Cell Transplantation 20(9) Abstracts

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Cell Transplantation, Vol. 20, pp.1321–1332, 2011
0963-6897/11 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368910X557146
E-ISSN 1555-3892
Copyright © 2011 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

 

Novel Culture Technique Involving an Histone Deacetylase Inhibitor Reduces the Marginal Islet Mass to Correct Streptozotocin-Induced Diabetes

Jun-Seop Shin,*†1 Byoung-Hoon Min,*‡1 Jong-Yeon Lim,* Byoung-Keun Kim,* Hyun-Ju Han,* Kun-Ho Yoon,§ Sang-Joon Kim,¶ and Chung-Gyu Park†

*Korea Islet Transplantation Institute, Inc., Seoul, Korea
†Department of Microbiology and Immunology, Cancer Research Institute, Xenotransplantation Research Center, Transplantation Research Institute, Seoul National University College of Medicine, Seoul, Korea
‡Department of Parasitology, Korea University School of Medicine, Korea University, Seoul, Korea
§Department of Endocrinology and Metabolism, The Catholic University of Korea, Seoul, Korea
¶Department of Surgery, Seoul National University Hospital, Seoul, Korea

Islet transplantation is limited by the difficulties in isolating the pancreatic islets from the cadaveric donor and maintaining them in culture. To increase islet viability and function after isolation, here we present a novel culture technique involving an histone deacetylase inhibitor (HDACi) to rejuvenate the isolated islets. Pancreatic islets were isolated from Sprague-Dawley (SD) rats and one group (FIs; freshly isolated islets) was used after overnight culture and the other group (RIs; rejuvenated islet) was subjected to rejuvenation culture procedure, which is composed of three discrete steps including degranulation, chromatin remodeling, and regranulation. FIs and RIs were compared with regard to intracellular insulin content, glucose-stimulated insulin secretion (GSIS) capacity, gene expression profile, viability and apoptosis rate under oxidative stresses, and the engraftment efficacy in the xenogeneic islet transplantation models. RIs have been shown to have 1.9 ± 0.28- and 1.7 ± 0.31-fold greater intracellular insulin content and GSIS capacity, respectively, than FIs. HDACi increased overall histone acetylation levels, with inducing increased expression of many genes including insulin 1, insulin 2, GLUT2, and Ogg1. This enhanced islet capacity resulted in more resistance against oxidative stresses and increase of the engraftment efficacy shown by reduction of twofold marginal mass of islets in xenogeneic transplantation model. In conclusion, a novel rejuvenating culture technique using HDACi as chromatin remodeling agents improved the function and viability of the freshly isolated islets, contributing to the reduction of islet mass for the control of hyperglycemia in islet transplantation.

Key words: Diabetes; Islet transplantation; Rejuvenation; Histone deacetylase inhibitor (HADCi); Marginal islet mass

1These authors provided equal contribution to this work.
Address correspondence to Chung-Gyu Park, M.D., Ph.D., Professor, Department of Microbiology and Immunology, Cancer Research Institute,
Xenotransplantation Research Center, Transplantation Research Institute, Seoul National University College of Medicine, 103 Daehak-ro, Jongnogu, Seoul 110-799, Korea. Tel: +82-2-740-8311; Fax: +82-2-743-0881; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 20, pp.1333–1342, 2011
0963-6897/11 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368910X557182
E-ISSN 1555-3892
Copyright © 2011 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Improvement of Rat Islet Viability During Transplantation: Validation of Pharmacological Approach to Induce VEGF Overexpression

A. Langlois,* W. Bietiger,* E. Seyfritz,* E. Maillard,* K. Vivot,* C. Peronet,* N. Meyer,† L. Kessler,‡§ N. Jeandidier,‡§ M. Pinget,*‡§ and S. Sigrist*

*Centre européen d’étude du Diabète, Strasbourg, France
†Faculté de Médecine de Strasbourg, Laboratoire de Biostatistique, Strasbourg, France
‡Service d’endocrinologie, de diabéte et des maladies métaboliques, Médicale B Hopital civil, Strasbourg cedex, France
§Université de Strasbourg (UdS), Strasbourg cedex, France

Delayed and insufficient revascularization during islet transplantation deprives islets of oxygen and nutrients, resulting in graft failure. Vascular endothelial growth factor (VEGF) could play a critical role in islet revascularization. We aimed to develop pharmacological strategies for VEGF overexpression in pancreatic islets using the iron chelator deferoxamine (DFO), thus avoiding obstacles or safety risks associated with gene therapy. Rat pancreatic islets were infected in vivo using an adenovirus (ADE) encoding human VEGF gene (4.108 pfu/pancreas) or were incubated in the presence of DFO (10 μmol/L). In vitro viability, functionality, and the secretion of VEGF were evaluated in islets 1 and 3 days after treatment. Infected islets or islets incubated with DFO were transplanted into the liver of syngenic diabetic rats and the graft efficiency was estimated in vivo by measuring body weight, glycemia, C-peptide secretion, and animal survival over a period of 2 months. DFO induced transient VEGF overexpression over 3 days, whereas infection with ADE resulted in prolonged VEGF overexpression lasting 14 days; however, this was toxic and decreased islet viability and functionality. The in vivo study showed a decrease in rat deaths after the transplantation of islets treated with DFO or ADE compared with the sham and control group. ADE treatment improved body weight and C-peptide levels. Gene therapy and DFO improved metabolic control in diabetic rats after transplantation, but this effect was limited in the presence of DFO. The pharmacological approach is an interesting strategy for improving graft efficiency during transplantation, but this approach needs to be improved with drugs that are more specific.

Key words: Islet transplantation; Angiogenesis; Vascular endothelial growth factor (VEGF); Gene therapy; Deferoxamine (DFO); Hypoxia-inducible factor-1α (HIF-1α)

Address correspondence to Allan Langlois, Centre européen d’étude du Diabète, Boulevard René Leriche, 67200 Strasbourg, France. Tel: (+33)390201212; Fax: (+33)390201219; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 20, pp.1343–1349, 2011
0963-6897/11 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368910X557263
E-ISSN 1555-3892
Copyright © 2011 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Combination Therapy With a Dipeptidyl Peptidase-4 Inhibitor and a Proton Pump Inhibitor Induces β-Cell Neogenesis From Adult Human Pancreatic Duct Cells Implanted in Immunodeficient Mice

Wilma L. Suarez-Pinzon* and Alex Rabinovitch†

*Department of Medicine, University of Alberta, Edmonton, AB, Canada
†Sanford Research, University of South Dakota, Sioux Falls, SD, USA

Combination therapy with a dipeptidyl peptidase-4 inhibitor (DPP-4i) and a proton pump inhibitor (PPI) raises endogenous levels of GLP-1 and gastrin, respectively, and restores pancreatic β-cell mass and normoglycemia in nonobese diabetic (NOD) mice with autoimmune diabetes. The aim of this study was to determine whether a DPP-4i and PPI combination could increase β -cell mass in the adult human pancreas. Pancreatic cells from adult human pancreas donors were implanted in NOD-severe combined immunodeficient (NODscid) mice and the mice were treated with a DPP-4i and a PPI for 16 weeks. Human grafts were examined for insulin content and insulin-stained cells. Graft β -cell function was assessed by intravenous glucose tolerance tests (IVGTT) and by glucose control in human cell-engrafted mice treated with streptozotocin (STZ) to delete mouse pancreatic β -cells. Plasma GLP-1 and gastrin levels were raised to two- to threefold in DPP-4i and PPI-treated mice. Insulin content and insulin-stained cells in human pancreatic cell grafts were increased 9- to 13-fold in DPP-4i and PPI-treated mice and insulin-stained cells were colocalized with pancreatic exocrine duct cells. Plasma human C-peptide responses to IVGTT were significantly higher and STZinduced hyperglycemia was more completely prevented in DPP-4i- and PPI-treated mice with grafts than in vehicle-treated mice with grafts. In conclusion, DPP-4i and PPI combination therapy raises endogenous levels of GLP-1 and gastrin and greatly expands the functional β -cell mass in adult human pancreatic cells implanted in immunodeficient mice, largely from pancreatic duct cells. This suggests that a DPP-4i and PPI combination treatment may provide a pharmacologic therapy to correct the β -cell deficit in type 1 diabetes.

Key words: Gastrointestinal hormones; Type 1 diabetes; Pancreas endocrine; Islet â-cell; Glucagon-like peptide-1; Gastrin

Address correspondence to Alex Rabinovitch, Sanford Research, University of South Dakota, 2301 East 60th Street North, Sioux Falls, SD 57104, USA. Tel: (605) 312-6015; Fax: (605) 328-0401; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 20, pp. 1351–1359, 2011
0963-6897/11 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368910X557173
E-ISSN 1555-3892
Copyright © 2011 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Enhanced Survival of Dopaminergic Neuronal Transplants in Hemiparkinsonian Rats by the p53 Inactivator PFT-α

J. Chou,* N. H. Greig,† D. Reiner,* B. J. Hoffer,* and Y. Wang*

*National Institute on Drug Abuse, Baltimore, MD, USA
†National Institute of Aging, Bethesda, MD, USA

A key limiting factor impacting the success of cell transplantation for Parkinson’s disease is the survival of the grafted cells, which are often short lived. The focus of this study was to examine a novel strategy to optimize the survival of exogenous fetal ventromesencephalic (VM) grafts by treatment with the p53 inhibitor, pifithrin-α (PFT-α), to improve the biological outcome of parkinsonian animals. Adult male Sprague- Dawley rats were given 6-hydroxydopamine into the left medial forebrain bundle to induce a hemiparkinsonian state. At 7 weeks after lesioning, animals were grafted with fetal VM or cortical tissue into the lesioned striatum and, thereafter, received daily PFT-α or vehicle injections for 5 days. Apomorphine-induced rotational behavior was examined at 2, 6, 9, and 12 weeks after grafting. Analysis of TUNEL or tyrosine hydroxylase (TH) immunostaining was undertaken at 5 days or 4 months after grafting. The transplantation of fetal VM tissue into the lesioned striatum reduced rotational behavior. A further reduction in rotation was apparent in animals receiving PFT-α and VM transplants. By contrast, no significant reduction in rotation was evident in animals receiving cortical grafts or cortical grafts + PFT-α. PFT-α treatment reduced TUNEL labeling and increased TH+ cell and fiber density in the VM transplants. In conclusion, our data indicate that early postgrafting treatment with PFT-α enhances the survival of dopamine cell transplants and augments behavioral recovery in parkinsonian animals.

Key words: Parkinson’s disease; Transplantation; Apoptosis; p53; Neuroprotection

Address correspondence to Yun Wang, M.D., Ph.D., National Institute on Drug Abuse, I.R.P., Neural Protection and Regeneration Section, 251 Bayview Boulevard, 06-721A, Baltimore, MD 21224, USA. Tel: 443-740-2587; Fax: 443-740-2840; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 20, pp. 1361–1379, 2011
0963-6897/11 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368910X557155
E-ISSN 1555-3892
Copyright © 2011 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Combination of Multifaceted Strategies to Maximize the Therapeutic Benefits of Neural Stem Cell Transplantation for Spinal Cord Repair

Dong H. Hwang,* Hyuk M. Kim,* Young M. Kang,* In S. Joo,† Chong-Su Cho,‡ Byung-Woo Yoon,§ Seung U. Kim,¶# and Byung G. Kim*†

*Brain Disease Research Center, Institute of Medical Sciences, Ajou University School of Medicine, Suwon, Republic of Korea
†Department of Neurology, Ajou University School of Medicine, Suwon, Republic of Korea
‡Department of Agricultural Biotechnology, Seoul National University, Seoul, Republic of Korea
§Departments of Neurology and Neuroscience Research Center, Clinical Research Institute, Seoul National University Hospital, Seoul, Republic of Korea
¶Department of Neurology, University of British Columbia, Vancouver, BC, Canada
#Medical Research Institute, Chungang University School of Medicine, Seoul, Republic of Korea

Neural stem cells (NSCs) possess therapeutic potentials to reverse complex pathological processes following spinal cord injury (SCI), but many obstacles remain that could not be fully overcome by NSC transplantation alone. Combining complementary strategies might be required to advance NSC-based treatments to the clinical stage. The present study was undertaken to examine whether combination of NSCs, polymer scaffolds, neurotrophin-3 (NT3), and chondroitinase, which cleaves chondroitin sulfate proteoglycans at the interface between spinal cord and implanted scaffold, could provide additive therapeutic benefits. In a rat hemisection model, poly(ε-caprolactone) (PCL) was used as a bridging scaffold and as a vehicle for NSC delivery. The PCL scaffolds seeded with F3 NSCs or NT3 overexpressing F3 cells (F3.NT3) were implanted into hemisected cavities. F3.NT3 showed better survival and migration, and more frequently differentiated into neurons and oligodendrocytes than F3 cells. Animals with PCL scaffold containing F3.NT3 cells showed the best locomotor recovery, and motor evoked potentials (MEPs) following transcranial magnetic stimulation were recorded only in PCL-F3.NT3 group in contralateral, but not ipsilateral, hindlimbs. Implantation of PCL scaffold with F3.NT3 cells increased NT3 levels, promoted neuroplasticity, and enhanced remyelination of contralateral white matter. Combining chondroitinase treatment after PCL-F3.NT3 implantation further enhanced cell migration and promoted axonal remodeling, and this was accompanied by augmented locomotor recovery and restoration of MEPs in ipsilateral hindlimbs. We demonstrate that combining multifaceted strategies can maximize the therapeutic benefits of NSC transplantation for SCI. Our results may have important clinical implications for the design of future NSC-based strategies.

Key words: Stem cells; Spinal cord injury (SCI); Tissue scaffold (Pclf polymer); Neurotrophin 3; Motor evoked potential; Chondroitinase ABC

Address correspondence to Dr. Byung G. Kim, M.D., Ph.D., Brain Disease Research Center, Institute of Medical Sciences, and Department of Neurology, Ajou University School of Medicine, San 5, Woncheon-Dong, Yeongtong-Gu, Suwon 443-721, Republic of Korea. Tel: 82-31-219-4495; Fax: 82-31-219-4530; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 20, pp. 1381–1393, 2011
0963-6897/11 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368910X550215
E-ISSN 1555-3892
Copyright © 2011 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

The Secretory Profiles of Cultured Human Articular Chondrocytes and Mesenchymal Stem Cells: Implications for Autologous Cell Transplantation Strategies

Martin Polacek,*† Jack-Ansgar Bruun,‡ Jan Elvenes,*† Yngve Figenschau,§¶ and Inigo Martinez†

*Orthopaedic Surgery Department, University Hospital of North Norway, Tromsø, Norway
†Orthopaedic Surgery Department, Institute of Clinical Medicine, University of Tromsø, Tromsø, Norway
‡Proteomic Platform, University of Tromsø, Tromsø, Norway
§Laboratory of Medicine, University Hospital of North Norway, Tromsø, Norway
¶Institute of Medical Biology, University of Tromsø, Tromsø, Norway

This study was undertaken to compare the phenotype of human articular chondrocytes (ACs) and bone marrow-derived mesenchymal stem cells (MSCs) after cell expansion by studying the spectrum of proteins secreted by cells into the culture medium. ACs and MSCs were expanded in monolayer cultures for some weeks, as done in standard cell transplantation procedures. Initially, the expression of cartilage signature genes was compared by real-time PCR. Metabolic labeling of proteins (SILAC) in combination with mass spectrometry (LC/MS-MS) was applied to investigate differences in released proteins. In addition, multiplex assays were carried out to quantify the amounts of several matrix metalloproteases (MMPs) and their natural inhibitors (TIMPs). Expanded chondrocytes showed a slightly higher expression of cartilage-specific genes than MSCs, whereas the overall spectra of released proteins were very similar for the two cell types. In qualitative terms MSCs seemed to secrete similar number of extracellular matrix proteins (43% vs. 45% of total proteins found) and catabolic agents (9% vs. 10%), and higher number of anabolic agents (12% vs. 7%) compared to ACs. Some matrix-regulatory agents such as serpins, BMP-1, and galectins were detected only in MSC supernatants. Quantitative analyses of MMPs and TIMPs revealed significantly higher levels of MMP-1, MMP-2, MMP-3, and MMP-7 in the medium of ACs. Our data show that after the expansion phase, both ACs and MSCs express a dedifferentiated phenotype, resembling each other. ACs hold a phenotype closer to native cartilage at the gene expression level, whereas MSCs show a more anabolic profile by looking at the released proteins pattern. Our data together with the inherent capability of MSCs to maintain their differentiation potential for longer cultivation periods would favor the use of these cells for cartilage reconstruction.

Key words: Mesenchymal stem cells; Chondrocytes; Secretome; SILAC; Proteomics

Address correspondence to Inigo Martinez, Orthopaedic Surgery Department, Institute of Clinical Medicine, University in Tromsø, 9037 Tromsø, Norway. Tel: +4777644686; Fax: +4777627164; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 20, pp. 1395–1408, 2011
0963-6897/11 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368910X557245
E-ISSN 1555-3892
Copyright © 2011 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Human Umbilical Cord Mesenchymal Stem Cells Ameliorate Mice Trinitrobenzene Sulfonic Acid (TNBS)-Induced Colitis

Lu Liang,* Chunlan Dong,* Xiaojun Chen,* Zhihong Fang,* Jie Xu,* Meng Liu,* Xiaoguang Zhang,* Dong Sheng Gu,* Ding Wang,* Weiting Du,* Delin Zhu,† and Zhong Chao Han*

*State Key Laboratory of Experimental Hematology, Institute of Hematology & Hospital of Blood Diseases, Chinese Academy of Medical Sciences & Peking Union Medical College (CAMS&PUMC), Tianjin, PR China
†National Engineering Research Center of Cell Products, AmCellGene Co. Ltd., Tianjin, PR China

Mesenchymal stem cells (MSCs), which are poorly immunogenic and have potent immunosuppressive activities, have emerged as a promising candidate for cellular therapeutics for the treatment of disorders caused by abnormal immune responses. In this study we investigated whether human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) could ameliorate colitis in a trinitrobenzene sulfonic acid (TNBS)-induced colitis model. TNBS-treated colitic mice were infused with hUC-MSCs or vehicle control. The mice were sacrificed on day 1, 3, and 5 after infusion, and their clinical and pathological conditions were evaluated by body weight, colon length, and histological analysis. The expression levels of proinflammatory cytokine proteins in colon were examined by ELISA. The homing of hUC-MSCs was studied by live in vivo imaging and immunofluorescent microscopy. hUC-MSCs were found to migrate to the inflamed colon and effectively treated the colitic mice with improved clinical and pathological signs. The levels of IL-17 and IL-23 as well as IFN-g and IL-6 were significantly lower in the colon tissues of the hUC-MSC-treated mice in comparison with the vehicle-treated mice. Coculture experiments showed that hUC-MSCs not only could inhibit IFN-g expression but also significantly inhibit IL-17 production by lamina propria mononuclear cells (LPMCs) or splenocytes of the colitic mice or by those isolated from normal animals and stimulated with IL-23. Systemically infused hUC-MSCs could home to the inflamed colon and effectively ameliorate colitis. In addition to the known suppressive effects on Th1-type immune responses, hUC-MSC-mediated modulation of IL-23/ IL-17 regulated inflammatory reactions also plays an important role in the amelioration of colitis.

Key words: Mesenchymal stem cells (MSCs); Colitis; Immunosuppression; IL-17

Address correspondence to Zhong Chao Han, M.D., Ph.D., Professor, Institute of Hematology Chinese Academy of Medical Sciences and Peking Union of Medical College, Tianjin, 288 Nan Jing Road 300020, PR China.Fax: 86-22-27317273; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it or Delin Zhu, Ph.D., Professor, National Engineering Research Center of Cell Products, AmCellGene Co. Ltd., Tianjin, PR China. Tel: 86-22-66211206; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 20, pp. 2409–1422, 2011
0963-6897/11 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368910X557218
E-ISSN 1555-3892
Copyright © 2011 Cognizant Comm. Corp.
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Expansion of Adipose Mesenchymal Stromal Cells Is Affected by Human Platelet Lysate and Plating Density

Dominik Cholewa,*1 Thomas Stiehl,†‡1 Anne Schellenberg,*1 Gudrun Bokermann,* Sylvia Joussen,* Carmen Koch,* Thomas Walenda,* Norbert Pallua,§ Anna Marciniak-Czochra,†‡ Christoph V. Suschek,§ and Wolfgang Wagner*

*Stem Cell Biology and Cellular Engineering, Medical Faculty, RWTH Aachen University, Aachen, Germany
†Interdisciplinary Center of Scientific Computing (IWR), Institute of Applied Mathematics, University of Heidelberg, Heidelberg, Germany
‡Heidelberg Academy of Sciences and Humanities, Heidelberg, Germany
§Department of Plastic and Reconstructive Surgery, Hand Surgery, Burn Center, Medical Faculty, RWTH Aachen University, Aachen, Germany

The composition of mesenchymal stromal cells (MSCs) changes in the course of in vitro culture expansion. Little is known how these cell preparations are influenced by culture media, plating density, or passaging. In this study, we have isolated MSCs from human adipose tissue in culture medium supplemented with either fetal calf serum (FCS) or human platelet lysate (HPL). In addition, culture expansion was simultaneously performed at plating densities of 10 or 10,000 cells/cm2 The use of FCS resulted in larger cells, whereas HPL significantly enhanced proliferation. Notably, HPL also facilitated expansion for more population doublings than FCS (43 ± 3 vs. 22 ± 4 population doubling; p < 0.001), while plating density did not have a significant effect on long-term growth curves. To gain further insight into population dynamics, we conceived a cellular automaton model to simulate expansion of MSCS. It is based on the assumptions that the number of cell divisions is limited and that due to contact inhibition proliferation occurs only at the rim of colonies. The model predicts that low plating densities result in more heterogeneity with regard to cell division history, and favor subpopulations of higher migratory activity. In summary, HPL is a suitable serum supplement for isolation of MSC from adipose tissue and facilitates more population doublings than FCS. Cellular automaton computer simulations provided additional insights into how complex population dynamics during long-term expansion are affected by plating density and migration.

Key words: Mesenchymal stromal cells (MSCs); Human platelet lysate (HPL); Adipose tissue; Plating density; Cellular automaton model

1These authors provided equal contribution to this work.
Address correspondence to Wolfgang Wagner, M.D., Ph.D., Helmholtz Institute for Biomedical Engineering-Cell Biology, RWTH Aachen University Medical School, Pauwelsstrasse 20, 52074 Aachen, Germany. Tel: +49 241 8088611; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 20, pp. 1423–1430, 2011
0963-6897/11 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368910X547444
E-ISSN 1555-3892
Copyright © 2011 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Simple and Highly Efficient Method for Production of Endothelial Cells From Human Embryonic Stem Cells

Rie Tatsumi,* Yutaka Suzuki,* Tomoyuki Sumi,† Masakatsu Sone,‡ Hirofumi Suemori,§ and Norio Nakatsuji¶#

*Stem Cell and Drug Discovery Institute, Kyoto Research Park, Kyoto, Japan
†Department of Cell Biology, Institute of Nephrology, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan
‡Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Kyoto, Japan
§Laboratory of Embryonic Stem Cell Research, Stem Cell Research Center, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan
¶Department of Development and Differentiation, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan
#Institute for Integrated Cell-Material Sciences (iCeMS), Kyoto University, Kyoto, Japan

Endothelial cells derived from human embryonic stem cells (hESC-ECs) hold much promise as a valuable tool for basic vascular research and for medical application such as cell transplantation or regenerative medicine. Here we have developed an efficient approach for the production of hESC-ECs. Using a differentiation method consisting of a stepwise combination of treatment with glycogen synthase kinase-3β (GSK-3β) inhibitor and culturing in vascular endothelial growth factor (VEGF)-supplemented medium, hESC-ECs are induced in 5 days with about 20% efficiency. These cells express vascular endothelial cadherin (VEcadherin), VEGF receptor-2 (VEGFR-2), CD34, and platelet endothelial cell adhesion molecule-1 (PECAM-1). These hESC-ECs can then be isolated with 95% purity using a magnetic sorting system, and expanded to more than 100-fold within a month. The hESC-ECs thus produced exhibit the endothelial morphological characteristics and specific functions such as capillary tube formation and acetylated low-density lipoprotein uptake. We propose that our methodology is useful for efficient and large-scale production of hESC-ECs.

Key words: Human embryonic stem cells; Endothelial cells; Differentiation; Glycogen synthase kinase-3β  (GSK-3β) inhibitor; Vascular endothelial cadherin (VE-cadherin)

Address correspondence to Yutaka Suzuki, Ph.D., Stem Cell and Drug Discovery Institute, Kyoto Research Park, Building 2, Room 412, 134 Chudoji Minami-machi, Shimogyo-ku, Kyoto 600-8813, Japan. Tel: +81-75-313-9535; Fax: +81-75-313-7019; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 20, pp. 1431–1443, 2011
0963-6897/11 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368910X557164
E-ISSN 1555-3892
Copyright © 2011 Cognizant Comm. Corp.
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A Novel and Simplified Method of Culture of Human Blood-Derived Early Endothelial Progenitor Cells for the Treatment of Ischemic Vascular Disease

M. Bouchentouf,* K. Forner,* J. Cuerquis,* M. R. Boulassel,† J. P. Routy,† E. K. Waller,‡ A. A. Quyyumi,§ P. Paradis,* E. L. Schiffrin,*¶ and J. Galipeau*‡#

*Lady Davis Institute for Medical Research, Jewish General Hospital, McGill University, Montreal, Quebec, Canada
†Royal Victoria Hospital, Division of Hematology, McGill University Health Center, Montreal, Quebec, Canada
‡Department of Hematology and Medical Oncology, Winship Cancer Institute of Emory University, Atlanta, GA, USA
§Department of Medicine, Emory University, Atlanta, GA, USA
¶Department of Medicine, Jewish General Hospital, McGill University, Montreal, Quebec, Canada
#Department of Pediatrics, Emory University, Atlanta, GA, USA

Endothelial progenitor cells (EPCs) consist of two different subpopulations named early (eEPCs) and late EPCs (lEPCs) that are derived from CD14+ and CD14- circulating cells, respectively. These cells are regularly cultured over fibronectin-coated surfaces in endothelial basal medium (EBM)-2 supplemented with insulin-like growth factor (IGF-1), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), and fibroblast growth factor (FGF). We have developed a new and simplified method for culturing human EPCs obtained from peripheral blood and tested their ability to preserve cardiac function following infarction. We first demonstrated that eEPCs derived from human peripheral blood mononuclear cells (PBMCs) and cultured in EBM-2 medium supplemented with autologous serum (10%) over fibronectincoated surfaces (10 μg/ml) in the presence of IGF-1 (50 ng/ml) only, have a secretome similar to eEPCs cultured under regular conditions with IGF-1, VEGF, EGF, and FGF. Our data also indicate that IGF-1 modulates PBMC secretome in a dose-dependent manner. In another series of experiments, we showed that PBMCs cultured in suspension in bags (S-PBMCs) in basal medium supplemented with fibronectin and IGF-1 secrete significant amounts of stem cell factor (SCF, 31.3 ± 3.1 pg/ml), hepatocyte growth factor (HGF, 438.6 ± 41.4 pg/ml), soluble tumor necrosis factor receptor 1 (sTNFR1, 127.1 ± 9.9 pg/ml), VEGF (139.3 ± 9.6 pg/ml), and IGF-1 (147.2 ± 46.1 pg/ml) but very low levels of TNF-α (13.4 ± 2.5 pg/ml). S-PBMCs injected intravenously into NOD SCID mice migrated to the injured myocardium, reduced cardiac fibrosis, enhanced angiogenesis, and preserved cardiac function after myocardial infarction (MI) in a manner similar to eEPCs cultured under standard conditions. In conclusion, we show in this study a refined and optimized method for culturing eEPCs. Our data indicate that S-PBMCs are composed of several cell populations including eEPCs and that they secrete high amounts of antiapoptotic, anti-inflammatory, and proangiogenic factors capable of preserving cardiac function following MI.

Key words: Endothelial progenitor cells (EPCs); Growth factors; Culture method; Angiogenesis; Myocardial infarction (MI)

Address correspondence to Jacques Galipeau, M.D., FRCPC, Winship Cancer Institute, 1365B Clifton Road, Atlanta, GA 30322, USA. Tel: 404-778-1779; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it or Tel: +1 (514)-340-8214; Fax: +1 (514)-340-8281; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 20, pp. 1445–1452, 2011
0963-6897/11 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368910X557272
E-ISSN 1555-3892
Copyright © 2011 Cognizant Comm. Corp.
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Enhancement of Human Peripheral Blood Mononuclear Cell Transplantation-Mediated Bone Formation

Hee Seok Yang,*1 Ga Hee Kim,*1 Wan-Geun La,† Suk Ho Bhang,† Tae-Jin Lee,* Jong Ho Lee,‡ and Byung-Soo Kim†

*Department of Bioengineering, Hanyang University, Seoul, Republic of Korea
†School of Chemical and Biological Engineering, Seoul National University, Seoul, Republic of Korea
‡Department of Oral and Maxillofacial Surgery, Collage of Dentistry, Seoul National University, Seoul, Republic of Korea

Recent studies have demonstrated the existence of osteoblast progenitor cells in circulating blood. Here we show that local delivery of bone morphogenetic protein-2 (BMP-2) to cell transplantation sites induces in situ osteogenic differentiation of transplanted human peripheral blood mononuclear cells (PBMNCs) and enhances in vivo bone formation mediated by PBMNC transplantation. Human PBMNCs were seeded on scaffolds with or without BMP-2 and implanted subcutaneously into athymic mice. Nonseeded scaffolds with BMP-2 were also implanted. Eight weeks later, radiographic and histological analyses showed that the PBMNC + BMP-2 group had undergone much more extensive bone formation than either the PBMNC group or BMP-2 group. Only the PBMNC + BMP-2 group expressed human Cbfa1, osteonectin, and osteocalcin, suggesting in situ osteogenic differentiation of and bone formation by transplanted human PBMNCs, while the other groups did not express these genes. This study provides a method to enhance human PBMNC transplantation-mediated bone formation.

Key words: Bone formation; Peripheral blood mononuclear cells (PBMNCs); Bone morphogenetic protein-2 (BMP-2)

1These authors provided equal contribution to this work.
Address correspondence to Byung-Soo Kim, School of Chemical and Biological Engineering, Seoul National University, San 56-1, Sillim-dong, Gwanak-gu, Seoul 151-744, Republic of Korea. Tel: +82-2-880-1509; Fax: +82-2-888-1604; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 20, pp. 1453–1463, 2011
0963-6897/11 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368910X552853
E-ISSN 1555-3892
Copyright © 2011 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Clinical-Scale Cultures of Cord Blood CD34+ Cells to Amplify Committed Progenitors and Maintain Stem Cell Activity

Zoran Ivanovic,*† Pascale Duchez,*† Jean Chevaleyre,*† Marija Vlaski,*† Xavier Lafarge,*† Bernard Dazey,* Elodie Robert-Richard,‡ Frédéric Mazurier,‡ and Jean-Michel Boiron*†

*Etablissement Français du Sang Aquitaine-Limousin, Bordeaux, France
†CNRS UMR 5164 Université Bordeaux Segalen, Bordeaux Segalen, France
‡INSERM U876, Université Bordeaux Segalen, Bordeaux, France

We developed a clinical-scale cord blood (CB) cell ex vivo procedure to enable an extensive expansion of committed progenitors—colony-forming cells (CFCs) without impairing very primitive hematopoietic stem cells (HSCs). CD34++ cells, selected from previously cryopreserved and thawed CB units, were cultured in two steps (diluted 1:4 after 6 days) in the presence of stem cell factor (SCF), fms-related tyrosine kinase 3 ligand (Flt-3L), megakaryocyte growth and development factor (MGDF) (100 ng/ml each), granulocytecolony stimulating factor (G-CSF) (10 ng/ml) in HP01 serum-free medium. HSC activity was evaluated in serial transplantation assay, by detection of human cells (CD45, CD33, CD19 and CFC of human origin) in bone marrow (BM) of primary and secondary recipient NOD/SCID mice 6–8 weeks after transplantation. A wide amplification of total cells (~350-fold), CD34+ cells (~100-fold), and CFC (~130-fold) without impairing the HSC activity was obtained. The activity of a particular HSC subpopulation (SRCCFC) was even enhanced. Thus, an extensive ex vivo expansion of CFCs is feasible without impairing the activity of HSCs. This result was enabled by associating antioxidant power of medium with an appropriate cytokine cocktail (i.e., mimicking physiologic effects of a weak oxygenation in hematopoietic environment).

Key words: Cord blood; CD34+ cells; Ex vivo expansion; Progenitors; Stem cells; NOD/SCID mice

Address correspondence to Zoran Ivanovic, Etablissement Franc¸ais du Sang Aquitaine-Limousin, PB 24, 5 Place Amélie Raba Léon 33035 Bordeaux CEDEX, France. Tel: 33 5 56 90 75 50; Fax: 33 5 56 90 75 51; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 20, pp. 1465–1477, 2011
0963-6897/11 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368910X550224
E-ISSN 1555-3892
Copyright © 2011 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Autologous Serum Improves Yield and Metabolic Capacity of Monocyte-Derived Hepatocyte-Like Cells: Possible Implication for Cell Transplantation

S. Ehnert,* C. Seeliger,* H. Vester,* A. Schmitt,* S. Saidy-Rad,* J. Lin,* M. Neumaier,* S. Gillen,*† J. Kleeff,† H. Friess,† J. Burkhart,‡ U. Stöckle,* and A. K. Nu¨ssler*

*Department of Traumatology, MRI, Technische Universita¨t Mu¨nchen, Munich, Germany
†Department of Surgery, MRI, Technische Universita¨t Mu¨nchen Munich, Germany
‡Blood Donor Service, Bavarian Red Cross, Munich, Germany

Hepatocyte-transplantation is a therapeutic approach for diverse acute and chronic liver diseases. As availability of primary cells is limited, there is an increasing demand for hepatocyte-like cells (e.g., neohepatocytes generated from peripheral blood monocytes). The aim of this study was to evaluate the effects of six different human AB sera, fetal calf serum, or autologous serum on production of neohepatocytes. The yield and quality of neohepatocytes varied considerably depending on the different sera. Using autologous sera for the whole production process we constantly generated the highest amount of cells with the highest metabolic activity for phase I (e.g., CYP1A1/2, CYP3A4) and phase II enzymes (e.g., glutathione-S-transferase). Moreover, similar effects were seen examining glucose and urea metabolism. Especially, glucose-6- phosphatase and PAS staining showed distinct serum-dependent differences. The role of macrophage activation was investigated by measuring the secretion of TNF-α, TGF-β, and RANKL, MMP activity, as well as mRNA levels of different interleukins in programmable cells of monocytic origin (PCMO). Our data clearly demonstrate that the use of autologous serum reduced initial macrophage activation in PCMOs and subsequently improved both yield and function of differentiated neohepatocytes. The autologous approach presented here might also be useful in other stem cell preparation processes where cell activation during generation shall be kept to a minimum.

Key words: Primary human hepatocytes; Programmable cells of monocytic origin (PCMOs); Neohepatocytes; Autologous cell therapy; Macrophage

Address correspondence to Andreas Nüssler at his current address: BG Unfallklinik, Eberhard-Karls University Tübingen, Schnarrenbergstr. 95, 72076 Tübingen, Germany. Tel: +49-7071-606-1001; Fax: +49-7071-606-1002; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 20, pp. 1479–1489, 2011
0963-6897/11 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368910X547453
E-ISSN 1555-3892
Copyright © 2011 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Replacement of Liver Parenchyma in Analbuminemic Rats With Allogenic Hepatocytes Is Facilitated by Intrabone Marrow-Bone Marrow Transplantation

Mitsuhiro Inagaki,*† Hiroyuki Furukawa,* Yoshiyasu Satake,* Yoko Okada,* Shinichi Chiba,‡ Yuji Nishikawa,† and Katsuhiro Ogawa†

*Department of Surgery, Asahikawa Medical University, Asahikawa, Japan
†Department of Pathology, Asahikawa Medical University, Asahikawa, Japan
‡Central Laboratory for Research and Education, Asahikawa Medical University, Asahikawa, Japan

Although hepatocyte transplantation (HCTx) is expected to become a useful therapy for human liver diseases, allogenic hepatocytes still tend to be rejected within a short period due to host immunosurveillance. In the present study, we investigated the effect of prior bone marrow transplantation (BMTx) for the engraftment of allogenic hepatocytes using the analbuminemic rat transplantation model. The hepatocytes of Lewis (LEW) rats were not accepted in the liver of retrorsine (RS)/partial hepatectomy (PH)-treated analbuminemic F344 (F344-alb) rats, which express the disparate major histocompatibility complex (MHC) against that of LEW rats. Prior BMTx with the LEW bone marrow cells (BMCs) after sublethal irradiation achieved acceptance and repopulation of LEW hepatocytes in the liver of the RS/PH-treated F344-alb rats, associated with elevation of serum albumin. The replacement of hepatic parenchyma with albumin positive (Alb+) donor hepatocytes and elevation of serum albumin levels were dependent on the bone marrow reconstitution by donor BMCs, which was more efficiently achieved by intrabone marrow (IBM)-BMTx than by intravenous (IV)-BMTx. Our results demonstrate that efficient bone marrow reconstitution by IBM-BMTx enables the replacement of the hepatic parenchyma with allogenic hepatocytes in RS/PH-treated analbuminemic rats without immunosuppressants.

Key words: Analbuminemic F344 rats; Intrabone marrow-bone marrow transplantation; Allogenic hepatocyte transplantation; Retrorsine; Partial hepatectomy

Address correspondence to Mitsuhiro Inagaki, M.D., Department of Surgery, Asahikawa Medical University, Midorigaoka-Higashi 2-1-1-1, Asahikawa, 078-8510 Japan. Tel: +81-166-68-2503; Fax: +81-166-68-2193; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 20, pp. 1491–1496, 2011
0963-6897/11 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368910X550189
E-ISSN 1555-3892
Copyright © 2011 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Brief Communication

Local Transplantation of G-CSF-Mobilized CD34+ Cells in a Patient With Tibial Nonunion: A Case Report

Ryosuke Kuroda,*†1 Tomoyuki Matsumoto,*†1 Masahiko Miwa,*† Atsuhiko Kawamoto,†‡ Yutaka Mifune,*† Tomoaki Fukui,*†‡ Yohei Kawakami,*†‡ Takahiro Niikura,*† Sang Yang Lee,*† Keisuke Oe,*† Taro Shoji,*†‡ Tomoya Kuroda,*†‡ Miki Horii,†‡ Ayumi Yokoyama,†‡ Takayuki Ono,§ Yasushi Koibuchi,¶ Shin Kawamata,¶ Masanori Fukushima,§ Masahiro Kurosaka,*† and Takayuki Asahara†‡§

*Department of Orthopedic Surgery, Kobe University Graduate School of Medicine, Kobe, Japan
†Department of Translational Research, Institute of Biomedical Research and Innovation, Kobe, Japan
‡Group of Vascular Regeneration Research, Institute of Biomedical Research and Innovation, Kobe, Japan
§Translational Research Informatics Center, Kobe, Japan
¶Foundation for Biomedical Research and Innovation, Kobe, Japan
#Department of Regenerative Medicine Science, Tokai University School of Medicine, Tokyo, Japan

Although implantation of crude bone marrow cells has been applied in a small number of patients for fracture healing, transplantation of peripheral blood CD34+ cells, the hematopoietic/endothelial progenitor cell-enriched population, in patients with fracture has never been reported. Here, we report the first case of tibial nonunion receiving autologous, granulocyte colony stimulating factor mobilized CD34+ cells accompanied with autologous bone grafting. No serious adverse event occurred, and the novel therapy performed 9 months after the primary operation resulted in bone union 3 months later without any symptoms including pain and gait disturbance.

Key words: Tibial nonunion; Peripheral blood CD34+ cells; Granulocyte colony stimulating factor (G-CSF)

1These authors provided equal contribution to this work.
Address correspondence to Ryosuke Kuroda, M.D., Department of Orthpaedic Surgery, Kobe University Graduate School of Medicine, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe, 650-0017 Japan. Tel: +81-78-382-5985; Fax: +81-78-351-6944; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it