Cell Transplantation 21(12) Abstracts

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Cell Transplantation, Vol. 21, pp. 2537-2554, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X637407
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Review

Guiding Differentiation of Stem Cells In Vivo by Tetracycline-Controlled Expression of Key Transcription Factors

Elena N. Kozlova* and Christian Berens†

*Department of Neuroscience, Uppsala University, Uppsala, Sweden
†Division of Microbiology, Department of Biology, Friedrich-Alexander-University Erlangen-Nuremberg, Erlangen, Germany

Transplantation of stem or progenitor cells is an attractive strategy for cell replacement therapy. However, poor long-term survival and insufficiently reproducible differentiation to functionally appropriate cells in vivo still present major obstacles for translation of this methodology to clinical applications. Numerous experimental studies have revealed that the expression of just a few transcription factors can be sufficient to drive stem cell differentiation toward a specific cell type, to transdifferentiate cells from one fate to another, or to dedifferentiate mature cells to pluripotent stem/progenitor cells (iPSCs). We thus propose here to apply the strategy of expressing the relevant key transcription factors to guide the differentiation of transplanted cells to the desired cell fate in vivo. To achieve this requires tools allowing us to control the expression of these genes in the transplant. Here, we describe drug-inducible systems that allow us to sequentially and timely activate gene expression from the outside, with a particular emphasis on the Tet system, which has been widely and successfully used in stem cells. These regulatory systems offer a tool for strictly limiting gene expression to the respective optimal stage after transplantation. This approach will direct the differentiation of the immature stem/progenitor cells in vivo to the desired cell type.

Key words: Cell survival; Central nervous system; Gene regulation; Transcription factor; Directed differentiation

Received July 15, 2011; final acceptance November 15, 2011. Online prepub date: March 28, 2012.
Address correspondence to Elena N. Kozlova, Department of Neuroscience, Uppsala University, Box 593, 751 24 Uppsala, Sweden. Tel: +46-18-471-4111; Fax: +46-18-51-15-40; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it or Christian Berens, Division of Microbiology, Department of Biology, Friedrich-Alexander-University Erlangen-Nuremberg, Staudtstr. 5, D-91058 Erlangen, Germany. Tel: +49-9131-85-28084; Fax: +49-9131-85-28082; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 2555-2567, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X653156
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Somatic Differentiation and MR Imaging of Magnetically Labeled Human Embryonic Stem Cells

Hossein Nejadnik,*1 Tobias D. Henning,†1 Rosalinda T. Castaneda,* Sophie Boddington,‡ Stefan Taubert,§ Priyanka Jha,¶ Sidhartha Tavri,# Daniel Golovko,** Larry Ackerman,†† Reinhard Meier,‡‡ and Heike E. Daldrup-Link*

*Department of Radiology, Molecular Imaging Program at Stanford, Stanford University, Stanford, CA, USA
†Department of Radiology, University of Cologne, Cologne, Germany
‡Department of Radiology, University of California, San Francisco, San Francisco, CA, USA
§Department of Medical Genetics, University of British Columbia, Vancouver, Canada
¶Department of Radiology, University of California Davis, Davis, CA, USA
#Department of Radiology, University of San Diego, San Diego, CA, USA
**Department of Medicine, University of Colorado School of Medicine, Aurora, CO, USA
††Department of Anatomy, University of California, San Francisco, San Francisco, CA, USA
‡‡Department of Radiology, Technical University of Munich, Munich, Germany

Magnetic resonance (MR) imaging of superparamagnetic iron oxide (SPIO)-labeled stem cells offers a noninvasive evaluation of stem cell engraftment in host organs. Excessive cellular iron load from SPIO labeling, however, impairs stem cell differentiation. The purpose of this study was to magnetically label human embryonic stem cells (hESCs) via a reduced exposure protocol that maintains a significant MR signal and no significant impairment to cellular pluripotency or differentiation potential. hESCs were labeled by simple incubation with Food and Drug Administration-approved ferumoxides, using concentrations of 50–200 μg Fe/ml and incubation times of 3–24 h. The most reduced exposure labeling protocol that still provided a significant MR signal comparable to accepted labeling protocols was selected for subsequent studies. Labeled hESCs were compared to unlabeled controls for differences in pluripotency as studied by fluorescence staining for SSEA-1, SSEA-4, TRA-60, and TRA-81 and in differentiation capacity as studied by quantitative real-time PCR for hOCT4, hACTC1, hSOX1, and hAFP after differentiation into embryoid bodies (EBs). Subsequent MR and microscopy imaging were performed to evaluate for cellular iron distribution and long-term persistence of the label. An incubation concentration of 50 μg Fe/ml and incubation time of 3 h demonstrated a significantly reduced exposure protocol that yielded an intracellular iron uptake of 4.50 ± 0.27 pg, an iron content comparable to currently accepted SPIO labeling protocols. Labeled and unlabeled hESCs showed no difference in pluripotency or differentiation capacity. Ferumoxide-labeled hESCs demonstrated persistent MR contrast effects as embryoid bodies for 21 days. Electron microscopy confirmed persistent lysosomal storage of iron oxide particles in EBs up to 9 days, while additional microscopy confirmed the iron distribution within single and multiple EBs. Labeling hESCs with ferumoxides by this tailored protocol reduces exposure of cells to the labeling agent while allowing for long-term visualization with MR imaging and the retention of cellular pluripotency and differentiation potential.

Key words: Human embryonic stem cells (hESCs); Superparamagnetic iron oxide (SPIO); Magnetic resonance (MR) imaging; Molecular imaging; Iron oxides; Cell labeling

Received May 10, 2010; final acceptance February 10, 2012. Online prepub date: August 2, 2012.
1These authors provided equal contribution to this work.
Address correspondence to Heike E. Daldrup-Link, M.D., Ph.D., Stanford University, 725 Welch Road, Room 1665, Stanford, CA 94305, USA. E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 2569-2585, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X636902
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Induced Pluripotent Stem Cells Without c-Myc Attenuate Acute Kidney Injury via Downregulating the Signaling of Oxidative Stress and Inflammation in Ischemia–Reperfusion Rats

Pei-Ying Lee,* Yueh Chien,†‡ Guang-Yuh Chiou,†‡ Chi-Hung Lin,†§ Chih-Hwa Chiou,†‡¶ and Der-Cherng Tarng*†#

*Department and Institute of Physiology, National Yang-Ming University, Taipei, Taiwan
†Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan
‡Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei, Taiwan
§Institute of Microbiology and Immunity, National Yang-Ming University, Taipei, Taiwan
¶Institute of Pharmacology, National Yang-Ming University, Taipei, Taiwan
#Division of Nephrology, Department of Medicine, Taipei Veterans General Hospital, Taipei, Taiwan

Induced pluripotent stem (iPS) cells have potential for multilineage differentiation and provide a resource for stem cell-based treatment. However, the therapeutic effect of iPS cells on acute kidney injury (AKI) remains uncertain. Given that the oncogene c-Myc may contribute to tumorigenesis by causing genomic instability, herein we evaluated the therapeutic effect of iPS cells without exogenously introduced c-Myc on ischemia–reperfusion (I/R)-induced AKI. As compared with phosphate-buffered saline (PBS)-treated group, administration of iPS cells via intrarenal arterial route into kidneys improved the renal function and attenuated tubular injury score at 48 h after ischemia particularly at the dose of 5 × 105 iPS cells. However, a larger number of iPS cells (5 × 107 per rat) diminished the therapeutic effects for AKI and profoundly reduced renal perfusion detected by laser Doppler imaging in the reperfusion phase. In addition, the green fluorescence protein-positive iPS cells mobilized to the peritubular area at 48 h following ischemia, accompanied by a significant reduction in infiltration of macrophages and apoptosis of tubular cells, and a remarkable enhancement in endogenous tubular cell proliferation. Importantly, transplantation of iPS cells reduced the expression of oxidative substances, proinflammatory cytokines, and apoptotic factors in I/R kidney tissues and eventually improved survival in rats of ischemic AKI. Six months after transplantation in I/R rats, engrafted iPS cells did not result in tumor formation in kidney and other organs. In summary, considering the antioxidant, anti-inflammatory, and antiapoptotic properties of iPS cells without c-Myc, transplantation of such cells may be a treatment option for ischemic AKI.

Key words: Acute kidney injury (AKI); Induced pluripotent stem (iPS) cells; Oxidative stress; Inflammatory cytokines

Received April 15, 2011; final acceptance January 8, 2012. Online prepub date: April 10, 2012.
Address correspondence to Der-Cherng Tarng, M.D., Ph.D., Department and Institute of Physiology, National Yang-Ming University, and Division of Nephrology, Department of Medicine, Taipei Veterans General Hospital, 201, Sec. 2, Shih-Pai Road, Taipei 11217, Taiwan. Tel: +886-2-2871 2121 ext. 2678; Fax: +886-2-2875 7824; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it or Chih-Hwa Chiou, M.D., Ph.D., Institute of Pharmacology, National Yang-Ming University, and Department of Medical Research and Education, Taipei Veterans General Hospital, 201, Sec. 2, Shih-Pai Road, Taipei 11217, Taiwan. Tel: +886-2-2875 7394; Fax: +886-2-2875 7396; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 2587-2602, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X653228
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
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Human Induced Pluripotent Stem Cells Improve Stroke Outcome and Reduce Secondary Degeneration in the Recipient Brain

Jérôme Polentes,* Pavla Jendelova,† Michel Cailleret,* Holger Braun,‡ Nataliya Romanyuk,† Philippe Tropel,*§ Marion Brenot,* Valerie Itier,*¶ Christine Seminatore,* Kathrin Baldauf,‡ Karolina Turnovcova,† Daniel Jirak,# Marius Teletin,** Julien Côme,* Johana Tournois,* Klaus Reymann,‡ Eva Sykova,† Stéphane Viville,§** and Brigitte Onteniente*

*I-Stem, INSERM UMR861, UEVE, Evry Cedex, France
†Institute of Experimental Medicine, ASCR, Department of Neuroscience and Center for Cell Therapy and Tissue Repair, Prague, Czech Republic
‡Leibniz Institut für Neurobiologie, Magdeburg, Germany
§IGBMC, Dpt Biologie Cellulaire & Développement, Illkirch-Graffenstaden Cedex, France
¶Université Paris-Est Créteil, Créteil Cedex, France
#Institute for Clinical and Experimental Medicine, Prague, Czech Republic
**Faculté de Médecine, Centre Hospitalier Universitaire, Strasbourg Cedex, France

Human induced pluripotent stem cells (hiPSCs) are a most appealing source for cell replacement therapy in acute brain lesions. We evaluated the potential of hiPSC therapy in stroke by transplanting hiPSC-derived neural progenitor cells (NPCs) into the postischemic striatum. Grafts received host tyrosine hydroxylase-positive afferents and contained developing interneurons and homotopic GABAergic medium spiny neurons that, with time, sent axons to the host substantia nigra. Grafting reversed stroke-induced somatosensory and motor deficits. Grafting also protected the host substantia nigra from the atrophy that follows disruption of reciprocal striatonigral connections. Graft innervation by tyrosine hydoxylase fibers, substantia nigra protection, and somatosensory functional recovery were early events, temporally dissociated from the slow maturation of GABAergic neurons in the grafts and innervation of substantia nigra. This suggests that grafted hiPSC-NPCs initially exert trophic effects on host brain structures, which precede integration and potential pathway reconstruction. We believe that transplantation of NPCs derived from hiPSCs can provide useful interventions to limit the functional consequences of stroke through both neuroprotective effects and reconstruction of impaired pathways.

Key words: Cell replacement therapy; Cerebral ischemia; Reprogramming; Intracerebral transplantation; Neurovascular lesions

Received November 16, 2011; final acceptance May 25, 2012. Online prepub date: August 10, 2012.
Address correspondence to Brigitte Onteniente, I-STEM; INSERM UMR861/UEVE/AFM, 5 rue Henri Desbrueres, F-91030 Evry Cedex, France. Tel: +33-1-6990-8538; Fax: +33-1-6990-8521; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it

Cell Transplantation, Vol. 21, pp. 2603-2619, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X653200
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Survival and Differentiation of Human Embryonic Stem Cell-Derived Neural Precursors Grafted Spinally in Spinal Ischemia-Injured Rats or in Naive Immunosuppressed Minipigs: A Qualitative and Quantitative Study

O. Kakinohana,* J. Juhasova,† S. Juhas,† J. Motlik,† O. Platoshyn,* J. Galik,‡§ M. Hefferan,* S. H. Yuan,¶# J. G. Vidal,** C. T. Carson,** S. van Gorp,†† D. Goldberg,* M. Leerink,* P. Lazar,‡‡ S. Marsala,* A. Miyanohara,§§ S. Keshavarzi,¶¶ J. D. Ciacci,¶¶ and M. Marsala*‡

*Neuroregeneration Laboratory, Department of Anesthesiology, University of California, San Diego, La Jolla, CA, USA
†Institute of Animal Physiology and Genetics, AS CR, Liběchov, Czech Republic
‡Institute of Neurobiology, Slovak Academy of Sciences, Košice, Slovakia
§Institute of Biology and Ecology, Faculty of Science, Pavol Jozef Safarik University, Košice, Slovakia
¶Howard Hughes Medical Institute and Department of Cellular and Molecular Medicine, School of Medicine, University of California, San Diego, La Jolla, CA, USA
#Department of Neurosciences, School of Medicine, University of California, San Diego, La Jolla, CA, USA
**BD Biosciences, La Jolla, CA, USA
††Department of Anesthesiology, School for Mental Health and Neuroscience, Maastricht University Medical Center, Maastricht, The Netherlands
‡‡University of Veterinary Medicine and Pharmacy, Department of Breeding and Diseases of Game and Fish, Košice, Slovakia
§§Vector Core Laboratory, Department of Pediatrics, University of California, San Diego, La Jolla, CA, USA
¶¶Division of Neurosurgery, University of California, San Diego, La Jolla, CA, USA

In previous studies, we have demonstrated that spinal grafting of human or rat fetal spinal neural precursors leads to amelioration of spasticity and improvement in ambulatory function in rats with spinal ischemic injury. In the current study, we characterize the survival and maturation of three different human embryonic stem (ES) cell line-derived neural precursors (hNPCs) once grafted into ischemia-injured lumbar spinal cord in rats or in naive immunosuppressed minipigs. Proliferating HUES-2, HUES-7, or HUES-9 colonies were induced to form embryoid bodies. During the nestin-positive stage, the rosettes were removed and CD184+/CD271/CD44/CD24+ population of ES-hNPCs FAC-sorted and expanded. Male Sprague–Dawley rats with spinal ischemic injury or naive immunosuppressed Gottingen–Minnesota minipigs received 10 bilateral injections of ES-NPCs into the L2–L5 gray matter. After cell grafting, animals survived for 2 weeks to 4.5 months, and the presence of grafted cells was confirmed after staining spinal cord sections with a combination of human-specific (hNUMA, HO14, hNSE, hSYN) or nonspecific (DCX, MAP2, CHAT, GFAP, APC) antibodies. In the majority of grafted animals, hNUMA-positive grafted cells were identified. At 2–4 weeks after grafting, double-labeled hNUMA/DCX-immunoreactive neurons were seen with extensive DCX+ processes. At survival intervals of 4–8 weeks, hNSE+ neurons and expression of hSYN was identified. Some hSYN-positive terminals formed putative synapses with the host neurons. Quantitative analysis of hNUMA+ cells at 2 months after grafting showed comparable cell survival for all three cell lines. In the presence of low-level immunosuppression, no grafted cell survival was seen at 4.5 months after grafting. Spinal grafting of proliferating pluripotent HUES-7 cells led to consistent teratoma formation at 2–6 weeks after cell transplantation. These data show that ES-derived, FACsorted NPCs can represent an effective source of human NPCs to be used in CNS cell replacement therapies.

Key words: Spinal cord ischemia; Human embryonic stem (ES) cells; Neuronal precursors (NPCs); Spinal cord grafting; Rat; Minipig

Received March 29, 2011; final acceptance February 22, 2012. Online prepub date: August 10, 2012.
Address correspondence to Martin Marsala, M.D., Neuroregeneration Laboratory, Department of Anesthesiology, University of California, San Diego, SCRM, Room 4009, 2880 Torrey Pines Scenic Drive, La Jolla, CA 92037, USA. Tel: +1-858-822-3249; Fax: +1-858-246-1483. E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 2621-2634, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X647153
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Application of Human Persistent Fetal Vasculature Neural Progenitors for Transplantation in the Inner Retina

Guochun Chen,*† Jie Ma,† Marie A. Shatos,† Huihui Chen,† Desirée Cyr,† and Kameran Lashkari†

*Department of Nephrology, The Second Xiangya Hospital, Central South University, Changsha, Hunan, P.R. China
†Schepens Eye Research Institute, Massachusetts Eye and Ear, Department of Ophthalmology, Harvard Medical School, Boston, MA, USA

Persistent fetal vasculature (PFV) is a potentially serious developmental anomaly in human eyes, which results from a failure of the primary vitreous and the hyaloid vascular systems to regress during development. Recent findings from our laboratory indicate that fibrovascular membranes harvested from subjects with PFV contain neural progenitor cells (herein called NPPFV cells). Our studies on successful isolation, culture, and characterization of NPPFV cells have shown that they highly express neuronal progenitor markers (nestin, Pax6, and Ki67) as well as retinal neuronal markers (β-III-tubulin and Brn3a). In the presence of retinoic acid and neurotrophins, these cells acquire a neural morphological appearance in vitro, including a round soma and extensive neurites, and express mature neuronal markers (β-III-tubulin and NF200). Further experiments, including real-time qRT-PCR to quantify characteristic gene expression profiles of these cells and Ca2+ imaging to evaluate the response to stimulation with different neurotransmitters, indicate that NPPFV cells may resemble a more advanced stage of retinal development and show more differentiation toward inner retinal neurons rather than photoreceptors. To explore the potential of inner retinal transplantation, NPPFV cells were transplanted intravitreally into the eyes of adult C57BL/6 mice. Engrafted NPPFV cells survived well in the intraocular environment in presence of rapamycin and some cells migrated into the inner nuclear layer of the retina 1 week posttransplantation. Three weeks after transplantation, NPPFV cells were observed to migrate and integrate in the inner retina. In response to daily intraperitoneal injections of retinoic acid, a portion of transplanted NPPFV cells exhibited retinal ganglion cell-like morphology and expressed mature neuronal markers (β-III-tubulin and synaptophysin). These findings indicate that fibrovascular membranes from human PFV harbor a population of neuronal progenitors that may be potential candidates for cell-based therapy for degenerative diseases of the inner retina.

Key words: NPPFV (persistent fetal vasculature-derived neural progenitor) cell; Transplantation; Differentiation; Retinal ganglion cell (RGC); Retina

Received September 8, 2010; final acceptance February 10, 2012. Online prepub date: June 15, 2012.
Address correspondence to Kameran Lashkari, M.D., Schepens Eye Research Institute, Massachusetts Eye and Ear, Harvard Medical School, 20 Staniford Street, Boston, MA 02114, USA. Tel: +1-617-912-7461; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 2635-2649, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X655181
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
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Synaptic Plasticity, But Not Hippocampal Neurogenesis, Mediated the Counteractive Effect of Wolfberry on Depression in Rats1

Endong Zhang,*† Suk Yu Yau,*† Benson Wui Man Lau,*† Henry Ma,*† Tatia M. C. Lee,†‡ Raymond Chuen-Chung Chang,*†§ and Kwok Fai So*†§¶

*Department of Anatomy, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong, China
†State Key Laboratory of Brain and Cognitive Sciences, The University of Hong Kong, Pokfulam, Hong Kong, China
‡Laboratory of Neuropsychology, The University of Hong Kong, Hong Kong, China
§Research Centre of Heart, Brain, Hormone, and Healthy Aging, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong, China
¶Joint Laboratory for Brain Function and Health (BFAH), Jinan University and The University of Hong Kong, Guangzhou, China

Depression is a life-threatening psychiatric disorder characterized with a long-term hypercortisolemia in depressed patients. Based on this clinical feature, hypercortisolemia was mimicked in experimental animals to understand the neuropathogy of depression and to explore new therapeutic strategies. Wolfberry, also known as Lycium barbarum, is a type of common fruit produced in mainland China. Accumulated evidence has shown that the extracts from Lycium barbarum (LBP) had a wide range of neuroprotective effects in various neurogenerative models. However, the antidepressant effect of LBP on depression and its mechanism has not yet been explored. In the present study, we investigated the effects of LBP on counteracting depression using an animal model injected with moderate dose (40 mg/kg) or severe dose (50 mg/kg) of corticosterone (CORT) treatments for 14 days. The results showed that CORT significantly increased immobility time and decreased hippocampal cell proliferation. LBP treatment significantly decreased the immobility time in forced swimming test, a test for the intensity of depressive behaviors, both in 40 and 50 mg/kg CORT stressed rats. Moreover, LBP treatment restored the reduced proliferation of neuroprogentior cells in the hippocampus in 40 mg/kg CORT stressed rats and the neuronal differentiation but not the proliferation in 50 mg/kg CORT stressed rats. After ablation of adult neurogenesis with Ara-c infusion, the beneficial effect of LBP treatment in reducing immobility time was not affected in 40 and 50 mg/kg CORT stressed rats. Golgi staining and Western blotting detection showed that LBP treatment restored the reduced spine density and the decreased level of PSD-95 in the hippocampus caused by 40 and 50 mg/kg CORT, respectively, indicating enhanced synaptic plasticity in the hippocampus. The data showed a novel effect of LBP on reducing depression-like behavior and its antidepressant effect may be mediated by enhanced synaptic plasticity, but not hippocampal neurogenesis.

Key words: Wolfberry; Depression; Corticosterone (CORT); Neurogenesis; Spine density

Online prepub date: October 31, 2012.
1This manuscript was originally submitted for inclusion in the special issue devoted to the Pan Pacific Symposium on Stem Cell Research (PPSSCR) but has been published in advance at the request of the authors.
Address correspondence to Prof. Kwok Fai So, Department of Anatomy, Li Ka Shing Faculty of Medicine, The University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong, China. Tel: +86 (852) 2819-9216; Fax: +86 (852) 2817-0857; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it or Dr. Raymond Cheung-Chung Chang, State Key Laboratory of Brain and Cognitive Sciences, The University of Hong Kong, Rm. L1-49, Laboratory Block, Faculty of Medicine Building, 21 Sassoon Road, Pokfulam, Hong Kong, China. E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 2651-2663, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X654975
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Neural Progenitor Cells Generate Motoneuron-Like Cells to Form Functional Connections With Target Muscles After Transplantation Into the Musculocutaneous Nerve

Huanxing Su,*1,2 Wenming Zhang,†1 Xiaoying Yang,*1 Dajiang Qin,* Yanhua Sang,* Chaoyang Wu,† Wai-Man Wong,* Qiuju Yuan,* Kwok-Fai So,*‡§ and Wutian Wu*‡§¶

*Department of Anatomy, The University of Hong Kong, Pokfulam, Hong Kong SAR, China
†Department of Orthopaedics, The First Affiliated Hospital of Fujian Medical University, Fujian, China
‡State Key Laboratory of Brain and Cognitive Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong SAR, China
§Joint Laboratory for Brain Function and Health (BFAH), Jinan University and The University of Hong Kong, Jinan University, Guangzhou, China
¶Research Center of Reproduction, Development and Growth, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong SAR, China

Neural progenitor cells (NPCs) are suggested to be a valuable source of cell transplant in treatment of various neurological diseases because of their distinct attributes. They can be expanded and induced to differentiate in vitro. However, it remains uncertain whether in vitro expanded NPCs have the capacity to give rise to functional motoneurons after transplantation in vivo. Here, we showed that in vitro expanded NPCs, when transplanted into the musculocutaneous nerve, generated motoneuron-like cells that exhibited typical morphology with large cell bodies, expressed specific molecules, and extended axons to form functional connections with the target muscle. In contrast, transplanted NPCs failed to yield motoneurons in the injured ventral horn of the spinal cord. The results of the study demonstrate that NPCs have the potential to generate functional motoneurons in an appropriate environment. The distinct differentiating fate of NPCs in the musculocutaneous nerve and the injured ventral horn suggests the importance and necessity of modifying the host microenvironment in use of NPCs for cell replacement therapies for motoneuron diseases.

Key words: Neural progenitor cells (NPCs); Motoneurons; Nerve injury; Spinal cord; Transplantation

Received July 15, 2011; final acceptance February 8, 2012. Online prepub date: September 7, 2012.
1These authors provided equal contribution to this work.
2Present address: State Key Laboratory of Quality Research in Chinese Medicine and Institute of Chinese Medical Sciences, University of Macau, Macao SAR, China.
Address correspondence to Wutian Wu, Department of Anatomy, Li Ka Shing Faculty of Medicine, The University of Hong Kong, 21 Sassoon Road, Hong Kong SAR, China. Tel: +852-2819-9187; Fax: +852-2817-0857; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 2665-2676, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X655028
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

AG490 Improves the Survival of Human Myoblasts In Vitro and In Vivo

Catherine Gérard, Christine Dufour, Sébastien Goudenege, Daniel Skuk, and Jacques P. Tremblay

Neurosciences Division-Human Genetics, CHUQ Research Centre-CHUL, Quebec, Canada

Cell therapies consist in transplanting healthy cells into a disabled tissue with the goal to repopulate it and restore its function at least partially. In muscular diseases, most of the time, myoblasts are chosen for their expansion capacity in culture. Nevertheless, cell transplantation has limitations, among them, death of the transplanted cells, during the days following the graft. One possibility to counteract this problem is to enhance the proliferation of the transplanted myoblasts before their fusion with the existing muscle fibers. AG490 is a specific inhibitor of janus tyrosine kinase 2 (JAK2). The hypothesis is to block myoblast differentiation with AG490, thus permitting their proliferation. The inhibition of myoblast fusion by AG490 was confirmed in this study by gene expression and with a myosin heavy chain staining (MyHC). Moreover, cell survival was estimated by flow cytometry. AG490 was found to protect myoblasts in vitro from apoptosis induced by H2O2 or by preventing attachment of cells to their substrate. Finally, in an in vivo model of muscle regeneration, when AG490 was coinjected with the myoblasts their survival was increased by 45% at 5 days after their transplantation.

Key words: Myoblast; AG490; Apoptosis; Survival; Cell therapy

Received February 9, 2011; final acceptance February 18, 2012. Online prepub date: September 7, 2012.
Address correspondence to Jacques P. Tremblay, Ph.D., Unité de Recherche en Génétique Humaine (RC 9300), Centre de Recherche du CHUL, 2705 Boulevard Laurier, Québec, QC, Canada, G1V 4G2. Tel: +1 (418) 654-2186; Fax: +1 (418) 654-2207; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 2677-2688, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X636993
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
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An Ectopic Stromal Implant Model for Hematopoietic Reconstitution and In Vivo Evaluation of Bone Marrow Niches

Flávio Henrique Paraguassú-Braga,* Ana Paula G. Alves,† Isabella Maria Alvim Andrade Santos,†1 Martin Bonamino,† and Adriana Bonomo†‡

*Banco de Sangue de Cordão Umbilical e Placentário, Centro de Transplante de Medula Óssea, Instituto Nacional de Câncer, Rio de Janeiro, Brazil
†Programa de Medicina Experimental, Coordenação Geral Técnico-Científica, Instituto Nacional de Câncer, Rio de Janeiro, Brazil
‡Departamento de Imunologia, Instituto de Microbiologia Professor Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil

In adults, hematopoiesis takes places in the bone marrow, where specialized niches containing mesenchymal nonhematopoietic cells (stroma) harbor the hematopoietic stem cell (HSC). These niches are responsible and essential for the maintenance of HSCs. Attempts to expand HSCs fail to keep the general properties of stem cells, which depend on several niche components difficult to reproduce in in vitro culture systems. Here, we describe a methodology for in vivo study of hematopoietic stroma. We use stroma-loaded macroporous microcarriers implanted in the subcutaneous tissue of experimental animals and show that the ectopic stroma implant (ESI) is able to support hematopoiesis. Moreover, lethally irradiated mice can be rescued by ESI preloaded with HSCs, showing that they function as an ectopic bone marrow. ESI is also shown as a good system to study the role of different niche components. As an example, we used stromas lacking connexin 43 (Cx43) and confirm the importance of this molecule in the maintenance of the HSC niche in vivo. We believe ESI can work as an ectopic bone marrow allowing in vivo testing of different niches components and opening new avenues for the treatment of a variety of hematologic conditions particularly when stromal cell defects are the main cause of disease.

Key words: Hematopoiesis; Stroma; Bone marrow; Cx43

Received July 26, 2011; final acceptance November 30, 2011. Online prepub date April 2, 2012.
1Supported with a Ministry of Health/INCA fellowship.
Address correspondence to Adriana Bonomo, Programa de Medicina Experimental, Instituto Nacional de Câncer, Rua André Cavalcanti, 37 - Centro, 20231-050 - Rio de Janeiro - RJ, Brazil. Tel: +55 21 3207-6598; Fax: +55 21 3207 6509; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 2689-2709, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X654920
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Local Transplantation of Ex Vivo Expanded Bone Marrow-Derived CD34-Positive Cells Accelerates Fracture Healing

Yohei Kawakami,*† Masaaki Ii,‡ Cantas Alev,*§ Atsuhiko Kawamoto,* Tomoyuki Matsumoto,*† Ryosuke Kuroda,† Taro Shoji,*† Tomoaki Fukui,*† Haruchika Masuda,¶ Hiroshi Akimaru,* Yutaka Mifune,*† Tomoya Kuroda,*† Miki Horii,* Ayumi Yokoyama,* Masahiro Kurosaka,† and Takayuki Asahara*¶

*Group of Vascular Regeneration, Institute of Biomedical Research and Innovation, Kobe, Hyogo, Japan
†Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, Kobe, Hyogo, Japan
‡Department of Pharmacology, Osaka Medical College, Takatsuki, Osaka, Japan
§Laboratory for Early Embryogenesis, RIKEN Center for Developmental Biology, Kobe, Hyogo, Japan
¶Department of Regenerative Medicine Science, Tokai University School of Medicine, Isehara, Kanagawa, Japan

Transplantation of bone marrow (BM) CD34+ cells, an endothelial/hematopoietic progenitor-enriched cell population, has shown therapeutic efficiency in the treatment of ischemic diseases enhancing neovascularization. However, the number of CD34+ cells obtained from bone marrow is not sufficient for routine clinical application. To overcome this issue, we developed a more efficient and clinically applicable CD34+ cell expansion method. Seven-day ex vivo expansion culture of BM CD34+ cells with a cocktail of five growth factors containing VEGF, SCF, IL-6, Flt-3 ligand, and TPO resulted in reproducible more than 20-fold increase in cell number. The favorable effect of the local transplantation of culture expanded (cEx)-BM CD34+ cells on rat unhealing fractures was equivalent or higher than that of nonexpanded (fresh) BM CD34+ cells exhibiting sufficient therapeutic outcome with frequent vasculogenic/osteogenic differentiation of transplanted cEx-BM CD34+ cells and fresh BM CD34+ cells as well as intrinsic enhancement of angiogenesis/osteogenesis at the treated fracture sites. Specifically, cEx-BM CD34+ cell treatment demonstrated the best blood flow recovery at fracture sites compared with the nonexpanded BM CD34+ cells. In vitro, cEx-BM CD34+ cells showed higher colony/tube-forming capacity than nonexpanded BM CD34+ cells. Both cells demonstrated differentiation potential into osteoblasts. Since fresh BM CD34+ cells can be easily collected from fracture sites at the time of primary operation and stored for future use, autologous cEx-BM CD34+ cell transplantation would be not only a simple but also a promising therapeutic strategy for unhealing fractures in the field of orthopedic trauma surgery.

Key words: Fracture healing; Osteogenesis; Angiogenesis; Stem/progenitor cell; Expansion

Received September 12, 2011; final acceptance February 25, 2012. Online prepub date: August 27, 2012.
Address correspondence to Takayuki Asahara, M.D., Ph.D., Group of Vascular Regeneration, Institute of Biomedical Research and Innovation, 2-2 Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan. Tel: +81-78-306-0727; Fax: +81-78-643-3685; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 2711-2721, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X654966
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Allogeneic Adipose-Derived Stem Cells With Low Immunogenicity Constructing Tissue-Engineered Bone for Repairing Bone Defects in Pigs

Ming-liang Ren,*†1 Wei Peng,*1 Zai-liang Yang,*1 Xin-jun Sun,* Shi-chang Zhang,* Zheng-guo Wang,* and Bo Zhang*

*Department 4, State Key Laboratory of Trauma, Burn and Combined Injury, Research Institute of Surgery and Daping Hospital, Third Military Medical University, Chongqing, China
†Department of Neurosurgery, Research Institute of Surgery and Daping Hospital, Third Military Medical University, Chongqing, China

The ideal cells for tissue engineering should have the following characteristics: easy obtainment, safety, immune privilege, the capability of self-renewal, and multipotency. Adipose-derived stem cells (ADSCs) are a promising candidate. However, the immunogenicity of allogeneic mesenchymal stem cells limits their long-term benefits. In this study, we introduced human cytomegalovirus US2/US3 gene into the ADSCs to decrease the expression of MHC I protein of ADSCs and reduce the activation of T-cells of the recipient animals. Moreover, the biosafety and biological characteristics of ADSCs transfected with the US2/US3 genes (ADSCs-US2/US3) were similar to normal ADSCs. Then we took ADSCs-US2/US3 to construct a tissue-engineered bone for repairing bone defects in pigs and found that there were no great differences in repair effects or healing time between the allogeneic ADSCs-US2/US3 group and the autologous ADSC group. These results suggest that allogeneic ADSCs-US2/US3 have the advantages of biological safety, low immunogenicity, and effective osteogenesis. Such barely immunogenic ADSCs will be crucial for the success of future tissue-regenerative approaches.

Key words: Adipose-derived stem cells (ADSCs); US2 and US3 genes; Immune escape; Tissue engineering; Repair of bone defects

Received March 25, 2011; final acceptance February 10, 2012. Online prepub date: September 7, 2012.
1These authors provided equal contribution to this work.
Address correspondence to Bo Zhang, Department 4, State Key Laboratory of Trauma, Burn and Combined Injury, Research Institute of Surgery and Daping Hospital, Third Military Medical University, 10 Changjiang Zhilu, Chongqing, 400042, China. Tel: +86-23-68757443; Fax: +86-23-68757443; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 2723-2722, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X638847
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Treatment of Reperfused Ischemia With Adipose-Derived Stem Cells in a Preclinical Swine Model of Myocardial Infarction

Manuel Mazo,* Salomon Hernandez,† Juan Jose Gavira,‡ Gloria Abizanda,* Miriam Arana,* Tania Lopez-Martinez,† Cristina Moreno,§ Juana Merino,§ Alba Martino-Rodriguez,¶ Alicia Uixeira,¶ Jose A. Garcia de Jalon,¶ Juan Pastrana,# Diego Martinez-Caro,‡ and Felipe Prosper*

*Hematology and Cell Therapy, Clinica Universidad de Navarra, Pamplona, Spain
†Universidad Panamericana, Mexico City, Mexico
‡Department of Cardiology and Cardiovascular Surgery, Clinica Universidad de Navarra, Pamplona, Spain
§Immunology Service, Clinica Universidad de Navarra, Pamplona, Spain
¶Department of Animal Pathology, Veterinary Faculty, University of Zaragoza, Spain
#Department of Internal Medicine, Clinica Universidad de Navarra, Pamplona, Spain

The aim of the study was to determine the long-term effect of transplantation of adipose-derived stromal cells (ADSCs) in a preclinical model of ischemia/reperfusion (I/R). I/R was induced in 28 Goettingen minipigs by 120 min of coronary artery occlusion followed by reperfusion. Nine days later, surviving animals were allocated to receive transendocardial injection of a mean of 213.6 ± 41.78 million green fluorescent protein (GFP)-expressing ADSCs (n = 7) or culture medium as control (n = 9). Heart function, cell engraftment, and histological analysis were performed 3 months after transplantation. Transplantation of ADSCs induced a statistically significant long-lasting (3 months) improvement in cardiac function and geometry in comparison with control animals. Functional improvement was associated with an increase in angiogenesis and vasculogenesis and a positive effect on heart remodeling with a decrease in fibrosis and cardiac hypertrophy in animals treated with ADSCs. Despite the lack of cell engraftment after 3 months, ADSC transplantation induced changes in the ratio between MMP/TIMP. Our results indicate that transplantation of ADSCs, despite the lack of long-term significant cell engraftment, increases vessel density and prevents adverse remodeling in a clinically relevant model of myocardial infarction, strongly suggesting a paracrine-mediated effect. ADSCs thus constitute an attractive candidate for the treatment of myocardial infarction.

Key words: Adipose-derived stem cells (ADSCs); Cardiac ischemia/reperfusion; Angiogenesis; Heart remodeling

Received August 16, 2011; final acceptance February 2, 2012. Online prepub date: April 17, 2012.
Address correspondence to Felipe Prosper, M.D., Ph.D., Hematology and Cell Therapy, Clinica Universitaria, Av. Pio XII 36, Pamplona 31008, Navarra, Spain. Tel: +34 948 255400; Fax: 34 948 296500; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 2735-2751, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X637497
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Genipin-Cross-Linked Microencapsulated Human Adipose Stem Cells Augment Transplant Retention Resulting in Attenuation of Chronically Infarcted Rat Heart Fibrosis and Cardiac Dysfunction

Arghya Paul,* Guangyong Chen,† Afshan Khan,* Vijayaraghava T. S. Rao,‡ Dominique Shum-Tim,† and Satya Prakash*

*Biomedical Technology and Cell Therapy Research Laboratory, Department of Biomedical Engineering and Artificial Cells and Organs Research Centre Faculty of Medicine, McGill University, Montreal, Québec, Canada
†Divisions of Cardiac Surgery and Surgical Research, McGill University Health Center, Montreal, Quebec, Canada
‡Institute of Parasitology, McGill University, Macdonald Campus, Ste. Anne de Bellevue, Quebec, Canada

Stem cell transplantation has been widely acknowledged for their immense potential in regenerative medicine. In these procedures, the implanted cells need to maintain both their viability and functional properties for effective therapeutic outcomes. This has long been a subject of major concern and intensive studies. Microencapsulation of stem cells within polymeric microcapsules can be an efficient approach to achieve this goal, particularly for heart diseases. This study reports the use of biocompatible, fluorogenic genipin-crosslinked alginate chitosan (GCAC) microcapsules in delivery of human adipose stem cells (hASCs) with an aim to increase the implant retention in the infarcted myocardium for maximum clinical benefits. In vitro results show, under hypoxic conditions, the microencapsulated cells overexpressed significantly higher amount of biologically active vascular endothelial growth factor (VEGF). We investigated on the in vivo potential using immunocompetent female rats after induction of myocardial infarction. For this, animal groups (n = 8) received empty control microcapsules, 1.5 × 106 free male hASCs, or 1.5 × 106 microencapsulated male hASCs. Results show significant retention (3.5 times higher) of microencapsulated hASCs compared to free hASCs after 10 weeks of transplantation. Microencapsulated hASCs showed significantly attenuated infarct size compared to free hASCs and empty microcapsule group (21.6% ± 1.1% vs. 27.2% ± 3.1% vs. 33.3% ± 3.2%; p < 0.05), enhanced vasculogenesis, and improved cardiac function (fractional shortening: 24.2% ± 2.1% vs. 19.1% ± 0.5% vs. 12.0% ± 4.0%; p < 0.05). These data suggest that microencapsulated hASCs can contribute significantly to the improvement in cardiac functions. Their greater retentions exhibit reduced fibrosis and cardiac dysfunction in experimental animals. However, further research is needed to fully comprehend the underlying biological and immunological effects of microencapsulated hASCs, which jointly play important roles in cardiac repair.

Key words: Cell transplant; Adipose stem cell therapy; Tissue engineering; Myocardial infarction; Microencapsulation; Regenerative medicine

Received November 25, 2010; final acceptance November 10, 2011. Online prepub date: April 10, 2012.
Address correspondence to Satya Prakash, Biomedical Technology and Cell Therapy Research Laboratory, Department of Biomedical Engineering and Artificial Cells and Organs Research Centre Faculty of Medicine, McGill University, 3775 University Street, Montreal, Québec, H3A 2B4, Canada. Tel: +1-514-398-3676; Fax: +1-514-398-7461; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 2753-2764, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X652959
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Adipose-Derived Stem Cells Can Abrogate Chemical-Induced Liver Fibrosis and Facilitate Recovery of Liver Function

Horng-Jyh Harn,*†1 Shinn-Zong Lin,‡§1 Shih-Hsiao Hung,¶ Yi-Maun Subeq,# Yuan-Sheng Li,¶ Wan-Sin Syu,¶ Dah-Ching Ding,** Ru-Ping Lee,# Dean-Kuo Hsieh,†† Po-Cheng Lin,‡‡ and Tzyy-Wen Chiou¶

*Department of Pathology, China Medical University Hospital, Taichung, Taiwan, ROC
†Department of Medicine, China Medical University, Taichung, Taiwan, ROC
‡Center for Neuropsychiatry, China Medical University and Hospital, Taichung, Taiwan, ROC
§Department of Neurosurgery, China Medical University Beigan Hospital, Yunlin, Taiwan, ROC
¶Department of Life Science and Graduate Institute of Biotechnology, National Dong Hwa University, Hualien, Taiwan, ROC
#Department of Nursing, Tzu Chi University, Hualien, Taiwan, ROC
**Department of Obstetrics and Gynecology, Buddhist Tzu Chi General Hospital, Tzu Chi University, Hualien, Taiwan, ROC
††Department of Applied Chemistry, Chaoyang University of Technology, Taichung, Taiwan, ROC
‡‡Department of Research and Development, Gwo Xi Stem Cell Applied Technology Co., Ltd., Hsinchu, Taiwan, ROC

Adipose-derived stem cells (ADSCs) are easy to harvest and have the ability for self-renewal and to differentiate into various cell types, including those of the hepatic lineage. Studies on the use of ADSCs for liver transplantation are, however, limited. The objective of this study was to investigate the feasibility of using human ADSCs and to better understand their mechanism of action for the repair of liver damage in a thioacetamide (TAA)-induced model of chronic liver damage in the rat. To induce liver damage, 200 mg/kg TAA was injected intraperitoneally into Wistar rats every 3 days for 60 days. For cell therapy, 1 × 106 human ADSCs suspended in 300 μl of phosphate-buffered saline were transplanted into each experimental rat by direct liver injection. Immunohistochemistry showed that the transplanted ADSCs differentiated into albumin- and α-fetoprotein-secreting liver-like cells 1 week after transplantation. In addition, liver function recovered significantly, as determined by biochemical analyses that analyzed total bilirubin, prothrombin time, and albumin levels. The Metavir score, derived from histopathological analysis, also showed a significant decrease in liver fibrosis and inflammatory activity after ADSC transplantation. Finally, we found a reduction in the expression of a-smooth muscle actin, a marker of hepatic stellate cells, which produce collagen fiber, and an increase in the expression of matrix metalloproteinase-9, which degrades collagen fiber, after ADSC transplantation. These findings are consistent with abrogation of liver fibrosis in the ADSC therapy group. Consequently, these results suggest that ADSC transplantation may facilitate recovery from chronic liver damage and thus may have clinical applications.

Key words: Adipose-derived stem cells (ADSCs); Thioacetamide; Liver fibrosis; Matrix metalloproteinase-9 (MMP-9)

Received November 13, 2011; final acceptance February 25, 2012. Online prepub date: July 5, 2012.
1These authors provided equal contribution to this work.
Address correspondence to Tzyy-Wen Chiou, Ph.D., Department of Life Science and Graduate Institute of Biotechnology, National Dong Hwa University, No. 1, Sec. 2, Da Hsueh Road, Shoufeng, Hualien 97401, Taiwan, Republic of China. Tel: +886-3-8630398; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 2765-2770, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X653255
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Hepatocyte Transplantation Using the Domino Concept in a Child With Tetrabiopterin Nonresponsive Phenylketonuria

X. Stéphenne,*† F. G. Debray,‡ F. Smets,*† N. Jazouli,*† G. Sana,*† T. Tondreau,*† R. Menten,§ P. Goffette,§ F. Boemer,¶ R. Schoos,¶ S. W. Gersting,# M. Najimi,† A. C. Muntau,# P. Goyens,** and E. M. Sokal*†

*Université Catholique de Louvain, Cliniques Universitaires St Luc, Service de Gastroentérologie et Hépatologie Pédiatrique, Banque D’hépatocytes, Bruxelles, Belgium
†Université Catholique de Louvain, Institut de Recherche Expérimentale et Clinique (IREC), Laboratoire D’hépatologie Pédiatrique & Thérapie Cellulaire, Brussels, Belgium
‡CHU & Université de Liège, Centre de Génétique Humaine, Liège, Belgium
§Université Catholique de Louvain, Cliniques Universitaires St Luc, Unité de Radiologie Pédiatrique, Bruxelles, Belgium
¶CHU & Université de Liège, Genetic Biochemistry Laboratory, Liège, Belgium
#Dr. von Hauner Children’s Hospital, Division of Molecular Pediatrics, Munich, Germany
**Université Libre de Bruxelles, Unité de Nutrition et Métabolisme & Laboratoire de Pédiatrie, Bruxelles, Belgium

Phenylketonuria is a metabolic disease caused by phenylalanine hydroxylase deficiency. Treatment is based on a strict natural protein-restricted diet that is associated with the risk of malnutrition and severe psychosocial burden. Oral administration of tetrahydrobiopterin can increase residual enzyme activity, but most patients with severe clinical phenotypes are nonresponders. We performed liver cell transplantation in a 6-year-old boy with severe tetrahydrobiopterin nonresponsive phenylketonuria who failed to comply with diet prescriptions. The transplanted hepatocytes were obtained in part from an explanted glycogen storage type 1b liver. Following two infusions, blood phenylalanine levels returned within the therapeutic target while the phenylalanine half-life assessed by loading tests decreased from 43 to 19 h. However, 3 months later, blood phenylalanine concentrations increased and the phenylalanine intake had to be reduced. Cell-based therapy is a promising therapeutic option in phenylketonuria, and the domino concept may solve the issue of cell sources for hepatocyte transplantation.

Key words: Liver cell transplantation; Phenylketonuria (PKU); Domino procedure

Received August 2, 2011; final acceptance January 15, 2012. Online prepub date: August 10, 2012.
Address correspondence to Pr. Etienne M. Sokal, M.D., Ph.D., Laboratoire D’hépatologie Pédiatrique & Thérapie Cellulaire, Université Catholique de Louvain, St Luc Clinics, 10 Hippocrate Avenue, B-1200 Bruxelles, Belgium. Tel: +3227641387; Fax: +3227648909; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 2771-2781, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X637046
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Mesenchymal Stem Cells and Islet Cotransplantation in Diabetic Rats: Improved Islet Graft Revascularization and Function by Human Adipose Tissue-Derived Stem Cells Preconditioned With Natural Molecules

Giuseppe Cavallari,*† Elena Olivi,‡§¶ Francesca Bianchi,§¶ Flavia Neri,*† Laura Foroni,‡ Sabrina Valente,# Gaetano La Manna,** Bruno Nardo,*† Sergio Stefoni,** and Carlo Ventura§¶

*Department of General Surgery and Transplantation, University of Bologna, Bologna, Italy
†Center for Applied Biomedical Research (CRBA), University of Bologna, Bologna, Italy
‡Department of Specialistic Surgery and Anaesthesiological Sciences, University of Bologna, Bologna, Italy
§Cardiovascular Department, University of Bologna, Bologna, Italy
¶Laboratory of Molecular Biology and Stem Cell Engineering - National Institute of Biostructures and Biosystems (NIBB), Bologna, Italy
#Department of Hematology, Oncology and Laboratory Medicine, University of Bologna, Bologna, Italy
**Nephrology, Dialysis and Renal Transplant Unit, S. Orsola -Malpighi Hospital, University of Bologna, Bologna, Italy

Hypoxia plays an important role in limiting the engraftment, survival, and function of intrahepatically transplanted islets. Mesenchymal stem cells (MSCs) were recently used in animal models of islet transplantation not only to reduce allograft rejection but also to promote revascularization. Among different possible origins, adipose tissue represents a novel and good source of MSCs. Moreover, the capability of adipose tissue-derived stem cells (ASCs) to improve islet graft revascularization was recently reported after hybrid transplantation in mice. Within this context, we have previously shown that hyaluronan esters of butyric and retinoic acids can significantly enhance the rescuing potential of human MSCs (hMSCs). Here we evaluated whether ex vivo preconditioning of human ASCs (hASCs) with a mixture of hyaluronic (HA), butyric (BU), and retinoic (RA) acids may result in optimization of graft revascularization after islet/stem cell intrahepatic cotransplantation in syngeneic diabetic rats. We demonstrated that hASCs exposed to the mixture of molecules are able to increase the secretion of vascular endothelial growth factor (VEGF) as well as the transcription of angiogenic genes, including VEGF, KDR (kinase insert domain receptor), and hepatocyte growth factor (HGF). Rats transplanted with islets cocultured with preconditioned hASCs exhibited a better glycemic control than rats transplanted with an equal volume of islets and control hASCs. Cotransplantation with preconditioned hASCs was also associated with enhanced islet revascularization in vivo, as highlighted by graft morphological analysis. The observed increase in islet graft revascularization and function suggests that our method of stem cell preconditioning may represent a novel strategy to remarkably improve the efficacy of islets-hMSCs cotransplantation.

Key words: Islet transplantation; Human mesenchymal stem cells (hMSCs); Hypoxia; Natural molecules

Received August 5, 2011; final acceptance January 5, 2012. Online prepub date: April 2, 2012.
Address correspondence to Prof. Carlo Ventura, Laboratory of Molecular Biology and Stem Cell Engineering-NIBB, Strada Maggiore 42, 40125 Bologna, Italy. Tel/Fax: +39-051-340339; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 2783-2795, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X654993
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Emergence of a Broad Repertoire of GAD65-Specific T-Cells in Type 1 Diabetes Patients With Graft Dysfunction After Allogeneic Islet Transplantation

Daisuke Chujo,* Emile Foucat,* Morihito Takita,† Takeshi Itoh,† Koji Sugimoto,† Masayuki Shimoda,† Kunimasa Yagi,‡ Masakazu Yamagishi,‡ Yoshiko Tamura,§ Liping Yu,¶ Bashoo Naziruddin,§ Marlon F. Levy,†§ Hideki Ueno,* and Shinichi Matsumoto*†1

*Baylor Institute for Immunology Research, Dallas, TX, USA
†Baylor Research Institute Islet Cell Laboratory, Dallas, TX, USA
‡Division of Cardiovascular Medicine, Kanazawa University Graduate School of Medical Science, Kanazawa, Japan
§Annette C. and Harold C. Simmons Transplant Institute, Dallas, TX, USA
¶Barbara Davis Center for Childhood Diabetes, University of Colorado Health Science Center, Aurora, CO, USA

Islet transplantation is one of the most promising therapies for type 1 diabetes (T1D). A major issue in islet transplantation is the loss of graft function at late phase. Several studies suggested the involvement of isletspecific T-cells in such islet graft dysfunction. In this study, we investigated the breadth and type of glutamic acid decarboxylase 65 (GAD65)-specific T-cells in T1D patients after allogeneic islet transplantation. Peripheral blood mononuclear cells (PBMCs) were obtained from islet-transplanted T1D patients during insulin-independent period and cultured for 7 days with pools of GAD65 overlapping peptides in the presence of IL-2. Cytokine secretion profiles of peptide-reactive T-cells were analyzed after a short-term restimulation with the same peptides by a multiplex bead-based cytokine assay and by an intracytoplasmic cytokine detection assay. Robust GAD65-specific CD4+ and CD8+ T-cell responses were detected in patients who eventually developed chronic graft dysfunction. Multiple GAD65 peptides were found to induce specific T-cell responses in these patients, indicating that the repertoire of GAD65-specific T-cells was broad. Furthermore, GAD65-specific CD4+ T-cells were composed of heterogeneous populations, which differentially expressed cytokines including IFN-γ and type 2 cytokines, but not IL-10. In contrast, patients who showed only marginal GAD65-specific T-cell responses maintained substantially longer graft survival and insulin independence. In conclusion, our study suggests that the emergence of islet-specific T-cells precedes the development of chronic graft dysfunction in islet-transplanted patients. Thus, our observations support the hypothesis that these islet-specific T-cells contribute to the development of chronic islet graft dysfunction.

Key words: Islet transplantation; Type 1 diabetes (T1D); Graft dysfunction; Glutamic acid decarboxylase 65 (GAD65); T-cells

Received September 23, 2011; final acceptance February 16, 2012. Online prepub date: September 7, 2012.
1Current address: National Center for Global Health and Medicine, Islet Transplantation Project, 1-21-1 Toyama Shinjuku-ku, Tokyo 162-8655, Japan. Address correspondence to Hideki Ueno, M.D., Ph.D., Baylor Institute for Immunology Research, 3434 Live Oak Street, Dallas, TX 75204, USA. Tel: +1-214-820-9917; Fax: +1-214-820-4813; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 2797-2804, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X653273
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

The Islet Size to Oxygen Consumption Ratio Reliably Predicts Reversal of Diabetes Posttransplant

Andrew R. Pepper,*† Craig P. Hasilo,† C. W. James Melling,‡ Delfina M. Mazzuca,† Greg Vilk,† Guangyong Zou,§ and David J. G. White*¶

*Department of Pathology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, ON, Canada
†Multi-Organ Transplantation Program, London Health Science Centre, London, ON, Canada
‡Department of Health Sciences, Schulich School of Medicine and Dentistry, University of Western Ontario, London, ON, Canada
§Department of Epidemiology and Biostatistics, Schulich School of Medicine and Dentistry, University of Western Ontario, London, ON, Canada
¶Department of Surgery, Schulich School of Medicine and Dentistry, University of Western Ontario, London, ON, Canada

β-Cell replacement therapy by either whole-organ pancreas or islets of Langerhans transplantation can restore carbohydrate control to diabetic patients and reduces complications associated with the disease. One of the variables inherent in islet transplantation is the isolation of functional islets from donor pancreata. Islet isolations fail to consistently produce good-quality functional islets. A rapid pretransplant assay to determine posttransplant function of islets would be an invaluable tool. We have tested the novel hypothesis that modified oxygen consumption rates (OCR), standardized to DNA quantity (nmol/min-mg DNA), would serve as a pretransplant assessment of the metabolic potency of the islets postisolation. This study compares the ability of current in vitro assays to predict in vivo restoration of normoglycemia in a diabetic nude mouse posttransplantation of adult pig islets. There is known to be a diversity of islet sizes within each preparation. This parameter has not heretofore been effectively considered a critical factor in islet engraftment. Our results suggest a surprising finding that islet size influences the probability of restoring carbohydrate control. Based on this observation, we thus developed a novel predictor of islet graft function that combines the effects of both islet OCR and size. When OCR was divided by the islet index (size), a highly significant predictor of graft function was established (p = 0.0002, n = 75). Furthermore, when OCR/islet index values exceeded 70.0 nmol/min-mg DNA/islet index, an effective threshold of diabetes reversal was observed. This assay can be performed with as few as 1,000 islet equivalents (IEQ) and conducted in less than 60 min. Our data suggest that, using this novel method to assess islet cell function prior to transplantation, OCR/islet index thresholds provide a valuable tool in identifying which islet preparations are most likely to restore glycemic control posttransplant.

Key words: Islets of Langerhans; Diabetes; Oxygen consumption rate (OCR); Viability testing; Transplantation

Received July 25, 2011; final acceptance February 23, 2012. Online prepub date: August 27, 2012.
Address correspondence to Dr. David J. G. White, Siebens-Drake Research Institute, 1400 Western Road, Room 200, London, ON, Canada N6G 2V4. Tel: +1-519-661-2111, ext. 82988; Fax: +1-519-858-5142; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 2805-2814, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X653138
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Supplements in Human Islet Culture: Human Serum Albumin Is Inferior to Fetal Bovine Serum

Efstathios S. Avgoustiniatos,* William E. Scott III,* Thomas M. Suszynski,* Henk-Jan Schuurman,* Rebecca A. Nelson,† Phillip R. Rozak,* Kate R. Mueller,* A. N. Balamurugan,* Jeffrey D. Ansite,* Daniel W. Fraga,* Andrew S. Friberg,* Gina M. Wildey,* Tomohiro Tanaka,* Connor A. Lyons,* David E. R. Sutherland,* Bernhard J. Hering,* and Klearchos K. Papas*

*Schulze Diabetes Institute, Department of Surgery, University of Minnesota, Minneapolis, MN, USA
†Administrative and Bioinformatics Coordinating Center (ABCC) for the Islet Cell Resource (ICR) Center Consortium, City of Hope National Medical Center and Beckman Research Institute, Duarte, CA, USA

Culture of human islets before clinical transplantation or distribution for research purposes is standard practice. At the time the Edmonton protocol was introduced, clinical islet manufacturing did not include culture, and human serum albumin (HSA), instead of fetal bovine serum (FBS), was used during other steps of the process to avoid the introduction of xenogeneic material. When culture was subsequently introduced, HSA was also used for medium supplementation instead of FBS, which was typically used for research islet culture. The use of HAS as culture supplement was not evaluated before this implementation. We performed a retrospective analysis of 103 high-purity islet preparations (76 research preparations, all with FBS culture supplementation, and 27 clinical preparations, all with HSA supplementation) for oxygen consumption rate per DNA content (OCR/DNA; a measure of viability) and diabetes reversal rate in diabetic nude mice (a measure of potency). After 2-day culture, research preparations exhibited an average OCR/DNA 51% higher (p < 0.001) and an average diabetes reversal rate 54% higher (p < 0.05) than clinical preparations, despite 87% of the research islet preparations having been derived from research-grade pancreata that are considered of lower quality. In a prospective paired study on islets from eight research preparations, OCR/DNA was, on average, 27% higher with FBS supplementation than that with HSA supplementation (p < 0.05). We conclude that the quality of clinical islet preparations can be improved when culture is performed in media supplemented with serum instead of albumin.

Key words: Islet culture; Islet viability; Islet potency; Fetal bovine serum (FBS); Human serum albumin (HSA)

Received June 26, 2011; final acceptance January 16, 2012. Online prepub date: August 2, 2012.
Address correspondence to Klearchos K. Papas, Ph.D., Professor, Institute for Cellular Transplantation, Department of Surgery, University of Arizona, 1656 E. Mabel Street, Room 121, Tucson, AZ 85724, USA. Tel: +1 (520) 626-4494; Fax: +1 (520) 626-7779; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it