Cell Transplantation 22(10) Abstracts

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Cell Transplantation, Vol. 22, pp. 1735-1751, 2013
0963-6897/13 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X657918
E-ISSN 1555-3892
Copyright © 2013 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Brain Repair in a Unilateral Rat Model of Huntington’s Disease: New Insights Into Impairment and Restoration of Forelimb Movement Patterns

Alexander Klein,* Emma L. Lane,† and Stephen B. Dunnett*

*Brain Repair Group, School of Biosciences, Cardiff University, Cardiff, Wales, UK
†Welsh School of Pharmacy, Cardiff University, Cardiff, Wales, UK

Huntington’s disease (HD) produces severe neurodegeneration in the striatum leading to disabling motor impairments, including the loss of control of skilled reaching movements. Fetal GABAergic transplants can physically replace the lost striatal cells but with only partial success in functional recovery. Here, we aimed to determine the extent and quality of the repair produced by fetal cell transplantation through an in-depth analysis of reaching behavior in the quinolinic acid-lesioned rat model of HD. Control, quinolinic acid-lesioned plus sham graft, and quinolinic acid-lesioned plus graft groups of rats were assessed in skilled reaching performance prior to and following lesion surgery and 3 months following injection of 400,000 fetal whole ganglionic eminence-derived cells into the striatum. This was compared to their performance in two more rudimentary tests of motor function (the adjusting step and vibrissae-evoked hand-placing tests). Grafted rats demonstrated a significant improvement in reaching success rate (graft +59%, shamTX +3%). Importantly, the quality of reaching behavior, including all components of the movement, was fully restored with no identifiable differences in the normal behavior shown by control rats. Postmortem immunohistochemical examination verified the survival of large intrastriatal grafts, and Fluoro-Gold tracing indicated appropriate outgrowth to the globus pallidus. Our study illustrates for the first time the detailed analysis of qualitative improvement of motor function following brain repair in a rat model of HD. The results demonstrate significant improvements not only in gross movements but also in the skilled motor patterns lost during HD. Fetal GABAergic cell transplantation showed a demonstrable ability to restore motor function to near normal levels, such that there were few differences from intact control animals, an effect not observed in standard tests of motor function.

Key words: Skilled reaching; Transplantation; Whole ganglionic eminence (wGE); Spontaneous behavior; Fluoro-Gold (FG); Adjusting step test; Vibrissae-evoked hand-placing test

Received January 28, 2012; final acceptance August 31, 2012. Online prepub date: October 12, 2012.
Address correspondence to Stephen B. Dunnett, Brain Repair Group, School of Biosciences, Cardiff University, Museum Avenue, Cardiff CF10 3AX, UK. Tel: +44 029 20875188; Fax: +44 029 20876749; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 22, pp. 1753-1766, 2013
0963-6897/13 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X658764
E-ISSN 1555-3892
Copyright © 2013 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Improved Method of Producing Human Neural Progenitor Cells of High Purity and in Large Quantities From Pluripotent Stem Cells for Transplantation Studies

Katja A. Puttonen,*1 Marika Ruponen,*†1 Riitta Kauppinen,* Sara Wojciechowski,* Outi Hovatta,‡§ and Jari Koistinaho*¶

*Department of Neurobiology, A. I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland
†School of Pharmacy, University of Eastern Finland, Kuopio, Finland
‡Department of Biotechnology and Molecular Medicine, A. I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland
§Department of Clinical Science, Intervention and Technology, Karolinska Institute, Stockholm, Sweden
¶Department of Oncology, Kuopio University Hospital, Kuopio, Finland

Transplantation of human neural progenitor cells (hNPCs) is a promising therapeutic approach for various diseases of the central nervous system (CNS). Reliable testing of hNPC transplantation in animal models of neurological diseases requires that these cells can be produced in sufficient amounts, show consistent homogeneity as a neural cell population, and be reliably labeled for in vivo tracking. In addition, the cells should be characterized as being at the optimal state of differentiation favoring successful engraftment. Here, we show that high numbers of purified hNPCs can be produced from human embryonic stem cells (hESCs) by manually selecting specifically sized and shaped spheres followed by fluorescence-activated cell sorting based on the relative cell size. In addition, we report that labeling of hNPCs with ultra-small superparamagnetic iron oxide (USPIO) particles does not affect the cellular morphology or growth. More importantly, we show that the transduction with lentiviral vector encoding green fluorescent protein (GFP) decreases the neurality of the cell population. We conclude that our cost-effective protocol of generating hNPCs is widely applicable for preclinical studies on CNS disorders. This improved method of producing large quantities of high-purity hNPCs maybe useful also when generating hNPCs from human induced pluripotent stem (hiPS) cell lines. However, caution should be used when lenti-GFP transduction is applied for hNPC labeling.

Key words: Human embryonic stem cells (hESCs); Neural progenitors; Neural differentiation; Cell transplantation

Received March 6, 2012; final acceptance September 9, 2012. Online prepub date: October 31, 2012.
1These authors provided equal contribution to this work.
Address correspondence to Jari Koistinaho, Department of Neurobiology, A. I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, P.O. Box 1627, FI-70211 Kuopio, Finland. Tel: +358-403552427; Fax: +358-17163030; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 22, pp. 1767-1783, 2013
0963-6897/13 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X657710
E-ISSN 1555-3892
Copyright © 2013 Cognizant Comm. Corp.
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Transplantation of Neural Crest-Like Cells Derived From Induced Pluripotent Stem Cells Improves Diabetic Polyneuropathy in Mice

Tetsuji Okawa,*† Hideki Kamiya,‡1 Tatsuhito Himeno,*† Jiro Kato,* Yusuke Seino,§ Atsushi Fujiya,*§ Masaki Kondo,† Shin Tsunekawa,* Keiko Naruse,¶ Yoji Hamada,§ Nobuaki Ozaki,# Zhao Cheng,† Tetsutaro Kito,† Hirohiko Suzuki,† Sachiko Ito,† Yutaka Oiso,* Jiro Nakamura,*1 and Ken-Ichi Isobe†

*Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan
†Department of Immunology, Nagoya University Graduate School of Medicine, Nagoya, Japan
‡Department of Chronic Kidney Disease Initiatives, Nagoya University Graduate School of Medicine, Nagoya, Japan
§Department of Metabolic Medicine, Nagoya University School of Medicine, Nagoya, Japan
¶Department of Internal Medicine, School of Dentistry, Aichi Gakuin University, Nagoya, Japan
#Research Center of Health, Physical Fitness and Sports, Nagoya University, Nagoya, Japan

Impaired vascularity and nerve degeneration are the most important pathophysiological abnormalities of diabetic polyneuropathy (DPN). Therefore, regeneration of both the vascular and nervous systems is required for the treatment of DPN. The neural crest (NC) is a transient embryonic structure in vertebrates that differentiates into a vast range of cells, including peripheral neurons, Schwann cells, and vascular smooth muscle cells. In this study, we investigated the ability of transplantation of NC-like (NCL) cells derived from aged mouse induced pluripotent stem (iPS) cells in the treatment of DPN. iPS cells were induced to differentiate into neural cells by stromal cell-derived inducing activity (SDIA) and subsequently supplemented with bone morphogenetic protein 4 to promote differentiation of NC lineage. After the induction, p75 neurotrophin receptor-positive NCL cells were purified using magnetic-activated cell sorting. Sorted NCL cells differentiated to peripheral neurons, glial cells, and smooth muscle cells by additional SDIA. NCL cells were transplanted into hind limb skeletal muscles of 16-week streptozotocin-diabetic mice. Nerve conduction velocity, current perception threshold, intraepidermal nerve fiber density, sensitivity to thermal stimuli, sciatic nerve blood flow, plantar skin blood flow, and capillary number-to-muscle fiber ratio were evaluated. Four weeks after transplantation, the engrafted cells produced growth factors: nerve growth factor, neurotrophin 3, vascular endothelial growth factor, and basic fibroblast growth factor. It was also confirmed that some engrafted cells differentiated into vascular smooth muscle cells or Schwann cell-like cells at each intrinsic site. The transplantation improved the impaired nerve and vascular functions. These results suggest that transplantation of NCL cells derived from iPS cells could have therapeutic effects on DPN through paracrine actions of growth factors and differentiation into Schwann cell-like cells and vascular smooth muscle cells.

Key words: Neural crest (NC); Induced pluripotent stem (iPS) cells; Diabetic polyneuropathy (DPN); Aging; Regenerative medicine

Received April 18, 2012; final acceptance September 20, 2012. Online prepub date: October 8, 2012.
1Current affiliation: Division of Diabetes, Department of Internal Medicie, Aichi Medical University School of Medicine, 21 Karimata, Yazako, Nagakute, Aichi 480-1195, Japan.
Address correspondence to Hideki Kamiya, 21 Karimata, Yazako, Nagakute, Aichi 480-1195, Japan. Tel: +81-561-63-1683; Fax: +81-561-63-1276; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 22, pp. 1785-1799, 2013
0963-6897/13 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X662462
E-ISSN 1555-3892
Copyright © 2013 Cognizant Comm. Corp.
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Therapeutic Potential of Human Induced Pluripotent Stem Cell-Derived Mesenchymal Stem Cells in Mice With Lethal Fulminant Hepatic Failure

Mohsen Moslem,* Mojtaba Rezazadeh Valojerdi,* Behshad Pournasr,† Ahad Muhammadnejad,‡ and Hossein Baharvand†§

*Department of Anatomical Sciences, Tarbiat Modares University, Tehran, Iran
†Department of Stem Cells and Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
‡Cancer Research Center, Iran Cancer Institute, Tehran University of Medical Sciences, Tehran, Iran
§Department of Developmental Biology, University of Science and Culture, ACECR, Tehran, Iran

Large-scale production and noninvasive methods for harvesting mesenchymal stem cells (MSCs), particularly in elderly individuals, has prompted researchers to find new patient-specific sources for MSCs in regenerative medicine. This study aims to produce MSCs from human induced pluripotent stem cells (hiPSCs) and to evaluate their therapeutic effects in a CCl4-induced mouse model of fulminant hepatic failure (FHF). hiPSC-MSCs have shown MSC morphology, antigen profile and differentiation capabilities, and improved hepatic function in our model. hiPSC-MSC-transplanted animals provide significant benefit in terms of survival, serum LDH, total bilirubin, and lipid peroxidation. hiPSC-MSC therapy resulted in a one-third reduction of histologic activity index and a threefold increase in the number of proliferating hepatocytes. This was accompanied by a significant decrease in the expression levels of collagen type I, Mmp13, Mmp2, and Mmp9 genes and increase in Timp1 and Timp2 genes in transplanted groups. hiPSC-MSCs secreted hepatocyte growth factor (HGF) in vitro and also expressed HGF in evaluated liver sections. Similar results were observed with human bone marrow (hBM)-derived MSCs. In conclusion, our results have demonstrated that hiPSC-MSCs might be valuable appropriate alternatives for hBM-MSCs in FHF liver repair and support liver function by cell therapy with a large-scale production capacity, patient-specific nature, and no invasive MSC harvesting.

Key words: Human induced pluripotent stem cells (hiPSCs); Mesenchymal stem cells (MSCs); Fulminant hepatic failure (FHF)

Received November 18, 2011; final acceptance August 24, 2012. Online prepub date: February 5, 2013.
Address correspondence to Hossein Baharvand, Department of Stem Cells and Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, P.O. Box 19395-4644, Tehran, Iran. Tel: +98-21-22306485; Fax: +98-21-22310406; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it or Mojtaba Rezazadeh Valojerdi, Department of Anatomy, Faculty of Medical Sciences, Tarbiat Modares University, P.O. Box 14115-111, Tehran, Iran. E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 22, pp. 1801-1811, 2013
0963-6897/13 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X657594
E-ISSN 1555-3892
Copyright © 2013 Cognizant Comm. Corp.
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Human Liver Endothelial Cells, But Not Macrovascular or Microvascular Endothelial Cells, Engraft in the Mouse Liver

Ebtisam El Filali,* Johan K. Hiralall,* Henk A. van Veen,† Donna B. Stolz,‡ and Jurgen Seppen*

*Tytgat Institute for Liver and Intestinal Research, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
†van Leeuwenhoek Center for Advanced Microscopy, Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
‡Department of Cell Biology and Physiology, Center for Biologic Imaging, University of Pittsburgh, Pittsburgh, Pennsylvania, PA, USA

Liver cell transplantation has had limited clinical success so far, partly due to poor engraftment of hepatocytes. Instead of hepatocytes. other cell types, such as endothelial cells, could be used in ex vivo liver gene therapy. The goal of the present study was to compare the grafting and repopulation capacity of human endothelial cells derived from various tissues. Human endothelial cells were isolated from adult and fetal livers using anti-human CD31 antibody-conjugated magnetic beads. Human macrovascular endothelial cells were obtained from umbilical vein. Human microvascular endothelial cells were isolated from adipose tissue. Cells were characterized using flow cytometry. Liver engraftment and repopulation of endothelial cells was studied after intrasplenic transplantation in monocrotaline-treated immunodeficient mice. Following transplantation, human liver endothelial cells engrafted throughout the mouse liver. With immunoscanning electron microscopy, fenestrae in engrafted human liver endothelial cells were identified, a characteristic feature of liver sinusoidal endothelial cells. In contrast, CD31-negative liver cells, human macrovascular and microvascular endothelial cells were not capable of repopulating mouse liver. Characterization of human liver, macrovascular, and microvascular endothelial cells demonstrated expression of CD31, CD34, and CD146 but not CD45. Our study shows that only human liver endothelial cells, but not macro- and microvascular endothelial cells, have the unique capacity to engraft and repopulate the mouse liver. These results indicate that mature endothelial cells cannot transdifferentiate in vivo and thus do not exhibit phenotypic plasticity. Our results have set a basis for further research to the potential of human liver endothelial cells in liver-directed cell and gene therapy.

Key words: Human fetal liver; Cell transplantation; Repopulation; Endothelium; Ex vivo gene therapy

Received October 12, 2011; final acceptance July 10, 2012. Online prepub date: October 3, 2012.
Address correspondence to Ebtisam El Filali, Tytgat Institute for Liver and Intestinal Research, Academic Medical Center, S1-172, Meibergdreef 69-71, 1105 BK Amsterdam, The Netherlands. Tel: +31-20-5668854; Fax: +31-20-5669190; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 22, pp. 1813-1829, 2013
0963-6897/13 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X657774
E-ISSN 1555-3892
Copyright © 2013 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Magnetic Resonance Imaging of Human Dental Pulp Stem Cells In Vitro and In Vivo

T. Struys,*† A. Ketkar-Atre,* P. Gervois,† C. Leten,* P. Hilkens,† W. Martens,† A. Bronckaers,† T. Dresselaers,* C. Politis,‡ I. Lambrichts,† and U. Himmelreich*

*Biomedical MRI Unit-MoSAIC, Department of Imaging and Pathology, KU Leuven, Leuven, Belgium
†Biomedical Research Institute, Lab of Histology, Hasselt University, Diepenbeek, Belgium
‡Ziekenhuis Oost-Limburg (ZOL), Genk, Belgium

Recent advances in stem cell research have shown the promising nature of mesenchymal stem cells as plausible candidates for cell-based regenerative medicine. Many studies reported the use of human dental pulp stem cells (hDPSCs), which possess self-renewal capacity, high proliferation potential, and the ability to undergo multilineage differentiation. Together with this therapeutic approach, development of effective, noninvasive and nontoxic imaging techniques for visualizing and tracking the cells in vivo is crucial for the evaluation and improvement of stem cell therapy. Magnetic resonance imaging (MRI) is one of the most powerful diagnostic imaging techniques currently available for in vivo diagnosis and has been proposed as the most attractive modality for monitoring stem cell migration. The aim of this study was to investigate the labeling efficiency of hDPSCs using superparamagnetic iron oxide (SPIO) particles in order to allow visualization using in vitro and in vivo MRI without influencing cellular metabolism. MRI and transmission electron microscopy (TEM) showed optimal uptake with low SPIO concentrations of 15 μg/ml in combination with 0.75 μg/ml poly-L-lysine (PLL) resulting in more than 13 pg iron/cell and an in vitro detection limit of 50 labeled cells/μl. Very low SPIO concentrations in the culture medium resulted in extremely high labeling efficiency not reported before. For these conditions, tetrazolium salt assays showed no adverse effects on cell viability. Furthermore, in vivo MRI was performed to detect labeled hDPSCs transplanted into the brain of Rag 2-g C immune-deficient mice. Transplanted cells did not show any signs of tumorgenecity or teratoma formation during the studied time course. We have reported on a labeling and imaging strategy to visualize human dental pulp stem cells in vivo using MRI. These data provide a solid base to allow cell tracking in future regenerative studies in the brain longitudinally.

Key words: Mesenchymal stem cells (MSCs); Dental pulp; Magnetic resonance imaging (MRI); Transmission electron microscopy (TEM); Cell imaging

Received September 12, 2011; final acceptance August 22, 2012. Online prepub date: October 8, 2012.
Address correspondence to Dr. Tom Struys, M.Sc., Ph.D., Biomedical NMR Unit/MoSAIC, Department of Medical Diagnostic Sciences, Division of Radiology, Katholieke Universiteit Leuven, O&N I, Herestraat 49, bus 00505, 3000 Leuven, Belgium. Tel: +3216 33 09 23; Fax: +3216 33 09 01; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 22, pp. 1831-1844, 2013
0963-6897/13 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X657945
E-ISSN 1555-3892
Copyright © 2013 Cognizant Comm. Corp.
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Accelerated Expansion of Epidermal Keratinocyte and Improved Dermal Reconstruction Achieved by Engineered Amniotic Membrane

Guofeng Huang,* Shizhao Ji,*1 Pengfei Luo,*1 Houqi Liu,† Shihui Zhu,* Guangyi Wang,* Panyu Zhou,* Shichu Xiao,* and Zhaofan Xia*

*Burns Institute of People’s Liberation Army, Affiliated Changhai Hospital of the Second Military Medical University, Shanghai, China
†Department of Histology and Embryology, College of Basic Medical Science, the Second Military Medical University, Shanghai, China

In this study, we used human amniotic membrane (AM) to prepare a dermal scaffold with intact basement membrane (BM) and good biostability for quick expansion and transplantation of epidermal keratinocytes (EKs). Fresh AM was treated by repeated freeze–thaw cycles and DNase digestion. This new method was able to cleanse the cell components effectively and retain the BM structure with continuous distributions of laminin, collagen IV, VI, and VII. Subsequently, the acellular amniotic membrane (AAM) was cross-linked with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) for 5 min, 30 min, and 6 h. With the time of cross-linking prolonging, the mechanical strength and biostability of AAM increased gradually, while its cytotoxicity to EKs also increased. The 5-min cross-linked AAM (5min-AAM) had no significant cytotoxicity with good histocompatibility. The relative cell viability of EKs seeded on the 5min-AAM surface was 367 ± 33% and 631 ± 43% at 7 and 14 days of culture, respectively, both higher than 294 ± 30% and 503 ± 41% of the conventional cell culture dish (CCD) group, and the proportion of P63-positive cells was significantly higher than that of the CCD group on day 7 (54.32 ± 4.27% vs. 33.32 ± 3.18%, p < 0.05). When the 5min-AAM loaded with EKs (EK-AAM) was grafted onto full-thickness skin defects in nude mice, the cells survived well and formed an epidermis similar to normal skin. The new epidermis was thicker, and reconstruction of the dermal structure was good with an intact BM. Four weeks after transplantation, the wound contraction rate in the EK-AAM group was 43.09 ± 7.05%, significantly lower than that in the EK sheet group (57.49 ± 5.93%) and control group (69.94 ± 9.47%) (p < 0.05). In conclusion, repeated freeze–thaw treatment with appropriate EDC cross-linking offers AAM an intact BM structure with good operability and biostability. It may prove to be an ideal dermal scaffold to promote expansion of EKs in vitro and be transplanted for reconstruction of the dermal structure.

Key words: Amniotic membrane (AM); Epidermal keratinocytes (EKs); Freeze–thaw; 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) cross-linking; Skin substitutes

Received January 8, 2012; final acceptance September 5, 2012. Online prepub date: October 12, 2012.
1These authors provided equal contribution to this work.
Address correspondence to Zhaofan Xia, M.D., Ph.D., Burns Institute of People’s Liberation Army, Affiliated Changhai Hospital of the Second Military Medical University, Shanghai 200433, China. Tel: +86-21-81873471; Fax: +86-21-65589829; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it or Shichu Xiao, M.D., Burns Institute of People’s Liberation Army, Affiliated Changhai Hospital of the Second Military Medical University, Shanghai 200433, China. Tel: +86-21-81873474; Fax: +86-21-65589829; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 22, pp. 1845-1858, 2013
0963-6897/13 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X657846
E-ISSN 1555-3892
Copyright © 2013 Cognizant Comm. Corp.
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Effects of Vitamin C on Cytotherapy-Mediated Muscle Regeneration

Ah-Young Kim,*† Eun-Mi Lee,*† Eun-Joo Lee,*† Chang-Woo Min,*† Kyung-Ku Kang,*† Jin-Kyu Park,* Il-Hwa Hong,* Akihito Ishigami,‡ Jacques P. Tremblay,§ and Kyu-Shik Jeong*†

*Department of Pathology, College of Veterinary Medicine, Kyungpook National University, Daegu, Republic of Korea
†Stem Cell Therapeutic Research Institute, Kyungpook National University, Daegu, Republic of Korea
‡Molecular Regulation of Aging, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan
§Department of Neurosciences, CHUL Research Center, Quebec City, QC, Canada

Skeletal muscles are the largest organs in the human body, and several therapeutic trials have been conducted that included stem cell transplantation to regenerate damaged or wasted muscles. It is well known that it is essential to make a favorable microenvironment (stem cell niche) to induce the proper differentiation of the transplanted stem cells. Some drugs, such as losartan (angiotensin II type I blocker), enhance the therapeutic effects of transplanted stem cells by inhibiting fibrosis. In this study, we hypothesized that another substance, vitamin C (ascorbic acid), might improve the niche for stem cell transplantation based on its potent antioxidant effects. In both gross and microscopic observations, vitamin C-depleted mice exhibited more incomplete regeneration of damaged muscles than those treated with vitamin C. Carbonylated protein groups, which are the end products of oxidative stress, were detected in all experimental groups; however, the vitamin C-depleted groups exhibited a more potent positive reaction than that of the vitamin C-supplied groups. The difference is clearer in the presence of transplanted stem cells. Moreover, the serum total vitamin C level and the ascorbic acid (AA) to dehydroascorbic acid (DHA) ratio also were decreased in the presence of transplanted adiposederived stem cells (ASCs). Taken together, these data can be considered as proof of vitamin C utilization by cells in vivo. The vitamin C-supplied groups displayed more severe fibrosis than that of the vitamin C-depleted groups. Since vitamin C is a major cofactor for the collagen synthesis, its deficiency resulted in reduced fibrosis. In conclusion, we demonstrated that vitamin C not only has a positive effect on adjusting the stem cell niche to boost muscle regeneration but also has an adverse aspect due to its profibrotic effect.

Key words: Adipose-derived stem cells (ASCs); Antioxidant; Muscle laceration; Stem cell niche; Vitamin C

Received January 20, 2012; final acceptance September 18, 2012. Online prepub date: October 8, 2012.
Address correspondence to Kyu-Shik Jeong, D.V.M., Ph.D., Department of Pathology, College of Veterinary Medicine, Kyungpook National University, Daegu 702-701, Republic of Korea. Tel: +82-53-950-5975; Fax: +82-52-950-5955; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 22, pp. 1859-1868, 2013
0963-6897/13 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X657206
E-ISSN 1555-3892
Copyright © 2013 Cognizant Comm. Corp.
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ABCG2 Protects Kidney Side Population Cells From Hypoxia/Reoxygenation Injury Through Activation of the MEK/ERK Pathway

Wei-Hui Liu,*†‡1 Hong-Bao Liu,*1 Da-Kuan Gao,§1 Guan-Qun Ge,¶1 Peng Zhang,* Shi-Ren Sun,* Han-Min Wang,* and Shui-Bing Liu#

*Department of Nephrology, Xijing Hospital, Fourth Military Medical University, Xi’an, China
†PLA Center of General Surgery, General Hospital of Chengdu Army Region, Chengdu, China
‡Department of Hepatobiliary Surgery, Xijing Hospital, Fourth Military Medical University, Xi’an, China
§Department of Neurosurgery, Xijing Hospital, Fourth Military Medical University, Xi’an, China
¶Department of Surgical Oncology, Comprehensive Cancer Center, The First Affiliated Hospital, Medical College, Xi’an Jiaotong University, Xi’an, China
#Department of Pharmacology, School of Pharmacy, Fourth Military Medical University, Xi’an, China

Breast cancer resistance protein 1 (BCRP1/ABCG2) is used to identify the side population (SP) within a population of cells, which is enriched for stem and progenitor cells in different tissues. Here, we investigated the role of extracellular signal-regulated kinase (ERK) 1/2 in the signaling mechanisms underlying ischemic/hypoxic conditions in kidney SP cells. Kidney SP cells were isolated using Hoechst 33342 dye-mediated fluorescein-activated cell sorting and then incubated under hypoxia/reoxygenation (H/R) with or without verapamil, a selective BCRP1/ABCG2 inhibitor. ABCG2 expression, ERK activity, cell viability, metabolic activity, and membrane damage were tested after H/R treatment. To evaluate the role of ERK 1/2 on the expression and function of ABCG2, the expression of mitogen-activated protein kinase (MAPK)/ERK kinase (MEK), which preferentially activates ERK, was upregulated by transfection with the recombinant sense expression vector pcDNA3.1-MEK and downregulated by pretreatment with U0126, a specific MEK inhibitor. We found that hypoxia activated ERK activity in the kidney SP cells but not in non-SP cells both in vitro and in vivo. Overexpression of MEK mimicked hypoxia-induced ABCG2 expression. Contrarily, U0126 inhibited hypoxia- and MEK-upregulated ABCG2 expression. Furthermore, H/R induced significant increases in nuclear, metabolic, and membrane damage in both SP cells and non-SP cells; however, this H/R-induced cytotoxicity was much more severe in non-SP cells than in SP cells. Notably, the viability of kidney SP cells was enhanced by MEK overexpression and inhibited by U0126. Verapamil treatment reversed MEK-induced viability of kidney SP cells. When administered systemically into animals with renal ischemia/reperfusion injury, the SP cells significantly improved renal function, accelerated mitogenic response, and reduced cell apoptosis. However, this improved therapeutic potential of SP cells was significantly reduced by pretreatment with verapamil. Collectively, these findings provide evidence for a crucial role for the MEK/ERK-ABCG2 pathway in protecting kidney SP cells from ischemic/hypoxic injury.

Key words: Stem cells; Kidney; Extracellular signal-regulated kinase (ERK); Side population; Breast cancer resistance protein (BCRP1/ABCG2)

Received December 12, 2011; final acceptance April 28, 2012. Online prepub date: October 2, 2012.
1These authors provided equal contribution to this work.
Address correspondence to Dr. Hong-Bao Liu, Department of Nephrology, Xijing Hospital, Fourth Military Medical University, Xi’an, 710032, China. Tel: +86-29-84773494; Fax: +86-29-84773494; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it or Dr. Shui-Bing Liu, Department of Pharmacology, School of Pharmacy, Fourth Military, Medical University, Xi’an, 710032, China. E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 22, pp. 1869-1882, 2013
0963-6897/13 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X657954
E-ISSN 1555-3892
Copyright © 2013 Cognizant Comm. Corp.
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Hypoxia-Induced Secretion of TGF-b1 in Mesenchymal Stem Cell Promotes Breast Cancer Cell Progression

Shun-Pei Hung,*† Muh-Hwa Yang,* Kuo-Fung Tseng,‡ and Oscar K. Lee*†§

*Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan
†Stem Cell Research Center, National Yang-Ming University, Taipei, Taiwan
‡Department of Orthopaedics, Cheng Hsin General Hospital, Taipei, Taiwan
§Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei, Taiwan

In solid tumors, a decreased oxygen and nutrient supply creates a hypoxic microenvironment in the central region. This hypoxic condition induces molecular responses of normal and cancer cells in the local area, including angiogenesis, metabolic changes, and metastasis. In addition, other cells including mesenchymal stem cells (MSCs) have been reported to be recruited into the hypoxic area of solid tumors. In our previous study, we found that hypoxic condition induces the secretion of growth factors and cytokines in MSCs, and here we demonstrate that elevated secretion of transforming growth factor-β1 (TGF-β1) by MSCs under hypoxia promotes the growth, motility, and invasive ability of breast cancer cells. It was found that TGF-β1 promoter activity was regulated by hypoxia, and the major hypoxia-regulated element was located between bp –1030 to –666 in front of the TGF-b1 promoter region. In ChIP assay, the results revealed that HIF-1 was bound to the hypoxia response element (HRE) of TGF-β1 promoter. Collectively, the results indicate that hypoxia microenvironment can enhance cancer cell growth through the paracrine effects of the MSCs by driving their TGF-β1 gene expression and secretion. Therefore, extra caution has to be exercised when considering hypoxia pretreatment of MSCs before cell transplantation into patients for therapeutic purposes, particularly in patients susceptible to tumor growth.

Key words: Mesenchymal stem cells (MSCs); Hypoxia; Breast cancer; Transforming growth factor-β1 (TGF-β1)

Received December 13, 2011; final acceptance September 4, 2012. Online prepub date: October 12, 2012.
Address correspondence to Oscar K. Lee, M.D., Ph.D., Department of Orthopaedics and Traumatology, Taipei Veterans General Hospital, 201, Sec. 2, Shipai Road, Beitou District, Taipei City, Taiwan 11217, Republic of China. Tel: +886-2-28757557; Fax: +886-2-2875-7657; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it or Kuo-Fung Tseng, M.D., Ph.D., Department of Orthopaedics, Cheng Hsin General Hospital, No. 45, Cheng Hsin Street, Beitou District, Taipei City, Taiwan 11217, Republic of China. Tel: +886-2-2826-4400; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 22, pp. 1883-1900, 2013
0963-6897/13 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X662444
E-ISSN 1555-3892
Copyright © 2013 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Common Expression of Stemness Molecular Markers and Early Cardiac Transcription Factors in Human Wharton’s Jelly-Derived Mesenchymal Stem Cells and Embryonic Stem Cells

Lian Ru Gao,* Ning K. Zhang,* Qing A. Ding,* Hai Y. Chen,* Xiang Hu,† Shu Jiang,† Tian C. Li,* Yu Chen,* Zhi G. Wang,* Yang Ye,* and Zhi Ming Zhu*

*Center of Cardiology, Navy General Hospital, Beijing, China
†Beike Cell Engineering Research Institute, Shenzhen, China

At present, there are still significant barriers that impede the clinical use of hESCs and iPS cells, including ethics, immunorejection, tumorigenesis from hESCs, and teratoma formation from iPS cells. It is therefore necessary to search for alternative sources of stem cells. WJ-MSCs originate from embryonic epiblasts and possess properties intermediate between hESCs and adult stem cells. However, the stemness properties of molecules in WJ-MSCs remain unclear compared to those of hESCs. In the present study, we isolated WJ-MSCs by a nonenzymatic method. Further, using microarray analysis by Affymetrix GeneChip and functional network analyses, we determined the degree of expression of stemness genes exhibited by the Human Stem Cell Pluripotency array. We also defined a wide range of stem cell gene expression in the WJ-MSCs in comparison with hESCs. At the same time, the definitive markers of early cardiac precursor cells and more committed progenitors were further characterized in WJ-MSCs. Our results demonstrated for the first time that WJ-MSCs had significant expression of undifferentiated human embryonic stem cell core markers, such as SOX2, NANOG, LIN28, SSEA1, SSEA3, SSEA4, KLF4, c-MYC, CRIPTO, and REX1, with a relatively lower level of expression than in hESCs. We also found WJ-MSCs have high expression of early cardiac transcription factors, such as Flk-1, Isl-1, and Nkx2.5. Functional analysis revealed signature genes of WJ-MSCs with specific roles involved in immune, cytoskeletal, and chemokine regulation, cell adhesion, and cell signaling. Our study indicated that there is a significant overlap between the stemness genes expressed by hESCs and WJ-MSCs. WJ-MSCs harbor a true stem cell population and are promising cells for stem cell-based therapies.

Key words: Stemness genes; Early cardiac transcription factors; Wharton’s jelly-derived mesenchymal stem cells (WJ-MSCs); Human embryonic stem cells (hESCs)

Received November 11, 2011; final acceptance September 10, 2012. Online prepub date: February 4, 2013.
Address correspondence to Dr. Lianru Gao, The Center of Cardiology, Navy General Hospital, 6 Fucheng Road, Beijing 100048, China. Tel: 011-86-10-88180197; Fax: 011-86-10-68780127; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 22, pp. 1901-1914, 2013
0963-6897/13 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X657837
E-ISSN 1555-3892
Copyright © 2013 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Development of Experimental Tumors Formed by Mouse and Human Embryonic Stem and Teratocarcinoma Cells After Subcutaneous and Intraperitoneal Transplantations Into Immunodeficient and Immunocompetent Mice

O. F. Gordeeva and T. M. Nikonova

Kol’tsov Institute of Developmental Biology, Russian Academy of Sciences, Moscow, Russia

Pluripotent stem cells represent an attractive cell source for regenerative medicine. However, the risk of teratoma formation after transplantation restricts their clinical application. Therefore, to adequately evaluate the potential risk of tumorigenicity after cell transplantation into human tissues, effective animal transplantation assays need to be developed. We performed a multiparameter (cell number, transplantation site, cell type, host) comparative analysis of the efficiency of tumor development after transplantation of mouse and human embryonic stem (ES) cells and their malignant counterparts, teratocarcinoma (EC) cells, into animal recipients and revealed several key correlations. We found that the efficiency of tumor growth was higher after intraperitoneal than after subcutaneous transplantations of all cell lines studied. The minimal cell numbers sufficient for tumor growth in immunodeficient nude mice were 100-fold lower for intraperitoneal than for subcutaneous transplantations of mouse and human ES cells (103 vs. 105 and 104 vs. 106, respectively). Moreover, mouse ES and EC cells formed tumors in immunodeficient and immunocompetent mice more effectively than human ES and EC cells. After intraperitoneal transplantation of 103, 104, and 105 mouse ES cells, teratomas developed in 83%, 100%, and 100% of nude mice, whereas after human ES cell transplantation, teratomas developed in 0%, 17%, and 60%, respectively. In addition, malignant mouse and human EC cells initiated tumor growth after intraperitoneal transplantation significantly faster and more effectively than ES cells. Mouse and human ES cells formed different types of teratomas containing derivatives of three germ layers but different numbers of undifferentiated cells. ES cell-like sublines with differentiation potential similar to the parental cell line were recloned only from mouse, but not from human, ES cell teratomas. These findings provide new information about the possibility and efficiency of tumor growth after transplantation of pluripotent stem cells. This information allows one to predict and possibly prevent the possible risks of tumorigenicity that could arise from stem cell therapeutics.

Key words: Pluripotent stem cells; Teratoma; Teratocarcinoma; Transplantation; Oct4; Nude mice

Received April 21, 2012; final acceptance September 3, 2012. Online prepub date: October 8, 2012.
Address correspondence to O. F. Gordeeva, Ph.D., Laboratory of Histogenesis, Kol’tsov Institute of Developmental Biology, Russian Academy of Sciences, 26 Vavilov Street, Moscow 119334, Russia. Tel: +7-499-135-8780; Fax: +7-499-135-1280; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 22, pp. 1915-1928, 2013
0963-6897/13 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X657369
E-ISSN 1555-3892
Copyright © 2013 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Human Multipotent Adult Progenitor Cells Are Nonimmunogenic and Exert Potent Immunomodulatory Effects on Alloreactive T-Cell Responses

Sandra A. Jacobs,*† Jef Pinxteren,‡ Valerie D. Roobrouck,§ Ariane Luyckx,¶ Wouter van’t Hof,# Robert Deans,# Catherine M. Verfaillie,§ Mark Waer,¶ An D. Billiau,¶ and Stefaan W. Van Gool*†

*Department of Experimental Medicine, Laboratory of Experimental Immunology, Catholic University of Leuven, Leuven, Belgium
†Department of Child and Woman, University Hospitals Leuven, Catholic University of Leuven, Leuven, Belgium
‡ReGenesys, Heverlee, Belgium
§Stem Cell Institute Leuven, Catholic University of Leuven, Leuven, Belgium
¶Department of Experimental Medicine, Laboratory of Experimental Transplantation, Catholic University of Leuven, Leuven, Belgium
#Athersys, Inc., Regenerative Medicine Program, Cleveland, OH, USA

Multipotent adult progenitor cells (MAPCs) are bone marrow-derived nonhematopoietic stem cells with a broad differentiation potential and extensive expansion capacity. A comparative study between human mesenchymal stem cells (hMSCs) and human MAPCs (hMAPCs) has shown that hMAPCs have clearly distinct phenotypical and functional characteristics from hMSCs. In particular, hMAPCs express lower levels of MHC class I than hMSCs and cannot only differentiate into typical mesenchymal cell types but can also differentiate in vitro and in vivo into functional endothelial cells. The use of hMSCs as cellular immunomodulatory stem cell products gained much interest since their immunomodulatory capacities in vitro became evident over the last decade. Currently, the clinical grade stem cell product of hMAPCs is already used in clinical trials to prevent graft-versus-host disease (GVHD), as well as for the treatment of acute myocardial infarct, ischemic stroke, and Crohn’s disease. Therefore, we studied the immune phenotype, immunogenicity, and immunosuppressive effect of hMAPCs in vitro. We demonstrated that hMAPCs are nonimmunogenic for T-cell proliferation and cytokine production. In addition, hMAPCs exert strong immunosuppressive effects on T-cell alloreactivity and on T-cell proliferation induced by mitogens and recall antigens. This immunomodulatory effect was not MHC restricted, which makes off-the-shelf use promising. The immunosuppressive effect of hMAPCs is partially mediated via soluble factors and dependent on indoleamine 2,3-dioxygenase (IDO) activity. At last, we isolated hMAPCs, the clinical grade stem cell product of hMAPCs, named MultiStem, and hMSCs from one single donor and observed that both the immunogenicity and the immunosuppressive capacities of all three stem cell products are comparable in vitro. In conclusion, hMAPCs have potent immunomodulatory properties in vitro and can serve as a valuable cell source for the clinical use of immunomodulatory cellular stem cell product.


Key words: Human multipotent adult progenitor cells (hMAPCs); Allogeneic T-cells; Immunomodulation; Cellular stem cell therapy

Received July 27, 2011; final acceptance July 24, 2012. Online prepub date: October 1, 2012.
Address correspondence to Sandra A. Jacobs, Laboratory of Experimental Immunology, O&N1 Box 811, Herestraat 49, 3000 Leuven, Belgium. Tel: +3216332211; Fax: +3216346035; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 22, pp. 1929-1941, 2013
0963-6897/13 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X658025
E-ISSN 1555-3892
Copyright © 2013 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Selective Depletion of Alloreactive T Cells Leads to Long-Term Islet Allograft Survival Across a Major Histocompatibility Complex Mismatch in Diabetic Mice

M. Hu,*† J. Wu,† G. Y. Zhang,* Y. M. Wang,* D. Watson,*‡ S. Yi,† W. J. Hawthorne,† P. J. O’Connell,† and S. I. Alexander*

*Centre for Kidney Research, the Children’s Hospital at Westmead, the University of Sydney, NSW, Australia
†Centre for Transplant and Renal Research, Westmead Millennium Institute, the University of Sydney, NSW, Australia
‡Centre for Medical Bioscience, University of Wollongong, NSW, Australia

Islet cell transplantation as a therapy for type 1 diabetes has been limited by progressive graft loss. Significant immunosuppression including T-cell ablation has been used in an attempt to limit islet rejection. Here, we show that CD3+ lymphocytes depleted of alloreactive T cells selected from a mixed lymphocyte reaction (MLR), where responder BALB/c splenocytes stained with carboxyfluorescein succinimidyl ester (CFSE) were stimulated with irradiated C57BL/6 splenocytes for 5 days, infused into diabetic immunodeficient mice are capable of restoring a broad T-cell repertoire and specifically do not reject islet transplants from the strain (C57BL/6) used in the original depletion. These mice demonstrate reconstitution with CD4+ and CD8+ T cells, the capacity to reject third-party grafts (CBA), and restoration of interferon-γ (IFN-γ) responses to third-party alloantigens. Over time, both forkhead box P3-positive (Foxp3+) T regulatory cells (Tregs) and γδ T cells expand, suggesting a role for peripheral tolerance, in addition to the initial depletion of alloreactive T cells, in long-term islet graft survival. Our results suggest that immune restoration with CD3+ lymphocytes where alloreactive T cells are removed can restore cognate immunity without islet allograft loss and recurrence of diabetes.

Key words: Islet cell transplantation; Long-term islet allograft survival; Depletion of alloreactive CD3+ T cells; T-cell repertoire; Type 1 diabetes

Received January 17, 2012; final acceptance September 5, 2012. Online prepub date: October 25, 2012.
Address correspondence to A/Prof. Stephen I. Alexander, Centre for Kidney Research, The Children’s Hospital at Westmead, University of Sydney, Westmead, NSW 2145, Australia. Tel: +612-9845-3408; Fax: +612-9845-3432; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 22, pp. 1943-1954, 2013
0963-6897/13 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X657963
E-ISSN 1555-3892
Copyright © 2013 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Development of Antibody-Tagged Nanoparticles for Detection of Transplant Rejection Using Biomagnetic Sensors

Kimberly S. Butler,* Debbie M. Lovato,* Natalie L. Adolphi,† Robert Belfon,‡ Danielle L. Fegan,§ Todd C. Monson,¶ Helen J. Hathaway,‡ Dale L. Huber,# T. E. Tessier,§ H. C. Bryant,§ Edward R. Flynn,§ and Richard S. Larson*

*Department of Pathology, University of New Mexico, and Cancer Research and Treatment Center, Albuquerque, NM, USA
†Department of Biochemistry and Molecular Biology, University of New Mexico, Albuquerque, NM, USA
‡Department of Cell Biology and Physiology, University of New Mexico, Albuquerque, NM, USA
§Senior Scientific, LLC, Albuquerque, NM, USA
¶Nanomaterials Sciences Department, Sandia National Laboratories, Albuquerque, NM, USA
#Center for Integrated Nanotechnologies, Sandia National Laboratories, Albuquerque, NM, USA

Organ transplantation is a life-saving procedure and the preferred method of treatment for a growing number of disease states. The advent of new immunosuppressants and improved care has led to great advances in both patient and graft survival. However, acute T-cell-mediated graft rejection occurs in a significant quantity of recipients and remains a life-threatening condition. Acute rejection is associated with decrease in long-term graft survival, demonstrating a need to carefully monitor transplant patients. Current diagnostic criteria for transplant rejection rely on invasive tissue biopsies or relatively nonspecific clinical features. A noninvasive way is needed to detect, localize, and monitor transplant rejection. Capitalizing on advances in targeted contrast agents and magnetic-based detection technology, we developed anti-CD3 antibody-tagged nanoparticles. T cells were found to bind preferentially to antibody-tagged nanoparticles, as identified through light microscopy, transmission electron microscopy, and confocal microscopy. Using mouse skin graft models, we were also able to demonstrate in vivo vascular delivery of T-cell targeted nanoparticles. We conclude that targeting lymphocytes with magnetic nanoparticles is conducive to developing a novel, noninvasive strategy for identifying transplant rejection.

Key words: Magnetic nanoparticles; Transplant rejection; Cluster of differentiation 3 (CD3); Superconducting Quantum Interference Device (SQUID) detection

Received August 31, 2011; final acceptance September 5, 2012. Online prepub date: October 12, 2012.
Address correspondence to Richard S. Larson, M.D., Ph.D., University of New Mexico School of Medicine, Albuquerque, NM 87112, USA. Tel: +505 272-9762; Fax: +505 272-5186; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it