|ognizant Communication Corporation|
The Regenerative Medicine Journal
VOLUME 16, NUMBER 8, 2007
Cell Transplantation, Vol. 16, pp. 765-774, 2007
0963-6897/07 $90.00 + 00
Copyright © 2007 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.
The Effects of Cell Density and Device Arrangement on the Behavior of Macroencapsulated b-Cells
Kristen E. La Flamme,1 Thomas J. LaTempa,2 Craig A. Grimes,2 and Tejal A. Desai3
1Department of Biomedical Engineering, Boston University,
Boston, MA 02215, USA
2Department of Electrical Engineering and Biomedical Engineering, The Pennsylvania State University, University Park, PA 16802, USA
3Department of Physiology and Division of Bioengineering, University of California San Francisco, San Francisco, CA 94158, USA
Over the last several decades, considerable research has focused on the development of cell encapsulation technology to treat a number of diseases, especially type 1 diabetes. One of the key advantages of cell encapsulation is that it permits the use of xenogenic tissue, particularly animal-derived cell lines. This is an attractive idea, because it circumvents the issue of a limited human organ supply. Furthermore, as opposed to whole islets, cell lines have a better proliferative capacity and can easily be amplified in culture to provide an endless supply of uniform cells. We have previously described a macroencapsulation device for the immunoisolation of insulin-secreting b-cells. The aim of this work was to optimize the viability and insulin secretion of cells encapsulated within this device. Specifically, the effects of cell packing density and device membrane configuration were investigated. The results indicated that cell density plays an important role in the secretory capacity of the cells, with higher cell density leading to increased insulin secretion. Increasing the transport area of the capsule by modifying the membrane configuration also led to an improvement in the insulin output of the device.
Key words: Immunoisolation; Diabetes; Cell encapsulation; b-Cells; Cell density
Address correspondence to Tejal A. Desai, Professor, Department of Physiology and Division of Bioengineering, UCSF MC 2520, Byers Hall Rm 203C, San Francisco, CA 94158-2330, USA. Tel: 1-415-514-4503; Fax: 1-415-514-4503; E-mail: Tejal.firstname.lastname@example.org
Expression of Transforming Growth Factor-b by Human Islets: Impact on Islet Viability and Function
Omaima M. Sabek,1 Daniel W. Fraga,1 James Henry,2 Lillian W. Gaber,1 Malak Kotb,3 and A. Osama Gaber1
1Department of Surgery, Methodist Hospital/Cornell University,
Physicians Organization, Houston, TX, USA
2Environmental Health and Safety, St. Jude Children's Research Hospital, Memphis, TN, USA
3Department of Surgery, Division of Transplantation, University of Tennessee, Memphis, TN, USA
Transforming growth factor-b1 (TGF-b1) is a pleotropic cytokine that promotes angiogenesis and extracellular matrix protein synthesis in addition to its immunosuppressive effects. The purpose of this study is to identify optimal conditions for in vivo expression of TGF-b1 by human islets to exploit the possible beneficial effects and minimize undesirable side effects. We transduced human islets with adenoviral vectors encoding the active form of Ad-TGF-b1 or Ad-LacZ to test the effects of TGF-b1 gene expression on islet in vivo function following their transplantation into a NOD-SCID mouse model. Islets were transduced with multiplicity of infection (MOI) of 20, 10, 5, and 2.5 per islet cell. At a MOI ranging from 2.5 to 20, expression of TGF-b1 in islet supernatant persisted for 1-2 months and ranged from 153 ± 5 to 2574 ± 1299 pg/ml, respectively. Transduction with the lowest MOI (2.5) did not compromise the in vivo production of human C-peptide. We conclude that TGF-b1 expression in transplanted islets does not compromise viability and that adenoviral transduction with the TGF-b1 gene has a dose-dependent effect, with larger MOIs being deleterious. The data also indicate that in vitro culture system and the in vivo NOD-SCID model could be used successfully to evaluate the nonimmune effects of gene transduction.
Key words: Human islets; Ad-TGF-b1; Viability; NOD-SCID
Address correspondence to Omaima M. Sabek, Ph.D., Research Scientist, Department of Surgery, The Methodist Hospital Research Institute, 6550 Fannin Street, SM1105, Houston, TX 77030, USA. Tel: 713-441-6309; Fax: 713-790-3085; E-mail: OMSabek@tmhs.org
Combination of Anti-CD4 With Anti-LFA-1 and Anti-CD154 Monoclonal Antibodies Promotes Long-Term Survival and Function of Neonatal Porcine Islet Xenografts in Spontaneously Diabetic NOD Mice
Hossein Arefanian,1,2 Eric B. Tredget,1 Ray V. Rajotte,1 Gregory S. Korbutt,1 Ron G. Gill,3 and Gina R. Rayat1
1Surgical-Medical Research Institute, Department of Surgery,
University of Alberta, Edmonton, Alberta, Canada T6G 2N8
2Endocrinology and Metabolism Research Center, Dr. Shariati Hospital, Tehran University of Medical Science, North Kargar Avenue, Tehran 14114, Iran
3Barbara Davis Center for Childhood Diabetes, University of Colorado Health Sciences Center, Aurora, CO 80010, USA
Type 1 diabetes mellitus (T1DM) is caused by the autoimmune destruction of pancreatic islet b-cells, which are required for the production of insulin. Islet transplantation has been shown to be an effective treatment option for T1DM; however, the current shortage of human islet donors limits the application of this treatment to patients with brittle T1DM. Xenotransplantation of pig islets is a potential solution to the shortage of human donor islets provided xenograft rejection is prevented. We demonstrated that a short-term administration of a combination of anti-LFA-1 and anti-CD154 monoclonal antibodies (mAbs) was highly effective in preventing rejection of neonatal porcine islet (NPI) xenografts in non-autoimmune-prone B6 mice. However, the efficacy of this therapy in preventing rejection of NPI xenografts in autoimmune-prone nonobese diabetic (NOD) mice is not known. Given that the current application of islet transplantation is for the treatment of T1DM, we set out to determine whether a combination of anti-LFA-1 and anti-CD154 mAbs could promote long-term survival of NPI xenografts in NOD mice. Short-term administration of a combination of anti-LFA-1 and anti-CD154 mAbs, which we found highly effective in preventing rejection of NPI xenografts in B6 mice, failed to promote long-term survival of NPI xenografts in NOD mice. However, addition of anti-CD4 mAb to short-term treatment of a combination of anti-LFA-1 and anti-CD154 mAbs resulted in xenograft function in 9/12 animals and long-term graft (>100 days) survival in 2/12 mice. Immunohistochemical analysis of islet grafts from these mice identified numerous insulin-producing b-cells. Moreover, the anti-porcine antibody as well as autoreactive antibody responses in these mice was reduced similar to those observed in naive nontransplanted mice. These data demonstrate that simultaneous targeting of LFA-1, CD154, and CD4 molecules can be effective in inducing long-term islet xenograft survival and function in autoimmune-prone NOD mice.
Key words: Neonatal porcine islets (NPI); CD154; LFA-1; CD4; Monoclonal antibodies (mAbs); Nonobese diabetic (NOD) mouse; Type 1 diabetes mellitus (T1DM)
Address correspondence to Dr. Gina R. Rayat, Surgical-Medical Research Institute, 1074 Dentistry/Pharmacy Centre, University of Alberta, Edmonton, Alberta, Canada T6G 2N8. Tel: (780) 492-6894; Fax: (780) 492-1627; E-mail: email@example.com
Neural Progenitor Cells Derived From the Adult Rat Subventricular Zone: Characterization and Transplantation
Kevin Chen, Stephanie M. Hughes, and Bronwen Connor
Department of Pharmacology and Clinical Pharmacology, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand
In order to fully characterize and determine the therapeutic potential of adult neural progenitor cells (NPCs), it is important to be able to isolate and study NPCs from animals such as rats, in which there are existing models of brain injury and disease. The focus of this study was to characterize the cultivation, differentiation, and transplantation of adult rat NPCs isolated from the subventricular zone of the lateral ventricles. We examined strategies for cell purification using a Percoll density gradient, and cell expansion using a range of maintenance medium and plating densities. Purification by Percoll gradient enriched a population of cells expressing nestin and SOX2, but resulted in a significant reduction in neurosphere generation. Culturing adult rat NPCs in Neurobasal-A media and plating at 200,000 cell/ml resulted in a higher percentage of cells surviving to generate neurospheres compared to culture in DMEM/F12 or NS-A media. On induction of differentiation, adult rat NPCs were capable of generating neurons, astrocytes, and oligodendrocytes in vitro that survived for up to 8 weeks, demonstrating multipotentiality of these cells. In addition, a population of cells continued to proliferate during the initial phase of differentiation, suggesting the presence of two populations of NPCs during differentiation. Cultured adult rat NPCs also survived and differentiated into astrocytes 6 weeks after transplantation into the striatum of the normal adult rat brain. In conclusion, we have optimized techniques that allow for the routine isolation, culture, and transplantation of multipotent NPCs derived from the adult rat SVZ.
Key words: Subventricular zone; Neural progenitor cells; Cultivation; Differentiation; Transplantation
Address correspondence to Dr. Bronwen Connor, Department of Pharmacology and Clinical Pharmacology, FMHS, University of Auckland, Private Bag 92019, Auckland, New Zealand. Tel: 64 9 373 7599, ext. 83037; Fax: 64 9 373 7556; E-mail: firstname.lastname@example.org
Nerve Regeneration Promoted in a Tube With Vascularity Containing Bone Marrow-Derived Cells
Tomoyuki Yamakawa, Ryosuke Kakinoki, Ryosuke Ikeguchi, Ken Nakayama, Yoshihide Morimoto, and Takashi Nakamura
Department of Orthopedic Surgery, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan
Bone marrow-derived cells (BMCs) are multipotent cells that have the potential to differentiate into bone, cartilage, fat, muscle, or neuronal lineages such as neurons and glial cells. A silicone tube model containing reverse-pedicled sural vessels was created in the sciatic nerves of Lewis rats. About 1 x 107 BMCs, removed from the bone marrow of synergetic rat femurs and cultured in vitro, were transplanted into the 15-mm-long chambers of the silicone tubes. Nerve regeneration in vessel-containing tubes that had received BMCs was significantly greater at 12 and 24 weeks after surgery than in tubes that did not receive cells. Transplantation of fibroblasts instead of BMCs into the vessel-containing tube resulted in reduced axonal regeneration, which was inferior to regeneration in the vessel-containing tube that did not receive cells. Polymerase chain reaction (PCR) studies revealed that in vessel-containing tubes containing transplanted BMCs, about 29% of cells in the regenerated nerve originated from BMCs. Cells identified by in situ hybridization and PKH26 prelabeling as being of BMC origin stained positively for S100 and GFAP. Transplanted BMCs differentiated into cells with phenotypes similar to those of Schwann cells under the influence of neurochemical factors and survived by obtaining nutrients from vessels that had been preinserted into the tube. They thus functioned similarly to Schwann cells, promoting nerve regeneration.
Key words: Bone marrow-derived cell; Nerve regeneration; Tubulation; Cell differentiation; Vascularity
Address correspondence to Ryosuke Kakinoki, Department of Orthopedic Surgery, Graduate School of Medicine, Kyoto University, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan. Tel: +81-75-751-3657; Fax: +81-75-751-8409; E-mail: email@example.com
Comparison of Chondral Defects Repair With In Vitro and In Vivo Differentiated Mesenchymal Stem Cells
Hongbin Fan,1 Haifeng Liu,2 Rui Zhu,3 Xusheng Li,1 Yuming Cui,1 Yunyu Hu,1 and Yongnian Yan4
1Institute of Orthopaedics & Traumatology, Xijing Hospital,
The Fourth Military Medical University, Xi'an, PR China
2Research Institute of Polymer Material, Tianjin University, Tianjin, PR China
3Department of Engineering, Military Engineering University, Xi'an, PR China
4Department of Mechanical Engineering, Tsinghua University, Beijing, PR China
The purpose of this study was to compare chondral defects repair with in vitro and in vivo differentiated mesenchymal stem cells (MSCs). A novel PLGA-gelatin/chondroitin/hyaluronate (PLGA-GCH) hybrid scaffold with transforming growth factor-b1 (TGF-b1)-impregnated microspheres (MS-TGF) was fabricated to mimic the extracellular matrix. MS-TGF showed an initial burst release (22.5%) and a subsequent moderate one that achieved 85.1% on day 21. MSCs seeded on PLGA-GCH/MS-TGF or PLGA-GCH were incubated in vitro and showed that PLGA-GCH/MS-TGF significantly augmented proliferation of MSCs and glycosaminoglycan synthesis compared with PLGA-GCH. Then MSCs seeded on PLGA-GCH/MS-TGF were implanted and differentiated in vivo to repair chondral defect on the right knee of rabbit (in vivo differentiation repair group), while the contralateral defect was repaired with in vitro differentiated MSCs seeded on PLGA-GCH (in vitro differentiation repair group). The histology observation demonstrated that in vivo differentiation repair showed better chondrocyte morphology, integration, and subchondral bone formation compared with in vitro differentiation repair 12 and 24 weeks postoperatively, although there was no significant difference after 6 weeks. The histology grading score comparison also demonstrated the same results. The present study implies that in vivo differentiation induced by PLGA-GCH/MS-TGF and the host microenviroment could keep chondral phenotype and enhance repair. It might serve as another way to induce and expand seed cells in cartilage tissue engineering.
Key words: Differentiation; Cartilage; Tissue engineering; Mesenchymal stem cells
Address correspondence to Yunyu Hu, Ph.D., M.D., Institute of Orthopaedics & Traumatology, Xijing Hospital, The Fourth Military Medical University, Xi'an, PR China. Tel: 86-29 84775291; Fax: 86-29 84775573; E-mail: Orth1_HuYY@yahoo.com
Subcultured Odontogenic Epithelial Cells in Combination With Dental Mesenchymal Cells Produce Enamel-Dentin-Like Complex Structures
M. J. Honda,1 Y. Shinohara,1 K. I. Hata,2 and M. Ueda13
1Tooth Regeneration, Division of Stem Cell Engineering, The
Institute of Medical Science, The University of Tokyo, Tokyo 108-8639,
2Japan Tissue Engineering Co. Ltd, Aichi 443-0022, Japan
3Department of Oral and Maxillofacial Surgery, Nagoya University Postgraduate School of Medicine, Aichi 466-8550, Japan
We showed in a previous study that odontogenic epithelial cells can be selectively cultured from the enamel organ in serum-free medium and expanded using feeder layers of 3T3-J2 cells. The subcultured odontogenic epithelial cells retain the capacity for ameloblast-related gene expression, as shown by semiquantitative RTPCR. The purpose of the present study was to evaluate the potential of subcultured odontogenic epithelial cells to form tooth structures in cell-polymer constructs maintained in vivo. Enamel organs from 6-monthold porcine third molars were dissociated into single odontogenic epithelial cells and subcultured on feeder layers of 3T3-J2 cells. Amelogenin expression was detected in the subcultured odontogenic epithelial cells by immunostaining and Western blotting. The subcultured odontogenic epithelial cells were seeded onto collagen sponge scaffolds in combination with fresh dental mesenchymal cells, and transplanted into athymic rats. After 4 weeks, enamel-dentin-like complex structures were present in the implanted constructs. These results show that our culture system produced differentiating ameloblast-like cells that were able to secrete amelogenin proteins and form enamel-like tissues in vivo. This application of the subculturing technique provides a foundation for further tooth-tissue engineering and for improving our understanding of ameloblast biology.
Key words: Ameloblast; Enamel-dentin complex; Odontogenic epithelial cells; Subculture; Tissue engineering
Address correspondence to M. J. Honda, Tooth Regeneration, Division of Stem Cell Engineering, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 Japan. Tel: +81 3 5449 5120; Fax: +81 3 5449 5121; E-mail: firstname.lastname@example.org
Efficient Intracytoplasmic Labeling of Human Umbilical Cord Blood Mesenchymal Stromal Cells With Ferumoxides
Jae Kwon Lee,1,2 Man Kyoung Lee,1 Hye Jin Jin,1 Dal-Soo Kim,1 Yoon Sun Yang,1 Wonil Oh,1 Sung-Eun Yang,1 Tae Seok Park,3 Soo Yeol Lee,3 Bum-Soo Kim,4 and Sin-Soo Jeun5
1Medipost Biomedical Research Institute, Medipost Co. Ltd.,
Asan Institute of Life Science, Seoul, 138-736, Republic of Korea
2School of Science Education (Biology Education), College of Education, Chungbuk National University, Chungcheongbuk-Do, 361-763, Republic of Korea
3Department of Biomedical Engineering, Kyung Hee University, Seoul, 130-701, Republic of Korea
4Department of Radiology, College of Medicine, The Catholic University of Korea, Seoul, 137-701, Republic of Korea
5Department of Neurosurgery, College of Medicine, The Catholic University of Korea, Seoul, 137-701, Republic of Korea
Mesenchymal stromal cells (MSCs) are multipotent cells found in several adult tissues; they have the capacity to differentiate into mesodermal, ectodermal, and endodermal tissues in vitro. There have been several reports that MSCs have therapeutic effects in a variety of diseases. Therefore, using a cell labeling technique, monitoring their temporal and spatial migration in vivo, would be useful in the clinical setting. Magnetic resonance imaging (MRI)-tracking of superparamagnetic iron oxide (SPIO)-labeled cells-is a noninvasive technique for determining the location and migration of transplanted cells. In the present study, we evaluated the influence and toxicity of SPIO (ferumoxides) labeling on multiple differentiated MSCs. To evaluate the influence and toxicity of ferumoxides labeling on differentiation of MSCs, a variety of concentrations of ferumoxides were used for labeling MSCs. We found that the cytoplasm of adherent cells was effectively labeled at low concentrations of ferumoxides. Compared with unlabeled controls, the ferumoxides-labeled MSCs exhibited a similar proliferation rate and apoptotic progression. The labeled MSCs differentiated into osteoblasts and adipocytes in an identical fashion as the unlabeled cells. However, chondrogenesis and neurogenesis were inhibited at high concentrations of ferumoxides. Our results suggest the effective concentration for ferumoxides use in tracking MSCs.
Key words: Umbilical cord blood; Mesenchymal stromal cells; Superparamagnetic iron oxide; Ferumoxides; Multilineage differentiation
Address correspondence to Sin-Soo Jeun, Department of Neurosurgery, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea. Tel: +82-2-590-2568; Fax: +82-2-594-4248; E-mail: email@example.com
Isolation of Mouse Hepatocytes for Transplantation: A Comparison Between Antegrade and Retrograde Liver Perfusion
Zhaohui Yin,1,2 Ewa C. S. Ellis,3 and Greg Nowak1
1Division of Transplantation Surgery, Department for Clinical
Science, Intervention and Technology CLINTEC, Karolinska University Hospital,
2Department of General Surgery, First Hospital of Shantou University Medical College, Guangdong Province, China
3Department of Pathology, University of Pittsburgh, Pittsburgh, PA 15161, USA
We compared antegrade with retrograde liver perfusion when isolating mouse hepatocytes for hepatocyte transplantation. Male mouse hepatocytes were isolated by different perfusion methods and transplanted into the spleen of congeneic female mice. Retrograde perfusion yielded a larger number of cells (4.90 x 107) than antegrade (4.09 x 107, p < 0.05), but hepatocytes obtained by antegrade perfusion gave higher engraftment efficiency (p < 0.05). More of the transplanted hepatocytes could be recovered from recipient liver with antegrade perfusion than with retrograde perfusion (p < 0.05). Our results indicate that hepatocytes isolated by antegrade perfusion gave a higher engraftment efficiency.
Key words: Hepatocyte transplantation; Liver perfusion; Real-time PCR; Sex-determining region Y (Sry); Mouse
Address correspondence to Greg Nowak, Division of Transplantation Surgery, Department for Clinical Science, Intervention and Technology CLINTEC, Karolinska University Hospital, Huddinge B56, 141 86 Stockholm, Sweden. E-mail: firstname.lastname@example.org
Stem Cell Research in Cell Transplantation: An Analysis of Geopolitical Influence by Publications
David J. Eve and Paul R. Sanberg
Center of Excellence for Aging and Brain Repair, Department of Neurosurgery, University of South Florida, College of Medicine, Tampa, FL, USA
One of the fastest growing fields in researching treatments for neurodegenerative and other disorders is the use of stem cells. These cells are naturally occurring and can be obtained from three different stages of an organism's life: embryonic, fetal, and adult. In the US, political doctrine has restricted use of federal funds for stem cells, enhancing research towards an adult source. In order to determine how this legislation may be represented by the stem cell field, a retrospective analysis of stem cell articles published in the journal Cell Transplantation over a 2-year period was performed. Cell Transplantation is considered a translational journal from preclinical to clinical, so it was of interest to determine the publication outcome of stem cell articles 6 years after the US regulations. The distribution of the source of stem cells was found to be biased towards the adult stage, but relatively similar over the embryonic and fetal stages. The fetal stem cell reports were primarily neural in origin, whereas the adult stem cell ones were predominantly mesenchymal and used mainly in neural studies. The majority of stem cell studies published in Cell Transplantation were found to fall under the umbrella of neuroscience research. American scientists published the most articles using stem cells with a bias towards adult stem cells, supporting the effect of the legislation, whereas Europe was the leading continent with a bias towards embryonic and fetal stem cells, where research is "controlled" but not restricted. Japan was also a major player in the use of stem cells. Allogeneic transplants (where donor and recipient are the same species) were the most common transplants recorded, although the transplantation of human-derived stem cells into rodents was the most common specific transplantation performed. This demonstrates that the use of stem cells is an increasingly important field (with a doubling of papers between 2005 and 2006), which is likely to develop into a major therapeutic area over the next few decades and that funding restrictions can affect the type of research being performed.
Key words: Stem cells; Transplants; Embryonic; Fetal; Adult; Trophic factors
Address correspondence to Dr. David Eve, Ph.D., Center of Excellence for Aging and Brain Repair, Department of Neurosurgery MDC-78, University of South Florida, 12901 Bruce B. Downs Blvd., Tampa, FL 33612, USA. Tel: 813-974-3154; Fax: 813-974-3078; E-mail: email@example.com