ognizant Communication Corporation


VOLUME 12, NUMBER 4, 2003

Cell Transplantation, Vol. 12, pp. 329-334, 2003
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Stem Cell Therapies in Reparative Medicine

Juan Domínguez-Bendala and Camillo Ricordi

Diabetes Research Institute, University of Miami School of Medicine, Miami, FL 33136

The future implementation of stem cell therapies to treat conditions thus far considered incurable has been envisioned as logical consequence of the fast-paced progress in stem cell research over the last few years. Still, many practical obstacles stand in the way to the routine application of these novel technologies in medicine. The conference "Stem Cell Therapies in Reparative Medicine," held aboard the cruise vessel Majesty of the Seas (Miami, USA- Nassau, Bahamas, April 19-22, 2002), focused on the analysis of these problems from different perspectives, including developmental biology (cell proliferation, fate determination, and enrichment), immunology (allorejection and prevention of autoimmunity recurrence), and clinical therapy, emphasizing the impact of stem cell technologies on the emerging field of tissue engineering and the treatment of alpha-1 antitrypsin deficiency.

Key words: Human embryonic stem cells; Diabetes; Alpha-1 antitrypsin; Development; Mesenchymal stem cells

Address correspondence to Camillo Ricordi, M.D., Diabetes Research Institute (R-134), 1450 NW 10 Avenue, Miami, FL 33136. Tel: (305) 243-6913; Fax: (305) 243-4404; E-mail: ricordi@miami.edu

Cell Transplantation, Vol. 12, pp. 335-349, 2003
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The Testicular-Derived Sertoli Cell: Cellular Immunoscience to Enable Transplantation

Dwaine F. Emerich,1 Richelle Hemendinger,2 and Craig R. Halberstadt2

1Sertoli Technologies, Inc, Cranston RI
2Department of General Surgery and The Transplant Center, Carolinas Medical Center, Charlotte, NC

There is a renewed enthusiasm for the potential of cellular transplantation as a therapy for numerous clinical disorders. The revived interest is largely due to the unprecedented success of the "Edmonton protocol," which produced a 100% cure rate for type I diabetics following the transplantation of human islet allografts together with a modified immunosuppressive regimen. While these data provide a clear and unequivocal demonstration that transplantation is a viable treatment strategy, the shortage of suitable donor tissue together with the debilitating consequences of lifelong immunosuppression necessitate a concerted effort to develop novel means to enable transplantation on a widespread basis. This review outlines the use of Sertoli cells to provide local immunoprotection to cografted discordant cells, including those from xenogeneic sources. Sertoli cells are normally found in the testes where one of their functions is to provide local immunologic protection to developing germ cells. Isolated Sertoli cells 1) engraft and self-protect when transplanted into allogeneic and xenogeneic environments, 2) protect cografted allogeneic and xenogeneic cells from immune destruction, 3) protect islet grafts to reverse diabetes in animal models, 4) enable survival and function of cografted foreign dopaminergic neurons in rodent models of Parkinson's disease (PD), and 5) promote regeneration of damaged striatal dopaminergic circuitry in those same PD models. These benefits are discussed in the context of several potential underlying biological mechanisms. While the majority of work to date has focused on Sertoli cells to facilitate transplantation for diabetes and PD, the generalized ability of these unique cells to potently suppress the local immune environment opens additional clinical possibilities.

Key words: Sertoli cells; Immune environment; Immunoprotection; Cografting; Allogeneic cells; Xenogeneic cells; Biological mechanisms; Diabetes; Parkinson's disease

Address correspondence to Dwaine F. Emerich, Ph.D., Sertoli Technologies, Inc., 766 Laten Knight Rd., Cranston, RI 02921. Tel: (401) 785-6961; E-mail: ED3FJM@aol.com

Cell Transplantation, Vol. 12, pp. 351-364, 2003
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Evaluation of Different Types of Alginate Microcapsules as Bioreactors for Producing Endostatin

A. M. Rokstad,1 B. Strand,2 K. Rian,1 B. Steinkjer,1 B. Kulseng,1 G. Skjåk-Bræk,2 and T. Espevik1

1Department of Clinical and Molecular Medicine and 2Institute of Biotechnology, Norwegian University of Science and Technology, Trondheim, Norway

The use of nonautologous cell lines producing a therapeutic substance encapsulated within alginate microcapsules could be an alternative way of treating different diseases in a cost-effective way. Malignant brain tumors have been proposed to be treated locally using engineered cells secreting proteins with therapeutic potential encapsulated within alginate microcapsules. Optimization of the alginate capsule bioreactors is needed before this treatment can be a reality. Recently, we have demonstrated that alginate-poly-L-lysine microcapsules made with high-G alginate and a gelled core disintegrated as cells proliferated. In this study we examined the growth and endostatin secretion of 293-EBNA (293 endo) cells encapsulated in six different alginate microcapsules made with native high-G alginate or enzymatically tailored alginate. Stability studies using an osmotic pressure test showed that alginate-poly-L-lysine-alginate microcapsules made with enzymatically tailored alginate was mechanically stronger than alginate capsules made with native high-G alginate. Growth studies showed that the proliferation of 293 endo cells was diminished in microcapsules made with enzymatically tailored alginate and gelled in a barium solution. Secretion of endostatin was detected in lower amounts from the enzymatically tailored alginate microcapsules compared with the native alginate microcapsules. The stability of the alginate microcapsules diminished as the 293 endo cells grew inside the capsules, while empty alginate microcapsules remained stable. By using microcapsules made of fluorescenamine-labeled alginate it was clearly visualized that cells perforated the alginate microcapsules as they grew, destroying the alginate network. Soluble fluorescence-labeled alginate was taken up by the 293 endo cells, while alginate was not detected in live spheroids within fluorescence-labeled alginate microcapsules. Despite that increased stability was achieved by using enzymatically tailored alginate, the cell proliferation destroyed the alginate microcapsules with time. It is therefore necessary to use cell lines that have properties more suited for alginate encapsulation before this technology can be used for therapy.

Key words: Enzymatically tailored alginate microcapsules; Stability; 293 cells; Endostatin; Fluorescence-labeled alginate

Address correspondence to Anne Mari Rokstad, Department of Clinical and Molecular Medicine, Medical Technical Center, Olav Kyrresgt. 3, 7489 Trondheim, Norway. Tel: (47) 73598666; Fax: (47) 73598801; E-mail: anne.m.rokstad@medisin.ntnu.no

Cell Transplantation, Vol. 12, pp. 365-377, 2003
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Overexpression of Telomerase Confers a Survival Advantage Through Suppression of TRF1 Gene Expression While Maintaining Differentiation Characteristics in K562 Cells

Osamu Yamada,1 Masaharu Akiyama,5 Kiyotaka Kawauchi,2 Tomoko Adachi,3 Hisashi Yamada,6 Naotoshi Kanda,7 and Eizo Aikawa4

1Medical Research Institute and Department of Hematology, 2Department of Medicine, 3Department of Gynecology, 4Department of Anatomy and Developmental Biology, Tokyo Women's Medical University
5Department of Pediatrics, 6Department of Molecular Genetics, Jikei University School of Medicine
7Department of Veterinary Anatomy, Tokyo University of Agriculture and Technology

Leukemic stem cells that expressed endogenous telomerase activity were induced to show overexpression of exogenous hTERT and were analyzed for biological changes in order to assess the possible influence of telomerase gene therapy on the transplantation of normal hematopoietic stem cells. Introduction of hTERT into K562, a telomerase-positive immortal cell line, resulted in a 2.5-fold elevation of telomerase activity and the lengthening of telomeres by 6 kb to 23 kb. Real-time fluorescent PCR, which could perform quantitative analysis of transcripts, revealed a 175-fold increase in hTERT expression, suggesting the posttranscriptional regulation of telomerase. Ectopic expression of hTERT in K562 cells showed a survival advantage during culture in the absence of serum. Expression of mRNA for the telomeric-repeat binding factor 1 (TRF1) and caspase-3 activity were both decreased in hTERT-transfected K562 cells. Transduced cells retained their usual phenotypic characteristics, differentiation ability, and signal transduction response to TPA. These data suggest that ectopic expression of hTERT by normal hematopoietic stem cells may confer a survival advantage without changing their innate biological characteristics.

Key words: Telomerase; Stem cell transplantation; Growth advantage; K562 cells

Address correspondence to Dr. Osamu Yamada, Medical Research Institute and Department of Hematology, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan. Tel: 81-3-3353-8111, ext. 30423; Fax: 81-3-5269-7308; E-mail: yamadao@lab.twmu.ac.jp

Cell Transplantation, Vol. 12, pp. 379-387, 2003
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Suppression of Allogeneic Response by Viral IL-10 Gene Transfer

Kenji Fujisawa, Shinya Saito, Yutaka Okada, Toshiyosi Fujiwara, Takahito Yagi, Hiromi Iwagaki, and Noriaki Tanaka

Department of Gastroenterological Surgery and Transplant, Okayama University Graduate School of Medicine and Dentistry, Okayama city, 700-8558 Japan

Th1 cell activation and cytokine production shift the balance between Th1 and Th2, favoring the upregulation of proinflammatory activity that leads to destruction of allogeneic hepatocytes following transplantation. Th2-type cytokines, such as IL-10, have immune regulatory function. The aim of this study was to determine the antirejection efficacy of allogeneic hepatocytes with spheroidal shape (spheroids) genetically modified with viral IL-10 (vIL-10). Allogeneic hepatocyte spheroids, transferred vIL-10 gene by using adenovirus as the vector, were transplanted into the spleen of Nagase's analbuminemic rats (NAR). NAR transplanted with vIL-10-transfected hepatocytes showed an abrupt rise in serum albumin levels that peaked on day 7 and remained at high levels up to day 21 after transplantation. The peak level of albumin on day 7 in vIL-10-transfected NAR was eminently higher than that in nontransfected NAR. Histopathological analysis revealed that in nontransfected NAR hepatocyte spheroids were more or less rejected on day 4, and, in contrast, vIL-10-transfected spheroids were still not rejected on day 14. This protective effect correlated with sustained high vIL-10 level in the splenic vein in NAR transplanted with vIL-10-transfected hepatocyte spheroids, suggesting that vIL-10 secreted from the transplanted hepatocytes induced an active suppression of allogeneic response. This study provides evidence to support the possibility of using vIL-10 gene therapy to prevent allogeneic response in hepatocyte transplantation.

Key words: Viral IL-10; Gene transfer; Allogeneic response; Hepatocyte transplantation

Address correspondence to Dr. Shinya Saito, Department of Gastroenterological Surgery and Transplant, Okayama University Graduate School of Medicine and Dentistry (Surgery I), 2-5-1 Shikata, Okayama 700-8558, Japan. Tel: +81-86-235-7255; Fax: +81-86-221-8775; E-mail: shinya@cc.okayama-u.ac.jp

Cell Transplantation, Vol. 12, pp. 389-398, 2003
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Transplants of Rat Chondrocytes Evoke Strong Humoral Response Against Chondrocyte-Associated Antigen in Rabbits

Anna Osiecka-Iwan, Anna Hyc, Jaroslaw Józwiak, Aldona Komar, Justyna Niderla, and Stanislaw Moskalewski

Department of Histology and Embryology, Medical University of Warsaw, Pl-02004 Warsaw, Poland

Rat chondrocytes transplanted intramuscularly in rabbits produced cartilage. In 1-day-old transplants chondrocytes remained viable. After 1 week peripheral chondrocytes of the transplant were dead and the cartilage was surrounded and resorbed by macrophages. In 2-week-old transplants cartilage deteriorated and was invaded by fibroblast-like cells and macrophages. Sera of rabbits that received two or three consecutive transplants of rat chondrocytes with 2-week intervals contained high titer of antichondrocyte cytotoxic antibodies. A part of the cytotoxic activity could be removed by absorption with rat splenocytes. Western blot analysis of lysates from fresh or 24-h cultured chondrocytes with absorbed sera detected antigen with Mr of ~74 and ~23 kDa. Only the latter remained after reduction in 2-mercaptoethanol. In lysates of fibroblasts and endotheliocytes the 23-kDa antigen was not found but the serum reacted with Mr 39-kDa antigen. In lysates of thymocytes a weak band corresponding to Mr of 35 kDa was present. Serum from rabbits receiving transplants of living chondrocytes followed by chondrocytes suspended in complete Freund's adjuvant contained antibodies directed against components of crude collagenase used for cell isolation. Such antibodies could not be detected in sera of rabbits receiving transplants of living chondrocytes only. Molecular weight of detected antigen differs from that of collagen type II, core of aggrecan, link proteins, and several other macromolecules of cartilage matrix. It could represent either a component of chondrocyte membrane or a membrane-bound substance resistant to enzymes used for isolation. Availability of antibodies against presumably chondrocyte-specific antigen produced during transplant rejection may help to characterize it more precisely and to ascertain whether its presence may influence results of autogenous chondrocyte transplants in humans.

Key words: Xenogeneic chondrocyte transplantation; Antichondrocyte cytotoxic antibodies; Chondrocyte-associated antigen

Address correspondence to Stanislaw Moskalewski, Department of Histology and Embryology, Medical University of Warsaw, Chalubinskiego 5, Pl-02004 Warsaw, Poland. Fax: 0048 22629-52-82; E-mail: smosk@ib.amwaw.edu.pl

Cell Transplantation, Vol. 12, pp. 399-406, 2003
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Fast Hematopoietic Recovery After Bone Marrow Engraftment Needs Physiological Proximity of Stromal and Stem Cells

Slawa Janczewska, Marcin Wisniewski, Stanislaw M. Stepkowski, and Barbara Lukomska

Surgical Research and Transplantology Department, Medical Research Center, Polish Academy of Sciences, Warsaw, Poland
Department of Surgery, The University of Texas Medical School, Houston, TX

Relatively slow hematopoietic recovery after isolated bone marrow (I-BM) engraftment is probably caused by a disrupted microenvironment of stromal and stem cells. Thus, we compared the kinetics of hematopoietic recovery of lethally irradiated rats that received I-BM versus vascularized BM (V-BM). Total body irradiated (TBI; 8 Gy) Lewis (LEW; RT11) rats were either injected IV with syngeneic sex-mismatched 80 x 106 I-BM or transplanted with 80 x 106 V-BM in orthotopic hind limb grafts. Ten days later, peripheral blood (PB) and mesenteric lymph nodes (MLN) of these recipients were examined for the presence of donor-derived hematopoietic cells with a panel of monoclonal antibodies by FACS. To detect male cells in sex-mismatched female recipients, PCR was performed using male Y chromosome primers. When examined in PB and MLN, recipients transplanted with V-BM displayed significantly faster recovery of leukocytes (CD43+), monocytes (CD14+), and T cells (CD5+) in comparison with I-BM recipients. In addition, only V-BM (but not I-BM) groups contained stroma-like male-positive cells in PB and MLN. Our results suggest that V-BM transplants provided superior hematopoietic recovery in comparison to I-BM transplants. We postulated that close proximity between stromal and stem cells in V-BM is essential for efficient repopulation with progenitors of different lines of leukocytes.

Key words: Hematopoiesis; Bone marrow stromal cells; Bone marrow transplantation

Address correspondence to Slawa Janczewska, Division of Immunology & Organ Transplantation, UT-Houston Medical School, 6431 Fannin St., Houston, TX 77030. Tel: (713) 500-7372; Fax: (713) 500-0784; E-mail: Slawa.Janczewska@uth.tmc.edu

Cell Transplantation, Vol. 12, pp. 407-412, 2003
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Improved Survival of Macroencapsulated Islets of Langerhans by Preimplantation of the Immunoisolating Device: A Morphometric Study

E. Rafael,1 G. S. Wu,1 K. Hultenby,2 A. Tibell,1 and A. Wernerson2

Departments of 1Transplantation Surgery and 2Pathology, Karolinska Institute, Huddinge University Hospital, Stockholm, Sweden

Encapsulation of cells in a semipermeable membrane may in the future provide an opportunity to treat a variety of endocrine and neurological disorders, without the need for lifelong immunosuppression. The physiological conditions in the device are crucial factors for graft survival. Previously, we have shown that the exchange across the immunoisolating membrane and the microcirculation around the TheraCyteT device increase around 3 months after implantation. The aim of this study was to determine whether preimplantation of the TheraCyteT device would improve the survival of a later transplanted islet graft. A TheraCyteT device was implanted SC on one side of the back of a nondiabetic SD rat. After 3 months, 1500 islets isolated from SD rats were transplanted via the device port. At the same time, another device, loaded with the same number of islets, was implanted on the other side of the back. Both devices were explanted 2 weeks after islet transplantation (i.e., 3.5 months and 0.5 month after device implantation, respectively). Six pairs of devices were evaluated by morphometery. The volume densities of viable islets were 0.22 ± 0.04 in the preimplanted device vs. 0.06 ± 0.03 in the nonpreimplanted one (p < 0.05). The corresponding volume densities of fibrosis and necrosis were 0.64 ± 0.13 vs. 0.85 ± 0.08 (p < 0.05) and 0.11 ± 0.14 vs. 0.09 ± 0.07 (ns), respectively. When the absolute volumes (mm3) were calculated, preimplanted devices contained 1.1 ± 0.7 endocrine cells while nonpreimplanted ones contained 0.4 ± 0.2 (p < 0.05). The percentages of insulin-positive b-cells in the preimplanted versus nonpreimplanted device were 80 ± 5% and 67 ± 6%, respectively (p < 0.01). The corresponding volumes of fibrotic tissue were 3.0 ± 1.8 vs. 5.2 ± 1.2 (p < 0.05), while the amount of necrotic tissue did not differ significantly (0.42 ± 0.5 vs. 0.50 ± 0.3). Preimplantation of the TheraCyteT device seems to improve the survival of an encapsulated islet graft and reduce fibroblast outgrowth in the device.

Key words: Islet transplantation; Macroencapsulation; Diabetes; Rat; Morphometry

Address correspondence to Dr. Ehab Rafael, Department of Transplantation Surgery, B56, Karolinska Institute, Huddinge University Hospital, 141 86 Stockholm, Sweden. Tel: + 46 8 58 58 00 00; Fax: + 46 8 774 31 91; E-mail: ehab.rafael@cfss.ki.se

Cell Transplantation, Vol. 12, pp. 413-421, 2003
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De novo Liver Tissue Formation in Rats Using a Novel Collagen-Polypropylene Scaffold

Yukinobu Takimoto,* Vivek Dixit,* Marika Arthur, and Gary Gitnick

University of California, Los Angeles, Department of Medicine, Division of Digestive Diseases, UCLA School of Medicine, Center for the Health Sciences, Los Angeles, CA 90095-7019

In experimental and clinical settings hepatocyte transplantation has provided limited benefit to patients with chronic liver disease because the transplanted hepatocytes were short-lived and were merely maintained for a brief period within the body. Except for whole-liver transplantation, creation of de novo liver tissue is necessary to treat this condition on a long-term basis. The aim of this study was to facilitate the formation of new tissue by actual self-regeneration, rather than by compensatory hypertrophy, or scar formation, with our collagen-polypropylene composite scaffold. Collagen-polypropylene composite scaffolds, not containing hepatocytes, were implanted into the median liver lobe and the dynamics of new liver tissue formation was analyzed immunohistochemically over a 6-month period. Control scaffolds consisted of polypropylene scaffolds without collagen matrix. The control scaffold implants remained hollow throughout the study period and became encapsulated with a hard connective tissue capsule 1 week after implantation. In contrast, the collagen-polypropylene composite scaffold was filled with regenerating tissue structures 3 weeks after implantation. At this time, the predominant cell type within the scaffold was sesmin-positive stellate cells. A week earlier, oval cells were identified using monoclonal antibody staining (OV-6). Subsequently, these cells differentiated into a-fetoprotein-positive immature hepatocytes. After 6 months, mature liver tissue, juxtaposed with bile ducts and blood vessels, was seen within the polypropylene scaffolds. We report the first evidence of de novo formation of liver tissue within a polypropylene scaffold, following implantation in the liver. This scaffold may play a role in treating chronic liver diseases requiring organ replacement therapy.

Key words: Hepatic stellate cells; Oval cells; Extracellular matrix; Nonparenchymal cells; Liver regeneration

*Co-first authors. Equal research and writing contribution were made by both of these authors.

Address correspondence to Vivek Dixit, Ph.D., Department of Medicine, University of California, Los Angeles, Division of Digestive Diseases, UCLA School of Medicine, Center for the Health Sciences, 675 Charles E. Young Drive, Room 1240, Los Angeles, CA 90095-7019. Tel: (310) 206-3326; Fax: (310) 206-7333; E-mail: vdixit@mednet.ucla.edu

Cell Transplantation, Vol. 12, pp. 423-438, 2003
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Evaluation of 2-Year-Old Intrasplenic Fetal Liver Tissue Transplants in Rats

Amelie Lupp,1 Manfred Danz,2 and Dieter Müller1

1Institute of Pharmacology and Toxicology, 2Department of Anatomy I, Friedrich Schiller University Jena, D-07743 Jena, Germany

Liver cell transplantation into host organs like the spleen may possibly provide a temporary relief after extensive liver resection or severe liver disease or may enable treatment of an enzyme deficiency. With time, however, dedifferentiation or malignant transformation of the ectopically transplanted cells may be possible. Thus, in the present study syngenic fetal liver tissue suspensions were transplanted into the spleen of adult male rats and evaluated 2 years thereafter in comparison to orthotopic livers for histopathological changes and (as markers for preneoplastic transformation) for cytochrome P450 (P450) and glutathione S-transferase (GST) isoform expression. Because inducibility of P450 and GST isoforms may be changed in preneoplastic foci, prior to sacrifice animals were additionally treated either with \GK\b-naphthoflavone, phenobarbital, dexamethasone, or the respective solvent. In the 2-year-old grafts more than 70% of the spleen mass was occupied by the transplant. The transplanted hepatocytes were arranged in cord-like structures. Also few bile ducts were present. Morphologically, no signs of malignancy were visible. With all rats, transplant recipients as well as controls, however, discrete nodular structures were seen in the livers. Due to age, both livers and transplants displayed only a low P450 2B1 and 3A2 and GST class a and m isoform expression. No immunostaining for P450 1A1 was visible. At both sites, b-naphthoflavone, phenobarbital, or dexamethasone treatment enhanced P450 1A1, P450 2B1 and 3A2, or P450 3A2 expression, respectively. No immunostaining for GST class p isoforms was seen in the transplants. The livers of both transplant recipients and control rats, however, displayed GST p-positive foci, corresponding to the nodular structures seen histomorphologically. Compared to the surrounding tissue, these foci also exhibited a more pronounced staining for GST class a and m isoforms and a stronger inducibility of the P450 1A1 expression due to b-naphthoflavone. In conclusion, in contrast to the livers, no preneoplastic foci seem to appear in the intrasplenic transplants even 2 years after transplantation. This may be due either to the protection of these transplants by the orthotopic livers or to the different humoral and nerval influences at the ectopic site.

Key words: Hepatocytes; Spleen; Transplantation; Carcinogenesis; Cytochrome P450; Glutathione S-transferase

Address correspondence to Dr. med. Amelie Lupp, Institute of Pharmacology and Toxicology, Friedrich Schiller University Jena, Nonnenplan 4, D-07743 Jena, Germany. Tel: +49-3641-938718; Fax: +49-3641-938702; E-mail: alup@mti-n.uni-jena.de

Cell Transplantation, Vol. 12, pp. 439-446, 2003
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Effect of Adhesion or Collagen Molecules on Cell Attachment, Insulin Secretion, and Glucose Responsiveness in the Cultured Adult Porcine Endocrine Pancreas: A Preliminary Study

Kazuya Edamura,1,2 Koko Nasu,1 Yukiko Iwami,1 Hiroyuki Ogawa,2 Nobuo Sasaki,3 and Hisako Ohgawara1

1Division of Cell Replacement and Regenerative Medicine, Medical Research Institute, School of Medicine, Tokyo Women's Medical University
2Laboratories of Veterinary Emergency Medicine and 3Veterinary Surgery, Graduate School of Agricultural and Life Sciences, The University of Tokyo

The effect of either adhesion or collagen molecules on cell attachment, insulin secretion, and glucose responsiveness was investigated in adult porcine pancreatic endocrine (PE) cells that were cultured for a longer term in vitro. Six different types of molecules--laminin, fibronectin, poly-L-lysine (PLL), type I collagen, gelatin, and Matrigel--were used. Approximately 2.0 x 105 cells per dish of each molecule type were cultured for 4 weeks. In the laminin group, the insulin accumulation was maintained at a significantly higher level than in the control group at 4 weeks of culture, and glucose-stimulated insulin secretion and the insulin-positive rate were also higher than in the control group. In the Matrigel group, islet-like cell clusters were formed, but insulin accumulation rapidly decreased at 3-4 weeks of culture. A large number of PE cells attached tightly and spread in the fibronectin group until the fourth week of culture, but their function was not better than those in the control group. In the PLL and gelatin groups, the PE cell function was not significantly different from that of the control group. In the type I collagen group, insulin secretion was inferior to that of the other groups. The results of this study suggest that laminin is the most suitable extracellular matrix for the long-term culture preservation of PE cells.

Key words: Adhesion molecules; Extracellular matrix; Laminin; Monolayer culture; Porcine pancreatic endocrine cells; Preservation

Address correspondence to Hisako Ohgawara, M.D., Division of Cell Replacement and Regenerative Medicine, Medical Research Institute, School of Medicine, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan. Tel: +81-3-5269-7364; Fax: +81-3-5269-7364; E-mail: hisakooh@ lab.twmu.ac.jp

Cell Transplantation, Vol. 12, pp. 449-454, 2003
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Mobilized Peripheral Blood Cells Administered Intravenously Produce Functional Recovery in Stroke

Alison E. Willing,1,2,3,4,5* Martina Vendrame,1,5 Jennifer Mallery,1,2 C. Jordan Cassady,1,2 Cyndy D. Davis,8 Juan Sanchez-Ramos,1,6* and Paul R. Sanberg1,2,4,5,7*

1Center of Excellence for Aging & Brain Repair, Departments of 2Neurosurgery, 3Anatomy, 4Pharmacology and Therapeutics, 5Pathology, 6Neurology, and 7Psychiatry, University of South Florida College of Medicine, 12901 Bruce B. Downs Blvd, Tampa, FL 33612
8Saneron CCEL Therapeutics, Inc., 13101 Telecom Park, Suite 105, Tampa FL 33617

Filgratism (granulocyte colony stimulating factor, G-CSF)-mobilized peripheral blood progenitor cells (PBPCs) have replaced bone marrow (BM) as a preferred source of autologous stem cells, in light of the faster hematologic recovery and lesser supportive care requirement exhibited by PBPC transplants. Other hematopoietic stem cells, like the human umbilical cord blood-derived stem cells (hUCBs), and nonhematopoietic stem cells have been shown to improve motor function in rodent models of injury and degenerative disease. In the present study we transplanted either G-CSF-mobilized PBPCs or hUCBs in rats 24 h after permanent middle cerebral artery occlusion (MCAO), and assessed their behavioral abnormalities in spontaneous activity and spontaneous motor asymmetry. In both transplanted groups of rats we observed a significant reduction of the stroke-induced hyperactivity compared with nontransplanted, stroked animals. In addition, transplantation of G-CSF PBPC and hUCB cells prevented the development of extensive motor asymmetry. Our findings raise the possibility that PBPCs could provide a novel transplantation therapy to treat stroke.

Key words: Peripheral blood; Stem cells; Filgrastim; G-CSF; Stroke; Middle cerebral artery occlusion; Behavioral analysis

Address correspondence to Alison E. Willing, Center of Excellence for Aging & Brain Repair, MDC 78, University of South Florida College of Medicine, 12901 Bruce B. Downs Blvd., Tampa, FL 33612. Tel: (813) 974-7812; Fax: (813) 974-6352; E-mail: awilling@hsc.usf.edu

*A.E.W. and J.S.-R. are consultants to Saneron CCEL Therapeutics, Inc. P.R.S. is cofounder of Saneron CCEL Therapeutics, Inc.