Cell Transplantation 21(1) Abstracts

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Cell Transplantation, Vol. 21, pp. 1–10, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X566208
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Review

Improving the Techniques for Human Hepatocyte Transplantation: Report From a Consensus Meeting in London

Juliana Puppi,* Stephen C. Strom,† Robin D. Hughes,* Sanjay Bansal,‡ Jose V. Castell,§¶ Ibrahim Dagher,#** Ewa C. S. Ellis,†† Greg Nowak,†† Bo-Goran Ericzon,†† Ira J. Fox,‡‡ M. José Gómez-Lecho´n,¶ Chandan Guha,§§ Sanjeev Gupta,¶¶ Ragai R. Mitry,* Kazuo Ohashi,## Michael Ott,*** Lola M. Reid,††† Jayanta Roy-Chowdhury,‡‡‡ Etienne Sokal,§§§ Anne Weber,** and Anil Dhawan*‡

*Institute of Liver Studies, King’s College London School of Medicine at King’s College Hospital, London, UK
†Department of Pathology, University of Pittsburgh, Pittsburgh, PA, USA
‡Paediatric Liver, GI & Nutrition Centre, King’s College Hospital, London, UK
§Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad de Valencia, Valencia, Spain
¶Unidad de Hepatología Experimental, Centro de Investigación, Hospital La Fe, Valencia, Spain
#Department of Surgery, Antoine Béclère Hospital, Assistance Publique-Hôpitaux de Paris (AP-HP), Clamart, France
**Institut National de la Santé et de la Recherche Mèdicale (INSERM) Unité 972, Institut Fédératif de Recherche 93, Bicêtre Hospital, Kremlin-Bicêtre, France
††Division of Transplantation Surgery, Department of Clinical Science, Intervention and Technology (CLINTEC), Liver Cell Laboratory, Karolinska University Hospital Huddinge, Stockholm, Sweden
‡‡Department of Surgery, and McGowan Institute for Regenerative Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
§§Department of Radiation Oncology, Montefiore Medical Center, Albert Einstein College of Medicine, New York, NY, USA
¶¶Departments of Medicine and Pathology, Marion Bessin Liver Research Center, Diabetes Research Center, and Cancer Research Center, Albert Einstein College of Medicine, New York, NY, USA
##Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University, Tokyo, Japan
***Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School and Twincore Centre for Experimental and Clinical Infection Research, Hannover, Germany
†††Department of Cell and Molecular Physiology and Department of Biomedical Engineering Program in Molecular Biology and Biotechnology, School of Medicine University of North Carolina, Chapel Hill, NC, USA
‡‡‡Departments of Medicine and Genetics, Marion Bessin Liver Research Center, Albert Einstein College of Medicine, New York, NY, USA
§§§Catholic University of Louvain and St Luc Clinics, Paediatric Department (HPED), PEDI Unit, Laboratory of Paediatric Hepatology and Cell Therapy, Brussels, Belgium

On September 6 and 7, 2009 a meeting was held in London to identify and discuss what are perceived to be current roadblocks to effective hepatocyte transplantation as it is currently practiced in the clinics and, where possible, to offer suggestions to overcome the blocks and improve the outcomes for this cellular therapy. Present were representatives of most of the active clinical hepatocyte transplant programs along with other scientists who have contributed substantial basic research to this field. Over the 2-day sessions based on the experience of the participants, numerous roadblocks or challenges were identified, including the source of cells for the transplants and problems with tracking cells following transplantation. Much of the discussion was focused on methods to improve engraftment and proliferation of donor cells posttransplantation. The group concluded that, for now, parenchymal hepatocytes isolated from donor livers remain the best cell source for transplantation. It was reported that investigations with other cell sources, including stem cells, were at the preclinical and early clinical stages. Numerous methods to modulate the immune reaction and vascular changes that accompany hepatocyte transplantation were proposed. It was agreed that, to obtain sufficient levels of repopulation of liver with donor cells in patients with metabolic liver disease, some form of liver preconditioning would likely be required to enhance the engraftment and/or proliferation of donor cells. It was reported that clinical protocols for preconditioning by hepatic irradiation, portal vein embolization, and surgical resection had been developed and that clinical studies using these protocols would be initiated in the near future. Participants concluded that sharing information between the groups, including standard information concerning the quality and function of the transplanted cells prior to transplantation, clinical information on outcomes, and standard preconditioning protocols, would help move the field forward and was encouraged.

Key words: Hepatocyte transplantation; Engraftment; Radiation; Stem cell; Metabolic liver disease

Received November 21, 2010; final acceptance February 18, 2011. Online prepub date: April 1, 2011.
Address correspondence to Professor Anil Dhawan, M.D., FRCPCH, Paediatric Liver Centre, King’s College Hospital, King’s College London School of Medicine, Denmark Hill, London SE 5 9RS, UK. Tel: +44 (0)20 3299 3578; Fax: +44 (0)20 3299 4228; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 11–22, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X580626
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
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Growth Ability and Repopulation Efficiency of Transplanted Hepatic Stem Cells, Progenitor Cells, and Mature Hepatocytes in Retrorsine-Treated Rat Livers

Norihisa Ichinohe,1 Junko Kon,1 Kazunori Sasaki, Yukio Nakamura, Hidekazu Ooe, Naoki Tanimizu, and Toshihiro Mitaka

Department of Tissue Development and Regeneration, Research Institute for Frontier Medicine, Sapporo Medical University School of Medicine, Sapporo, Japan

Cell-based therapies as an alternative to liver transplantation have been anticipated for the treatment of potentially fatal liver diseases. Not only mature hepatocytes (MHs) but also hepatic stem/progenitor cells are considered as candidate cell sources. However, whether the stem/progenitor cells have an advantage to engraft and repopulate the recipient liver compared with MHs has not been comprehensively assessed. Therefore, we used Thy1+ (oval) and CD44+ (small hepatocytes) cells isolated from GalN-treated rat livers as hepatic stem and progenitor cells, respectively. Cells from dipeptidylpeptidase IV (DPPIV)+ rat livers were transplanted into DPPIV livers treated with retrorsine following partial hepatectomy. Both stem and progenitor cells could differentiate into hepatocytes in host livers. In addition, the growth of the progenitor cells was faster than that of MHs until days 14. However, their repopulation efficiency in the long term was very low, since the survival period of the progenitor cells was much shorter than that of MHs. Most foci derived from Thy1+ cells disappeared within 2 months. Many cells expressed senescence-associated β-galactosidase in 33% of CD44-derived foci at day 60, whereas the expression was observed in 13% of MH-derived ones. The short life of the cells may be due to their cellular senescence. On the other hand, the incorporation of sinusoidal endothelial cells into foci and sinusoid formation, which might be correlated to hepatic maturation, was completed faster in MH-derived foci than in CD44-derived ones. The survival of donor cells may have a close relation to not only early integration into hepatic plates but also the differentiated state of the cells at the time of transplantation.

Key words: CD44; Thy1; Small hepatocytes; Maturation; Sinusoidal endothelial cells; Cellular senescence

Received December 5, 2010; final acceptance April 2, 2011. Online prepub date: June 9, 2011.
1These authors provided equal contribution to this work.
Address correspondence to Toshihiro Mitaka, M.D., Ph.D., Department of Tissue Development and Regeneration, Research Institute for Frontier Medicine, Sapporo Medical University School of Medicine, South-1, West-17, Chuo-ku, Sapporo 060-8556, Japan. Tel: +81-11-611-2111 ext. 2390; Fax: +81-11-615-3099; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 23–37, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X580509
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Improvement of the Cold Storage of Isolated Human Hepatocytes

Gesine Pless,* Igor M. Sauer,† and Ursula Rauen*

*Institut für Physiologische Chemie, Universitätsklinikum Essen, Essen, Germany
†AG Experimentelle Chirurgie, Klinik für Allgemein-, Viszeral- und Transplantationschirurgie, Charité - Universitätsmedizin Berlin, Berlin, Germany

Increasing amounts of human hepatocytes are needed for clinical applications and different fields of research, such as cell transplantation, bioartificial liver support, and pharmacological testing. This demand calls for adequate storage options for isolated human liver cells. As cryopreservation results in severe cryoinjury, short-term storage is currently performed at 2–8°C in preservation solutions developed for the storage of solid organs. However, besides slowing down cell metabolism, cold also induces cell injury, which is, in many cell types, iron dependent and not counteracted by current storage solutions. In this study, we aimed to characterize storage injury to human hepatocytes and develop a customized solution for cold storage of these cells. Human hepatocytes were isolated from material obtained from partial liver resections, seeded in monolayer cultures, and, after a preculture period, stored in the cold in classical and new solutions followed by rewarming in cell culture medium. Human hepatocytes displayed cold-induced injury, resulting in >80% cell death (LDH release) after 1 week of cold storage in University of Wisconsin solution or cell culture medium and 3 h of rewarming. Cold-induced injury could be significantly reduced by the addition of the iron chelators deferoxamine and LK 614. Experiments with modified solutions based on the new organ preservation solution Custodiol-N showed that ion-rich variants were better than ion-poor variants, chloride-rich solutions better than chloride-poor solutions, potassium as main cation superior to sodium, and pH 7.0 superior to pH 7.4. LDH release after 2 weeks of cold storage in the thus optimized solution was below 20%, greatly improving cold storage of human hepatocytes. The results were confirmed by the assessment of hepatocellular mitochondrial membrane potential and functional parameters (resazurin reduction, glucagon-stimulated glucose liberation) and thus suggest the use of a customized hepatocyte storage solution for the cold storage of these cells.

Key words: Cold storage; Hypothermia; Human hepatocytes; Preservation solution; Mitochondrial fission

Received April 2, 2010; final acceptance March 1, 2011. Online prepub date: June 7, 2011.
Address correspondence to Prof. Dr. Ursula Rauen, Institut für Physiologische Chemie, Universitätsklinikum Essen, Hufelandstr. 55, 45122 Essen, Germany. Tel: +49-201-723 1620; Fax: +49-201-723 1623; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 39–47, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X582732
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
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Evaluation of Viable β-Cell Mass Is Useful for Selecting Collagenase for Human Islet Isolation: Comparison of Collagenase NB1 and Liberase HI

R. Misawa,*† C. Ricordi,*‡§¶#** A. Miki,* S. Barker,* R. D. Molano,* A. Khan,* S. Miyagawa,† L. Inverardi,*§** R. Alejandro,* A. Pileggi,*‡ and H. Ichii*‡§

*Cell Transplant Center, Diabetes Research Institute, University of Miami Leonard M. Miller School of Medicine, Miami, FL, USA
†Division of Transplantation, Department of Surgery, Shinshu University School of Medicine, Matsumoto, Japan
‡DeWitt Daughtry Family Department of Surgery, University of Miami Leonard M. Miller School of Medicine, Miami, FL, USA
§Department of Medicine, University of Miami Leonard M. Miller School of Medicine, Miami, FL, USA
¶Department of Microbiology and Immunology, University of Miami Leonard M. Miller School of Medicine, Miami, FL, USA
#Miami Transplant Institute, University of Miami Leonard M. Miller School of Medicine, Miami, FL, USA
**Department of Biomedical Engineering, University of Miami Leonard M. Miller School of Medicine, Miami, FL, USA

The selection of enzyme blend is critical for the success of human islet isolations. Liberase HI collagenase (Roche) was introduced in the 1990s and had been widely used for clinical islet transplantation. More recently, a blend collagenase NB1 has been rendered available. The aim of this study was to evaluate the isolation outcomes and islet quality comparing human islet cells processed using NB1 and Liberase HI. A total of 90 isolations processed using NB1 (n = 40) or Liberase HI (n = 50) was retrospectively analyzed. Islet yield, function in vitro and in vivo, cellular (including β-cell-specific) viability and content, as well as isolation-related factors were compared. No significant differences in donor-related factors were found between the groups. There were also no significant differences in islet yields (NB1 vs. Liberase: 263,389 ± 21,550 vs. 324,256 ± 27,192 IEQ; p = n.s., respectively). The pancreata processed with NB1 showed a significantly longer digestion time (18.6 ± 0.7 vs. 14.5 ± 0.5 min, p < 0.01), lower -cell viability (54.3 ± 3.4% vs. 72.0 ± 2.1%, p < 0.01), β -cell mass (93,671 ± 11,150 vs. 148,961 ± 12,812 IEQ, p < 0.01), and viable β-cell mass (47,317 ± 6,486 vs. 106,631 ± 10,228 VβIEQ, p < 0.01) than Liberase HI. In addition, islets obtained with Liberase showed significantly better graft function in in vivo assessment of islet potency. The utilization of collagenase NB1 in human islet isolation was associated with significantly lower β-cell viability, mass, and islet potency in vivo in our series when compared to Liberase HI, even though there was no significant difference in islet yields between the groups. Evaluation of viable β-cell mass contained in human islet preparations will be useful for selecting enzyme blends.

Key words: Islet; Transplantation; Isolation; Collagenase; Cell viability; Potency; Nude mice

Received October 4, 2010; final acceptance March 25, 2011. Online prepub date: September 16, 2011.
Address correspondence to Hirohito Ichii, M.D., Ph.D., at his current address: Division of Transplantation, Department of Surgery, University of California, Irvine, 333 City Boulevard West Suite 1205, Orange, CA 92868, USA. Tel: (714) 456-8429; Fax: (714) 456-8796; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 49–60, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X566262
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Expression of Pro- and Antiapoptotic Molecules of the Bcl-2 Family in Human Islets Postisolation

Peter D. Campbell,*† Anita Weinberg,‡ Jonathan Chee,* Lina Mariana,* Rochelle Ayala,* Wayne J. Hawthorne,§ Philip J. O’Connell,§ Thomas Loudovaris,* Mark J. Cowley,¶ Thomas W. Kay,*† Shane T. Grey,‡ and Helen E. Thomas*†

*St Vincent’s Institute, Fitzroy, Australia
†The University of Melbourne Department of Medicine, St. Vincent’s Hospital, Fitzroy, Australia
‡Immunology Program, The Garvan Institute of Medical Research, Sydney, Australia
§The Centre for Transplant and Renal Research, Westmead Hospital, Westmead, Australia
¶Peter Wills Bioinformatics Centre, The Garvan Institute of Medical Research, Sydney, Australia

Human islets are subjected to a number of stresses before and during their isolation that may influence their survival and engraftment after transplantation. Apoptosis is likely to be activated in response to these stresses. Apoptosis due to intrinsic stresses is regulated by pro- and antiapoptotic members of the Bcl-2 family. While the role of the Bcl-2 family in apoptosis of rodent islets is becoming increasingly understood, little is known about which of these molecules are expressed or required for apoptosis of human islets. This study investigated the expression of the Bcl-2 family of molecules in isolated human islets. RNA and protein lysates were extracted from human islets immediately postisolation. At the same time, standard quality control assays including viability staining and β-cell content were performed on each islet preparation. Microarrays, RT-PCR, and Western blotting were performed on islet RNA and protein. The prosurvival molecules Bcl-xl and Mcl-1, but not Bcl-2, were highly expressed. The multidomain proapoptotic effector molecule Bax was expressed at higher levels than Bak. Proapoptotic BH3-only molecules were expressed at low levels, with Bid being the most abundant. The proapoptotic molecules BNIP3, BNIP3L, and Beclin-1 were all highly expressed, indicating exposure of islets to oxygen and nutrient deprivation during isolation. Our data provide a comprehensive analysis of expression levels of pro- and antiapoptotic Bcl-2 family members in isolated human islets. Knowledge of which molecules are expressed will guide future research to understand the apoptotic pathways activated during isolation or after transplantation. This is crucial for the design of methods to achieve improved transplantation outcomes.

Key words: Human islet transplantation; Bcl-2; Apoptosis

Received September 13, 2010; final acceptance February 16, 2011. Online prepub date: April 29, 2011.
Address correspondence to Dr. Helen Thomas, St. Vincent’s Institute, 41 Victoria Parade, Fitzroy, VIC, 3065, Australia. Tel: 61-3-9288-3282; Fax: 61-3-9416-2676; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it

Cell Transplantation, Vol. 21, pp. 61–71, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X580563
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Tissue Factor Knockdown in Porcine Islets: An Effective Approach to Suppressing the Instant Blood-Mediated Inflammatory Reaction

Xiaoqian Ma, Bin Ye, Feng Gao, Qi Liang, Qiong Dong, Yin Liu, Pengfei Rong, Wei Wang, and Shounan Yi

Cell Transplantation and Gene Therapy Institute, The Third Xiang Ya Hospital of Central South University, Changsha, Hunan, China

Tissue factor (TF) expression on islets has been shown to trigger instant blood-mediated inflammatory reaction (IBMIR), leading to rapid islet loss in portal vein islet transplantation. This study investigated whether antisense RNA-mediated TF gene knockdown in islets could suppress IBMIR as a strategy to overcome IBMIR. Neonatal porcine islet cell clusters (NICCs) were transfected with or without TF-specific antisense RNA or a nonspecific RNA by a lipid-based method. Expression of both TF gene and protein in NICCs was analyzed after transfection by real-time PCR, Western blot, and FACS, respectively. The impact of antisense RNA transfection on NICC viability and in vitro function was examined by FACS and insulin release test, respectively. The effect of TF knockdown in NICCs on IBMIR was assessed with an in vitro tubing loop assay using human blood. A significant reduction in TF gene and protein expression was achieved in TF antisense RNA but not control RNA transfected NICCs, which did not affect NICCs’ viability or their insulin secreting capacity. Incubation of TF antisense RNA transfected with human blood resulted in a considerable reduction in blood clot formation, platelet consumption, and complement and coagulation activation compared to that observed in the loops containing human blood and untreated or control RNA transfected NICCs. Consistent with these findings, infiltrating neutrophils in the blood clots with entrapped TF antisense RNA transfected NICCs was also reduced substantially compared to that seen in the clots containing untreated or control RNA transfected NICCs. This study presents a nontoxic TF antisense RNA-mediated TF knockdown in porcine islets that leads to an effective suppression of IBMIR, suggesting a potentially new strategy to improve islet transplantation outcomes.

Key words: Instant blood-mediated inflammatory reaction (IBMIR); Tissue factor; Porcine islets; Islet transplantation; Antisense RNA transfection; Gene knockdown

Received September 8, 2010; final acceptance March 10, 2011. Online prepub date: June 7, 2011.
Address correspondence to Wei Wang, M.D., Ph.D., Cell Transplantation and Gene Therapy Institute, The Third Xiang Ya Hospital of Central South University, Changsha, Hunan 410013, China. Tel: 86-731-88618411; Fax: 86-731-8852936; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it or Shounan Yi, M.D., Ph.D., Cell Transplantation and Gene Therapy Institute, The Third Xiang Ya Hospital of Central South University, Changsha, Hunan 410013, China. Tel: 86-731-88618411; Fax: 86-731-8852936; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 73–90, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X580635
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
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In Vitro Generation of Pancreatic Endocrine Cells From Human Adult Fibroblast-Like Limbal Stem Cells

Angela Criscimanna,* Giovanni Zito,* Annalisa Taddeo,* Pierina Richiusa,* Maria Pitrone,* Daniele Morreale,† Gaetano Lodato,† Giuseppe Pizzolanti,* Roberto Citarrella,* Aldo Galluzzo,* and Carla Giordano*‡

*Sezione di Endocrinologia, Dipartimento Biomedico di Medicina Interna e Specialistica (Di.Bi.M.I.S.), Università degli Studi di Palermo, Palermo, Italy
†Sezione di Oftalmologia, Dipartimento di Biomedicina Sperimentale e Neuroscienze Cliniche (BioNeC), Università degli Studi di Palermo, Palermo, Italy
‡Institute of Biomedicine and Molecular Immunology “A. Monroy” (CNR-IBIM), Palermo, Italy

Stem cells might provide unlimited supply of transplantable cells for β-cell replacement therapy in diabetes. The human limbus is a highly specialized region hosting a well-recognized population of epithelial stem cells, which sustain the continuous renewal of the cornea, and the recently identified stromal fibroblast-like stem cells (f-LSCs), with apparent broader plasticity. However, the lack of specific molecular markers for the identification of the multipotent limbal subpopulation has so far limited the investigation of their differentiation potential. In this study we show that the human limbus contains uncommitted cells that could be potentially harnessed for the treatment of diabetes. Fourteen limbal biopsies were obtained from patients undergoing surgery for ocular diseases not involving the conjunctiva or corneal surface. We identified a subpopulation of f-LSCs characterized by robust proliferative capacity, expressing several pluripotent stem cell markers and exhibiting self-renewal ability. We then demonstrated the potential of f-LSCs to differentiate in vitro into functional insulin-secreting cells by developing a four-step differentiation protocol that efficiently directed f-LSCs towards the pancreatic endocrine cell fate. The expression of specific endodermal, pancreatic, islet, and β-cell markers, as well as functional properties of f-LSC-derived insulin-producing cells, were evaluated during differentiation. With our stage-specific approach, up to 77% of f-LSCs eventually differentiated into cells expressing insulin (also assessed as C-peptide) and exhibited phenotypic features of mature β-cells, such as expression of critical transcription factors and presence of secretory granules. Although insulin content was about 160-fold lower than what observed in adult islets, differentiated cells processed ~98% of their proinsulin content, similar to mature β-cells. Moreover, they responded in vitro in a regulated manner to multiple secretory stimuli, including glucose. In conclusion, f-LSCs represent a possible relevant source of autologous, transplantable, insulin-producing cells that could be tested for the reversal of diabetes.

Key words: Limbus; Fibroblast-like stem cells; β-Cells; Diabetes

Received October 22, 2010; final acceptance March 14, 2011. Online prepub date: June 9, 2011.
Address correspondence to Carla Giordano, Sezione di Endocrinologia, Dipartimento Biomedico di Medicina Interna e Specialistica (Di.Bi.M.I.S.), Università degli Studi di Palermo, Piazza delle Cliniche 2, 90127 Palermo, Italy. Tel: +39 0916552109; Fax: +39 0916552123; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 91–98, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X580617
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
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Secretory Unit of Islet Transplant Objects (SUITO) Index Can Predict Severity of Hypoglycemic Episodes in Clinical Islet Cell Transplantation

Morihito Takita,* Shinichi Matsumoto,* Huanying Qin,† Hirofumi Noguchi,* Masayuki Shimoda,‡ Daisuke Chujo,§ Takeshi Itoh,* Koji Sugimoto,* Nicholas Onaca,¶ Bashoo Naziruddin,¶ and Marlon F. Levy¶

*Islet Cell Laboratory, Baylor Research Institute Fort Worth Campus, Fort Worth, TX, USA
†Institute for Health Care Research and Improvement, Baylor Health Care System, Dallas, TX, USA
‡Baylor University Medical Center, Dallas, TX, USA
§Baylor Research Institute for Immunology Research, Dallas, TX, USA
¶Baylor Regional Transplant Institute, Dallas, TX, USA

One endpoint of clinical islet cell transplantation for type 1 diabetic patients is the elimination or reduction of hypoglycemia. We previously developed a simple tool to evaluate islet graft function: the secretory unit of islet transplant objects (SUITO) index. The aim of this study is to clarify the association between the SUITO index and hypoglycemic episodes. Data from 310 clinical evaluations of 11 islet recipients were included in this study. Fasting plasma C-peptide and glucose levels were measured at every evaluation. The SUITO index was calculated according to the following formula: 1500 × C-peptide level (ng/ml)/[blood glucose level (mg/dl) − 63]. The number of hypoglycemic events (<3.8 mmol/L) and severe hypoglycemic events (<2.2 mmol/L or hypoglycemic unawareness) was assessed on the basis of interviews and self-monitoring of blood glucose (SMBG). Receiver operating characteristic (ROC) analysis was performed to determine the cut-off values of the SUITO index for hypoglycemic events. Based on the ROC study, follow-up data after transplantations were divided into the following three groups: low-SUITO (SUITO index <10, n = 91), middle-SUITO (10 ≤SUITO index <26, n = 83), high-SUITO (SUITO index ≤26, n = 125). The frequency of total hypoglycemia in the high-SUITO group was significantly decreased when compared to the other groups (value with Kruskal-Wallis test p < 0.001). The frequency of total severe hypoglycemia was significantly decreased in the low-SUITO group compared to pretransplant status and further decreased in the middle- and high-SUITO group. Spearman correlation coefficients were −0.663 (p < 0.001) between the number of total hypoglycemic events per one month and the SUITO index and −0.521 (p < 0.001) between that of severe events and the SUITO index. The SUITO index could predict the severity of hypoglycemic episodes in type 1 diabetic patients who received islet cell transplantations.

Key words: Islet graft function; Hypoglycemia; Type 1 diabetes; SUITO index

Received August 10, 2010; final acceptance March 15, 2011. Online prepub date: June 9, 2011.
Address correspondence to Shinichi Matsumoto, M.D., Ph.D., Islet Cell Laboratory, Baylor Research Institute Fort Worth Campus, 1400 8th Ave., Fort Worth, TX 76104, USA. Tel: 817-922-2570; Fax: 817-922-4655; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it or Morihito Takita, M.D., Islet Cell Laboratory, Baylor Research Institute Fort Worth Campus, 1400 8th Ave., Forth Worth, TX 76104, USA. Tel: 817-922-2573; Fax: 817-922-4655; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 99–111, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X582750
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
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B7-H4 Induces Donor-Specific Tolerance in Mouse Islet Allografts

Xiaojie Wang,* Jianqiang Hao,* Daniel L. Metzger,† Alice Mui,* Ziliang Ao,* C. Bruce Verchere,‡ Lieping Chen,§ Dawei Ou,* and Garth L. Warnock*

*Department of Surgery, University of British Columbia, Vancouver, BC, Canada
†Department of Paediatrics, University of British Columbia, Vancouver, BC, Canada
‡Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada
§Department of Immunobiology, Yale University School of Medicine, New Haven, CT, USA

Negative cosignaling molecules play an important role in regulating T-cell responses to alloantigen stimulation. We recently reported that adenoviral-mediated transduction of islet allografts with B7-H4 inhibits allograft rejection. In this study, we investigate the mechanism for B7-H4-induced prolongation of mouse islet allograft survival. Streptozotocin-induced diabetic C57BL/6 mice were rendered normoglycemic by renal subcapsular implants of B7-H4-transduced BALB/c islets. Grafts and spleens were removed after days 2, 10, and 60 (n = 8 each) for characterization of kinetics of Foxp3 and interleukin 10 (IL-10) expression. Mixed lymphocyte reaction (MLR) was done at day 60. Ten mice were subjected to nephrectomy at 60 days and then five were implanted with secondary BALB/c islets and five were given third-party CBA/J islets. An increase in Foxp3 and IL-10 mRNA expression was detected in recipients’ spleens at day 60 and this was associated with increased quantities of Foxp3+ cells. Splenocytes at day 60 showed hyporesponsiveness during MLR to alloantigen stimulation. Proliferation was partially restored after CD25+ T-cell depletion. Secondary BALB/c islets survived for 79 ± 29 days compared with 21 ± 3.6 days for CBA/J islets (p < 0.001). Local expression of B7-H4 induces long-term unresponsiveness to donor-specific alloantigens, and is associated with T regulatory cells, suggesting the development of tolerance.

Key words: B7-H4; Islet allograft tolerance; Regulatory T cells; Secondary transplantation

Received November 8, 2010; final acceptance April 5, 2011. Online prepub date: September 16, 2011.
Address correspondence to Garth L. Warnock, M.D., Department of Surgery, University of British Columbia, 3100, 910 West 10th Avenue, Vancouver, BC V5Z 4E3, Canada. Tel: (604) 875-4136; Fax: (604) 875-4376; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 113–125, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X586747
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
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Quantitative In Situ Analysis of FoxP3+ T Regulatory Cells on Transplant Tissue Using Laser Scanning Cytometry

Hidenori Takahashi,*† Phillip Ruiz,*† Camillo Ricordi,*†‡ Victor Delacruz,*† Atsushi Miki,‡ Atsuyoshi Mita,‡ Ryosuke Misawa,‡ Scott Barker,‡ George W. Burke,*† Andreas G. Tzakis,*† and Hirohito Ichii*†‡

*Miami Transplant Institute, University of Miami Leonard M. Miller School of Medicine, Miami, FL, USA
†Department of Surgery, University of Miami Leonard M. Miller School of Medicine, Miami, FL, USA
‡Cell Transplant Center, Diabetes Research Institute, University of Miami Leonard M. Miller School of Medicine, Miami, FL, USA

There is abundant evidence that immune cells infiltrating into a transplanted organ play a critical role for destructive inflammatory or regulatory immune reactions. Quantitative in situ analysis (i.e., in tissue sections) of immune cells remains challenging due to a lack of objective methodology. Laser scanning cytometry (LSC) is an imaging-based methodology that performs quantitative measurements on fluorescently and/or chromatically stained tissue or cellular specimens at a single-cell level. In this study, we have developed a novel objective method for analysis of immune cells, including Foxp3+ T regulatory cells (Tregs), on formalin-fixed /paraffin-embedded (FFPE) transplant biopsy sections using iCys® Research Imaging Cytometer. The development of multiple immunofluorescent staining was established using FFPE human tonsil sample. The CD4/CD8 ratio and the population of Tregs among CD4+cells were analyzed using iCys and compared with the results from conventional flow cytometry analysis (FCM). Our multiple immunofluorescent staining techniques allow obtaining clear staining on FFPE sections. The CD4/CD8 ratio analyzed by iCys was concordant with those obtained by FCM. This method was also applicable for liver, small intestine, kidney, pancreas, and heart transplant biopsy sections and provide an objective quantification of Tregs within the grafts.

Key words: T regulatory cells (Tregs); Foxp3; Laser scanning cytometry (LSC); iCys; Transplant biopsy; Quantification

Received February 24, 2010; final acceptance April 6, 2011. Online prepub date: September 16, 2011.
Address correspondence to Hirohito Ichii, M.D., Ph.D., at his current address: Division of Transplantation, Department of Surgery, University of California, Irvine, 333 City Boulevard West Suite 1205, Orange, CA 92868, USA. Tel: (714) 456-8429; Fax: (714) 456-8796; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 127–137, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X576018
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Fibrin Gel Improves the Survival of Transplanted Myoblasts

Catherine Gerard, Marie Anne Forest, Genevieve Beauregard, Daniel Skuk, and Jacques P. Tremblay

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

Duchenne muscular dystrophy (DMD) is the most frequent muscular dystrophy in children and young adults. Currently, there is no cure for the disease. The transplantation of healthy myoblasts is an experimental therapeutic strategy, since it could restore the expression of dystrophin in DMD muscles. Nevertheless, this cellular therapy is limited by immune reaction, low migration of the implanted cells, and high early cell death that could be at least partially due to anoikis. To avoid the lack of attachment of the cells to an extracellular matrix after the transplantation, which is the cause of anoikis, we tested the use of a fibrin gel for myoblast transplantation. In vitro, three concentrations of fibrinogen were compared (3, 20, and 50 mg/ml) to form a fibrin gel. A stiffer fibrin gel leads to less degradability and less proliferation of the cells. A concentration of 3 mg/ml fibrin gel enhanced the differentiation of the myoblasts earlier as a culture in monolayer. Human myoblasts were also transplanted in muscles of Rag/mdx mice in a fibrin gel or in a saline solution (control). The use of 3 mg/ml fibrin gel for cell transplantation increased not only the survival of the cells as measured after 5 days but also the number of fibers expressing dystrophin after 21 days, compared to the control. Moreover, the fibrin gel was also compared to a prosurvival cocktail. The survival of the myoblasts at 5 days was increased in both conditions compared to the control but the efficacy of the prosurvival cocktail was not significantly higher than the fibrin gel.

Key words: Duchenne muscular dystrophy (DMD); Fibrin gel; Cell transplantation; Cell survival; Graft success

Received July 19, 2010; final acceptance February 16, 2011. Online prepub date: April 29, 2011.
Address correspondence to Jacques P. Tremblay Ph.D., Unité de Recherche en Génétique Humaine (RC 9300), Centre de recherche du CHUL, 2705 boulevard Laurier, Québec, QC, Canada, G1V 4G2. Tel: (418) 654-2186; Fax: (418) 654-2207; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 139–152, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X576045
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
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Losartan Enhances the Success of Myoblast Transplantation

Raouia Fakhfakh, Yann Lamarre, Daniel Skuk, and Jacques P. Tremblay

Unité de recherche de recherche en Génétique Humaine, Centre de recherche de CHUL, CHUQ, Faculté de médecine, Université Laval, Québec, QC, Canada

Duchenne muscular dystrophy is a recessive X-linked genetic disease caused by dystrophin gene mutations. Cell therapy can be a potential approach aiming to introduce a functional dystrophin in the dystrophic patient myofibers. However, this strategy produced so far limited results. Transforming growth factor-β (TGF-β) is a negative regulator of skeletal muscle development and is responsible for limiting myogenic regeneration. The combination of TGF-β  signaling inhibition with myoblast transplantation can be an effective therapeutic approach in dystrophin-deficient patients. Our aim was to verify whether the success of human myoblast transplantation in immunodeficient dystrophic mice is enhanced with losartan, a molecule that downregulates TGF-β  expression. In vitro, blocking TGF-β  activity with losartan increased proliferation and fusion and decreased apoptosis in human myoblasts. In vivo, human myoblasts were transplanted in mice treated with oral losartan. Immunodetection of human dystrophin in tibialis anterior cross sections 1 month posttransplantation revealed more human dystrophin-positive myofibers in these mice than in nontreated dystrophic mice. Thus, blocking the TGF-β  signal with losartan treatment improved the success of myoblast transplantation probably by increasing myoblast proliferation and fusion, decreasing macrophage activation, and changing the expression of myogenic regulator factors.

Key words: Duchenne muscular dystrophy (DMD); Transforming growth factor- β1 (TGF- β1); Myoblast transplantation; Apoptosis; Inflammation

Received October 4, 2010; final acceptance March 8, 2011. Online prepub date: April 29, 2011.
Address correspondence to Jacques P. Tremblay, Ph.D., Unité de recherche en Génétique Humaine, Centre de recherche du CHUL,2705, Boulevard Laurier, Québec, QC, G1V 4G2, Canada. Tel: (418) 654-2186; Fax: (418) 654-2207; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 153–173, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X580554
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
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Myogenic Properties of Human Mesenchymal Stem Cells Derived From Three Different Sources

Anabel S. de la Garza-Rodea, Ietje van der Velde-van Dijke, Hester Boersma, Manuel A. F. V. Gonçalves, Dirk W. van Bekkum, Antoine A. F. de Vries, and Shoshan Knaän-Shanzer

Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, the Netherlands

Mesenchymal stem cells (MSCs) of mammals have been isolated from many tissues and are characterized by their aptitude to differentiate into bone, cartilage, and fat. Differentiation into cells of other lineages like skeletal muscle, tendon/ligament, nervous tissue, and epithelium has been attained with MSCs derived from some tissues. Whether such abilities are shared by MSCs of all tissues is unknown. We therefore compared for three human donors the myogenic properties of MSCs from adipose tissue (AT), bone marrow (BM), and synovial membrane (SM). Our data show that human MSCs derived from the three tissues differ in phenotype, proliferation capacity, and differentiation potential. The division rate of AT-derived MSCs (ATMSCs) was distinctly higher than that of MSCs from the other two tissue sources. In addition, clear donor-specific differences in the long-term maintenance of MSC proliferation ability were observed. Although similar in their in vitro fusogenic capacity with murine myoblasts, MSCs of the three sources contributed to a different extent to skeletal muscle regeneration in vivo. Transplanting human AT-, BM-, or SM-MSCs previously transduced with a lentiviral vector encoding β-galactosidase into cardiotoxin-damaged tibialis anterior muscles (TAMs) of immunodeficient mice revealed that at 30 days after treatment the frequency of hybrid myofibers was highest in the TAMs treated with AT-MSCs. Our finding of human-specific β-spectrin and dystrophin in hybrid myofibers containing human nuclei argues for myogenic programming of MSCs in regenerating murine skeletal muscle. For the further development of MSC-based treatments of myopathies, AT-MSCs appear to be the best choice in view of their efficient contribution to myoregeneration, their high ex vivo expansion potential, and because their harvesting is less demanding than that of BM- or SM-MSCs.

Key words: Mesenchymal stem cells (MSCs); Myogenic differentiation; Cell therapy; Skeletal muscle regeneration; NOD/SCID mice

Received September 6, 2010; final acceptance March 10, 2011. Online prepub date: June 7, 2011.
Address correspondence to Shoshan Knaän-Shanzer, Ph.D., Virus and Stem Cell Biology Laboratory, Department of Molecular Cell Biology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, the Netherlands. Tel: +31-(0)71-526-9246; Fax: +31-(0)71-526-8270; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it or as This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 175–189, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368910X550233
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
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Autologous Lung-Derived Mesenchymal Stem Cell Transplantation in Experimental Emphysema

Edward P. Ingenito,* Larry Tsai,* Shankar Murthy,* Shivraj Tyagi,* Melissa Mazan,† and Andrew Hoffman†

*Division of Pulmonary & Critical Care Medicine, Brigham and Women’s Hospital, Boston, MA, USA
†Tufts Cummings School of Veterinary Medicine, N. Grafton, MA, USA

Autologous lung-derived mesenchymal stem cells (LMSCs) were transplanted endoscopically into sheep with experimental emphysema to assess their capacity to regenerate functional tissue. LMSC lines were derived from transbronchial biopsies, cloned at passage 2, expanded in culture, and labeled. A delivery scaffold containing 1% fibrinogen, 20 μg/ml of fibronectin, and 20 μg/ml of poly-L-lysine was used to promote cell attachment and spreading. Treatment animals received scaffold containing 5–10 × 106 cells/site; control animals received scaffold alone. Phenotypic markers, differentiation capacity, extracellular matrix protein expression, and paracrine function of LMSCs were characterized in vitro. Responses to LMSC transplantation in vivo were assessed in terms of clinical toxicity, lung physiology, change in tissue mass (measured by CT scanning) and perfusion (measured by scintigraphy scanning), and tissue histology. At 4-week follow-up, transplants were well tolerated and associated with increased tissue mass and lung perfusion compared to control treatment. Histology confirmed cell retention, increased cellularity, and increased extracellular matrix content following LMSC treatment. Labeled cells were distributed in the alveolar septum and peribronchiolar interstitium. Some label was also present within phagocytes, indicating that a fraction of autologous LMSCs do not survive transplantation. These results suggest that endobronchial delivery of autologous LMSCs has potential therapeutic utility for regenerating functional lung in emphysema.

Key words: Cell therapy; Mesenchymal stem cells; Cell engraftment

Received February 13, 2010; final acceptance December 4, 2010. Online prepub date: February 3, 2011.
Address correspondence to Edward P. Ingenito, M.D., Ph.D., Pulmonary and Critical Care Medicine, Brigham and Women’s Hospital, 75 Francis Street, Boston, MA 02115, USA. Tel: 617-833-8531; Fax: 617-732-7421; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 191–205, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X593118
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
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Cationic Gd-DTPA Liposomes for Highly Efficient Labeling of Mesenchymal Stem Cells and Cell Tracking With MRI

Jamal Guenoun,* Gerben A. Koning,† Gabriela Doeswijk,* Lizanne Bosman,* Piotr A. Wielopolski,* Gabriel P. Krestin,* and Monique R. Bernsen*

*Department of Radiology, Erasmus MC-University Medical Center Rotterdam, Rotterdam, The Netherlands
†Laboratory of Experimental Surgical Oncology, Section Surgical Oncology, Department of Surgery, Erasmus MC-University Medical Center Rotterdam, Rotterdam, The Netherlands

In the current study cell labeling was performed with water-soluble gadolinium (Gd)-DTPA containing liposomes, to allow for cell tracking by MRI. Liposomes were used to assure a highly concentrated intracellular build up of Gd, aiming to overcome the relatively low MRI sensitivity of Gd (compared to T2 contrast agents). Liposomes were positively charged (cationic) to facilitate uptake by binding to anionic charges in the cell membrane of bone marrow-derived mesenchymal stem cells (MSCs). We determined the cellular Gd load by variations in labeling time (1, 4, and 24 h) and liposome concentration (125, 250, 500, 1000 μM lipid), closely monitoring effects on cell viability, proliferation rate, and differentiation ability. Labeling was both time and dose dependent. Labeling for 4 h was most efficient regarding the combination of processing time and final cellular Gd uptake. Labeling for 4 h with low-dose concentration (125 μM lipid, corresponding to 52 ± 3 μM Gd) yielded an intracellular load of 30 ± 2.5 pg Gd cell−1, without any effects on cell viability, proliferation, and cell differentiation. Gd liposomes, colabeled with fluorescent dyes, exhibited a prolonged cellular retention, with an endosomal distribution pattern. In vitro assay over 20 days demonstrated a drop in the average Gd load per cell, as a result of mitosis. However, there was no significant change in the sum of the Gd load in all daughter cells at endpoint (20 days), indicating an excellent cellular retention of Gd. MSCs labeled with Gd liposomes were imaged with MRI at both 1.5T and 3.0T, resulting in excellent visualization both in vitro and in vivo. Prolonged in vivo imaging of 500,000 Gd-labeled cells was possible for at least 2 weeks (3.0T). In conclusion, Gd-loaded cationic liposomes (125 μM lipid) are an excellent candidate to label cells, without detrimental effects on cell viability, proliferation, and differentiation, and can be visualized by MRI.

Key words: Cell labeling; Gadolinium; Liposomes; Magnetic resonance imaging (MRI); Cell tracking; Cell transplantation

Received October 19, 2010; final acceptance April 16, 2011. Online prepub date: September 16, 2011.
Address correspondence to Monique R. Bernsen, Ph.D., Department of Radiology Ee-2338, Erasmus MC-University Medical Center Rotterdam, Dr. Molewaterplein 50, 3015 GE Rotterdam, The Netherlands. Tel: +31-10-7044075; Fax: +31-10-7044742; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 207–216, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X586756
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Banking Human Umbilical Cord-Derived Mesenchymal Stromal Cells for Clinical Use

Wei Gong,*1 Zhibo Han,†1 Hui Zhao,*‡1 Youwei Wang,* Jiming Wang,* Jian Zhong,* Bin Wang,* Shanshan Wang,* Yongjuan Wang,* Lingyun Sun,§ and Zhongchao Han*†

*National Engineering Research Center of Cell Products, AmCellGene Co. Ltd, TEDA, Tianjin, China
†Institute of Hematology and Blood Disease Hospital, Chinese Academy of Medical Sciences and Peking Union of Medical College, Tianjin, China
‡Tianjin Key Laboratory of Food and Biotechnology, School of Biotechnology and Food Science, Tianjin University of Commerce, Tianjin, China
§Department of Rheumatology and Immunology, The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing, China

A great deal of interest has arisen recently with respect to human mesenchymal stem cells (MSCs), due to their broad therapeutic potential. However, the safety and efficacy of MSCs expanded ex vivo for clinical applications remain a concern. In this article, we establish a standardized process for manufacture of human umbilical cord-derived MSCs (UC-MSCs), which encompasses donor screening and testing, recovery, two-stage expansion, and administration. The biological properties and safety of UC-MSCs were then characterized and tested. The safety data from use in human patients have also been reported. After clinical-scale expansion, a yield of 1.03–3.78 × 108 MSCs was achieved in 10 batch manufacturing runs. The biological properties, such as plastic adherence, morphology, specific surface antigen (CD105, CD73, CD90, positive ≥95%; CD45, CD34, CD31, CD11b, CD19, HLA-DR, negative ≤2%), and multipotent differentiation potential (osteogenesis and adipogenesis) were retained. Bacterial and mycoplasma tests were negative and endotoxin levels were lower than 2 EU/ml. No adverse events were noted in two patients treated with intravenously and/or intrathecally administered MSCs. The data obtained indicate that banking UC-MSCs for clinical use is feasible.

Key words: Mesenchymal stem cells; Umbilical cord; Bank; Multiple sclerosis; Graft versus host disease

Received March 17, 2010; final acceptance April 18, 2011. Online prepub date: September 16, 2011.
1These authors contributed equally to this work.
Address correspondence to Zhongchao Han, M.D., Ph.D., Professor, Institute of Hematology Chinese Academy of Medical Sciences and Peking Union of Medical College, 288 Nanjing Road, Tianjin, China 300020. E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 217–234, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X582723
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
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Derivation of High-Purity Definitive Endoderm From Human Parthenogenetic Stem Cells Using an In Vitro Analog of the Primitive Streak

Nikolay Turovets,* Jeffrey Fair,† Richard West,‡ Alina Ostrowska,* Ruslan Semechkin,* Jeffrey Janus,* Li Cui,§ Vladimir Agapov,* Irina Turovets,* Andrey Semechkin,* Marie Csete,§ and Larissa Agapova*

*International Stem Cell Corporation, Oceanside, CA, USA
†Cedars-Sinai Medical Center, Los Angeles, CA, USA
‡West Labs Scientific, Grand Rapids, MI, USA
§Organovo, San Diego, CA, USA

Human parthenogenetic stem cells (hpSCs) are pluripotent stem cells with enormous potential as cell sources for cell-based therapies: hpSCs may have histocompatibilty advantages over human embryonic stem cells (hESCs) and derivation of hpSCs does not require viable blastocyst destruction. For translation of all pluripotent stem cell-based therapies, derivation of differentiated cell products that are not contaminated with undifferentiated cells is a major technical roadblock. We report here a novel method to derive high-purity definitive endoderm (DE) from hpSCs, based on reproducing features of the normal human embryonic microenvironment. The method mimics the developmental process of transition through a primitive streak, using a differentiation device that incorporates a three-dimensional extracellular matrix (ECM) combined with a porous membrane. Treatment of undifferentiated hpSCs above the membrane results an epithelial-tomesenchymal transition (EMT); thus, responsive cells acquire the ability to migrate through the membrane into the ECM, where they differentiate into DE. Importantly, the resultant DE is highly purified, and is not contaminated by undifferentiated cells, as assessed by OCT4 expression using immunocytochemistry and flow cytometry. The functional properties of the DE are also preserved by the process: DE differentiated in the device can generate a highly enriched population of hepatocyte-like cells (HLCs) characterized by expression of hepatic lineage markers, indocyanine green clearance, glycogen storage, cytochrome P450 activity, and engraftment in the liver after transplantation into immunodeficient mice. The method is broadly applicable and we obtained purified DE using hESCs, as well as several hpSC lines. The novel method described here represents a significant step toward the efficient generation of high-purity cells derived from DE, including hepatocytes and pancreatic endocrine cells, for use in regenerative medicine and drug discovery, as well as a platform for studying cell fate specification and behavior during development.

Key words: Human parthenogenetic stem cells (hpSCs); Human embryonic stem cells (hESCs); Differentiation; Definitive endoderm (DE); Extracellular matrix (ECM); Hepatocytes

Received September 28, 2010; final acceptance April 3, 2011. Online prepub date: June 9, 2011.
Address correspondence to Larissa Agapova, International Stem Cell Corporation, 2595 Jason Court, Oceanside, CA 92056, USA. Tel: 1-760-940-6383; Fax: 1-760-940-6387; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 235–250, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X580518
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
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Human Adipose Tissue as a Source of Cells With Angiogenic Potential

Krisztina Szöke, Karen Johanne Beckstrøm, and Jan E. Brinchmann

Norwegian Center for Stem Cell Research, Institute of Immunology, Oslo University Hospital, Rikshospitalet and Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway

Endothelial cells (ECs) are involved in the process of angiogenesis, the outgrowth of new vessels from preexisting blood vessels. If available in sufficiently large numbers, ECs could be used therapeutically to establish blood flow through in vitro engineered tissues and tissues suffering from severe ischemia. Adipose tissue (AT) is an easily available source of large number of autologous ECs. Here we describe the isolation, in vitro expansion, and characterization of human AT derived ECs (AT-ECs). AT-ECs proliferated rapidly through 15–20 population doublings. The cultured cells showed cobblestone morphology and expressed EC markers including CD31, CD144, eNOS, CD309, CD105, von Willebrand factor, CD146, CD54, and CD102. They bound Ulex europaeus agglutinin I lectin and took up DiI-Ac-LDL. The AT-ECs formed capillary-like tubes in Matrigel in vitro and formed functional blood vessels in Matrigel following subcutaneous injection into immunodeficient mice. In conclusion, AT-ECs reach clinically significant cell numbers after few population doublings and are easily accessible from autologous AT, which also contains mesenchymal stem cells/pericytes. Thus, AT yields two cell populations that may be used together in the treatment of tissue ischemia and in clinical applications of tissue engineering.

Key words: Adipose tissue (AT); Endothelial cells (ECs); Vasculogenesis; Tissue ischemia; Tissue engineering

Received November 8, 2010; final acceptance March 5, 2011. Online prepub date: June 7, 2011.
Address correspondence to Jan E. Brinchmann, Norwegian Center for Stem Cell Research, Institute of Immunology, Oslo University Hospital, Rikshospitalet and Institute of Basic Medical Sciences, University of Oslo, PO Box 1121 Blindern, 0317 Oslo, Norway. Tel: +47 22 84 04 89; Fax: +47 22 85 10 58; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 251–267, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X580581
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
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Transcriptional Profiling of the Hematopoietic Support of Interleukin-Stimulated Human Umbilical Vein Endothelial Cells (HUVECs)

Gürkan Bal,* Julian Kamhieh-Milz,* Matthias Futschik,† Thomas Häupl,‡ Abdulgabar Salama,* and Anja Moldenhauer*

*Institute for Transfusion Medicine, Charité-Universitätsmedizin Berlin, Berlin, Germany
†Institute for Theoretical Biology, Humboldt-University Berlin, Berlin, Germany
‡Department of Rheumatology and Clinical Immunology, Charité-Universitätsmedizin Berlin, Berlin, Germany

Endothelial cells can be successfully used to maintain or increase the number of hematopoietic stem cells in vitro. Previously we identified hematopoietic progenitor cell (HPC) expansion or survival benefit induced by IL-1β-, IL-3-, and IL-6-stimulated human umbilical vein endothelial cell (HUVEC) supernatants. In order to identify molecular mechanisms that support hematopoiesis, we examined the time-dependent expression profiles of IL-1β -, IL-3-, and IL-6-stimulated HUVECs via microarray. Here, we present 24 common upregulated elements and three common downregulated elements of IL-1β - and IL-3-stimulated HUVECs, with these factors exhibiting great potential for the observed HPC expansion. Furthermore, metabolic pathway analysis resulted in the identification of nonproteinogenic factors such as prostaglandin E2 (PGE2) and nitric oxide (NO) and determined their HPC expansion potential via delta, methylcellulose, and cobblestone assays. We confirmed PGE2 and spermine as hematopoietic expansion factors. Furthermore, we identified several factors such as SSAT, extracellular matrix components, microRNA21, and a microvesicle-mediated crosstalk between the endothelium and HPCs that may play a crucial role in determining stem cell fate. Our results suggest that microarray in combination with functional annotations is a convenient method to identify novel factors with great impact on HPC proliferation and differentiation.

Key words: Microarray; Human umbilical vein endothelial cells (HUVECs); Interleukin; Hematopoietic growth factors; Hematopoietic stem cells

Received September 10, 2010; final acceptance March 14, 2011. Online prepub date: June 7, 2011.
Address correspondence to Gürkan Bal, Institute for Transfusion Medicine, Campus Virchow-Klinikum, Charité-Universitätsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany. Tel: +49-30-450 553139; Fax: +49-30-450 565904; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 269–283, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368910X564535
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
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Bypassing the Patient: Comparison of Biocompatible Models for the Future of Vascular Tissue Engineering

L. Khait and R. K. Birla

Section of Cardiac Surgery, University of Michigan, Ann Arbor, MI, USA

The objective of vascular tissue engineering is to develop tissue-engineered, biocompatible, small-diameter vessels suitable to withstand in vivo systolic pressures as well as be immunologically compatible with the patient, in order to minimize graft rejection. In this study, we present and compare two models of biocompatible, tissue-engineered vascular grafts (TEVG), using chitosan and acellularized rat aortas as options for scaffolds. Human aortic adventitial smooth muscle cells and fibroblasts were seeded onto a fibrin gel and subsequently wrapped around either of the two scaffolds. After several weeks of maturation in standard culturing conditions, the graft models were analyzed and compared by mechanical testing, cell viability, and histology. Histological and viability data showed that both models were viable and developed similarly, with a scaffold surrounded by two layers of cells, the fibroblasts lying on top of the smooth muscle cells. Both models responded to 200 mM potassium chloride (KCl) (tensions of 38 ± 3, 78 ± 13, and 52 ± 7 μN) and 25 mM 8-bromo-cyclic AMP (tensions of −23 ± 4, −39 ± 10, and −31 ± 12 μN) stimulation by vasoconstriction and vasorelaxation (n = 3), respectively; however, the chitosan model was unable to maintain the contracted and relaxed tension. Because the acellularized aorta TEVGs were able to maintain stimulated tension better than chitosan TEVGs, we concluded that the acellularized aorta model was better suited for further development.

Key words: Vascular graft; Tissue engineering; Fibrinogen/fibrin; Smooth muscle cells; Fibroblasts; Endothelial cells; Vasocontraction; Vasorelaxation

Received July 10, 2008; final acceptance December 15, 2010. Online prepub date: March 9, 2011.
Address correspondence to Ravi K. Birla, at his current address: Assistant Professor, Paul H. and Donna D. Flower Early Career Professor, Department of Biomedical Engineering, Tulane University, KLindy Boggs Building, Rm. 536, New Orleans, LA, 70118, USA. Tel: 504-865-5851; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 285–297, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X580608
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
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Seeding of Endothelial Cells on the Luminal Surface of a Sheet Model of Cold-Stored (at 4°C) Sheep Carotid Arteries

Arthur Smardencas* and Helena C. Parkington†

*Department of Forensic Medicine, Monash University, Clayton, Victoria, Australia
†Department of Physiology, Monash University, Clayton, Victoria, Australia

Cold-stored arteries are biomaterials that potentially represent an abundant “off-the-shelf” source of vascular grafts for use in vascular surgery. One of the keys to reestablishing the antithrombogenic endothelial cell (EC) lining of cold-stored arterial grafts is to maximize the number of ECs that attach following seeding. In this study, the cold-stored sheep carotid artery is used as a substrate to determine the conditions that maximize EC adherence following seeding. The effect of serum concentration, duration of seeding incubation, seeding density, and period of cold storage on attachment of ECs following seeding of 4-week cold-stored sheep carotid arteries (n = 5 arteries), 8-week cold-stored sheep carotid arteries (n = 5 arteries), and 12-week cold-stored sheep carotid arteries (n = 5 arteries) was examined. Three experiments (serum concentration, time of incubation, and seeding density) were conducted to determine the conditions that maximized the number of cultured sheep carotid artery ECs that attached to cold-stored sheep carotid artery following seeding. A flat sheet model was used. Serum concentration (0%, 10%, 20%, and 30%) in the seeding suspension did not have a significant effect on overall EC adherence on 4-, 8-, and 12-week cold-stored arteries. Time of seeding incubation (30, 60, and 90 min) did not have a significant effect on overall EC adherence on 4-, 8-, and 12-week cold-stored arteries. Seeding density (500,000 cells/ml, 1,000,000 cells/ml, and 2,000,000 cells/ml) had a significant effect (p = 0.036) on overall EC adherence on 4-, 8-, and 12-week cold-stored arteries. The period of cold storage (4, 8, and 12 weeks) of the artery had a significant effect (p = 0.002, p < 0.0001, p < 0.0001 in serum, time, and seeding density experiments, respectively) on overall EC adherence following seeding. Pairwise comparisons of EC adherence revealed the following. In the serum experiment, EC adherence on 4-week cold-stored arteries was significantly greater than on 8-week cold-stored arteries (p = 0.003) and 12-week cold-stored arteries (p = 0.002). This effect was due largely to the significant difference between EC adherence on 4-week and 8-week cold-stored arteries (p = 0.0002) and between EC adherence on 4-week and 12-week cold-stored arteries (p = 0.0091) at 20% serum. In the time experiment, EC adherence on 4-week cold-stored arteries was significantly greater than on 12-week cold-stored arteries (p < 0.0001). In the seeding density experiment, EC adherence on 4-week cold-stored arteries was significantly greater than on 8-week cold-stored arteries (p < 0.0001) and 12-week cold-stored arteries (p < 0.0001). In the same experiment, EC adherence following seeding at a density of 1,000,000 cells/ml and 2,000,000 cells/ml was significantly greater (p = 0.03 and p = 0.02, respectively) than EC adherence following seeding at a density of 500,000 cells/ml. Thus, it was determined that 4-week cold-stored arteries were superior to 8- and 12-week cold-stored arteries in terms of the number of ECs that adhered. It was also determined that a seeding density of 1,000,000 or 2,000,000 cells/ml was superior to a seeding density of 500,000 cells/ml in terms of producing maximal EC attachment. The ideal conditions, from those examined, for maximizing EC attachment to cold-stored arteries were 4 weeks of cold storage, a serum concentration of 20%, a seeding density of 2,000,000 cells/ml, and a duration of incubation of 30–90 min.

Key words: Endothelial cell (EC); Cold-stored artery; Seeding density; Time of incubation; Serum

Received April 27, 2009; final acceptance March 28, 2011. Online prepub date: June 9, 2011.
Address correspondence to Arthur Smardencas, Florey Neuroscience Institutes, University of Melbourne, Parkville, Victoria, Australia, 3010. Tel: 0417 386 139; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 299–312, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X580527
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
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In Situ Cardiomyogenic Differentiation of Implanted Bone Marrow Mononuclear Cells by Local Delivery of Transforming Growth Factor-β1

Hee Seok Yang,* Suk Ho Bhang,† Il-Kwon Kim,‡ Tae-Jin Lee,* Jin-Muk Kang,* Dae-Hee Lee,§ Soo-Hong Lee,§ Ki-Chul Hwang,‡ and Byung-Soo Kim†

*Department of Bioengineering, Hanyang University, Seoul, Republic of Korea
†School of Chemical and Biological Engineering, Bio-Max Institute, Institute of Chemical Processes, Engineering Research Institute, Seoul National University, Seoul, Republic of Korea
‡Cardiovascular Research Institute, Yonsei University College of Medicine, Seoul, Republic of Korea
§CHA Stem Cell Institute & CHA Biotech, Pochon CHA University, Seoul, Republic of Korea

Bone marrow mononuclear cells (BMMNCs) can be used to treat patients with myocardial infarction, since BMMNCs can differentiate in vitro toward cardiomyogenic lineages when treated with transforming growth factor-β1 (TGF-β1). However, the in vitro cardiomyogenic differentiation culture process is costly and laborious, and the patients should wait during the culture period. In this study, we hypothesize that BMMNCs implanted in cardiomyogenically undifferentiated state to myocardial infarction site would differentiate cardiomyogenically in situ when exogenous TGF-β1 is delivered to the cell implantation site. Heparinconjugated poly(lactic-co-glycolic acid) nanospheres (HCPNs) suspended in fibrin gel were used as a TGF-β1 delivery system. BMMNCs were labeled with a green fluorescent dye (PKH67) and implanted into the infarction border zone of rat myocardium using fibrin gel containing HCPNs and TGF-β1. BMMNC implantation using fibrin gel and HCPNs without TGF-β1 served as a control. Four weeks after implantation, the expression of cardiomyogenic marker proteins by the implanted BMMNCs was dramatically greater in the TGF-β1 delivery group than in the control group. This method can significantly improve the stem cell therapy technology for myocardial regeneration, since it can remove in vitro cell culture step for cardiomyogenic differentiation prior to cell implantation.

Key words: Bone marrow mononuclear cells (BMMNCs); Myocardial infarction; Transforming growth factor-β1 (TGF-β1); Cardiomyogenic differentiation

Received January 20, 2010; final acceptance February 26, 2011. Online prepub date: June 7, 2011.
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. 21, pp. 313–332, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X580572
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Docosahexaenoic Acid Promotes Dopaminergic Differentiation in Induced Pluripotent Stem Cells and Inhibits Teratoma Formation in Rats With Parkinson-Like Pathology

Yuh-Lih Chang,*† Shih-Jen Chen,†‡ Chung-Lan Kao,†‡ Shih-Chieh Hung,†‡ Dah-Ching Ding,‡§ Cheng-Chia Yu,¶# Yi-Jen Chen,**†† Hung-Hai Ku,¶ Chin-Po Lin,‡‡ Kun-Hsiung Lee,§§ Yu-Chih Chen,†‡ Jhi-Joung Wang,¶¶ Chuan-Chih Hsu,¶¶ Liang-Kung Chen,‡## Hsin-Yang Li,¶**†† and Shih-Hwa Chiou*†‡

*Institute of Pharmacology, Faculty of Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan
†Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei, Taiwan
‡Institute of Clinical Medicine, Faculty of Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan
§Department of Obstetrics and Gynecology, Buddhist Tzu Chi General Hospital & Tzu Chi University, Taipei, Taiwan
¶Institute of Anatomy and Cell Biology, Faculty of Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan
#Institute of Oral Biology and Biomaterial Science, Chung-Shan Medical University & Department of Dentistry, Chung Shan Medical University Hospital, Taipei, Taiwan
**Division of Obstetrics and Gynecology, Faculty of Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan
††Department of Obstetrics and Gynecology, Taipei Veterans General Hospital, Taipei, Taiwan
‡‡Brain Research Center, Faculty of Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan
§§Division of Biotechnology, Animal Technology Institute Taiwan, Chunan, Miaoli, Taiwan
¶¶Department of Surgery, Chi-Mei Medical Center & Chia Nan University of Pharmacy & Science, Taipei, Taiwan
##Center for Geriatrics and Gerontology, Taipei Veterans General Hospital, Taipei, Taiwan

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by the degeneration of dopaminergic (DA) neurons in the midbrain. Induced pluripotent stem (iPS) cells have shown potential for differentiation and may become a resource of functional neurons for the treatment of PD. However, teratoma formation is a major concern for transplantation-based therapies. This study examined whether functional neurons could be efficiently generated from iPS cells using a five-step induction procedure combined with docosahexaenoic acid (DHA) treatment. We demonstrated that DHA, a ligand for the RXR/Nurr1 heterodimer, significantly activated expression of the Nurr1 gene and the Nurr1-related pathway in iPS cells. DHA treatment facilitated iPS differentiation into tyrosine hydroxylase (TH)-positive neurons in vitro and in vivo and functionally increased dopamine release in transplanted grafts in PD-like animals. Furthermore, DHA dramatically upregulated the endogenous expression levels of neuroprotective genes (Bcl-2, Bcl-xl, brain-derived neurotrophic factor, and glial cell-derived neurotrophic factor) and protected against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced apoptosis in iPS-derived neuronal precursor cells. DHA-treated iPS cells significantly improved the behavior of 6-hydroxydopamine (6-OHDA)-treated PD-like rats compared to control or eicosapentaenoic acid-treated group. Importantly, the in vivo experiment suggests that DHA induces the differentiation of functional dopaminergic precursors and improves the abnormal behavior of 6-OHDAtreated PD-like rats by 4 months after transplantation. Furthermore, we found that DHA treatment in iPS cell-grafted rats significantly downregulated the mRNA expression of embryonic stem cell-specific genes (Oct-4 and c-Myc) in the graft and effectively blocked teratoma formation. Importantly, 3 Tesla-magnetic resonance imaging and ex vivo green fluorescence protein imaging revealed that no teratomas were present in transplanted grafts of DHA-treated iPS-derived DA neurons 4 months after implantation. Therefore, our data suggest that DHA plays a crucial role in iPS differentiation into functional DA neurons and that this approach could provide a novel therapeutic approach for PD treatment.

Key words: Parkinson’s disease; Dopamine; Induced pluripotent stem cells (iPS); Dopaminergic neurons; Docosahexaenoic acid (DHA); 3T-MRI

Received October 23, 2009; final acceptance February 22, 2011. Online prepub date: June 7, 2011.
Address correspondence to Hsin-Yang Li, M.D., Ph.D., Department of Obstetrics and Gynecology, Taipei Veterans General Hospital, #201 Sec. 2, Shih-Pai Road, Taipei 11217, Taiwan. Tel: 886-2-28757826; Fax: 886-2-28734101; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it or Shih-Hwa Chiou, M.D., Ph.D., Department of Medical Research and Education, Taipei Veterans General Hospital, #201 Sec. 2, Shih-Pai Road, Taipei 11217, Taiwan. Tel: 886-2-28757394; Fax: 886-2-28757396; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 333–343, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X576036
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
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Targeted Intra-arterial Transplantation of Stem Cells to the Injured CNS Is More Effective Than Intravenous Administration: Engraftment Is Dependent on Cell Type and Adhesion Molecule Expression

Johan Lundberg,*† Erik Södersten,‡ Erik Sundström,§ Katarina Le Blanc,¶# Tommy Andersson,*† Ola Hermanson,‡ and Staffan Holmin*†

*Department of Clinical Neuroscience, Karolinska Institutet, Stockholm, Sweden
†Department of Neuroradiology, Karolinska University Hospital, Stockholm, Sweden
‡DBRM, Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden
§Division of Neurodegeneration, Department of Neurobiology, Care Sciences and Society, Karolinska Institutet and Stiftelsen Stockholms Sjukhem, Stockholm, Sweden
¶Department of Laboratory Medicine, Division of Clinical Immunology, Karolinska Institutet, Stockholm, Sweden
#Hematology Center, Karolinska University Hospital, Stockholm, Sweden

Stem cell transplantation procedures using intraparenchymal injections cause tissue injury in addition to associated surgical risks. Intravenous cell administration give engraftment in parenchymal lesions although the method has low efficacy and specificity. In pathological conditions with inflammation, such as traumatic brain injury, there is a transient up-regulation of ICAM-1 and VCAM-1 which might provide environmental cues for migration of stem cells from blood to parenchyma. The aim of this study was to i) analyze the effect of intra-arterial administration on cellular engraftment, ii) compare engraftment and side effects between three different stem cell systems, and iii) analyze gene expression in these three systems. We performed specific intra-arterial transplantations with human mesenchymal stem cells (hMSCs), human neural progenitor cells (hNPCs), and rat neural progenitor cells (rNPCs) in a rat model of traumatic brain injury. These results were compared to the intravenous route for each cell type, respectively. Analysis of engraftment and recipient characterization was performed by immunohistochemistry. We further characterized the different types of cells by microarray and RT-qPCR analysis. Specific intra-arterial transplantations produced significantly higher engraftment compared to intravenous transplantation with hMSCs and rNPCs. No engraftment was detected after intra-arterial or intravenous administration of hNPCs. Characterization of integrin expression indicated that CD49dVCAM-1 and possibly ICAM-1 interactions through CD18 and CD11a, respectively, are important for engraftment after intravascular cell administration. No side effects, such as thromboembolic complications, were detected. When translating stem cell therapies to clinical practice, the route of transplantation and the properties of the cell lines (homing, diapedesis, and migration) become important. This study supports the use of selective intra-arterial transplantation for improving engraftment after traumatic brain injury. In addition, we conclude that careful analysis of cells intended for local, intra-arterial transplantation with respect to integrin expression is important.

Key words: Endovascular; Transplantation; Human mesenchymal stem cells (hMSCs); Rat neural progenitor cells (rNPCs); Human neural progenitor cells (hNPCs); Vascular cell adhesion molecule-1 (VCAM-1)

Received June 29, 2010; final acceptance February 28, 2011. Online prepub date: June 7, 2011.
Address correspondence to Staffan Holmin, M.D., Ph.D., Department of Neuroradiology, Karolinska University Hospital, Solna, 171 76 Stockholm, Sweden. Tel: +46 8 517 77188; Fax: +46 8 517 73557; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 345–354, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X582741
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Suppression of Etk/Bmx Protects Against Ischemic Brain Injury

Kai-Yun Chen,*†‡ Chung-Che Wu,*§ Cheng-Fu Chang,¶ Yuan-Hao Chen,¶ Wen-Ta Chiu,*† Ya-Hsin Lou,¶ Yen-Hua Chen,†‡ Hsiu-Ming Shih,# and Yung-Hsiao Chiang*†‡

*Department of Neurosurgery, Taipei Medical University Hospital, Taipei, Taiwan
†Department of Surgery, College of Medicine, Taipei Medical University, Taipei, Taiwan
‡Translational Research Laboratory, Cancer Center, Taipei Medical University Hospital, Taipei, Taiwan
§Graduate Institute of Medical Sciences, National Defense Medical Center, Taipei, Taiwan
¶Department of Neurological Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan
#Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan

Etk/Bmx (epithelial and endothelial tyrosine kinase, also known as BMX), a member of the Tec (tyrosine kinase expressed in hepatocellular carcinoma) family of protein-tyrosine kinases, is an important regulator of signal transduction for the activation of cell growth, differentiation, and development. We have previously reported that activation of Etk leads to apoptosis in MDA-MB-468 cells. The purpose of this study was to examine the role of Etk in neuronal injury induced by H2O2 or ischemia. Using Western blot analysis and immunohistochemistry, we found that treatment with H2O2 significantly enhanced phosphorylation of Etk and its downstream signaling molecule Stat1 in primary cortical neurons. Inhibiting Etk activity by LFMA13 or knocking down Etk expression by a specific shRNA increased the survival of primary cortical neurons. Similarly, at 1 day after a 60-min middle cerebral artery occlusion (MCAo) in adult rats, both phosphorylated Etk and Stat1 were coexpressed with apoptotic markers in neurons in the penumbra. Pretreatment with LFM-A13 or an adenoviral vector encoding the kinase deletion mutant EtkΔk attenuated caspase-3 activity and infarct volume in ischemic brain. All together, our data suggest that Etk is activated after neuronal injury. Suppressing Etk activity protects against neurodegeneration in ischemic brain.

Key words: Etk/Bmx; Cerebral ischemia; Stat1; Apoptosis

Received January 29, 2011; final acceptance April 15, 2011. Online prepub date: September 16, 2011.
Address correspondence to Yung-Hsiao Chiang, Department of Neurosurgery, Taipei Medical University Hospital, 252, Wu Hsing Street, Taipei, Taiwan 110. Tel: 886-2-27372181, #8016; Fax: 886-2-27390946; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 355–364, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X580545
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Stem Cells Decreased Neuronal Cell Death After Hypoxic Stress in Primary Fetal Rat Neurons In Vitro1

Tetsuro Sakai* and Yan Xu†

*Department of Anesthesiology, The McGowan Institute for Regenerative Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
†Departments of Anesthesiology, Pharmacology & Chemical Biology, Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

To explore stem cell-mediated neuronal protection through extracellular signaling pathways by transplanted stem cells, we sought to identify potential candidate molecules responsible for neuronal protection using an in vitro coculture system. Primary fetal rat hippocampal neurons underwent hypoxia (≤1% oxygen) for 96 h and then were returned to a normoxic condition. The study group then received rat umbilical cord matrixderived stem cells, while the control group received fresh media only. The experimental group showed decreased neuronal apoptosis compared to the control group [44.5 ± 1.6% vs. 71.0 ± 4.2% (mean ± SD, p = 0.0005) on day 5] and higher neuronal survival (4.9 ± 1.2 cells/100× field vs. 2.2 ± 0.3, p = 0.02 on day 5). Among 90 proteins evaluated using a protein array, stem cell coculture media showed increased protein secretion of TIMP-1 (5.61-fold), TIMP-2 (4.88), CNTF-Rá (3.42), activin A (2.20), fractalkine (2.04), CCR4 (2.02), and decreased secretion in MIP-2 (0.30-fold), AMPK á1 (0.43), TROY (0.48), and TIMP-3 (0.50). This study demonstrated that coculturing stem cells with primary neurons in vitro decreased neuronal cell death after hypoxia with significantly altered protein secretion. The results suggest that stem cells may offer neuronal protection through extracellular signaling.

Key words: Stem cell; Neuronal protection; Rat; Protein array; Hypoxia

Received June 3, 2010; final acceptance March 2, 2011. Online prepub date: June 7, 2011.
1Presented in part at 104th Annual Meeting of the American Society of Anesthesiologists, New Orleans, LA, USA, October 17–21, 2009.
Address correspondence to Tetsuro Sakai, M.D., Ph.D., Associate Professor, Department of Anesthesiology, The McGowan Institute for Regenerative Medicine, University of Pittsburgh School of Medicine, UPMC Montefiore, M469.11, 200 Lothrop Street, Pittsburgh, P, 15213, USA. Tel: 1-412-648-6077; Fax: 1-412-648-6014 E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 365–371, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X586765
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Brief Communication

Human Neural Stem Cells Overexpressing Choline Acetyltransferase Restore Cognitive Function of Kainic Acid-Induced Learning and Memory Deficit Animals

Dongsun Park,* Seong Soo Joo,† Tae Kyun Kim,* Sun Hee Lee,* Hyomin Kang,* Hong Jun Lee,‡§ Inja Lim,¶ Akinori Matsuo,# Ikuo Tooyama,# Yun-Bae Kim,*§ and Seung U. Kim‡§

*College of Veterinary Medicine, Chungbuk National University, Cheongju, Republic of Korea
†Division of Marine Molecular Biotechnology, Gangneung-Wonju National University, Gangneung, Republic of Korea
‡Medical Research Institute, Chung-Ang University Medical School, Seoul, Republic of Korea
§Division of Neurology, Department of Medicine, University of British Columbia Hospital, Vancouver, Canada
¶Department of Physiology, Chung-Ang University Medical School, Seoul, Republic of Korea
#Molecular Neuroscience Research Center, Shiga University of Medical Science, Ohtsu, Japan

Alzheimer disease (AD) is a progressive neurodegenerative disease, which is characterized by loss of memory and cognitive function. In AD patients dysfunction of the cholinergic system is the main cause of cognitive disorders, and decreased activity of choline acetyltransferase (ChAT), an enzyme responsible for acetylcholine (ACh) synthesis, is observed. In the present study we investigated if brain transplantation of human neural stem cells (NSCs) genetically modified to encode ChAT gene improves cognitive function of kainic acid (KA)-induced learning deficit rats. Intrahippocampal injection of KA to hippocampal CA3 region caused severe neuronal loss, resulting in profound learning and memory deficit. F3.ChAT human NSCs transplanted intracerebroventricularly improved fully the learning and memory function of KA-induced learning deficit animals, in parallel with the elevation of ACh levels in cerebrospinal fluid. F3.ChAT human NSCs migrated to the KA-induced injury site (CA3) and differentiated into neurons and astrocytes. The present study demonstrates that human NSCs expressing ChAT have lesion-tropic property and improve cognitive function of learning deficit model rats with hippocampal injury by increasing ACh level.

Key words: Learning and memory; Alzheimer disease; Kainic acid; Neural stem cells; Choline acetyltransferase (ChAT) gene; Acetylcholine

Received January 12, 2011; final acceptance March 31, 2011. Online prepub date: September 16, 2011.
Address correspondence to Yun-Bae Kim, College of Veterinary Medicine, Chungbuk National University, 52 Naesudongro (Gaesin-dong), Cheongju, Chungbuk 361-763, Republic of Korea. Tel: +82-43-261-3358; Fax: +82-43-271-3246; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it or Seung U. Kim, Division of Neurology, Department of Medicine, University of British Columbia Hospital, 2211 Wesbrook Mall, Vancouver, BC V6T 2B5, Canada. Tel: +1-604-822-7145; Fax: +1-604-822-7897; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 373–378, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X565010
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Brief Communication

GMP-Compliant Culture of Human Hair Follicle Cells for Encapsulation and Transplantation

Mario Marazzi,* Francesca Crovato,* Massimo Bucco,†1 Maria Chiara Sironi,* Marta Cecilia Tosca,* Barbara Antonioli,* Theodora Chlapanidas,† Giulia Lucconi,† Vincenzo Rapisarda,§ Alessandro Scalise,‡ Daniele Vigo,¶ Massimo Faustini,¶ and Maria Luisa Torre†

*Struttura Semplice Terapia Tissutale, A.O. Ospedale Niguarda Ca’ Granda, Milan, Italy
†Dipartimento di Scienze del Farmaco, Università di Pavia, Pavia, Italy
‡Dipartimento di Scienze Mediche e Chirurgiche, Università Politecnica delle Marche, Ancona, Italy
§Centro Grandi Ustionati, A.O. Ospedale Niguarda Ca’ Granda, Milan, Italy
¶Dipartimento di Scienze e Tecnologie Veterinarie per la Sicurezza Alimentare, Università di Milano, Milan, Italy

Human hair follicle cells, both bulge and dermal papilla cells, were isolated and cultured in a GMP cell factory, in order to obtain an in vitro hair follicle source for encapsulation end transplantation in alopecia regenerative cell therapy. An in vitro model, constituted by organotypic cultures of human skin sample, was set up to simulate the dermal–epidermal interaction between bulge cells and dermal papilla cells, evaluating the possible new follicles formation and the regenerative potentiality of these hair follicle cells. Both the bulge and dermal papilla cells show an excellent cellular proliferation as well as an abundant extracellular matrix production. The immunofluorescence investigation revealed the positivity of both cell lines to CK15 and CD200, whereas both cell lines were negative to CD71 and Oct-4. The pool of cultured bulge and dermal papilla cells was injected into the deep dermis; at day 28 of culture, some organized areas with a higher cell density can be observed: the cells self-organize into papilla-like lengthened aggregates. In samples in which the follicular cells have been seeded on the dermis surface, an epidermis-like homogeneous monolayer on the dermis surface can be seen, therefore showing a potentiality of these cells for epidermis regeneration. These data show the efficacy of a cellular isolation and amplification approach to obtain an in vitro human hair follicle regenerative source on industrial scale in a GMP cell factory. The results also proved an intrinsic potentiality of follicular cells to in vitro recreate the epidermis for tissue engineering purposes. Thus, it is feasible to produce bioengineered hair follicles in a GMP cell factory, for encapsulation and transplantation in alopecic patients.

Key words: Hair follicle; Bulge; GMP culture; Alopecia; Regenerative therapy

Received December 14, 2009; final acceptance February 22, 2011. Online prepub date: March 25, 2011.
1Present address: Bioscience Institute, Via Rovereta, 42 47891 Falciano, Repubblica di San Marino.
Address correspondence to Prof. Massimo Faustini, Dipartimento di Scienze e Tecnologie Veterinarie per la Sicurezza Alimentare, Via Celoria 10, Milano, Italia. Tel: +39 02 50317938; Fax: +39 02 50317941; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it