bluuebunny/crossref_metadata_2025 huggingface dataset:
The Use of Multiparametric Monitoring during Islet Cell Isolation and Culture: A Potential Tool for In-Process Corrections of Critical Physiological Factors
Authors: Fraker Chris , Montelongo Jorge , Szust Joel , Khan Aisha , Ricordi Camillo
Published: April 2008

Variables such as pH, pCO2, and pO2 have been established in the literature as critical factors that could affect the outcome of the islet cell processing and, therefore, the quality of the cells that could be transplanted. This report describes a highly accurate continuous multiparametric monitoring system and its evaluation for continuous monitoring of physiological variables during critical steps of the islet isolation procedure as well as during in vitro culture of the insulin-producing cells. Close monitoring of these variables could be of assistance to improve the outcome of islet cell processing, allowing to identify as soon as possible problems that could be corrected during the procedure, as well as during in vitro preservation, or shipment to remote sites.
Factors Influencing Functional Survival of Microencapsulated Islet Grafts
Authors: De Vos Paul , De Haan Bart J. , De Haan Aalzen , Van Zanten Jacoba , Faas Marijke M.
Published: April 2008

Graft function of encapsulated islets is restricted in spite of the fact that inflammatory responses against capsules are limited to a portion less than 10%. It has been shown that dysfunction is accompanied by a gradual decrease in the glucose-induced insulin response (GIIR), a hyperproliferation of islet cells, and gradual necrosis. Also, limited survival is associated with the presence of macrophages in the overgrowth. In the present study, we investigate whether macrophages are the inducers of dysfunction of encapsulated grafts. Four weeks after successful transplantation of microencapsulated rat allografts we determined the GIIR, the rate of islet cell replication, and islet cell death. Also, we quantified the number of macrophages on the overgrown capsules. This assessment was applied to set up an in vitro coculture system of macrophages and encapsulated islets. We retrieved 93 ± 6.2% of the capsules of which 9.2 ± 0.3% was overgrown. The GIIR of the retrieved nonovergrown islets was reduced when compared with freshly encapsulated islets. The replication rate of the retrieved islet cells was eightfold higher than in the normal pancreas. Apoptosis was rarely observed but 37 ± 4% of the total islet surface was composed of necrosis. We found a mean of 1542 ± 217 macrophages per capsule. Coculture of 1500 NR8383 macrophages per encapsulated islets induced a substantial reduction in GIIR but a decrease instead of increase in replication. Necrosis was restricted to 13 ± 1.3% of the islet cells and was not increased by the presence of macrophages. Our observations indicate that we should focus on reduction of macrophage activation and on improving the nutrition of encapsulated islets to prevent islet cell death.
Vigorous Neuronal Differentiation of Amplified and Grafted Basic Fibroblast Growth Factor-Responsive Neurospheres Derived from Neuroepithelial Stem Cells
Authors: Yamada Motoyuki , Uchida Koichi , Hayashi Takuro , Mine Yutaka , Kawase Takeshi
Published: April 2008

Neuroepithelial stem cells (NESCs) have emerged as a possible donor material aimed at neural transplantation for the repair of damaged neural circuitry, particularly because of their propensity to differentiate into neurons. We previously ascertained in vitro that NESCs derived from rat early embryos could be amplified in culture containing basic fibroblast growth factors (bFGF), and that neurospheres grown for 7 days in the culture had a strong tendency to differentiate into neurons. In this report, we analyze immunohistochemically the biological nature of bFGF-responsive neurospheres derived from NESCs. We first succeeded in amplifying the number of NESCs from the mesencephalic neural plate of embryonic day 10 Wistar rats with the addition of bFGF. Grown neurospheres were labeled with bromodeoxyuridine (BrdU) in vitro and were stereotactically transplanted into the right striatum of the normal adult Wistar rat. Two weeks after transplantation, a viable graft in the host brain was observed. While many BrdU/Hu double positive cells were seen in the graft, and a few BrdU/nestin double positive cells were also seen, no BrdU/GFAP double positive cells could be identified. These results suggested that bFGF-responsive neurospheres derived from NESCs demonstrated a propensity to differentiate into neurons in the adult brain environment. Furthermore, following in vitro amplification of the original stem cell number with bFGF, the grown neurospheres preserved their propensity to differentiate vigorously into neurons. NESCs are thus suggested as a feasible candidate for intracerebral grafting donor materials aimed at reconstruction of damaged neural circuits.
Functional Activity of Human Hepatoma Cells Transfected with Adenovirus-Mediated Hepatocyte Nuclear Factor (HNF)-4 Gene
Authors: Naiki Takafumi , Nagaki Masahito , Shidoji Yoshihiro , Kojima Hisanori , Moriwaki Hisataka
Published: April 2008

Fulminant hepatic failure (FHF) is still associated with high mortality despite recent advances in medical management. There is need of an effective and safe bioartificial liver (BAL) support to help keep patients with FHF alive until an organ becomes available for transplantation or the native liver recovers. The aim of this study was to establish highly functional liver cells by means of transfecting hepatocyte nuclear factor (HNF)-4 gene for the development of BAL. We constructed adenovirus vector carrying rat HNF-4 cDNA, and transfected to hepatoma-derived cell lines, HepG2 and HuH-7, to enforce expression of the exogenous HNF-4 gene. We analyzed expression of HNF-4, HNF-1, and liver-specific genes in cells infected by the adenovirus vector expressing HNF-4. Adenovirus-mediated HNF-4 gene transfer resulted in increases in expressions of HNF-4, HNF-1, and liver-specific genes such as apolipoproteins, α1-antitrypsin (α1-AT), phosphoenolpyruvate carboxy-kinase, cytochrome P450 families, and glutamine synthetase in transfected hepatoma cells. Cells overexpressing HNF-4 removed ammonia from medium supplemented with NH4Cl to a greater extent than control cells. These findings demonstrated that transfected cell lines restored differentiated gene expressions and liver-specific function by the overproduction of HNF-4. HNF-4-overexpressing hepatocyte cell lines are useful for bioreactor of BAL systems.
Liposomal Formulations of Tacrolimus and Rapamycin Increase Graft Survival and Fiber Outgrowth of Dopaminergic Grafts
Authors: Alemdar Aylin Y. , Sadi Damaso , Mcalister Vivian C. , Mendez Ivar
Published: April 2008

The immunosuppressive drugs tacrolimus (TAC) and rapamycin (RAPA) have both been found to have neuroprotective effects on dopaminergic neurons. The purpose of the present study was to investigate whether liposomal formulations of these drugs administered directly into the brain improve cell survival and fiber outgrowth. Rats with unilateral 6-hydroxydopamine lesions were transplanted with 800,000 fetal rat ventral mesencephalic cells and randomly divided to one of four groups. Group 1 received a transplant containing cells only; group 2 received a cell suspension containing 0.68 μM liposomal RAPA (LRAPA); group 3 received a cell suspension containing 2.0 μM liposomal TAC (LTAC); and group 4 received a cell suspension containing a liposomal formulation of both 0.68 μM RAPA and 2.0 μM TAC (LRAPATAC). Rats were sacrificed after 6 weeks, and cell survival and fiber outgrowth were assessed using tyrosine hydroxylase (TH) immunohistochemistry. The animals receiving a cell suspension containing either LTAC or LRAPATAC were found to have significantly more surviving TH-immunoreactive (TH-ir) cells than the control group receiving cells only. The group receiving LTAC had significantly longer fibers, the group receiving LRAPA had significantly more fibers close to the graft, and the group receiving LRAPATAC had significantly more fibers at all distances. This study shows the feasibility of using liposomal formulations of neuroimmunophilins directly in the brain at the time of implantation to improve graft survival and fiber outgrowth. Furthermore, we have shown that the combination of LTAC and LRAPA has a synergistic effect. These compounds may play an important role in optimizing graft survival and host reinnervation in cellmediated brain repair strategies for the treatment of neurological conditions.
Neuroprotective Effects of Encapsulated CNTF-Producing Cells in a Rodent Model of Huntington's Disease are Dependent on the Proximity of the Implant to the Lesioned Striatum
Authors: Emerich Dwaine F. , Winn Shelley R.
Published: April 2008

Huntington's disease (HD) is a devastating genetic disorder with no effective treatments for preventing or lessening the underlying neuronal degeneration. Intracerebral delivery of CNTF in animal models of HD has shown considerable promise as a means of protecting striatal neurons that would otherwise be destined to die. The present study examines whether the neuroprotective effects of CNTF require that the delivery be immediately proximal to the lesion site or whether protective effects can be exerted when the delivery site is more distal to the site of injury. Encapsulated CNTF-producing cells were implanted into the lateral ventricle either ipsilateral or contralateral to an intrastriatal quinolinic acid (QA) injection. A robust neuroprotective effect was observed only in those animals receiving CNTF implants ipsilateral to the QA injection. In these animals, the loss of striatal ChAT and GAD activity as well as the behavioral impairments that resulted from QA were completely prevented. In contrast, no neurochemical or behavioral benefits were produced by implants of CNTF-producing cells in the contralateral ventricle. These data continue to support the use of cellular delivery of CNTF for HD but caution that delivery directly to the striatum may be needed if any clinical benefits are to be seen.
Comparison of Bioenergetic Activity of Primary Porcine Hepatocytes Cultured in Four Different Media
Authors: Dabos Konstantinos J. , Nelson Leonard J. , Hewage Chandralal H. , Parkinson John A. , Howie A. Forbes , Sadler Ian H. , Hayes Peter C. , Plevris John N.
Published: April 2008

Primary hepatocytes have extensively been used in biochemical, pharmacological, and physiological research. Recently, primary porcine hepatocytes have been regarded as the cells of choice for bioartificial liver support systems. The optimum culture medium for hepatocytes to be used in such devices has yet to be defined. In this study we investigated the effectiveness of four culture media in driving energy metabolism of primary porcine hepatocytes. The media selected were William's E medium, medium 1640, medium 199, and hepatocyte medium. Cells (3 × 1010; viability 87 ± 6%) were isolated from weanling piglets and seeded on 90-mm plates in the above media supplemented with antibiotics and hormones at a density of 8 × 106 viable cells per plate. Using 1H NMR spectroscopy we looked at indices of glycolysis, gluconeogenesis, ketogenesis, and ureagenesis on days 2, 4, and 6 of the experiments (n = 9). We also studied urea and albumin synthesis and total P450 content. The examined metabolic pathways of the hepatocytes were maintained by all media, although there were statistically significant differences between them. All media performed well in glycolysis, ureagenesis, and albumin synthesis. William's E medium and medium 199 outperformed the rest in gluconeogenesis. Medium 199 was best in ketogenesis. Overall, medium 199 was the best at driving energy metabolism from its constituent substrates and we think that it preferentially should be used in the culture of primary porcine hepatocytes.
AN69 Hollow Fiber Membrane will Reduce but Not Abolish the Risk of Transmission of Porcine Endogenous Retroviruses
Authors: Pakhomov Oleg , Martignat Lionel , Honiger Jiri , Clémenceau Béatrice , Säi Pierre , Darquy Sylviane
Published: April 2008

As the risk of porcine endogenous retrovirus (PERV) infection is a major obstacle to the xenotransplantation of porcine tissue, we investigated whether an AN69 hollow fibre membrane, used for islets of Langerhans transplantation, could prevent the transfer of PERVs and thus reduce the risk of PERV infection. PK15 cells were used as a PERV source. A specific and highly sensitive RCR was used for detection of a PERV provirus DNA (gag region) and a porcine mtDNA. Human U293 cells were incubated in vitro with encapsulated PK15 cells, concentrated encapsulated PK15 supernatant, or concentrated PK15 supernatant as a control. CD1 mice were implanted in vivo with encapsulated PK15 cells or injected with PK15 supernatant. We found no infection in human cells incubated with either encapsulated PK15 supernatant or in 10 out of 11 samples after coincubation with encapsulated PK15 cells. Infection of human cells was, however, detected in 1 out of 11 samples after coincubation with encapsulated PK15 cells. The presence of PERV provirus DNA and porcine mtDNA was detected in all the investigated tissues of the mice injected with PK15 supernatant and in various tissues of the mice implanted with encapsulated PK15 cells. Four weeks after the last injection of PK15 supernatant or a fiber explantation, no mouse showed any presence of PERV provirus DNA or porcine mtDNA. Our results demonstrate that AN69 hollow fiber membrane will reduce but not abolish the risk of PERV infection. Because the real risk of PERV infection still remains unknown, it is necessary to investigate further the real protection that could be provided by hollow fibers to ensure the safety of clinical xenotransplantation.
The Effects of Bone Marrow-Derived Mesenchymal Stem Cells and Fascia Wrap Application to Anterior Cruciate Ligament Tissue Engineering
Authors: Ge Zigang , Goh James Cho Hong , Lee Eng Hin
Published: April 2008

After an anterior cruciate ligament (ACL) injury, surgical reconstructions are necessary in most cases, either with autografts, allografts, or artificial ligaments. Potential tissue-engineered ligaments would circumvent the disadvantages apparent in these methods. While seeding of mesenchymal stem cells (MSCs) and fascia wrap could potentially improve tissue regeneration and mechanical properties, their exact roles were evaluated in the current study. Knitted biodegradable scaffolds of poly-L-lactic acid (PLLA) and poly-glycolic-lactic acid (PGLA) yarns were used to reconstruct ACL in 48 rabbits. These were divided into four equal groups: only knitted scaffolds were used in group I; knitted scaffolds and mesenchymal stem cells were used in group II; knitted scaffolds, MSCs, and fascia lata were used in group III; knitted scaffolds and fascia lata were used in group IV. Carboxyfluorescein diacetate (CFDA)-labeled MSCs were used to trace the fate of seeded cells in groups II and III. Histology, Western blot analysis, and mechanical properties of reconstructed ACL were analyzed after 20 weeks. Fibroblast ingrowths were seen in all four groups while CFDA-labeled MSCs could be found after 8 weeks of implantation in groups II and III. Both the amount of collagen type I and collagen type III in groups III and IV were significantly higher than in group II, which was much higher than in group I. Both maximal tensile loads and stiffness of the reconstructed ACLs in groups I, II, III, and IV were significantly lower than normal controls after 20 weeks of implantation. It is concluded that MSCs could promote synthesis of collagen type I and collagen type III in tissue-engineered ligaments, while fascia wraps have stronger effects. Both MSC seeding and fascia wrap could not enhance ultimate tensile load and stiffness.
Influence of Preservation Solution on the Isolation and Culture of Human Hepatocytes from Liver Grafts
Authors: Serralta Alfonso , Donato Maria Teresa , Martinez Amparo , Pareja Eugenia , Orbis Francisco , Castell Jose Vicente , Mir Jose , Gómez-Lechón Maria Jose
Published: April 2008

A major problem for the isolation and transplantation of hepatocytes is the lack of resources for obtaining viable hepatocytes. Improving this situation would enhance hepatic cell transplantation programs. Our objective was to evaluate the influence of the preservation solutions used during organ retrieval on the quality of hepatocytes isolated from liver tissue. We compared the results of the collagenase perfusion technique for isolation of hepatocytes in human livers flushed with University of Wisconsin (UW) and Celsior preservation solutions. Yield (number of viable cells per gram of tissue), cellular viability, efficiency of cells to attach to culture plates and form a monolayer, and drug metabolizing competence of the hepatocytes were measured. Successful isolation was achieved in 63% of the procedures using the UW solution and 100% of the procedures using the Celsior solution. In the UW group, significantly lower cell viability (38 ± 41% vs. 79 ± 14%, p < 0.05), yield of cells (4.0 ± 5.2 × 106 vs. 8.2 ± 5.6 × 106 cells/g, p < 0.05), and protein content at 24 h of culture (0.6 ± 0.6 vs. 1.2 ± 0.3 mg protein per plate, p < 0.05) than in Celsior solution were found. However, similar values of P450 activities were found in both groups. The more successful isolation, better yield, and higher cell viability obtained from human liver grafts preserved in Celsior solution, in comparison to UW solution, suggest Celsior solution as the most appropriate for preserving cadaveric hepatic tissue to be used for hepatocyte harvesting.
Synergistic Effect of Keratinocyte Transplantation and Epidermal Growth Factor Delivery on Epidermal Regeneration
Authors: Gwak So-Jung , Kim Sang-Soo , Sung Kyungeun , Han Joungho , Choi Cha Yong , Kim Byung-Soo
Published: April 2008

Both keratinocyte transplantation and epidermal growth factor (EGF) delivery stimulate epidermal regeneration. In this study, we hypothesized that the combined therapy of keratinocyte transplantation and EGF delivery accelerates epidermal regeneration compared to the single therapy of either keratinocyte transplantation or EGF delivery. To test this hypothesis, we utilized fibrin matrix as a keratinocyte/EGF delivery vehicle for epidermal regeneration. Full-thickness wounds were created on the dorsum of athymic mice, and human keratinocytes and EGF in fibrin matrix were sprayed onto the wounds to regenerate epidermal layers (group 1). As controls, human keratinocytes in fibrin matrix (group 2), EGF in fibrin matrix (group 3), or fibrin matrix alone (group 4) was sprayed onto the wounds. Spraying keratinocytes suspended in fibrin matrix did not affect the keratinocyte viability, as the cell viabilities before and after spraying were not different. EGF was released from fibrin matrix for 3 days. The wounds were analyzed with histology and immunohistochemistry at 1 and 3 weeks after treatments. Compared with the control groups, initial wound closure rate was highest in group 1. Histological analyses indicated that group 1 exhibited faster and better epidermal regeneration than the other groups. Immunohistochemical analyses showed that regenerated epithelium in groups 1 and 2 stained positively for human involucrin at 3 weeks, whereas the tissue sections of the groups 3 and 4 stained negatively. Human laminin was detected at the dermal–epidermal junction of the regenerated tissues in groups 1 and 2 at 3 weeks and was not detected in groups 3 and 4. The epidermal thickness of the regenerated tissues in group 1 was significantly thicker than that of the other groups at all time points. These results suggest that the combined therapy of keratinocyte transplantation and EGF delivery is more efficacious for epidermal regeneration than each separate therapy alone.
PDX-1 Protein is Internalized by Lipid Raft-Dependent Macropinocytosis
Authors: Noguchi Hirofumi , Matsumoto Shinichi , Okitsu Teru , Iwanaga Yasuhiro , Yonekawa Yukihide , Nagata Hideo , Matsushita Masayuki , Wei Fan-Yan , Matsui Hideki , Minami Kohtaro , Seino Susumu , Masui Yumi , Futaki Shiroh , Tanaka Koichi
Published: April 2008

PDX-1 plays a central role in regulating insulin gene transcription and differentiation of insulin-producing cells. It was previously reported that, due to its own Antennapedia-like protein transduction domain (PTD), exogenous PDX-1 protein can permeate cells and induces insulin gene expression in pancreatic ducts, thought to be islet progenitor cells. These data suggest that PDX-1 protein transduction could be a safe and valuable strategy for facilitating differentiation of progenitor cells into insulin-producing cells without requiring gene transfer technology. Here it is shown that after an initial ionic cell–surface interaction, PDX-1 proteins are rapidly internalized by lipid raft-dependent macropinocytosis. HeLa cells were treated with both FITC-conjugated PDX-1 PTD and FM 4–64, a general fluorescent marker of endocytosis. A punctate cytoplasmic distribution of PDX-1 PTD, which colocalized with FM 4–64, was observed in treated cells. Because expression of dominant-negative dynamin-1 did not block PDX-1 PTD uptake, PDX-1 protein transduction is independent on phagocytosis and clathrin- or caveolar-mediated endocytosis. Cells were pretreated with amiloride, a specific inhibitor of the Na+/H+ exchange required for macropinocytosis, or cytochalasin D, an F-actin elongation inhibitor. Treatment of cells with both macropinosome inhibitors resulted in the reduction in PDX-1 PTD transduction into vesicles, suggesting that PDX-1 PTD-mediated cellular entry occurs by lipid raft-mediated macropinocytosis. Taken together, these observations provide the mechanism of PDX-1 protein transduction and suggest that the protein transduction system could work for experimental and therapeutic strategies.
In Vivo PET Measurements with [11C]PE2I to Evaluate Fetal Mesencephalic Transplantations to Unilateral 6-OHDA-Lesioned Rats
Authors: Inaji Motoki , Yoshizaki Takahito , Okauchi Takashi , Maeda Jun , Nagai Yuji , Nariai Tadashi , Ohno Kikuo , Ando Kiyoshi , Okano Hideyuki , Obayashi Shigeru , Suhara Tetsuya
Published: April 2008

Positron emission tomography (PET) is a useful tool to assess and visualize neurotransmissions in vivo. In this study, we performed repeated PET scans with [11C]PE2I, a tracer of the dopamine transporter, to evaluate the alteration of the expression of dopamine (DA) transmission component after a fetal mesencephalic transplantation. The fetal mesencephalic cells were transplanted into the striatum of unilateral 6-OHDA-lesioned rats. PET scans with [11C]PE2I were performed to evaluate the DA transporter before and 2 and 4 weeks after the transplantation. Rotation behavior tests, in vitro autoradiography, measurements of DA contents in the striatum by high-performance liquid chromatography (HPLC), and tyrosine hydroxylase (TH) immuno-histological examinations were performed at the same time points and examined for their relationship to changes in the dopamine transporter. The number of ipsilateral rotations induced by methamphetamine injections decreased. DA contents in the striatum measured with HPLC significantly increased. In the PET study, the binding potential of [11C]PE2I increased at 4 weeks. The results of the in vitro autoradiography study corresponded with those of the PET study. The degrees of the change in the binding potentials correlated with those of the numbers of rotations in the behavioral study and the DA contents in the striatum. In the histological examination, TH-positive cells with axons were observed at 2 and 4 weeks after the transplantation. As the dopamine transporter exists only in the axon terminal of DA neurons, these results suggested that PET measurements of [11C]PE2I binding indicated not only survival, but maturity and functioning of the transplanted cells. Repeated PET measurements of DA transporters are a useful tool in assessing the effectiveness of neural transplantations.
A Novel Method of Cryopreservation of Rat and Human Hepatocytes by Using Encapsulation Technique and Possible Use for Cell Transplantation
Authors: Aoki Takeshi , Koizumi Tomotake , Kobayashi Yasuna , Yasuda Daisuke , Izumida Yoshihiko , Jin Zhenghao , Nishino Nobukazu , Shimizu Yoshinori , Kato Hirohisa , Murai Noriyuki , Niiya Takashi , Enami Yuta , Mitamura Keitaro , Yamamoto Toshihiro , Kusano Mitsuo
Published: April 2008

Encapsulated hepatocyte transplantation is a promising approach to cell transplantation without immunosuppression as an alternative to whole organ liver transplantation. However, the shortage of donor cells for hepatocyte transplantation has not been resolved, and at this critical point, it seems necessary to establish a method of hepatocyte cryopreservation to allow clinical application of hepatocyte transplantation and the development of a bioartificial liver system in the near future. In this study we demonstrated that cryopreserved microencapsulated rat and human hepatocytes can retain their hepatic function and that cryopreserved microencapsulated human hepatocytes transplanted into rat spleen remain viable without immunosuppression. Rat and human hepatocytes were isolated by a collagenase digestion method, and they were microencapsulated with poly-L-lysine. The microencapsulated rat hepatocytes were transferred to culture medium (DMEM containing 10% FBS and 10% DMSO) and immediately frozen in liquid nitrogen. A warm water bath (37°C) was used to thaw the microencapsulated hepatocytes. Hepatic function, drug metabolism, and cell morphology were assessed after 90 days of cryopreservation. After 1 week of cryopreservation, microencapsulated hepatocytes were cultured for up to 2 weeks to assess their hepatic function and morphology. The morphology of human hepatocytes was assessed after 30 days of cryopreservation. Cryopreserved human hepatocytes were transplanted into rat spleen to assess their morphology. Cryopreserved microencapsulated hepatocytes retained their viability and were strongly positive for expression of albumin, OAT2, CYP3A2, and CYP3A9. Two weeks after cultivation, the cryopreserved microencapsulated rat hepatocytes had retained their hepatic function (urea synthesis). Cryopreserved microencapsulated human hepatocytes also mainly survived and retained their hepatic function for at least 30 days after cryopreservation. Moreover, entrapped cryopreserved human hepatocytes also survived and expressed albumin in rat spleen after transplantation. We demonstrated a novel method of long-term cryopreservation of rat and human hepatocytes by using an encapsulation technique, with retention of biological activity and excellent survival of the cryopreserved microencapsulated human hepatocytes transplanted into rat spleen. We believe that this novel approach to hepatocytes cryopreservation provides a new direction in encapsulated cell therapy with the goal of clinical application in the near future.
Survival of Rat or Mouse Ventral Mesencephalon Neurons after Cotransplantation with Rat Sertoli Cells in the Mouse Striatum
Authors: Shamekh R. , Newcomb J. , Mallery J. , Cassady C. J. , Saporta S. , Cameron D. F. , Sanberg P. R. , Willing A. E.
Published: April 2008

Transplanting cells across species (xenotransplantation) for the treatment of Parkinson's disease has been considered an option to alleviate ethical concerns and shortage of tissues. However, using this approach leads to decreased cell survival; the xenografted cells are often rejected. Sertoli cells (SCs) are testis-derived cells that provide immunological protection to developing germ cells and can enhance survival of both allografted and xenografted cells. It is not clear whether these cells will maintain their immunosuppressive support of cografted cells if they are transplanted across species. In this study, we investigated the immune modulatory capacity of SCs and the feasibility of xenografting these cells alone or with allografted and xenografted neural tissue. Transplanting xenografts of rat SCs into the mouse striatum with either rat or mouse ventral mesencephalon prevented astrocytic infiltration of the graft site, although all transplants showed activated microglia within the core of the graft. Surviving tyrosine hydroxylase-positive neurons were observed in all conditions, but the size of the grafts was small at best. SCs were found at 1 and 2 weeks posttransplant. However, few SCs were found at 2 months posttransplant. Further investigation is under way to characterize the immune capabilities of SCs in a xenogeneic environment.
Creatine Supplementation Improves Dopaminergic Cell Survival and Protects against MPP+ Toxicity in an Organotypic Tissue Culture System
Authors: Andres Robert H. , Ducray Angelique D. , Pérez-Bouza Alberto , Schlattner Uwe , Huber Alexander W. , Krebs Sandra H. , Seiler Rolf W. , Wallimann Theo , Widmer Hans R.
Published: April 2008

Cell replacement therapy using mesencephalic precursor cells is an experimental approach for the treatment of Parkinson's disease (PD). A significant problem associated with this procedure is the poor survival of grafted neurons. Impaired energy metabolism is considered to contribute to neuronal cell death after transplantation. Creatine is a substrate for mitochondrial and cytosolic creatine kinases (CK) and buffers cellular ATP resources. Furthermore, elevated cellular creatine levels facilitate metabolic channeling and show anti-apoptotic properties. Exogenous creatine supplementation therefore might offer a tool for improvement of dopaminergic neuron survival. The present study aimed at investigating the effects of creatine on cell survival of rat embryonic day 14 (E14) ventral mesencephalic neurons grown as organotypic free-floating roller tube (FFRT) cultures. We found that the brain-specific isoform of CK (BB-CK) and the ubiquitous mitochondrial isoform (uMt-CK) are expressed at high levels in FFRT cultures and colocalize with tyrosine hydroxylase immunoreactive (TH-ir) cells. Exposure of these cultures to creatine induced an increase in the content of the BB-CK isotype. Creatine (5 mM) administration starting at day in vitro (DIV) 7 resulted in a significant increase (+35%) in TH-ir cell density at DIV21. In addition, we observed that creatine treatment provided neuroprotection against 1-methyl-4-phenyl pyridinium ion (MPP+)-induced TH-ir cell loss in the FFRT culture system, resulting in a significantly higher density (+19%) of TH-ir neurons in creatine-treated cultures compared to corresponding controls. The decrease of TH-ir neurons in the MPP+-treated group corresponded with an increase in immunoreactivity for active caspase-3, an effect that was not seen in the group receiving creatine supplementation. In conclusion, our data imply that creatine administration is beneficial for the survival of TH-ir neurons encountering harmful conditions.
Seeding of Corneal Wounds by Epithelial Cell Transfer from Micropatterned PDMS Contact Lenses
Authors: Pino Christopher J. , Haselton Frederick R. , Chang Min S.
Published: April 2008

Persistent corneal wounds result from numerous eye disorders, and to date, available treatments often fail to accelerate reepithelialization, the key initial step in wound healing. To speed reepithelialization, we explored a cell-transfer transplant method utilizing polydimethylsiloxane (PDMS) contact lenses to deliver epithelial cells derived from limbal explants directly within a corneal wound. Human primary epithelial cells and an immortalized corneal epithelial cell line (HCE-SV40) grew well on PDMS contact lenses and their morphology and growth rates where similar to cells grown on tissue culture polystyrene. To initially study cell transfer from PDMS, HCE-SV40 cells were seeded onto PDMS with or without micropatterned posts. After a day in culture, HCE-SV40 cells attached to the unpatterned PDMS uniformly, whereas on micropatterned PDMS they appeared to attach primarily between posts. The cell-covered PDMS contacts were then placed cell-side down onto tissue culture plastic and, after 1, 2, or 3 days, the PDMS contact was removed and the transferred cells were trypsinized and counted. Micropatterned PDMS contact lenses with 100-μm-diameter posts and a post height of 40 μm transferred three times as many cells as unpatterned PDMS. Cell transfer to a wounded cornea was tested in a pig cornea organ culture model deepithelialized by alkali treatment. Post micropatterned PDMS contact lenses were seeded with labeled HCE-SV40 cells at a density 50,000 cells/cm2 and applied to the wounded pig corneas. After 24, 48, or 96 h of application, PDMS contact lenses were removed, corneas fixed with formaldehyde, and sectioned. After 48 h, epithelial cells transferred from post micropatterned contact lenses to provide 35% epithelial coverage of denuded pig corneas; after 96 h coverage was 65%. We conclude that cell transfer from epithelial-coated PDMS contact lenses micropatterned with posts provides a promising approach to reepithelialize corneal surfaces.
The Use of Pancreas Biopsy Scoring Provides Reliable Porcine Islet Yields While Encapsulation Permits the Determination of Microbiological Safety
Authors: Gazda Lawrence S. , Adkins Hollie , Bailie Johannah A. , Byrd Wendy , Circle Lisa , Conn Bryan , Diehl Carolyn H. , Hall Richard D. , Rubin Albert L. , Smith Barry H.
Published: April 2008

For clinical xenogenic islet transplantation to be successful, several requirements must be met. Among them is a sizeable and reliable source of fully functional and microbiologically safe islets. The inherent variability among porcine pancreases, with respect to islet yield, prompted us to develop a Biopsy Score technique to determine the suitability of each pancreas for islet isolation processing. The Biopsy Score consists of an assessment of five variables: warm ischemia time, pancreas color, fat content, islet size, and islet demarcation, each of which is assigned a value of −1 or +1, depending on whether or not the established criteria is met. For determination of islet size and demarcation, fresh biopsies of porcine pancreases are stained with dithizone (DTZ) solution and examined under a dissecting microscope. Based on the scoring of such biopsies in pancreases from 26—56-month-old sows, we report here that the presence of large (>100 μm diameter), well-demarcated islets in the pancreas biopsy is a reliable predictor of isolation success. Encapsulation of the isolated porcine islets within the inner layer of a 1.5% agarose and an outer layer of 5.0% agarose macrobead, containing 500 equivalent islet number (EIN), provides for extended in vitro functional viability (>6 months of insulin production in response to glucose), as well as for comprehensive microbiological testing and at least partial isolation of the xenogeneic islets from the host immune system. All microbiological testing to date has been negative, except for the presence of porcine endogenous retrovirus (PERV). Taken together, we believe that the Biopsy Score enhancement of our islet isolation technique and our agarose-agarose macroencapsulation methodology bring us significantly closer to realizing clinical porcine islet xenotransplantation for the treatment of insulin-dependent diabetic patients.
Survival Analysis of Escherichia coli Encapsulated in a Hollow Fiber Membrane In Vitro and In Vivo: Preliminary Report
Authors: Granicka L. H. , Wdowiak M. , Kosek A. , Świezewski S. , Wasilewska D. , Jankowska E. , Weryński A. , Kawiak J.
Published: April 2008

The purpose of the observations was the viability and quality evaluation of E. coli bacteria encapsulated in hollow fiber membranes (HF) in short in vivo and in vitro experiments. A polypropylene, surface-modified hollow fiber was applied for immunoisolation of E. coli bacteria transfected with a green fluorescent protein (E. coli GFPI). The presence of GFP fluorescence of organisms was assessed with the use of flow cytometry. The E. coli GFPIs were then observed for the period of 5 days in in vitro experiments in the culture medium. A single IPTG (isopropyl β-D-1-thiogalactopyranoside) induction of GFP gene appeared to be adequate for an expression of GFP protein for 5 days. The GFP expression values observed for E. coli GFPs encapsulated in HF during culture in different culture media were comparable. The survival of E. coli GFPIs encapsulated in HF after 1, 2, 4, or 5 days of subcutaneous implantation into mice was evaluated. The explanted E. coli GFPIs exhibited mean expression 603 ± 17 (n = 32) units of fluorescence during the implantation period. The values obtained were comparable for selected days of observation. It was observed that the membranes applied ensured the bacteria growth within the HF's space only.
Combining Neuroprotective Treatment of Embryonic Nigral Donor Tissue with Mild Hypothermia of the Graft Recipient
Authors: Karlsson Jenny , Petersén ÅSa , Gidö Gunilla , Wieloch Tadeusz , Brundin Patrik
Published: April 2008

Around 80–95% of the immature dopaminergic neurons die when embryonic ventral mesencephalic tissue is transplanted. Cell death occurs both during the preparation of donor tissue and after graft implantation, but the effect of combining successful neuroprotective treatments before and after transplantation has not been extensively investigated. We therefore treated embryonic rat mesencephalic tissue with a combination of the lipid peroxidation inhibitor tirilazad mesylate (3 μM) and the caspase inhibitor Ac.YVAD.cmk (500 μM) and transplanted the tissue into hemiparkinsonian rats kept hypothermic (32–33°C) or normothermic (37°C) during, and 90 min following, graft surgery. Suspension cell number did not differ between untreated or tirilazad/YVAD-treated preparations prior to transplantation. When graft survival was evaluated 6 weeks after implantation, both tirilazad/YVAD pretreatment and mild hypothermia increased the survival of transplanted dopaminergic neurons. Approximately 50–57% of the embryonic dopaminergic neurons survived the dissociation and grafting procedure in rats rendered hypothermic, but there was no significant additive effect on graft survival with a combined treatment. All groups of rats exhibited behavioral recovery in the amphetamine-induced rotation test. There was a significantly enhanced functional capacity of grafts placed in hypothermic as compared to normothermic rats. However, tirilazad/YVAD pretreated implants did not afford greater behavioral improvement than control-treated grafts. Our results suggest that neuroprotective treatments administered prior to and immediately after neural graft implantation may under certain conditions rescue, at least in part, the same subset of dopaminergic neurons. The study also emphasizes the importance of the immediate time after grafting for transplant survival, with relevance both for primary mesencephalic implants and stem cell grafts.
NMDA Antagonist Peptide Supplementation Enhances Pain Alleviation by Adrenal Medullary Transplants
Authors: Nasirinezhad Farinaz , Sagen Jacqueline
Published: April 2008

Spinal transplantation of adrenal medullary chromaffin cells has been shown to decrease pain responses in several animal models. Improved potency may be possible by engineering cells to produce greater levels of naturally derived analgesics. As an initial screen for potential candidates, adrenal medullary transplants were evaluated in combination with exogenously administered neuropeptides in rodent pain models. Histogranin is a 15-amino acid peptide that exhibits NMDA receptor antagonist activity. The stable derivative [Ser1]histogranin (SHG) can attenuate pain symptoms in some animal models. The formalin model for neurogenic inflammatory pain and the chronic constriction injury (CCI) model for neuropathic pain were used to evaluate the combined effects of chromaffin cell transplantation and intrathecal (IT) SHG injections. Animals were implanted with either adrenal medullary or control striated muscle tissue in the spinal subarachnoid space. For evaluation of formalin responses, animals were pretreated with SHG (0.5, 1.0, 3.0 μg) followed by an intraplantar injection of formalin, and flinching responses were quantified. Pretreatment with SHG had no significant effect on flinching behavior in control animals at lower doses, with incomplete attenuation only at the highest dose. In contrast, 0.5 μg SHG significantly reduced flinching responses in animals with adrenal medullary transplants, and 1.0 μg nearly completely eliminated flinching in these animals in the tonic phase. For evaluation of effects on neuropathic pain, animals received transplants 1 week following CCI, and were tested for thermal and mechanical hyperalgesia and cold allodynia before and following SHG treatment. The addition of low doses of SHG nearly completely eliminated neuropathic pain symptoms in adrenal medullary transplanted animals, while in control transplanted animals only thermal hyperalgesia was attenuated, at the highest dose of SHG. These results suggest that SHG can augment adrenal medullary transplants, and the combination may result in improved effectiveness and range in the treatment of chronic pain syndromes.
Brain Transplantation of Neural Stem Cells Cotransduced with Tyrosine Hydroxylase and GTP Cyclohydrolase 1 in Parkinsonian Rats
Authors: Ryu M. Y. , Lee M. A. , Ahn Y. H. , Kim K. S. , Yoon S. H. , Snyder E. Y. , Cho K. G. , Kim S. U.
Published: April 2008

Neural stem cells (NSCs) of the central nervous system (CNS) recently have attracted a great deal of interest not only because of their importance in basic research on neural development, but also in terms of their therapeutic potential in neurological diseases, such as Parkinson's disease (PD). To examine if genetically modified NSCs are a suitable source for the cell and gene therapy of PD, an immortalized mouse NSC line, C17.2, was transduced with tyrosine hydroxylase (TH) gene and with GTP cyclohydrolase 1 (GTPCH1) gene, which are important enzymes in dopamine biosynthesis. The expression of TH in transduced C17.2-THGC cells was confirmed by RT-PCR, Western blot analysis, and immunocytochemistry, and expression of GTPCH1 by RT-PCR. The level of L-DOPA released by C17.2-THGC cells, as determined by HPLC assay, was 3793 pmol/106 cells, which is 760-fold higher than that produced by C17.2-TH cells, indicating that GTPCH1 expression is important for L-DOPA production by transduced C17.2 cells. Following the implantation of C17.2-THGcC NSCs into the striata of parkinsonian rats, a marked improvement in amphetamine-induced turning behavior was observed in parkinsonian rats grafted with C17.2-THGC cells but not in the control rats grafted with C17.2 cells. These results indicate that genetically modified NSCs grafted into the brain of the parkinsonian rats are capable of survival, migration, and neuronal differentiation. Collectively, these results suggest that NSCs have great potential as a source of cells for cell therapy and an effective vehicle for therapeutic gene transfer in Parkinson's disease.
Human Marrow Stromal Cells Enhance Connexin43 Gap Junction Intercellular Communication in Cultured Astrocytes
Authors: Gao Qi , Katakowski Mark , Chen Xiaoguang , Li Yi , Chopp Michael
Published: April 2008

Human marrow stromal cells (hMSCs) provide functional benefit in rats subjected to stroke. Astrocytes are coupled into a cellular network via gap junction channels, predominantly composed of connexin-43 (Cx43) proteins. Astrocytes are believed to play a vital role in neuroprotection by providing energy substrates to neurons and by regulating the concentrations of K+ and neurotransmitters via gap junctions. We therefore investigated the effect of factors secreted by hMSCs on gap junction intercellular communication (GJIC), expression of Cx43, and phosphorylation of Cx43 in an astrocyte cell culture system. Exposing rat cortical astrocytes to various concentrations of hMSC conditioned medium, we demonstrate that hMSCs produce soluble factors that significantly increase astrocytic GJIC, measured by the scrape-loading dye transfer method. Immunohistochemistry and Western blot showed increased Cx43 expression concomitant with altered GJIC. As the PI3K/Akt signaling pathway has been demonstrated to alter gap junction expression and GJIC, we selectively blocked phosphoinositide 3-kinase (PI3K). Addition of the PI3K inhibitor LY294002 decreased GJIC and Cx43 expression in astrocytes. These inhibitory effects of LY294002 were countered by the addition of hMSC conditioned media. Furthermore, coculturing hMSCs with rat astrocytes increased astrocyte GJIC in a manner dependent upon the hMSC/astrocyte ratio. These findings demonstrate that hMSCs secrete soluble factors that increase GJIC of astrocytes through upregulation of Cx43, and indicate a mechanistic role for PI3K.
Dopaminergic Reinnervation of the Globus Pallidus by Fetal Nigral Grafts in the Rodent Model of Parkinson's Disease
Authors: Bartlett L. E. , Mendez I.
Published: April 2008

The current neural transplantation strategy for Parkinson's disease (PD) involves the dopaminergic reinnervation of the striatum (STR). Although up to 85% reinnervation of the STR has been attained by neural transplantation, functional recovery in animal models and transplanted patients is incomplete. This limitation may be due to an incomplete restoration of the dopaminergic input to other basal ganglia structures such as the external segment of the globus pallidus (GPe, homologue of the rodent GP), which normally receives dopaminergic input from the substantia nigra (SN). As part of our investigation into a multiple grafting strategy for PD, we have explored the effects of dopaminergic grafts in the GP of rodents with unilateral 6-hydroxydopamine (6-OHDA) lesions. In this experiment, lesioned rats received either 300,000 fetal ventral mesencephalic (FVM) cells or a sham injection into the GP. Functional assessment consisted of rotational behavior at 3 and 6 weeks posttransplantation. A fluorogold tracer study was conducted to rule out any behavioral improvement due to striatal outgrowth of the GP graft. Sections were stained for glial fibrillary acidic protein (GFAP) to assess the degree of trauma in the GP by the graft in comparison to the sham injection. Immunohistochemistry for tyrosine hydroxylase (TH) was performed after transplantation to assess graft survival. Animals with GP grafts demonstrated a significant improvement in rotational behavior at 3 and 6 weeks posttransplantation (p < 0.05) while sham control animals did not improve. All animals receiving FVM cells showed TH-immunoreactive grafts in the GP posttransplantation. TH-positive neurons in the GP showed no double labeling with an intrastriatal injection of fluorogold, indicating that behavioral improvement was not due to striatal innervation by the GP graft. These observations suggest that functional recovery was the result of dopaminergic reinnervation of the GP and that this nucleus may be a potential target for neural transplantation in clinical PD.