bluuebunny/crossref_metadata_2025 huggingface dataset:
Quantum Dots for Labeling Adipose Tissue-Derived Stem Cells
Authors: Yukawa Hiroshi , Mizufune Shogo , Mamori Chiharu , Kagami Yukimasa , Oishi Koichi , Kaji Noritada , Okamoto Yukihiro , Takeshi Manabu , Noguchi Hirofumi , Baba Yoshinobu , Hamaguchi Michinari , Hamajima Nobuyuki , Hayashi Shuji
Published: June 2017

Adipose tissue-derived stem cells (ASCs) have a self-renewing ability and can be induced to differentiate into various types of mesenchymal tissue. Because of their potential for clinical application, it has become desirable to label the cells for tracing transplanted cells and for in vivo imaging. Quantum dots (QDs) are novel inorganic probes that consist of CdSe/ZnS-core/shell semiconductor nanocrystals and have recently been explored as fluorescent probes for stem cell labeling. In this study, negatively charged QDs655 were applied for ASCs labeling, with the cationic liposome, Lipofectamine. The cytotoxicity of QDs655-Lipofectamine was assessed for ASCs. Although some cytotoxicity was observed in ASCs transfected with more than 2.0 nM of QDs655, none was observed with less than 0.8 nM. To evaluate the time dependency, the fluorescent intensity with QDs655 was observed until 24 h after transfection. The fluorescent intensity gradually increased until 2 h at the concentrations of 0.2 and 0.4 nM, while the intensity increased until 4 h at 0.8 nM. The ASCs were differentiated into both adipogenic and osteogenic cells with red fluorescence after transfection with QDs655, thus suggesting that the cells retain their potential for differentiation even after transfected with QDs655. These data suggest that QDs could be utilized for the labeling of ASCs.
Cell Transplantation of Adipose Tissue-Derived Stem Cells in Combination with Heparin Attenuated Acute Liver Failure in Mice
Authors: Yukawa Hiroshi , Noguchi Hirofumi , Oishi Koichi , Takagi Soichi , Hamaguchi Michinari , Hamajima Nobuyuki , Hayashi Shuji
Published: June 2017

The effect of adipose tissue-derived stem cells (ASCs) in combination with heparin transplantation on acute liver failure mice with carbon tetrachloride (CCl4) injection was investigated. CCl4 is a well-known hepatotoxin and induces hepatic necrosis. Heparin did not affect the viability of ASCs for at least 24 h. The injection of heparin into the caudal tail vein decreased slightly the activities of the alanine aminotransferase (ALT), asparate aminotransferase (AST), and lactate dehydrogenase (LDH) in plasma. In the transplantation of ASCs (1 × 106 cells) group, there was a trend toward decreased activities of all markers. However, four out of six mice died of the lung infarction. In the transplantation of ASCs in combination with heparin group, there was also a trend toward decreased activities of all markers. In addition, all mice survived for at least the duration of the study period. In conclusion, the transplantation of ASCs in combination with heparin was thus found to effectively treat acute liver failure.
Dystrophin Cytochemistry in Mdx Mouse Muscles Injected with Labeled Normal Myoblasts
Authors: Chen Ming , Li Hua-Ju , Fang Qiuwen , Goodwin Tena G. , Florendo J. Ann , Law Peter K.
Published: June 2017

A new technique enables correlation of dystrophin expression with the location of donor versus host nuclei in the same sections of mdx mouse muscle injected with normal myoblasts. Myoblasts from C57BL/6J mice or from humans were labeled with 0.01% fluoro-gold (FG) in Dulbecco's Modified Eagles Medium (DMEM) for 16 h at 37°C before myoblast transfer. About 3 × 104 myoblasts were injected into the quadriceps muscles of mdx mice immunosuppressed with cyclosporine A (CsA). At 11, 21, or 25 days after myoblast transfer, injected muscles were dissected out and sectioned. These mouse sections were processed for dystrophin and then labeled with a fluorescent nucleus counterstain, 5 μg% Hoechst 33342 in phosphate-buffered saline (PBS), for 10 min at room temperature. Fluoro-gold labeling corresponding with Hoechst 33342 staining indicated survival of normal nuclei in dystrophic muscle. Dystrophin was found in the sarcolemma of myofibers containing FG-labeled nuclei but not of myofibers containing only Hoechst 33342-labeled nuclei. Control muscle samples showed neither FG labeling nor dystrophin. This study demonstrates that the donor human and mouse myoblasts survived and developed in host mouse muscles for at least 25 days after myoblast transfer, and that the localization of their normal nuclei correlates with dystrophin expression in muscle fibers of immunosuppressed mdx host mice.
Long-Term Functional Recovery of Hepatocytes after Cryopreservation in a Three-Dimensional Culture Configuration
Authors: Rinkes Inne H.M. Borel , Toner Mehmet , Sheehan Sean J. , Tompkins Ronald G. , Yarmush Martin L.
Published: June 2017

Hepatocyte cryopreservation is essential to ensure a ready supply of cells for use in transplantation or as part of an extracorporeal liver assist device to provide on-demand liver support. To date, most of the work on hepatocyte cryopreservation has been performed on isolated hepatocytes, and has generally yielded cells which display low viability and greatly reduced short-term function. This report presents the development of a freezing procedure for hepatocytes cultured in a sandwich configuration. A specially designed freezing unit was used to provide controlled temperatures throughout the freeze-thaw cycle. Cooling rate, warming rate, and final freezing temperature were evaluated as to their effect on hepatocyte function as judged by albumin secretion. Under optimized conditions (cooling at 5°C/min and warming at ≥400°C/min), freezing to −40°C resulted in full recovery of albumin secretion within 2-3 days post-freezing, whereafter albumin secretion levels remained normal for the duration of the experiments (2 wks). Freezing to −80°C lead to an approximate 70% recovery of long-term protein secretion when compared to control cultures. In addition, the overall hepatocyte morphology as judged by light microscopy, closely followed the functional results. The sandwich culture configuration, thus, enables hepatocytes to maintain a satisfactory level of long-term protein secretion after a freeze-thaw cycle under optimized conditions, and offers an attractive tool for further studies into the mechanisms of freezing injury and subsequent hepatocellular recovery. These results are a promising step in the development of satisfactory storage procedures for hepatocytes.
Factors Affecting in Vitro Growth of Harvested Enterocytes
Authors: Santos M. , Nguyen B. T. , Thompson J.S.
Published: June 2017

Selective enterocyte transplantation may be an alternative to whole organ transplantation for increasing absorptive capacity. Our aim was to determine the effect of initial cell number and viability, proportion of intact crypts, and basement membrane components (BMC) on the in vitro growth of rabbit enterocytes. Enterocytes were harvested using warm trypsinization from ileal segments in 40 rabbits. Initial cell viability was 92 ± 4% (mean ± SD), cell yield was 7.7 ± 3.6 × 106 cells/cm, and there were 0.51 ± 0.33 crypts/100 cells. Initial cell viability correlated with cell yield (r = −0.508, p < 0.001) and % crypts (r = 0.313, p < 0.05). Cell yield also correlated with % crypts (r = −0.645, p < 0.001). Enterocytes (5 × 106) were incubated in growth media in plain or BMC coated growth culture vessels for 14 days. There was a correlation between both number of cells seeded (r = 0.824, p < 0.001) and cell viability (r = −0.696, p < 0.01) and % growth colonies containing epithelial cells at 14 days. Both total growth colonies (r = −0.565, p < 0.05) and colonies with epithelial cells (r = −0.589, p < 0.05) had a negative correlation with % crypts. Incubating cells in BMC coated vessels (n = 6) resulted in significantly more dispase liberated cells after 14 days than in plain vessels (n = 6) (6.6 ± 1.1 vs. 3.8 ± 1.0 × 106, p < 0.05) but viability was similar (97 ± 2% vs. 96 ± 2%). Diamine oxidase (DAO) activity was significantly increased in the growth media of cells cultured on BMC compared to controls (0.76 ± 0.24 vs. 0.43 ± 0.28 units, p < 0.05) although DAO act/cell was similar. Conclusions: 1) cell yield using warm trypsinization is increased at the expense of viability and intact crypts; 2) in vitro growth is increased by increasing the number of cells but not intact crypts seeded; 3) BMC increase the yield of cells with viability and maturity similar to those cultured in plain vessels.
Expression of Platelet-Derived Growth Factor in and around Intrastriatal Embryonic Mesencephalic Grafts
Authors: Smits Anja , Odin Per , Duan Wei-Ming , Brundin Patrik , Widner Håkan , Heldin Carl-Henrik , Lindvall Olle , Funa Keiko
Published: June 2017

The expression of platelet-derived growth factor (PDGF) was investigated in the embryonic donor tissue and surrounding host brain before and after intracerebral transplantation in a rat model of Parkinson's disease (PD). Ventral mesencephalic tissue from E13-E15 rat embryos was dissociated and implanted into adult rats with unilateral lesions of the mesostriatal dopamine system. Immunohistochemical studies showed that the majority of the grafted cells were PDGF-positive at early time points after grafting. However, the immunostaining gradually decreased, and had disappeared almost completely 3 wk after transplantation. These results were in agreement with in situ hybridization data demonstrating detectable levels of mRNA for PDGF chains in graft cells after 1, but not after 6 wk. In contrast, a large number of PDGF-immunoreactive cells was observed in the host brain adjacent to the grafts from 1 wk after transplantation, and increasing with time. Increased expression of PDGF was also observed in response to a sham-operation (injection of vehicle), although the number of PDGF-positive cells seemed lower than after grafting of embryonic tissue. Double immunofluorescence labeling of these cells with an anti-glial fibrillary acidic protein (GFAP) antiserum and a monoclonal antibody against PDGF B-chain, indicated that the PDGF-positive cells were astrocytes. The dynamic expression of PDGF in and around intrastriatal embryonic mesencephalic implants has several, potentially important, implications for graft survival and function. Glial cells could utilize the elevated levels of PDGF to proliferate in a reactive gliosis, and PDGF might also augment immune responses. It is also possible that PDGF increases the survival of, and promotes neurite outgrowth from, grafted dopaminergic neurons.
Metabolic Activity and Proliferation of Cho Cells in Hydroxyethyl Methacrylate-Methyl Methacrylate (Hema-Mma) Microcapsules
Authors: Uludag Hasan , Sefton Michael V.
Published: June 2017

To better understand encapsulated cell behaviour, Chinese Hamster Ovary (CHO) fibroblasts were encapsulated in HEMA-MMA microcapsules and short-term (<2 wks) proliferation and changes in metabolic activity were investigated in vitro. CHO cells were observed to undergo rapid proliferation in the first week following encapsulation after which a growth arrest was obtained at ~3500 cells/capsule. The cell growth was localized in aggregates in the capsule core, resulting in high local cell density but low cell density in the whole capsule interior (~107 cells/mL). The total metabolic activity, as determined by the MTT (3-14,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium) assay, within the microcapsules increased in the first week, with no significant change afterwards. A broad variation in metabolic activity among the individual capsules was obtained. Supplementing the cell suspension with 20% Ficoll 400 during the encapsulation process resulted in significantly higher morphological uniformity among the individual capsules (with reduced capsule wall thickness and eccentricity); however, this did not change the extent of heterogeneity in metabolic activity. We conclude that viability and proliferation ability (at least to a limited extent) of CHO cells are maintained in HEMA-MMA microcapsules. The local cell growth and subsequent growth arrest remain issues to be addressed in order to obtain better utilization of the microcapsule core volume. Alternatively, small diameter (<400 μm as opposed to the present ~750 μm diameter) capsules are necessary.
Synthetic Polymer Matrices for Neural Cell Transplantation
Authors: Woerly S. , Ulbrich K. , Chytrý V. , Smetana K. , Petrovický P. , Říhová B. , Morassutti D.J.
Published: June 2017

This study proposes a strategy to promote the integration of a neural graft into the host brain tissue. It involves the attachment of donor cells to a polymeric matrix, and the implantation of this cell-polymer matrix. We have synthesized hydrogels based on N-(2-hydroxypropyl)-methacrylamide (HPMA) to produce highly porous matrices. As preliminary steps, we have examined: 1) The response of the brain tissue to the implantation of PHPMA/collagen hydrogels; 2) adhesion, growth, differentiation, and viability of embryonic neuronal cells, and embryonal carcinoma-derived neurons seeded onto PHPMA substrates containing hexosamine residues (glucosamine and N-acetylglucosamine), and after entrapment of cells within the hydrogels. Histological analysis seven wk after implantation showed the tolerance of PHPMA hydrogels, and the penetration of host cells into the pore structures. However, cellular ingrowth requires the presence of collagen, and is dependent upon porosity. In vitro data showed that PHPMA substrates supported neuronal cell attachment and neuritic growth, but the biocompatibility of the substrate was enhanced after incorporation of N-acetylglucosamine into the hydrogel. The data also showed the feasibility of entrapping cells into the polymer matrices, and that these “cellular” hydrogel matrices could be maintained in vitro with preservation of cell viability and differentiation. These findings suggest that PHPMA-based hydrogels can serve as carriers for neural transplant, and as a support to guide tissue ingrowth and organization.
Beneficial Effect of Hepatic Stimulatory Substances on the Survival of Intrasplenically Transplanted Hepatocytes
Authors: Jiang Bojian , Kasai Shinichi , Sawa Masayuki , Hirai Shuji , Yamamoto Tetsu , Ebata Hidetaka , Mito Michio
Published: June 2017

Intrasplenic hepatocyte transplantation has been demonstrated to have a tentative role in treating experimental liver disease, but methods for promoting the rapid proliferation of intrasplenic hepatocytes are still quite limited. In this study, hepatic stimulatory substances (HSS) obtained from regenerating porcine livers were injected directly into the subcutaneously translocated spleens of recipient rats that had received intrasplenic hepatocyte transplantation. The clusters of intrasplenic hepatocytes contained more than 100 cells, and formed cord structures at 2 wk after transplantation, and the hepatocytes still survived at 6 wk in the HSS-treated rats. In contrast, the clusters contained less than 10 hepatocytes at 2 wk after transplantation, and no surviving hepatocytes was observed at 4 and 6 wk in control rats. Additionally, marked proliferation of bile ductular-like structures appeared around the clusters of surviving hepatocytes in the splenic red pulp of the HSS-treated rats, but were not found in control rats at 4 and 6 wk after transplantation.
Induction of Donor-Specific Transplantation Tolerance to Skin and Cardiac Allografts Using Mixed Chimerism in (A + B → A) in Rats
Authors: Markus Peter M. , Selvaggi Gennaro , Cai Xin , Fung John J. , Starzl Thomas E.
Published: June 2017

Mixed allogeneic chimerism (A + B → A) was induced in rats by reconstitution of lethally irradiated LEW recipients with a mixture of T-cell depleted (TCD) syngeneic and TCD allogeneic ACI bone marrow. Thirty-seven percent of animals repopulated as stable mixed lymphopoietic chimeras, while the remainder had no detectable allogeneic chimerism. When evaluated for evidence of donor-specific transplantation tolerance, only those recipients with detectable allogeneic lymphoid chimerism exhibited acceptance of donor-specific skin and cardiac allografts. Despite transplantation over a major histocompatibility complex (MHC)- and minor-disparate barrier, animals accepted donor-specific ACI skin and primarily vascularized cardiac allografts permanently, while rejecting third party Brown Norway (BN) grafts. The tolerance induced was also donor-specific in vitro as evidenced by specific hyporeactivity to the allogeneic donor lymphoid elements, yet normal reactivity to MHC-disparate third party rat lymphoid cells. This model for mixed chimerism in the rat will be advantageous to investigate specific transplantation tolerance to primarily vascularized solid organ grafts that can be performed with relative ease in the rat, but not in the mouse, and may provide a method to study the potential existence of organ- or tissue-specific alloantigens in primarily vascularized solid organ allografts.
Cellular Organization in Retinal Transplants Using Cell Suspensions or Fragments of Embryonic Retinal Tissue
Authors: Juliusson Bengt , Bergström Anders , Van Veen Theo , Ehinger Berndt
Published: June 2017

We have investigated the cellular organization in two different types of retinal transplants using cell type-specific monoclonal antibodies. Both fragments and cell suspensions of E17-E19 Sprague–Dawley rat retina were transplanted to a subretinal site in congenic adult rat hosts. After a survival time of 28 days, the transplants were stained by immunocytochemistry with antibodies against rhodopsin, which stained rods; with antibodies against HPC-1, which stained amacrine cells and outer and inner plexiform layers; and with antibodies against vimentin, which stained Müller cell fibers and horizontal cells. In the host retina, the distribution of immunocytochemical staining was similar, irrespective of transplantation technique. In the transplants, the antirhodopsin staining showed that fragment transplants developed photoreceptors in rosettes, whereas in cell suspension transplants, this staining showed a scattered distribution of photoreceptors. The HPC-1 staining showed that regions corresponding to the inner nuclear layer surrounded both types of transplants and made large invaginations into them. In one case, using the cell suspension technique, fibres were found to run from the inner plexiform layer of the transplant to the outer plexiform layer of the host. The vimentin staining revealed a disorganized array of Müller cell fibres in both types of transplants, but with some concentration to the regions corresponding to the inner plexiform layer.
Determination of the Osmotic Characteristics of Hamster Pancreatic Islets and Isolated Pancreatic Islet Cells
Authors: Benson C.T. , Liu C. , Gao D.Y. , Critser E.S. , Critser J.K.
Published: June 2017

The ability to store pancreatic islets using cryopreservation methodology would greatly assist the application of clinical islet transplantation to Type 1 (insulin-dependent) diabetics. It is our working thesis that the illumination of fundamental biophysical characteristics of these cells will lead to increased cryosurvival rates through theoretically predicted and experimental testing of optimal freezing protocol; as has been found for cells and tissues such as mammalian and Drosophila embryos. Pancreatic islets were isolated from Golden hamsters and their osmometric behavior, including inactive cell volume (Vb), was determined for either whole islets or isolated individual islet cells. When islets or islet cells were exposed to various concentrations of NaCl, they were found to exhibit a “classic” “Boyle-Van't Hoff” osmometric response. The Boyle-Van't Hoff representation of the volume curve (relative cell volume vs. 1/osmolality) yields a linear response with r values of .99 for each curve. Extrapolations to the normalized osmotically inactive volumes (Vb) were .43 and .22 for whole islets and individual islet cells, respectively. These data regarding the fundamental cryobiological characteristics of islets and islet cells should provide the foundation upon which to further the investigation of osmotic parameters of these cells and eventually lead to the determination of optimal freezing protocols.
Reversal of Graft-Versus-Host Disease with Infusion of Autologous Bone Marrow
Authors: Ricordi Camillo , Tzakis Anreas G. , Zeevi Adriana , Rybka Witold B. , Demetris Anthony J. , Fontes Paulo A.C. , Nalesnik Michael A. , Trucco Massimo , Ukah Ferdinand O. , Ball Edward D. , Mullen Edward E. , Marino Ignazio R. , Fung John J. , Starzl Thomas E.
Published: June 2017

Graft-versus-host disease (GVHD) remains a major complication of bone marrow transplantation. This report describes reversal of GVHD by infusion of stored recipient bone marrow following combined liver-bone marrow allotransplantation. Graft-versus-host disease developed at the end of the first postoperative week. The skin involvement progressively spread to approximately 80% of the body surface and was not affected by modification of the immunosuppressive treatment. On the 42nd and 43rd postoperative day 1.23 × 108 and 1.6 × 108 autologous bone marrow cells per kg of recipient body weight were infused. The skin rush began to dramatically improve and resolved within 2 wk from the autologous marrow infusion. Autologous bone marrow storage previous to allogeneic bone marrow transplantation for tolerance induction could constitute a safety net in case of occurrence of GVHD.
Cell Preservation in University of Wisconsin Solution during Isolation of Canine Islets of Langerhans
Authors: Van Der Burg Michael P.M. , Guicherit Onno R. , Frölich Marijke , Prins Frans A. , Bruijn Jan Anthonie , Gooszen Hein G.
Published: June 2017

Allogeneic islet transplantation in Type I diabetic patients is considerably hampered by the variable outcome of islet isolation and purification. After collagenase digestion of the pancreas, islet isolation is traditionally performed under hypothermic conditions in physiological solutions such as Hanks and RPMI. The University of Wisconsin solution (UWS) has been shown superior for hypothermic preservation of the pancreas. We, therefore, compared the UWS and RPMI for canine islet isolation and subsequent purification in either a conventional hyperosmotic density gradient of dextran in Hanks, or a novel normosmotic density gradient of Percoll in UWS. The isolation solution did not affect islet yield before purification (51% of the native islet mass). Loss of amylase (30%) and swelling of the acinar cells were observed in RPMI. In contrast, no loss of amylase and slight shrinkage of the acinar cells were observed in the UWS. Cell swelling affected the density separation and viability of the cells. Dextran density separation resulted in a 15% purity and 41% recovery of the islets isolated in RPMI, as compared to a 93% purity and 52% recovery of islets isolated in UWS. Percoll density separation improved the purity (99%) and recovery (74%) of islets isolated in UWS. Islets isolated in UWS demonstrated a superior basal and glucose stimulated insulin release during perifusion. Electron microscopy demonstrated a well-preserved islet ultrastructure after isolation in both solutions — except for slightly swollen mitochondria after isolation in RPMI. Autotransplantation of islets in pancreatectomised dogs was successful both after isolation in UWS and RPMI. We conclude that prevention of cell swelling during isolation and purification in the UWS resulted in an improved yield of viable and consistent virtually pure islets. Prevention of cell swelling during islet isolation should facilitate the analysis and control of other factors affecting outcome in man.
Studies on Small (<300 μm) Microcapsules: II — Parameters Governing the Production of Alginate Beads by High Voltage Electrostatic Pulses
Authors: Hallé Jean-Pierre , Leblond François A. , Pariseau Jean-François , Jutras Paul , Brabant Marie-Josée , Lepage Yves
Published: June 2017

The size of microcapsules is a critical parameter in the immunoisolation of islets of Langerhans by microencapsulation. The use of smaller capsules decreases the total implant volume and improves insulin kinetics and oxygen supply. A high voltage electrostatic pulse system was used for the production of small (<300 μm) alginate beads, the first step of the encapsulation technique. However, islets often protruded from capsules that were too small, further emphasizing the need for a method to control bead size. A study of 7 parameters [electrostatic pulse amplitude (A), duration (D) and wavelength (λ), pump flow rate (P), needle gauge, alginate viscosity and distance between electrodes] showed that P (r = 0.981, p = 0.003) and λ (r = 0.988, p = 0.0002) were the principal determinants of bead size. To detect potential interactions between parameters, 270 combinations of different levels of A, D, λ, and P were studied. A multivariate regression analysis of these data confirmed that P and λ are the prime determinants of bead size, and showed that a 2-parameter (P, λ) model could be used to precisely predict bead size (R2 = 0.84), while keeping the application simple. The precision of the predictive model is only slightly improved by the use of additional parameters. The reliability of the data used to elaborate this model was demonstrated (p = 0.6226) by comparing them with a second data set obtained under the same conditions. A third set of experiments confirmed the applicability of the model. This work has major implications on the preclinical application of microencapsulation since it showed that it is possible to predetermine the bead size.
Carboxyfluorescein (CFSE) Labelling of Hepatocytes for Short-Term Localization following Intraportal Transplantation
Authors: Fujioka Hikaru , Hunt Peter J. , Rozga Jacek , Wu Guo-Du , Cramer Donald V. , Demetriou Achilles A. , Moscioni Albert D.
Published: June 2017

Renewed interest in the transplantation of isolated hepatocytes into the liver as a potential therapy for liver disease has stimulated the development of methods for the identification of donor cells within the recipient organ. We describe a method for cellular tagging and in vivo identification of intraportally transplanted hepatocytes using an intracellular fluorescent dye, 5(6)-carboxyfluorescein diacetate, succinimidyl-ester (CFSE). Rat and porcine hepatocytes were isolated and labelled with CFSE. The optimal conditions for labelling consisted of a buffered saline suspension of hepatocytes (5 × 106 cells/mL) in 20.0 μM CFSE incubated for 15 min at 37°C. In vitro, labelled hepatocytes were cultured either on fibronectin-coated chamber slides or in culture flasks. Cultures were evaluated in situ by fluorescence photomicrography or by fluorescence-activated cell sorting (FACS) after cell detachment. Cell viability was assessed serially and cultured, labelled hepatocytes retained the dye for up to 3 wk (last day of study). CFSE did not effect hepatocyte viability and there was no evidence of intercellular diffusion of the dye. In vivo, syngeneic Lewis rats underwent selective portal vein infusion of freshly isolated, labelled hepatocytes (2.0 × 107 cells/2.0 mL saline/animal) into the posterior liver lobes. All recipients were sacrificed 48 h and 96 h later and their livers examined. Transplanted hepatocytes were identified by fluorescence microscopy in tissue sections and by FACS following collagenase digestion of the liver tissue. CFSE persisted in a population of viable, engrafted hepatocytes. FACS analysis demonstrated that 9 ± 3% of the hepatocytes in the posterior liver lobes were labelled 48 and 96 h after transplantation. At 96 h following transplantation, multiple engrafted hepatocytes could be observed by fluorescence microscopy around the central veins. CFSE labelling allows for both in vitro identification and in vivo localization of donor hepatocytes. Furthermore, it appears to be more stable and specific for labelling hepatocytes than other tested dyes (especially DiI).
Platelet-Derived Growth Factor Receptor Expression after Neural Grafting in a Rat Model of Parkinson's Disease
Authors: Ballagi Andrea E. , Odin Per , Othberg-Cederström Agneta , Smits Anja , Duandr Wei-Ming , Lindvall Olle , Funa Keiko
Published: June 2017

Platelet-derived growth factor (PDGF) has trophic effect on dopaminergic neurons in vitro. We have previously shown dynamic changes in the expression of PDGF in embryonic mesencephalic grafts and surrounding host striatal tissue following intracerebral transplantation in a rat model of Parkinson's disease. In this study the expression of the PDGF receptors was examined in the same model using immunohistochemistry. Most ventral mesencephalic (VM) cells from E13–E15 rat embryos possessed both PDGF α-and β-receptors before implantation. Double immunofluorescence staining revealed that about 10% of the cells also expressed tyrosine hydroxylase (TH). The PDGF α-receptor was detectable in the graft up to 1 wk after transplantation but had disappeared at 3 wk. In the host tissue, scattered glial cells were positive for the α-receptor but the expression was unchanged following transplantation. The β-receptor expression almost completely disappeared from the grafted tissue by 4 h following transplantation, and only a few cells of the host striatum showed immunoreactivity. However, after 3 wk β-receptor positive cells were again detectable in the graft. These cells appeared to be endothelial cells as identified by an antibody against von Willebrand's factor. Our data suggest that PDGF might act locally on embryonic dopaminergic cells in an autocrine or juxtacrine manner before and shortly after transplantation, and on surrounding glial cells in a paracrine manner after transplantation. Furthermore, PDGF-BB might influence neovascularization in the graft.
Mechanical Stress-Induced Orientation and Ultrastructural Change of Smooth Muscle Cells Cultured in Three-Dimensional Collagen Lattices
Authors: Kanda Keiichi , Matsuda Takehisa
Published: June 2017

The effect of tensile stress on the orientation and phenotype of arterial smooth muscle cells (SMCs) cultured in three-dimensional (3D) type I collagen gels was morphologically investigated. Ring-shaped hybrid tissues were prepared by thermal gelation of a cold mixed solution of type I collagen and SMCs derived from bovine aorta. The tissues were subjected to three different modes of tensile stress. They were floated (isotonic control), stretched isometrically (static stress) and periodically stretched and recoiled by 5% above and below the resting tissue length at 60 RPM frequency (dynamic stress). After incubation for up to four wk, the tissues were investigated under a light microscope (LM) and a transmission electron microscope (TEM). Hematoxylin and eosinstained LM samples revealed that, irrespective of static or dynamic stress loading, SMCs in stress-loaded tissues exhibited elongated bipolar spindle shape and were regularly oriented parallel to the direction of the strain, whereas those in isotonic control tissues were polygonal or spherical and had no preferential orientation. In Azan-stained samples, collagen fiber bundles in isotonic control tissues were somewhat retracted around the polygonal SMCs to form a random network. On the other hand, those in statically and dynamically stressed tissues were accumulated and prominently oriented parallel to the stretch direction. Ultrastructural investigation using a TEM showed that SMCs in control and statically stressed tissues were almost totally filled with synthetic organelles such as rough endoplasmic reticulums, free ribosomes, Golgi complexes and mitochondria, indicating that the cells remained in the synthetic phenotype. On the other hand, SMCs in dynamically stressed tissues had increased fractions of contractile apparatus, such as myofilaments, dense bodies and extracellular filamentous materials equivalent to basement membranes, that progressed with incubation time. These results indicate that periodic stretch, in concert with 3-D extracellular collagen matrices, play a significant role in the phenotypic modulation of SMCs from the synthetic to the contractile state, as well as cellular and biomolecular orientation.
The Fasciovascular Flap: A New Vehicle for Islet Transplantation
Authors: Borud Loren J. , Shaw William W. , Passaro Edward , Brunicardi F. Charles , Mullen Yoko
Published: June 2017

In order to determine whether pancreatic islets could be neovascularized by a fasciovascular flap (FVP), islet transplant studies were conducted in Lewis rats. Islets from two donors were isolated by collagenase digestion and discontinuous gradient centrifugation on Ficoll. These islets were injected in syngeneic recipients either into random groin SC fat as a control, or into a flap composed of fascia and fat elevated from the groin based on the superficial inferior epigastric vessels. After two wk, islet viability was assessed by histological analysis. The degree of neovascularization of the islet tissue was evaluated with India ink injection through the vascular pedicle. Whereas control islets degenerated and did not show clear signs of neovascularization, FVP-islets showed rich neovascularization and viability as a large sheet of islet clusters. These results have demonstrated that the FVP-flap is a novel recipient site which can support a large quantity of islet tissue. This model constitutes a unique neo-endocrine pancreas flap, which can be subsequently transplanted at will to transfer the established neo-endocrine pancreas to a desired site using microvascular surgical technique.
Encapsulated PC 12 Cell Transplants into Hemiparkinsonian Monkeys: A Behavioral, Neuroanatomical, and Neurochemical Analysis
Authors: Kordower Jeffrey H. , Liu Yue-Ting , Winn Shelley , Emerich Dwaine F.
Published: June 2017

Four cynomolgus monkeys were trained on a hand reaching task and then rendered hemiparkinsonian with an intracarotid injection of n-methyl 4 phenyl 1,2,3,6, tetrahydropyridine (MPTP). Performance on this task with the limb contralateral to the MPTP injection was significantly impaired following the lesion. Three monkeys received implants of polymer-encapsulated containing PC12 cells into the caudate nucleus and putamen. One monkey received identical implants of empty capsules and served as a control. After a transient improvement, limb use in the control monkey dissipated and returned to post-MPTP disability. Two of the three PC12 cell grafted monkeys recovered performance on the hand reach task to near normal levels for up to 6.5 mo posttransplantation. Capsules retrieved from the monkeys who recovered limb function postimplantation contained numerous viable PC12 cells that continued to release levodopa, basal dopamine, and potassium evoked dopamine. In contrast, capsules retrieved from the PC12 cell-grafted monkey which did not recover limb use on the hand reach task contained few cells which secreted negligible or undetectable levels of levodopa and dopamine. Interestingly, functional disability was not reinstated following removal of the capsules. Neuroanatomical and neurochemical evaluation of the grafted striatum did not reveal a host-derived sprouting response of catecholaminergic or indolaminergic fibers. These data indicate that xenografts of PC12 cells can survive for up to 6.5 mo in nonimmunosuppressed monkeys when immunoisolated via polymer encapsulation. Moreover, these cells continue to secrete high levels of levodopa and dopamine and induce recovery of motor function in parkinsonian nonhuman primates.
A Comparative Study of Preparation Techniques for Improving the Viability of Nigral Grafts using Vital Stains, in Vitro Cultures, and in Vivo Grafts
Authors: Barker Roger A. , Fricker Rosemary A. , Abrous D. Nora , Fawcett James , Dunnett Stephen B.
Published: June 2017

The intracerebral transplantation of embryonic dopaminergic nigral neurons, although relatively successful, leads to a fairly low yield of surviving cells. Many factors may influence the viability of dopaminergic grafts and one of these is the preparation of the tissue prior to transplantation. We have investigated the effects of different steps during the preparation and storage of embryonic rat nigral cell suspensions on their subsequent survival at a variety of different time points using a combination of techniques and studies. For studies concerned with the first 24 h we employed vital stains, in the period covering the next 7 days we used in vitro cultures, and in the long term experiment we used in vivo grafts. The results suggest that nigral cell suspensions may remain sufficiently viable for grafting for much longer periods than previously reported. In addition a number of parameters which affect cell survival have been characterised, including the age of the embryonic donor tissue, the use of proteolytic enzymes and the trituration procedure used during the preparation of the suspension. The optimal preparation technique, therefore, uses E13-E14 embryos with the dissected ventral mesencephalon being incubated in purified 0.1% trypsin solutions for 60 min and triturated using a flame polished Pasteur pipette. This may have important implications in improving intracerebral transplantation for Parkinson's disease.
Synthetic Vascular Grafts Seeded with Genetically Modified Endothelium in the Dog: Evaluation of the Effect of Seeding Technique and Retroviral Vector on Cell Persistence in Vivo
Authors: Sackman Jill E. , Freeman Michael B. , Petersen Mark G. , Allebban Zuhair , Niemeyer Glenn P. , Lothrop Clinton D.
Published: June 2017

Unique characteristics of endothelium make it an attractive target cell for gene transfer. Genetically modified endothelial cells (ECs) seeded on synthetic vascular grafts offer the potential to control neointimal hyperplasia, decrease graft thrombogenicity and improve small diameter graft patency. This study addresses the issue of synthetic vascular graft colonization with endothelial cells transduced with noninducible retroviral marker genes in the dog. Autologous endothelial cells were enzymatically harvested and transduced with either the bacterial NeoR gene or human growth hormone gene using retroviral vectors. All transduced cells were positive by polymerase chain reaction (PCR) amplification for the transduced gene sequence prior to graft seeding. Transduced ECs were seeded on Dacron grafts (n = 3) pre-clotted with autologous blood. These grafts exhibited complete endothelialization at times from 250 to 360 days. Recovered DNA, however, was negative for the transduced gene sequence when analyzed by PCR and Southern blotting. Expanded polytetrafluoroethylene (ePTFE) was evaluated (n = 8) using several different cell seeding protocols. Grafts were seeded at 3 densities (ranging from 6 × 103 to 1.5 × 105 cells/cm2) and 2 different adherence times. Seeding substrate was also evaluated. Grafts were either preclotted with whole blood or incubated with 20 or 120 μg/ml fibronectin for 60 min. Graft biopsies were evaluated from 2 to 52 wk. Limited endothelialization was present in 4 dogs as early as 2 wk, but never progressed to full luminal coverage. The remaining dogs failed to ever exhibit any luminal EC adherence. Two dogs with limited EC coverage had positive DNA by PCR for the NeoR gene sequence at 2 and 3 wk. In contrast to transduced EC's, nontransduced EC colonization of ePTFE was complete at 2 wk when seeded under conditions that transduced cells had failed to persist. Neither seeding density, adherence time, seeding substrate or retroviral vector used influenced the uniformly poor graft coverage seen with transduced cells. Results of this study indicate that despite successful gene transfer using 4 different retroviral vectors, transduced endothelial cells seeded under varying conditions appear altered in their ability to stably adhere and colonize synthetic vascular grafts in vivo.
Limitations of In-Vitro Labeling of Endothelial Cells with Indium-111 Oxine
Authors: Carr H.M.H. , Smyth J.V. , Rooney O.B. , Dodd P.D. , Sharma H. , Walker M.G.
Published: June 2017

Indium-111 oxine labeling is widely used as a marker of endothelial cell attachment to vascular prostheses. The long term effect of labeling human adult endothelial cells (HAECs) with this isotope has not been determined. In this study the viability of labeled HAECs, leakage of isotope from labeled cells and adherence of circulating isotope to fibronectin coated prostheses were investigated over 24 h. The effect of incubation time on labeling efficiency was also assessed. There were significant differences in cell viability between the labeled and unlabeled groups beyond 4 h (p < 0.005, 2-tailed, unpaired t-test). In the control group cell numbers increased by 42% while in the labeled group this had decreased by 20% at 24 h. Spontaneous leakage increased with time but was maximal in the first 2 h. Adherence of circulating isotope to fibronectin coated expanded polytetrafluoroethylene (ePTFE) grafts was minimal but was significantly greater to gelatin impregnated Dacron (GEL-SEAL) beyond 1 hour (p < 0.05). Incubation times greater than 5 minutes during labeling do not significantly improve labeling efficiency, and may contribute to toxicity by prolonging exposure to oxine. Indium-111 oxine labeling of HAECs is a suitable technique for acute studies of endothelial cell kinetics up to 4 h, but its use in chronic studies may lead to significant underestimations of cell retention.
Isolation and Manipulation of Rostral Mesencephalic Tegmental Progenitor Cells from Rat
Authors: Ptak Louis R. , Hart Katherine R. , Lin Donghui , Carvey Paul M.
Published: June 2017

A technique for isolating mitotic progenitor cells from the embryonic rostral mesencephalon is described. Culture of the progenitor cells in complete media with subsequent staining for neuron specific enolase (NSE) revealed that only 0.6% of the cells were NSE immunoreactive. Coculturing the progenitor cells with established striatal cultures did not result in conversion of any of the cells to the dopamine neuron phenotype (tyrosine hydroxylase immunoreactive (THir) neurons). In contrast, co-culture of progenitor cells with established mesencephalic cultures produced a statistically significant, and in some cases (three of twelve), dramatic increase in the number of THir cells. The THir cells that were present had more pronounced process extension than those observed in mesencephalic mono-cultures. Culturing progenitor cells in transwell baskets that were continuously exposed to media but physically separated from established mesencephalic cultures growing underneath the baskets led to the conversion of only a few progenitor cells to THir neurons in four of twelve transwell studies suggesting that cell-cell contact between progenitor cells and mesencephalic cells is required for the conversion. This co-culture technique also increased the number of THir neurons in the mesencephalic cultures although the increase was not profound enough to explain the increase observed in traditional co-culture. These data suggest that mitotic progenitor cells can be isolated from fetal rat tissue and successfully converted to the dopamine neuron phenotype. Progenitor-mesencephalic co-culture appears to increase the number of THir cells in both tissue sources mediated in part by soluble factor(s) although cell-cell contact and presumably extracellular matrix proteins play a more substantial role. These progenitor cells may prove useful as a tissue source for transplantation procedures in Parkinson's disease.
Cell Transplantation and Federal Regulation
Authors: Noguchi Philip D.
Published: June 2017

Federal regulation of tissues and organs has proceeded slowly, with main emphasis on safety of the procured material. More recently with the development of somatic cell therapies, the Food and Drug Administration has issued some guidance documents that establish that some classes of cells that are manufactured will be subject to not only safety but efficacy requirements. Cell transplantation presents several aspects that are similar to somatic cell therapies, and the purpose of this presentation is to explore those relationships.
Infectious Disease Transmission through Cell, Tissue, and Organ Transplantation: Reducing the Risk through Donor Selection
Authors: Eastlund Ted
Published: June 2017

The incidence of cell transplant-transmitted infection is unknown and can only be inferred from prospective studies–that have not yet been performed and reported. The possibility of donor-to-recipient disease transmission through cell transplant therapy can be considered by reviewing the risk associated with other transplanted tissues and organs. Viral, bacterial, and fungal infections have been transmitted via transplantation of organs, tissue allografts such as bone, skin, cornea, and heart valves, and cells such as islets, hematopoietic stem cells, and semen. Several types of protozoan and worm parasites have been transferred via organ transplants. Bone allografts have transmitted hepatitis, tuberculosis, and human immunodeficiency virus (HIV-1). Corneas have transmitted rabies, Creutzfeldt-Jakob disease (CJD), hepatitis B (HBV), cytomegalovirus (CMV), herpes simplex virus (HSV), bacteria, and fungi. Heart valves have been implicated in transmitting tuberculosis and hepatitis B. HIV-1 and CMV seroconversion has been reported in patients receiving skin from seropositive donors. CJD has been transmitted by dura and pericardium transplants. Over the past several years, improvements in donor screening criteria, such as excluding potential donors with infection and those with behaviors risky for HIV-1 and hepatitis infection, and introduction of new donor blood tests have greatly reduced the risk of HIV-1 and hepatitis and may have nearly eliminated the risk of tuberculosis and CJD. Prior to use, many tissues are exposed to antibiotics, disinfectants, and sterilants, which further reduce or remove the risk of transmitted disease. Because organs, cells, and some tissue grafts cannot be subjected to sterilization steps, the risk of infectious disease transmission remains and thorough donor screening and testing is especially important.
Ethical and Policy Issues in Human Fetal Tissue Transplants
Authors: Vawter Dorothy E. , Gervais Karen G.
Published: June 2017

A review of human fetal tissue policies over the past 40 years reveals an increasing awareness that the use of this tissue raises unique ethical and policy issues. Initially it was believed that the use of fetal tissue was no different from using any other abandoned surgical waste specimen. Then state laws were passed requiring fetal tissue to be treated with the same respect and protection as tissue obtained from cadaver donors generally. Most recently, a few state and federal policies suggest that the use of fetal tissue requires a unique set of provisions, that it is unlike either the use of tissue from a routine cadaver donor or the use of tissue from abandoned waste specimens. In this paper we identify four central features of fetal tissue donation that make it a unique type of tissue donation requiring its own framework of respect and protection for the parties involved. We then critically review the most important current state and federal fetal tissue policies, including the NIH Revitalization Act of 1993.