Cell Transplantation 21(9) Abstracts

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Cell Transplantation, Vol. 21, pp. 1805–1820, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X652986
E-ISSN 1555-3892
Copyright ©2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Effects of Iron Oxide Nanoparticle Labeling on Human Endothelial Cells

Fu-Yuan Yang,* Ming-Xi Yu,* Quan Zhou,† Wen-Li Chen,* Peng Gao,† and Zheng Huang‡

*MOE Key Laboratory of Laser Life Science, South China Normal University, Guangzhou, China
†Medical Imaging Center, the First Affiliated Hospital of Jinan University, Guangzhou, China
‡School of Medicine and School of Engineering and Applied Science, University of Colorado-Denver, Denver, CO, USA

Iron oxide nanoparticles (INOPS) are a potential contrast agent for magnetic resonance (MR) tracking of transplanted endothelial cells. The objective of this study was to examine the effect of INOPS labeling on endothelial cells. The mixture of INOPS and poly-L-lysine (PLL) was used to label human endothelial cells. Labeling efficiency was examined by Prussian blue staining, transmission electron microscopy, and atomic absorption spectrometry. The effect of iron oxide concentration on cell viability and proliferation were determined. The correlation of reactive oxygen species (ROS) and apoptosis was also examined. In vitro MRI scanning was carried out using a 1.5T MR system. INOPS-PLL could be readily taken up by endothelial cells and subsequently induce MRI signal intensity changes. However, higher labeling concentration (>50 μg/ml) and longer incubation (48 h) can affect cell viability and proliferation. Mitochondrial damage, apoptosis, and autolysosmes were observed under high INOPS-PLL concentrations, which were correlated to ROS production. INOPS-PLL nanoparticles can be used to label transplanted endothelial cells. However, high concentration of INOPS can impair cell viability, possibly through ROS-mediated apoptosis and autophagy.

Key words: Iron oxide nanoparticles (INOPS); Magnetic resonance imaging (MRI); Cell tracking; Cell labeling

Received February 21, 2011; final acceptance November 11, 2011. Online prepub date: July 5, 2012.
Address correspondence to Quan Zhou, Medical Imaging Center, the First Affiliated Hospital of Jinan University, Guangzhou, 510630, China. Tel: +86-20-38688416; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it or Wen-Li Chen, MOE Key Laboratory of Laser Life Science, South China Normal University, Guangzhou, 510631, China. Tel: +86-20-85224007; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 1821–1835, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X637416
E-ISSN 1555-3892
Copyright ©2012 Cognizant Comm. Corp.
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18F-FDG Cell Labeling May Underestimate Transplanted Cell Homing: More Accurate, Efficient, and Stable Cell Labeling With Hexadecyl-4-[18F]Fluorobenzoate for In Vivo Tracking of Transplanted Human Progenitor Cells by Positron Emission Tomography

Yan Zhang,*†‡§ Jean N. DaSilva,†‡§ Tayebeh Hadizad,†‡ Stephanie Thorn,†‡§ Drew Kuraitis,*‡§ Jennifer M. Renaud,†‡ Ali Ahmadi,*‡§ Myra Kordos,†‡ Robert A. deKemp,†‡ Rob S. Beanlands,†‡§ Erik J. Suuronen,*‡§ and Marc Ruel*‡§

*Division of Cardiac Surgery, University of Ottawa Heart Institute, Ottawa, Canada
†Cardiac PET Centre, Division of Cardiology, University of Ottawa Heart Institute, Ottawa, Canada
‡Molecular Function and Imaging Program, University of Ottawa Heart Institute, Ottawa, Canada
§Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Canada

Cell therapy is expected to restore perfusion and improve function in the ischemic/infarcted myocardium; however, the biological mechanisms and local effects of transplanted cells remain unclear. To assess cell fate in vivo, hexadecyl-4-[18F]fluorobenzoate (18F-HFB) cell labeling was evaluated for tracking human circulating progenitor cells (CPCs) with positron emission tomography (PET) and was compared to the commonly used 2-[18F]fluoro-2-deoxy-D-glucose (18F-FDG) labeling method in a rat myocardial infarction model. CPCs were labeled with 18F-HFB or 18F-FDG ex vivo under the same conditions. 18F-HFB cell-labeling efficiency (23.4 ± 7.5%) and stability (4 h, 88.4 ± 6.0%) were superior to 18F-FDG (7.6 ± 4.1% and 26.6 ± 6.1%, respectively; p < 0.05). Neither labeling approach significantly altered cell viability, phenotype or migration potential up to 24 h postlabeling. Two weeks after left anterior descending coronary artery ligation, rats received echo-guided intramyocardial injection in the infarct border zone with 18F-HFB-CPCs, 18F-FDG-CPCs, 18F-HFB, or 18F-FDG. Dynamic PET imaging of both 18F-HFB-CPCs and 18F-FDG-CPCs demonstrated that only 16–37% of the initial injection dose (ID) was retained in the injection site at 10 min postdelivery, and remaining activity fell significantly over the first 4 h posttransplantation. The 18F-HFB-CPC signal in the target area at 2 h (23.7 ± 14.7% ID/g) and 4 h (17.6 ± 13.3% ID/g) postinjection was greater than that of 18F-FDG-CPCs (5.4 ± 2.3% ID/g and 2.6 ± 0.7% ID/g, respectively; p < 0.05). Tissue biodistribution confirmed the higher radioactivity in the border zone of 18F-HFB-CPC rats. Immunostaining of heart tissue sections revealed no significant difference in cell retention between two labeled cell transplantation groups. Good correlation with biodistribution results was observed in the 18F-HFB-CPC rats (r = 0.81, p < 0.05). Compared to 18F-FDG, labeling human CPCs with 18F-HFB provides a more efficient, stable, and accurate way to quantify the distribution of transplanted cells. 18FHFB cell labeling with PET imaging offers a better modality to enhance our understanding of early retention, homing, and engraftment with cardiac cell therapy.

Key Words: Stem cells; Myocardial infarction; Transplantation; Cell tracking; Positron emission tomography

Received March 21, 2010; final acceptance November 11, 2011. Online prepub date: March 28, 2012.
Address correspondence to Marc Ruel or Erik J. Suuronen, Division of Cardiac Surgery, University of Ottawa Heart Institute, 40 Ruskin Street, Ottawa K1Y 4W7, Canada. Tel: +1-613-798-5555 ext. 14893 (M.R.) or ext. 19087 (E.J.S.); Fax: +1-613-761-5367; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it or This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 1837–1851, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X627426
E-ISSN 1555-3892
Copyright ©2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

The Lewis GFP Transgenic Rat Strain Is a Useful Cell Donor for Neural Transplantation

Martin Krause,*1 Claudia Ganser,*1 Eiji Kobayashi,† Anna Papazoglou,* and Guido Nikkhah*

*Laboratory of Molecular Neurosurgery, Department of Stereotactic and Functional Neurosurgery, Neurocentre, University Hospital Freiburg, Freiburg, Germany
†Division of Development of Advanced Treatment, Center for Development of Advanced Medical Technology, Jichi Medical University, Tochigi, Japan

Stem cell transplantation is a promising therapeutic approach in neurodegenerative diseases. Studying graft survival and development has important implications for the further development of experimental and clinical transplantation protocols. Cellular elements in neural transplants are sometimes difficult to identify. The existing labeling methods cannot reliably provide stably labeled cells that can be detected in long-term experiments. Transgenic (tg) Lewis rats ubiquitously expressing green fluorescent protein (GFP) provide an ideal donor source. The aim of this project was to investigate the potential of GFP-tg Lewis rats to serve as donor tissue for neural stem cell transplantation. Ventral mesencephalon (VM) GFP-tg E14.5-derived cells were compared to wild-type (wt) in vitro and in vivo. Firstly, cells from GFP and non-GFP VM tissue were compared with regard to their proliferation and response towards 6-OHDA-toxicity in culture. Secondly, 6-OHDA-lesioned hemiparkinsonian Sprague–Dawley/Crl:CD(SD) rats received intrastriatal grafts derived from VM of E14.5 GFP-tg rats. Due to the fact that donor and recipient belong to two different rat strains, we focused on graft survival in correlation with immunosuppression and graft GFP and tyrosine hydroxylase (TH) expression. In summary, in vitro tg cells exhibited 98% GFP expression and did not differ from wt cells in any of the measured parameters. In vivo, all experimental groups showed a significant compensation in rotation behavior after transplantation. Furthermore, there was no difference on rotation behavior or graft morphology and survival pattern as well as GFP expression between immunosuppressed and nonimmunosuppressed animals. The GFP-positive population of the graft was composed of 13.3% GFAP-positive, 56.1% NeuN-positive, and 1.9% TH-positive cells. Analysis of graft subpopulations manifested that 70.6% of GFAP-positive, 86.9% of NeuN-positive, and 80.1% of TH-positive cells coexpressed GFP. In conclusion, our data show that the Lewis GFP-tg rats serve as an excellent cell source for studying primary neural precursor cells in the transplantation paradigm.

Key words: Green fluorescent protein; MFB model for PD; Ventral mesencephalon; Parkinson’s disease; Dopamine; 6-Hydroxydopamine; Neural transplantation

Received September 30, 2010; final acceptance July 5, 2011. Online prepub date: March 8, 2012.
1These authors contributed equally to this study and share first authorship.
Address correspondence to Anna Papazoglou, Laboratory of Molecular Neurosurgery, Department of Stereotactic and Functional Neurosurgery, Neurocentre, Albert-Ludwigs-University, Breisacher Str. 64, D-79106 Freiburg, Germany. Tel: +49-761-270-5056; Fax: +49-761-270-9303; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 1853–1865, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X627471
E-ISSN 1555-3892
Copyright ©2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Noninvasive Bioluminescence Imaging of Olfactory Ensheathing Glia and Schwann Cells Following Transplantation Into the Lesioned Rat Spinal Cord

Kasper C. D. Roet,* Ruben Eggers,* and Joost Verhaagen*†

*Department of Neuroregeneration, Netherlands Institute for Neuroscience, An Institute of the Royal Netherlands Academy of Arts and Sciences, Amsterdam, The Netherlands
†Department of Molecular and Cellular Neurobiology, Center for Neurogenomics and Cognitive Research, Neuroscience Campus Amsterdam, VU University, Amsterdam, The Netherlands

In this study, we assess the feasibility of bioluminescence imaging to monitor the survival of Schwann cells (SCs) and olfactory ensheathing glia cells (OECs) after implantation in the lesioned spinal cord of adult rats. To this end, purified SCs and OECs were genetically modified with lentiviral vectors encoding luciferase-2 and GFP and implanted in the lesioned dorsal column. The bioluminescent signal was monitored for over 3 months, and at 7 and 98 days postsurgery, the signal was compared to standard histological analysis of GFP expression in the spinal cords. The temporal profile of the bioluminescent signal showed three distinct phases for both cell types. (I) A relatively stable signal in the first week. (II) A progressive decline in signal strength in the second and third week. (III) After the third week, the average bioluminescent signal stabilized for both cell types. Interestingly, in the first week, the peak of the bioluminescent signal after luciferin injection was delayed when compared to later time points. Similar to in vitro, our data indicated a linear relationship between the in vivo bioluminescent signal and the GFP signal of the SCs and OECs in the spinal cords when the results of both the 7 and 98 day time points are combined. This is the first report of the use of in vivo bioluminescence to monitor cell survival in the lesioned rat spinal cord. Bioluminescence could be a potentially powerful, noninvasive strategy to examine the efficacy of treatments that aim to improve the survival of proregenerative cells transplanted in the injured rat spinal cord.

Key words: Bioluminescence; Spinal cord; Noninvasive; Olfactory ensheathing cell; Schwann cell; Luciferase; Survival

Received April 13, 2011; final acceptance September 20, 2011. Online prepub date: March 22, 2012.
Address correspondence to Kasper C. D. Roet, M.Sc., Department of Neuroregeneration, Netherlands Institute for Neuroscience, Meibergdreef 47, 1105BA Amsterdam, The Netherlands. Tel: +31(20)5665511; Fax: +31(20)5666121; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 1867–1881, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X636920
E-ISSN 1555-3892
Copyright ©2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Cell Type-Associated Differences in Migration, Survival, and Immunogenicity Following Grafting in CNS Tissue

Jelle Praet,*†‡ Kristien Reekmans,*† Dan Lin,§ Nathalie De Vocht,*†‡ Irene Bergwerf,‡ Bart Tambuyzer,*† Jasmijn Daans,*† Niel Hens,†§ Herman Goossens,† Patrick Pauwels,# Zwi Berneman,*† Annemie Van der Linden,‡ and Peter Ponsaerts*†

*Laboratory of Experimental Hematology, University of Antwerp, Antwerp, Belgium
†Vaccine and Infectious Disease Institute (Vaxinfectio), University of Antwerp, Antwerp, Belgium
‡BioImaging Laboratory, University of Antwerp, Antwerp, Belgium
§Center for Statistics, I-Biostat, University of Hasselt, Hasselt, Belgium
#Laboratory of Pathology, University of Antwerp, Antwerp, Belgium

Cell transplantation has been suggested to display several neuroprotective and/or neuroregenerative effects in animal models of central nervous system (CNS) trauma. However, while most studies report on clinical observations, currently little is known regarding the actual fate of the cell populations grafted and whether or how the brain’s innate immune system, mainly directed by activated microglia and astrocytes, interacts with autologous cellular implants. In this study, we grafted well-characterized neural stem cell, mouse embryonic fibroblast, dendritic cell, bone marrow mononuclear cell, and splenocyte populations, all isolated or cultured from C57BL/6-eGFP transgenic mice, below the capsula externa (CE) of healthy C57BL/6 mice and below the inflamed/demyelinated CE of cuprizone-treated C57BL/6 mice. Two weeks postgrafting, an extensive quantitative multicolor histological analysis was performed in order (i) to quantify cell graft localization, migration, survival, and toxicity and (ii) to characterize endogenous CNS immune responses against the different cell grafts. Obtained results indicate dependence on the cell type grafted: (i) a different degree of cell graft migration, survival, and toxicity and (ii) a different organization of the endogenous immune response. Based on these observations, we warrant that further research should be undertaken to understand—and eventually control—cell graft-induced tissue damage and activation of the brain’s innate immune system. The latter will be inevitable before cell grafting in the CNS can be performed safely and successfully in clinical settings.

Key words: Cell transplantation; Survival; Migration; Toxicity; Immunogenicity

Received June 23, 2011; final acceptance November 26, 2011. Online prepub date: April 2, 2012.
Address correspondence to Dr. Peter Ponsaerts, Laboratory of Experimental Hematology, Vaccine and Infectious Disease Institute (Vaxinfectio), University of Antwerp, Campus Drie Eiken (CDE-S6.51), Universiteitsplein 1, 2610 Antwerp (Wilrijk), Belgium. Tel.: +32-3-2652428; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 1883–1897, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X637443
E-ISSN 1555-3892
Copyright ©2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Intraventricularly Injected Olig2-NSCs Attenuate Established Relapsing–Remitting EAE in Mice

Falak Sher,* Sandra Amor,†‡ Wouter Gerritsen,† David Baker,‡ Samuel L. Jackson,‡ Erik Boddeke,* and Sjef Copray*

*Department of Neuroscience, University Medical Centre Groningen, Groningen, The Netherlands
†Department of Pathology, VU University Medical Centre, Amsterdam, The Netherlands
‡Department of Neuroscience and Trauma Centre, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, UK

In multiple sclerosis (MS), a chronic inflammatory relapsing demyelinating disease, failure to control or repair damage leads to progressive neurological dysfunction and neurodegeneration. Implantation of neural stem cells (NSCs) has been shown to promote repair and functional recovery in the acute experimental autoimmune encephalomyelitis (EAE) animal model for MS; the major therapeutic mechanism of these NSCs appeared to be immune regulation. In the present study, we examined the efficacy of intraventricularly injected NSCs in chronic relapsing experimental autoimmune encephalomyelitis (CREAE), the animal disease model that is widely accepted to mimic most closely recurrent inflammatory demyelination lesions as observed in relapsing–remitting MS. In addition, we assessed whether priming these NSCs to become oligodendrocyte precursor cells (OPCs) by transient overexpression of Olig2 would further promote functional recovery, for example, by contributing to actual remyelination. Upon injection at the onset of the acute phase or the relapse phase of CREAE, NSCs as well as Olig2-NSCs directly migrated toward active lesions in the spinal cord as visualized by in vivo bioluminescence and biofluorescence imaging, and once in the spinal cord, the majority of Olig2-NSCs, in contrast to NSCs, differentiated into OPCs. The survival of Olig2-NSCs was significantly higher than that of injected control NSCs, which remained undifferentiated. Nevertheless, both Olig2-NSCs and NSC significantly reduced the clinical signs of acute and relapsing disease and, in case of Olig2-NSCs, even completely abrogated relapsing disease when administered early after onset of acute disease. We provide the first evidence that NSCs and in particular NSC-derived OPCs (Olig2-NSCs) ameliorate established chronic relapsing EAE in mice. Our experimental data in established neurological disease in mice indicate that such therapy may be effective in relapsing–remitting MS preventing chronic progressive disease.

Key words: Oligodendrocyte; Remyelination; Multiple sclerosis

Received March 9, 2011; final acceptance October 29, 2011. Online prepub date: March 28, 2012.
Address correspondence to Dr. Sjef Copray, Department of Neuroscience, University Medical Centre Groningen, A. Deusinglaan 1, 9713 AV Groningen, The Netherlands. Tel: +31 50 3632785; Fax: +31 50 3632751; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 1899–1907, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X582769
E-ISSN 1555-3892
Copyright ©2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Stem Cell Transplantation in Amyotrophic Lateral Sclerosis Patients: Methodological Approach, Safety, and Feasibility

Héctor R. Martínez,*†‡ Juan Francisco Molina-Lopez,† María Teresa González-Garza,† Jorge E. Moreno-Cuevas,† Enrique Caro-Osorio,†§ Alfonso Gil-Valadez,† Eugenio Gutierrez-Jimenez,† Oscar E. Zazueta-Fierro,† J. Alfonso Meza,† Patricio Couret-Alcaraz,† and Martin Hernandez-Torre¶

*Neurology Service, Hospital San Jose Tec de Monterrey, Monterrey, Nuevo Leon, Mexico
†Stem Cell Therapy Service, Tecnologico de Monterrey School of Medicine, Monterrey, Nuevo Leon, Mexico
‡Neurology Service, Hospital Universitario UANL, Monterrey, Nuevo Leon, Mexico
§Neurosurgery Service, Hospital San Jose Tec de Monterrey, Monterrey, Nuevo Leon, Mexico
¶Tecnologico de Monterrey School of Biotechnology and Health, Monterrey, Nuevo Leon, Mexico

Amyotrophic lateral sclerosis is characterized by the selective death of motor neurons. Stem cells have been proposed as a potential therapeutic strategy. The safety of stem cell transplantation into the frontal motor cortex to improve upper motor neuron function is described. Sixty-seven patients with definite amyotrophic lateral sclerosis were included. After giving their informed consent, the patients underwent magnetic resonance imaging, functional rating, pulmonary function test, and laboratory tests. Their bone marrow was stimulated with daily filgrastim (300 μg) given subcutaneously for 3 days. Peripheral blood mononuclear cells were obtained by leukapheresis. Isolated CD133+ stem cells were suspended in 300 μl of the patient’s cerebrospinal fluid and implanted into the motor cortex. Adverse events were recorded at each step of the procedure and were classified according to the Common Terminology Criteria for Adverse Events v3.0. The survival at 1 year was 90% after transplantation. with a mean long-term survival rate of 40.17 months from diagnosis. The most common adverse events were in grades I–II and involved transient skin pain (19.5% of patients) attributed to the insertion of the Mahurkar catheter into the subclavian vein, minor scalp pain (15.9%), and headache (12.2%) from the surgical procedure. Several patients (1.5–4.5%) reported diverse grade I adverse events. There were two deaths, one considered to be associated with the procedure (1.5%) and the other associated with the disease. Autologous stem cell transplantation into the frontal motor cortex is safe and tolerated well by patients. Further controlled studies are required to define the efficacy of this procedure.

Key words: ALS; Safety; Stem cell: Transplant

Received January 15, 2011; final acceptance October 7, 2011. Online prepub date: February 13, 2012.
Address correspondence to Héctor R. Martínez, M.D., FACP, Tecnologico de Monterrey, School of Medicine, CITES, 3rd Floor, 3000 Morones Prieto Pte, 64710 Monterrey, Nuevo Leon, Mexico. Tel: +52 (81) 8888 2177; Fax: +52 (81) 8888 2148; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 1909–1918, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X636948
E-ISSN 1555-3892
Copyright ©2012 Cognizant Comm. Corp.
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No Difference in Intra-Arterial and Intramuscular Delivery of Autologous Bone Marrow Cells in Patients With Advanced Critical Limb Ischemia

Andrej Klepanec,*† Martin Mistrik,Cestmir Altaner,§ Martina Valachovicova,* Ingrid Olejarova,Roman Slysko,Tibor Balazs,Terezia Urlandova,Daniela Hladikova,Branislav Liska,Jan Tomka,Ivan Vulev,*† and Juraj Madaric*†

*Slovak Medical University, Bratislava, Slovakia
†National Cardiovascular Institute, Bratislava, Slovakia
‡Clinic of Haematology and Transfusiology, Faculty Hospital, Bratislava, Slovakia
§Institute of Experimental Oncology, Slovak Academy of Science, Bratislava, Slovakia

Stem cell therapy has been proposed to be an alternative therapy in patients with critical limb ischemia (CLI), not eligible for endovascular or surgical revascularization. We compared the therapeutic effects of intramuscular (IM) and intra-arterial (IA) delivery of bone marrow cells (BMCs) and investigated the factors associated with therapeutic benefits. Forty-one patients (mean age, 66 ± 10 years; 35 males) with advanced CLI (Rutherford category, 5 and 6) not eligible for revascularization were randomized to treatment with 40 ml BMCs using local IM (n = 21) or selective IA infusion (n = 20). Primary endpoints were limb salvage and wound healing. Secondary endpoints were changes in transcutaneous oxygen pressure (tcpO2), quality-of-life questionnaire (EQ5D), ankle–brachial index (ABI), and pain scale (0–10). Patients with limb salvage and wound healing were considered to be responders to BMC therapy. At 6-month follow-up, overall limb salvage was 73% (27/37) and 10 subjects underwent major amputation. Four patients died unrelated to stem cell therapy. There was significant improvement in tcpO2 (15 ± 10 to 29 ± 13 mmHg, p < 0.001), pain scale (4.4 ± 2.6 to 0.9 ± 1.4, p < 0.001), and EQ5D (51 ± 15 to 70 ± 13, p < 0.001) and a significant decrease in the Rutherford category of CLI (5.0 ± 0.2 to 4.3 ± 1.6, p < 0.01). There were no differences among functional parameters in patients undergoing IM versus IA delivery. Responders (n = 27) were characterized by higher CD34+ cell counts in the bone marrow concentrate (CD34+ 29 ± 15×106 vs. 17 ± 12×106, p < 0.05) despite a similar number of total nucleated cells (4.3 ± 1.4×109 vs. 4.1 ± 1.2×109, p = 0.66) and by a lower level of C-reactive protein (18 ± 28 vs. 100 ± 96 mg/L, p < 0.05) as well as serum leukocytes (8.3 ± 2.1×109/L vs. 12.3 ± 4.5×109/L, p < 0.05) as compared with nonresponders (10 patients). Both IM and IA delivery of autologous stem cells are effective therapeutic strategies in patients with CLI. A higher concentration of CD34+ cells and a lower degree of inflammation are associated with better clinical therapeutic responses.

Key words: Autologous stem cells; Critical limb ischemia; Angiogenesis; Intramuscular delivery; Intra-arterial delivery

Received June 13, 2011; final acceptance October 22, 2011. Online prepub date: April 2, 2012.
Address correspondence to Juraj Madaric, M.D., Ph.D., Department of Cardiology and Angiology, National Cardiovascular Institute, Pod Krasnou horkou 1, 833 48 Bratislava, Slovakia. Tel: 00421259320276; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 1919–1930, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X627552
E-ISSN 1555-3892
Copyright ©2012 Cognizant Comm. Corp.
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Embryonic Stem Cells Improve Cardiac Function in Doxorubicin-Induced Cardiomyopathy Mediated Through Multiple Mechanisms

Dinender K. Singla, Aisha Ahmed, Reetu Singla, and Binbin Yan

Biomolecular Science Center, Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL, USA

Doxorubicin (DOX) is an effective antineoplastic agent used for the treatment of a variety of cancers. Unfortunately, its use is limited as this drug induces cardiotoxicity and heart failure as a side effect. There is no report that describes whether transplanted embryonic stem (ES) cells or their conditioned medium (CM) in DOX-induced cardiomyopathy (DIC) can repair and regenerate myocardium. Therefore, we transplanted ES cells or CM in DIC to examine apoptosis, fibrosis, cytoplasmic vacuolization, and myofibrillar loss and their associated Akt and ERK pathway. Moreover, we also determined activation of endogenous c-kit+ve cardiac stem cells (CSCs), levels of HGF and IGF-1, growth factors required for c-kit cell activation, and their differentiation into cardiac myocytes, which also contributes in cardiac regeneration and improved heart function. We generated DIC in C57Bl/6 mice (cumulative dose of DOX 12 mg/kg body weight, IP), and animals were treated with ES cells, CM, or cell culture medium in controls. Two weeks post-DIC, ES cells or CM transplanted hearts showed a significant (p < 0.05) decrease in cardiac apoptotic nuclei and their regulation with Akt and ERK pathway. Cardiac fibrosis observed in the ES cell or CM groups was significantly less compared with DOX and cell culture medium groups (p < 0.05). Next, cytoplasmic vacuolization and myofibrillar loss was reduced (p < 0.05) following treatment with ES cells or CM. Moreover, our data also demonstrated increased levels of c-kit+ve CSCs in ES cells or CM hearts and differentiated cardiac myocytes from these CSCs, suggesting endogenous cardiac regeneration. Importantly, the levels of HFG and IGF-1 were significantly increased in ES cells or CM transplanted hearts. In conclusion, we reported that transplanted ES cells or CM in DIC hearts significantly decreases various adverse pathological mechanisms as well as enhances cardiac regeneration that effectively contributes to improved heart function.

Key words: Stem cells; Heart; Doxorubicin (DOX); Apoptosis; Fibrosis

Received June 29, 2011; final acceptance October 22, 2011. Online prepub date: March 22, 2012.
Address correspondence to Dinender K. Singla, Ph.D. FAHA, Biomolecular Science Center, College of Medicine, University of Central Florida, 4000 Central Florida Blvd., Room 224, Orlando, FL, 32816, USA. Tel: +1 407-823-0953; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 1931–1944, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X627561
E-ISSN 1555-3892
Copyright ©2012 Cognizant Comm. Corp.
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Overexpression of TIMP-1 in Embryonic Stem Cells Attenuates Adverse Cardiac Remodeling Following Myocardial Infarction

Carley Glass and Dinender K. Singla

Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL, USA

Transplanted embryonic stem (ES) cells, following myocardial infarction (MI), contribute to limited cardiac repair and regeneration with improved function. Therefore, novel strategies are still needed to understand the effects of genetically modified transplanted stem cells on cardiac remodeling. The present study evaluates whether transplanted mouse ES cells overexpressing TIMP-1, an antiapoptotic and antifibrotic protein, can enhance cardiac myocyte differentiation, inhibit native cardiac myocyte apoptosis, reduce fibrosis, and improve cardiac function in the infarcted myocardium. MI was produced in C57BL/6 mice by coronary artery ligation. TIMP-1-ES cells, ES cells, or culture medium (control) were transplanted into the peri-infarct region of the heart. Immunofluorescence, TUNEL staining, caspase-3 activity, ELISAs, histology, and echocardiography were used to identify newly differentiated cardiac myocytes and assess apoptosis, fibrosis, and heart function. Two weeks post-MI, significantly (p < 0.05) enhanced engraftment and cardiac myocyte differentiation was observed in TIMP-1-ES cell-transplanted hearts compared with hearts transplanted with ES cells and control. Hearts transplanted with TIMP-1-ES cells demonstrated a reduction in apoptosis as well as an increase (p < 0.05) in p-Akt activity compared with ES cells or culture media controls. Infarct size and interstitial and vascular fibrosis were significantly (p < 0.05) decreased in the TIMP-1-ES cell group compared to controls. Furthermore, MMP-9, a key profibrotic protein, was significantly (p < 0.01) reduced following TIMP-1-ES cell transplantation. Echocardiography data showed fractional shortening and ejection fraction were significantly (p < 0.05) improved in the TIMP-1-ES cell group compared with respective controls. Our data suggest that transplanted ES cells overexpressing TIMP-1 attenuate adverse myocardial remodeling and improve cardiac function compared with ES cells that may have therapeutic potential in regenerative medicine.

Key words: Stem cells; Heart; TIMP-1; MMP; Apoptosis; Fibrosis

Received August 2, 2011; final acceptance October 22, 2011. Online prepub date: March 22, 2012.
Address correspondence to Dinender K. Singla, Ph.D., Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, 4000 Central Florida Blvd., Room 224, Orlando, FL, 32816, USA. Tel: +1 407-823-0953; Fax: 407-823-0956; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 1945–1967, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X638919
E-ISSN 1555-3892
Copyright ©2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Alteration of Cardiac Progenitor Cell Potency in GRMD Dogs

M. Cassano,* E. Berardi,* S. Crippa,* J. Toelen,† I. Barthelemy,‡ R. Micheletti,* M. Chuah,§ T. VandenDriessche,§ Z. Debyser,† S. Blot,‡ and M. Sampaolesi*¶

*Laboratory of Translational Cardiomyology, Stem Cell Institute, Department of Development and Regeneration, University of Leuven (KU Leuven), Belgium
‡Laboratoire de Neurobiologie, Ecole Nationale Vétérinaire d’Alfort, Maisons-Alfort, France
†Molecular Virology and Gene Therapy, Department of Molecular and Cellular Medicine, University of Leuven (KU Leuven), Belgium
§Department of Gene Therapy & Regenerative Medicine, Free University of Brussels (VUB), Brussles, Belgium and Center for Molecular and Vascular Biology, Department of Cardiovascular Sciences, University of Leuven (KU Leuven), Belgium

¶Human Anatomy Institute, Department of Public Health, Neuroscience, Experimental and Forensic Medicine, University of Pavia, Pavia, Italy

Among the animal models of Duchenne muscular dystrophy (DMD), the Golden Retriever muscular dystrophy (GRMD) dog is considered the best model in terms of size and pathological onset of the disease. As in human patients presenting with DMD or Becker muscular dystrophies (BMD), the GRMD is related to a spontaneous X-linked mutation of dystrophin and is characterized by myocardial lesions. In this respect, GRMD is a useful model to explore cardiac pathogenesis and for the development of therapeutic protocols. To investigate whether cardiac progenitor cells (CPCs) isolated from healthy and GRMD dogs may differentiate into myocardial cell types and to test the feasibility of cell therapy for cardiomyopathies in a preclinical model of DMD, CPCs were isolated from cardiac biopsies of healthy and GRMD dogs. Gene profile analysis revealed an active cardiac transcription network in both healthy and GRMD CPCs. However, GRMD CPCs showed impaired self-renewal and cardiac differentiation. Population doubling and telomerase analyses highlighted earlier senescence and proliferation impairment in progenitors isolated from GRMD cardiac biopsies. Immunofluorescence analysis revealed that only wt CPCs showed efficient although not terminal cardiac differentiation, consistent with the upregulation of cardiac-specific proteins and microRNAs. Thus, the pathological condition adversely influences the cardiomyogenic differentiation potential of cardiac progenitors. Using PiggyBac transposon technology we marked CPCs for nuclear dsRed expression, providing a stable nonviral gene marking method for in vivo tracing of CPCs. Xenotransplantation experiments in neonatal immunodeficient mice revealed a valuable contribution of CPCs to cardiomyogenesis with homing differences between wt and dystrophic progenitors. These results suggest that cardiac degeneration in dystrophinopathies may account for the progressive exhaustion of local cardiac progenitors and shed light on cardiac stemness in physiological and pathological conditions. Furthermore, we provide essential information that canine CPCs may be used to alleviate cardiac involvement in a large preclinical model of DMD.

Key words: Cardiac progenitor; Muscular dystrophy; GRMD model; Cell therapy

Received May 16, 2011; final acceptance October 21, 2011. Online prepub date: April 10, 2012.
Address correspondence to Maurilio Sampaolesi, Ph.D., Laboratory of Translational Cardiomyology, Stem Cell Institute, Department of Development and Regeneration, Katholieke Universiteit Leuven, Herestraat 49 O&N1 bus 814, 3000 Leuven, Belgium. Tel: +32-(0)163-30295; Fax: +32-(0)163-30294; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 1969–1984, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X647252
E-ISSN 1555-3892
Copyright ©2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Alginate Scaffolds for Mesenchymal Stem Cell Cardiac Therapy: Influence of Alginate Composition

Caroline Ceccaldi,*† Sophie Girod Fullana,* Chiara Alfarano,† Olivier Lairez,† Denis Calise,† Daniel Cussac,†‡ Angelo Parini,†‡§ and Brigitte Sallerin†‡§

*Université de Toulouse, CIRIMAT, UPS-INPT-CNRS, Faculté de Pharmacie, Toulouse, France
†INSERM, U858, Toulouse, France
‡Université de Toulouse, UPS, Faculté des Sciences Pharmaceutiques, Toulouse, France
§CHU Toulouse, Service de Pharmacie Hospitalière, Toulouse, France

Despite the success of alginate scaffolds and mesenchymal stem cells (MSCs) therapy in cardiac failure treatment, the impact of the physicochemical environment provided by alginate matrices on cell behavior has never been investigated. The purpose of this work was double: to determine the alginate composition influence on (1) encapsulated rat MSC viability, paracrine activity, and phenotype in vitro and (2) cardiac implantability and in vivo biocompatibility of patch shape scaffolds. Two alginates, differing in composition and thus presenting different mechanical properties when hydrogels, were characterized. In both cases, encapsulated MSC viability was maintained at around 75%, and their secretion characteristics were retained 28 days postencapsulation. In vivo study revealed a high cardiac compatibility of the tested alginates: cardiac parameters were maintained, and rats did not present any sign of infection. Moreover, explanted hydrogels appeared surrounded by a vascularized tissue. However, scaffold implantability was highly dependent on alginate composition. G-type alginate patches, presenting higher elastic and Young moduli than M-type alginate patches, showed a better implantation easiness and were the only ones that maintained their shape and morphology in vivo. As a consequence of alginate chemical composition and resulting hydrogel structuration, G-type alginate hydrogels appear to be more adapted for cardiac implantation.

Key words: Alginate; Cell encapsulation; Mesenchymal stem cells; Biomimetic materials; Heart

Received October 13, 2010; final acceptance October 28, 2011. Online prepub date: July 5, 2012.
Address correspondence to Caroline Ceccaldi, CIRIMAT, UPS-INPT-CNRS, Faculté de Pharmacie, 118 route de Narbonne, 31062 Toulouse Cedex 4, France. Tel: +33-5-62-25-68-37, Fax: +33-5-62-25-68-40; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 1985–1996, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X636858
E-ISSN 1555-3892
Copyright ©2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Adipose-Derived Stromal Cell Therapy Improves Cardiac Function After Coronary Occlusion in Rats

Luiza L. S. Bagno,* João Pedro S. Werneck-de-Castro,*† Patrícia F. Oliveira,‡ Márcia S. Cunha-Abreu,* Nazareth N. Rocha,*¶ Taís H. Kasai-Brunswick,* Vivian M. Lago,* Regina C. S. Goldenberg,* and Antonio C. Campos-de-Carvalho*§#

*Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
†Departamento de Biociências da Atividade Física, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
‡Área de Ciências Fisiológicas, Instituto de Ciências Biomédicas, Universidade Federal de Uberlândia, Brazil
§Instituto Nacional de Cardiologia, Rio de Janeiro, RJ, Brazil
¶Instituto Biomédico, Universidade Federal Fluminense, Rio de Janeiro, RJ, Brazil
#Albert Einstein College of Medicine, Bronx, NY, USA

Recent studies have identified adipose tissue as a new source of mesenchymal stem cells for therapy. The purpose of this study was to investigate the therapy with adipose-derived stromal cells (ASCs) in a rat model of healed myocardial infarction (MI). ASCs from inguinal subcutaneous adipose tissue of male Wistar rats were isolated by enzymatic digestion and filtration. Cells were then cultured until passage 3. Four weeks after ligation of the left coronary artery of female rats, a suspension of either 100 μl with phosphate-buffered saline (PBS) + Matrigel + 2 × 106 ASCs labeled with Hoechst (n = 11) or 100 μl of PBS + Matrigel (n = 10) was injected along the borders of the ventricular wall scar tissue. A sham-operated group (n = 5) was submitted to the same surgical procedure except permanent ligation of left coronary artery. Cardiac performance was assessed by electro- and echocardiogram. Echo was performed prior to injections (baseline, BL) and 6 weeks after injections (follow-up, FU), and values after treatment were normalized by values obtained before treatment. Hemodynamic measurements were performed 6 weeks after injections. All infarcted animals exhibited cardiac function impairment. Ejection fraction (EF), shortening fractional area (SFA), and left ventricular akinesia (LVA) were similar between infracted groups before treatment. Six weeks after therapy, ASC group showed significant improvement in all three ECHO indices in comparison to vehicle group. In anesthetized animals dp/dt+ was also significantly higher in ASCs when compared to vehicle. In agreement with functional improvement, scar area was diminished in the ASC group. We conclude that ASCs improve cardiac function in infarcted rats when administered directly to the myocardium.

Key words: Adipose-derived stromal cell; Myocardial infarction; Cell therapy and heart healing

Received July 18, 2011; final acceptance October 23, 2011. Online prepub date: April 2, 2012.
Address correspondence to Prof. Antônio Carlos Campos de Carvalho, Universidade Federal do Rio de Janeiro (UFRJ), Instituto de Biofísica Carlos Chagas Filho, CCS, bloco G, Ilha do Fundão, 21941-902, Rio de Janeiro, RJ, Brazil. Tel: +55-21/2562-6558; Fax: +55-21/2280-8193; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 1997–2008, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X637452
E-ISSN 1555-3892
Copyright ©2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Analysis of Biodistribution and Engraftment Into the Liver of Genetically Modified Mesenchymal Stromal Cells Derived From Adipose Tissue

Giuliana Di Rocco,*† Antonietta Gentile,‡ Annalisa Antonini,‡ Silvia Truffa,‡ Giulia Piaggio,† Maurizio C. Capogrossi,‡ and Gabriele Toietta†‡

*Vascular Biology and Regenerative Medicine, Centro Cardiologico Monzino IRCCS, Milan, Italy
†Experimental Oncology Department, Istituto Regina Elena IRCCS, Rome, Italy
‡Vascular Pathology, Istituto Dermopatico dell’Immacolata IRCCS, Rome, Italy

Presently, orthotopic liver transplant is the major therapeutic option for patients affected by primary liver diseases. This procedure is characterized by major invasive surgery, scarcity of donor organs, high costs, and lifelong immunosuppressive treatment. Transplant of hepatic precursor cells represents an attractive alternative. These cells could be used either for allogeneic transplantation or for autologous transplant after ex vivo genetic modification. We used stromal cells isolated from adipose tissue (AT-SCs) as platforms for autologous cell-mediated gene therapy. AT-SCs were transduced with lentiviral vectors expressing firefly luciferase, allowing for transplanted cell tracking by bioluminescent imaging (BLI). As a complementary approach, we followed circulating human α1-antitrypsin (hAAT) levels after infusion of AT-SCs overexpressing hAAT. Cells were transplanted into syngeneic mice after CCl4-induced hepatic injury. Luciferase bioluminescence signals and serum hAAT levels were measured at different time points after transplantation and demonstrate persistence of transplanted cells for up to 2 months after administration. These data, along with immunohistochemical analysis, suggest engraftment and repopulation of injured livers by transplanted AT-SCs. Moreover, by transcriptional targeting using cellular tissue-specific regulatory sequences, we confirmed that AT-SCs differentiate towards a hepatogenic-like phenotype in vitro and in vivo. Additionally, in transplanted cells reisolated from recipient animals’ livers, we detected activation of the α-fetoprotein (AFP) promoter. This promoter is normally transcriptionally silenced in adult tissues but can be reactivated during liver regeneration, suggesting commitment towards hepatogenic-like differentiation of engrafted cells in vivo. Our data support AT-SC-mediated gene therapy as an innovative therapeutic option for disorders of liver metabolism.

Key words: Cell biology; Gene therapy; Bioluminescent imaging; Liver; Stem cells

Received January 27, 2011; final acceptance November 15, 2011. Online prepub date: March 28, 2012.
Address correspondence to Gabriele Toietta, Ph.D., Experimental Oncology Department, Instituto Regina Elena IRCCS, via delle Messi d’Oro, 156, 00158, Rome, Italy. Tel: +39-06-52662563; Fax: +39-06-52662505; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 2009–2019, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X640448
E-ISSN 1555-3892
Copyright ©2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Intraparenchymal Injection of Bone Marrow Mesenchymal Stem Cells Reduces Kidney Fibrosis After Ischemia-Reperfusion in Cyclosporine-Immunosuppressed Rats

C. Alfarano,* C. Roubeix,* R. Chaaya,*†‡ C. Ceccaldi,*§ D. Calise,* C. Mias,*† D. Cussac,*† J. L. Bascands,* and A. Parini*†

*Inserm, UMR 1048, Institute of Metabolic and Cardiovascular Diseases (I2MC), Toulouse, France
†Université Toulouse III Paul Sabatier, Toulouse, France
‡Université Saint Joseph, Beirut, Lebanon
§CIRIMAT-UMR 5085 UPS-INPT-CNRS, Toulouse, France

Ischemia-reperfusion and immunosuppressive therapy are a major cause of progressive renal failure after kidney transplantation. Recent studies have shown that administration of bone marrow mesenchymal stem cells (MSCs) improves kidney functional recovery in the acute phase of post ischemia-reperfusion injury. In the present study, we used an original model of renal ischemia-reperfusion in immunosuppressed rats (NIRC) to investigate the effects of bone marrow MSCs on progression of chronic renal failure and the mechanisms potentially involved. Left renal ischemia-reperfusion (IR) was induced in unilateral nephrectomized Lewis rats. After IR, rats were treated daily with cyclosporine (10 mg/kg SC) for 28 days. MSCs were injected into the kidney at day 7 after IR. At day 28 after IR, kidneys were removed for histomorphological, biochemical, and gene expression analysis. The effect of conditioned media from MSCs on epithelial–mesenchymal transition was studied in vitro on HK2 cells. Our results show that, as compared to untreated NIRC rats, rats treated by intrarenal injection of MSCs 7 days after IR displayed improvement in renal function, reduction of interstitial fibrosis, and decrease in chronic tubule injury. These effects were associated with a decrease in interstitial a-SMA accumulation and MMP2 activity, markers of fibroblast/fibroblast-like cell activation, and renal remodeling, respectively. Finally, experiments in vitro showed that MSC-conditioned medium prevented epithelial–mesenchymal transition induced by TGF-β in HK2 cells. In conclusion, our results show that, in immunosuppressed animals, a single intrarenal administration of MSCs reduced renal fibrosis and promoted the recovery of renal function.

Key words: Mesenchymal stem cells; Kidney; Ischemia-reperfusion; Cyclosporine

Received Febuary 15, 2011; Final acceptance September 25, 2011. Online prepub date: April 17, 2012.
Address correspondence to Angelo Parini, UMR 1048, Institute of Metabolic and Cardiovascular diseases (I2MC), CHU Rangueil, 1 avenue Jean Poulhès, BP 84225, 31432 Toulouse Cedex 4, France. Tel: +33 561325601; Fax: +33 562172554; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 2021–2032, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X636939
E-ISSN 1555-3892
Copyright ©2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Characterization of Adult Stem/Progenitor Cell Populations From Bone Marrow in a Three-Dimensional Collagen Gel Culture System

Silvia Claros,* Noela Rodriguez-Losada,† Encarnacion Cruz,‡Enrique Guerado,‡ Jose Becerra,*§ and Jose A. Andrades*

*Department of Cell Biology, Genetics and Physiology, Faculty of Sciences, Networking Biomedical Research Center in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), University of Malaga, Malaga, Spain
†Research Laboratory, Heart Unit, Virgen de la Victoria University Hospital, Campus de Teatinos, Malaga, Spain
‡Hospital Costa del Sol, Division of Orthopaedic Surgery and Traumatology, Marbella, Spain
§University of Malaga, BIONAND, Malaga, Spain

Stem cell transplantation therapy using mesenchymal stem cells (MSCs) is considered a useful strategy. Although MSCs are commonly isolated by exploiting their plastic adherence, several studies have suggested that there are other populations of stem and/or osteoprogenitor cells that are removed from primary culture during media replacement. Therefore, we developed a three-dimensional (3D) culture system in which adherent and nonadherent stem cells are selected and expanded. Here, we described the characterization of 3D culture-derived cell populations in vitro and the capacity of these cells to differentiate into bone and/or cartilage tissue when placed inside of demineralized bone matrix (DBM) cylinders, implanted subcutaneously into the backs of rat for 2, 4, and 8 weeks. Our results demonstrates that 3D culture cells were a heterogeneous population of uncommitted cells that express pluripotent-, hematopoietic-, mesenchymal-, and endothelial-specific markers in vitro and can undergo osteogenic differentiation in vivo.

Key words: Stem/progenitor cells; Three-dimensional (3D) culture; Transforming growth factor-β1 (TGF-β1); Osteogenesis; Demineralized bone matrix

Received February 24, 2011; final acceptance October 22, 2011. Online prepub date: April 2, 2012.
Address correspondence to Jose A. Andrades, Ph.D., Department of Cell Biology, Genetics and Physiology, Faculty of Sciences, University of Malaga, Campus Universitario de Teatinos, 29071-Malaga, Spain. Tel: +34 952 131872; Fax: +34 952 131937; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 2033–2045, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X653110
E-ISSN 1555-3892
Copyright ©2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Cytokine Profiles in Various Graft-Versus-Host Disease Target Organs Following Hematopoietic Stem Cell Transplantation

Hsiu-Yu Lai,*† Teh-Ying Chou,‡ Cheng-Hwai Tzeng,§ and Oscar Kuang-Sheng Lee*†¶

*Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan
†Stem Cell Research Center, National Yang-Ming University, Taipei, Taiwan
‡Department of Pathology, Taipei Veterans General Hospital, Taiwan
§Section of Transfusion Medicine, Department of Medicine, Taipei Veterans General Hospital, Taiwan
¶Department of Orthopaedics and Traumatology, Taipei Veterans General Hospital, Taiwan

Previous studies using genetic-deficient murine models suggest that different T-helper subsets may contribute to different types of tissue damages in graft-versus-host disease (GvHD). However, there is limited information available on the distribution of T-helper cytokines in the various GvHD target tissues. In the current study, an acute GvHD murine model was set up to directly assess the in situ cytokine profiles in various GvHD tissue lesions; in addition, we also studied GvHD tissues from patients who had undergone bone marrow transplantation procedures. We observed that interferon-γ (IFN-γ was dominant in murine liver and gastrointestinal tissue lesions, whereas IFN-γ and interleukin 17 (IL-17) were abundant in murine skin lesions. Furthermore, in human GvHD tissues, interleukin 4 (IL-4) and IFN-γ were predominant in liver lesions and colon lesions, respectively, while no specific cytokine was prevalent in human GvHD skin lesions. In addition, a low ratio of CD4+ T helper (Th) versus CD8+ T cytotoxic (Tc) cells in human GvHD tissue lesions, especially in the liver, was detected, and this contrasts with the situation in murine GvHD tissues where CD4+ Th cells were predominant. Dual staining for CD markers and cytokine expression showed that IFN- γ-secreting T cells were enriched in all murine GvHD target tissue lesions, and Tc1 and Tc2 cells were predominant in human GvHD colon and liver sections, respectively. However, IFN-γ + Th1, IL-17+ Th17, IFN-γ+ Tc1, and IL-17+ Tc17 cells were slightly more frequent in human skin lesions compared to IL-4+ Th2 and IL-4+ Tc2 cells. To sum up, these results suggest that differences in cytokine imbalances may significantly contribute to tissue-specific pathogenesis in GvHD target organs, and CD8+ Tc cells may play an important role in human GvHD induction.

Key words: Graft-versus-host disease (GvHD); Bone marrow transplantation (BMT); T-helper (Th) cell; Cytotoxic T (Tc) cell

Received April 10, 2011; final acceptance November 15, 2011. Online prepub date: July 26, 2012.
Address correspondence to Oscar Kuang-Sheng Lee, M.D., Ph.D., Institute of Clinical Medicine, National Yang-Ming University, Department of Orthopaedics and Traumatology, Taipei Veterans General Hospital, 201, Sec. 2, Shih-Pai Road, Taipei 112, Taiwan. Tel: +886-2-28757557 ext. 128; Fax: +886-2-28757657; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 2047–2061, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X637479
E-ISSN 1555-3892
Copyright ©2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Ex Vivo Alloanergization With Belatacept: A Strategy to Selectively Modulate Alloresponses After Transplantation

Jeff K. Davies,*†‡1 Christine M. Barbon,*1 Annie Voskertchian,* Lee M. Nadler,*† and Eva C. Guinan§

*Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA
†Brigham and Women’s Hospital, Boston, MA, USA
‡Centre for Haemato-Oncology, Barts Cancer Institute, Queen Mary University of London, London, UK
§Departments of Radiation Oncology and Pediatrics, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, USA

Ex vivo alloanergization of human immune cells, via allostimulation in the presence of costimulatory blockade with either a combination of anti-B7.1 and anti-B7.2 antibodies or first-generation cytotoxic T-lymphocyte antigen 4-immunoglobulin (CTLA4-Ig), induces alloantigen-specific hyporesponsiveness and expands alloantigen-specific regulatory T cells (Treg). We have successfully used this approach in the clinical setting of haploidentical hematopoietic stem cell transplantation. Recently, the in vivo use of a new second-generation CTLA4-Ig, belatacept, has shown promise in controlling alloresponses after transplantation of both human kidneys and islet cells. We therefore compared the efficiency of first- and second-generation CTLA4-Ig in alloanergizing human peripheral blood mononuclear cells (PBMCs) and investigated whether ex vivo alloanergization with belatacept could be used to engineer an alloantigen-specific immunoregulatory population of autologous cells suitable for administration to recipients of cellular or solid organ transplant recipients. Alloanergization of HLA-mismatched human PBMCs with belatacept resulted in a greater reduction in subsequent alloresponses than alloanergization with first generation CTLA4-Ig. Moreover, subsequent ex vivo re-exposure of alloanergized cells to alloantigen in the absence of belatacept resulted in a significant expansion of Tregs with enhanced alloantigen-specific suppressive function. Alloanergized PBMCs retained functional Epstein-Barr virus (EBV)-specific T-cell responses, and expanded Tregs did not suppress EBV-specific proliferation of autologous cells. These results suggest that ex vivo alloanergization with belatacept provides a platform to engineer populations of recipient Treg with specificity for donor alloantigens but without nonspecific suppressive capacity. The potential advantages of such cells for solid organ transplantation include (1) reduction of the need for nonspecific immunosuppression, (2) retention of pathogen-specific immunity, and (3) control of graft rejection, if used as an intervention.

Key words: Transplantation; Belatacept; Costimulatory blockade; Alloreactivity; Anergization; Regulatory T cells

Received April 27, 2011; final acceptance November 12, 2011. Online prepub date: April 10, 2012.
1These authors provide equal contribution to this work.
Address correspondence to Eva C. Guinan, Dana-Farber Cancer Institute, Boston, MA 02115, USA. Tel: +1-617-632-4932; Fax: +1-617-632-3770; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 2063–2078, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X627372
E-ISSN 1555-3892
Copyright ©2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Human Islets Express a Marked Proinflammatory Molecular Signature Prior to Transplantation

Mark J. Cowley,*†1 Anita Weinberg,†‡1 Nathan W. Zammit,†‡ Stacey N. Walters,†‡ Wayne J. Hawthorne,§ Thomas Loudovaris,¶ Helen Thomas,¶ Tom Kay,¶ Jenny E. Gunton,† Stephen I. Alexander,# Warren Kaplan,*† Jeremy Chapman,** Philip J. O’Connell,§ and Shane T. Grey†‡2

*Peter Wills Bioinformatics Centre, Darlinghurst, Australia
†Garvan Institute, Darlinghurst, Australia
‡Gene Therapy and Autoimmunity Group, Darlinghurst, Australia
§The Centre for Transplant and Renal Research, Westmead Hospital, Westmead, Australia
¶St. Vincent’s Institute, Westmead, Australia
#Paediatrics and Child Health, Children’s Hospital, Westmead Hospital, Westmead, Australia
**Acute Interventional Medicine (SWAHS) and Renal Services at Westmead Hospital, Westmead, Australia

In the context of islet transplantation, experimental models show that induction of islet intrinsic NF-κB-dependent proinflammatory genes can contribute to islet graft rejection. Isolation of human islets triggers activation of the NF-κB and mitogen-activated kinase (MAPK) stress response pathways. However, the downstream NF-κB target genes induced in human islets during the isolation process are poorly described. Therefore, in this study, using microarray, bioinformatic, and RTqPCR approaches, we determined the pattern of genes expressed by a set of 14 human islet preparations. We found that isolated human islets express a panel of genes reminiscent of cells undergoing a marked NF-κB-dependent proinflammatory response. Expressed genes included matrix metallopeptidase 1 (MMP1) and fibronectin 1 (FN1), factors involved in tissue remodeling, adhesion, and cell migration; inflammatory cytokines IL-1β and IL-8; genes regulating cell survival including A20 and ATF3; and notably high expression of a set of chemokines that would favor neutrophil and monocyte recruitment including CXCL2, CCL2, CXCL12, CXCL1, CXCL6, and CCL28. Of note, the inflammatory profile of isolated human islets was maintained after transplantation into RAG−/− recipients. Thus, human islets can provide a reservoir of NF-κB-dependent inflammatory factors that have the potential to contribute to the anti-islet graft immune response. To test this hypothesis, we extracted rodent islets under optimal conditions, forced activation of NF-κB, and transplanted them into allogenic recipients. These NF-κB activated islets not only expressed the same chemokine profile observed in human islets but also struggled to maintain normoglycemia posttransplantation. Further, NF-κB-activated islets were rejected with a faster tempo as compared to non-NF-κB-activated rodent islets. Thus, isolated human islets can make cell autonomous contributions to the ensuing allograft response by elaborating inflammatory factors that contribute to their own demise. These data highlight the potential importance of islet intrinsic proinflammatory responses as targets for therapeutic intervention.

Key words: Islet; NF-κB; Human islet; Islet transplantation; Inflammation; Gene expression; Gene set enrichment analysis

Received June 7, 2010; final acceptance November 25, 2011. Online prepub date: March 8, 2012.
1These authors provide equal contribution to this work.
2On behalf of “The Australian Islet Transplant Consortium.”
Address correspondence to Shane T. Grey, Gene Therapy and Autoimmunity Group, Immunology Program, Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, NSW 2010, Australia. Tel: +11-61-2-9295 8104; Mobile: 0413 443 729; Fax: +11-61-2-9295 8404; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 2079–2088, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X638928
E-ISSN 1555-3892
Copyright ©2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Concentration and Chain Length of Polyethylene Glycol in Islet Isolation Solution: Evaluation in a Pancreatic Islet Transplantation Model

S. Giraud,*†‡ D. Bon,*‡ Y. Neuzillet,§¶ R. Thuillier,*†‡ M. Eugene,*†‡ T. Hauet,*†‡ and B. Barrou*#**

*INSERM U1082, Poitiers, France
†Service de Biochimie, CHU Poitiers, Poitiers, France
‡Université de Poitiers, Faculté de Médicine et de Pharmacie, Poitiers, France
§Service d’urologie et transplantation rénale, Hôpital Foch, Suresnes, France
¶Faculté de médecine Paris-Ile de France ouest, UVSQ, Versailles, France
#Service d’Urologie, GH Pitié-Salpêtrière, AP-HP, Paris, France
**UPMC Université Paris VI, Paris, France

To improve graft preservation and consequently reduce conservation injuries, the composition of preservation solution is of outmost importance. It was demonstrated that the colloid polyethylene glycol (PEG), used in SCOT solution, has protective effects on cell membranes and immunocamouflage properties. The aim of this study was to optimize the concentration and chain length of PEG to improve pancreatic islet preservation and outcome. In a model of murine islet allotransplantation, islets were isolated with SCOT containing various concentrations of PEG 20 kDa or 35 kDa. Better islet yield (IEQ) was obtained with SCOT + PEG at 15–30 g/L versus other PEG concentrations and control CMRL-1066 + 1% BSA solution (p < 0.05). Allograft survival was better prolonged (up to 20 days) in the groups SCOT + PEG 20 kDa 10–30 g/L compared to PEG 35 kDa (less than 17.8 days) and to control solutions (less than 17.5 days). In terms of graft function recovery, the use of PEG 20 kDa 15–30 g/L induced no primary nonfunction and delayed graft function contrary to CMRL-1066 and other PEG solutions. The use of the extracellular-type solution SCOT containing PEG 20 kDa 15 g/L as colloid could be a new way to optimize graft integrity preservation and allograft outcome.

Keys words: Ischemia reperfusion injury; Preservation solution; Polyethelene glycol (PEG); Graft preservation; Graft outcome

Received November 3, 2010; final acceptance November 18, 2011. Online prepub date April 10, 2012.
Address correspondence to Pr. Benoît Barrou, Service d’Urologie, Unité de Transplantation Rénale et Pancréatique, GH Pitié-Salpetriere, 83 Boulevard de L’Hôpital, 75013 Paris, France. Tel: +33-1-42-17-71-14; Fax: +33 1 42 17 71 93; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 2089–2098, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X623835
E-ISSN 1555-3892
Copyright ©2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Muscle Precursor Cells for the Restoration of Irreversibly Damaged Sphincter Function

Daniel Eberli,1 Tamer Aboushwareb, Shay Soker, James J. Yoo, and Anthony Atala

Department of Urology and Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Winston-Salem, NC, USA

Multiple modalities, including injectable bulking agents and surgery, have been used to treat stress urinary incontinence. However, none of these methods is able to fully restore normal striated sphincter muscle function. In this study, we explored the possibility of achieving functional recovery of the urinary sphincter muscle using autologous muscle precursor cells (MPCs) as an injectable, cell-based therapy. A canine model of striated urinary sphincter insufficiency was created by microsurgically removing part of the sphincter muscle in 24 dogs. Autologous MPCs were obtained, expanded in culture, and injected into the damaged sphincter muscles of 12 animals. The animals were followed for up to 6 months after injection, and urodynamic studies, functional organ bath studies, ultrastructural and histological examinations were performed. Animals receiving MPC injections demonstrated sphincter pressures of approximately 80% of normal values, while the pressures in the control animals without cells dropped and remained at 20% of normal values. Histological analysis indicated that the implanted cells survived and formed tissue, including new innervated muscle fibers, within the injected region of the sphincter. These results indicate that autologous muscle precursor cells may be able to restore otherwise irreversibly damaged urinary sphincter function clinically.

Key words: Muscle precursor cells; Cellular therapy; Sphincter; Muscle regeneration; Incontinence

Received January 3, 2011; final acceptance June 27, 2011. Online prepub date: January 10, 2012.
1Current address: University Hospital, Department of Urology, Frauenklinikstr. 10, CH-8091 Zürich, Switzerland.
Address correspondence to Anthony Atala, M.D., Wake Forest Institute for Regenerative Medicine, Medical Center Boulevard, Winston-Salem, NC 27154-1094, USA. Tel: +1 336-716-5701; Fax: +1 336-713-7290; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it