ognizant Communication Corporation


VOLUME 12, NUMBER 8, 2003

Cell Transplantation, Vol. 12, pp. 827-837, 2003
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Neural Precursor Cells as Carriers for a Gene Therapeutical Approach in Tumor Therapy

S. Arnhold,1 M. Hilgers,1 D. Lenartz,3 I. Semkova,2 S. Kochanek,2 J. Voges,3 C. Andressen,1 and K. Addicks1

1Department of Anatomy I, 2Center for Molecular Medicine (ZMMK), 3Clinic for Stereotaxy and Functional Neurosurgery, University of Cologne, Joseph-Stelzmann Str. 9, 50931 Köln, Germany

Conventional therapeutical approaches such as surgery, radiotherapy, or chemotherapy have been shown to be rather unsuccessful in the treatment of infiltrative growing tumors such as the malignant glioblastoma multiforme. Thus, new therapeutical strategies have to be developed that are suitable for inducing cell death also in migrating tumor cells. These new therapeutical stategies include cell and/or gene therapeutical approaches. We demonstrate that glial-restricted progenitor cells as well as embryonic stem cell-derived neural stem cells belong to cell populations applicable to such therapeutical concepts. Both cell types can be efficiently transduced using a third-generation high-capacity "gutless" adenoviral vector, and show a tropism for the F98 glioma cells by migrating towards a spheroid of F98 glioma cells with a tendency to form a barrier around the tumor spheroid in an in vitro tumor confrontation model. Moreover, in a migration assay, secretion products of glial-restricted precursor cells have shown a potency to inhibit the migratory activity of glioma cells in vitro. In vivo, F98 glioma cell-derived tumor formation in the right striatum resulted in migration of glial as well as neural precursor cells towards the tumor area when cotransplanted in the corpus callosum of the contralateral hemisphere. After arrival, both cell types surround the tumor mass and even invade the experimentally induced tumor. These data indicate that glial-restricted as well as embryonic stem cell-derived neural precursor cells are good candidates as carriers for an ex vivo gene therapeutical approach in tumor therapy.

Key words: Glial-restricted precursor cells; Embryonic stem cells; High-capacity adenoviral vector; F98 glioma cells

Address correspondence to Dr. Stefan Arnhold, Department of Anatomy I, Joseph-Stelzmann Str. 9, 50931 Köln, Germany. Tel: ++49 221 4786095; Fax: ++49 221 4786711; E-mail: stefan.arnhold@uni-koeln.de

Cell Transplantation, Vol. 12, pp. 839-848, 2003
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Expression of Neuronal Markers in Differentiated Marrow Stromal Cells and CD133+ Stem-Like Cells

Claudio S. Padovan, Klaus Jahn, Tobias Birnbaum, Petra Reich, Petra Sostak, Michael Strupp, and Andreas Straube

Department of Neurology, Ludwig-Maximilian-University, Munich, Germany

Bone marrow stromal cells, which normally give rise to bone, cartilage, adipose tissue, and hematopoiesis-supporting cells, have been shown to differentiate in vitro and in vivo into neural-like cells. In this study, we examined the expression of neuronal and glial markers in human marrow stromal cells under culture conditions appropriate for neural stem cells, and compared the unsorted cell population to bone marrow CD133+ stem-like cells using immunofluorescence, Western blot, and functional patch-clamp analysis. Overall, the expression of the early neuronal marker b3-tubulin was most pronounced in the presence of DMEM/F12 and neurotrophin 3 (NT3) or brain-derived neurotrophic factor (BDNF), when marrow stromal cells were cultured onto fibronectin. Electrophysiological examination, however, could not show fast sodium currents or functional neurotransmitter receptors in differentiated marrow stromal cells. CD133+ mesenchymal stem-like cells, but not CD34+/CD133- cells, generally showed a higher expression of neuronal markers than did unsorted marrow stromal cells, and differentiated CD133+ cells more resembled neuron-like cells.

Key words: Marrow stromal cells; Mesenchymal cells; CD133; Neural transdifferentiation; Adult stem cells; Stem cell plasticity

Address correspondence to Claudio S. Padovan, M.D., Department of Neurology, Ludwig-Maximilian-University, Marchioninistr. 15, 81377 Munich, Germany. Tel: 0049-89-7095-1; Fax: 0049-89-7095-3677; E-mail: claudio.padovan@med.uni-muenchen.de

Cell Transplantation, Vol. 12, pp. 849-857, 2003
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Insulin-Induced Normoglycemia Reduces Islet Number Needed to Achieve Normoglycemia After Allogeneic Islet Transplantation in Diabetic Mice

Juan C. Ferrer-Garcia,1 Juan F. Merino-Torres,1 Gema Pérez Bermejo,1 Carmen Herrera-Vela,2 Jose L. Ponce-Marco,2 and Francisco Piñon-Selles1

1Laboratory of Experimental Diabetes, Endocrinology Department and 2Surgery Department, University Hospital "La Fe," Av. Campanar 21, Valencia 46009, Spain

The Edmonton protocol established that insulin independence could be reached with the transplantation of an appropriate number of islet cells. However, to effect a cure, islets from two or three pancreases are needed. The aim of this study was to examine whether normoglycemia, with insulin treatment before and after transplantation, reduces the islet number needed to achieve normoglycemia in allogeneic islet transplantation. Swiss mice were used as donors and recipients. Diabetes was induced by IP administration of streptozotocin (180 mg/kg BW). Diabetic mice were transplanted with 300 (n = 16), 400 (n = 16), or 500 (n = 16) islets under the left kidney capsule. For every group, half the animals were kept normoglycemic with insulin treatment from day 4 before transplantation to day 10 after transplantation. At the end of the study, all normoglycemic mice were given an IP glucose tolerance test (IPGTT). For statistical analysis, paired or unpaired Student's t-test or ANOVA was used. Only insulin-treated mice achieved normoglycemia by the end of the study (37.5% of animals transplanted with 400 islets and 50% transplanted with 300 or 500 islets). At the end of the study, normoglycemic mice transplanted with 300 allogeneic islets showed better glycosylated hemoglobin (HbA1C) than did normoglycemic mice transplanted with 500 islets (300 islets: 2.7 ± 0.2%; 500 islets: 3.6 ± 0.2%; p < 0.05). After the IPGTT, insulin-treated mice transplanted with 500 islets showed abnormal glucose tolerance; however, insulin-treated mice transplanted with 300 or 400 islets showed normal glucose tolerance. Insulin treatment reduced the islet number needed to achieve normoglycemia in allogeneic islet transplantation. The HbA1C and IPGTT results suggest that transplanting smaller numbers of allogeneic islets improves b-cell function; some studies suggest that this may be due to lower immunogenicity, hypoxia, and inflammation.

Key words: Islet transplantation; Insulin; Glucose tolerance; Islet number

Address correspondence to Dr. Juan F. Merino-Torres, Endocrinology Department, University Hospital "La Fe," Av. Campanar 21, Valencia 46009, Spain. Tel: 34 96 197 31 65; Fax: 34 96 197 32 96; E-mail: merino_jfr@gva.es

Cell Transplantation, Vol. 12, pp. 859-865, 2003
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Hyperthermic Preconditioning Protects Pig Islet Grafts From Early Inflammation But Enhances Rejection in Immunocompetent Mice

Daniel Brandhorst, Heide Brandhorst, Vidya Kumarasamy, Adel Maataoui, Alexandra Alt, Mathias D. Brendel, and Reinhard G. Bretzel

Third Medical Department, Justus-Liebig-University, Giessen, Germany

The induction of heat shock proteins (HSP) protects isolated islet cells against the cytotoxicity of inflammatory mediators in vitro. Very little information is available about the effect of HSP overexpression on function of preconditioned islet grafts. The present study investigated the function of heat-exposed pig islets after transplantation into immunocompetent mice in comparison with in vitro resistance against inflammatory mediators. Pig islets were preconditioned at 43°C or sham treated prior to subcapsular transplantation into diabetic C57/Bl6j mice. Nondiabetic mice simultaneously receiving preconditioned and control islets were subjected to bilateral nephrectomy for determination of pig insulin. Resistance against H2O2, NO, human Il-1b, IFN-g, or TNF-a was assessed by trypan blue exclusion and insulin determination. Heat-induced protein expression was confirmed by Western blot analysis. Graft preconditioning increased resistance against H2O2, NO, or cytokines (p < 0.05) but decreased survival in nondiabetic mice (p < 0.05) and function in diabetic mice (p < 0.01). Upregulation of caspase-3 activity as well as Bax, Fas, FasL, and DFF expression (p < 0.05) indicated simultaneous induction of apoptosis. The coexpression of HSP and proapoptotic proteins reveals the dual character of the stress response simultaneously starting mechanisms for protection and apoptosis. In vitro assays seem to reflect only insufficiently the situation of islets after transplantation.

Key words: Heat shock proteins; Porcine islets; Apoptosis; Islet transplantation

Address correspondence to Daniel Brandhorst, Ph.D., Third Medical Department, Justus-Liebig-University, Rodthohl 6, 35385 Giessen, Germany. Tel: +49-641-99-42846; Fax: +49-641-99-42849; E-mail: daniel.brandhorst@innere.med.uni-giessen.de

Cell Transplantation, Vol. 12, pp. 867-875, 2003
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Response of Encapsulated Rat Pancreatic Islets to Hypoxia

M. de Groot, T. A. Schuurs, P. P. M. Keizer, S. Fekken, H. G. D. Leuvenink, and R. van Schilfgaarde

Surgical Research Laboratory, Department of Surgery, Groningen University Hospital, Groningen, The Netherlands

Hypoxia contributes to encapsulated pancreatic islet graft failure. To gain insight into the mechanisms that lead to hypoxia-induced graft failure, encapsulated islet function, vitality, and cell replication were assessed after 2 and 5 days of hypoxic (1% O2) and normoxic (20% O2) culture. The mRNA expression levels of Bcl-2, Bax, inducible nitric oxide synthase (iNOS), and monocyte chemoattractant protein 1 (MCP-1) were assessed, as well as the amount of nitrite and MCP-1 in the culture medium. Hypoxia was associated with loss of encapsulated islet function and vitality, but not with an increase in islet cell replication. Loss of vitality was due to necrosis, and only modestly due to apoptosis. Hypoxia was not associated with changes in the Bcl-2/Bax mRNA ratio, but it did increase the expression of iNOS and MCP-1 mRNA. The increased mRNA levels were, however, not associated with elevated concentrations of nitrite nor with elevated levels of MCP-1 protein. The increased iNOS mRNA levels imply a role for NO in the completion of cell death by hypoxia. The increased MCP-1 mRNA levels suggest that encapsulated islets in vivo contribute to their own graft failure by attracting cytokine-producing macrophages. The discrepancy between iNOS mRNA and nitrite is explained by the longer half-life of NO during hypoxia. MCP-1 protein levels are underestimated as a consequence of the lower number of vital cells in combination with a higher proteolytic activity due to necrosis. Thus, strategies to eliminate hypoxia may not only improve islet function and vitality, but may also reduce the attraction of macrophages by encapsulated islets.

Key words: Hypoxia; Islets of Langerhans; Encapsulation; Cell death; Replication; Monocyte chemoattractant protein 1 (MCP-1)

Address correspondence to M. de Groot, Department of Surgery, Surgical Research Laboratory, Groningen University Hospital, Hanzeplein 1, 9713 GZ Groningen, The Netherlands. Tel: +31 (0) 50 3619028; Fax: +31 (0) 50 3632796; E-mail: M.de.Groot@med.rug.nl

Cell Transplantation, Vol. 12, pp. 877-881, 2003
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Improvement of Pancreatic Islet Isolation Outcomes Using Glutamine Perfusion During Isolation Procedure

J. G. Avila,1,2 T. Tsujimura,1,2 J. Oberholzer,2 T. Churchill,1 P. Salehi,1 A. M. James Shapiro,1,2 and J. R. T. Lakey1,2

1Surgical-Medical Research Institute, 2Department of Surgery, University of Alberta, 1074 Dentistry/Pharmacy Centre, Edmonton, Canada T6G 2N8

During procurement, isolation, and transplantation, islets are exposed to high levels of oxidative stress triggering a variety of signaling pathways that can ultimately lead to cell death. Glutamine is an important cellular fuel and an essential precursor for the antioxidant glutathione. The aim of this study was to examine the role of intraductal glutamine administration in facilitating recovery of isolated rat islets from pancreases subjected to a clinically relevant period of warm ischemia. Islets were isolated in Sprague-Dawley (SD) rats (n = 18 per group). Pancreata in groups 1 and 2 were procured immediately while groups 3 and 4 were subjected to 30-min warm ischemia. Groups 2 and 4 were treated intraductally with 5 mM glutamine prior to pancreatectomy. Exposure to 30-min warm ischemia significantly reduced islet yield [groups 1 & 2 (nonischemia): 503 ± 29 islets/rat vs. groups 3 & 4 (ischemia): 247 ± 26 islets/rat; p < 0.05]. Intraductal glutamine treatment significantly improved islet yield when pancreata were subjected to 30-min warm ischemia [144 ± 16 islets/rat without glutamine (group 3) vs. 343 ± 36 islets/rat with glutamine (group 4), p < 0.05]. Glutamine also significantly improved islet viability (values were 50 ± 4% in group 4 vs. 27 ± 3% in group 3, p < 0.05). Similarly, glutathione (reduced) levels were significantly elevated in both glutamine-treated groups; however, this increase was greatest in tissues exposed to ischemia (2.76 ± 0.04 nmol/mg protein in group 4 vs. 1.66 ± 0.04 nmol/mg protein in group 3, p < 0.05). Intraductal glutamine administration considerably improves the islet yield, viability, and augments endogenous glutathione levels in pancreata procured after a clinically relevant period of ischemia. Intraductal administration of glutamine at the time of digestive enzyme delivery into the harvested pancreas may represent a simple yet effective tool to improve islet yields in clinical isolations.

Key words: Islet isolation; Oxidative stress; Antioxidants; Glutamine; Glutathione

Address correspondence to Dr. Jonathan R. T. Lakey, Assistant Professor of Surgery, CDA and AHFMR Scholar, Director, Clinical Islet Isolation Laboratory, University of Alberta, Surgical-Medical Research Institute, 1074 Dentistry/Pharmacy Centre, Edmonton, Canada T6G 2N8. Tel: (780) 492-3077; E-mail:jlakey@ualberta.ca

Cell Transplantation, Vol. 12, pp. 883-890, 2003
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A Simple and Cost-Effective Method for the Isolation of Islets From Nonhuman Primates

John J. O'Neil,1 Vaja Tchipashvili,1 Richard J. Parent,1 Obinna Ugochukwu,1 Gaurav Chandra,1 Maria Koulmanda,2 Dicken Ko,2 and Tatsuo Kawai2

1Joslin Diabetes Center and 2Massachusetts General Hospital, Harvard Medical School, Boston, MA

Recent advances in islet cell transplantation have led to insulin independence in a majority of islet transplant recipients. However, there exists a need to overcome the shortage of donor tissue and the necessity for life-long immunosuppression. Preclinical studies in large animal models are necessary to evaluate the safety and efficacy of alternative approaches for clinical islet transplantation. The nonhuman primate serves as an appropriate animal model for such investigations; however, a major impediment in performing such preclinical research has been the difficulty in isolating islets of sufficient quantity and quality. The current study describes a simple and cost-effective method to isolate nonhuman primate islets to support preclinical islet transplantation research. The results of islet isolations from 54 cynomolgus monkeys and 4 baboons are reported. The pancreas was infused with Liberase HI and subjected to static digestion. The digested tissue was shaken, filtered through a mesh screen, applied to a discontinuous gradient, and centrifuged in much the same manner as with conventional rodent islet isolations. Islets were collected from the two interfaces, washed, and transplanted. Following purification, cynomolgus monkey islet isolation yields were 50,100 ± 3120 IE total or 8760 ± 420 IE/g pancreas with the percent purity and viability of 90.8 ± 0.9 and 90.7 ± 0.7, respectively. Total insulin content of the isolated islets was 405 ± 53 mg insulin with DNA content being and 976 ± 117 mg DNA, corresponding to a ratio of 0.57 mg insulin/mg DNA. STZ-induced diabetes was reversed in both mouse and nonhuman primate recipients, which possessed significant levels of c-peptide following transplantation and well-granulated islet grafts. The technique yields sufficient numbers of pure and viable islets to support preclinical research to develop improved strategies to prevent the immune destruction of the transplanted islet graft.

Key words: Nonhuman primate; Islet isolation; Assessment; Transplantation

Address correspondence to John J. O'Neil, Johnson & Johnson, 199 Grandview Road, Skillman, NJ 08558. Tel: (908) 874-1356; Fax: (908) 874-1170; E-mail: jjoneil@cpcus.jnj.com.

Cell Transplantation, Vol. 12, pp. 891-896, 2003
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Hepatic Cells Via Cava Vein Can Influence Allogenic Islet Rat Transplantation

Antonino Jara-Albarrán,1 M. Luisa Soto-Montenegro,1 Ana Zugasti,1 Eduardo Rollán,2 Ysmael Alvarez,3 and Granada Alvarez3

Unidad de Medicina y Cirugía Experimental, 1Servicio de Endocrinología, 2Facultad de Veterinaria UCM, 3Laboratorio de Investigación Biomédica, Cantoblanco, Hospital General Universitario Gregorio Marañón, Madrid, Spain

We have reported, previously, some effect of allogenic hepatic cells for islet tolerance when they are injected mixed (hepatic cells and islets) in different proportions via portal vein, in diabetic Wistar rats. Now we have studied the role of allogenic hepatic cells injected sequentially 15 min before islets, comparing via the portal vein (A and B groups) and via the cava vein (C and D groups) with a control group of islets alone. The allogenic islets were always injected via portal vein, in similar conditions, while the ratio of hepatic cells/islets was 100:1 (A, C groups) or 200:1 (B, D groups). Islets and hepatic cells were obtained from several different rats. The transplanted rats were observed during 30 days and results compared among the different rat groups: porta-porta (P/P), cava-porta (C/P), and control group. Statistically, a significant interaction between type of transplant and proportion of hepatic cells was observed. Also, C plus D groups showed statistical difference with the control group (p < 0.017) and also all the groups together (p < 0.047). These results suggest that hepatic cells can induce, in some cases, islet graft prolongation in Wistar rats. Better results were obtained when hepatic cells were injected via the cava vein than via the portal vein. Because we used a liver cell suspension integrated for several kinds of cells, the study does not clarify if this effect can be related to some specific hepatic cell subpopulation. To confirm the results and to determine if the hypothetical mechanism can be attributed to a block of the immune system or to some factor secreted by hepatic cells, more studies must be performed.

Key words: Islet allografts; Cotransplantation; Hepatic cells; Rats

Address correspondence to Prof. Antonino Jara-Albarrán, Servicio de Endocrinología, Hospital General Universitario Gregorio Marañón, C/Doctor Esquerdo 46, 28007 Madrid, Spain. Tel: 0034-91-5868112; Fax: 0034-91-5868113; E-mail: ajara@radonc.hggm.es

Cell Transplantation, Vol. 12, pp. 897-906, 2003
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In Vitro Expansion of Human Hepatocytes Is Restricted by Telomere-Dependent Replicative Aging

Henning Wege,1 Michael S. Chui,1 Hai T. Le,1 Stephen C. Strom,2 and Mark A. Zern1

1Transplant Research Institute, University of California, Davis Medical Center, Sacramento, CA 95817
2Department of Pathology, University of Pittsburgh Medical Center, Pittsburgh, PA 15261

Currently, different techniques to expand human hepatocytes in vitro are being investigated to generate enough cells for liver-directed cell therapies. However, based on observations in fibroblasts and other cell types, telomere attrition limits the proliferative capacity of normal somatic cells. Therefore, we explored whether telomere-dependent replicative aging restricts the in vitro proliferation of human hepatocytes. Subpopulations of cells isolated from a neonatal liver and characterized as hepatocyte derived by RT-PCR and flow cytometry started to proliferate 5-7 days after plating and were termed proliferating human hepatocytes (PHH). Following retroviral-mediated transduction of the catalytic telomerase subunit, telomerase reverse transcriptase (hTERT), telomerase activity increased from almost undetectable levels to levels as high as in HepG2 and other telomerase-positive cell lines. As expected, untransduced PHH progressively lost telomeric repeats and arrested after 30-35 cell divisions with telomeres of less than 5 kilo bases. In comparison, telomerase-reconstituted PHH maintained elongated telomeres and continued to proliferate as shown by colorimetric assays and cell counts. In this study, telomere stabilization extended the proliferative capacity of in vitro proliferating human neonatal hepatocytes. Therefore, telomere attrition needs to be addressed when developing techniques to expand human hepatocytes.

Key words: Hepatocyte; Immortality; Proliferation; Senescence; Telomerase

Address correspondence to Mark A. Zern, M.D., University of California, Davis Medical Center, Research Building One, Suite 1001, 4635 Second Avenue, Sacramento, CA 95817. Tel: (916) 734-8063; Fax: (916) 734-8097; E-mail: mazern@ucdavis.edu

Cell Transplantation, Vol. 12, pp. 907-919, 2003
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p27Kip1 Inactivation Provides a Proliferative Advantage to Transplanted Hepatocytes in DPPIV/Rag2 Double Knockout Mice After Repeated Host Liver Injury

Ray-Hwang Yuan,1,2 Atsushi Ogawa,1 Emi Ogawa,1 David Neufeld,1 Liang Zhu,1 and David A. Shafritz1

1Marion Bessin Liver Research Center, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461
2Department of Surgery, National Taiwan University Hospital, Taipei, Taiwan

Studies were conducted to develop a new DPPIV-/-/Rag2-/- mouse model for hepatocyte transplantation by allogeneic and xenogeneic cells and to compare the proliferative capacity of p27 null hepatocytes versus normal hepatocytes in this system. Dipeptidyl peptidase IV (DPPIV) gene knockout mice, in which wild-type (wt) DPPIV+ donor hepatocytes can be readily identified by enzyme histochemistry, were bred with Rag2 null mice to prepare immunotolerant DPPIV-/-/Rag2-/- double knockout mice. DPPIV-/-/Rag-/- mice were transplanted with wt hepatocytes or p27 null mouse hepatocytes, which show enhanced cell cycle activity due to disruption of the Kip1 cyclin kinase inhibitor gene, and liver repopulation was assessed under nonproliferative versus proliferative experimental conditions. After their initial engraftment, transplanted wt hepatocytes did not proliferate in untreated livers or increase significantly in response to an acute liver regenerative stimulus. p27 null hepatocytes engrafted with the same efficiency as wt hepatocytes, but showed a noticeable, although not statistically significant, increase in proliferation in response to partial hepatectomy or acute CCl4 administration. Repeated treatments with CCl4 substantially increased proliferation and liver repopulation by p27 null hepatocytes but not by wt hepatocytes. These results suggest that p27 gene inactivation does not overcome proliferative restrictions imposed on hepatocytes by the normal liver, but that after repeated episodes of toxic liver injury, the augmented proliferative capacity of p27 null hepatocytes leads to significant liver repopulation compared with wt hepatocytes. These properties of p27-deficient hepatocytes could prove useful as a target for liver repopulation in patients with intermittent or a low level of chronic liver injury.

Key words: Hepatocyte transplantation; Proliferation; p27 null hepatocytes; Liver injury; Kip 1

Address correspondence to David A. Shafritz, M.D., Marion Bessin Liver Research Center, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. Tel: (718) 430-2098; Fax: (718) 430-8975; E-mail: shafritz@aecom.yu.edu