Cell Transplantation 20(8) Abstracts

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Cell Transplantation, Vol. 20, pp. 1153–1161, 2011
0963-6897/11 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368910X564553
E-ISSN 1555-3892
Copyright © 2011 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Optimization of Immunosuppressive Therapy for Spinal Grafting of Human Spinal Stem Cells in a Rat Model of ALS

Michael P. Hefferan,* Karl Johe,† Thomas Hazel,† Eva L. Feldman,‡ J. Simon Lunn,‡ and Martin Marsala*

*Neuroregeneration Laboratory, Department of Anesthesiology, University of California-San Diego, La Jolla, CA, USA
†Neuralstem Inc., Gaithersburg, MD, USA
‡Department of Neurology, University of Michigan, Ann Arbor, MI, USA

Previous rodent studies employing monotherapy or combined immunosuppressive regimens have demonstrated a variable degree of spinal xenograft survival in several spinal neurodegenerative models including spinal ischemia, trauma, or amyotrophic lateral sclerosis (ALS). Accordingly, the characterization of optimal immunosuppressive protocols for the specific neurodegenerative model is critical to ensure reliable assessment of potential long-term therapeutic effects associated with cell replacement. In the present study we characterized the survival of human spinal stem cells when grafted into the lumbar spinal cords of a rodent model of ALS, SOD1 (G93A) male and female rats (60–67 days old). Four different immunosuppressive protocols were studied: i) FK506 (q12h); ii) FK506 (qd) + mycophenolate (PO; q12h, up to 7 days postop); iii) FK506 (qd) + mycophenolate (IP; q12h, up to 7 days postop); and iv) FK506 (qd) + mycophenolate (IP; qd, up to 7 days postop). Three weeks after cell grafting the number of surviving human cells was then systematically assessed. The highest density of grafted cells was seen in animals treated with FK506 (qd) and mycophenolate (IP; qd; an average 915 ± 95 grafted cells per spinal cord section). The majority of hNUMA-positive cells colocalized with doublecortin (DCX) immunoreactivity. DCX-positive neurons showed extensive axodendritic sprouting toward surrounding host neurons. In addition, migrating grafted cells were identified up to 500 μm from the graft. In animals treated with FK506 (q12h), FK506 (qd) + mycophenolate (PO; q12h) or FK506 (qd) + mycophenolate (IP; q12h), 11.8 ± 3.4%, 61.2 ± 7.8%, and 99.4 ± 8.9% [expressed as percent of the FK506 (qd) and mycophenolate (IP; qd)] cell survival was seen, respectively. In contrast to animals treated with a combination of FK506 + mycophenolate, robust CD4/8 immunoreactivity was identified in the vicinity of the injection tract in animals treated with FK506 only. These data suggest that a combined, systemically delivered immunosuppression regimen including FK506 and mycophenolate can significantly improve survival of human spinal stem cells after intraspinal transplantation in SOD1 (G93A) rats.

Key words: Amyotrophic lateral sclerosis (ALS); Superoxide dismutase 1 (SOD1); Human spinal stem cells; FK506; Mycophenolate

Address correspondence to Michael P. Hefferan, Ph.D., Neuroregeneration Laboratory, Department of Anesthesiology, University of California-San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0695, USA. Tel: 858-534-7380; Fax: 858-822-3249; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 20, pp. 1163–1178, 2011
0963-6897/11 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368910X564544
E-ISSN 1555-3892
Copyright © 2011 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Transplantation of Rat Synapsin-EGFP-Labeled Embryonic Neurons Into the Intact and Ischemic CA1 Hippocampal Region: Distribution, Phenotype, and Axodendritic Sprouting

K. Kucharova,*† M. P. Hefferan,† P. Patel,‡§ S. Marsala,†¶ T. Nejime,† A. Miyanohara,# M. Marsala,†** and J. C. Drummond‡§

*Sanford-Burnham Medical Research Institute, La Jolla, CA, USA
†Neuroregeneration Laboratory, Department of Anesthesiology, University of California-San Diego, La Jolla, CA, USA
‡Department of Anesthesiology, University of California-San Diego, La Jolla, CA, USA
§Veterans Affairs San Diego Health Care System, San Diego, CA, USA
¶Department of Pathology, University of California-San Diego, La Jolla, CA, USA
#Human Gene Therapy Program, Department of Pediatrics, University of California-San Diego, La Jolla, CA, USA
**Institute of Neurobiology, Slovak Academy of Sciences, Kosice, Slovakia

A major limitation of neural transplantation studies is assessing the degree of host–graft interaction. In the present study, rat hippocampal/cortical embryonic neurons (E18) were infected with a lentivirus encoding enhanced green fluorescent protein (GFP) under control of the neuron-specific synapsin promoter, thus permitting robust identification of labeled neurons after in vivo grafting. Two weeks after transient forebrain ischemia or sham-surgery, GFP-expressing neurons were transplanted into CA1 hippocampal regions in immunosuppressed adult Wistar rats. The survival, distribution, phenotype, and axonal projections of GFP-immunoreactive (IR) positive transplanted neurons were evaluated in the sham-operated and ischemia-damaged CA1 hippocampal regions 4, 8, and 12 weeks after transplantation. In both experimental groups, we observed that the main phenotype of host-derived afferents projecting towards grafted GFP-IR neurons as well as transplant-derived GFP-IR efferents were glutamatergic in both animal groups. GFP axonal projections were seen in the nucleus accumbens, septal nuclei, and subiculum—known target areas of CA1 pyramidal neurons. Compared to sham-operated animals, GFP-IR neurons grafted into the ischemia-damaged CA1 migrated more extensively throughout a larger volume of host tissue, particularly in the stratum radiatum. Moreover, enhanced axonal sprouting and neuronal plasticity of grafted cells were evident in the hippocampus, subiculum, septal nuclei, and nucleus accumbens of the ischemia-damaged rats. Our study suggests that the adult rat brain retains some capacity to direct newly sprouting axons of transplanted embryonic neurons to the correct targets and that this capacity is enhanced in previously ischemia-injured forebrain.

Key words: Neuronal transplantation; Green fluorescent protein (GFP); Axonal projection; Hippocampus; Ischemia; Rat

Address correspondence to Karolina Kucharova, M.D., Ph.D., Sanford-Burnham Medical Research Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA. Tel: 858-646-3100; Fax: 858-646-3197; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 20, pp. 1179–1193, 2011
0963-6897/11 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368910X552826
E-ISSN 1555-3892
Copyright © 2011 Cognizant Comm. Corp.
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Bone Marrow Contributes Simultaneously to Different Neural Types in the Central Nervous System Through Different Mechanisms of Plasticity

Javier S. Recio,*1 Manuel Álvarez-Dolado,†1 David Díaz,* Fernando C. Baltana´s,* Marina Piquer-Gil,† José R. Alonso,*‡ and Eduardo Weruaga*

*Laboratory of Neuronal Plasticity and Neurorepair, Institute for Neuroscience of Castilla y León, Universidad de Salamanca, Salamanca, Spain
†Laboratory of Cell Therapy for Neuropathologies, Andalucian Center for Molecular Biology and Regenerative Medicine, CABIMER, Seville, Spain
‡Universidad de Tarapacá en Arica, Arica, Chile

Many studies have reported the contribution of bone marrow-derived cells (BMDC) to the CNS, raising the possibility of using them as a new source to repair damaged brain tissue or restore neuronal function. This process has mainly been investigated in the cerebellum, in which a degenerative microenvironment has been suggested to be responsible for its modulation. The present study further analyzes the contribution of BMDC to different neural types in other adult brain areas, under both physiological and neurodegenerative conditions, together with the mechanisms of plasticity involved. We grafted genetically marked green fluorescent protein/Cre bone marrow in irradiated recipients: a) the PCD (Purkinje Cell Degeneration) mutant mice, suffering a degeneration of specific neuronal populations at different ages, and b) their corresponding healthy controls. These mice carried the conditional lacZ reporter gene to allow the identification of cell fusion events. Our results demonstrate that BMDC mainly generate microglial cells, although to a lesser extent a clear formation of neuronal types also exists. This neuronal recruitment was not increased by the neurodegenerative processes occurring in PCD mice, where BMDC did not contribute to rescuing the degenerated neuronal populations either. However, an increase in the number of bone marrow-derived microglia was found along the life span in both experimental groups. Six weeks after transplantation more bone marrow-derived microglial cells were observed in the olfactory bulb of the PCD mice compared to the control animals, where the degeneration of mitral cells was in process. In contrast, this difference was not observed in the cerebellum, where Purkinje cell degeneration had been completed. These findings demonstrated that the degree of neurodegenerative environment can foster the recruitment of neural elements derived from bone marrow, but also provide the first evidence that BMDC can contribute simultaneously to different encephalic areas through different mechanisms of plasticity: cell fusion for Purkinje cells and differentiation for olfactory bulb interneurons.

Key words: Bone marrow transplantation; Cell fusion; Neural repair; Neurodegeneration; Neural differentiation

1These authors provided equal contribution to this work.
Address correspondence to Eduardo Weruaga, Ph.D., Laboratorio de Plasticidad Neuronal y Neurorreparación, Instituto de Neurociencias de Castilla y León, Universidad de Salamanca, C/ Pintor Fernando Gallego, No. 1, E-37007 Salamanca, Spain. Tel: +34-923-294500, ext. 5324; Fax: +34-923294549; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 20, pp. 1193–1208, 2011
0963-6897/11 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368910X543394
E-ISSN 1555-3892
Copyright © 2011 Cognizant Comm. Corp.
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Mesenchymal Stem Cells in Renal Function Recovery After Acute Kidney Injury: Use of a Differentiating Agent in a Rat Model

Gaetano La Manna,*†1 Francesca Bianchi,†‡1 Maria Cappuccilli,*† Giovanna Cenacchi,†§ Lucia Tarantino,†§ Gianandrea Pasquinelli,†¶ Sabrina Valente,†¶ Elena Della Bella,*† Silvia Cantoni,†‡ Cavallini Claudia,†‡ Flavia Neri,† Matvey Tsivian,† Bruno Nardo,† Carlo Ventura,‡ and Sergio Stefoni*

*Department of Internal Medicine, Aging and Renal Disease-Section of Nephrology, University of Bologna, Bologna, Italy
†Center for Applied Biomedical Research (CRBA), S. Orsola-Malpighi University Hospital, Bologna, Italy
‡Laboratory of Molecular Biology and Stem Cell Engineering, Cardiovascular Department-National Institute of Biostructures and Biosystems, University of Bologna, Bologna, Italy
§Clinical Department of Radiological and Histocytopathological Sciences-Section of Anatomical Pathology, University of Bologna, Bologna, Italy
¶Clinical Department of Radiological and Histocytopathological Sciences-Section of Clinical Pathology, University of Bologna, Bologna, Italy

Acute kidney injury (AKI) is a major health care condition with limited current treatment options. Within this context, stem cells may provide a clinical approach for AKI. Moreover, a synthetic compound previously developed, hyaluronan monoesters with butyric acid (HB), able to induce metanephric differentiation, formation of capillary-like structures, and secretion of angiogenic cytokines, was tested in vitro. Thereafter, we investigated the effects of human mesenchymal stem cells from fetal membranes (FMhMSCs), both treated and untreated with HB, after induction of ischemic AKI in a rat model. At reperfusion following 45-min clamping of renal pedicles, each rat was randomly assigned to one of four groups: CTR, PBS, MSC, and MSC-HB. Renal function at 1, 3, 5, and 7 days was assessed. Histological samples were analyzed by light and electron microscopy and renal injury was graded. Cytokine analysis on serum samples was performed. FMhMSCs induced an accelerated renal functional recovery, demonstrated by biochemical parameters and confirmed by histology showing that histopathological alterations associated with ischemic injury were less severe in cell-treated kidneys. HB-treated rats showed a minor degree of inflammation, both at cytokine and TEM analyses. Better functional and morphological recovery were not associated to stem cells’ regenerative processes, but possibly suggest paracrine effects on microenvironment that induce retrieval of renal damaged tissues. These results suggest that FMhMSCs could be useful in the treatment of AKI and the utilization of synthetic compounds could enhance the recovery induction ability of cells.

Key words: Stem cells; Acute kidney injury (AKI); Mesenchymal stem cells (MSCs); Renal function; Renal cell biology

1These authors provided equal contribution to this work.
Address correspondence to Prof. Gaetano La Manna, Dipartimento di Medicina Interna, dell’Invecchiamento e Malattie Nefrologiche, c/o U.O. Nefrologia, Dialisi e Trapianto, Policlinico S. Orsola-Malpighi, via Massarenti 9, 40138 Bologna, Italy. Tel: 051-6364577; Fax: 051-6363720; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 20, pp. 1209–1220, 2011
0963-6897/11 $90.00 + .00
DOI: http://dx.doi.org/10.3727/0963689109X546562
E-ISSN 1555-3892
Copyright © 2011 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Cell Contact Accelerates Replicative Senescence of Human Mesenchymal Stem Cells Independent of Telomere Shortening and p53 Activation: Roles of Ras and Oxidative Stress

Jennifer H. Ho,*†‡ Yu-Fan Chen,§ Wei-Hsien Ma,‡ Tzu-Ching Tseng,§ Ming-Hsiang Chen,§ and Oscar K. Lee§¶#

*Graduate Institute of Clinical Medicine, Taipei Medical University, Taipei, Taiwan
†Department of Ophthalmology, Taipei Medical University-Wan Fang Hospital, Taipei, Taiwan
‡Center for Stem Cell Research, Taipei Medical University-Wan Fang Hospital, Taipei, Taiwan
§Stem Cell Research Center, National Yang-Ming University, Taipei, Taiwan
¶Department of Orthopaedics and Traumatology, Taipei Veterans General Hospital, Taipei, Taiwan
#Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan

Mesenchymal stem cells (MSCs) are of great therapeutic potentials due to their multilineage differentiation capabilities. Before transplantation, in vitro culture expansion of MSCs is necessary to get desired cell number. We observed that cell contact accelerated replicative senescence during such process. To confirm the finding as well as to investigate the underlying mechanisms, we cultured both human bone marrow- and umbilical cord blood-derived MSCs under noncontact culture (subculture performed at 60–70% of confluence), or contact culture (cell passage performed at 100% of confluence). It was found that MSCs reached cellular senescence earlier in contact culture, and the doubling time was significantly prolonged. Marked increase of senescence-associated β-galactosidase-positive staining was also observed as a result of cell contact. Cell cycle analysis revealed increased frequency of cell cycle arrest after contact culture. It was noted, however, that the telomere length was not altered during contact-induced acceleration of senescence. Moreover, cell cycle checkpoint regulator P53 expression was not affected by cell contact. Marked increase in intracellular reactive oxygen species (ROS) and a concomitant decrease in the activities of antioxidative enzymes were also observed during contact-induced senescence. Importantly, increased p16INK4a following Ras upregulation was found after contact culture. Taken together, cell contact induced accelerated senescence of MSCs, which is telomere shortening and p53 independent. ROS accumulation due to defective ROS clearance function together with Ras and p16INK4a upregulation play an important role in contact-induced senescence of MSCs. Overconfluence should therefore be avoided during in vitro culture expansion of MSCs in order to maintain their qualities for clinical application purposes. The contact-induced senescence model reported in this study will serve as a useful model system that allows further study of the molecular mechanisms of senescence in MSCs.

Key words: Mesenchymal stem cells; Senescence; Ras; Oxidative stress; p16INK4a

Address correspondence to Oscar K. Lee, M.D., Ph.D., Department of Orthopaedics and Traumatology, Taipei Veterans General Hospital201, Sec. 2, Shih-Pai Road, Taipei 112, Taiwan. Tel: 886-2-2875-7391; Fax: 886-2-2875-7657; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 20, pp. 1221–1230, 2011
0963-6897/11 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368910X546553
E-ISSN 1555-3892
Copyright © 2011 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Inhibition of T-Cell Proliferation by Murine Multipotent Mesenchymal Stromal Cells Is Mediated by CD39 Expression and Adenosine Generation

Christine Sattler,*1 Manuela Steinsdoerfer,*1 Monika Offers,* Elke Fischer,† Rudolf Schierl,† Kathrin Heseler,‡ Walter Däubener,‡ and Jochen Seissler*

*Diabetes Center, Medical Clinic-Innenstadt, Ludwig-Maximilians-University, Munich, Germany
†Institute and Outpatient Clinic for Occupational, Social and Environmental Medicine, Ludwig-Maximilians-University, Munich, Germany
‡Institute for Medical Microbiology and Hospital Hygiene, Heinrich-Heine-University, Duesseldorf, Germany

Multipotent mesenchymal stromal cells (MSCs) are bone marrow-derived cells of nonhematopoietic origin with immunoregulatory properties. Although some functions of MSCs have been identified, there are still features that are not explained thus far. The aim of the present study was to identify novel factors involved in MSC-mediated inhibition of T-cell proliferation. We here demonstrate that the surface molecule CD39 is coexpressed in concert with CD73 on murine MSCs catalyzing the generation of adenosine, which can directly act on activated T cells via the adenosine A2A receptor. Blocking of the adenosine pathway either by the A2A receptor antagonist SCH58261 or the specific CD39 inhibitor polyoxotungstate 1 (POM-1) blocked MSC-mediated suppression of T-cell proliferation almost completely. We conclude that CD39/CD73 coexpression is a novel important component of the immunoregulatory functions of murine MSCs. Our findings may both be important to improve our understanding of MSC function and for the development of immunomodulatory cellular therapies.

Key words: Mesenchymal stromal cells (MSCs); Immunoregulation; CD39/CD73; T-cell inhibition; Adenosine

1These authors provided equal contribution to this work.
Address correspondence to Jochen Seissler, M.D., Diabetes Center, Medical Clinic-Innenstadt, Ludwig-Maximilians-University Munich, Ziemssenstraße 1, 80336 Munich, Germany. Tel: (+49) 89 5160 2186; Fax: (+49) 89 5160 4956; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 20, pp. 1231–1240, 2011
0963-6897/11 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368910X552835
E-ISSN 1555-3892
Copyright © 2011 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Effect of Dimethyl Sulfoxide (DMSO) on Cryopreservation of Porcine Mesenchymal Stem Cells (pMSCs)

Sun-A Ock*† and Gyu-Jin Rho*‡

*College of Veterinary Medicine, Gyeongsang National University, Jinju, Republic of Korea
†Institute of Animal Medicine, Gyeongsang National University, Jinju, Republic of Korea
‡Institute of Life Science, Gyeongsang National University, Jinju, Republic of Korea

Dimethyl sulfoxide (DMSO), a commonly used cryoprotectant in cryopreservation procedures, is detrimental to viability of cells. In this view point, a comparative study was carried out to evaluate the effect of DMSO on porcine mesenchymal stem cells (pMSCs). We compared the viability, colony forming unit-fibroblast (CFU-F) assay, expression of Bak and Bcl2 genes, Bcl2 protein antigen, and CD90 in pMSCs cryopreserved with 5%, 10%, and 20% DMSO. pMSCs isolated from bone marrow were characterized by alkaline phosphatase activity and the expression of transcription factors, such as Oct 3/4, Nanog, and Sox2. The cells were then cryopreserved by cooling at a rate of −1°C/min in a programmable freezer and stored in liquid nitrogen. The results of survival of pMSCs cryopreserved at 5% DMSO were comparable to control group (fresh pMSCs). The survival and the number of colonies formed in cryopreserved pMSCs were inversely proportional to the concentration of DMSO. The number of colonies formed in pMSCs cryopreserved with all concentrations of DMSO was significantly (p < 0.05) lower than the control group. An increased tendency for Bak and Bcl2 gene expression was noticed in cryopreserved pMSCs at 3 h postthawing compared to control group. There was a close resemblance in higher level of expression of CD90 between control and cryopreserved pMSCs. Because there was no considerable difference in the results of pMSCs cryopreserved at 5% and 10% DMSO, this study strongly suggests the use of 5% DMSO in cryopreservation of pMSCs as an alternative to conventional 10% DMSO.

Key words: Mesenchymal stem cells; Porcine; Cryopreservation; Dimethyl sulfoxide (DMSO)

Address correspondence to Gyu-Jin Rho, D.V.M., Ph.D., Professor, College of Veterinary Medicine, Gyeongsang National University, 900 Gazwa, Jinju, Republic of Korea 660-701. Tel: 82-55-751-5824; Fax: 82-55-751-5803; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 20, pp. 1241–1257, 2011
0963-6897/11 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368910X547426
E-ISSN 1555-3892
Copyright © 2011 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Biological and Physicochemical Characterization of a Serumand Xeno-Free Chemically Defined Cryopreservation Procedure for Adult Human Progenitor Cells

Steffen M. Zeisberger,*†1 Julia C. Schulz,‡1 Mario Mairhofer,§1 Peter Ponsaerts,¶ Guy Wouters,# Daniel Doerr,‡ Alisa Katsen-Globa,‡ Martin Ehrbar,* Jurgen Hescheler,** Simon P. Hoerstrup,† Andreas H. Zisch,*2 Andrea Kolbus,§1 and Heiko Zimmermann‡††1

*Department of Obstetrics, University Hospital Zurich, Zurich, Switzerland
†Swiss Center for Regenerative Medicine, University Hospital Zurich and University Zurich, Zurich, Switzerland
‡Fraunhofer Institute for Biomedical Engineering, St. Ingbert, Germany
§Department of Obstetrics and Gynecology, Medical University of Vienna, Vienna, Austria
¶Laboratory of Experimental Hematology, Vaccine and Infectious Disease Institute (Vaxinfectio), University of Antwerp, Antwerp, Belgium
#Cryo-Save Group, Niel, Belgium
**Center of Physiology and Pathophysiology, Institute of Neurophysiology, and Center of Molecular Medicine (CMMC), University of Cologne, Cologne, Germany
††University of Saarland/Chair of Molecular and Cellular Biotechnology/Nanotechnology, Saarbrucken, Germany

While therapeutic cell transplantations using progenitor cells are increasingly evolving towards phase I and II clinical trials and chemically defined cell culture is established, standardization in biobanking is still in the stage of infancy. In this study, the EU FP6-funded CRYSTAL (CRYo-banking of Stem cells for human Therapeutic AppLication) consortium aimed to validate novel Standard Operating Procedures (SOPs) to perform and validate xeno-free and chemically defined cryopreservation of human progenitor cells and to reduce the amount of the potentially toxic cryoprotectant additive (CPA) dimethyl sulfoxide (DMSO). To achieve this goal, three human adult progenitor and stem cell populations—umbilical cord blood (UCB)-derived erythroid cells (UCB-ECs), UCB-derived endothelial colony forming cells (UCB-ECFCs), and adipose tissue (AT)-derived mesenchymal stromal cells (AT-MSCs)—were cryopreserved in chemically defined medium supplemented with 10% or 5% DMSO. Cell recovery, cell repopulation, and functionality were evaluated postthaw in comparison to cryopreservation in standard fetal bovine serum (FBS)-containing freezing medium. Even with a reduction of the DMSO CPA to 5%, postthaw cell count and viability assays indicated no overall significant difference versus standard cryomedium. Additionally, to compare cellular morphology/membrane integrity and ice crystal formation during cryopreservation, multiphoton laser-scanning cryomicroscopy (cryo-MPLSM) and scanning electron microscopy (SEM) were used. Neither cryo-MPLSM nor SEM indicated differences in membrane integrity for the tested cell populations under various conditions. Moreover, no influence was observed on functional properties of the cells following cryopreservation in chemically defined freezing medium, except for UCB-ECs, which showed a significantly reduced differentiation capacity after cryopreservation in chemically defined medium supplemented with 5% DMSO. In summary, these results demonstrate the feasibility and robustness of standardized xeno-free cryopreservation of different human progenitor cells and encourage their use even more in the field of tissue-engineering and regenerative medicine.

Key words: Human progenitor and mesenchymal stem cells; Cryopreservation; Serum free; Xeno free; Chemically defined; Current good manufacturing practice; Cryomicroscopy

1These authors provided equal contribution to this work.
2Deceased during preparation of the manuscript.
Address correspondence to Steffen M. Zeisberger, Swiss Center for Regenerative Medicine, Moussonstrasse 13, 8091 Zurich, Switzerland. Tel: +41 (0)44 634 5606; Fax: +41 (0)44 634 5608; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 20, pp. 1259–1270, 2011
0963-6897/11 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368910X547417
E-ISSN 1555-3892
Copyright © 2011 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Hepatocyte Transplantation Through the Hepatic Vein: A New Route of Cell Transplantation to the Liver

Yuichiro Goto,*† Kazuo Ohashi,*† Rie Utoh,* Masakazu Yamamoto,† and Teruo Okano*

*Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University, Tokyo, Japan
†Department of Surgery, Institute of Gastroenterology, Tokyo Women’s Medical University, Tokyo, Japan

The efficiency of hepatocyte transplantation into the liver varies with the method of administration. This study investigated whether retrograde infusion via the hepatic vein provides a sufficient number of donor cells for the liver. Donor hepatocytes were isolated from dipeptidyl peptidase IV (DPPIV+) rats and transplanted into DPPIV− rat livers either by antegrade portal vein infusion or retrograde hepatic vein infusion. Hepatocyte engraftment ratios and localization were evaluated by histological DPPIV enzymatic staining at 1 week and 8 weeks after the transplantation. No significant differences in engraftment efficiency were observed at either 1 week or 8 weeks after transplantation by either route. However, the localization of the transplanted hepatocytes differed with the administration route. Portal vein infusion resulted in predominantly periportal engraftment, whereas hepatic vein infusion led to pericentral zone engraftment. Immunohistochemical analysis showed that the transplanted hepatocytes engrafted in the pericentral zone after retrograde infusion displayed intense CYP2E1 staining similar to the surrounding native hepatocytes. CYP2E1 staining was further enhanced by administration of isosafrole, an inducing agent for various cytochrome P450 enzymes, including CYP2E1. This study demonstrates a novel approach of transplanting hepatocytes into the liver through retrograde hepatic vein infusion as the means to target cell implantation to the pericentral zone.

Key words: Hepatocyte transplantation; Hepatocyte function; Hepatic veins; Experimental transplantation

Address correspondence to Kazuo Ohashi, M.D., Ph.D., Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan. Tel: +81-3-3353-8111, ext. 66214; Fax: +81-3-3359-6046; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 20, pp. 1271–1283, 2011
0963-6897/11 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368910X545077
E-ISSN 1555-3892
Copyright © 2011 Cognizant Comm. Corp.
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Effects of Acute Cytomegalovirus Infection on Rat Islet Allograft Survival

M. J. Smelt,* B. J. de Haan,* M. M. Faas,* B. N. Melgert,* A. de Haan,† and P. de Vos*

*Department of Pathology and Medical Biology, Division of Medical Biology, Section Immunoendocrinology, University Medical Center Groningen and University of Groningen, Groningen, The Netherlands
†Department of Medical Microbiology, Division Molecular Virology, University Medical Center Groningen and University of Groningen, Groningen, The Netherlands

Transplantation of pancreatic islets is a promising therapy for the treatment of type 1 diabetes mellitus. However, long-term islet graft survival rates are still unsatisfactory low. In this study we investigated the role of cytomegalovirus (CMV) in islet allograft failure. STZ-diabetic rats received an allogenic islet graft in combination with either an acute CMV infection or control infection. A third group received ganciclovir treatment in addition to the CMV infection. Graft function was assessed by measuring basal blood glucose levels. After sacrifice, the islet grafts were retrieved for analysis of infection and leukocyte infiltration. CMV-infected recipients demonstrated accelerated islet graft failure compared to noninfected controls. CMV infection of the graft was only observed prior to complete graft failure. Quantification of the leukocyte infiltration demonstrated increased CD8+ T-cell and NK cell infiltration in the CMV-infected grafts compared to the controls. This suggests that CMV infection accelerates immune-mediated graft destruction. Antiviral ganciclovir treatment did not prevent accelerated graft failure, despite effectively decreasing the grade of infection. Our data confirm the recently published CITR data, which state that CMV is an independent risk factor for failure of islet grafts. Also, our data demonstrate that new approaches for preventing virus-induced islet allograft failure may be required.

Key words: Islet transplantation; Cytomegalovirus (CMV) infection; Graft failure; CD8+; T cells; NK cells

Address correspondence to M. J. Smelt, Ph.D., Pathology and Medical Biology, Division of Medical Biology, Section of Immunoendocrinology, University of Groningen, Hanzeplein 1, 9700 RB Groningen, The Netherlands. Tel: (31) (50) 361 01 09; Fax: (31) (50) 33619911; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 20, pp. 1285–1297, 2011
0963-6897/11 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368910X550242
E-ISSN 1555-3892
Copyright © 2011 Cognizant Comm. Corp.
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Pdx1- and Ngn3-Cre-Mediated PLAG1 Expression in the Pancreas Leads to Endocrine Hormone Imbalances That Affect Glucose Metabolism

Jeroen Declercq,*† Anujith Kumar,* Conny Gysemans,‡ Caterina Di Pietro,* Anica Schraenen,§ Marie Chintinne,¶ Katleen Lemaire,§ Leentje Van Lommel,§ Marc Van De Casteele,¶ Harry Heimberg,¶ Daniel Pipeleers,¶ Frans C. Schuit,§ Chantal Mathieu,‡ Nadine Ectors,# Wim J. M. Van de Ven,† and Catherine M. Verfaillie*

*Stem Cell Institute, Katholieke Universiteit Leuven, Leuven, Belgium
†Laboratory for Molecular Oncology, Department of Human Genetics, Katholieke Universiteit Leuven, Leuven, Belgium
‡The Laboratory for Experimental Medicine and Endocrinology (LEGENDO), Katholieke Universiteit Leuven, Leuven, Belgium
§Gene Expression Unit, Department of Molecular Cell Biology, Katholieke Universiteit Leuven, Leuven, Belgium
¶Diabetes Research Center, Brussels Free University-VUB, JDRF Center for Beta Cell Therapy in Diabetes, Brussels, Belgium
#Department of Pathology, University Hospital Gasthuisberg, Leuven, Belgium

Pleomorphic adenoma gene-like 1 (PLAGL1) has been linked to transient neonatal diabetes mellitus. Here, we investigated the role of the related pleomorphic adenoma gene 1 (PLAG1) in glucose homeostasis. PLAG1 transgenic mice in which expression of the PLAG1 transgene can be targeted to different organs by Cre-mediated modulation were crossed with Pdx1-Cre or Ngn3-Cre mice, resulting in double transgenic P1-Pdx1Cre or P1-Ngn3Cre mice, respectively. P1-Pdx1Cre and P1-Ngn3Cre mice developed hyperplasia of pancreatic islets due to increased β- and δ- but not α-cell proliferation. In young P1-Pdx1Cre mice (less than 15 weeks) there was a balanced increase in the pancreatic content of insulin and somatostatin, which was associated with normoglycemia. In older P1-Pdx1Cre mice the pancreatic somatostatin content far exceeded that of insulin, leading to the progressive development of severe hypoglycemia beyond 30 weeks. In contrast, in older P1-Ngn3Cre mice the relative increase of the pancreatic insulin content exceeded that of somatostatin and these mice remained normoglycemic. In conclusion, forced expression of PLAG1 under the control of the Pdx1 or Ngn3 promoter in murine pancreas induces different degrees of endocrine hormone imbalances within the pancreas, which is associated with hypoglycemia in P1-Pdx1Cre mice but not P1-Ngn3Cre mice. These results suggest that once stem cell-derived islet transplantations become possible, the appropriate balance between different hormone-producing cells will need to be preserved to prevent deregulated glucose metabolism.

Key words: Pleomorphic adenoma gene 1 (PLAG1); Islet hyperplasia; Hypoglycemia; Hyperinsulinemia

Address correspondence to Jeroen Declercq, Stem Cell Institute, O&N1, Herestraat 49 box 804, B-3000 Leuven, Belgium. Tel: +32 16 330292; Fax: +32 16 330294; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 20, pp. 1299–1313, 2011
0963-6897/11 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368910X546571
E-ISSN 1555-3892
Copyright © 2011 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Mesoangioblasts From Facioscapulohumeral Muscular Dystrophy Display In Vivo a Variable Myogenic Ability Predictable by Their In Vitro Behavior

Roberta Morosetti,*† Teresa Gidaro,* Aldobrando Broccolini,*† Carla Gliubizzi,*† Cristina Sancricca,* Pietro Attilio Tonali,*† Enzo Ricci,*† and Massimiliano Mirabella*†

*Department of Neurosciences, Catholic University School of Medicine “A. Gemelli,” Rome, Italy
†Don Carlo Gnocchi Onlus Foundation, Italy

Facioscapulohumeral muscular dystrophy (FSHD) is the third most frequent inherited myopathy. We previously demonstrated that mesoangioblasts can be efficiently isolated from FSHD muscles, although their differentiation ability into skeletal muscle was variably impaired. This correlates with overall disease severity and degree of histopathologic abnormalities, since mesoangioblasts from morphologically normal muscles did not show any myogenic differentiation block. The aim of our present study was to verify whether mesoangioblasts from differentially affected FSHD muscles reproduce in vivo the same differentiation ability shown in vitro by studying their capability to form new muscle fibers during muscle regeneration of experimentally damaged muscles. We show that a diverse ability of FSHD mesoangioblasts to engraft and differentiate into skeletal muscle of SCID mice is strictly related to the characteristics of the muscle of origin, closely replicating in vivo what was previously observed in vitro. Moreover, we demonstrate that mesoangioblasts obtained from severely affected muscles scarcely integrate into muscle fibers, remaining mainly localized in the connective tissue. This suggests a defective migration in response to chemoattractants released by damaged fibers, as indicated by cell migration assays in response to HMGB1 and very low levels of RAGE expression, along with a decreased ability to fuse or to appropriately trigger the myogenic program. Our study indicates that FSHD mesoangioblasts from unaffected muscles can be used as selective treatment to halt muscle degeneration in severely affected muscles, and suggests that pharmacological and molecular interventions aimed to ameliorate homing and engraftment of transplanted autologous mesoangioblasts may open the way to cell therapy for FSHD patients, without requiring immunosuppression or genetic correction in vitro.

Key words: Facioscapulohumeral muscular dystrophy (FSHD); Mesoangioblasts; Transplantation; Myogenic stem cells

Address correspondence to Roberta Morosetti, M.D., Department of Neurosciences, Catholic University School of Medicine, Largo A. Gemelli 8, 00168 Rome, Italy. Tel: +39-06-30154303; Fax: +39-06-35501909; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it or Massimiliano Mirabella, M.D., Ph.D., Department of Neurosciences, Catholic University School of Medicine, Largo A. Gemelli 8, 00168 Rome, Italy. Tel: +39-06-30154303; Fax: +39-06-35501909; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 20, pp. 1315–1319, 2011
0963-6897/11 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X593127
E-ISSN 1555-3892
Copyright © 2011 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Commentary

Technology and Innovation: 2010 a Year in Review

Paul R. Sanberg,*† Cecilia Vindrola-Padros,‡ David J. Eve,† and Howard J. Federoff§

*Office of Research & Innovation, University of South Florida, Tampa, FL, USA
†Center of Excellence for Aging & Brain Repair, Department of Neurosurgery and Brain Repair, University of South Florida, Tampa, FL, USA
‡Department of Anthropology, University of South Florida, Tampa, FL, USA
§Georgetown University Medical Center, Washington, DC, USA

The following commentary provides a discussion of the articles published in Technology and Innovation in 2010 and where possible places them into context with those reported in Cell Transplantation. These articles can be divided into the following topics: a) models for innovation and technological commercialization, b) the ethical and legal consequences of the emergence of new technologies, c) research on novel technologies and methods, and d) the difficulties involved in peer review and scientific assessment. The articles shed light on the effects of technological innovation and commercialization on scientific ethical regulation, the establishment of legal standards for the protection of intellectual property, and the development of financial models.

Key words: Technology and Innovation; Technological commercialization; Innovation; Peer review; Invention