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The Regenerative Medicine Journal
VOLUME 17, NUMBER 9, 2008
Cell Transplantation, Vol. 17, pp. 997-1003, 2008
0963-6897/08 $90.00 + 00
Copyright © 2008 Cognizant Comm. Corp.
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Biohybrid Devices and Encapsulation Technologies for Engineering a Bioartificial Pancreas
Alexander Fort,1 Nicholas Fort,1 Camillo Ricordi,1,2,3,4,5 and Cherie L. Stabler1,2,3
1Diabetes Research Institute, University of Miami, Miami,
2Department of Surgery, University of Miami, Miami, FL, USA
3Department of Biomedical Engineering, University of Miami, Miami, FL, USA
4Karolinska Institute, Stockholm, Sweden
5Wake Forest Institute for Regenerative Medicine, Winston-Salem, NC, USA
The use of cell-based treatments in the field of metabolic organs, particularly the pancreas, has seen tremendous growth in recent years. The transplantation of islet of Langerhans cells for the treatment of type 1 diabetes mellitus (T1DM) has allowed for natural glycemic control for patients plagued with hypoglycemia unawareness. The transplantation of islet cells into the portal vein of the liver, however, has presented challenges to the survival of the cells due to inflammation, vascularization, the need for systemic immunosuppression, and physical stress on the graft. New advances in the engineering of appropriate biohybrid devices and encapsulation technologies have led to promising alternatives to traditional methods.
Key words: Diabetes; Bioartificial pancreas; Biohybrid device; Encapsulation
Address correspondence to Cherie Stabler, Ph.D., Assistant Professor, Department of Biomedical Engineering, Director, Tissue Engineering and Nanotechnology Laboratory, Diabetes Research Institute, University of Miami, 1450 NW 10th Avenue, Miami, FL 33136, USA. Tel: 305-243-9768; Fax: 305-243-4404; E-mail: email@example.com
The Choice of Anatomical Site for Islet Transplantation
Dirk J. van der Windt,1,2 Gabriel J. Echeverri,3,4 Jan N. M. Ijzermans,2 and David K. C. Cooper3
1Division of Immunogenetics, Department of Pediatrics, Children's
Hospital of Pittsburgh, Pittsburgh, PA, USA
2Department of Surgery, Erasmus University Medical Center Rotterdam, Rotterdam, Netherlands
3Thomas E. Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh, PA, USA
4Transplantation Unit, Fundacion Valle del Lili, Cali, Colombia
Islet transplantation into the portal vein is the current clinical practice. However, it has now been recognized that this implantation site has several characteristics that can hamper islet engraftment and survival, such as low oxygen tension, an active innate immune system, and the provocation of an inflammatory response (IBMIR). These factors result in the loss of many transplanted islets, mainly during the first hours or days after transplantation, which could in part explain the necessity for the transplantation of islets from multiple pancreas donors to cure type 1 diabetes. This increases the burden on the limited pool of donor organs. Therefore, an alternative anatomical site for islet transplantation that offers maximum engraftment, efficacious use of produced insulin, and maximum patient safety is urgently needed. In this review, the experience with alternative sites for islet implantation in clinical and experimental models is discussed. Subcutaneous transplantation guarantees maximum patient safety and has become clinically applicable. Future improvements could be achieved with innovative designs for devices to induce neovascularization and protect the islets from cellular rejection. However, other sites, such as the omentum, offer drainage of produced insulin into the portal vein for direct utilization in the liver. The use of pigs would not only overcome the shortage of transplantable islets, but genetic modification could result in the expression of human genes, such as complement regulatory or "anticoagulation" genes in the islets to overcome some site-specific disadvantages. Eventually, the liver will most likely be replaced by a site that allows long-term survival of islets from a single donor to reverse type 1 diabetes.
Key words: Immune privilege; Intraportal; Islet transplantation; Omental pouch; Subcutaneous; Xenotransplantation
Address correspondence to David K. C. Cooper, M.D., Ph.D., FRCS, Thomas E. Starzl Transplantation Institute, University of Pittsburgh Medical Center, Biomedical Science Tower, W1543, 200 Lothrop Street, Pittsburgh, PA 15261, USA. Tel: 412-383-6961; Fax: 412-624-1172; E-mail: firstname.lastname@example.org
Comparison of the Portal Vein and Kidney Subcapsule as Sites for Primate Islet Autotransplantation
Amer Rajab,1 Jill Buss,1 Elizabeth Diakoff,2 Gregg A. Hadley,1 Kwame Osei,2 and Ronald M. Ferguson1
1Department of Surgery, Division of Transplantation, The
Ohio State University, Columbus, OH, USA
2Department of Endocrinology, Diabetes and Metabolism, The Ohio State University, Columbus, OH, USA
To date, the portal vein has been the primary site for clinical islet transplantation. Despite success, potential complications such as portal vein thrombosis still exist. The kidney subcapsule has been used successfully in rodent models of islet transplantation. We hypothesized that the kidney subcapsule as a site for islet transplantation in the nonhuman primate model would be as effective as the portal vein. Diabetes was induced in the primate Macaca fascicularis via a total pancreatectomy. Animals were kept under anesthesia during the isolation procedure. Islet isolation was performed using intraductal infusion with LiberaseTM HI and mechanical digestion in the Ricordi chamber, and were purified using a continuous Ficoll gradient. Purified islets were autotransplanted either into the portal vein (n = 6) or the left kidney subcapsule (n = 5) of pancreatectomized animals. Intravenous glucose tolerance tests were performed prior to pancreatectomy and 10 days following transplantation. Three animals underwent pancreatectomy and served as diabetic controls. Of the six animals receiving islets in the portal vein, one developed portal vein thrombosis. All remaining autotransplanted animals in this group remained normoglycemic with glucose-induced insulin secretion that was not different from that prior to pancreatectomy. Of the five animals undergoing transplantation into the kidney subcapsule, only one maintained normoglycemia and elicited insulin secretion in response to glucose stimulation. The other four animals remained hyperglycemic. We conclude that the portal vein is superior to the kidney subcapsule as a site for islet transplantation in nonhuman primates 10 days posttransplantation.
Key words: Islet transplantation; Kidney subcapsule; Nonhuman primate; Portal vein
Address correspondence to Amer Rajab, M.D., Ph.D., Director, Pancreas/Islet Transplantation, The Ohio State University, 355 Means Hall, 1654 Upham Drive, Columbus, OH 43210, USA. Tel: (614) 293-8545; Fax: (614) 293-4541; E-mail: Amer.Rajab@osumc.edu
Islet Graft Response to Transplantation Injury Includes Upregulation of Protective as Well as Apoptotic Genes
Silvia Rodríguez-Mulero1 and Eduard Montanya1,2,3
1Laboratory of Diabetes and Experimental Endocrinology, Clinical
Science Department, IDIBELL-University of Barcelona, Barcelona, Spain
2CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Barcelona, Spain
3Endocrine Unit, IDIBELL-Hospital Universitari de Bellvitge, Barcelona, Spain
Pancreatic islets are particularly vulnerable in the initial days after transplantation when multiple factors converge to damage the islet graft. The aim of this study was to investigate the expression profile of genes involved in damage and protection of b-cells in the initial days after syngeneic islet transplantation. We studied the expression of a set of selected genes involved in apoptosis (Bcl2, BclxL, Bax, Bad, Bid, and CHOP), cytokine defense, (SOCS-1 and SOCS-3), or free radical protection (Hmox1, Cu/Zn-SOD, Mn-SOD, and Hsp70). Because hyperglycemia has deleterious effects on islet transplantation outcome, we studied its effect on the expression of these genes. Five hundred islets were syngeneically transplanted under the kidney capsule of normoglycemic or streptozotocin-induced diabetic Lewis rats. Gene expression was analyzed by quantitative real-time RT-PCR in grafts 1, 3, and 7 days after transplantation, and in freshly isolated islets. The expression of proapoptotic genes Bid and CHOP, as well as protective genes BclxL, Socs1, Socs3, Hmox1, and MnSod, was maximally increased 1 day after transplantation, and in most cases it remained increased 7 days later, indicating the presence of a protective response against cell damage. In contrast, the expression of Bcl2, Bax, Bad, Cu/ZnSod, and Hsp70 genes did not change. Hyperglycemia did not modify the expression of most studied genes. However, MnSod and Ins2 expression was increased and reduced, respectively, on day 7 after transplantation to diabetic recipients, suggesting that hyperglycemia increased oxidative stress and deteriorated b-cell function in transplanted islets.
Key words: Islet transplantation; Apoptosis; Oxidative stress; ER stress; Gene expression; Hyperglycemia
Address correspondence to Eduard Montanya, Endocrine Unit, Hospital Universitari Bellvitge, Feixa Llarga, s/n, 08907 L'Hospitalet de Llobregat, Barcelona, Spain. Tel: +34-934037265; Fax: +34-934035804; E-mail: email@example.com
Myoblast Xenotransplantation as a Tool to Evaluate the Appropriateness of Nanoparticular Versus Cellular Trackers
C. Praud,1 K. Vauchez,1,2 A. Lombes,1 M. Y. Fiszman,1 and J.-T. Vilquin1
1Inserm U582, Institut de Myologie, UPMC Univ Paris 06, Paris,
2Genzyme S.A., Saint Germain en Laye, France
Myoblast transplantation is being considered as a potential strategy to improve muscle function in myopathies; hence, it is important to identify the transplanted cells and to have available efficient reagents to track these cells. We first validated a human to mouse xenotransplantation model warranting the complete and rapid rejection of the cells. We then used this model to assess the appropriateness of a nanoparticle reagent to track the transplanted cells. Human myoblasts were loaded with ferrite nanoparticles and injected into the tibialis muscle of immunocompetent mice. Upon collection and histological analysis of muscle sections at different time points, we observed the total disappearance of the human cells within 6 days while ferrite particles remained detectable and colocalized with mouse infiltrating and neighboring cells at the injection site. These results suggest that the use of exogenous markers such as ferrite nanoparticles may lead to falsepositive results and misinterpretation of cell fate.
Key words: Human myoblasts; Ferrites; Macrophages; COX2; Xenotransplantation
Address correspondence to C. Praud, Ph.D., Inserm U582, Institut de Myologie, Groupe hospitalier Pitié-Salpêtrière, Bâtiment Babinski, 47 Boulevard de l'Hôpital, 75651 Paris Cedex 13, France. Tel: +33 1 42 16 57 12; Fax: +33 1 42 16 57 00; E-mail: Christophe.Praud@tours.inra.fr
Mesenchymal Stem Cells for Ischemic Stroke: Changes in Effects After Ex Vivo Culturing
Wen Yu Li,1 Yun Jung Choi,1 Phil Hyu Lee,1 Kyoon Huh,1 Yoon Mi Kang,2 Hyun Soo Kim,2 Young Hwan Ahn,3 Gwang Lee,4 and Oh Young Bang5
1Department of Neurology, Ajou University School of Medicine,
Suwon, South Korea
2FCB-Pharmicell, Sungnam-si, South Korea
3Department of Neurosurgery, Ajou University School of Medicine, Suwon, South Korea
4Brain Disease Research Center, Ajou University School of Medicine, Suwon, South Korea
5Department of Neurology, Sungkyunkwan University School of Medicine, Seoul, South Korea
Although ex vivo culture expansion is necessary to use autologous mesenchymal stem cells (MSCs) in treating stroke patients, and several researchers have utilized culture-expanded cells in their studies, the effects of culture expansion on neurogenesis and trophic support are unknown. Thus, we evaluated the impact of the passage of MSCs on their effects in a rat stroke model. The IV application of ex vivo-cultured human MSCs, earlier (passage 2) or later passage (passage 6), was performed in a rat stroke model. Behavioral tests, immunohistochemical studies, and quantitative analysis using the CAST-grid system were performed to evaluate the degree of neurogenesis. We also evaluated the levels of trophic factors in both control and MSC-treated rat brain extract. Compared to rats that received later-passage human MSCs, behavioral recovery and neurogenesis as revealed by bromodeoxyuridine staining were more pronounced in rats that received earlier-passage human MSCs (p < 0.01 in both cases). Double staining showed that most of the endogenous neuronal progenitor cells, but few human MSCs, expressed neuronal and glial phenotypes. Tissue levels of trophic factors, including glial cell line-derived neurotrophic factor, nerve growth factor, vascular endothelial growth factor, and hepatocyte growth factor, were higher in earlier-passage MSC-treated brains than in control or later-passage MSC-treated brains (p < 0.01 in all cases). Our results indicate that ischemia-induced neurogenesis was enhanced by the IV administration of human MSCs. The effects were more pronounced with earlier-passage than with later-passage human MSCs, which may be related to the differential capacity in trophic support, depending on their passage.
Key words: Cerebral infarction; Mesenchymal stem cells; Neurogenesis; Stem cell
Address correspondence to Oh Young Bang, M.D., Ph.D., Department of Neurology, the Stroke and Cerebrovascular Center, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Irwon-dong, Gangnam-gu, Seoul, 135-710, South Korea. Tel: 82-2-3410-3599; Fax: 82-2-3410-0052; E-mail: firstname.lastname@example.org
Hypothalamic Proline-Rich Polypeptide Enhances Bone Marrow Colony-Forming Cell Proliferation and Stromal Progenitor Cell Differentiation
A. A. Galoyan,1 L. I. Korochkin,2* E. J. Rybalkina,2,4 G. V. Pavlova,2 I. N. Saburina,2 E. I. Zaraiski,5 N. A. Galoyan,6 T. K. Davtyan,1,7 K. B. Bezirganyan,1 and A. V. Revishchin2,3
1H. Buniatian Institute of Biochemistry, NAS RA, Yerevan,
Republic of Armenia
2Institute of Gene Biology, RAS, Moscow, Russia
3Institute of Ecology and Evolution, RAS, Moscow, Russia
4Institute of Carcinogenesis of N.N. Blokhin Russian Cancer Research Center, Moscow, Russia
5Institute of Morphology of Human, Moscow, Russia
6Polfarma, Moscow, Russia
7"Armenicum" Research Center, Republic of Armenia
The AGAPEPAEPAQPGVY proline-rich peptide (PRP-1) was isolated from neurosecretory granules of the bovine neurohypophysis; it is produced by N. supraopticus and N. paraventricularis. It has been shown that PRP-1 has many potentially beneficial biological effects, including immunoregulatory, hematopoietic, antimicrobial, and antineurodegenerative properties. Here we showed that PRP increased colony-forming cell (CFC) proliferation in rat bone marrow (BM) cells in vivo. In PRP-treated rat BM, the CFU number at day 7 and day 14 was considerably increased in comparison with untreated rat BM and no difference was found at day 21 and day 28. The related peptide [arg8]vasopressin did not reveal CFC proliferation. PRP failed to farther increase CFC proliferation in vitro in BM obtained from PRP-treated or untreated rats. After 3-4 days of human BM stromal cell cultivation in the presence of 2-20 mg/ml PRP the appearance of cells expressing CD15, CD10, CD11a, CD11b, CD3, CD4, and CD16 surface antigens did not differ from the untreated cells. PRP increased the appearance of CD14-positive cells upon 3-4-day incubation with both adult and fetal BM stromal cells. Our results suggest a previously undescribed role for the hypothalamic peptide within neurosecretory hypothalamus-bone marrow humoral axis, because PRP enhances BM colonyforming cell proliferation and stromal cell differentiation.
Key words: Proline-rich polypeptides; Neurohypophysis; Myelopoiesis; Bone marrow; Colony-forming cells; Stromal cells
Address correspondence to Professor Armen A. Galoyan, Buniatian Institute of Biochemistry of NAS RA, 5/1 Sevag Street, Yerevan, 375014, Republic of Armenia. E-mail: email@example.com
*Dedicated to Professor L. I. Korochkin, who died in February 2007.
Introduction of hIGF-1 Gene Into Bone Marrow Stromal Cells and Its Effects on the Cell's Biological Behaviors
Chengjun Hu,1 Yan Wu,1 Yu Wan,2 Qiao Wang,1 and Jian Song1,2
1Department of Anatomy and Embryology, Wuhan University School
of Medicine, Wuhan, P.R. China
2Key Laboratory of Allergy and Immune-Related Diseases, and Center for Medical Research, Wuhan University, Wuhan, P.R. China
Autologous and gene-modified bone marrow stromal cells (MSCs) have shown a bright future in clinical applications. However, does a gene-modified MSC still maintain its stem cell-like properties? To answer this question, human IGF-1 was introduced into rat MSCs using a recombinant retroviral vector and the effects of the gene manipulation on the cells' behaviors were investigated. The MSCs transfected with hIGF-1 could secrete 6.7-fold higher IGF-1 than the native cells. These MSCs had an elevated baseline activity of ERK signaling, an enhanced proliferation, increased accumulative numbers of cell doublings, and a reduced apoptosis; they showed upregulated expressions of OCT-4, CYP51, and SM22a, and a downregulated expression of nestin. This indicates that the overexpressed IGF-1 enhances the MSCs' self-renewal, endodermal and mesodermal differentiation, but weakens their neuronal potential. Although a puromycin selection after hIGF-1 gene transfection could produce a purer transfected MSC population with stronger ability to express functional hIGF-1, it induced premature senescence of the selected cells by activating oncogene Ras, leading to a shortened replicative life span and a weakened multipotency.
Key words: Bone marrow stromal cell; hIGF-1; Proliferation; Self-renewal; Multipotency
Address correspondence to Prof. Jian Song, Ph.D., Department of Anatomy and Embryology, Wuhan University School of Medicine, 135 Donghu Road, Wuhan, Hubei 430071, P.R. China. Tel: +86 27 68759697; Fax: +86 27 687578766; E-mail: firstname.lastname@example.org
Establishment of Immortalized Human Hepatocytes by Introduction of HPV16 E6/E7 and hTERT as Cell Sources for Liver Cell-Based Therapy
Yosuke Tsuruga,1 Tohru Kiyono,2 Michiaki Matsushita,1 Tohru Takahashi,1 Hironori Kasai,1 Shuichiro Matsumoto,1 and Satoru Todo1
1Department of General Surgery, Hokkaido University Graduate
School of Medicine, Sapporo 060-8648, Japan
2Virology Division, National Cancer Center Research Institute, Tokyo 104-0045, Japan
For future cell-based therapies for liver diseases, the shortage of cell sources must be resolved. Immortalized human hepatocytes are expected to be among the new sources. In addition to telomerase activation by the introduction of human telomerase reverse transcriptase (hTERT), inactivation of the p16/RB pathway and/or p53 by E6/E7 of human papillomavirus type 16 (HPV16) has been shown to be useful for efficient immortalization of several human cell types. Here we report the immortalization of human hepatocytes by the introduction of HPV16 E6/E7 and hTERT. Human adult hepatocytes were lentivirally transduced with HPV16 E6/E7 and hTERT. Two human immortalized hepatocyte cell lines were established and were named HHE6E7T-1 and HHE6E7T-2. Those cells proliferated in culture beyond 200 population doublings (PDs). Albumin synthesis and expression of liver-enriched genes were confirmed, but gradually decreased as passages progressed. Karyotype analysis showed that HHE6E7T-1 cells remained near diploid but that HHE6E7T-2 cells showed severe aneuploidy at 150 PDs. Subcutaneous injection of these cells into severe combined immunodeficiency (SCID) mice did not induce tumor development. Intrasplenic transplantation of dedifferentiated HHE6E7T-1 cells over 200 PDs significantly improved the survival of acetaminopheninduced acute liver failure SCID mice. In conclusion, we successfully established immortalized human hepatocytes that retain the characteristics of differentiated hepatocytes. We also showed the reduction of hepatocyte-specific functions in long-term culture. However, the results of intrasplenic transplantation to SCID mice with acetaminophen-induced acute liver failure showed the possibility of HHE6E7T-1 serving as a cell source for hepatocyte transplantation.
Key words: Hepatocyte transplantation; Acetaminophen-induced acute liver failure; Dedifferentiation; hromosomal instability; Tumorigenicity
Address correspondence to Yosuke Tsuruga, M.D., Department of General Surgery, Hokkaido University Graduate School of Medicine, N-14, -5, Kita-ku, Sapporo 060-8648, Japan. Tel: (81)11-706-5927; Fax: (81)11-717-7515; E-mail: email@example.com
Variation in the Incidence of Teratomas After the Transplantation f Nonhuman Primate ES Cells Into Immunodeficient Mice
Yukiko Kishi,1 Yujiro Tanaka,1 Hiroaki Shibata,1,4 Shinichiro Nakamura,5 Koichi Takeuchi,3 Shigeo Masuda,1 Tamako Ikeda,1 Shin-ichi Muramatsu,2 and Yutaka Hanazono1
1Division of Regenerative Medicine, Center for Molecular
Medicine, Jichi Medical University, Tochigi 329-0498, Japan
2Division of Neurology, Department of Internal Medicine, Jichi Medical University, Tochigi 329-0498, Japan
3Department of Anatomy, Jichi Medical University, Tochigi 329-0498, Japan
4Tsukuba Primate Research Center, National Institute of Biomedical Innovation, Ibaraki 305-0843, Japan
5The Corporation for Production and Research of Laboratory Primates, Ibaraki 300-2658, Japan
Embryonic stem (ES) cells have the ability to generate teratomas when transplanted into immunodeficient mice, but conditions affecting the generation remain to be elucidated. Nonhuman primate cynomolgus ES cells were transplanted into immunodeficient mice under different conditions; the number of transplanted cells, physical state (clumps or single dissociated cells), transplant site, differentiation state, and immunological state of recipient mice were all varied. The tumorigenicity was then evaluated. When cynomolgus ES cells were transplanted as clumps into the lower limb muscle in either nonobese diabetic/severe combined immunodeficiency (NOD/SCID) or NOD/SCID/gcnull (NOG) mice, teratomas developed in all the animals transplanted with 1 x 105 or more cells, but were not observed in any mouse transplanted with 1 x 103 cells. However, when the cells were transplanted as dissociated cells, the number of cells necessary for teratomas to form in all mice increased to 5 x 105. When the clump cells were injected subcutaneously (instead of intramuscularly), the number also increased to 5 x 105. When cynomolgus ES cell-derived progenitor cells (1 x 106), which included residual pluripotent cells, were transplanted into the lower limb muscle of NOG or NOD/SCID mice, the incidence of teratomas differed between the strains; teratomas developed in five of five NOG mice but in only two of five NOD/SCID mice. The incidence of teratomas varied substantially depending on the transplanted cells and recipient mice. Thus, considerable care must be taken as to tumorigenicity.
Key words: Nonhuman primate embryonic stem cells; NOD/SCID mouse; NOG mouse; Teratoma
Address correspondence to Yutaka Hanazono, M.D., Ph.D., Professor, Division of Regenerative Medicine, Center for Molecular Medicine, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke, Tochigi 329-0498, Japan. Tel: +81-285-58-7451; Fax: +81-285-44-5205; E-mail: firstname.lastname@example.org