ognizant Communication Corporation

The Regenerative Medicine Journal

VOLUME 19, NUMBER 8, 2010

Cell Transplantation, Vol. 19, pp. 919-936, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368910X494623
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Effects of MRI Contrast Agents on the Stem Cell Phenotype

Annelies Crabbe,1* Caroline Vandeputte,2* Tom Dresselaers,3 Angel Ayuso Sacido,4 Jose Manuel Garcia Verdugo,4 Jeroen Eyckmans,5 Frank P. Luyten,5 Koen Van Laere,2 Catherine M. Verfaillie,1 and Uwe Himmelreich3

1Stem Cell Institute, K.U. Leuven, Leuven, Belgium
2Division of Nuclear Medicine, K.U. Leuven, Leuven, Belgium
3Biomedical NMR Unit/MOSAIC, K.U. Leuven, Leuven, Belgium
4Centro de Investigación Príncipe Felipe, CIBERNED, Universidad de Valencia, Valencia, Spain
5Laboratory for Skeletal development and Joint Disorders, K.U. Leuven, Leuven, Belgium

The ultimate therapy for ischemic stroke is restoration of blood supply in the ischemic region and regeneration of lost neural cells. This might be achieved by transplanting cells that differentiate into vascular or neuronal cell types, or secrete trophic factors that enhance self-renewal, recruitment, long-term survival, and functional integration of endogenous stem/progenitor cells. Experimental stroke models have been developed to determine potential beneficial effect of stem/progenitor cell-based therapies. To follow the fate of grafted cells in vivo, a number of noninvasive imaging approaches have been developed. Magnetic resonance imaging (MRI) is a high-resolution, clinically relevant method allowing in vivo monitoring of cells labeled with contrast agents. In this study, labeling efficiency of three different stem cell populations [mouse embryonic stem cells (mESC), rat multipotent adult progenitor cells (rMAPC), and mouse mesenchymal stem cells (mMSC)] with three different (ultra)small superparamagnetic iron oxide [(U)SPIO] particles (Resovist®, Endorem®, Sinerem®) was compared. Labeling efficiency with Resovist® and Endorem® differed significantly between the different stem cells. Labeling with (U)SPIOs in the range that allows detection of cells by in vivo MRI did not affect differentiation of stem cells when labeled with concentrations of particles needed for MRI-based visualization. Finally, we demonstrated that labeled rMAPC could be detected in vivo and that labeling did not interfere with their migration. We conclude that successful use of (U)SPIOs for MRI-based visualization will require assessment of the optimal (U)SPIO for each individual (stem) cell population to ensure the most sensitive detection without associated toxicity.

Key words: Stem cells; Magnetic resonance imaging (MRI); Stroke; Ultrasmall superparamagnetic iron oxide [(U)SPIO] particles; Animal models

Address correspondence to Annelies Crabbe, Stamcelinstituut Leuven, O&N1-Herestraat 49, bus 804, 3000 Leuven, Belgium. Tel: 003216330292; Fax: 003216330294; E-mail: Annelies.crabbe@med.kuleuven.be

*These authors provided equal contribution to this work.

Cell Transplantation, Vol. 19, pp. 937-948, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368910X494911
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Factors Affecting Residence Time of Mesenchymal Stromal Cells (MSC) Injected Into the Myocardium

Jason Westrich,1* Peter Yaeger,1 Chufa He,1 Jeff Stewart,1 Raymond Chen,1 Gitta Seleznik,2# Shane Larson,3 Bruce Wentworth,1 Michael O'Callaghan,1 Sam Wadsworth,1 Geoffrey Akita,1 and Gyongyi Molnar1

1Cardiovascular Department of Genzyme Corporation, Framingham, MA, USA
2HAESF Graduate Scholarship Program, Budapest, Hungary
3Worcester Polytechnic Institute, Worcester, MA, USA

The therapeutic mechanism of mesenchymal stromal/stem cells (MSC) for the treatment of acute myocardial infarction is not well understood. Our goal was to get insights into this mechanism by analyzing the survival kinetics of allogeneic and syngeneic cell transplants under different tissue conditions. Two MSC cell banks, stably and equally expressing the luciferase reporter construct, were developed for these studies and injected directly to the myocardium of Lewis rat recipients under syngeneic or allogeneic transplantation conditions. Cell survival was monitored by real-time fashion for up to 2 weeks, using optical imaging device (IVIS, Xenogen Corp.). We found that both syngeneic and allogeneic grafts reduced significantly in size during the first week of transplantation, either in the normal or in the late infarcted heart (5 days after MI) and allotransplants became always smaller than syngeneic grafts during this period. Low dose of cyclosporine A treatment had a benefit on both allo- and syngeneic graft sizes, suggesting that multiple mechanisms play a role in early graft reduction. The MSC characteristic factors IL-6, IL-8, MCP-1, and VEGF were well above the control level in the heart tissue at 4 days after cell injection, suggesting that the peak therapeutic effect of MSC can be expected during the first week of the administration. Although allogeneic cells induced immunoglobulin production, their biological effects (cell survival, factor productions) are very similar to the syngeneic transplants and therefore they could deliver the same therapeutic effect as the syngeneic cells. Finally, freshly infarcted tissue (30 min) supported better the survival of MSC than late postischemic tissue (5 days) but only "off the shelf" allogeneic cell transplants fits with this treatment strategy.

Key words: Bone marrow stem/stromal cells; Heart; Cell survival; IVIS; Luciferase reporter

Address correspondence to Gyongyi Molnar, Cardiovascular DepartmentGenzyme Corporation, 49 New York Ave, Framingham, MA 01701, USA. Tel: 1-508-271-3725; Fax: 1-508-271-4917; E-mail: gyongyi.molnar@genzyme.com

*Current address: Environmental Health Sciences, University of Georgia, Athens, GA, USA.
#Current address: Transplantation Medicine and Immunology, University of Zurich, Switzerland.

Cell Transplantation, Vol. 19, pp. 949-958, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368910X504450
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

The Apelin-APJ Pathway Exists in Cardiomyogenic Cells Derived From Mesenchymal Stem Cells In Vitro and In Vivo

Lian Ru Gao,1 Nin Kun Zhang,1 Jing Bai,2 Qing Ai Ding,1 Zhi Guo Wang,1 Zhi Ming Zhu,1 Yu Xing Fei,1 Ye Yang,1 Ru Yi Xu,1 and Yu Chen1

1Department of Cardiology, Navy General Hospital, Beijing, China
2The First Affiliated Hospital of Chinese PLA General Hospital, Beijing, China

Our previous study demonstrated that apelin level increased significantly after the treatment of intracoronary implantation of bone marrow mononuclear cells (BMMCs), followed by the improvement of cardiac function in patients with severe ischemic heart failure. The present studies both in vivo and in vitro explored whether mesenchymal stem cells derived from bone marrow (BMSCs) activate the apelin-APJ pathway when differentiating into cardiomyogenic cells. Isolated BMSCs from rat femurs and tibias were cultured and expanded for three passages, labeled with DAPI, and treated with 5-azacytidine (5-AZ). BMSCs labeled with ad-EGFP were injected intramyocardially into the peri-infarct area of rat models with acute myocardial infarction. Immunofluorescence staining exposed that CMGs expressed apelin together with myogenic-specific proteins such as a-actin, troponin T, GATA-4, and connexin-43 at 7 days after 5-AZ treatment or EGFP-BMSC injection. RT-PCR revealed that mRNA in CMGs started to express apelin and APJ from day 7 and progressively increased until day 28. Cardiac function, as measured by echocardiography in vivo, was significantly improved in parallel with the extent of apelin expression after BMSC transplantation. Our finding indicated that the expression of the apelin-APJ pathway during differentiation of BMSCs into CMGs may be an important mechanism in regulation of myocardial regeneration and functional recovery after BMSC transplantation.

Key words: Myocardial infarct; Cell transplantation; Mesenchymal stem cell; Cardiomyogenic cell; Apelin-APJ pathway

Address correspondence to Dr. Yu Chen or Dr. Lian Ru Gao, Department of Cardiology, Navy General Hospital, 6 Fucheng Road, Beijing 100048, China. Tel: 011-86-10-88180197; Fax: 011-86-10-68780185;E-mail: yuchen911@hotmail.com or lianru@yahoo.com.cn

Cell Transplantation, Vol. 19, pp. 959-964, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368910X514648
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Global Contractility Increment in Nonischemic Dilated Cardiomyopathy After Free Wall-Only Intramyocardial Injection of Autologous Bone Marrow Mononuclear Cells: An Insight Over Stem Cells Clinical Mechanism of Action

Roberto T. Sant'Anna,1 Renato A. K. Kalil,1,2 Angelo S. Pretto Neto,1,2 Fernando Pivatto Júnior,1,2 James Fracasso,1 João R. M. Sant'Anna,1 Maurício Marques,1 Melissa Markoski,1 Paulo R. Prates,1 Nance B. Nardi,1,3 and Ivo A. Nesralla1

1Instituto de Cardiologia do Rio Grande do Sul/FUC, Porto Alegre, RS, Brazil
2Universidade Federal de Ciências da Saúde de Porto Alegre, Porto Alegre, RS, Brazil
3Universidade Luterana do Brasil, Canoas, RS, Brazil

Bone marrow mononuclear cells (BMMC) effects have been investigated in small series of nonischemic dilated cardiomyopathy (NIDC). Left ventricular myocardial contractility improvements occur, but doubt remains about their mechanism of action. We compared contractility changes in areas treated (free wall) and nontreated (septal wall) with BMMC, in selected patients who have showed significant ventricular improvement after free wall-only intramyocardial stem cells injection. From 15 patients with functional class III/IV (NYHA) and LVEF inferior to 35%, who received 9.6 ± 2.6 x 107 BMMC divided into 10 points over the left ventricular free wall, 7 (46.7%) showed LVEF relative improvement greater than 15%. Those patients were selected for further contractility study. BMMC were collected from iliac bone and isolated with Ficoll-Hypaque. Magnetic resonance imaging was used to measure the systolic thickening of the septal (nontreated) and free wall (treated) before injection and 3 months postoperatively. Mean systolic septal wall thickening increased from 0.46 to 1.23 mm (an absolute 0.77 ± 1.3 mm and relative 167.4% increase) and in the free wall from 1.13 to 1.87 mm (an absolute 0.74 ± 1.5 mm and relative increase of 65.5%). There was no difference in the rate of absolute or relative systolic thickening between the two walls (p = 0.866 and 1.0, respectively), when cells were injected only in the left ventricular free wall. BMMC transplantation in nonischemic dilated cardiomyopathy can improve ventricular function by an overall effect, even in areas that are not directly injected. This finding favors the existence of a diffuse mechanism of action, rather than a local effect, and should be reminded when the pathophysiology of stem cells is considered.

Key words: Stem cells; Autologous transplantation; Dilated cardiomyopathy; Heart fail

Address correspondence to Renato A. K. Kalil, Instituto de Cardiologia do Rio Grande do Sul/Fundação Universitária de Cardiologia (IC/FUC) and Universidade Federal de Ciências da saúde de Porto Alegre (UFCSPA)Av. Princesa Isabel, 395 90620-000 Santana-Porto Alegre, RS, Brasil. Tel: 55 51 32172729; Fax: 55 51 32192802; E-mail: kalil.pesquisa@cardiologia.org.br

Cell Transplantation, Vol. 19, pp. 965-971, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368910X506845
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Bone Marrow Mononuclear Cell Therapy Led to Alveolar-Capillary Membrane Repair, Improving Lung Mechanics in Endotoxin-Induced Acute Lung Injury

Luiz Felipe M. Prota,1 Roberta M. Lassance,1 Tatiana Maron-Gutierrez,1,2 Raquel C. Castiglione,1 Cristiane Souza Baez Garcia,2 Maria Cristina Ebole Santana,2 Jackson Souza-Menezes,1,6 Soraia C. Abreu,2 Vivian Samoto,4 Marcelo Felipe Santiago,3 Vera Luiza Capelozzi,5 Christina Maeda Takiya,4 Patricia R. M. Rocco,2 and Marcelo M. Morales1

1Laboratory of Cellular and Molecular Physiology, Carlos Chagas Filho Biophysics Institute, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
2Laboratory of Pulmonary Investigation, Carlos Chagas Filho Biophysics Institute, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
3Laboratory of Cellular and Molecular Neurology, Carlos Chagas Filho Biophysics Institute, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
4Laboratory of Cell Pathology, Biomedical Science Institute, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
5Department of Pathology, University of São Paulo, São Paulo, Brazil
6Federal University of Rio de Janeiro, Macaé, Brazil

The aim of this study was to test the hypothesis that bone marrow mononuclear cell (BMDMC) therapy led an improvement in lung mechanics and histology in endotoxin-induced lung injury. Twenty-four C57BL/6 mice were randomly divided into four groups (n = 6 each). In the acute lung injur;y (ALI) group, Escherichia coli lipopolysaccharide (LPS) was instilled intratracheally (40 mg, IT), and control (C) mice received saline (0.05 ml, IT). One hour after the administration of saline or LPS, BMDMC (2 x 107 cells) was intravenously injected. At day 28, animals were anesthetized and lung mechanics [static elastance (Est), resistive (DP1), and viscoelastic (DP2) pressures] and histology (light and electron microscopy) were analyzed. Immunogold electron microscopy was used to evaluate if multinucleate cells were type II epithelial cells. BMDMC therapy prevented endotoxin-induced lung inflammation, alveolar collapse, and interstitial edema. In addition, BMDMC administration led to epithelial and endothelial repair with multinucleated type II pneumocytes. These histological changes yielded a reduction in lung Est, DP1, and DP2 compared to ALI. In the present experimental ALI model, the administration of BMDMC yielded a reduction in the inflammatory process and a repair of epithelium and endothelium, reducing the amount of alveolar collapse, thus leading to an improvement in lung mechanics.

Key words: Lung mechanics; Bone marrow-derived mononuclear cells (BMDMC); Electron microscopy; Inflammation

Address correspondence to Marcelo Marcos Morales, Universidade Federal do Rio de Janeiro, Instituto de Biofísica Carlos Chagas Filho-C.C.S., Laboratório de Fisiologia Celular e Molecular, Ilha do Fundão, 21949-902 Rio de Janeiro-RJ, Brazil. Tel: +55 21 2562 6572; Fax: +55 21 2280 8193; E-mail: mmorales@biof.ufrj.br

Cell Transplantation, Vol. 19, pp. 973-984, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368910X494885
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Human Mesenchymal Stromal Cells and Their Derivative, SB623 Cells, Rescue Neural Cells via Trophic Support Following In Vitro Ischemia

Ciara C. Tate, Carlos Fonck, Michael McGrogan, and Casey C. Case

SanBio, Inc., Mountain View, CA, USA

Cell transplantation is a promising treatment strategy for many neurological disorders, including stroke, which can target multiple therapeutic mechanisms in a sustained fashion. We investigated the ability of human mesenchymal stromal cells (MSCs) and MSC-derived SB623 cells to rescue neural cells via trophic support following an in vitro stroke model. Following oxygen glucose deprivation, cortical neurons or hippocampal slices were cocultured with either MSCs or SB623 cells separated by a semiporous membrane (prohibits cell-cell contact) or with MSC- or SB623 cell-conditioned medium. MSCs, SB623 cells, MSC-conditioned media, and SB623 cell-conditioned media all significantly reduced neural cell damage/death compared to untreated conditions, and the rescue effect of the conditioned media was dose dependent. We identified 11 neurotrophic factors secreted by MSCs and/or SB623 cells. This study emphasizes the importance of trophic support provided by marrow-derived cells, which likely contributes to the efficacy of cell therapy for brain injury.

Key words: Cerebral ischemia; Mesenchymal stromal cells; Oxygen glucose deprivation; SB623 cells; Trophic support

Address correspondence to Ciara C. Tate, SanBio Inc., 231 S. Whisman Road, Mountain View, CA 94041, USA. Tel: 650-625-8965, ext. 35; Fax: 650-625-8969; E-mail: ciara.tate@san-bio.com

Cell Transplantation, Vol. 19, pp. 985-998, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368910X498944
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Embryonic Stem Cell-Derived Glial Precursors as a Vehicle for Sulfamidase Production in the MPS-IIIA Mouse Brain

Aaron J. Robinson,1 Guoying Zhao,1 Joy Rathjen,2* Peter D. Rathjen,2* Rhonda G. Hutchinson,3 Helen J. Eyre,3 Kim M. Hemsley,1,4 and John J. Hopwood1,4

1Lysosomal Diseases Research Unit, SA Pathology at the Women's and Children's Hospital, North Adelaide, Australia
2School of Molecular and Biomedical Science, University of Adelaide, Adelaide, Australia
3Department of Cytogenetics, Women's and Children's Hospital, North Adelaide, Australia
4Department of Paediatrics, University of Adelaide, Women's and Children's Hospital, North Adelaide, Australia

Pluripotent stem cells, including human embryonic stem cells and induced pluripotent stem cells, have generated much excitement about their prospects for use in cell transplantation therapies. This is largely attributable to their virtually unlimited growth potential, their ability to be precisely genetically altered in culture, and their utility for forming differentiated cell populations with potential clinical applications. Lysosomal storage diseases such as Sanfilippo syndrome (MPS-IIIA) represent ideal candidate diseases for the evaluation of cell therapies in the central nervous system (CNS). These diseases exhibit widespread pathology yet result from a single gene deficiency, in the case of Sanfilippo syndrome the lysosomal enzyme sulfamidase. The aim of this study was to investigate mouse embryonic stem (ES) cell-derived glial precursor cells as a vehicle for sulfamidase delivery in the MPS-IIIA mouse brain. In this study we have created a mouse ES cell line genetically modified to stably express and secrete high levels of human sulfamidase and a protocol for the in vitro derivation of large numbers glial precursors from ES cells. Differentiation of sulfamidase-expressing ES cells resulted in cell populations with sustained secretion of high levels of sulfamidase, comprised primarily of glial precursor cells with minor contaminants of other neural cell phenotypes but not residual pluripotent cells. CNS implantation studies demonstrated that ES cell-derived glial precursor cells formed using this differentiation method were able to engraft and survive for at least 12 weeks following implantation. The percentage of engraftment was quantified in different regions of the brain in 2-, 4-, and 8-week-old normal and MPS-IIIA mice. No teratomas were observed in any of the cell-transplanted animals. The results of this study support the further investigation of sulfamidase-expressing glial precursor cells as a vehicle for delivery of deficient enzyme into the CNS of MPS-IIIA mice.

Key words: Cell therapy; Glial precursor cell; Embryonic stem cell; Lysosomal storage disease; Central nervous system

Address correspondence to Dr. Aaron Robinson at his current address: Division of Molecular Medicine, The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville Victoria 3052, Australia. E-mail: arobinson@wehi.edu.au

*Current address: Department of Zoology, University of Melbourne, Parkville VIC, 3010.

Cell Transplantation, Vol. 19, pp. 999-1006, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368910X515854
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Evaluation of Tumorigenic Risk of Tissue-Engineered Oral Mucosal Epithelial Cells by Using Combinational Examinations

A. Thépot,1,2 A. P. Morel,3 V. Justin,4 A. Desanlis,1 L. Thivillier,1 E. Hoffman,1 M. Till,5 R. Accardi,2 M. Tommasino,2 P. Breton,6 P. Hainaut,2 and O. Damour1

1Banque de Tissus et Cellules, Hôpital E. Herriot, Lyon Cedex 03, France
2Centre International de Recherche sur le cancer, Lyon, France
3Centre Léon Bérard, Lyon Cedex 08, France
4CellSeed France S.A.R.L., Lyon, France
5Laboratoire de cytogénétique HFME, Bron Cedex, France
6Service de Chirurgie Maxillo-faciale, Hospices Civils de Lyon, Lyon, France

Recently, oral mucosal epithelial cells were proposed as a cell source of the autologous cell transplant therapy for corneal trauma or disease. The question addressed is to know if the biological conditions of grafting could induce certain cellular, molecular, and genetic alterations that might increase the risk of mutations and possibly of cellular transformation. Recent progress in cancer research enables us to depict the generation mechanisms and basic characteristics of human cancer cells from molecular, cytological, and biological aspects. The aim of this study is to evaluate the risk of tumorigenicity of the oral mucosal epithelial culture process in order to mitigate that risk, if any, before clinical application. Oral mucosal epithelial cells from three different human donors were investigated by combinational examinations to detect possible tumorigenic transformation. We investigated (i) clonogenic and karyology types, (ii) the validation of proliferation rate, (iii) the epithelial-mesenchymal transition, (iv) anchorage-independent growth potential, and (v) tumorigenicity on nude mice. Results show that the culture process used in this study presents no risk of tumorigenicity.

Key words: Tissue engineering; Epithelial cells; Tumorigenicity; Cornea

Address correspondence to Dr. Odile Damour, Banque de Tissus et cellules, Hôpital E. Herriot, 5 place d'Arsonval 69437, Lyon Cedex 03, France. Tel: +33472110618; Fax: +33472110619; E-mail: odile.damour@chu-lyon.fr or Dr. Amélie Thépot, Banque de Tissus et cellules, Hôpital E. Herriot, 5 place d'Arsonval 69437, Lyon Cedex 03, France. Tel: +33472110618; Fax: +33472110619; E-mail: thepot.amelie@gmail.com

Cell Transplantation, Vol. 19, pp. 1007-1020, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368910X492571
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Prevascular Structures Promote Vascularization in Engineered Human Adipose Tissue Constructs Upon Implantation

Femke Verseijden,1 Sandra J. Posthumus-van Sluijs,1 Eric Farrell,2,3 Johan W. van Neck,1 Steven E. R. Hovius,1 Stefan O. P. Hofer,4 and Gerjo J. V. M. van Osch2,3

1Department of Plastic and Reconstructive Surgery, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands
2Department of Orthopedics, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands
3Department of Otorhinolaryngology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands
4Division of Plastic Surgery, University Health Network, University of Toronto, Toronto, Ontario, Canada

Vascularization is still one of the most important limitations for the survival of engineered tissues after implantation. In this study, we aim to improve the in vivo vascularization of engineered adipose tissue by preforming vascular structures within in vitro-engineered adipose tissue constructs that can integrate with the host vascular system upon implantation. Different cell culture media were tested and different amounts of human adipose tissue-derived mesenchymal stromal cells (ASC) and human umbilical vein endothelial cells (HUVEC) were combined in spheroid cocultures to obtain optimal conditions for the generation of prevascularized adipose tissue constructs. Immunohistochemistry revealed that prevascular structures were formed in the constructs only when 20% ASC and 80% HUVEC were combined and cultured in a 1:1 mixture of endothelial cell medium and adipogenic medium. Moreover, the ASC in these constructs accumulated lipid and expressed the adipocyte-specific gene fatty acid binding protein-4. Implantation of prevascularized ASC/HUVEC constructs in nude mice resulted in a significantly higher amount of vessels (37 ± 17 vessels/mm2) within the constructs compared to non-prevascularized constructs composed only of ASC (3 ± 4 vessels/mm2). Moreover, a subset of the preformed human vascular structures (3.6 ± 4.2 structures/mm2) anastomosed with the mouse vasculature as indicated by the presence of intravascular red blood cells. Our results indicate that preformed vascular structures within in vitro-engineered adipose tissue constructs can integrate with the host vascular system and improve the vascularization upon implantation.

Key words: Angiogenesis; Tissue engineering; Adipose tissue; Mesenchymal stem cells; Endothelial cells

Address correspondence to Femke Verseijden, Department of Plastic and Reconstructive Surgery, ErasmusMC, University Medical Center Rotterdam, P.O. Bbox 2040, 3000CA Rotterdam, The Netherlands. Tel: +31107043242; Fax: +317044685; E-mail: f.verseijden@erasmusmc.nl

Cell Transplantation, Vol. 19, pp. 1021-1029, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368910X491789
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Limited Penetration of Perfluorocarbon in Porcine Pancreas Preserved by Two-Layer Method With 19Fluorine Magnetic Resonance Spectroscopy and Headspace Gas Chromatography

Aditya Agrawal,1,5 Po-Wah So,2 Andy Penman,3 Steve Powis,5 Brian Davidson,1 Martin Press,4 and Barry Fuller1

1Department of HPB Surgery and Liver Transplantation, Royal Free Hospital and Royal Free and University College Medical School of Medicine, University College London, London, UK
2Biological Imaging Centre, Imperial College London, Hammersmith Hospital, London, UK
3F2 Chemicals Ltd, Preston, UK
4Department of Endocrinology, Royal Free Hospital and Royal Free and University College Medical School of Medicine, University College London, London, UK
5Department of Nephrology, Royal Free Hospital and Royal Free and University College Medical School of Medicine, University College London, London, UK

The mechanism of the two-layer method (TLM) of pancreas preservation is unclear. Facilitating oxygen diffusion into preserved pancreas has been suggested, but direct measurements of tissue pO2 have yielded conflicting results. The degree of penetration of perfluorocarbon (PFC) into the pancreas during TLM storage is unknown. Segments of porcine pancreas (7.5 cm in length) were preserved either in University of Wisconsin solution (UW) alone (n = 6) or in TLM for 24 h (n = 6). Pancreatic samples were analyzed using Varian INOVA 9.4T MR scanner. External PFC standard was introduced for quantification. Four consecutive transverse images of 4 mm thickness were obtained using a spin-echo sequence. 19Fluorine magnetic resonance spectroscopy (19F MRS) was performed with the same parameters except with more averages. MR data were confirmed by headspace chromatography. PFC standard was readily detected in 19F MR images. There was no signal from pancreas in 19F MR images following either UW or TLM storage. 19F MR spectra typical of PFC were not obtained from either UW- or TLM-preserved pancreas with nonlocalized 19F MRS. Mean concentration of PFC in TLM pancreas measured by head space chromatography was 0.011 nl/g (SD ± 0.006), not significantly different from background concentration (0.012 nl/g, SD ± 0.006) in UW pancreas (p = 0.42). There was no evidence of penetration of PFC into pancreas tissues investigated either by MR or chromatography in organs preserved at hypothermia by TLM, and mechanisms of TLM remain speculative.

Key words: 19F magnetic resonance spectroscopy; Headspace gas chromatography; Organ preservation; Pancreatic islet transplantation; Perfluorocarbon (PFC); Two-layer method

Address correspondence to Aditya Agrawal, Department of HPB Surgery and Liver Transplantation, Royal Free Hospital, Pond Street London NW3 2QG, UK. Tel: +44 207 830 2757; Fax: +44 207 830 2688; E-mail: agrawal_aditya@hotmail.com or a.agrawal@medsch.ucl.acuk

Cell Transplantation, Vol. 19, pp. 1031-1046, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368910X514639
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Role of CCL2/MCP-1 in Islet Transplantation

Raffaella Melzi,1 Alessia Mercalli,1 Valeria Sordi,1 Elisa Cantarelli,1 Rita Nano,1 Paola Maffi,2 Giovanni Sitia,3 Luca G. Guidotti,3 Antonio Secchi,2 Ezio Bonifacio,4 and Lorenzo Piemonti1

1Beta Cell Biology Unit, Diabetes Research Institute, San Raffaele Scientific Institute, Milan, Italy
2Transplant Unit, Department of Medicine, San Raffaele Scientific Institute, Milan, Italy
3Virology & Immunopathology Unit, Diabetes Research Institute, San Raffaele Scientific Institute, Milan, Italy
4Center for Regenerative Therapies Dresden, Dresden University of Technology, Dresden, Germany

High levels of donor-derived CCL2 have been associated with poor islet allograft outcome in patients with type 1 diabetes. The aim of our work was to determine whether CCL2 secreted by the islet has independent proinflammatory effects that influence engraftment and graft acceptance. Both in mice and humans CCL2 is significantly positively associated with other cytokines/chemokines, in particular with the highly released "proinflammatory" IL-6 and CXCL8 or CXCL1. Transplantation of CCL2-/- islets into syngenic recipients did not improve the transplant function. Transplantation of islets into CCL2-/- syngenic recipients led to a significant improvement of transplant function and partial abrogation of local hepatic inflammation. When evaluated in human islets CCL2 release was strongly related to the immediate local inflammatory response in the liver and impacted short-term human islet function dependently by the induced inflammatory response and independently by the immunosuppressive therapy. The data showed that islet CCL2 release is a sign of "inflamed" islets without having a direct role in graft failure. On the other hand, a causal effect for developing detrimental proinflammatory conditions after transplant was proved for recipient CCL2. Strategies to selectively decrease recipient, but not donor, CCL2 release may increase the success of islet transplantation.

Key words: Islet transplantation; Engraftment; Portal vein; Mouse model; Human model

Address correspondence to Lorenzo Piemonti, Beta Cell Biology Unit, Diabetes Research Institute, S. Raffaele Scientific Institute, Via Olgettina 60, 20132 Milan, Italy. Tel: 39 02 26432706; Fax: 39 02 26433960; E-mail: piemonti.lorenzo@hsr.it

Cell Transplantation, Vol. 19, pp. 1047-1054, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368910X500643
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Endotoxin Deactivation by Transient Acidification

Melina M. Ribeiro,1,2 Xiumin Xu,1 Dagmar Klein,1 Norma S. Kenyon,1 Camillo Ricordi,1 Maria Sueli S. Felipe,2 and Ricardo L. Pastori1

1Diabetes Research Institute, University of Miami Leonard Miller School of Medicine, Miami, FL, USA
2Depto de Biologia Celular, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, Brazil

Recombinant proteins are an important tool for research and therapeutic applications. Therapeutic proteins have been delivered to several cell types and tissues and might be used to improve the outcome of the cell transplantation. Recombinant proteins are propagated in bacteria, which will contaminate them with the lypopolysacharide endotoxin found in the outer bacterial membrane. Endotoxin could interfere with in vitro biological assays and is the major pathological factor, which must be removed or inactivated before in vivo administration. Here we describe a one-step protocol in which the endotoxin activity on recombinant proteins is remarkably reduced by transient exposure to acidic conditions. Maximum endotoxin deactivation occurs at acidic pH below their respective isoelectric point (pI). This method does not require additional protein purification or separation of the protein from the endotoxin fraction. The endotoxin level was measured both in vitro and in vivo. For in vitro assessment we have utilized Limulus Amebocyte Lysate method for in vivo the pyrogenic test. We have tested the above-mentioned method with five different recombinant proteins, including a monoclonal antibody clone 5c8 against CD154 produced by hybridomas. More than 99% of endotoxin was deactivated in all of the proteins; the recovery of the protein after deactivation varied between maximum 72.9% and minimum 46.8%. The anti-CD154 clone 5c8 activity remained unchanged as verified by the measurement of binding capability to activated lymphocytes. Furthermore, the effectiveness of this method was not significantly altered by urea, commonly used in protein purification. This procedure provides a simple and cost-efficient way to reduce the endotoxin activity in antibodies and recombinant proteins.

Key words: Endotoxin; Recombinant protein; Pyrogen; Lypopolysaccharide

Address correspondence to Ricardo L. Pastori, Ph.D., Diabetes Research Institute, University of Miami Leonard Miller School of Medicine, 1450 NW 10th Avenue (R-134), Miami, FL 33136, USA. Tel: (305) 243-5349; Fax: (305) 243-4404; E-mail: rpastori@med.miami.edu

Cell Transplantation, Vol. 19, pp. 1055-1062, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368910X494632
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Brief Communication
Comparison of Transplant Efficiency Between Spontaneously Derived and Noggin-Primed Human Embryonic Stem Cell Neural Precursors in the Quinolinic Acid Rat Model of Huntington's Disease

Elena M. Vazey,1 Mirella Dottori,2 Pegah Jamshidi,3 Doris Tomas,4 Martin F. Pera,5 Malcolm Horne,3,4 and Bronwen Connor1

1Department of Pharmacology and Clinical Pharmacology, FMHS, University of Auckland, Auckland, New Zealand
2Centre for Neuroscience and Department of Pharmacology, University of Melbourne, Parkville, Australia
3Monash Institute of Medical Research, Monash University, and The Australian Stem Cell Centre, Victoria, Australia
4Howard Florey Institute of Experimental Physiology and Medicine, University of Melbourne, Victoria, Australia
5Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA

Human neural precursors (hNP) derived from embryonic stem cells (hESC) may provide a viable cellular source for transplantation therapy for Huntington's disease (HD). However, developing effective transplantation therapy for the central nervous system (CNS) using hESC relies on optimizing the in vitro production of hNP to control appropriate in vivo posttransplantation neuronal differentiation. The current study provides the first direct in vivo comparison of the transplant efficiency and posttransplantation characteristics of spontaneously derived and noggin-primed hNP following transplantation into the quinolinic acid (QA) rat model of HD. We show that spontaneously derived and noggin-primed hNP both survived robustly up to 8 weeks after transplantation into the QA-lesioned striatum of the adult rat. Transplanted hNP underwent extensive migration and large-scale differentiation towards a predominantly neuronal fate by 8 weeks posttransplantation. Furthermore, in vitro noggin priming of hNP specifically increased the extent of neuronal differentiation at both 4 and 8 weeks posttransplantation when compared to spontaneously derived hNP grafts. The results of this study suggest that in vitro noggin priming provides an effective mechanism by which to enhance hNP transplant efficiency for the treatment of HD.

Key words: Stem cell transplantation; Noggin; Neural progenitors; Huntington's disease

Address correspondence to Associate Professor Bronwen Connor, Department of Pharmacology & Clinical Pharmacology, School of Medical Sciences, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag 92019, Auckland 1142, New Zealand. Tel: +64 9 373 7599, ext. 83037; Fax: +64 9 373 7556; E-mail: b.connor@auckland.ac.nz