ognizant Communication Corporation

Cell Transplantation, Vol. 16, pp. 587-598, 2008
0963-6897/08 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2008 Cognizant Comm. Corp.
Printed in the USA. All rights reserved..

¹CORIT (Consorzio per la Ricerca sul Trapianto d’Organi), Padua, Italy
²Department of Medical and Surgical Sciences, University of Padua, Padua, Italy
³Department of Internal Medicine, Section of Internal Medicine and Endocrine and Metabolic Sciences, University of Perugia, Perugia, Italy
⁴Department of Clinical Veterinary Science, University of Padua, Legnaro, Italy
⁵Direzione Sanitaria, Padua General Hospital, Padua, Italy
⁶Clinica Chirurgica III, Padua General Hospital, Padua, Italy

Apoptotic phenomena observed in vitro following isolation and following transplantation contribute significantly to islet graft loss. Strategies to reduce apoptosis of islet tissue prior to and posttransplantation may improve graft survival and function and reduce the amount of tissue necessary to achieve insulin independence. The expression of cytoprotective proteins is one such strategy that may prolong islet survival. In this light, heme-oxygenase 1 (HO-1) upregulation has been studied in both allo- and xenotransplantation models. In this study, the effect of HO-1 on apoptosis in neonatal porcine islet-like cell clusters (NPICC) was assessed. In in vitro assessments of NPICC apoptosis, NPICC showed a high sensitivity to apoptotic stimulation using a combination of TNF-a and cycloheximide. Stimulation with TNF-a alone was sufficient to induce reproducible apoptotic responses as demonstrated by caspase-3,-7 activation and subdiploid DNA analysis. Dose-dependent, high-level HO-1 protein expression was achieved following culture of NPICC in medium containing either cobalt protoporphyrin (CoPP) or cobalt mesoporphyrin (CoMP). CoPP treatment resulted in the reduction of caspase-3,-7 enzyme activity following TNF-a stimulation. However, such an effect was not associated with a reduction in the levels of cell death. Indeed, the inhibition of caspase enzyme activity resulted in decreased PARP-1 cleavage, which may lead to heightened levels of necrosis in treated NPICC cultures, possibly explaining the observed commitment of NPICC to the death pathway.

Key words: Islet; Porcine; Apoptosis; Necrosis; Metalloporphyrin

Address correspondence to Erika Bosio, Ph.D., Laboratori CORIT, Via dell’Università, 10, Legnaro (PD), 35020 Italy. Tel: +39 049 808 4364; Fax: +39 049 808 4361; E-mail: erika.bosio@unipd.it

¹Surgical-Medical Research Institute, Department of Surgery, University of Alberta, Edmonton, Alberta, Canada
²Barbara Davis Centre for Childhood Diabetes, University of Colorado Health Sciences Center, Aurora, CO, USA

Previously we demonstrated that anti-LFA-1 monoclonal (mAb) could promote long-term survival of discordant porcine islet xenografts in mice. The aim of this study, therefore, was to determine whether a short-term administration of anti-LFA-1 mAb would promote long-term survival of concordant rat islet xenografts in mice, and whether combining short-term administration of anti-LFA-1 mAb therapy with an immunosuppressive drug, rapamycin, would facilitate islet xenograft survival. Streptozotocin-induced diabetic BALB/c mice were transplanted with 500 Wistar-Furth rat islets under the kidney capsule and were either left untreated or treated with short-term administration of rapamycin (0.2 mg/kg) alone, anti-LFA-1 mAb (0.2 mg/ dose) alone, or a combination of rapamycin and anti-LFA-1 mAb using the same doses. All untreated mice rejected their grafts by 24 days posttransplantation with a mean graft survival time of 18.8 ± 2.5 days posttransplantation (n = 5). All mice treated with rapamycin alone had prolonged islet graft survival but eventually rejected their islet grafts by 81 days posttransplantation. In contrast, the majority of the mice (27/28) treated with anti-LFA-1 mAb alone maintained long-term normoglycemia (>100 days). Rapamycin in combination with anti-LFA-1 mAb proved equally effective with 29 of 30 mice maintaining normoglycemia for more than 100 days posttransplantation. Low levels of mouse anti-rat antibodies, as well as a decrease in the degree of mononuclear cell infiltration of the islet graft, closely correlated with long-term islet xenograft survival. These results demonstrate that monotherapy with anti-LFA-1 mAb is highly effective in promoting long-term survival of rat islet xenografts and that combination of anti-LFA-1 mAb with rapamycin does not facilitate nor abrogate the induction of long-term xenograft survival by anti-LFA-1 mAb therapy in BALB/c mice. Our study indicates that immunomodulation through mAb therapy could form a significant component of future antirejection therapies in clinical islet xenotransplantation.

Key words: Rapamycin (sirolimus); Leukocyte function associated antigen-1 (LFA-1); Islet transplantation; Xenograft; Type 1 diabetes mellitus (T1DM); Rat islets

Address correspondence to Gina R. Rayat, Ph.D., Surgical-Medical Research Institute, Department of Surgery, 1074 Dentistry/Pharmacy Building, University of Alberta, Edmonton, Canada, T6G 2N8. Tel: 780-492-6894; Fax: 780-492-1627; E-mail: grayat@ualberta.ca

¹Department of Surgery, University of Ulsan College of Medicine and Asan Medical Center, Seoul, 138-736, Korea
²Institute for Cancer Research, Yonsei University College of Medicine, Seoul, 120-749, Korea

Islet transplantation is a potential cure for diabetes. However, allotransplant rejection severely limits its clinical application. In this study, we sought to transfect rat islets with an adenoviral vector containing the viral IL-10 (vIL-10) gene and examine its efficacy in preventing graft rejection. The immunosuppressive effect of vIL-10 is reported but its efficacy is somehow debatable in transplantation model. vIL-10 transfected islets were transplanted into streptozotocin-induced diabetic rats. Blood glucose, serum vIL-10 concentration, graft histology, and graft cytokine expression were used to monitor graft function up to day 21 after transplantation. Transfected islets released a large amount of vIL-10 protein without affecting their viability and functional integrity. When we transplanted the transfected islets into allogeneic hosts, the survival of grafted islets was not significantly increased. However, the combined use of vIL-10 and subtherapeutic doses of CsA (cyclosporine) significantly prolonged graft survival beyond that achieved with either agent alone (p < 0.001). vIL-10 and CsA-treated rats contain high level of vIL-10 in serum, which is evidenced by the inhibition of allogeneic mixed lymphocyte reaction (MLR). Histological analysis additionally revealed the presence of viable islets up to 21 days. IL-10 mRNA expression in grafted liver was higher and IFN-g mRNA was lower in vIL-10 and CsA-treated animals, compared with other groups. The synergistic effect of this combination therapy is potentially correlated with the induction of inhibitory cytokine secretion and downregulation of proinflammatory cytokine secretion from host cells.

Key words: Graft survival; Viral IL-10; Adenovirus; Islets; Immunosuppression

Address correspondence to Duck Jong Han, M.D., Department of Surgery, Ulsan University College of Medicine, 388-1 Poongnab-dong, Songpagu, Seoul, 138-736, Korea. Tel: 82-2-3010-3487; Fax: 82-2-3010-4182; E-mail: djhan@amc.seoul.kr

Department of Surgery I, Fukushima Medical University, Fukushima, 960-1295, Japan

One of the goals of islet transplantation is to transplant viable islets without host immunosuppression. The present study was designed to determine whether pretreatment of islets with mitomycin-C (MMC) followed by culture enhances islet survival in a rat-to-mouse xenogeneic combination. WS(RT1k) rat islets pretreated with various concentrations of MMC (0, 3.2, 10, 32, 100, 320, and 1000 mg/ml) were tested for viability by in vitro insulin secretory capacity and vital staining of islets. The MMC-treated islets (10 mg/ml) cultured for various periods (4, 20, or 40 h, 3 or 7 days) were transplanted into the renal subcapsular space of STZ-induced diabetic C57BL/6 (B6: H-2b) mice. MMC-treated or nontreated islets were subjected to microarray gene analysis and immunohistological study. Evaluation of in vitro insulin secretory capacity and vital staining of islets indicated that MMC at a dose <=32 mg/ml is nontoxic and preserves islet function. Marked prolongation of graft survival was noted with half of islet grafts surviving indefinitely (>100 days) when 10 mg/ml of MMC-treated islets was transplanted after 40 h or 3 days in culture, but not when they were transplanted within 4 h following treatment or at 7 days following treatment, indicating that there is a critical culture period necessary for successful islet graft survival. Microarray analysis suggested possible genes for this prolongation with TGF-b highly expressed in MMC-treated islets subjected to culture for 3 days. Our results indicate that MMC treatment followed by a critical culture period induces marked prolongation of rat islet xenograft survival in nonimmunosuppressed recipient mice, offering a strategy for islet transplantation without immunosuppression.

Key words: Mitomycin-C; Culture; Islet xenograft; Insulin secretion

Address correspondence to Mitsukazu Gotoh, M.D., Department of Surgery I, Fukushima Medical University, 1 Hikarigaoka, Fukushima, 960-1295, Japan. Tel: +81-24-547-1252; Fax: +81-24-548-2735; E-mail: mgotoh@fmu.ac.jp

¹Department of Medicine, University of Alberta, Edmonton, Alberta, T6G 2S2, Canada
²Clinical Islet Transplantation Group Inc., Edmonton, Alberta T6G 2C8, Canada

Pancreatic islet transplantation as a treatment for type 1 diabetes is limited by human donor tissue availability. We investigated whether the b-cell mass in human isolated islets could be expanded by treatments with glucagon-like peptide-1 (GLP-1) and gastrin, peptides reported to stimulate b-cell growth in mice and rats with deficits in b-cell mass. Human islets with low endocrine cell purity (7% b-cells, 4% a-cells) and abundant exocrine cells (29% duct cells and 25% acinar cells) were implanted under the renal capsule of nonobese diabetic-severe combined immune deficiency (NOD-scid) mice made diabetic with streptozotocin. The mice were treated with GLP-1 and gastrin, separately and together, daily for 5 weeks. Blood glucose was significantly reduced only in mice implanted with human pancreatic cells and treated with GLP-1 plus gastrin. Correction of hyperglycemia was accompanied by increased insulin content in the human pancreatic cell grafts as well as by increased plasma levels of human C-peptide in the mice. Immunocytochemical examination revealed a fourfold increase in insulin-positive cells in the human pancreatic cell grafts in GLP-1 plus gastrin-treated mice, and most of this increase was accounted for by the appearance of cytokeratin 19-positive pancreatic duct cells expressing insulin. We conclude that combination therapy with GLP-1 and gastrin expands the b-cell mass in human islets implanted in immunodeficient diabetic mice, largely from pancreatic duct cells associated with the islets, and this is sufficient to ameliorate hyperglycemia in the mice.

Key words: Diabetes; Islet transplantation; GLP-1; Gastrin

Address correspondence to Alex Rabinovitch, M.D., 430 Heritage Medical Research Centre, University of Alberta, Edmonton, Alberta T6G 2S2, Canada. Tel: 780-492-6791; Fax: 780-492-4666; E-mail: alex.rabinovitch@ualberta.ca

¹Division of Endocrinology and Metabolism, Department of Internal Medicine, Chang Gung University and Memorial Hospital, Taoyuan, Taiwan
²Department of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taiwan

Exendin-4 stimulates insulin secretion, suppresses glucagons secretion, increases b-cell replication and neogenesis, and reduces b-cell apoptosis. However, it has been shown that posttransplant exendin-4 treatment did not improve glucose homeostasis in diabetic mice transplanted with a large number of freshly isolated islets. The aim of this study was to test if exendin-4 is beneficial for hyperglycemic recipients with a marginal number of fresh islets. We transplanted 150 C57BL/6 mouse islets under the kidney capsule of inbred streptozotocin-diabetic mice, and then treated the recipients with and without exendin-4 for 6 weeks. Before and after transplantation, recipients’ blood glucose, body weight, and intraperitoneal glucose tolerance test were measured. At 6 weeks, the grafts were removed to determine b-cell mass. Blood glucose levels in both groups decreased progressively after transplantation, and the exendin-4-treated group had had lower blood glucose than controls since day 3. By 6 weeks, euglycemia was achieved more in mice treated with exendin-4 than in controls (100% vs. 62.5%, p = 0.018). The time to obtain normoglycemia was shorter in the exendin-4-treated group than in controls (12 ± 8 vs. 29 ± 13 days, p < 0.001). Blood glucose at 6 weeks was 123 ± 18 and 170 ± 62 mg/dl in the exendin-4-treated group and controls, respectively (p = 0.008). Additionally, the exendin-4-treated group had better glucose tolerance than controls at 2 and 4 weeks (p < 0.02). However, both groups exhibited increased body weight over time, and weight changes did not significantly differ between the two groups throughout the study period. At 6 weeks after transplantation, grafts in the exendin-4-treated group were more prominent and contained more insulin-stained cells than those of controls. They had 2.3-fold b-cell mass of the graft compared with controls (0.30 ± 0.11 vs. 0.13 ± 0.03 mg, p = 0.012). These results indicate posttransplant exendin-4 treatment in the diabetic recipient with a marginal number of fresh islets expands graft b-cell mass and improves transplantation outcome.

Key words: Diabetes mellitus; Islet transplantation; Exendin-4; b-Cell mass

Address correspondence to Jyuhn-Huarng Juang, M.D., Division of Endocrinology and Metabolism, Chang Gung Memorial Hospital, 5 Fu-Shin Street, Kweishan, Taoyuan, Taiwan. Tel/Fax: 886-3-3277976; E-mail: jjuang@adm.cgmh.org.tw

¹General Surgery, First Clinical College of Harbin Medical University, Harbin, P. R. China
²Second Surgery, Fujita Health University, Toyoake, Japan
³Baylor All Saints Islet Cell Laboratory, Baylor Research Institute, Fort Worth, TX, USA
⁴Kyoto University Hospital Transplantation Unit, Japan
⁵Diabetes Institute for Immunology and Transplantation, University of Minnesota, Minneapolis, MN, USA
⁶The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
⁷Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan

Recently, we demonstrated that islet transplantation from non-heart-beating donors (NHBDs) using the Kyoto islet isolation method (KIIM) successfully reversed patients’ diabetes state. In this study, we evaluated the effects of donor- and isolation-related variables on islet isolation results from NHBDs by KIIM. Twenty-one islet preparations from the pancreata of NHBDs were isolated by KIIM. Islet preparations that met transplantation criteria and achieved improved patient diabetes control after transplantation were defined as successful isolations. Potential risk factors deemed to affect islet isolation results, such as age, gender, body mass index, hospital stay, donors’ blood biochemical tests, a modified pancreata procurement method, and isolation and purification procedure-related variables, were analyzed. Seventeen out of 21 islet isolations (81%) were successful isolations. Postpurification islet yield was 447,639 ± 39,902 islet equivalents (IE) in the successful isolation group and 108,007 ± 31,532 IE in the failure group. Donor age was significantly younger in the success group (41.9 ± 4.0 years old in the success group vs. 57.5 ± 2.2 years old in the failure group, p = 0.003). Chronic pancreatitis significantly decreased islet yields (p = 0.006). Phase I time was significantly shorter (p = 0.010) and undigested tissue volume was significantly smaller (p = 0.020) in the success group. Purity was in positive correlation to postpurification islet yield, while donor age was in reverse correlation to postpurification islet yield. KIIM enables us to perform islet transplantation from NHBDs; however, the decision to use pancreata from older donors or those with chronic pancreatitis requires careful consideration.

Key words: Non-heart-beating donor; Kyoto islet isolation method; Pancreatic islet transplantation; Type 1 diabetes

Address correspondence to Shinichi Matsumoto, M.D., Ph.D., Baylor All Saints Islet Cell Laboratory, 1400 8th Avenue, Fort Worth, TX 76104, USA. Tel: 817-922-2570; Fax: 817-922-4645; E-mail: shinichm@baylorhealth.edu

¹Institute of Toxicology, College of Medicine, National Taiwan University, Taipei, Taiwan
²Division of Plastic and Reconstructive Surgery, Sun Yat-Sen Cancer Center, Taipei, Taiwan
³Department of Surgery, National Taiwan University Hospital, Taipei, Taiwan

Islet-like cell clusters (ICCs) have been suggested to be a source of insulin-producing tissue for xenotransplantation in type 1 diabetes. We designed an approach to maintain the cultured rat pancreatic ICC survival and function, when cocultured with human umbilical cord mesenchymal stem cells (HUMSCs). HUMSCs in coculture have the ability to maintain ICC survival and function, for which number and insulin secretion of ICCs are increasing and lasting for 3 months, while ICCs gradually crash, which results in cell death after a period of 12 days of culture without HUMSCs. Cytokine protein array showed it has more than a twofold increase in levels of several cytokines (interleukin-6, tissue inhibitor of metalloproteinases-1, tissue inhibitor of metalloproteinases-2, monocyte chemoattractant protein-1, growth related oncogene, hepatocyte growth factor, insulin-like growth factor binding proteins 4, and interleukin-8) on coculture medium, implying an important role of these cytokines in this coculture system. These findings suggest that coculture with HUMSCs may have a significant potential to protect ICCs from damage during culture, and may be employed in a novel culture approach to maintain islet cell survival and function before transplantation.

Key words: Coculture; Islet-like cell clusters; Human umbilical cord mesenchymal stem cells; Cytokines

*These three authors contributed equally to this work.

Address correspondence to Shing-Hwa Liu, Ph.D., Institute of Toxicology, College of Medicine, National Taiwan University, No. 1, Section 1, Jen-Ai Road, Taipei, 10051, Taiwan. Fax: (+886)-2-23410217; E-mail: shinghwaliu@ntu.edu.tw

¹Foundation onlus "Staminali e Vita", Institute of Biomedical Technologies, C.N.R., 56127 Pisa, Italy
²Department of Evolution and Experimental Biology, University of Bologna, 40126 Bologna, Italy
³Audiology Unit, Bioacoustic Center and National Institute of Neuroscience, University Hospital S. Anna, University of Ferrara, 44100 Ferrara, Italy
⁴Animal Model Facility, National Institute for Cancer Research (IST), 16132 Genua, Italy
⁵Bone Marrow Transplantation Center, Department of Haematology, University Hospital, 50134 Florence, Italy
⁶Histopathology Unit, Cancer Research UK, London Research Institute, London WC2A 3PX, UK

We investigated the fate of human cord blood CD133⁺ hematopoietic stem cells (HSC) transplanted intravenously (IV) into irradiated nod-scid mice previously made deaf by ototoxic treatment with kanamycin and/or intense noise, to verify whether HSC engraft the cochlea and contribute to inner ear restoration, in vivo. We tested the presence of HLA.DQa1 by PCR, used for traceability of engrafted cells, finding evidence that HSC migrated to various host tissues, including the organ of Corti (OC). By histology, antibody and lectin-staining analysis, we confirmed that HSC IV transplantation in mice previously damaged by ototoxic agents correlated with the repair process and stimulation ex novo of morphological recovery in the inner ear, while the cochlea of control oto-injured, nontransplanted mice remained seriously damaged. Dual color FISH analysis also provided evidence of positive engraftment in the inner ear and in various mouse tissues, also revealing small numbers of heterokaryons, probably derived from fusion of donor with endogenous cells, for up to 2 months following transplantation. These observations offer the first evidence that transplanted human HSC migrating to the inner ear of oto-injured mice may provide conditions for the resumption of deafened cochlea, emerging as a potential strategy for inner ear rehabilitation.

Key words: Cochlea repair; Organ of Corti; Stem cell; Transplantation; Deafness

Address correspondence to Roberto P. Revoltella, M.D., Ph.D., Foundation onlus, "Staminali e Vita", Institute of Biomedical Technologies, C.N.R., Via L. L. Zamenhof, 8- 56127 Pisa, Italy. Tel: (+39) 050 544047; Fax: (+39) 050 2218606; E-mail: rrevoltella@yahoo.it or roberto.revoltella@itb.cnr.it

¹Department of Biomedical Sciences, University of Padua School of Medicine, Padua, Italy
²Department of Pediatrics, University of Padua School of Medicine, Padua, Italy
³Department of Cardiologic, Thoracic and Vascular Sciences, University of Padua School of Medicine, Padua, Italy
⁴Surgery Unit, Great Ormond Street Hospital and Institute of Child Health, University College London, London, UK

Efficacy of adult (bone marrow, BM) versus fetal (amniotic fluid, AF) mesenchymal stem cells (MSCs) to replenish damaged rat heart tissues with new cardiovascular cells has not yet been established. We investigated on the differentiation potential of these two rat MSC populations in vitro and in a model of acute necrotizing injury (ANI) induced by cryoinjury. Isolated BM-MSCs and AF-MSCs were characterized by flow cytometry and cytocentrifugation and their potential for osteogenic, adipogenic, and cardiovascular differentiation assayed in vitro using specific induction media. The left anterior ventricular wall of syngeneic Fisher 344 (n = 48) and athymic nude (rNu) rats (n = 6) was subjected to a limited, nontransmural epicardial ANI in the approximately one third of wall thickness without significant hemodynamic effects. The time window for in situ stem cell transplantation was established at day 7 postinjury. Fluorochrome (CMTMR)-labeled BM-MSCs (2 × 10⁶) or AF-MSCs (2 × 10⁶) were injected in syngeneic animals (n = 26) around the myocardial lesion via echocardiographic guidance. Reliability of CMTMR cell tracking in this context was ascertained by transplanting genetically labeled BM-MSCs or AF-MSCs, expressing the green fluorescent protein (GFP), in rNu rats with ANI. Comparison between the two methods of cell tracking 30 days after cell transplantation gave slightly different values (1420,58 ± 129,65 cells/mm² for CMTMR labeling and 1613.18 ± 643.84 cells/mm² for genetic labeling; p = NS). One day after transplantation about one half CMTMR-labeled AF-MSCs engrafted to the injured heart (778.61 ± 156.28 cells/mm²) in comparison with BM-MSCs (1434.50 ± 173.80 cells/mm², p < 0.01). Conversely, 30 days after cell transplantation survived MSCs were similar: 1275.26 ± 74.51/mm² (AF-MSCs) versus 1420.58 ± 129.65/mm² for BM-MSCs (p = NS). Apparent survival gain of AF-MSCs between the two time periods was motivated by the cell proliferation rate calculated at day 30, which was lower for BM-MSCs (6.79 ± 0.48) than AF-MSCs (10.83 ± 3.50; p < 0.01), in the face of a similar apoptotic index (4.68 ± 0.20 for BM-MSCs and 4.16 ± 0.58 for AF-MSCs; p = NS). These cells were also studied for their expression of markers specific for endothelial cells (ECs), smooth muscle cells (SMCs), and cardiomyocytes (CMs) using von Willebrand factor (vWf), smooth muscle (SM) a-actin, and cardiac troponin T, respectively. Grafted BM-MSCs or AF-MSCs were found as single cell/small cell clusters or incorporated in the wall of microvessels. A larger number of ECs (227.27 ± 18.91 vs. 150.36 ± 24.08 cells/mm², p < 0.01) and CMs (417.91 ± 100.95 vs. 237.43 ± 79.99 cells/mm², p < 0.01) originated from AF-MSCs than from BM-MSCs. Almost no SMCs were seen with AF-MSCs, in comparison to BM-MSCs (98.03 ± 40.84 cells/mm²), in concordance with lacking of arterioles, which, instead, were well expressed with BM-MSCs (71.30 ± 55.66 blood vessels/mm²). The number of structurally organized capillaries was slightly different with the two MSCs (122.49 ± 17.37/mm² for AF-MSCs vs. 148.69 ± 54.41/mm² for BM-MSCs; p = NS). Collectively, these results suggest that, in the presence of the same postinjury microenvironment, the two MSC populations from different sources are able to activate distinct differentiation programs that potentially can bring about a myocardial–capillary or myocardial–capillary–arteriole reconstitution.

Key words: Cardiac injury; Experimental model; Stem cells; Cardiac cell regeneration; Fetal stem cells; Adult stem cells

Address correspondence to Saverio Sartore, Ph.D., Department of Biomedical Sciences, University of Padua, Viale G. Colombo, 3, 35121 Padua, Italy. Tel: +390498276481; Fax: +390498276040; E-mail: saverio.sartore@unipd.it

Department of Surgery and Clinical Science, Department of Orthopaedic Surgery, Yamaguchi University Graduate School of Medicine, Yamaguchi 755-8505, Japan

Various cytokines produced by bone marrow cells can protect adult cardiomyocytes against apoptosis. Thus, we investigated the feasibility of implanting adult cardiomyocytes in combination with bone marrow cells for myocardial repair. Ventricular cardiomyocytes were isolated from adult rats and cocultured with bone marrow cells. Using a rat model of doxorubicin-induced cardiomyopathy, we injected 6 × 10⁵ adult cardiomyocytes, 3 × 10⁷ bone marrow cells, or both into damaged hearts, for myocardial repair. Coculture of the cardiomyocytes with the bone marrow cells enhanced the expression of integrin-b1D and focal adhesion kinase in cardiomyocytes, resulting in increased survival and decreased apoptosis of the cardiomyocytes after 7 days of culture. Compared with the baseline levels, cardiac function was preserved by the implantation of bone marrow cells alone and by the implantation of cardiomyocytes in combination with bone marrow cells, but it was decreased significantly 28 days after the implantation of cardiomyocytes alone. Furthermore, apoptosis of the host cardiomyocytes was decreased significantly after the implantation of bone marrow cells alone, or in combination with cardiomyocytes, compared with that after the implantation of cardiomyocytes alone (p < 0.01). Interestingly, the implantation of adult cardiomyocytes in combination with bone marrow cells resulted in a dramatic increase in the survival of donor cardiomyocytes, and induced the myogenic differentiation of donor bone marrow stem cells. Our findings indicate that cardiomyocytes and bone marrow cells can assist and compliment each other; thus, the implantation of adult cardiomyocytes in combination with bone marrow cells shows promise as a feasible new strategy for myocardial repair.

Key words: Bone marrow cell; Cardiomyocyte; Cell transplantation; Myocardial regeneration

Address correspondence to Tao-Sheng Li, M.D., Ph.D., Department of Surgery and Clinical Science, Yamaguchi University Graduate School of Medicine, 1-1-1 Minami-Kogushi, Ube, Yamaguchi 755-8505, Japan. Tel: 836-222260; Fax: 836-222423; E-mail: litaoshe@yamaguchi-u.ac.jp

Research Institute for Cell Engineering (RICE), National Institute of Advanced Industrial Science and Technology (AIST), Hyogo 661-0974, Japan

Marrow mesenchymal stem cells (MSCs) are multipotent progenitor cells and reported to be immunoprivileged as well as immunosuppressive. Hence, MSCs might be ideal candidates for allogeneic transplantation to induce regeneration of damaged tissues/organs. To confirm the differentiation capability of allogeneic MSCs in vivo is important for the acceleration of regenerative medicine. Consequently, we have established an in vivo rat model using subcutaneous implantation of a hydroxyapatite (HA) ceramic/MSCs composite. Osteogenic differentiation was used as an indicator of differentiation. When syngeneic MSCs were implanted, MSCs showed osteogenic differentiation as evidenced by new bone formation as well as high alkaline phosphatase (ALP) activity. When allogeneic MSCs were implanted, none of the allografts survived or showed osteogenic differentiation. However, when the recipient rats were treated with FK506 immunosuppressant, allogeneic MSCs showed osteogenic differentiation. Although this finding might not be adequate for the acceleration of regenerative medicine, these results did confirm that MSCs are not intrinsically immunoprivileged but that under appropriate immunosuppressant treatment, allogeneic MSCs can survive and show differentiation capability in vivo.

Key words: Mesenchymal stem cells (MSCs); Osteoblasts; Bioengineering; Implants; Bone mineralization

Address correspondence to Motohiro Hirose, Ph.D., Research Institute for Cell Engineering (RICE), National Institute of Advanced Industrial Science and Technology (AIST), 3-11-46 Nakouji, Amagasaki, Hyogo 661-0974 Japan. Tel: 81-6-6494-7869; Fax: 81-6-6494-7861; E-mail: moto hiro-hirose@aist.go.jp

¹Division of Transplant Immunology, Department of Surgery, University of Alabama at Birmingham, Birmingham, AL, USA
²Division of Plastic Surgery, Department of Surgery, University of Alabama at Birmingham, Birmingham, AL, USA
³Division of Cardiothoracic Surgery, Department of Surgery, University of Alabama at Birmingham, Birmingham, AL, USA

The mechanisms mediating T-cell depletion plus 15-deoxyspergualin (DSG)-induced prolonged allograft survival or tolerance are uncertain. The purpose of this study is to evaluate the role of IL-4 and IL-10 in prolonged allograft survival induced by T-cell depletion plus DSG. MHC mismatched skin allograft transplantation was performed, using wild-type and three separate knockout (i.e., IL-4-/-, Stat6-/-, or IL-10-/-) mice as recipients. Induction therapy consisted of T-cell depletion and or brief course of DSG. The data demonstrate that monotherapy with T-cell-depleting mAbs or DSG prolonged skin allograft survival, compared to controls, in wild-type Balb/c recipients [median survival time (MST) = 25 and 21 vs. 10 days, p < 0.007]. T-cell depletion plus DSG further augmented skin allograft survival in wild-type animals relative to monotherapy (MST = 35 days vs. 25 and 21 days, p < 0.006 vs. mAbs or DSG only), and was equally effective in IL-4-/- and Stat6-/-recipients. In contrast, combined therapy was no better than monotherapy in IL-10-/- animals (p > 0.05). Furthermore, skin allograft survival after combined therapy was shorter in IL-10-/- versus wild-type recipients (MST 20 and 41 days, respectively, p < 0.001). IL-4-mediated signaling through Stat6 is dispensable for prolonged allograft survival induced by T-cell depletion plus DSG. In contrast, IL-10 appears to be important for prolonged allograft survival induced by combined therapy in this model.

Key words: Mice; Depletion; Deoxyspergualin (DSG); Cytokines; Allografts

Address correspondence to Clement Asiedu, 1670 University Blvd., Volker Hall, Suite G82, Birmingham, AL 35294-0019, USA. Tel: 205-975-2325; Fax: 205-934-9039; E-mail: Clement.Asiedu@ccc.uab.edu