faith A. , Fiore Deborah M. , Hazlett Tyrone F. , Bell William J. , Flanagan Thomas R.
Published: June 2017

Two PC12 cell-derived lines have been studied following encapsulation into polymer-based hollow fibers with respect to secreted catecholamines and their metabolites. Cellular encapsulation provides a chronic microperfusion environment within which basally secreted PC12 products can be readily measured. Encapsulated PC12 cells grown and held under the conditions specified in this report basally release amounts exceeding their total cellular stores of the dopamine precursor L-DOPA and the electrochemically active dopamine metabolites DOPAC and HVA during 45-min static incubations. Under these same conditions, these cells release less than 0.1% of their total cellular store of dopamine. Depolarizing incubations enhance dopamine secretion eightyfold and enhance secretion of L-DOPA, HVA, and DOPAC about twofold. The relative composition of products basally secreted differs between PC12-derived cell lines, and an inverse relationship exists between basal release of L-DOPA and total cellular store of dopamine. These results further indicate that selected PC12 cell lines are potentially a source of both dopamine and L-DOPA in therapeutic cellular replacement applications.
Reinnervation of Isolated Islets of Langerhans Transplanted beneath the Kidney Capsule in the Rat
Authors: Portis Andrew J. , Rajotte Ray V. , Krukoff Teresa L.
Published: June 2017

Neural regulation of islets of Langerhans mediates responses to stress and food ingestion. Transplantation of isolated islets offers hope to patients with insulin dependent diabetes mellitus but denervation of isolated islets may affect the capacity for appropriate metabolic control. Previous examination of the endocrine response to stress in islet autografted dogs revealed differences consistent with loss of neural regulation. Therefore, in the present study, islets grafted in rats were examined for extent and nature of reinnervation. Islets isolated from syngeneic donors were grafted under the kidney capsule of Wistar-Furth rats (n = 7) after 3 wk of streptozotocin induced diabetes. After 4 mo, graft-bearing kidneys were recovered and processed for double immunofluorescence. Antibodies were directed against (a) neuron associated proteins: synapsin (SYN) and L1; (b) neurotransmitters; tyrosine hydroxylase (TH), neuropeptide Y (NPY), vasoactive intestinal peptide (VIP), and calcitonin gene-related peptide (CGRP); and (c) islet hormones: insulin and somatostatin. SYN and L1 immunoreactivities in nerve fibres suggested reinnervation of the grafted islets although fibres were not associated with structures within the transplanted islets as in intact islets. CGRP immunoreactivity was observed in fibres and in a subpopulation of cells within intact islets but only in cells of the grafted islets. VIP, TH, and NPY immunoreactivities were found in nerve fibres of intact islets but only VIP was observed in fibres of grafted islets suggesting an absence of sympathetic reinnervation. In conclusion, transplanted islets of Langerhans become reinnervated but with a distribution and complement of neurotransmitters distinct from intact islets.
Design and Fabrication of Biodegradable Polymer Devices to Engineer Tubular Tissues
Authors: Mooney D. J. , Organ G. , Vacanti J. P. , Langer R.
Published: June 2017

Engineering new tissues by transplanting cells on polymeric delivery devices is one approach to alleviate the vast shortage of donor tissue. However, it will be necessary to fabricate cell delivery devices that deliver cells to a given location and promote the formation of specific tissue structures from the transplanted cells and the host tissue. This report describes the design and fabrication of a polymeric device for guiding the development of tubular vascularized tissues, which may be useful for engineering a variety of tissues including intestine, blood vessels, tracheas, and ureters. Porous films of poly (d, l-lactic-co-glycolic acid) have been formed and fabricated into tubes capable of resisting compressional forces in vitro and in vivo. These devices promote the ingrowth of fibrovascular tissue following implantation into recipient animals, resulting in a vascularized, tubular tissue. To investigate the utility of these devices as cell delivery devices, enterocytes (intestinal epithelial cells) were seeded onto the devices in vitro. Enterocytes were found to attach to these devices and form an organized epithelial cell layer. These results suggest that these devices may be an appropriate delivery vehicle for transplanting cells and engineering new tubular tissues.
Effects of Iso and Xeno Fetal Liver Fragments Transplantation on Acute and Chronic Liver Failure in Rats
Authors: Hagihara M. , Shimura T. , Takebe K. , Munkhbat B. , Tsuji K.
Published: June 2017

Isogeneic (rat) and xenogeneic (swine) fetal liver fragments (FLF) transplantation into the omentum was performed for d-galactosamine (d-Gal)-induced acute and carbon tetrachloride (CCI4)-induced chronic hepatic failure in rats. The recipients that had iso or xeno FLF showed higher survival rates than the nontransplanted controls on a lethal dose (2.6 g/kg body weight) of d-Gal (survival rates: Iso 70%, Xeno 80%, and control 9.1%). On a sublethal dose (1.0 or 1.2 g/kg) of d-Gal, iso, or xeno FLF caused marked improvement of the values of GPT, GOT, and total bilirubin (T. Bil); at 72 h after d-Gal injection they went significantly lower than those of controls (Iso vs. control; p < 0.01, Xeno vs. control; p < 0.05). Histological examination of the livers revealed severe damage in controls, however, only a slight damage was found in iso or xeno FLF transplanted rats. Iso grafts were fairly well preserved in the omentum at 72 h posttransplants, however, xeno graft had almost changed into a necrotic tissue. CCI4 was administered subcutaneously for 14 wk to induce chronic hepatic failure and then iso FLF were transplanted 3 days after the last CCI4 injection. Iso FLF transplanted rats showed higher improvement of GPT and GOT values at 12 days posttransplants compared with controls (GPT p < 0.01, GOT p < 0.05), although histological improvement was not so remarkable in both group. Iso grafts formed nodules with many hepatocytes in the omentum 12 days posttransplant. The results indicate that iso or xeno FLF transplantation could be an alternative approach for incurable liver insufficiencies.
Cold-Preservation of Pancreatic Beta Cells
Authors: Korbutt Gregory S. , Pipeleers Daniel G.
Published: June 2017

Clinical islet transplantation requires graft preservation conditions with a minimal loss in viable beta cells. The present study examines whether rat pancreatic beta cells survive a 96 h storage period at 4°C. In a single cell viability assay, less than 20% viable cells were counted after 48 h storage in physiological HAM's F10 medium; cell survival was better in the high potassium solutions UW or Collins and further improved by supplementing Collins with albumin and benzamidine (CAB - 77% viability after 96 h). Suspended beta cell aggregates were also well preserved during 4 days in CAB-4°C as judged from DNA recovery and electron microscopy. After cold-storage in CAB, beta cells exhibited a higher insulin content than after culture in HAM's F10 at 20° or 37°C, but their capacity for subsequent insulin synthesis and release was comparable. When isolated islets were stored in CAB-4°C for 96 h, they yielded slightly higher numbers of dissociated cells than after culture in HAM's F10 at 20°C. Implantation of cold stored islets from 2 donor pancreata corrected hyperglycemia in streptozotocin-diabetic rats. It is concluded that rat beta cells can be stored in a supplemented Collins solution for at least 4 days at 4°C, with preservation of initial cell number, hormone content and glucose responsiveness. During short-term periods, this new storage condition is at least equivalent to cultures at 20° or 37°C. Further studies are needed to assess any advantage for long-term storage of islet cell grafts.
Pore Morphology Effects on the Fibrovascular Tissue Growth in Porous Polymer Substrates
Authors: Wake M. Conley , Patrick Charles W. , Mikos Antonios G.
Published: June 2017

The feasibility of developing biodegradable polymer scaffolds to engineer tissues was investigated by studying the effects of pore size on the dynamics of fibrovascular tissue ingrowth. Tissue advanced into amorphous poly(l-lactic acid) porous substrates faster as the pore diameter increased. Porous cylindrical devices of 13.5 mm diameter, 5 mm thickness, and approximately 500 μm pore size were filled completely by tissue 5 days postimplantation. Although prevascularized devices possessed minimal void volume for cell seeding to regenerate metabolic organs, they hold promise in the regeneration of tubular tissues by relying on the epithelization of prevascularized grafts.
A Minimally Invasive Technique for Intrathymic Cell Transplantation in the Dog
Authors: Schachner Robert D. , Qian Tie , Strasser Sheryl , Brendel Mathias D. , Alejandro Rodolfo , Mintz Daniel H.
Published: June 2017

As an alternative to drug immunosuppression, attempts at inducing donor-specific tolerance by intrathymic (IT) inoculations to transplant recipient of donor origin alloantigenic products has proven very promising. Using fiber optic thoracoscopy, a technique for the study of this phenomena was developed for the dog. We show an approach to the dog thymus using fiber optics for injection of bone marrow (BM) cells as the tolerogen. Bone marrow was retrieved from the donor beagles and purified using an automated Ficoll-Paque gradient technique. The purified cellular suspension was injected into the thymus through a small intercostal incision with the use of an injection needle port guided by the use of a rigid fiberoptic scope. To demonstrate engraftment, supravital staining with Fluorescein Diacetate of the BM cells was performed prior to inoculation. Immunofluorescence of cryostat sections obtained at necropsy confirmed the presence of viable BM cells up to several days after transplantation. Results of this study show that the thoracoscopic approach to the thymus can be safely and effectively used for IT inoculation studies in dogs.
Hollow Fibers for Hepatocyte Encapsulation and Transplantation: Studies of Survival and Function in Rats
Authors: Yang Michael B. , Vacanti Joseph P. , Ingber Donald E.
Published: June 2017

In this study, the feasibility of transplanting hepatocytes using hollow fibers (HF) was investigated. Experiments were carried out in vitro and in vivo to determine the viability and function of hepatocytes encapsulated in four different types of commercially available HF: regenerated cellulose HF (RCHF), polysulfone HF of two different sizes (PSHF-1 and PSHF-2), and polyvinylidine HF (PVDF). Hepatocytes remained viable in all types of HF for at least 1 wk in vitro as measured by light microscopy and their ability to synthesize protein and secrete albumin. However, the levels of protein synthesis and albumin secretion in these cells varied significantly between different HF (RCHF > PSHF-2 > PVDF ã PSHF-1) and appeared to be inversely related to their internal diameters (215, 500, 1000, and 1100 μm for RCHF, PSHF-2, PVDF, and PSHF-1, respectively). While PSHF-2, PVDF, and PSHF-1 did not support long term viability in vivo, hepatocytes in RCHF survived after implantation in the mesentery. After 24 h in vivo, the hepatocytes appeared morphologically intact and exhibited a similar rate of protein synthesis when compared with cells cultured in parallel. The hepatocytes in RCHF also maintained the ability to synthesize protein after 7 days in vivo. These results suggest that HF of appropriate size may be useful for hepatocyte transplantation applications in which prevascularization is not possible.
Immune Privilege of the Testis for Islet Xenotransplantation (Rat to Mouse)
Authors: Ar'rajab Aamer , Dawidson Ingemar J.A. , Harris Richard B. , Sentementes James T.
Published: June 2017

The testis has been suggested as an immune privileged site for islet transplantation. The present study evaluated this hypothesis by transplanting islets from Wistar Furth rats into (a) the testes; (b) the subcapsular space of the kidneys; or (c) the cryptorchid abdominal testes of streptozotocin-induced diabetic Swiss ND4 mice. Transplantation of 800 rat islets into the cryptorchid testes normalized blood glucose for 9.3 ± 1.4 (Mean ± SD) days, not significantly different from that of the scrotal testis site (12.4 ± 1.3), or when the subcapsular space of the kidneys was used (11.5 ± 1.2). When mouse islets were isotransplanted into the cryptorchid testes of diabetic mice, normoglycemia was maintained for the entire 3 month study period. Histologic examination of the islet xenograft-bearing cryptorchid testes at day 7 post transplantation and 2 days after returning to hyperglycemia revealed lymphocyte infiltration surrounding and inside the graft. No lymphocyte infiltration was seen in the isograft bearing-testes at 3 mo after transplantation. Cyclosporine A (CsA, 15 mg/kg/day) administration to the islet xenograft recipient slightly prolonged the normoglycemic period to 13.7 ± 1.8 days (p < 0.01). Increasing CsA dose to 25 mg/kg induced a 66% (4/6) mortality, and did not further prolong the normoglycemic period. Using a lower number of rat islets (200 or 400 islets), prolonged graft survival was achieved in some (4 out of 20) animals when the cryptorchid testis was used. In contrast, transplantation of 400 rat islets into the subcapsular space of the kidneys was not associated with prolonged graft survival. When a second rat islet xenograft was transplanted into the subcapsular space of the kidneys of diabetic mice, subsequent to a rejected first intratesticular xenograft, the normoglycemic period was markedly shortened to 3.2 ± 0.5 days (p < 0.01). It is concluded that the testis may act as an immune privileged site for islet xenotransplantation, however, only when a small number of islets is transplanted. A large number of islets will override the immune privilege status of the testis. CsA slightly but significantly delays islet xenograft rejection. Finally, islets transplanted in the testes do sensitize the host against a second islet graft.
Transplantation of Mesencephalic Cell Suspensions from Wild-Type and Heterozygous Weaver Mice into the Denervated Striatum: Assessing the Role of Graft-Derived Dopaminergic Dendrites in the Recovery of Function
Authors: Witt Thomas C. , Triarhou Lazaros C.
Published: June 2017

The Weaver (wv) mutation leads to a loss of mesencephalic dopamine cells and nigrostriatal dopamine axons in homozygosity (wv/wv) and to a deficiency of nigral dopaminergic dendrites without a concomitant loss of dopamine cell somata or axons in heterozygosity (wv/+). Previous studies have shown that grafts of foetal dopamine cells from wild-type (+/+) donors can survive when implanted into the wv/wv striatum, supply both an axonal and a dendritic innervation to the host, establish synaptic connections with host striatal neurons, and bring about a functional recovery evidenced by rotational asymmetry tests. The aims of the present study were to examine whether wv/+ dopamine cells maintain a “dendrite-poor” phenotype after transplantation to the denervated striatum, and to compare their functional effects with those of wild-type (+/+) grafts in reversing amphetamine-induced turning behaviour. To that end, +/+ and wv/+ ventral mesencephalic tissue (dissected out from E10-E12 foetal mice and made into a cell suspension by enzymatic and mechanical dissociation) was stereotactically grafted into the right striatum of either wv/wv hosts or +/+ hosts subjected in advance to 6-OHDA lesions of the right substantia nigra. Viability and morphology of grafted neurons were assessed by tyrosine hydroxylase immunocytochemistry on serial sections of the host forebrains. Dopamine cell bodies survived in comparable numbers in the grafts regardless of donor genotype; however, grafts of either genotype contained fewer dopaminergic cells when they were hosted in the wv/wv striatum as compared to the striatum of +/+ mice with 6-OHDA lesions. Despite the survival of cell somata, the dendritic arborisation of wv/+ cells was strikingly poorer than that of +/+ cells in grafts placed into both host types, most likely reflecting their in situ phenotypic abnormality. Recipient wv/wv mice with +/+ and wv/+ grafts exhibited 88% and 83% left rotations, respectively; 6-OHDA hosts with +/+ and wv/+ grafts showed 178% and 165% reversals of asymmetry, respectively. The differences between the effects of +/+ and wv/+ grafts were not statistically significant. We conclude that (i) wv/+ and +/+ dopamine cell somata survive in comparable numbers after intrastriatal grafting; (ii) grafted wv/+ dopamine cells express an anatomical phenotype consistent with that seen in the wv/+ substantia nigra in situ; and (iii) the axonal innervation supplied by wv/+ grafts to the denervated striatum induces a functional recovery comparable to that brought about by +/+ cells, which in addition supply a substantial dendritic innervation to the host; (iv) the wv/wv host environment may be associated with smaller numbers of graft dopamine neurons compared to the environment of +/+ mouse hosts with 6-OHDA lesions.
Immunostaining of Human Amniotic Epithelial Cells: Possible use as a Transgene Carrier in Gene Therapy for Inborn Errors of Metabolism
Authors: Sakuragawa Norio , Tohyama Jun , Yamamoto Hiroshi
Published: June 2017

Immunoreactivity of human cultured amniotic epithelial (AE) cells was investigated to evaluate the possible use of these cells as a transgene carrier in gene therapy for inborn errors of metabolism. AE cells were prepared and cultured by the methods described previously. Flow cytometry analysis revealed that these cells did not express any class II antigen at all on their surfaces. But the class I antigen was slightly expressed on their surfaces. Immunoperoxidase staining was slightly positive as to the class I antigen but not to the class II antigen at all. pSV-β-galactosidase was transfected into AE cells by means of electroporation, followed by staining of the cells with X-gal. Several cells in 60 mm dish expressed β-galactosidase activity. The possible gene transfer of β-galactosidase into cultured AE cells may suggest that these cells could be used as a transgene carrier in gene therapy for inborn errors of metabolism.
Immunobiology of Cellular Transplantation
Authors: Gill Ronald G. , Wolf Leslie
Published: June 2017

The goal of cellular transplantation is to allow long-term function of the grafted cells using minimal host immunosuppression. To this end, the major strategies to implant cells and tissues are through: (i) the pretreatment of the graft to reduce tissue immunogenicity; (ii) the application of immunoisolation technologies to prevent host sensitization to implanted cells; and (iii) the induction of immunological tolerance to the donor tissues. Further, a major dilemma facing clinical tissue grafting is the shortage of donor tissue for transplantation. This problem requires the consideration of tissues from other species (xenografts) as a potential source of donor material. In light of these issues, the focus of this discussion is on the T cell-dependent response to allogeneic and xenogeneic transplants and the implications of this reactivity on the field of cellular replacement therapy.
Implantable Biohybrid Artificial Organs
Authors: Colton Clark K.
Published: June 2017

Biohybrid artificial organs encompass all devices which substitute for an organ or tissue function and incorporate both synthetic materials and living cells. This review concerns implantable immunoisolation devices in which the tissue is protected from immune rejection by enclosure within a semipermeable membrane. Two critical areas are discussed in detail: (i) Device design and performance as it relates to maintenance of cell viability and function. Attention is focussed on oxygen supply limitation and how it is affected by tissue density and the development of materials that induce neovascularization at the host tissue-membrane interface; and (ii) Protection from immune rejection. Our current knowledge of the mechanisms that may be operative in immune rejection in the presence of a semipermeable membrane barrier is limited. Nonetheless, recent studies shed light on the role played by membrane properties in preventing immune rejection, and many studies demonstrate substantial progress towards clinically useful implantable immunoisolation devices.
Morphometric Study of Fetal Brain Transplants in the Insular Cortex and NGF Effects on Neuronal and Glial Development
Authors: Díaz-Cintra S. , Rivas P. , Cintra L. , Aguilar A. , Gutiérrez G. , Pérez E. , Escobar M. , Bermúdez-Rattoni F.
Published: June 2017

Homotopic grafts supplemented with nerve growth factor (NGF) speed the recovery from learning deficits observed following electrolytic lesions of the insular cortex in rats. NGF also reduces the time in which the activity of choline acetyltransferase (ChAT) is first detected inside the graft by histochemical techniques. It is not known whether this behavioral and biochemical recovery correlates with an advanced maturation of the cellular elements within the graft, presumably induced by NGF. To investigate the degree of maturation of neurons, glial cells and blood vessels in NGF-supplemented grafts, adult rats were lesioned electrolytically in the insular cortex, and homotopic embryonic grafts (E16) with or without NGF supplementation were transplanted into the lesion. Fifteen days post grafting, the rats were perfused and the brains stained using silver impregnation techniques. Our results showed that neuronal maturation, as evaluated through several morphometric parameters, was advanced in NGF-supplemented grafts when compared with other experimental groups. Furthermore, grafts supplemented with NGF also showed significant increases in the number of neurons, oligodendrocytes, astrocytes and blood vessels. These observations indicated that the addition of NGF to insular cortex grafts promoted the maturation of neuronal and glial elements within the graft. They also support the possibility that the advanced morphological maturation of insular cortex grafts supplemented with NGF underlies the accelerated functional and biochemical recovery of animals with lesions of the insular cortex.
Migration of Macrophage-Like Cells within Encapsulated Islets of Langerhans Maintained in Tissue Culture
Authors: Fraser Robert B. , Macaulay Malcolm A. , Wright James R. , Sun Anthony M. , Rowden Geoffrey
Published: June 2017

Islets of Langerhans isolated from the pancreas and encapsulated in alginate-polylysine-alginate microspheres can potentially serve as a self-regulating supply of insulin in response to glucose loads. A longitudinal ultrastructural and immunohistochemical study of encapsulated rat islets cultured in CMRL-1969 media at a constant glucose concentration of 5.5 mmol/L (100 mg%) allowed several observations. First, acinar cells, which remain attached to isolated islets, disappeared within 1 wk in tissue culture. Damaged endocrine cells also disappeared at this time. Phagocytic cells having ultrastructural features suggesting that they are macrophages emerged from the islets within about a week and ingested portions of the inner layer of capsule polymer. These macrophage-like cells retained these polymers until their death which occurred at around 1-2 mo after isolation; at no time did we observe phagocytic cells actually breaching the microsphere capsules. Beta cells remained well-granulated over 90 days of culture but accumulated lipofuscin-like residual bodies. Under these conditions, these bodies began to accumulate appreciably after about one week in culture.
Effects of Hibernation or Cryopreservation on the Survival and Integration of Striatal Grafts Placed in the Ibotenate-Lesioned Rat Caudate-Putamen
Authors: Frodl Eva M. , Sauer Hansjörg , Lindvall Olle , Brundin Patrik
Published: June 2017

Tissue storage prior to intracerebral transplantation would represent a major advantage when conducting clinical transplantation trials in that the procurement of the embryonic donor tissue and the timing of neurosurgery could be planned more efficiently. In the present study, the effects of storing rat embryonic striatal tissue at either +4°C or below freezing temperature prior to grafting to the adult striatum, were assessed with regard to transplant survival, morphology and integration. Eleven days following a unilateral injection of ibotenic acid into the head of the caudate-putamen, a control group of rats received grafts of striatal primordium prepared immediately after dissection from rat embryos (embryonic day 16). A second group of rat embryonic striatal tissue was stored at 4°C (hibernation) for 5 days and then transplanted. A third group of the striatal donor tissue was cryopreserved in liquid nitrogen for 5 days before implantation surgery. Six to seven weeks following transplantation surgery, the grafts were analysed in brain sections processed for acetylcholinesterase histochemistry, DARPP-32 (dopamine and cyclic AMP regulated phosphoprotein with a molecular weight of 32 kDa) and tyrosine hydroxylase (TH) immunocytochemistry. The mean total graft volume and the relative size of the AChE-positive regions were not significantly different between the three groups. Striatal-specific graft regions, positively stained for AChE and DARPP-32, generally exhibited TH immunoreactivity, suggesting that they had received dopaminergic afferents from the host brain. We conclude that embryonic rat striatal tissue can be cryopreserved or hibernated over 5 days without significant impairment in the yield of striatal neurons following intrastriatal implantation and without markedly affecting transplant morphology.
The Effect of UW Solution and Its Components on the Collagenase Digestion of Human and Porcine Pancreas
Authors: Contractor Harold H. , Johnson Paul R. V. , Chadwick David R. , Robertson Gavin S. M. , London Nicholas J. M.
Published: June 2017

University of Wisconsin (UW) solution is used extensively as a cold storage solution during the procurement and transport of the pancreas prior to islet isolation. However, it has been observed that UW inhibits the collagenase digestion phase of human but not porcine islet isolation, resulting in poor islet yields and islets of poor viability. The aim of this study was, therefore, to confirm this species difference and to determine which components of UW are responsible for the inhibition in the human. In the initial experiment, blocks of human and porcine pancreas (n = 7) were incubated in test tubes containing collagenase at a concentration of 4 mg/mL at 37°C dissolved in 4 mL of either Hanks' solution or UW. Every 5 min the tubes were manually shaken and the degree of tissue dissociation scored on a scale of + and +++. Our results confirm the inhibition of collagenase digestion in the human but not the pig. Using the same methodology, we then investigated the components of UW that were causing the observed inhibition in the human pancreas (n = 7). This time the collagenase was dissolved in individual or combinations of UW components. Using Hank's as a control, the results were then expressed as a median ratio. The components found to be most inhibitory were magnesium, the Na+/K+ ratio, hydroxyethyl starch (HES), and adenosine. Allopurinol in combination with either lactobionate or glutathione was markedly inhibitory (i.e., median ratio 1.8 and 1.9, respectively). The most inhibitory solution tested was a combination of the three components raffinose, glutathione, and lactobionate (median ratio 2.1). This combination was almost as inhibitory as UW itself (median ratio 2.7). These findings are essential for the development of effective cold-storage solutions for the human pancreas that do not inhibit the subsequent collagenase digestion phase of islet isolation.
Induction of Immunological Tolerance to Islet Allografts
Authors: Rossini Aldo A. , Parker David C. , Phillips Nancy E. , Durie Fiona H. , Noelle Randolph J. , Mordes John P. , Greiner Dale L.
Published: June 2017

T-cell dependent activation of resting B cells involves the interaction of gp39 on T cells with its receptor, CD40, on B cells. We administered either a combination of T-cell-depleted splenic lymphocytes and anti-gp39 monoclonal antibody or antibody alone to establish islet allografts in mice without continuous immunosuppression. Fully allogeneic H-2q FVB islets were permanently accepted by chemically diabetic H-2b C57BL/6 mice provided that the recipients were pretreated with both T-cell-depleted donor spleen cells and anti-gp39 antibody. Antibody alone was less effective in prolonging allograft survival, but we did observe that anti-gp39 mAb alone can exert an independent, primary effect on islet allograft survival that was dose dependent. Targeting gp39, in combination with lymphocyte transfusion, might prove suitable for tolerance induction and allotransplantation without immunosuppression.
Genetically Modified Pc 12 Brain Grafts: Survivability and Inducible Nerve Growth Factor Expression
Authors: Rohrer Daniel C. , Nilaver Gajanan , Nipper Valerie , Machida Curtis A.
Published: June 2017

Neural transplantation of genetically modified cells has been successfully employed to reverse functional deficits in animal models of neurodegenerative disorders, including Parkinson's disease. While implanted PC12 cells secrete dopamine in vivo and can ameliorate dopamine deficiency in parkinsonian rat model systems, these cells either degenerate within 2-3 wk postimplantation (presumably due to the lack of neural trophic factor support at the site of implantation), or in some cases, form a tumor mass leading to the death of the host animal. To address these limitations, we have developed a genetically modified PC12 cell line that can synthesize nerve growth factor (NGF) under the control of a zinc-inducible metallothionein promoter. When implanted in the rat striatum and under in vivo zinc stimulation, these cells will neurodifferentiate, express tyrosine hydroxylase, and will undergo survival through potential autocrine trophic support. This regulatable cell line and general approach may provide additional insight on the potential utilization of cell transplants for treatment of Parkinson's disease and other neurodegenerative disorders.
Intracerebroventricularly Transplanted Embryonic Neuronal Tissue from Inflammatory-Resistant F344/N Rats Decreases Acoustic Startle Responses in Inflammatory-Susceptible Lew/N Rats
Authors: Misiewicz B. , Poltorak M. , Gomez M. , Glowa J.R. , Gold P.W. , Sternberg E.M.
Published: June 2017

Recently, we have shown that intracerebral transplantation of fetal F344/N hypothalamic tissue into LEW/N rats converts the LEW/N inflammatory-susceptible phenotype into an inflammatory-resistant phenotype in LEW/N hosts. Because LEW/N rats also exhibit relatively high acoustic startle responses (ASRs) compared to F344/N rats, in the present study we examined the effects on ASR of transplantation of F344/N hypothalamic tissue into the third ventricle of LEW/N rats. Dissected neuronal tissue from F344/N rats (Day E15-16) was implanted into the third ventricle of LEW/N rats. After 4 wk of postoperative survival, the animals' responses to acoustic startle stimuli were tested. Compared to naive and sham-operated animals, LEW/N rats transplanted with hypothalamic tissue exhibited significant decreases in ASR amplitudes. A similar decrease in ASR amplitude was observed in the group of LEW/N rats transplanted with embryonic striatal tissue. Our results indicate that the third ventricular neuronal grafts may modulate behavioral responses in the LEW/N rats. Although the mechanism of this effect is unknown, these studies suggest that intracerebral neuronal transplantation is a viable method with which to explore mechanisms of behavioral, neuroendocrine, and inflammatory response associations.
Blood Stem Cell Collection: Factors Influencing the Recovery of Granulocyte-Macrophage Colony Forming Cells
Authors: Imrie Kevin , Stewart A. Keith , Crump R. Michael , Prince H. Miles , Trip Kathy , Keating Armand
Published: June 2017

We evaluated data from all blood cell (BC) collections performed in our institution between 1989 and 1995 to determine factors influencing the outcome of collection. One hundred and thirty-three collections were performed on 106 patients. Malignant diagnoses were: non-Hodgkins lymphoma (NHL) in 35%, multiple myeloma in 31%, breast cancer in 26%, and Hodgkin's disease in 8%. Collections were obtained routinely in myeloma and breast cancer and due to bone marrow involvement with malignancy or inaspirable bone marrow in lymphoma patients. Collections were obtained on a Cobe Spectra or Baxter-Fenwall CS3000+. Engraftment potential was determined by methylcellulose colony assay (CFU-GM), with a target of >10 × 104 CFU-GM/kg. Apheresis nucleated cell count correlated significantly, albeit weakly (r = 0.26), with CFU-GM with a cell count of >5 × 10s/kg resulting in an adequate number of CFU-GM in 78% of patients. In univariant analysis outcome of collection was significantly influenced by the patients age (p = 0.01), malignant diagnosis (p < 0.001), reason for collection (p = 0.002), and the mobilization regimen (p = 0.01). The nature of the apheresis device used did not influence outcome. Only malignant diagnosis was significant (p < 0.001) in multivariate analysis. We conclude that the outcome of BC is most strongly influenced by patient factors such as malignant diagnosis. These factors must be considered when comparing the outcome of different mobilization regimens and when planning collection strategies.
A Mathematical Model for the Estimation of Human Embryonic and Fetal Age
Authors: Evtouchenko L. , Studer L. , Spenger C. , Dreher E. , Seiler R.W.
Published: June 2017

Precise determination of donor age in human embryonic and fetal tissue is crucial for cell transplantation due to the existence of distinct time windows within which successful grafting is possible. This study demonstrates that between 4-12 wk postconception embryonic and fetal age can be estimated based on various morphometric parameters measured on a routine basis in suction abortion material. The greatest length, the neck-rump length, the foot length, and the proximal and distal arm and leg length were correlated with the anamnestic and ultra-sonographically estimated age. Multivariate regression analyses showed a linear correlation between age and the logarithmic value of the various morphometric parameters. The best correlation was found for a mathematical model combining the limb parameters (r = 0.904; p < 0.001; n −37). A prospective follow-up study (n = 40) was carried out to test the validity of the mathematical model. A high correlation was found between the calculated age and the estimated age based on anamnestic data (r = 0.749, p < 0.001). Outliers due to errors in the anamnestic data were readily identified by comparing anamnestic with calculated age. This method allows determination of embryonic and fetal age within and beyond the age group of the Carnegie classification and may, therefore, be useful for the needs of experimental and clinical cell transplantation.