ognizant Communication Corporation

The Regenerative Medicine Journal

VOLUME 18, NUMBER 8, 2009

Cell Transplantation, Vol. 18, pp. 827-832, 2009
0963-6897/09 $90.00 + 00
DOI: 10.3727/096368909X472278
E-ISSN 1555-3892
Copyright © 2009 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Brief Communication
Laser Capture Microdissection as a New Tool to Assess Graft-Infiltrating Lymphocytes Gene Profile in Islet Transplantation

A. Vergani,1,2 B. Clissi,2 F. Sanvito,2 C. Doglioni,2,3 P. Fiorina,1,2 and R. Pardi2,3

1Transplantation Research Center(TRC)-Nephrology, Children's Hospital-Harvard Medical School, Boston, MA, USA
2Department of Biological and Technological Research, Scientific Institute San Raffaele, Milan, Italy
3School of Medicine, Vita-Salute San Raffaele University, Milan, Italy

Innovative tolerogenic protocols in transplantation would take advantage of the development of new tools capable of evaluating the impact of these treatments on the immune system. These assays have potential for clinical application. Currently, many of these studies are based on the analysis of peripheral lymph nodes and blood-derived cells, where the percentage of alloantigen-specific cells can be low or even unpredictable. We combined a laser capture microdissection (LCM) technique with real-time PCR (RT-PCR) to evaluate gene profile of islet-infiltrating lymphocytes. Donor Lewis rats islets were transplanted under the kidney capsule in diabetic Brown Norway rats. Administration of anti-LFA1 mAb or anti-CD28 F(Ab)´ was able to prolong islet survival, while the combined treatment resulted in indefinite survival. The analysis of gene expression profile for IL-2, IFN-g, and IL-10 production of graft-infiltrating cells revealed high IL-2, IFN-g, and IL-10 in untreated rats; on the contrary, the combined treatment selectively abrogated IL-2- and IFN-g-producing cells infiltrate. The comparison between cytokine profile in periphery (even during an allogenic extra stimulus) and in the graft revealed the dichotomy between graft and peripheral cytokine assessment. We thus propose that direct analysis of graft-infiltrating cells should be used whenever possible to evaluate the effects of a new immunomodulatory protocol.

Key words: Islet transplantation; Laser capture microdissection (LCM); Gene expression; Graft-infiltrating lymphocytes; Costimulation blockade

Address correspondence to Ruggero Pardi, M.D., Professor of Pathology, Head, Unit of Leukocyte Biology, Vita-Salute University School of Medicine DIBIT-Scientific Institute, San Raffaele via Olgettina 58, I-20132 Milano, Italy. Tel: ?39 02 2643 4731 (personal) or +39 02 2643 4763 (secretary); Fax: +39 02 2643 4723; E-mail: pardi.ruggero@hsr.it

Cell Transplantation, Vol. 18, pp. 833-845, 2009
0963-6897/09 $90.00 + 00
DOI: 10.3727/096368909X471198
E-ISSN 1555-3892
Copyright © 2009 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Different Effects of FK506, Rapamycin, and Mycophenolate Mofetil on Glucose-Stimulated Insulin Release and Apoptosis in Human Islets

James D. Johnson,1,2* Ziliang Ao,1* Peter Ao,1,2 Hong Li,1 Long-Jun Dai,2 Zehua He,2 May Tee,1,2 Kathryn J. Potter,3 Agnieszka M. Klimek,2,3 R. Mark Meloche,2 David M. Thompson,2 C. Bruce Verchere,2,3 and Garth L. Warnock2

1Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, Canada
2Department of Surgery, University of British Columbia, Vancouver, Canada
3Department of Pathology & Laboratory Medicine, Child & Family Research Institute, University of British Columbia, Vancouver, Canada

Pancreatic islet transplantation has the potential to be an effective treatment for type 1 diabetes mellitus. While recent improvements have improved 1-year outcomes, follow-up studies show a persistent loss of graft function/survival over 5 years. One possible cause of islet transplant failure is the immunosuppressant regimen required to prevent alloimmune graft rejection. Although there is evidence from separate studies, mostly in rodents and cell lines, that FK506 (tacrolimus), rapamycin (sirolimus), and mycophenolate mofetil (MMF; CellCept) can damage pancreatic b-cells, there have been few side-by-side, multiparameter comparisons of the effects of these drugs on human islets. In the present study, we show that 24-h exposure to FK506 or MMF impairs glucose-stimulated insulin secretion in human islets. FK506 had acute and direct effects on insulin exocytosis, whereas MMF did not. FK506, but not MMF, impaired human islet graft function in diabetic NOD.scid mice. All of the immunosuppressants tested in vitro increased caspase-3 cleavage and caspase-3 activity, whereas MMF induced ER-stress to the greatest degree. Treating human islets with the GLP-1 agonist exenatide ameliorated the immunosuppressant-induced defects in glucosestimulated insulin release. Together, our results demonstrate that immunosuppressants impair human b-cell function and survival, and that these defects can be circumvented to a certain extent with exenatide treatment.

Key words: Diabetes; Islet transplantation; FK506; Rapamycin; Mycophenolate mofetil

Address correspondence to James D. Johnson, Ph.D., Assistant Professor, Department of Cellular and Physiological Sciences, University of British Columbia, 5358 Life Sciences Building, 2350 Health Sciences Mall,Vancouver, BC, Canada, V6T 1Z3. Tel: (604) 822-7187; Fax: (604) 822-2316; E-mails: jimjohn@interchange.ubc.ca or Garth L. Warnock, M.D., Department of Surgery, Faculty of Medicine, University of British Columbia, Rom 3100, 910 West 10th Avenue, Vancouver, BC, Canada V5Z 4E3. E-mail: Garth.Warnock@vch.ca

*Contributed equally to the project.

Cell Transplantation, Vol. 18, pp. 847-853, 2009
0963-6897/09 $90.00 + 00
DOI: 10.3727/096368909X471134
E-ISSN 1555-3892
Copyright © 2009 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Influence of Cell Treatment With PDGF-BB and Reperfusion on Cardiac Persistence of Mononuclear and Mesenchymal Bone Marrow Cells After Transplantation Into Acute Myocardial Infarction in Rats

Benjamin Krausgrill,1,3 Marius Vantler,1 Volker Burst,2 Martin Raths,1 Marcel Halbach,1,3 Konrad Frank,1 Silke Schynkowski,1 Kerstin Schenk,1 Jürgen Hescheler,3 Stephan Rosenkranz,1 and Jochen Müller-Ehmsen1

1Department III of Internal Medicine, University Hospital of Cologne, Köln, Germany
2Department IV of Internal Medicine, University Hospital of Cologne, Köln, Germany
3Institute of Neurophysiology, University Hospital of Cologne, Köln, Germany

Bone marrow cells are used for cell therapy after myocardial infarction (MI) with promising results. However, cardiac persistence of transplanted cells is rather low. Here, we investigated strategies to increase the survival and cardiac persistence of mononuclear (MNC) and mesenchymal (MSC) bone marrow cells transplanted into infarcted rat hearts. MNC and MSC (male Fischer 344 rats) were treated with different doses of PDGF-BB prior to intramyocardial injection into border zone of MI (syngeneic females, permanent LAD ligation) and hearts were harvested after 5 days and 3 weeks. In additional experiments, untreated MNC and MSC were injected immediately after permanent or temporary LAD ligation and hearts were harvested after 48 h, 5 days, 3 weeks, and 6 weeks. DNA of the hearts was isolated and the number of donor cells was determined by quantitative real-time PCR with Y chromosome-specific primers. There was a remarkable though not statistically significant (p = 0.08) cell loss of ~46% between 5 days and 3 weeks in the control group, which was completely inhibited by treatment with high dose of PDGF-BB. Forty-eight hours after reperfusion only 10% of injected MSC or 1% for MNC were found in the heart, decreasing to 1% for MSC and 0.5% for MNC after 6 weeks. These numbers were lower than after permanent LAD ligation for both MNC and MSC at all time points studied. Treatment with PDGF-BB seems to prevent loss of transplanted bone marrow cells at later times presumably by inhibition of apoptosis, while reperfusion of the occluded artery enhances cell loss at early times putatively due to enhanced early wash-out. Further investigations are needed to substantially improve the persistence and survival of grafted bone marrow cells in infarcted rat hearts, in order to fully explore the therapeutic potential of this novel treatment modality for myocardial repair.

Key words: Myocardial infarction; Cell therapy; Stem cells; Transplantation; Bone marrow cells; Growth factors

Address correspondence to PD Dr. med. Jochen Müller-Ehmsen, Department III of Internal Medicine, University Hospital of Cologne, Kerpener Str. 62, 50937 Köln, Germany. Tel: +49 221 478 32396; Fax: +49 221 478 32397; E-mail: muller.ehmsen@uni-koeln.de

Cell Transplantation, Vol. 18, pp. 855-868, 2009
0963-6897/09 $90.00 + 00
DOI: 10.3727/096368909X471170
E-ISSN 1555-3892
Copyright © 2009 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Autologous Umbilical Cord Blood Mononuclear Cell Transplantation Preserves Right Ventricular Function in a Novel Model of Chronic Right Ventricular Volume Overload

Can Yerebakan,1 Eugen Sandica,1 Stephanie Prietz,1 Christian Klopsch,1 Murat Ugurlucan,1 Alexander Kaminski,1 Sefer Abdija,2 Björn Lorenzen,3 Johannes Boltze,4 Björn Nitzsche,4 Dietmar Egger,5 Malte Barten,6 Dario Furlani,1 Nan Ma,1 Brigitte Vollmar,7 Andreas Liebold,1 and Gustav Steinhoff1

1Department of Cardiac Surgery, Medical Faculty, University of Rostock, Rostock, Germany
2Department of Cardiology, Ruppiner Clinics, Neuruppin, Germany
3Department of Diagnostic and Interventional Radiology, Medical Faculty, University of Rostock, Rostock, Germany
4Fraunhofer Institute for Cell Therapy and Immunology, Translational Center of Regenerative Medicine, Leipzig, Germany
5Vita 34 AG, Leipzig, Germany
6Institute for Pathology, Medical Faculty, University of Rostock, Rostock, Germany
7Institute for Experimental Surgery, Medical Faculty, University of Rostock, Rostock, Germany

We aimed to evaluate the feasibility and efficacy of autologous umbilical cord blood mononuclear cell (UCMNC) transplantation on right ventricular (RV) function in a novel model of chronic RV volume overload. Four-month-old sheep (n = 20) were randomized into cell (n = 10) and control groups (n = 10). After assessment of baseline RV function by the conductance catheter method, a transannular patch (TAP) was sutured to the right ventricular outflow tract (RVOT). Following infundibulotomy the ring of the pulmonary valve was transected without cardiopulmonary bypass. UCMNC implantation (8.22 ± 6.28 × 107) in the cell group and medium injection in the control group were performed into the RV myocardium around the TAP. UCMNCs were cultured for 2 weeks after fluorescence-activated cell sorting (FACS) analysis for CD34 antigen. Transthoracic echocardiography (TTE) and computed tomography were performed after 6 weeks and 3 months, respectively. RV function was assessed 3 months postoperatively before the hearts were excised for immunohistological examinations. FACS analysis revealed 1.2 ± 0.22% CD34+ cells within the isolated UCMNCs from which AcLDL+ endothelial cells were cultured in vitro. All animals survived surgery. TTE revealed grade II-III pulmonary regurgitation in both groups. Pressure-volume loops under dobutamine stress showed significantly improved RV diastolic function in the cell group (dP/dtmin: p = 0.043; Eed: p = 0.009). CD31 staining indicated a significantly enhanced number of microvessels in the region of UCMNC implantation in the cell group (p < 0.001). No adverse tissue changes were observed. TAP augmentation and pulmonary annulus distortion without cardiopulmonary bypass constitutes a valid large animal model mimicking the surgical repair of tetralogy of Fallot. Our results indicate that the chronically volumeoverloaded RV profits from autologous UCMNC implantation by enhanced diastolic properties with a probable underlying mechanism of increased angiogenesis.

Key words: Tetralogy of Fallot; Right ventricular dysfunction; Pulmonary insufficiency; Umbilical cord blood; Stem cells

Address correspondence to Can Yerebakan, M.D., Department of Cardiac Surgery, Medical Faculty, University of Rostock, Schillingallee 35, 18057, Rostock, Germany. Tel: +49 494 381 6101; Fax: +49 494 381 6102; E-mail: can.yerebakan@med.uni-rostock.de

Cell Transplantation, Vol. 18, pp. 869-886, 2009
0963-6897/09 $90.00 + 00
DOI: 10.3727/096368909X471189
E-ISSN 1555-3892
Copyright © 2009 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Attenuate Hyperoxia-Induced Lung Injury in Neonatal Rats

Yun Sil Chang,1,2* Wonil Oh,3* Soo Jin Choi,3 Dong Kyung Sung,2 Soo Yoon Kim,2 Eun Yang Choi,2 Saem Kang,2 Hye Jin Jin,3 Yoon Sun Yang,3 and Won Soon Park 1,2

1Department of Pediatrics, Samsung Medical Center, Seoul, Korea
2Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Seoul, Korea
3Biomedical Research Institute, MEDIPOST Co., Ltd., Seoul, Korea

Recent evidence suggests mesenchymal stem cells (MSCs) can downmodulate bleomycin-induced lung injury, and umbilical cord blood (UCB) is a promising source for human MSCs. This study examined whether intratracheal or intraperitoneal transplantation of human UCB-derived MSCs can attenuate hyperoxia-induced lung injury in immunocompetent newborn rats. Wild-type Sprague-Dawley rats were randomly exposed to 95% oxygen or air from birth. In the transplantation groups, a single dose of PKH26-labeled human UCBderived MSCs was administered either intratracheally (2 x 106 cells) or intraperitoneally (5 x 105 cells) at postnatal day (P) 5. At P14, the harvested lungs were examined for morphometric analyses of alveolarization and TUNEL staining, as well as the myeoloperoxidase activity, the level of tumor necrosis factor (TNF)-a, interleukin (IL)-6, and transforming growth factor (TGF)-b mRNA, a-smooth muscle actin (SMA) protein, and collagen levels. Differentiation of MSCs to the respiratory epithelium was also evaluated both in vitro before transplantation and in vivo after transplantation. Despite one fourth dosage of MSCs, significantly more PKH26-labeled donor cells were recovered with intratracheal administration than with intraperitoneal administration both during normoxia and hyperoxia. The hyperoxia-induced increase in the number of TUNELpositive cells, myeloperoixdase activity, and the level of IL-6 mRNA were significantly attenuated with both intratracheal and intraperitoneal MSCs transplantation. However, the hyperoxia-induced impaired alveolarization and increased the level of TNF-a and TGF-b mRNA, a-SMA protein, and collagen were significantly attenuated only with intratracheal MSCs transplantation. MSCs differentiated into respiratory epithelium in vitro and a few PKH26-positive donor cells were colocalized with pro surfactant protein C in the damaged lungs. In conclusion, intratracheal transplantation of human UCB-derived MSCs is more effective than intraperitoneal transplantation in attenuating the hyperoxia-induced lung injury in neonatal rats.

Key words: Hyperoxic lung injury; Transplantation; Animal; Newborn; Inflammation; Cell differentiation

Address correspondence to Won Soon Park, M.D., Ph.D., Department of Pediatrics, Samsung Medical Center, 50 Irwon-dong, Kangnam-gu, Seoul, 135-710, Republic of Korea. Tel: +82-2-3410-3523; Fax: +82-2-3410-0043; E-mail: wonspark@skku.edu or ws123.park@samsung.com

*Co-first authors.

Cell Transplantation, Vol. 18, pp. 887-897, 2009
0963-6897/09 $90.00 + 00
DOI: 10.3727/096368909X471152
E-ISSN 1555-3892
Copyright © 2009 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Copper Modulates the Differentiation of Mouse Hematopoietic Progenitor Cells in Culture

Xiaosong Huang,1* L. Jeanne Pierce,2 Paul A. Cobine,2** Dennis R. Winge,2 and Gerald J. Spangrude1,2

1Department of Pathology, School of Medicine, University of Utah, Salt Lake City, UT, USA
2Department of Medicine, Division of Hematology, School of Medicine, University of Utah, Salt Lake City, UT, USA

Copper chelation has been shown to favor the expansion of human hematopoietic stem/progenitor cells in vitro. To further understand the effects of copper modulation on defined subsets of stem cells versus progenitor cells, we extended the studies in a mouse system. We isolated mouse hematopoietic stem cells (HSCs) or hematopoietic progenitor cells (HPCs) and cultured them with or without the copper chelator tetraethylenepentamine (TEPA) or CuCl2. Cytokine-stimulated HPC cultures treated with TEPA for 7 days generated about two to three times more total and erythroid colony-forming cells (CFCs) compared to control cultures. In contrast, CuCl2 treatment decreased the CFC numbers. Similar results were seen with HSC after 14, but not 7, days of culture. Transplant studies showed that HPCs cultured for 7 days in TEPA had about twofold higher short-term erythroid repopulation potential compared to control cultures, while CuCl2 decreased the erythroid potential of cultured HPCs compared to control cultures. HSCs cultured with TEPA for 7 days did not exhibit significantly higher repopulation potential in either leukocyte or erythrocyte lineages compared to control cultures in short-term or long-term assays. Based on JC-1 staining, the mitochondrial membrane potential of HPCs cultured with TEPA was lower relative to control cultures. Our data suggest that decreasing the cellular copper content with TEPA results in preferential expansion or maintenance of HPC that are biased for erythroid differentiation in vivo, but does not enhance the maintenance of HSC activity in culture.

Key words: Hematopoietic stem cells; Progenitor cells; Adult stem cells; Bone marrow; Erythropoiesis

Address correspondence to Gerald J. Spangrude, Ph.D., Division of Hematology, University of Utah, RM 5C402, 30 N 1900 East, Salt Lake City, UT 84132-2408, USA. Tel: (801) 585 5544; Fax: (801) 585 3778; E-mail: gspangrude@mac.com

*Current address: Department of Pediatrics, University of Rochester Medical Center, Rochester, NY, USA.
**Current address: Department of Microbiology, Auburn University, Auburn, AL, USA.

Cell Transplantation, Vol. 18, pp. 899-914, 2009
0963-6897/09 $90.00 + 00
DOI: 10.3727/096368909X471314
E-ISSN 1555-3892
Copyright © 2009 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Amniotic Mesenchymal Tissue Cells Inhibit Dendritic Cell Differentiation of Peripheral Blood and Amnion Resident Monocytes

Marta Magatti,1 Silvia De Munari,1 Elsa Vertua,1 Claudia Nassauto,1 Alberto Albertini,2 Georg S. Wengler,1 and Ornella Parolini1

1Centro di Ricerca E. Menni, Fondazione Poliambulanza Istituto Ospedaliero, Brescia, Italy
2Istituto di Tecnologie Biomediche, CNR, Milano, Italy

Cells derived from the amniotic membranes of human term placenta have drawn much interest for their characteristics of multipotency and low immunogenicity, supporting a variety of possible clinical applications in the field of cell transplantation and regenerative medicine. We have previously shown that cells derived from the mesenchymal region of human amnion (AMTC) can strongly inhibit T-lymphocyte proliferation. In this study, we demonstrate that AMTC can block differentiation and maturation of monocytes into dendritic cells (DC), preventing the expression of the DC marker CD1a and reducing the expression of HLA-DR, CD80, and CD83. The monocyte maturation block resulted in impaired allostimulatory ability of these cells on allogeneic T cells. In attempting to define the mechanisms responsible for these findings, we have observed that the presence of AMTC in differentiating DC cultures results in the arrest of the cells to the G0 phase and abolishes the production of inflammatory cytokines such as TNF-a, CXCL10, CXCL9, and CCL5. Finally, we also demonstrate that the monocytic cells present in the amniotic mesenchymal region fail to differentiate toward the DC lineage. Taken together, our data suggest that the mechanisms by which AMTC exert immumodulatory effects do not only relate directly to T cells, but also include inhibition of the generation and maturation of antigen-presenting cells. In this context, AMTC represent a very attractive source of multipotent allogeneic cells that promise to be remarkably valuable for cell transplantation approaches, not only due to their low immunogenicity, but also because of the added potential of modulating immune responses, which could be fundamental both for controlling graft rejection after transplantation and also for controlling diseases characterized by inflammatory processes.

Key words: Amnion; Mesenchymal stromal cell; Human placenta; Cell transplantation; Immunosuppression; Dendritic cell; Monocyte

Address correspondence to Ornella Parolini, Ph.D., Centro di Ricerca E. Menni, Fondazione Poliambulanza Istituto Ospedaliero, Via Bissolati, 57, I-25124 Brescia, Italy. Tel: +390302455754; Fax: +390302455704; E-mail: ornella.parolini@tin.it or ornella.parolini@poliambulanza.it

Cell Transplantation, Vol. 18, pp. 915-921, 2009
0963-6897/09 $90.00 + 00
DOI: 10.3727/096368909X471161
E-ISSN 1555-3892
Copyright © 2009 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Effects of Mechanical Stretch on Collagen and Cross-Linking in Engineered Blood Vessels

Amy Solan,1 Shannon L. M. Dahl,2 and Laura E. Niklason3

1Mentor Corporation, Irving, TX, USA
2Humacyte Corporation, Durham, NC, USA
3Departments of Biomedical Engineering and Anesthesia, Yale University, New Haven, CT, USA

It has been shown that mechanical stimulation affects the physical properties of multiple types of engineered tissues. However, the optimum regimen for applying cyclic radial stretch to engineered arteries is not well understood. To this end, the effect of mechanical stretch on the development of engineered blood vessels was analyzed in constructs grown from porcine vascular smooth muscle cells. Cyclic radial distension was applied during vessel culture at three rates: 0 beats per minute (bpm), 90 bpm, and 165 bpm. At the end of the 7-week culture period, harvested vessels were analyzed with respect to physical characteristics. Importantly, mechanical stretch at 165 bpm resulted in a significant increase in rupture strength in engineered constructs over nonstretched controls. Stress-strain data and maximal elastic moduli from vessels grown at the three stretch rates indicate enhanced physical properties with increasing pulse rate. In order to investigate the role of collagen cross-linking in the improved mechanical characteristics, collagen cross-link density was quantified by HPLC. Vessels grown with mechanical stretch had somewhat more collagen and higher burst pressures than nonpulsed control vessels. Pulsation did not increase collagen cross-link density. Thus, increased wall thickness and somewhat elevated collagen concentrations, but not collagen cross-link density, appeared to be responsible for increased burst strength.

Key words: Mechanical stretch; Tissue engineering; Blood vessels; Rupture strength; Collagen cross-links

Address correspondence to Laura Niklason, M.D., Ph.D., Department of Biomedical Engineering, Yale University, P.O. Box 208260, New Haven, CT 06520-8620, USA. Tel: (203) 737-1422; Fax: (203) 737-1484; E-mail: laura.niklason@yale.edu

Cell Transplantation, Vol. 18, pp. 923-932, 2009
0963-6897/09 $90.00 + 00
DOI: 10.3727/096368909X471297
E-ISSN 1555-3892
Copyright © 2009 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Enhanced Early Tissue Regeneration After Matrix-Assisted Autologous Mesenchymal Stem Cell Transplantation in Full Thickness Chondral Defects in a Minipig Model

Martin Jung,1 Balazs Kaszap,1 Anna Redöhl,3 Eric Steck,1 Steffen Breusch,2 Wiltrud Richter,1 and Tobias Gotterbarm1

1Department of Orthopaedic Surgery, University of Heidelberg, Heidelberg, Germany
2Department of Orthopaedics, University of Edinburgh, Edinburgh, Scotland
3Department of Small Animal Medicine, University of Veterinary Medicine, Hannover, Germany

Adult mesenchymal stem cells (MSCs) are an attractive cell source for new treatment strategies in regenerative medicine. This study investigated the potential effect of matrix assisted MSC transplantation for articular cartilage regeneration in a large-animal model 8 weeks postoperatively. MSCs from bone marrow aspirates of eight Goettingen minipigs were isolated and expanded prior to surgery. Articular cartilage defects of 5.4 mm were created bilaterally in the medial patellar groove without penetrating the subchondral bone plate. Defects were either left empty (n = 4), covered with a collagen type I/III membrane (n = 6) or additionally treated with autologous MSC transplantation (2 × 106; n = 6). After 8 weeks animals were euthanized and the defect area was assessed for its gross appearance. Histomorphological analysis of the repair tissue included semiquantitative scoring (O'Driscoll score) and quantitative histomorphometric analysis for its glycosaminoglycan (GAG) and collagen type II content. All membranes were found to cover the defect area 8 weeks postoperatively. Median defect filling was 115.8% (membrane), 117.8% (empty), and 100.4% (MSC), respectively (not significant). Histomorphological scoring revealed significantly higher values in MSC-treated defects (median 16.5) when compared to membrane treatment (median 9.5) or empty defects (median 11.5; p = 0.015 and p = 0.038). Histomorphometric analysis showed larger GAG/collagen type II-positive areas in the MSC-treated group (median 24.6%/29.5% of regeneration tissue) compared to 13.6%/33.1% (empty defects) and 1.7%/6.2% (membrane group; p = 0.066). Cell distribution was more homogeneous in MSC compared to membrane-only group, where cells were found mainly near the subchondral zone. In conclusion, autologous matrix-assisted MSC transplantation significantly increased the histomorphological repair tissue quality during early articular cartilage defect repair and resulted in higher GAG/collagen type II-positive cross-sectional areas of the regenerated tissue.

Key words: Cartilage defect; Mesenchymal stem cells; Minipig model; Cartilage repair; Collagen membrane

Address correspondence to Tobias Gotterbarm, M.D., Department of Orthopaedic Surgery, University of Heidelberg, Schlierbacher Landstr. 200a, 69118 Heidelberg, Germany. Tel: +49/6221/965; Fax: +49/6221/966356; E-mail: tobias.gotterbarm@ok.uni-heidelberg.de

Cell Transplantation, Vol. 18, pp. 933-940, 2009
0963-6897/09 $90.00 + 00
DOI: 10.3727/096368909X471143
E-ISSN 1555-3892
Copyright © 2009 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Small Agarose Microcapsules With Cell-Enclosing Hollow Core for Cell Therapy: Transplantation of Ifosfamide-Activating Cells to the Mice With Preestablished Subcutaneous Tumor

Shinji Sakai,1 Ichiro Hashimoto,1 Shinji Tanaka,2 Brian Salmons,3 and Koei Kawakami1

1Department of Chemical Engineering, Faculty of Engineering, Kyushu University, Fukuoka-city, Fukuoka, Japan
2Department of Hepato-Biliary-Pancreatic Surgery, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo, Japan
3Austrianova Singapore Pte Ltd, Centros, Biopolis, Singapore

Cell transplantation after enclosing in microcapsules has been studied as an alternative approach for treatment of wide variety of diseases. In the present study, we examined the feasibility of using agarose microcapsules, having a cell-enclosing hollow core of 100-150 mm in diameter and agarose gel membrane of about 20 mm in thickness, as a device for the methodology. We enclosed cells that had been genetically engineered to express cytochrome P450 2B1, an enzyme that activates the anticancer prodrug ifosfamide. The enclosed cells were shown to express the enzymatic function in the microcapsules in that they suppressed the growth of tumor cells in medium containing ifosfamide. In addition, a more significant regression of preformed tumors was observed in the nude mice implanted with the cell-enclosing microcapsules compared with those implanted with empty capsules after administration of ifosfamide. Preformed tumors shrank by less than 40% in volume in 6 of the 10 recipients implanted with cell-enclosing microcapsules. In contrast, only 1 in 10 of the preformed tumors in the recipient implanted with empty microcapsules shrank by this amount. These results suggest that agarose microcapsules containing cytochrome P450 2B1 enzyme-expressing cells are feasible devices for improving the chemotherapy of tumors. Thus, agarose microcapsule having hollow cores are generally a good candidate as vehicles for cell-encapsulation approaches to cell therapy.

Key words: Local therapy; Ifosfamide; Hollow-core agarose microcapsule; Cell therapy; Cytochrome P450

Address correspondence to Shinji Sakai, Department of Chemical Engineering, Faculty of Engineering, Kyushu University, 744 Motooka, Nishiku, Fukuoka, 819-0395, Japan. Tel: +81-92-802-2768; Fax: +81-92-802-2768; E-mail: sakai@chem-eng.kyushu-u.ac.jp

Cell Transplantation, Vol. 18, pp. 941-947, 2009
0963-6897/09 $90.00 + 00
DOI: 10.3727/096368909X471323
E-ISSN 1555-3892
Copyright © 2009 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Experience of Microbiological Screening of Human Hepatocytes for Clinical Transplantation

Sharon C. Lehec,1 Robin D. Hughes,1 Ragai R. Mitry,1 Michelle A. Graver,2 Anita Verma,2 Jim J. Wade,2 and Anil Dhawan1

1Institute of Liver Studies, King's College London School of Medicine, King's College Hospital, London, UK
2Health Protection Agency London Region Laboratory, King's College Hospital, London, UK

Hepatocyte transplantation is being used in patients with liver-based metabolic disorders and acute liver failure. Hepatocytes are isolated from unused donor liver tissue under GMP conditions. Cells must be free of microbiological contamination to be safe for human use. The experience of microbiological screening during 72 hepatocyte isolation procedures at one center is reported. Samples were taken at different stages of the process and tested using a blood culture bottle system and Gram stain. Bacterial contamination was detected in 37.5% of the UW organ preservative solutions used to transport the liver tissue to the Cell Isolation Unit. After tissue processing the contamination was reduced to 7% overall in the final hepatocyte product, irrespective of the presence of initial contamination of the transport solution. The most common organisms recovered were coagulase-negative staphylococci, a skin commensal. A total of 41 preparations of fresh or cryopreserved hepatocytes were used for cell transplantation in children with liver-based metabolic disorders without any evidence of sepsis due to infusion of hepatocytes. In conclusion, the incidence of bacterial contamination of the final product was low, confirming the suitability of the organs used, hepatocyte isolation procedure, and the environmental conditions of the clean room.

Key words: Human hepatocytes; Contamination; Bacteria; Transplantation

Address correspondence to Professor Anil Dhawan, Paediatric Liver Centre, King's College London School of Medicine, King's College Hospital, Denmark Hill, London SE5 9RS, UK. Tel: +44 (0)20-3299-3578; Fax: +44 (0)20-3299-4228; E-mail: anil.dhawan@kcl.ac.uk