Cell Transplantation 22(2) Abstracts

Return to Cell Transplantation main page>

Cell Transplantation, Vol. 22, pp. 189-204, 2013
0963-6897/13 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X656117
E-ISSN 1555-3892
Copyright © 2013 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Review

Advances and Pitfalls of Cell Therapy in Metabolic Leukodystrophies

Catarina Oliveira Miranda,*† Pedro Brites,* Mónica Mendes Sousa,* and Carla Andreia Teixeira*

*Nerve Regeneration Group, IBMC-Instituto de Biologia Molecular e Celular, Porto, Portugal
†ICBAS, Universidade do Porto, Porto, Portugal

Leukodystrophies are a group of disorders characterized by myelin dysfunction, either at the level of myelin formation or maintenance, that affect the central nervous system (CNS) and also in some cases, to a lesser extent, the peripheral nervous system (PNS). Although these genetic-based disorders are generally rare, all together they have a significant impact in the society, with an estimated overall incidence of 1 in 7,663 live births. Currently, there is no cure for leukodystrophies, and the development of effective treatments remains challenging. Not only leukodystrophies generally progress very fast, but also most are multifocal needing the simultaneous targeting at multiple sites. Moreover, as the CNS is affected, the blood–brain barrier (BBB) limits the efficacy of treatment. Recently, interest on cell therapy has increased, and the leukodystrophies for which metabolic correction is needed have become first-choice candidates for cell-based clinical trials. In this review, we present and discuss the available cell transplantation therapies in metabolic leukodystrophies including fucosidosis, X-linked adrenoleukodystrophy, metachromatic leukodystrophy, Canavan disease, and Krabbe’s disease. We will discuss the latest advances of cell therapy and its pitfalls in this group of disorders, taking into account, among others, the limitations imposed by reduced cell migration in multifocal conditions, the need to achieve corrective enzyme threshold levels, and the growing awareness that not only myelin but also the associated axonopathy needs to be targeted in some leukodystrophies.

Key words: Leukodystrophy; Fucosidosis; X-linked adrenoleukodystrophy; Metachromatic leukodystrophy; Canavan disease; Krabbe’s disease; Transplantation; Stem cell

Received November 30, 2011; final acceptance April 13, 2012. Online prepub date: September 21, 2012.
Address correspondence to Mónica Mendes Sousa, Nerve Regeneration Group-IBMC, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal. Tel: +35 122 607 4900; Fax: +35 122 609 9157; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 22, pp. 205-229, 2013
0963-6897/13 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X656171
E-ISSN 1555-3892
Copyright © 2013 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Review

Translational Research and Therapeutic Applications of Stem Cell Transplantation in Periodontal Regenerative Medicine

Hong Lu,*1 Cheng Xie,†1 Yi-Min Zhao,†‡ and Fa-Ming Chen*‡

*Department of Periodontology and Oral Medicine, School of Stomatology, Fourth Military Medical University, Xi’an, People’s Republic of China
†Department of Prosthodontics, School of Stomatology, Fourth Military Medical University, Xi’an, People’s Republic of China
‡Translational Research Team, School of Stomatology, Fourth Military Medical University, Xi’an, People’s Republic of China

Stem cells have received a great deal of interest from the research community as potential therapeutic “tools” for a variety of chronic debilitating diseases that lack clinically effective therapies. Stem cells are also of interest for the regeneration of tooth-supporting tissues that have been lost to periodontal disease. Indeed, substantial data have demonstrated that the exogenous administration of stem cells or their derivatives in preclinical animal models of periodontal defects can restore damaged tissues to their original form and function. As we discuss here, however, considerable hurdles must be overcome before these findings can be responsibly translated to novel clinical therapies. Generally, the application of stem cells for periodontal therapy in clinics will not be realized until the best cell(s) to use, the optimal dose, and an effective mode of administration are identified. In particular, we need to better understand the mechanisms of action of stem cells after transplantation in the periodontium and to learn how to preciously control stem cell fates in the pathological environment around a tooth. From a translational perspective, we outline the challenges that may vary across preclinical models for the evaluation of stem cell therapy in situations that require periodontal reconstruction and the safety issues that are related to clinical applications of human stem cells. Although clinical trials that use autologous periodontal ligament stem cells have been approved and have already been initiated, proper consideration of the technical, safety, and regulatory concerns may facilitate, rather than inhibit, the clinical translation of new therapies.

Key words: Stem cell-based therapy; Periodontal regeneration; Translational research; Preclinical models; Safety issues; Regulatory concerns; Clinical trials

Received September 8, 2011; final acceptance April 27, 2012. Online prepub date: October 1, 2012.
1These authors provided equal contribution to this work.
Address correspondence to Fa-Ming Chen, Ph.D., Department of Periodontology and Oral Medicine, School of Stomatology, Fourth Military Medical University, 145th West Chang-le Road, Xi’an 710032, Shaanxi, People’s Republic of China. Tel: +86 029 84776096; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it or Yi-Min Zhao, Ph.D., Department of Prosthodontics, School of Stomatology, Fourth Military Medical University, 145th West Chang-le Road, Xi’an 710032, Shaanxi, People’s Republic of China. Tel: +86 029 84776001; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 22, pp. 231-242, 2013
0963-6897/13 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X654939
E-ISSN 1555-3892
Copyright © 2013 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Human-Scale Whole-Organ Bioengineering for Liver Transplantation: A Regenerative Medicine Approach

Hiroshi Yagi,* Ken Fukumitsu,† Kazumasa Fukuda,* Minoru Kitago,* Masahiro Shinoda,* Hideaki Obara,* Osamu Itano,* Shigeyuki Kawachi,* Minoru Tanabe,* Gina M. Coudriet,‡ Jon D. Piganelli,‡ Thomas W. Gilbert,§ Alejandro Soto-Gutierrez,†§ and Yuko Kitagawa*

*Department of Surgery, School of Medicine, Keio University, Shinjuku-ku, Tokyo, Japan
†Department of Pathology, Center for Innovative Regenerative Therapies, Department of Surgery, Transplantation Section, Children’s Hospital of Pittsburgh and the Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh, PA, USA
‡Department of Pediatrics, Division of Immunogenetics /Immunology, Children’s Hospital of Pittsburgh of UPMC, Pittsburgh, PA, USA
§McGowan Institute for Regenerative Medicine, Departments of Surgery, Cardiothoracic Surgery, and Bioengineering, University of Pittsburgh, Pittsburgh, PA, USA

At this time, the only definitive treatment of hepatic failure is liver transplantation. However, transplantation has been limited by the severely limited supply of human donor livers. Alternatively, a regenerative medicine approach has been recently proposed in rodents that describe the production of three-dimensional whole-organ scaffolds for assembly of engineered complete organs. In the present study, we describe the decellularization of porcine livers to generate liver constructs at a scale that can be clinically relevant. Adult ischemic porcine livers were successfully decellularized using a customized perfusion protocol, the decellularization process preserved the ultrastructural extracellular matrix components, functional characteristics of the native microvascular and the bile drainage network of the liver, and growth factors necessary for angiogenesis and liver regeneration. Furthermore, isolated hepatocytes engrafted and reorganized in the porcine decellularized livers using a human-sized organ culture system. These results provide proof-of-principle for the generation of a human-sized, three-dimensional organ scaffold as a potential structure for human liver grafts reconstruction for transplantation to treat liver disease.

Key words: Whole-organ scaffold; Liver tissue engineering; Decellularization; Bile duct; Porcine liver; Regenerative medicine; Bioengineering in organ transplantation

Received August 5, 2011; final acceptance May 7, 2012. Online prepub date: August 27, 2012.
Address correspondence to Alejandro Soto-Gutierrez, M.D., Ph.D., Department of Pathology and Surgery, Transplantation Section, Children’s Hospital of Pittsburgh, University of Pittsburgh, 3511 Rangos Research Building 530 45th Street, Pittsburgh, PA 15201, USA. Tel: +1 (412) 692-5562; Fax: +1 (412) 692-6599; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it or Yuko Kitagawa, M.D., Ph.D., FACS, Department of Surgery, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. Tel: +(81)-3-3353-1211 ext. 62334; Fax: +(81)-3-3355-4707; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 22, pp. 243-251, 2013
0963-6897/13 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X640637
E-ISSN 1555-3892
Copyright © 2013 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Islet Engraftment and Revascularization in Clinical and Experimental Transplantation

Christian Molnár,*† Magnus Essand,* Lars Wennberg,† Christian Berne,‡ Erik Larsson,* Gunnar Tufveson,§ and Olle Korsgren*

*Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden
†Department of Transplantation Surgery, Karolinska Institute, Karolinska University Hospital, Stockholm, Sweden
‡Department of Medical Sciences, Division of Medicine, Uppsala University Hospital, Uppsala, Sweden
§Department of Surgical Sciences, Division of Transplantation Surgery, Uppsala University Hospital, Uppsala, Sweden

Proper revascularization after transplantation is assumed to be crucial for appropriate islet graft function. We developed a novel noninvasive imaging method, based on adenoviral transduction of islets with a hypoxia responsive reporter gene, for continuous in vivo monitoring of hypoxia in islet grafts in a mouse model. In addition, morphological data were obtained from a deceased patient previously subject to intraportal transplantation. We detected only transient hypoxia in a minority of the animals transplanted. Importantly, a clear response to hypoxia was observed in vitro after removal of the islet grafts on day 28 after transplantation. Also, the morphological data from the deceased patient demonstrated an extensive revascularization of the transplanted islets. In fact, no differences could be seen between native islets, in pancreas biopsies taken prior to islet isolation, and transplanted islets regarding the number, distribution, and shape of the blood vessels. However, fewer small islets (diameter <39 μm) were found in the liver compared to those found in native pancreases. Notably, an absolute majority of the transplanted islets were found remaining within the venous lumen, in direct contact with the vessel wall. In conclusion, the results presented show less pronounced islet graft hypoxia after subcapsular transplantation than previously reported using more invasive methods. Also, formation of an extensive intraislet capillary network, similar to that seen in native islets in the pancreas, was seen after clinical islet transplantation.

Key words: Islets; Transplantation; Hypoxia; Revascularization; Real time

Received March 25, 2011; final acceptance January 31, 2012. Online prepub date: May 8, 2012.
Address correspondence to Christian Molnár, Department of Oncology, Radiology and Clinical Immunology, Uppsala University, Uppsala, Sweden. Tel: +46 18 611 4189; Fax: +46 18 6110222; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 22, pp. 253-266, 2013
0963-6897/13 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X647180
E-ISSN 1555-3892
Copyright © 2013 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Hypoxia-Inducible Factor-1α (HIF-1α) Potentiates β-Cell Survival After Islet Transplantation of Human and Mouse Islets

Rebecca A. Stokes,* Kim Cheng,* Natasha Deters,* Sue Mei Lau,* Wayne J. Hawthorne,† Philip J. O’Connell,† Jessica Stolp,‡ Shane Grey,§ Thomas Loudovaris,¶ Thomas W. Kay,¶ Helen E. Thomas,¶ Frank J. Gonzalez,# and Jenny E. Gunton*,**††‡‡

*Diabetes and Transcription Factors Group, Garvan Institute of Medical Research (GIMR), Sydney, Australia
†National Pancreas Transplant Unit, Westmead Hospital, Sydney, Australia
‡Immunology: B Cell Tolerance, GIMR, Sydney, Australia
§Gene Therapy and Autoimmunity Group, GIMR, Sydney, Australia
¶Tom Mandel Islet Transplant Program, St. Vincent’s Institute, Melbourne, Australia
#Laboratory of Metabolism, National Cancer Institute, Bethesda, MA, USA
**St. Vincent’s Clinical School, University of New South Wales, Sydney, Australia
††Faculty of Medicine, University of Sydney, Sydney, Australia
‡‡Department of Diabetes and Endocrinology, Westmead Hospital, Sydney, Australia

A high proportion of β-cells die within days of islet transplantation. Reports suggest that induction of hypoxiainducible factor-1α (HIF-1α) predicts adverse transplant outcomes. We hypothesized that this was a compensatory response and that HIF-1α protects β-cells during transplantation. Transplants were performed using human islets or murine β-cell-specific HIF-1α-null (β-HIF-1α-null) islets with or without treatment with deferoxamine (DFO) to increase HIF-1α. β-HIF-1α-null transplants had poor outcomes, demonstrating that lack of HIF-1α impaired transplant efficiency. Increasing HIF-1α improved outcomes for mouse and human islets. No effect was seen in β-HIF-1α-null islets. The mechanism was decreased apoptosis, resulting in increased β-cell mass posttransplantation. These findings show that HIF-1α is a protective factor and is required for successful islet transplant outcomes. Iron chelation with DFO markedly improved transplant success in a HIF-1α-dependent manner, thus demonstrating the mechanism of action. DFO, approved for human use, may have a therapeutic role in the setting of human islet transplantation.

Key words: Hypoxia-inducible factor-1α (HIF-1α); β-Cell function; Islet transplantation; Deferoxamine (DFO); Diabetes; Hypoxia

Received August 10, 2011; final acceptance February 29, 2012. Online prepub date: June 15, 2012.
Address correspondence to Dr. Jenny E. Gunton, Diabetes and Transcription Factors Group, Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, Sydney, NSW 2010, Australia. Tel: +61-2-9295-8474; Fax: +61-2-9295-8404; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 22, pp. 267-278, 2013
0963-6897/13 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X655055
E-ISSN 1555-3892
Copyright © 2013 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Establishment of Immortalized Human Amniotic Mesenchymal Stem Cells

Zan Teng,*1 Toshiko Yoshida,* Motonori Okabe,* Ayaka Toda,* Osamu Higuchi,† Makiko Nogami,‡ Noriko Yoneda,§ Kaixuan Zhou,* Satoru Kyo,¶ Touru Kiyono,# and Toshio Nikaido*

*Department of Regenerative Medicine, University of Toyama, Graduate School of Medicine and Pharmaceutical Sciences, Toyama, Japan
†Department of Pediatrics, University of Toyama, Graduate School of Medicine and Pharmaceutical Sciences, Toyama, Japan
‡Department of Orthopaedic Surgery, University of Toyama, Graduate School of Medicine and Pharmaceutical Sciences, Toyama, Japan
§Department of Obstetrics and Gynecology, University of Toyama, Graduate School of Medicine and Pharmaceutical Sciences, Toyama, Japan
¶Department of Obstetrics and Gynecology, School of Medicine, Kanazawa University, Kanazawa, Japan
#Virology Division, National Cancer Center Research Institute, Tokyo, Japan

Human amniotic mesenchymal cells (HAM cells) are known to contain somatic stem cells possessing the characteristics of pluripotency. However, little is known about the biology of these somatic cells because isolated HAM cells from amniotic membrane have a limited lifespan. To overcome this problem, we attempted to prolong the lifespan of HAM cells by infecting retrovirus encoding human papillomavirus type16E6 and E7 (HPV16E6E7), bmi-1, and/or human telomerase reverse transcriptase (hTERT) genes and investigated their characteristics as stem cells. We confirmed the immortalization of the four lines of cultured HAM cells for about 1 year. Immortalized human amnion mesenchymal cells (iHAM cells) have continued to proliferate over 200 population doublings (PDs). iHAM cells were positive for CD73, CD90, CD105, and CD44 and negative for CD34, CD14, CD45, and HLA-DR. They expressed stem cell markers such as Oct3/4, Sox2, Nanog, Klf4, SSEA4, c-myc, vimentin, and nestin. They showed adipogenic, osteogenic, and chondrogenic differentiation abilities after induction. These results suggested that immortalized cell lines with characteristics of stem cells can be established. iHAM cells with an extended lifespan can be used to produce good experimental models both in vitro and in vivo.

Key words: Amniotic; Mesenchymal cells; Human; Immortalized; Establishment; Stem cells

Received February 14, 2010; final acceptance March 25, 2012. Online prepub date: September 21, 2012.
1Present address: Department of Pharmaceutical Toxicology, School of Pharmaceutical, Sciences, China Medical University, China.
Address correspondence to Toshio Nikaido, Ph.D., Department of Regenerative Medicine, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Sugitani 2630, Toyama 930-0194, Japan. Tel: +81-76-434-7210; Fax: +81-76-434-5011; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 22, pp. 279-285, 2013
0963-6897/13 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X656045
E-ISSN 1555-3892
Copyright © 2013 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Autologous Adipose Tissue-Derived Stem Cells for the Treatment of Crohn’s Fistula: A Phase I Clinical Study

Yong Beom Cho,*1 Woo Yong Lee,*1 Kyu Joo Park,† Mihyung Kim,‡ Hee-Won Yoo,§ and Chang Sik Yu¶

*Department of Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea
†Department of Surgery, Seoul National University College of Medicine, Seoul, Korea
‡Anterogen Co., Ltd., Seoul, Korea
§Bukwang Pharm. Co., Ltd., Seoul, Korea
¶Department of Colon and Rectal Surgery, University of Ulsan College of Medicine and Asan Medical Center, Seoul, Korea

The present study was designed to evaluate the safety and potential of adipose tissue-derived stem cells (ASCs) for the treatment of Crohn’s fistula. In this dose escalation study, patients were sequentially enrolled into three dosing groups with at least three patients per group. The first three patients (group 1) were given 1 × 107 cells/ml. After 4 weeks, this dose was deemed safe, and so an additional four patients (group 2) were given 2 × 107 cells/ml. Four weeks later, after which this second dose was deemed safe, a third and final group of three patients were given 4 × 107 cells/ml. Each patient was followed for a minimum of 8 weeks. Patients who showed complete healing at week 8 were followed up for an additional 6 months. Efficacy endpoint was complete healing at week 8 after injection, defined as complete closure of the fistula track and internal and external openings without drainage or signs of inflammation. There were no grade 3 or 4 severity adverse events, and there were no adverse events related to the study drug. Two patients in group 2, treated with 2 × 107 ASCs/ml, showed complete healing at week 8 after injection. Of the three patients enrolled in group 3, treated with 4 × 107 ASCs/ml, one showed complete healing. Outcome in another patient was assessed as partial healing due to incomplete closure of the external opening, although the inside of fistula track was filled considerably and there was no drainage. All three patients with complete healing at week 8 showed a sustained effect without recurrence 8 months after injection. In conclusion, this study demonstrates the tolerability, safety, and potential efficacy of ASCs for the treatment of Crohn’s fistula and provides support for further clinical study.

Key words: Adipose tissue-derived stem cells (ASCs); Autologous stem cells; Crohn’s fistula; Complete healing

Received September 8, 2011, final acceptance March 31, 2012. Online prepub date: September 21, 2012.
1These authors provided equal contribution to this work.
Address correspondence to Prof. Chang Sik Yu, M.D., Ph.D., Department of Colon and Rectal Surgery, University of Ulsan College of Medicine and Asan Medical Center, 388-1 Pungnap-2 dong, Songpa-gu, Seoul 138-736, Korea. Tel: +82-2-3010-3494; Fax: +82-2-3010-6701; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 22, pp. 287-297, 2013
0963-6897/13 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X655019
E-ISSN 1555-3892
Copyright © 2013 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Low Serum Cultured Adipose Tissue-Derived Stromal Cells Ameliorate Acute Kidney Injury in Rats

Takayuki Katsuno, Takenori Ozaki, Yosuke Saka, Kazuhiro Furuhashi, Hangsoo Kim, Kaoru Yasuda, Tokunori Yamamoto, Waichi Sato, Naotake Tsuboi, Masashi Mizuno, Yasuhiko Ito, Enyu Imai, Seiichi Matsuo, and Shoichi Maruyama

Department of Nephrology, Nagoya University Graduate School of Medicine, Nagoya, Japan

Current studies suggest that mesenchymal stromal cells (MSCs) improve acute kidney injury (AKI) via paracrine/endocrine effects. We established human adipose tissue-derived stromal cells (hASCs) cultured in low (2%) serum (hLASCs), which have great potential of tissue regeneration. The present study was performed to investigate the therapeutic effects of hLASCs on AKI and to clarify the mechanisms involved. In low serum, hASCs proliferated well, while human bone marrow-derived stromal cells (hBMSCs) did not. hLASCs secreted higher levels of hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) than did hASCs cultured in high (20%) serum (hHASCs) or hBMSCs cultured in high serum (hHBMSCs). AKI was induced in nude rats by folic acid, and hLASCs, hHASCs or control medium were administered into the renal subcapsules. hLASCs significantly attenuated acute renal damage, while hHASCs showed far less effect. Furthermore, interstitial fibrosis observed on day 14 was less pronounced in the hLASCs group. Cell tracking experiment showed no evidence of transdifferentiation. Intravenous injection of hLASCs or hHBMSCs or subcapsular injection of hHBMSCs did not ameliorate AKI. Concerning the mechanisms, our in vivo experiments showed that HGF knockdown by siRNA impaired the ability of hLASCs to protect the kidney from acute injury whereas VEGF knockdown did not. In conclusion, hLASCs, but not hHASCs or hHBMSCs, ameliorated AKI via paracrine effects, and HGF is one of the key mediators.

Key words: Acute renal failure; Stem cells; Cell transfer; Interstitial fibrosis

Received August 5, 2011; final acceptance March 30, 2012. Online prepub date: September 7, 2012.
Address correspondence to Shoichi Maruyama, M.D., Department of Nephrology, Nagoya University Graduate School of Medicine, 65 Tsurumaicho, Showaku, Nagoya, Japan 466-8550. E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 22, pp. 299-308, 2013
0963-6897/13 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X656054
E-ISSN 1555-3892
Copyright © 2013 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Serum-Free Medium and Mesenchymal Stromal Cells Enhance Functionality and Stabilize Integrity of Rat Hepatocyte Spheroids

Ji Bao,*† James E. Fisher,† Joseph B. Lillegard,† William Wang,† Bruce Amiot,‡ Yue Yu,† Allan B. Dietz,§ Yaakov Nahmias,¶ and Scott L. Nyberg†

*Department of Pathology, West China Hospital, Chengdu, Sichuan, People’s Republic of China
†Department of Surgery, Division of Experimental Surgery, Mayo Clinic, Rochester, MN, USA
‡Brami Biomedical Inc., Minneapolis, MN, USA
§Division of Transfusion Medicine, Mayo Clinic, Rochester, MN, USA
¶Center for Engineering in Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA

Long-term culture of hepatocyte spheroids with high ammonia clearance is valuable for therapeutic applications, especially the bioartificial liver. However, the optimal conditions are not well studied. We hypothesized that liver urea cycle enzymes can be induced by high protein diet and maintain on a higher expression level in rat hepatocyte spheroids by serum-free medium (SFM) culture and coculture with mesenchymal stromal cells (MSCs). Rats were feed normal protein diet (NPD) or high protein diet (HPD) for 7 days before liver digestion and isolation of hepatocytes. Hepatocyte spheroids were formed and maintained in a rocked suspension culture with or without MSCs in SFM or 10% serum-containing medium (SCM). Spheroid viability, kinetics of spheroid formation, hepatic functions, gene expression, and biochemical activities of rat hepatocyte spheroids were tested over 14 days of culture. We observed that urea cycle enzymes of hepatocyte spheroids can be induced by high protein diet. SFM and MSCs enhanced ammonia clearance and ureagenesis and stabilized integrity of hepatocyte spheroids compared to control conditions over 14 days. Hepatocytes from high protein diet-fed rats formed spheroids and maintained a high level of ammonia detoxification for over 14 days in a novel SFM. Hepatic functionality and spheroid integrity were further stabilized by coculture of hepatocytes with MSCs in the spheroid microenvironment. These findings have direct application to development of the spheroid reservoir bioartificial liver.

Key words: Bioartificial liver; Spheroid; Hepatocyte; Medium; Ureagenesis

Received August 29, 2011; final acceptance April 7, 2012. Online prepub date: September 21, 2012.
Address correspondence to Scott L. Nyberg, M.D., Ph.D., Professor of Surgery, Mayo Clinic 200 First Street SW, Rochester, MN 55905, USA. Tel: +1-507-266-6772; Fax: +1-507-266-2810; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 22, pp. 309-329, 2013
0963-6897/13 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X653291
E-ISSN 1555-3892
Copyright © 2013 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Induction of Functional Mesenchymal Stem Cells From Rabbit Embryonic Stem Cells by Exposure to Severe Hypoxic Conditions

Takeshi Teramura,* Yuta Onodera,* Toshiyuki Takehara,* John Frampton,† Toshiki Matsuoka,† Syunsuke Ito,‡ Koichi Nakagawa,§ Yoshihisa Miki,§ Yoshihiko Hosoi,¶ Chiaki Hamanishi,§ and Kanji Fukuda*§

*Institute of Advanced Clinical Medicine, Kinki University Faculty of Medicine, Osaka, Japan
†Department of Biomedical Engineering, University of Michigan, Michigan, USA
‡Center for Developmental Biology, Riken, Kobe, Japan
§Department of Orthopaedic Surgery, Kinki University Faculty of Medicine, Osaka, Japan
¶Department of Biology-Oriented Science and Technology, Kinki University, Wakayama, Japan

Embryonic stem cells (ESCs) have the potential to be used as an unlimited cell source for cell transplantation therapy, as well as for studying mechanisms of disease and early mammalian development. However, applications involving ESCs have been limited by the lack of reliable differentiation methods in many cases. Mesenchymal stem cells (MSCs) have also emerged as a promising cell source, but as suggested in recent studies, these cells display limited potential for proliferation and differentiation, thereby limiting their usefulness in the clinic and in the laboratory. Unfortunately, effective methods for induction of MSCs from pluripotent stem cells have not been established, and the development of such methods remains a major challenge facing stem cell biologists. Oxygen concentration is one of the most important factors regulating tissue development. It has profound effects on cell metabolism and physiology and can strongly influence stem cell fate. Here we demonstrate that severe low O2 concentrations (1%) can function as a selective pressure for removing undifferentiated pluripotent cells during the induction of MSCs from rabbit ESCs (rESCs) and that MSCs induced under severe hypoxic conditions function as normal MSCs; that is, they repopulate after cloning, express specific markers (vimentin, CD29, CD90, CD105, and CD140a) and differentiate into adipocytes, osteoblasts, and chondrocytes. Furthermore, we demonstrate that these cells can contribute to cartilage regeneration in an in vivo rabbit model for joint cartilage injury. These results support the notion that exposing ESCs to severe hypoxic conditions during differentiation can be used as a strategy for the preparation of functional MSCs from ESCs.

Key words: Rabbit embryonic stem cell (rESC); Mesenchymal stem cells (MSCs); Hypoxia; Chondrocyte

Received October 17, 2011; final acceptance April 21, 2012. Online prepub date: August 27, 2012.
Address correspondence to Takeshi Teramura, Division of Cell Biology for Regenerative Medicine, Institute of Advanced Clinical Medicine, Kinki University Faculty of Medicine, 377-2 Ohno-higashi, Osaka-sayama, Osaka 5898511, Japan. Tel: +81 72 3660221; Fax: +81 72 3660206; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 22, pp. 331-340, 2013
0963-6897/13 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X657648
E-ISSN 1555-3892
Copyright © 2013 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Chondrogenically Tuned Expansion Enhances the Cartilaginous Matrix-Forming Capabilities of Primary, Adult, Leporine Chondrocytes

Daniel J. Huey, Jerry C. Hu, and Kyriacos A. Athanasiou

Department of Biomedical Engineering, University of California-Davis, Davis, CA, USA

When expanded through passage, chondrocytes lose their ability to produce high-quality cartilaginous matrix. This study attempts to improve the properties of constructs formed with expanded chondrocytes through alterations in the expansion protocol and the ratio of primary to expanded chondrocytes used to form cartilage constructs. A chondrogenically tuned expansion protocol provided similar monolayer growth rates as those obtained using serum-containing medium and enhanced cartilaginous properties of resultant constructs. Various ratios of primary to chondrogenically expanded chondrocytes were then self-assembled to form neocartilage. Biochemical analysis showed that constructs formed with only expanded cells had twice the GAG per wet weight and collagen II/collagen I ratio compared to constructs formed with primary chondrocytes. Biomechanically, compressive properties of constructs formed with only passaged cells matched the instantaneous modulus and exceeded the relaxation modulus of constructs formed with only primary cells. These counterintuitive results show that, by applying proper expansion and three-dimensional culture techniques, the cartilage-forming potential of adult chondrocytes expanded through passage can be enhanced over that of primary cells.

Key words: Cartilage; Tissue engineering; Serum free; Chondrocyte; Self-assembly

Received June 22, 2011; final acceptance April 25, 2012. Online prepub date: October 4, 2012.
Address correspondence to Kyriacos A. Athanasiou, Ph.D., P.E., Department of Biomedical Engineering, University of California-Davis, One Shields Avenue, Davis, CA 95616, USA. Tel: +1 (530) 754-6645; Fax: +1 (530) 754-8375; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 22, pp. 341-353, 2013
0963-6897/13 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X653147
E-ISSN 1555-3892
Copyright © 2013 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Evaluation of the Use of Induced Pluripotent Stem Cells (iPSCs) for the Regeneration of Tracheal Cartilage

Mitsuyoshi Imaizumi,* Yukio Nomoto,* Yuka Sato,† Takashi Sugino,‡ Masao Miyake,§ Ikuo Wada,¶ Tatsuo Nakamura,# and Koichi Omori*

*Department of Otolaryngology, School of Medicine, Fukushima Medical University, Fukushima City, Japan
†Department of Microbiology, School of Medicine, Fukushima Medical University, Fukushima City, Japan
‡Department of Basic Pathology, School of Medicine, Fukushima Medical University, Fukushima City, Japan
§Department of Physiology, School of Medicine, Fukushima Medical University, Fukushima City, Japan
¶Department of Cell Science, Institute of Biomedical Sciences, School of Medicine, Fukushima Medical University, Fukushima City, Japan
#Department of Bioartificial Organs, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan

The treatment of laryngotracheal stenosis remains a challenge as treatment often requires multistaged procedures, and successful decannulation sometimes fails after a series of operations. Induced pluripotent stem cells (iPSCs) were generated in 2006. These cells are capable of unlimited symmetrical self-renewal, thus providing an unlimited cell source for tissue-engineering applications. We have previously reported tracheal wall regeneration using a three-dimensional (3D) scaffold containing iPSCs. However, the efficiency of differentiation into cartilage was low. In addition, it could not be proven that the cartilage tissues were in fact derived from the implanted iPSCs. The purpose of this study was to evaluate and improve the use of iPSCs for the regeneration of tracheal cartilage. iPSCs were cultured in vitro in a 3D scaffold in chondrocyte differentiation medium. After cultivation, differentiation into chondrocytes was examined. The ratio of undifferentiated cells was analyzed by flow cytometry. The 3D scaffolds were implanted into tracheal defects, as an injury site, in 24 nude rats. Differentiation into chondrocytes in vitro was confirmed histologically, phenotypically, and genetically. Flow cytometric analysis demonstrated that the population of undifferentiated cells was decreased. Cartilage tissue was observed in the regenerated tracheal wall in 6 of 11 rats implanted with induced iPSCs, but in none of 13 rats implanted with the control and noninduced iPSCs. The expression of cartilage-specific protein was also demonstrated in vivo in 3D scaffolds containing iPSCs. The presence of the GFP gene derived from iPSCs was confirmed in samples of cartilage tissue by the combination of laser microdissection (LMD) and polymerase chain reaction (PCR) techniques. Our study demonstrated that iPSCs have the potential to differentiate into chondrogenic cells in vitro. Cartilage tissue was regenerated in vivo. Our results suggest that iPSCs could be a new cell source for the regeneration of tracheal cartilage.

Key words: Induced pluripotent stem cells (iPSCs); Chondrogenesis; Bioengineered 3D scaffold; Regeneration; Tumor formation; Undifferentiated cells

Received May 26, 2011; final acceptance January 20, 2012. Online prepub date: August 2, 2012.
Address correspondence to Koichi Omori, M.D., Department of Otolaryngology, School of Medicine, Fukushima Medical University, 1 Hikarigaoka, Fukushima 960-1295, Japan. Tel: +81-24-547-1321; Fax: +81-24-548-3011; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 22, pp. 355-367, 2013
0963-6897/13 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X656108
E-ISSN 1555-3892
Copyright © 2013 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Toward Defining the Regenerative Potential of Olfactory Mucosa: Establishment of Schwann Cell-Free Adult Canine Olfactory Ensheathing Cell Preparations Suitable for Transplantation

Susanne Ziege,* Wolfgang Baumgartner,*† and Konstantin Wewetzer*†‡

*Department of Pathology, University of Veterinary Medicine, Hannover, Germany
†Center for Systems Neuroscience, Hannover, Germany
‡Department of Functional and Applied Anatomy, Center of Anatomy, Hannover Medical School, Hannover, Germany

Olfactory mucosa (OM)-derived olfactory ensheathing cells (OECs) are attractive candidates for autologous cell transplantation-based therapy of nervous system injury. However, defining the regenerative capacity of OM-derived OECs is impeded by the fact that cell cultures used for transplantation may contain significant amounts of contaminating trigeminal nerve Schwann cells that escape identification by sharing in vitro expression of OEC markers. The aim of the present study, therefore, was to quantify contaminating Schwann cells in OEC preparations and to develop a protocol for their specific depletion. Based on the observation that freshly dissociated, but not cultured, OECs and Schwann cells display differential expression of HNK-1 and p75NTR, magnet-activated cell sorting (MACS) was used to deplete myelinating (HNK-1-positive) and nonmyelinating (p75NTR-positive) Schwann cells from primary cell suspensions containing HNK-1-/p75NTR-negative OECs. Upregulation of p75NTR expression in OECs during culturing allowed their subsequent MACS-based separation from fibroblasts. Immunofluorescence analysis of freshly dissociated OM prior to MACS depletion revealed that 21% of the total and 56% of all CNPase-positive cells, representing both OECs and Schwann cells, expressed the Schwann cell antigens HNK-1 or p75NTR, indicating that freshly dissociated OM prior to culturing contained as many Schwann cells as OECs, while olfactory bulb (OB) primary cell suspensions revealed lower levels of Schwann cell contamination. Interestingly, neurite growth of neonatal rat dorsal root ganglion (DRG) neurons cocultured with OM-OECs, OB-OECs, and fibular nerve (FN) Schwann cells used as control was significantly higher in the presence of OECs than of Schwann cells. The first report on identification and specific depletion of Schwann cells from OEC preparations provides a solid basis for future efforts to fully define the regenerative potential of nasal mucosa OECs.

Key words: Olfactory ensheating cells (OECs); Purification; Large animal model; Translational medicine

Received November 11, 2011; final acceptance March 12, 2012. Online prepub date: September 21, 2012.
Address correspondence to Prof. Dr. Konstantin Wewetzer, Department of Functional and Applied Anatomy, Hannover Medical School, Carl-Neuberg-Str. 1, D-30625, Hannover, Germany. Tel: +49-511-532-5397; Fax: +49-511-532-166741; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 22, pp. 369-379, 2013
0963-6897/13 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X582769
E-ISSN 1555-3892
Copyright © 2013 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

The Immunosuppressive Effect of Mesenchymal Stromal Cells on B Lymphocytes Is Mediated by Membrane Vesicles

Manuela Budoni,*1 Alessandra Fierabracci,*1 Rosa Luciano,* Stefania Petrini,* Vincenzo Di Ciommo,† and Maurizio Muraca*

*Research Laboratories, Children’s Hospital “Bambino Gesù” Research Institute, Rome, Italy
†Service of Epidemiology and Biostatistics, Children’s Hospital “Bambino Gesù” Research Institute, Rome, Italy

The immunomodulatory properties of mesenchymal stromal cells are the subject of increasing interest and of widening clinical applications, but the reproducibility of their effects is controversial and the underlying mechanisms have not been fully clarified. We investigated the transfer of membrane vesicles, a recently recognized pathway of intercellular communication, as possible mediator of the interaction between mesenchymal stromal cells and B lymphocytes. Mesenchymal stromal cells exhibited a strong dose-dependent inhibition of B-cell proliferation and differentiation in a CpG-stimulated peripheral blood mononuclear cell coculture system. We observed that these effects could be fully reproduced by membrane vesicles isolated from mesenchymal stromal cell culture supernatants in a dose-dependent fashion. Next, we evaluated the localization of fluorescently labeled membrane vesicles within specific cell subtypes both by flow cytometry and by confocal microscopy analysis. Membrane vesicles were found to be associated with stimulated B lymphocytes, but not with other cell phenotypes (T lymphocytes, dendritic cells, natural killer cells), in peripheral blood mononuclear cell culture. These results suggest that membrane vesicles derived from mesenchymal stromal cells are the conveyors of the immunosuppressive effect on B lymphocytes. These particles should be further evaluated as immunosuppressive agents in place of the parent cells, with possible advantages in term of standardization, safety, and feasibility.

Key words: Mesenchymal stromal cells (MSCs); Membrane vesicles (MVs); Immunomodulation; B lymphocytes; Proliferation; Differentiation

Received August 2, 2011; final acceptance March 12 ,2012. Online prepub date: August 27, 2012.
1These authors provided equal contribution to this work.
Address correspondence to Prof. Maurizio Muraca, Research Laboratories, Children’s Hospital “Bambino Gesù,” Piazza Sant’Onofrio, 4, 00165 Rome, Italy. Tel: +39 06 6859 2210; Fax +39 06 6859 2014; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it