|ognizant Communication Corporation|
The Regenerative Medicine Journal
VOLUME 17, NUMBER 5, 2008
Cell Transplantation, Vol. 16, pp. 489-494, 2008
0963-6897/08 $90.00 + 00
Copyright © 2008 Cognizant Comm. Corp.
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A Preliminary Gene Expression Profile of Acute Graft-Versus-Host Disease
Matthew P. Buzzeo,1 Jie Yang,2 George Casella,2 and Vijay Reddy1
1Department of Medicine, Division of Hematology/Oncology,
University of Florida, Gainesville, FL, USA
2Department of Statistics, University of Florida, Gainesville, FL, USA
Allogeneic hematopoietic stem cell transplantation (HSCT) is the treatment of choice for high-risk hematological malignancies, yet a major complication associated with this therapy is acute graft-versus-host disease (GVHD). Despite a well-defined pathophysiological mechanism, there are no definitive markers for predicting acute GVHD development or progression to advanced stages. In the current study, we enrolled four acute GVHD and four acute GVHD-free recipients of allogeneic HSCT and collected peripheral blood just prior to onset of clinical acute GVHD for analysis on Affymetrix GeneChip® Human Genome U133 Plus 2.0 microarrays. We noted significant differences in expression of 1,658 genes between control and acute GVHD patients, based on an analysis of covariance (ANCOVA) by type of transplant, a pooled error estimate, and a false discovery rate (FDR) of 10%. In conclusion, we offer the first report of a preliminary molecular signature of acute GVHD in allogeneic HSCT patients.
Key words: Graft-versus-host disease (GVHD); Hematopoietic stem cell transplantation (HSCT); Microarray; Gene expression
Address correspondence to Vijay Reddy, M.D. Ph.D., at his current address: Florida Center for Cellular Therapy, Florida Hospital Cancer Institute, 2501 N. Orange Ave., Suite 581, Orlando, FL 32804, USA. Tel: 407-303-2070; E-mail: firstname.lastname@example.org
Prenatal Tolerance Induction: Relationship Between Cell Dose, Marrow T-Cells, Chimerism, and Tolerance
Jeng-Chang Chen,1 Ming-Ling Chang,2 Shiu-Feng Huang,3 Pei-Yeh Chang,1 Marcus O. Muench,4 Ren-Huei Fu,5 Liang-Shiou Ou,5 and Ming-Ling Kuo6
1Department of Surgery, Chang Gung Children's Hospital, Chang
Gung University College of Medicine, Taoyuan, Taiwan
2Department of Hepatogastroenterology, Chang Gung Memorial Hospital, Taoyuan, Taiwan
3Division of Molecular and Genomic Medicine, National Health Research Institutes, Miaoli, Taiwan
4Blood Systems Research Institute and Department of Laboratory Medicine, University of California, San Francisco, CA, USA
5Department of Pediatrics, Chang Gung Children's Hospital, Taoyuan, Taiwan
6Department of Microbiology and Immunology, Graduate Institute of Basic Medical Sciences, Chang Gung University College of Medicine, Taoyuan, Taiwan
It was reported that the dose of self-antigens can determine the consequence of deletional tolerance and donor T cells are critical for tolerance induction in mixed chimeras. This study aimed at assessing the effect of cell doses and marrow T cells on engraftment and tolerance induction after prenatal bone marrow transplantation. Intraperitoneal cell transplantation was performed in FVB/N (H-2Kq) mice at gestational day 14 with escalating doses of adult C57BL/6 (H-2Kb) marrows. Peripheral chimerism was examined postnatally by flow cytometry and tolerance was tested by skin transplantation. Transplantation of light-density marrow cells showed a dose response. High-level chimerism emerged with a threshold dose of 5.0 × 106 and host leukocytes could be nearly replaced at a dose of 7.5-10.0 × 106. High-dose transplants conferred a steady long-lasting donor-specific tolerance but were accompanied by >50% incidence of graft-versus-host disease. Depletion of marrow T cells lessened graft-versus-host disease to the detriment of engraftment. With low-level chimerism, tolerance was a graded phenomenon dependent upon the level of chimerism. Durable chimerism within 6 months required a threshold of >2% chimerism at 1 month of age and predicted a 50% chance of long-term tolerance, whereas transient chimerism (<2%) only caused hyporesponsiveness to the donor. Tolerance induction did not succeed without peripheral chimerism even if a large amount of injected donor cells persisted in the peritoneum. Neither did an increase in cell doses or donor T-cell contents benefit skin graft survivals unless it had substantially improved peripheral chimerism. Thus, peripheral chimerism level can be a simple and straightforward test to predict the degree of prenatal immune tolerance.
Key words: Cell dose; Chimerism; Graft-versus-host disease; Immune tolerance; In utero transplantation; T cell
Address correspondence to Jeng-Chang Chen, M.D., Department of Surgery, Chang Gung Children's Hospital, 5, Fu-Shin Street, Kweishan, 333, Taoyuan, Taiwan. Tel: +886-3-3281200, ext. 8227; Fax :+886-3-3287261; E-mail: email@example.com
Improved Xenogenic Hepatocyte Implantation Into Nude Mouse Liver Parenchyma With Acute Liver Failure When Followed by Repeated Anti-Fas Antibody (Jo2) Treatment
Isabelle Vidal,1,2 Nadège Blanchard,2,3 Eliane Alexandre,2 Arnaud Gandillet,2* Marie-Pierre Chenard-Neu,4 Frank Staedtler,5 Martin Schumacher,5 Philippe Bachellier,2,6 Daniel Jaeck,2,6 Huseyin Firat,7 Bruno Heyd,1,8 and Lysiane Richert1,2,3
1EA 3921, IFR 133, Faculté de Médecine et de
Pharmacie, Besançon, France
2Laboratoire de Chirurgie Expérimentale, Fondation Transplantation, Strasbourg, France
3KaLy-Cell, Témis Innovation, Besançon, France
4Service d'Anatomo-Pathologie, Hôpital de Hautepierre, Strasbourg, France
5Novartis Pharma AG, Biomarker Development, Basel, Switzerland
6Centre de Chirurgie Viscérale et de Transplantation, Hôpital de Hautepierre, Strasbourg, France
7Firalis SAS, Huningue, France
8Service de Chirurgie Digestive et Vasculaire, Hôpital Jean Minjoz, Besançon, France
Hepatocyte transplantation is a promising therapy for acute liver failure in humans. Recently, we succeeded in inducing various acute and chronic liver failures in nude mice. Engraftment of transplanted xenogeneic rat hepatocytes, visualized in the host liver by anti-MHC class I immunohistochemistry, revealed that liver repopulation was limited, and equivalent in nude mice with and without acute liver failure. In the present study, acute liver failure was induced in nude mice by a single injection of sublethal anti-Fas antibody Jo2, followed 24 h later by rat hepatocyte transplantation and than by a weekly repeated injection of Jo2. Rat hepatocyte engraftment into the recipient liver parenchyma 3 weeks following hepatocyte transplantation was about sevenfold increased when nude mice were subsequently subjected to weekly repeated Jo2 injection. Genomic analysis of these mice showed an overall transcriptome profile of upregulation of cellular cycle blocking transcripts, activation of liver injury inducing IFN-g/STAT1 pathway, and circadian transcript signature of antiproliferative cell status compared to mice submitted to hepatocyte transplantation only. The findings of the present study suggest that the induction of cell proliferation blockade in recipient livers could promote sufficient engraftment of transplanted hepatocytes to allow transient or definitive treatment of liver failure in humans.
Key words: Nude mice; Anti-Fas antibody treatment; Xenogeneic hepatocyte engraftment; Circadian genes; p21
Address correspondence to Lysiane Richert, Laboratoire de Biologie Cellulaire, EA 3921, IFR 133, Faculté de Pharmacie, 4, place Saint Jacques, 25030 Besançon Cedex, France. Tel: +33 3 81 66 55 52; Fax: +33 3 81 66 56 79; E-mail: firstname.lastname@example.org
*Present address: Stem Cell Research, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, UK.
Sertoli Cell Line Lacks the Immunoprotective Properties Associated With Primary Sertoli Cells
Jannette M. Dufour,1 Brinda Dass,1 Katie R. Halley,1 Gregory S. Korbutt,2,3 Doreen E. Dixon,2 and Ray V. Rajotte2,3
1Cell Biology and Biochemistry, Texas Tech University Health
Sciences Center, Lubbock, TX, USA
2Surgical-Medical Research Institute, University of Alberta, Edmonton, Alberta, Canada
3Department of Surgery, University of Alberta, Edmonton, Alberta, Canada
Sertoli cells are important for maintenance of the immune privileged environment of the testis and prolong survival of cotransplanted cells. The objective of the current study was to examine the immunoprotective properties of a mouse Sertoli cell line (MSC-1) in order to identify a Sertoli cell line that could be used to aid in investigation of the immunoprotective abilities of Sertoli cells. BALB/c islets were cotransplanted with 0-9 million primary BALB/c Sertoli cells or MSC-1 cells into diabetic C3H or BALB/c mice and protection of grafted islets was examined by monitoring blood glucose levels and immunohistochemical analysis. Additionally, expression of potential immunoprotective factors in MSC-1 cells was examined. Cotransplantation of islets with 3 million primary Sertoli cells significantly prolonged islet allograft survival (61.1 ± 6.9 days; p < 0.05) compared with control mice that received allogeneic islets alone (26.9 ± 2.1 days). Grafts collected from normoglycemic C3H mice at 100 days posttransplant contained insulin-positive b-cells adjacent to allogeneic Sertoli cells arranged in tubule-like structures. In contrast, cotransplantation of islet allografts with MSC-1 cells did not prolong islet survival (average 29.8 ± 3.3 days) regardless of the number of MSC-1 cells transplanted and the rejected grafts contained very few b-cells and randomly arranged MSC-1 cells. The lack of islet cell survival was not due to detrimental effects of MSC-1 cells because syngneic islets cotransplanted with MSC-1 cells were functional throughout the study. MSC-1 cells were found to express known Sertoli cell-expressed, immunoprotective factors, clusterin, Fas ligand, and transforming growth factor-b1, suggesting additional factors may be involved in Sertoli cell immune privilege. These data indicate the MSC-1 cell line lacks the immunoprotective properties associated with primary Sertoli cells. Further study of this cell line could be useful in examining the mechanisms that enable Sertoli cells to provide immune privilege.
Key words: Sertoli cell; Immune privilege; Islet; Transplantation; MSC-1
Address correspondence to Dr. Jannette M. Dufour, Cell Biology and Biochemistry, 3601 4th St. STOP 6540, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA. Tel: (806) 743-2616; Fax: (806) 743-2990; E-mail: email@example.com
A Rat Embryo Staging Scale for the Generation of Donor Tissue for Neural Transplantation
E. M. Torres,1 U. M. Weyrauch,1 R. Sutcliffe,2 and S. B. Dunnett1
1Department of Biosciences, Cardiff University, Cardiff CF10
2Charles River Laboratories, Research Models & Services, UK
In rat models of Parkinson's and Huntington's diseases, embryonic neural cells obtained from embryos of specified ages can be implanted into the brain to partially restore both physiology and function. However, in litters produced using overnight mating protocols (often from commercial suppliers), the embryonic age can be difficult to determine precisely. As a result, embryonic size based on crown to rump length (CRL) is usually a more reliable method of embryo staging than the day of mating. This approach is not without difficulty. There are a number of rat staging scales in the literature, none of which deal with donor ages younger than E13, and there are discrepancies between scales at some donor ages. In the present article, we have devised a short mating-period protocol to produce precisely aged embryos. We show that CRL is a highly accurate, reproducible index of donor age and we present an updated embryonic staging scale for Sprague-Dawley (CD) rats that includes donor ages younger than those previously reported.
Key words: Dopamine graft; Donor age; Embryo staging
Address correspondence to E. M. Torres, Cardiff University, Biomedical Sciences Building, Museum Avenue, PO box 911, Cardiff CF10 3US, UK. Tel: 00 44 (0)2920 874115; E-mail: firstname.lastname@example.org
Electrical Stimulation-Induced Release of <gk>b-Endorphin From Genetically Modified Neuro-2a Cells
Volker Storn,1*+ Michael Kirschbaum,1*# Burkhard Schlosshauer,1 Andreas F. Mack,2 and Cornelia Fricke1
1NMI Natural and Medical Sciences Institute, University of
Tuebingen, 72770 Reutlingen, Germany
2Anatomisches Institut, University of Tuebingen, 72074 Tuebingen, Germany
The quantity of therapeutic gene products released from genetically engineered cells can be controlled externally at different levels. The widely used approach of controlling expression, however, generally has the disadvantage that chemical substances must be applied for stimulation. An alternative strategy aims at controlling gene products at posttranslational levels such as secretion. The secretion of a therapeutic agent can be regulated if the agent is targeted to the regulated secretory pathway and stored in the secretory granules until its release. In this article we address the question of whether the release of b-endorphin, an opioid with a potent analgesic effect, could be induced by electrically stimulating stably transfected Neuro-2a cells. Throughout this study we used the human proopiomelanocortin (POMC) gene, which is the precursor molecule for human -endorphin. We analyzed its subcellular localization and found it in the regulated secretory pathway in Neuro-2a cells. Using electrical field stimulation we were able to identify a stimulation pattern that significantly increased the release of b-endorphin-immunoreactive material, although to a limited extent. This result indicates that electrical stimulation of secretion could be used to manipulate the amount of a therapeutic agent released from transplanted cells.
Key words: Electrical stimulation of secretion; Endorphin; Genetically engineered cells; Neuro-2a; Pain; Regulated secretory pathway
Address correspondence to Prof. Burkhard Schlosshauer, NMI Markwiesenstr. 55, 72770 Reutlingen, Germany. Tel: +49-7121-51530-20; Fax: +49-7121-51530-62; E-mail: email@example.com
*These authors contributed equally.
+Current address: Department of Dermatology, University of Heidelberg, Vossstr. 11; 69115 Heidelberg, Germany.
#Current address: Fraunhofer Institute for Biomedical Engineering, Am Mühlenberg 13; 14476 Potsdam, Germany.
Comparison of Human Serum With Fetal Bovine Serum for Expansion and Differentiation of Human Synovial MSC: Potential Feasibility for Clinical Applications
K. Tateishi,1 W. Ando,2 C. Higuchi,2 D. A. Hart,3 J. Hashimoto,2 K. Nakata,2 H. Yoshikawa,2 and N. Nakamura2
1Department of Orthopedics, National Hospital Organization
Kure Medical Center, Hiroshima, Japan
2Department of Orthopedics, Osaka University Graduate School of Medicine, Osaka, Japan
3McCaig Centre for Joint Injury and Arthritis Research, The University of Calgary, Calgary, Canada
The aim of this study was to evaluate the effect of human serum (HS) on growth and differentiation capacity of human synovium-derived mesenchymal stem cells (MSC) in comparison to cells grown in fetal bovine serum (FBS). Human MSCs were isolated from the synovium of knee joints of three donors and the cells were cultured individually in varying concentrations of allogenic HS or FBS. Bovine MSCs were isolated from synovium and cultured in the same manner. Cell proliferation was assessed by the tetrazolium assay after passage 3. The capacity for chondrogenic and osteogenic differentiation was investigated in specific media followed by 1,9-dimethylmethylene blue assay and alcian blue staining, or by alizarin red staining, respectively. Human MSCs proliferated significantly more rapidly in the presence of HS than with equivalent levels of FBS. Chondrogenic or osteogenic differentiation occurred to nearly identical levels in HS or FBS. The results of this study indicate that HS is superior for the culture of human MSCs compared with FBS in terms of cellular expandability, without losing chondrogenic or osteogenic differentiation capacity. Coupled with the advantage in eliminating the potential risk accompanied with the use of xeno-derived materials, pooled, well-characterized HS could be a useful reagent to promote cellular expansion for clinical synovial stem cell-based therapy.
Key words: Human synovial mesenchymal stem cells; Human serum; Cell proliferation; Differentiation
Address correspondence to Norimasa Nakamura, M.D., Ph.D., Department of Orthopedics, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. Tel: +81-6-6879-3552; Fax: +81-6-6879-3559; E-mail: firstname.lastname@example.org
Riboflavin Inhibits IL-6 Expression and p38 Activation in Islet Cells
Lorenzo Cobianchi,1,2* Alessia Fornoni,1,3,4* Antonello Pileggi,1,5 R. Damaris Molano,1 Nahir Y. Sanabria,1 Jorge Gonzalez-Quintana,1 Nicola Bocca,1 Simona Marzorati,1 Elsie Zahr,1 Anthony R. Hogan,1 Camillo Ricordi,1,5 and Luca Inverardi1,3,5
1Cell Transplant Center, Diabetes Research Institute, Leonard
M. Miller School of Medicine, University of Miami, Miami, FL, USA
2Department of Surgery, Fondazione IRCCS "San Matteo" Hospital, University of Pavia, Italy
3Department of Medicine, Leonard M. Miller School of Medicine, University of Miami, Miami, FL USA
4Division of Nephrology, Leonard M. Miller School of Medicine, University of Miami, Miami, FL USA
5DeWitt Daughtry Family Department of Surgery, Division of Cellular Transplantation, Leonard M. Miller School of Medicine, University of Miami, Miami, FL USA
6Department of Microbiology and Immunology, Leonard M. Miller School of Medicine, University of Miami, Miami, FL USA
Riboflavin is a water-soluble vitamin that reduces the production of proinflammatory mediators and oxygen radicals. Because islet <gk>b-cells are very sensitive to oxidative stress and to cytokines, we investigated the possible cytoprotective effects of riboflavin on insulinoma NIT-1 cells and on isolated rodent islets. NIT-1 cells and islets cultured in the presence or absence of 10 mM riboflavin were studied at baseline and after exposure to cytokines (TNF-a, IL-1b, INF-g). Riboflavin treatment did not affect islet cell viability as assessed by flow cytometry for caspases activation. However, riboflavin prevented the cytokine-induced increase in IL-6 mRNA expression and p38 phosphorylation analyzed by real-time PCR and immunoassay, respectively. In summary, nontoxic doses of riboflavin prevent cytokines-induced p38 phosphorylation and IL-6 upregulation in islet cells. This observation, together with the safety profile of riboflavin in the clinical setting, makes it an appealing agent for islet cytoprotection in islet transplantation protocols.
Key words: Riboflavin; IL-6 p38; Cytoprotection; Pancreatic islets; Inflammation
Address correspondence to Luca Inverardi, M.D., Cell Transplant Center, Diabetes Research Institute, University of Miami Leonard M. Miller School of Medicine, 1450 NW 10th Avenue (R-134), Miami, FL 33136, USA. Tel: (305) 243-5347; Fax: (305) 243-4404; E-mail: email@example.com
*These two authors equally contributed to this work.
Improved Glucose Regulation on a Low Carbohydrate Diet in Diabetic Rats Transplanted With Macroencapsulated Porcine Islets
Horatiu V. Vinerean,1 Lawrence S. Gazda,1,2 Richard D. Hall,1,4 Albert L. Rubin,2,3 and Barry H. Smith1,2,3
1The Rogosin Institute-Xenia Division, Xenia, OH, USA
2The Rogosin Institute, New York, NY, USA
3New York Presbyterian Hospital and Weill Medical College of Cornell University, New York, NY, USA
4Bob Evans Farms, Inc., Columbus, OH, USA
Islet xenografts from porcine donors can reverse diabetes in experimental animal models and may be an alternative to human islet transplantation. We have recently reported the ability of porcine islets encapsulated in a double layer of hydrophilic agarose to maintain in vitro functional ability for >6 months. Although b-cells are capable of adapting their secretory capacity in response to glucose levels, evidence has shown that prolonged hyperglycemia can compromise this ability. The aim of the present study was to determine the effects of diet manipulation on the long-term regulation of blood glucose levels, and the preservation of functional islet in the macrobeads. Twenty-one streptozotocin-induced diabetic Wistar-Furth male rats were randomly assigned to two diets containing 64% carbohydrate (CHO) or 20% CHO. Groups of five to six animals assigned to either diet were implanted with either empty (EM) or porcine islet-containing macrobeads (PIM) and followed for 333 days. Observations included general condition, body weight, blood glucose, and food and water intakes. Monthly blood samples were collected for insulin and C-peptide analysis. The 20% CHO diet significantly lowered blood glucose values when compared with those of the 64% CHO groups for both the empty (14.94 ± 0.41 vs. 16.26 ± 0.42 mmol/L, respectively, p < 0.001) and islet macrobead recipients (12.88 ± 0.39 vs. 15.57 ± 0.85 mmol/L, respectively, p < 0.001). The different diets, however, had no statistically significant effects on the preservation of islet mass in the macrobead. Serum porcine C-peptide was detected throughout the experiment in animals receiving porcine islet macrobeads, regardless of diet. Diabetic rats fed a low carbohydrate level diet and transplanted with porcine islet macrobeads had improved total tissue glucose disposal and improved clinical parameters associated with diabetes.
Key words: Porcine islets; Xenotransplantation; Encapsulation; Diet
Address correspondence to Horatiu V. Vinerean, The Rogosin Institute-Xenia Division, 740 Birch Rd, Xenia, OH 45385, USA. Tel: (937) 374-3116; Fax: (937) 374-3261; E-mail: firstname.lastname@example.org
Efficient Delivery of Human Single Fiber-Derived Muscle Precursor Cells Via Biocompatible Scaffold
Luisa Boldrin,1,5*+ Alberto Malerba,1,5* Libero Vitiello,2 Elisa Cimetta,3 Martina Piccoli,1 Chiara Messina,1 Pier Giorgio Gamba,4 Nicola Elvassore,3 and Paolo De Coppi1,5
1Stem Cell Processing Laboratory, Department of Paediatrics,
University of Padova, Padova, Italy
2Gene Transfer Laboratory, Department of Biology, University of Padova, Padova, Italy
3Department of Chemical Engineering, University of Padova, Padova, Italy
4Paediatric Surgery, Department of Paediatrics, University of Padova, Padova, Italy
5Surgery Unit, Institute of Child Health, University College of London, London, UK
The success of cell therapy for skeletal muscle disorders depends upon two main factors: the cell source and the method of delivery. In this work we have explored the therapeutic potential of human muscle precursor cells (hMPCs), obtained from single human muscle fibers, implanted in vivo via micropatterned scaffolds. hMPCs were initially expanded and characterized in vitro by immunostaining and flow cytometric analysis. For in vivo studies, hMPCs were seeded onto micropatterned poly-lactic-glycolic acid 3D-scaffolds fabricated using soft-lithography and thermal membrane lamination. Seeded scaffolds were then implanted in predamaged tibialis anterior muscles of CD1 nude mice; hMPCs were also directly injected in contralateral limbs as controls. Similarly to what we previously described with mouse precursors cells, we found that hMPCs were able to participate in muscle regeneration and scaffold-implanted muscles contained a greater number of human nuclei, as revealed by immunostaining and Western blot analyses. These results indicate that hMPCs derived from single fibers could be a good and reliable cell source for the design of therapeutic protocols and that implantation of cellularized scaffolds is superior to direct injection for the delivery of myogenic cells into regenerating skeletal muscle.
Key words: Human single fibers; Muscle precursor cells; Biocompatible scaffold; Muscle regeneration
Address correspondence to Paolo De Coppi, M.D., Ph.D., Surgery Unit, Institute of Child Health, 30 Guilford Street, London, WC1N 1EH, UK. Tel: +44 020-7905-2641; Fax: +44 020-7404-6181; E-mail: email@example.com
*L. Boldrin and A. Malerba equally contributed to this work.
+Current address: The Dubowitz Neuromuscular Unit, Hammersmith Hospital, Imperial College London, Du Cane Road, London W12 0NN, UK.