|ognizant Communication Corporation|
The Regenerative Medicine Journal
VOLUME 15, NUMBER 5, 2006
Cell Transplantation, Vol. 15, pp. 369-380, 2006
0963-6897/06 $90.00 + 00
Copyright © 2006 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.
Fate of Embryonic Stem Cells Transplanted Into the Deafened Mammalian Cochlea
B. Coleman,1,2,5 J. Hardman,2 A. Coco,2 S. Epp,2 M. de Silva,3 J. Crook,4 and R. Shepherd1,2,5
1Department of Otolaryngology, University of Melbourne, 3002,
2Bionic Ear Institute, Melbourne, 3002, Australia
3Murdoch Children's Research Institute, Melbourne, 3052, Australia
4ES Cell International, Singapore, 138667
5Royal Victorian Eye and Ear Hospital, Melbourne, 3002, Australia
Spiral ganglion neurons (SGNs), the primary afferent neurons of the cochlea, degenerate following a sensorineural hearing loss (SNHL) due to lack of trophic support normally received from hair cells. Cell transplantation is emerging as a potential strategy for inner ear rehabilitation, as injected cells may be able to replace damaged SGNs in the deafened cochlea. An increase in the number of surviving SGNs may result in improved efficacy of cochlear implants (CIs). We examined the survival of partially differentiated mouse embryonic stem cells (MESCs), following xenograft transplantation into the deafened guinea pig cochlea (n = 15). Cells were delivered directly into the left scala tympani via microinjection through the round window. Small numbers of MESCs were detected in the scala tympani for up to 4 weeks following transplantation and a proportion of these cells retained expression of neurofilament protein 68 kDa in vivo. While this delivery method requires refinement for effective long-term replacement of damaged SGNs, small numbers of MESCs were capable of survival in the deafened mammalian cochlea for up to 4 weeks, without causing an inflammatory tissue response.
Key words: Cell-based therapy; Cochlea; Deafness; Embryonic stem cell; Transplantation
Address correspondence to Bryony Coleman, Department of Otolaryngology, University of Melbourne, Level 2, Royal Victorian Eye and Ear Hospital, 32 Gisborne Street, East Melbourne, 3002, Australia. Tel: +61 3 9929 8462; Fax: +61 3 9663 1958; E-mail: email@example.com
Porcine Fetal Ventral Mesencephalic Cells Are Targets for Primed Xenoreactive Human T Cells
Jan Koopmans,1 Aalzen de Haan,2 Elinda Bruin,2 Ieneke van der Gun,2 Henk van Dijk,3 Jan Rozing,4 Lou de Leij,2 and Michiel Staal1
1Department of Neurosurgery, University Medical Center Groningen,
Hanzeplein 1, 9700 RB Groningen, The Netherlands
2Medical Biology Section of Pathology and Laboratory Medicine, University Medical Center Groningen, Hanzeplein 1, 9700 RB Groningen, The Netherlands
3Department of Veterinary Anatomy and Physiology, University of Utrecht, Yalelaan 1, 3584 CL, The Netherlands
4Department of Cell Biology, Section Immunology, University Medical Center Groningen, A. Deusinglaan 1, 9713 AV Groningen, The Netherlands
Xenotransplantation of porcine fetal ventral mesencephalic (pfVM) cells to overcome the dopamine shortage in the striatum of patients with Parkinson's disease seems a viable alternative to allotransplantion of human fetal donor tissue, especially because the latter is complicated by both practical and ethical issues. There is, however, little known about the xenospecific immune responses involved in such an intracerebral xenotransplantation. The aim of our study was to investigate whether 1) naive human peripheral blood mononuclear cells (PMBC) display cytotoxicity against pfVM cells of E28 pig fetuses, and 2) priming of human PBMC by xenogeneic antigen presenting cells (APC) modulates pfVM-directed cellular cytotoxicity. For this purpose fresh PMBC from nine individual donors were primed by incubation with either irradiated pfVM cells or porcine spleen cells (PSC) as APC in the presence of IL-2 for 1 week before assessing cytotoxicity in a 51Cr release assay. Also, direct NK reactivity and antibody-dependent cellular cytotoxicity (ADCC) of fresh PMBC against pfVM cells was assessed. No direct cytotoxicity of naive cells (either NK reactivity or ADCC) against pfVM cells could be determined. Only PMBC primed with PSC were capable of lysing pfVM cells. PBMC primed with pfVM cells did not show cytolytic capacity towards pfVM. Interestingly, large differences in xenospecific T-cell responses exist between individual donor PBMC. Thus, human T cells are capable of killing pfVM cells in a xenoreactive response, but only after priming by donor APC. The large interindividual differences between human donors in their xenoreactive response may influence patient selection for xenotransplantation and chances of graft survival for individual patients.
Key words: Xenotransplantation; Ventral mesencephalon; Parkinson's disease; T cells; Antibody-dependent cellular cytotoxicity (ADCC); Porcine
Address correspondence to J. Koopmans, Department of Neurosurgery, University Medical Center Groningen, Hanzeplein 1, 9700 RB Groningen, The Netherlands. Tel: +31-50-3613218; Fax: +31-50-3611715; E-mail: J.Koopmans@nchir.umcg.nl
Ectopic Ossification in the Scar Tissue of Rats With Myocardial Infarction
Karla Consort Ribeiro,1 Elisabete César Mattos,2 João Pedro Saar Werneck-de-Castro,1 Vanessa Pinho Ribeiro,1 Ricardo Henrique Costa-e-Sousa,1 Amarildo Miranda,1 Emerson Lopes Olivares,1 Marcos Farina,3 José Geraldo Mill,5 Regina Coeli dos Santos Goldenberg,1 Masako Oya Masuda,1 and Antônio Carlos Campos de Carvalho1,4
1Instituto de Biofísica Carlos Chagas Filho
UFRJ-CCS, Bloco G, Ilha do Fundão, Rio de Janeiro, Brasil
2Ecodata Exames Médicos LTDA, Rio de Janeiro, Brasil
3Instituto de Ciências Biomédicas UFRJ-CCS, Bloco F, Ilha do Fundão, Rio de Janeiro, Brasil
4Instituto Nacional de Cardiologia Laranjeiras, Rio de Janeiro, Brasil
5Departamento de Ciências Fisiológicas UFES, Vitória, Brasil
We describe the occurrence of bone-like formations in the left ventricular wall of infarcted rats treated or not with bone marrow cells injected systemically or locally into the myocardium. The incidence of ectopic calcification in hearts has been reported in rare cases in children with infarcts without previous coronary artery disease. Recently, ventricular calcification has been correlated with unselected bone marrow cell transplantation into infarcted rat hearts. Echocardiographic analysis of large infarction in rats frequently reveals the presence of echogenic structures in the left ventricular wall, sometimes projecting to the lumen of the chamber. The histological examination of these echogenic structures exhibited bone, cartilage, and marrow-like formations extending from the collagen-rich matrix of the ventricle wall. Microanalytical techniques verified the presence of hydroxyapatite in the mineral phase. Ossification was found in 25 out of 30 hearts evaluated 90 days postinfarct, being observed in 14 out of 17 animals submitted to cell therapy and in 11 out of 13 infarcted rats not submitted to cell therapy. Our study indicates that chondro-osteogenic differentiation can take place in the pathological rat heart independent of animal treatment with marrow cells.
Key words: Myocardial infarction; Osteogenesis; Correlative microscopy; Ectopic ossification
Address correspondence to Antonio Carlos Campos de Carvalho, Instituto de Biofísica Carlos Chagas Filho, UFRJ CCS, Bloco G, Ilha do Fundão 21949-900 Rio de Janeiro, RJ, Brasil. Tel: 552125626568; Fax: 552122808193; E-mail: firstname.lastname@example.org
Suppression of Human T-Cell Responses to β-Cells by Activation of B7-H4 Pathway
Dawei Ou,1 Xiaojie Wang,2 Daniel L. Metzger,2 Ziliang Ao,1 Paolo Pozzilli,3 Roger F. L. James,4 Lieping Chen,5 and Garth L. Warnock1
1Department of Surgery, University of British Columbia, Vancouver,
BC, V5Z 1L8, Canada
2Department of Pediatrics, University of British Columbia, Vancouver, BC, V5Z 4H4, Canada
3St. Bartholomew's Hospital, Royal London School of Medicine, London, C1A 7BE, UK
4Department of Infection, Immunity and Inflammation, University of Leicester, Leicester, LE2 7LX, UK
5Department of Dermatology and Oncology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
B7-H4, a recently described member of the B7 family of cosignal molecules, is thought to be involved in the regulation of cellular and humoral immune responses through receptors on activated T and B cells. Human islet cells express positive B7-H4 mRNA in RT-PCR assays, but not B7-H4 protein on cell surface in flow cytometric analyses. To investigate the regulatory effects of activation of the B7-H4 pathway on the function of activated T cells of patients with type 1 diabetes (T1D), we have used our in vitro human experimental system, including human b-cell antigen-specific T-cell clones and human b-cell lines CM and HP62, as well as primary islet cells. B7-H4.Ig protein was purified from the culture supernatant of 293T cells transfected by a B7-H4.Ig plasmid (pMIgV, containing a human B7-H4 cDNA and a mouse IgG2a Fc cDNA). Our preliminary studies showed that immobilized fusion protein human B7-H4.Ig (coated with 5 mg/ml for 2 h at 37°C), but not control Ig, clearly inhibited the proliferation of activated CD4+ and CD8+ T cells of patients induced by anti-CD3 antibody in CFSE assays. B7-H4.Ig also arrested cell cycle progression of T cells in G0/G1 phase and induced T-cell apoptosis as measured by BrdU-7-AAD flow cytometric analysis. To determine the cytoprotective effects of B7-H4, we developed transfectants of human b-cell lines CM and HP62 and islet cells transfected with the B7-H4.Ig plasmid, using empty vector transfectants as controls. The results demonstrate that cell-associated B7-H4.Ig expressed on human b-cells clearly inhibits the cytotoxicity of the T-cell clones to targeted human b-cells in 51Cr release cytotoxicity assays. Activation of the B7-H4 pathway may represent a novel immunotherapeutic approach to inhibit T-cell responses for the prevention of b-cell destruction in T1D.
Key words: B7-H4; Type 1 diabetes; Costimulatory molecules; Suppression of T-cell responses; b-Cell destruction; Islet cell transplantation
Address correspondence to Dr. Dawei Ou, Department of Surgery, Faculty of Medicine, The University of British Columbia, VGH Research Pavilion, 400-828 West 10th Avenue, Vancouver, B.C. Canada, V5Z 1L8. Tel: (604)-875-4111, ext. 62474; Fax: (604)-875-4317; E-mail: email@example.com
Regeneration of Osteonecrosis of Canine Scapho-lunate Using Bone Marrow Stromal Cells: Possible Therapeutic Approach for Kienböck Disease
Ryosuke Ikeguchi,1,2* Ryosuke Kakinoki,2* Tomoki Aoyama,1* Kotaro Roberts Shibata,1,2 Seiji Otsuka,1,4 Kennichi Fukiage,1 Koichi Nishijo,1,2 Tatsuya Ishibe,1,2 Yasuko Shima,1,2 Bungo Otsuki,2 Takashi Azuma,3 Sadami Tsutsumi,3 Tomitaka Nakayama,2 Takanobu Otsuka,4 Takashi Nakamura,2 and Junya Toguchida1
1Department of Tissue Regeneration, Institute for Frontier
Medical Sciences, Kyoto University, Kyoto, Japan
2Department of Orthopaedic Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan
3Department of Medical Simulation Engineering, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan 4Department of Orthopaedic Surgery, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan
We evaluated the ability of canine bone marrow stromal cells (cBMSCs) to regenerate bone in a cavity of the scapholunate created by curretage and freeze-thawing with liquid nitrogen (LN). Autologous BMSCs were harvested from the iliac crest and expanded in vitro. Their potential to differentiate into osteo-, chondro-, and adipogenic lineages was confirmed using a standard differentiation induction assay. LN-treated scapholunates showed no regeneration of bone tissue when the cavity was left alone, demonstrating severe collapse and deformity as observed in human Kienböck disease. A combination of b-tri-calcium phosphate and a vascularized bone graft with autologous fibroblasts failed to regenerate bone in the LN-treated cavity. When the same procedure was performed using BMSCs, however, LN-treated scapholunates showed no collapse and deformity, and the cavity was completely filled with normal cancerous bone within 4 weeks. These results suggested the potential of using BMSCs to treat Kienböck disease.
Key words: Osteonecrosis; Kienböck disease; Bone marrow stromal cells; Vascularized bone graft; b-Tri-calcium phosphate (β-TCP)
Address correspondence to Junya Toguchida, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. Tel: +81-75-751-4134; Fax: +81-75-751-4646; E-mail: firstname.lastname@example.org
*These three authors contributed equally to this study.
Assessment of In Vitro Applicability of Reversibly Immortalized NKNT-3 Cells and Clonal Derivatives
Ruurdtje Hoekstra,1,2 Tanja Deurholt,2 Lysbeth ten Bloemendaal,1,2 Mireille Desille,3 Albert C. W. A. van Wijk,1 Bruno Clement,3 Ronald P. J. Oude Elferink,2 Thomas M. van Gulik,1 and Robert A. F. M. Chamuleau2
1Surgical Laboratory, Academic Medical Center, University
of Amsterdam, The Netherlands
2AMC Liver Center, Academic Medical Center, University of Amsterdam, The Netherlands
3INSERM U456, Detoxication and Tissue Repair Unit, University of Rennes I, Rennes, France
In vitro applications of human hepatocytes, such as bioartificial livers and toxicity assays, require thoroughly testing of human cell lines prior to using them as alternative cell sources. The reversibly immortalized NKNT-3 cell line was reported to show clear in vivo functionality. Here, NKNT-3 cells were tested for their in vitro applicability. Low-passage (P2) and high-passage (P28) NKNT-3 cells and clonal derivatives were characterized for reversion of immortalization, heterogeneity, and hepatic functionality. Reversion with reduced expression of immortalizing agent could be established. However, during culturing the cells lost the capacity to be selected for completed reversion. The phenotypic instability is probably associated with heterogeneity in the culture, as clonal derivatives of P2 cells varied in morphology, growth, and reversion characteristics. The mRNA levels of genes related with hepatic differentiation increased 4-20-fold after reversion. However, the levels never exceeded 0.1% of that detected in liver and no urea production nor ammonia elimination was detected. Additionally, activities of different cytochrome P450s were limited. In conclusion, the NKNT-3 culture is heterogeneous and unstable and the in vitro functionality is relatively low. These findings emphasize that in vivo testing of hepatic cell lines is little informative for predicting their value for in vitro applications.
Key words: Hepatocyte; Immortalization; Telomerase; SV40 large T antigen; Liver
Address correspondence to R. Hoekstra, Department of Surgery (Surgical Laboratory), IWO-1-172, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. Tel: 0031-20-5666683; Fax: 0031-20-6976621; E-mail: email@example.com
A Novel Bioreactor Microcarrier Cell Culture System for High Yields of Proliferating Autologous Human Keratinocytes
Jin Yu Liu, Jürg Hafner, Gayla Dragieva, and Günter Burg
Department of Dermatology, University Hospital of Zurich, Zurich, Switzerland
Rapid and efficient resurfacing of various skin defects by autologous keratinocyte transplantation is significant in skin wound healing. We developed a novel bioreactor microcarrier cell culture system (Bio-MCCS) to produce autologous human keratinocytes on a large scale. In this Bio-MCCS we used porcine gelatin microbeads as microcarriers for autolgous keratinocytes and spinning bottles as fermentation tanks. First, the microbeads were modified by culturing them with autologous dermal fibroblasts that were subsequently killed when they proliferated to confluence on the microbeads. We then performed the Bio-MCCS by expanding ketatinocytes on the microbeads in spinning bottles at 37°C, 5% CO2. Our results showed that keratinocytes rapidly attached to and actively proliferated on the modified microbeads in the Bio-MCCS, achieving high cell densities on the modified microbeads (MTT assay and PI staining). Keratinocytes cultured on the modified microbeads in the Bio-MCCS remained proliferating potentials as shown by positive PCNA staining and BrdU labeling. In contrast, keratinocytes cultured on nonmodified microbeads in the Bio-MCCS proliferated slowly, rapidly ceased to proliferate, and finally dislodged from the microbeads. When removed from the Bio-MCCS and cultured under static conditions, keratinocytes were able to leave the modified microbeads and formed a multilayered epidermal equivalent on the culture surfaces. While stored at room temperature, keratinocytes remained at higher viabilities on the modified microbeads when compared to those on nonmodified microbeads. The achievement of high yields of proliferating autologous keratinocytes by this Bio-MCCS offers a practical potential of resurfacing various skin defects by direct administration of autologous keratinocyte microbeads on various skin defects.
Key words: Keratinocyte; Cell transplantation; Microcarrier; Wound healing
Address correspondence to Prof. Dr. Med. Günter Burg, M.D., Chairman, Department of Dermatology, University Hospital of Zurich, Glorriastr. 31., CH-8091 Zürich, Switzerland. Tel: 41-1-255-2550; Fax: 41-1-255-3999; E-mail: firstname.lastname@example.org
High Yields of Autologous Living Dermal Equivalents Using Porcine Gelatin Microbeads as Microcarriers for Autologouos Fibroblasts
Jin Yu Liu, Jürg Hafner, Gayla Dragieva, and Günter Burg
Department of Dermatology, University Hospital of Zurich, Zurich, Switzerland
Permanent skin replacement requires a dermal component to ensure adequate long-term graft stability and to prevent wound contraction. This study was to construct a bioreactor microcarrier cell culture system (Bio-MCCS) to produce autologous living dermal equivalents on a large scale. Autologous fibroblasts were isolated from split-thickness skin biopsy from a leg ulcer patient, inoculated onto macroporous porcine gelatin microbeads, and incubated in a bioreactor (Cellspin) in serum-free fibroblast growth medium or in DMEM medium containing 10% fetal calf serum (FCS). Fibroblasts rapidly adhered to and actively proliferated on the microbeads in the bioreactor in both serum-free and serum-containing medium. MTT assay showed the number of fibroblasts on the microbeads reached up to 5.3- or 4.0-fold the cells seeded in DMEM medium containing 10% FCS or serum-free medium, respectively. When removed from Bio-MCCS and cultured under static conditions, fibroblasts were able to leave the microbeads and proliferate to confluence on the bottom of tissue culture flasks. When stored at room temperature in DMEM containing 10% FBS, fibroblast cultured on the microbeads retained highest viabilities for at least 3 weeks, up to 82% of originals. This Bio-MCCS using porcine gelatin microbeads as carriers for fibroblasts offers a new option of mass production of autologous living dermal equivalents.
Key words: Fibroblast; Cell culture; Tissue engineering; Bioreactor; Microbeads
Address correspondence to Prof. Dr. med. Günter Burg, M.D.,
Chairman, Department of Dermatology, University Hospital of Zurich, GlorriaStr.
31. CH-8091, Zürich, Switzerland. Tel: 41-1-255-2550; Fax: 41-1-2553999;