Cell Transplantation 23(12) Abstracts

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Cell Transplantation, Vol. 23, pp. 1475-1487, 2014
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DOI: http://dx.doi.org/10.3727/096368913X670192
E-ISSN 1555-3892
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Chromosomal Instability but Lack of Transformation in Human Myoblast Preparations

Aurélie Bisson,*†‡1 Stéphanie Le Corre,*§1 Géraldine Joly-Helas,¶ Pascal Chambon,¶# Laetitia Demoulins,§ Laetitia Jean,*† Sahil Adriouch,*† Laurent Drouot,*† Camille Giverne,§ Francis Roussel,** Serge Jacquot,*†§ Christelle Doucet,‡ Francis Michot,†††‡‡ Marek Lamacz,*† Thierry Frébourg,†#§§ Jean-Michel Flaman,†#§§ and Olivier Boyer*†§

*Inserm, U905, Rouen, France
†Normandy University, IRIB, Rouen, France
Celogos, Paris, France
§Rouen University Hospital, Laboratory of Biotherapy, Rouen, France
¶Rouen University Hospital, Department of Cytogenetics, Rouen, France
#Inserm, U1079, Rouen, France
**Rouen University Hospital, Department of Pathology, Rouen, France
††Inserm, U1073, Rouen, France
‡‡Rouen University Hospital, Department of Digestive Surgery, Rouen, France
§§Rouen University Hospital, Department of Genetics, Rouen, France

Genetic alterations have recently been described as emerging during the culture of embryonic stem cells or induced pluripotent stem cells, raising concerns about their safety in future clinical use. Myoblasts are adult stem cells with important therapeutic potential that have been used in clinical trials for almost 20 years, but their genome integrity has not yet been established. Here we produced 10 human myoblast preparations and investigated their genomic stability. At the third passage, half of the preparations had a normal karyotype and half showed one to four alterations/30 metaphases. Chromosome 2 trisomy was found in 1-2/30 metaphases and/or 2/100 nuclei by FISH in 3/10 samples, and there was no other recurrent anomaly. When prolonging cultures, these erratic abnormalities were never associated with a growth advantage. Cellular senescence was manifested in all samples by growth arrest before passage 15. Expression of TERT was always negative. Molecular analysis of individual p53 transcripts did not reveal tumorigenic mutations. CGH array (10 samples) and exome sequencing (one sample) failed to detect copy number variations or accumulation of mutations, respectively. Myoblasts did not grow either in soft agar or in vivo after injection inimmunodeficient mice. Hence, occasional genomic abnormalities may occur during myoblast culture but are not associated with risk of transformation.

Key words: Cell culture; Cell therapy; Genomic stability; KaryotypeMyoblasts

Received November 14, 2012; final acceptance July 9, 2013. Online prepub date: July 17, 2013.
1These authors provided equal contribution to this work.
Address correspondence to Olivier Boyer, Inserm U905, Faculté de Médecine et Pharmacie, 22 bd Gambetta, F-76000 Rouen, France. Tel: +33 2 35 148535; Fax: +33 2 32 888186; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 23, pp. 1489-1500, 2014
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DOI: http://dx.doi.org/10.3727/096368913X672037
E-ISSN 1555-3892
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Radioelectric Asymmetric Conveyed Fields and Human Adipose-Derived Stem Cells Obtained With a Nonenzymatic Method and Device: A Novel Approach to Multipotency

Margherita Maioli,*†‡ Salvatore Rinaldi,‡§ Sara Santaniello,*† Alessandro Castagna,‡§ Gianfranco Pigliaru,*† Alessandro Delitala,* Francesca Bianchi,†¶**†† Carlo Tremolada,# Vania Fontani,‡§ and Carlo Ventura†‡¶**††

*Department of Biomedical Sciences, University of Sassari, Sassari, Italy
†Laboratory of Molecular Biology and Stem Cell Engineering - National Institute of Biostructures and Biosystems, Bologna, Italy
‡Department of Regenerative Medicine, Rinaldi Fontani Institute, Florence, Italy
§Department of Neuro Psycho Physical Optimization, Rinaldi Fontani Institute, Florence, Italy
¶Cardiovascular Department, S. Orsola - Malpighi Hospital, University of Bologna, Bologna, Italy
#Istituto Image, Milano, Italy
**Current affiliation: National Institute of Biostructures and Biosystems, Bologna, Italy
††Current affiliation: Stem Wave Institute for Tissue Healing (SWITH), Gruppo Villa Maria (GVM) Care & Research - Ettore Sansavini Health Science Foundation, Lugo (Ravenna), Italy

Human adipose-derived stem cells (hASCs) have been recently proposed as a suitable tool for regenerative therapies for their simple isolation procedure and high proliferative capability in culture. Although hASCs can be committed into different lineages in vitro, the differentiation is a low-yield and often incomplete process. We have recently developed a novel nonenzymatic method and device, named Lipogems, to obtain a fat tissue derivative highly enriched in pericytes/mesenchymal stem cells by mild mechanical forces from human lipoaspirates. When compared to enzymatically dissociated cells, Lipogems-derived hASCs exhibited enhanced transcription of vasculogenic genes in response to provasculogenic molecules, suggesting that these cells may be amenable for further optimization of their multipotency. Here we exposed Lipogems-derived hASCs to a radioelectric asymmetric conveyer (REAC), an innovative device asymmetrically conveying radioelectric fields, affording both enhanced differentiating profiles in mouse embryonic stem cells and efficient direct multilineage reprogramming in human skin fibroblasts. We show that specific REAC exposure remarkably enhanced the transcription of prodynorphin, GATA-4, Nkx-2.5, VEGF, HGF, vWF, neurogenin-1, and myoD, indicating the commitment toward cardiac, vascular, neuronal, and skeletal muscle lineages, as inferred by the overexpression of a program of targeted marker proteins. REAC exposure also finely tuned the expression of stemness-related genes, including NANOG, SOX-2, and OCT-4. Noteworthy, the REAC-induced responses were fashioned at a significantly higher extent in Lipogems-derived than in enzymatically dissociated hASCs. Therefore, REAC-mediated interplay betweenradioelectric asymmetrically conveyed fields and Lipogemsderived hASCs appears to involve the generation of an ideal “milieu” to optimize multipotency expression from human adult stem cells in view of potential improvement of future cell therapy efforts.

Key words: Radioelectric asymmetric conveyed fields; Human stem cells; Cell multipotency

Received November 1, 2012; final acceptance August 17, 2013. Online prepub date: August 30, 2013.
Address correspondence to Prof. Carlo Ventura, Stem Wave Institute for Tissue Healing (SWITH), Gruppo Villa Maria (GVM) Care and Research - Ettore Sansavini Health Science Foundation, Via Provinciale per Cotignola 9, 48022 Lugo (Ravenna), Italy. E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 23, pp. 1501-1515, 2014
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DOI: http://dx.doi.org/10.3727/096368914X678553
E-ISSN 1555-3892
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Human Second Trimester Amniotic Fluid Cells Are Able to Create Embryoid Body-Like Structures In Vitro and to Show Typical Expression Profiles of Embryonic and Primordial Germ Cells

Ivana Antonucci,*† Roberta Di Pietro,†‡ Melissa Alfonsi,§ Maria Antonietta Centurione,†‡ Lucia Centurione,†‡ Silvia Sancilio,§ Francesca Pelagatti,* Maria Angela D’Amico,‡ Angela Di Baldassarre,‡ Adriano Piattelli,§ Stefano Tetè,†¶ Giandomenico Palka,¶ Cesar V. Borlongan,# and Liborio Stuppia*†

*Laboratory of Molecular Genetics, Department of Psychological, Humanities and Territorial Sciences, School of Medicine and Health Sciences, G. d’Annunzio University Chieti-Pescara, Chieti, Italy
†Stem Tech Group, Center for Aging Studies (CESI), “G. d’Annunzio” University, Chieti-Pescara, Italy
‡Department of Medicine and Aging Sciences, School of Medicine and Health Sciences
§Pharmacy Department, “G. d’Annunzio” University, Chieti-Pescara, Italy
¶Department of Clinical, Oral and Biotechnological Sciences, School of Medicine and Health Sciences, “G. d’Annunzio” University, Chieti-Pescara, Italy
#Department of Neurosurgery and Brain Repair, Center of Excellence for Aging and Brain Repair, University of South Florida Morsani College of Medicine, Tampa, FL, USA

Human amniotic fluid-derived stem cells (AFSCs) represent a novel class of broadly multipotent stem cells sharing characteristics of both embryonic and adult stem cells. However, both the origin of these cells and their actual properties in terms of pluripotentdifferentiation potential are still debated. In order to verify the presence of features of pluripotency in human second trimester AFSCs, we have investigated the ability of these cells to form in vitro three-dimensional aggregates, known as embryoid bodies (EBs), and to express specific genes of embryonic stem cells (ESCs) and primordial germ cells (PGCs). EBs were obtained after 5 days of AFSC culture in suspension and showed positivity for alkaline phosphatase (AP) staining and for specific markers ofpluripotency (OCT4 and SOX2). Moreover, EB-derived cells showed the expression of specific transcripts of the three germ layers. RT-PCR analysis, carried out at different culture times (second, third, fourth, fifth, and eighth passages), revealed the presence of specific markers of ESCs (such as FGF4 and DAPPA4), as well as of markers typical of PGCs and, in particular, genes involved in early stages of germ cell development (Fragilis, Stella, Vasa, c-Kit, Rnf17). Finally, the expression of genes related to the control of DNA methylation (DNMT3A, DNMT3b1, DNMT1, DNMT3L, MBD1, MBD2, MBD3, MDB4, MeCP2), as well as the lack of inactivation of the X-chromosome in female samples, was also demonstrated. Taken together, these data provide further evidence for the presence of common features among human AFSCs, PGCs, and ESCs.

Key words: Amniotic fluid stem cells (AFSCs); Primordial germ cells (PGCs); Pluripotency

Received August 9, 2013; final acceptance December 30, 2013. Online prepub date: January 29, 2014.
Address correspondence to Liborio Stuppia, M.D., Laboratory of Molecular Genetics, Department of Psychological, Humanities and Territorial Sciences, School of Medicine and Health Sciences, G. d’Annunzio University Chieti-Pescara, Via dei Vestini 31, 66013 Chieti, Italy. Tel: (+39) 08713555300; Fax: (+39) 08713555341; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 23, pp. 1517-1523, 2014
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DOI: http://dx.doi.org/10.3727/096368913X674071
E-ISSN 1555-3892
Copyright ©
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Role of Serum Levels of Angiogenic Cytokines in Assessment of Angiogenesis After Stem Cell Therapy of Diabetic Patients With Critical Limb Ischemia

Michal Dubsky,*† Alexandra Jirkovska,* Robert Bem,* Vladimira Fejfarova,* Martin Varga,‡ Libor Kolesar,§ Libuse Pagacova,¶ Eva Sykova,# and Edward. B. Jude**

*Diabetes Centre, Institute for Clinical and Experimental Medicine, Prague, Czech Republic
†First Medical Faculty, Charles University, Prague, Czech Republic
‡Clinic of Transplant Surgery, Institute for Clinical and Experimental Medicine, Prague, Czech Republic
§Department of Immunogenetics, Institute for Clinical and Experimental Medicine, Prague, Czech Republic
Autotransfusion Unit, Institute for Clinical and Experimental Medicine, Prague, Czech Republic
#Institute of Experimental Medicine, Czech Academy of Science, Prague, Czech Republic
**Diabetes Centre, Tameside Hospital NHS Foundation Trust and University of Manchester, Ashton-Under-Lyne, Lancashire, UK

The release of proangiogenic cytokines into the circulation after stem cell (SC) therapy and compensatory increase of angiogenesis inhibitors may reflect local vasculogenesis but also can increase the risk of side effects. The aim of our study was to evaluate serum levels of angiogenic cytokines with regard to the assessment of local and systemic vasculogenesis in diabetic patients with no-option critical limb ischemia (NO-CLI). Twenty-five diabetic patients with NO-CLI treated with SCs isolated from bone marrow or stimulated peripheral blood were included in the study. Serum levels of proangiogenic cytokines (VEGF, bFGF, Ang-1, PDGF-AA, and PDGF-BB) and an antiangiogenic cytokine (endostatin) were assessed 6 months after cell treatment, compared to baseline values, and correlated with the number of injected CD34+
cells. The clinical effect of SC therapy (assessed by changes in TcPO2) and potential systemic vasculogenesis (assessed by eye fundus examination) were evaluated after 6 months. Serum levels of angiogenic inhibitor endostatin increased significantly after 1 and 3 months (p = 0.0003), but no significant increase in serum levels of proangiogenic cytokines was observed. A significant correlation between number of injected CD34+ cells and serum levels of endostatin was observed (r = 0.41, p < 0.05); however, proangiogenic cytokines did not correlate with CD34+ cells. No correlation between increase in TcPO2 after treatment and serum levels of any of the angiogenic cytokines were seen, and no signs of systemic vasculogenesis in the retina were observed after 6 months. Despite the significant increase in the levels of the angiogenic inhibitor endostatin following SC treatment, there was no risk of systemic vasculogenesis after SC therapy as documented by serum levels of proangiogenic cytokines or changes in the retina.

Key words: Systemic vasculogenesis; Stem cell (SC) therapy; Angiogenic cytokines; Critical limb ischemia (CLI)

Received April 24, 2013; final acceptance October 3, 2013. Online prepub date: October 21, 2013.
Address correspondence to Michal DubskyVidenska 1958/9, 14021 Prague 4, Czech Republic. Tel: +420261362150; Fax: +420261362820; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 23, pp. 1525-1535, 2014
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DOI: http://dx.doi.org/10.3727/096368913X664874
E-ISSN 1555-3892
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Efficiency of Statin Treatment on EPC Recruitment Depends on Baseline EPC Titer and Does Not Improve Angiographic Outcome in Coronary Artery Disease Patients Treated With the Genous™ Stent

Wijnand K. den Dekker,* Jaco H. Houtgraaf ,* Stephen M. Rowland,† Erik Ligtenberg,† Sanneke P. M. de Boer,* Renate de Jong,* Robbert J. de Winter,‡ Peter den Heijer,§ Felix Zijlstra,* Patrick W.Serruys,* Caroline Cheng,*¶ and Henricus J. Duckers*#

*Thoraxcenter Rotterdam, Erasmus University Medical Center, Rotterdam, The Netherlands
OrbusNeich, Fort Lauderdale, FL, USA
Academisch Medisch Centrum, Amsterdam, The Netherlands
§Amphia Ziekenhuis, Breda, The Netherlands
¶Regenerative Medicine and Stem Cells, Vascular Tissue Regeneration, Division of Internal Medicine and Dermatology, University Medical Center Utrecht, The Netherlands
#Department of Cardiology, University Medical Center Utrecht, The Netherlands

The objective of this study was to assess the effect of high-dose atorvastatin treatment on endothelial progenitor cell (EPC) recruitment and angiographic and clinical outcome in coronary artery disease (CAD) patients treated with the GenousTM
EPC-capturing stent. The HEALING IIB study was a multicenter, open-label, prospective trial that enrolled 100 patients. Patients were started on 80 mg atorvastatin qd, at least 2 weeks before the index procedure and continued for at least 4 weeks after the index procedure. Eighty-seven patients were included in this analysis. EPC levels significantly increased as early as 2 weeks after the start of atorvastatin. Remarkably, among this group, 31 patients proved to be nonresponders to atorvastatin treatment (i.e., no increase in EPC levels), while 56 patients were responders (i.e., rise in EPC count between week −2 and 0). Compared to responders, nonresponders had a significantly higher baseline EPC count (76 ± 10 vs. 41 ± 5, p < 0.01) with a lower late luminal loss (LLL) at 6- and 18-month follow-up (FU) (0.61 ± 0.07 vs. 0.88 ± 0.08, p < 0.05, and 0.50 ± 0.08 vs. 0.82 ± 0.08, p < 0.01 respectively). Furthermore, baseline EPC count was inversely correlated with LLL at 6-month FU (R = −0.42, p < 0.001). Patients with a higher EPC count at baseline showed no increase in EPC recruitment in response to statin treatment but had favorable LLL at 6- and 18-month FU, whereas patients with lower EPC count were responsive to statin therapy, but EPCs might be less functional as they had higher LLL at 6- and 18-month FU. These data imply that although statin treatment can enhance EPC titer in patients with low baseline levels, there is no indication for a possible beneficial clinical effect with EPC capture stents.

Keywords: Endothelial progenitor cells; Restenosis; Stent; Statin; Late luminal loss

Received April 21, 2012; final acceptance March 5, 2013. Online prepub date: April 3, 2013.
Address correspondence to Dr. H. J. Duckers, M.D., Ph.D. FESC, Division of Cardiology & Pulmonology, Department of Interventional Cardiology, University Medical Center Utrecht, Room E04-201, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands. Tel: +31-(0)88 755 6167; Fax: +31 10 463 2857; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 23, pp. 1537-1544, 2014
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DOI: http://dx.doi.org/10.3727/096368913X672046
E-ISSN 1555-3892
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Potential Barriers to Human Hepatocyte Transplantation in MUP–uPAtg(+/+)Rag2−/−γC−/− Mice

Junji Komori,* Aaron D. DeWard,* Roberto Gramignoli,† Stephen C. Strom,† Paulo Fontes,‡ and Eric Lagasse*

*McGowan Institute for Regenerative Medicine, Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
Karolinska Institutet and Hospital, Department of Laboratory Medicine, Division of Pathology, Stockholm, Sweden
‡McGowan Institute for Regenerative Medicine, Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

Primary human fetal and adult hepatocytes have been considered feasible donor cell sources for cell transplantation. We compared the engraftment efficiencies between adult human, fetal human, and adult porcine hepatocytes after transplantation into MUP–uPAtg(+/+)Rag2−/−γC−/− mice. Transplantation of adult human hepatocytes yielded a 1,000-fold higher serum albumin level compared to transplantation of fetal human hepatocytes, while transplantation of adult porcine hepatocytes resulted in a 100-fold higher serum albumin level than adult human hepatocytes. These results suggest that adult liver cells are superior to fetal liver cells for transplantation, and caution should be applied if porcine hepatocytes are used for preclinical studies as a proof of concept for human hepatocytes.

Key words: Liver cell transplantation; Fetal human hepatocytes; Adult human hepatocytes; Adult porcine hepatocytes

Received April 16, 2013; final acceptance August 24, 2013. Online prepub date: August 30, 2013.
Address correspondence to Eric LagassePharmD, Ph.D., McGowan Institute for Regenerative Medicine, University of Pittsburgh School of Medicine, 450 Technology Drive, Room 329, Pittsburgh, PA 15219, USA. Tel: +1-412-624 5285; Fax: +1-412-624-5363; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 23, pp. 1545-1556, 2014
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DOI: http://dx.doi.org/10.3727/096368914X680064
E-ISSN 1555-3892
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Rapid and Sensitive Assessment of Human Hepatocyte Functions

Roberto Gramignoli,* Veysel Tahan,† Kenneth Dorko,‡ Raman Venkataramanan,§ Ira J. Fox,¶ Ewa C. S. Ellis,# Massoud Vosough,* and Stephen C. Strom*

*Department of Laboratory Medicine, Division of Pathology, Karolinska Institutet, Stockholm, Sweden
†Department of Gastroenterology, University of Iowa, Iowa City, IA, USA
‡Department of Pharmacology, Toxicology and Therapeutics, KU Medical Center, Kansas City, KS, USA
§Department of Pharmaceutical Sciences, University of Pittsburgh, Pittsburgh, PA, USA
¶Department of Surgery, Children’s Hospital of Pittsburgh of UPMC, and McGowan Institute for Regenerative Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
#Department of Clinical Science, Intervention and Technology (CLINTEC), Division of Transplantation Surgery, Karolinska University Hospital Huddinge, Stockholm, Sweden

Transplantation of human hepatocytes (HTx) has gained recognition as a bridge to, or an alternative to, orthotopic liver transplantation for patients with acute liver failure or genetic defects in liver function. Although the quality of the hepatocytes used for cell transplantation is critical, no consensus exists on protocols to assess the function of hepatocytes prior to HTx. Application of this cell therapy in clinical practice could be aided by fast and reliable assays to evaluate the functional competence of isolatedhepatocytes prior to clinical transplantation. Traditional assays for measuring metabolic functions in primary hepatocytes frequently involve highly technical equipment, time-consuming methods, and large numbers of cells. We describe a novel approach for the rapid assessment of the metabolic capabilities of human hepatocytesThis report details simple procedures to evaluate 11 endpoints from cells isolated from human liver that can be performed by a single operator within approximately 2 h of isolation. Longer term cultured hepatocytes were also analyzed to determine if the results from the 2-h tests were predictive of long-term hepatic function. The assays simultaneously measure five cytochrome 450 activities, one phase II activity, plating efficiency, and ammonia metabolism in addition to viability and cell yield. The assays require fewer than 20 million cells and can be completed using commonly available and inexpensive laboratory equipment. The protocol details methods that can be used in a time frame that would allow analysis of hepatic functions in freshly isolated hepatocytes prior to their use for clinical transplantation.

Key words: Hepatocyte transplantation; Human hepatocytes; Hepatic functions

Received November 19, 2013; final acceptance December 30, 2013. Online prepub date: March 24, 2014.
Address correspondence to Stephen C. Strom, Ph.D., Department of Laboratory Medicine, Division of Pathology, Karolinska Institutet, 141 86 Stockholm, Sweden. E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 23, pp. 1557-1572, 2014
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DOI: http://dx.doi.org/10.3727/096368913X667501
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Increased Survival Despite Failure of Transplanted Human Hepatocyte Implantation Into Liver Parenchyma of Nude Mice With Repeated Lethal Jo2-Induced Liver Deficiency

Isabelle Vidal,*† Nadege Blanchard,‡ Marie-Pierre Chenard-Neu,§ Philippe Bachellier,¶ Bruno Heyd,*# Frank Staedtler,** Martin Schumacher,** Eliane Alexandre,‡ and Lysiane Richert*‡

*EA 3921, IFR 133, Faculte de Medecine et de Pharmacie, Besancon, France
†Unit of Pediatric Surgery, University Hospital, Geneva, Switzerland
KaLy-Cell, Plobsheim, France
§Service d’Anatomo-PathologieHopital de Hautepierre, Strasbourg, France
¶Centre de Chirurgie Viscerale et de Transplantation, Hopital de Hautepierre, Strasbourg, France
#Service de Chirurgie Digestive et VasculaireHopital Jean Minjoz, Besancon, France
**Novartis Pharma AG, Biomarker Development, Basel, Switzerland

We recently found that rat hepatocyte transplantation was efficient (liver repopulation: 2.4%) in a sublethal nude mouse model (less than 33% mortality) of repeated liver injury generated using Jo2, a mouse-specific anti-Fas antibody, at sublethal dose of 250μg/kg for 3 weeks. Genomic analysis of the livers revealed cell cycle blockade and an antiproliferative status of circadian genes, suggesting a selective advantage. By contrast, in the present study, freshly isolated human hepatocyte transplantation performed in the same mouse model resulted in implantation of less than 6,000 cells per liver (about 0.006% repopulation) in all animals. Genomic analysis of nude mouse livers revealed a lack of P21 upregulation, while a signature of stimulation of liver regeneration was observed, including upregulation of early response genes and upregulation of circadian genes. When we translated this sublethal model to a lethal model (65% mortality) by increasing the Jo2 repeated doses to 375 μg/kg, human hepatocyte engraftment was still very low; however, animal mortality was corrected by transplantation (only 20% mortality). Genomic findings in livers from the mice of the lethal Jo2 transplanted group were similar to those of the sublethal Jo2 transplanted group, that is, no selective advantage genomic signature and signature of mouse liver regeneration. In conclusion, transplanted human hepatocytes acted as if they modified nude mouse liver responses to Jo2 by stimulating liver regeneration, leading to an increased survival rate.

Key words: Human hepatocytes; Jo2; Hepatocyte transplantation; Genomic analysis; Nude mouse

Received July 23, 2011; final acceptance April 19, 2013. Online prepub date: April 29, 2013.
Address correspondence to Lysiane RichertKaLy-Cell, 20A rue du General Leclerc, 67115 Plobsheim, France. Tel: +33 3 88 10 88 30; Fax: +33 3 88 43 56 71; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 23, pp. 1573-1584, 2014
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DOI: http://dx.doi.org/10.3727/096368913X673432
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Enabling Autologous Human Liver Regeneration With Differentiated Adipocyte Stem Cells

Dan Xu,* Toshihiko Nishimura,* Ming Zheng,* Manhong Wu,* Hua Su,† Noboru Sato,† Gordon Lee,‡ Sara Michie,§ Jeffrey Glenn,¶ and Gary Peltz*

*Department of Anesthesia, Stanford University School of Medicine, Stanford, CA, USA
†Department of Biochemistry, University of California-Riverside, Riverside, CA, USA
‡Department of Plastic Surgery, Stanford University School of Medicine, Stanford, CA, USA
§Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA
¶Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA

We developed a novel method for differentiating adipocyte-derived stem cells (ASCs) into hepatocyte-like cells (iHeps). ASCs are cultured as spherical cellular aggregates and are then induced by culture in chemically defined media for a short time period to differentiate into spherical culture iHeps (SCi-Heps). SCi-Heps have many of the in vitro functional properties of mature hepatocytes, and they can stably reconstitute functioning human liver in vivo in a murine model system. Implantation studies demonstrate that SCi-Heps have a very low malignant potential. All human liver regenerative procedures, including ultrasound-guided direct liver implantation, are scalable and appropriate for human clinical use. These methods can be used to achieve the major promise of regenerative medicine. It may now be possible to regenerate human liver using autologous stem cells obtained from a readily accessible tissue.

Key words: Liver regeneration; Adipocyte stem cells; Chimeric mice; Induced hepatocyte-like cells

Received July 25, 2013; final acceptance September 3, 2013. Online prepub date: October 21, 2013.
Address correspondence to Gary Peltz, Department of Anesthesia, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305, USA. Tel: +1 650 721 2487; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 23, pp. 1585-1597, 2014
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DOI: http://dx.doi.org/10.3727/096368913X673450
E-ISSN 1555-3892
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Transplantation of Human Adipose Tissue-Derived Stem Cells Delays Clinical Onset and Prolongs Life Span in ALS Mouse Model

Kwang S. Kim,*†1 Hong J. Lee,*‡1 Jin An,* Yun B. Kim Jung Chan Ra,¶ Inja Lim,† and Seung U. Kim*‡1

*Medical Research Institute, Chung-Ang University College of Medicine, Seoul, Korea
†Department of Physiology, Chung-Ang University College of Medicine, Seoul, Korea
‡Division of Neurology, Department of Medicine, UBC Hospital University of British Columbia, Vancouver, BC, Canada
§Department of Toxicology, Chungbuk National University School of Veterinary Medicine, Cheungju, Korea
¶Stem Cell Research Center, K-STEMCELL Co., Ltd., Seoul, Korea

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that selectively affects motor neurons in the cortex, brain stem, and spinal cord. The precise pathogenic mechanism remains unknown, and there is currently no effective therapy. We evaluated the therapeutic effects of human adipose tissue-derived stem cells (ASCs) in an animal model of ALS. Human abdominal subcutaneous fat tissues were obtained by simple liposuction from donors, and ASCs were isolated from the fat stromal vascular fraction. ASCs were found to differentiate into adipocyteschondrocytesosteocytes, and neurons. SOD1G93A ALS mice were divided into three groups: sham, intravenous (IV), and intracerebroventricular (ICV) groups. Human ASCs were transplanted in the ALS mice at 70 postnatal days before the appearance of clinical symptoms. Behavior of transplanted animals was assessed by rotarod test, paw grip endurance (PaGE), and reflex index. Mice in every group were sacrificed after 4 weeks posttransplantation. Transplanted ASCs were identified in the lumbar spinal cords with an antihuman mitochondria antibody and cell type-specific markers for neurons or astrocytes. Delayed onset of clinical symptoms (26 days) and extended survival of animals (24 days) were observed in ALS mice transplanted with ASCs via ICV route. ASCs were found to secrete high levels of neurotrophic factors such as NGF, BDNF, IGF-1, and VEGF. Reduction of apoptotic cell death by these factors was confirmed in cultured CNS cells and in the ALS spinal cord. These results indicate that transplantation of ASCs in ALS mice provides neuroprotective effects by production of cytokines/growth factors, delays disease progression, and prolongs the life span of ALS mice.

Key words: Adipose tissue-derived stem cells (ASCs); Mesenchymal stem cells (MSCs); Transplantation; Cell therapy; Cytokines; Neurotrophic factor; Neuroprotection; Amyotrophic lateral sclerosis (ALS); Cu/Zn superoxide dismutase (SOD1); Transgenic mouse

Received May 5, 2013; final acceptance September 5, 2013. Online prepub date: September 18, 2013.
1These authors provided equal contribution to this work.
Address correspondence to Seung U. Kim, M.D., Ph.D., Division of Neurology, Department of Medicine, UBC Hospital, University of British Columbia, 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. 23, pp. 1599-1612, 2014
0963-6897/14 $90.00
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DOI: http://dx.doi.org/10.3727/096368914X678562
E-ISSN 1555-3892
Copyright ©
2014 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Intracerebral Implantation of Autologous Peripheral Blood Stem Cells in Stroke Patients: A Randomized Phase II Study

Der-Cherng Chen,*†‡1 Shinn-Zong Lin,*§1 Jia-Rong Fan,* Chen-Huan Lin,* Wei Lee,* Chao-Chun Lin,¶# Yi-Jui Liu,** Chon-Haw Tsai,†† Jui-Cheng Chen,†† Der-Yan Cho,‡§ Chau-Chin Lee,‡‡ and Woei-Cherng Shyu*§††§§

*Center for Neuropsychiatry, China Medical University Hospital, Taichung, Taiwan
†Graduate Institute of Clinical Medical Science, China Medical University, Taichung, Taiwan
‡Department of Neurosurgery, China Medical University Hospital, Taichung, Taiwan
§Graduate Institute of Immunology, China Medical University, Taichung, Taiwan
¶Department of Radiology, China Medical University Hospital, Taichung, Taiwan
#Graduate Institute of Biomedical Electronics and Bioinformatics, National Taiwan University, Taipei, Taiwan
**Department of Automatic Control Engineering, Feng-Chia University, Taichung, Taiwan
††Department of Neurology, China Medical University Hospital, Taichung, Taiwan
‡‡Department of Radiology, Buddhist Tzu-Chi General Hospital, Hualien, Taiwan
§§Translational Medicine Research Center, China Medical University Hospital, Taichung, Taiwan

In our previous study, intracerebral implantation of peripheral blood stem cells (PBSCs) improved functional outcome in rats with chronic cerebral infarction. Based on this finding, a randomized, single blind controlled study was conducted in 30 patients [PBSC group (n = 15) and control group (n = 15)] with middle cerebral artery infarction confirmed on a T2-weighted MRI 6 months to 5 years after a stroke. Only subjects with neurological deficits of intermediate severity based on the National Institute of Health Stroke Scale (NIHSS; range: 9–20) that had been stable for at least 3 months were enrolled. Those in the PBSC group received subcutaneous G-CSF injections (15 μg/kg/day) for 5 consecutive days, and then stereotaxic implantation of 3–8 × 106
CD34+ immunosorted PBSCs. All 30 patients completed the 12-month follow-up. No serious adverse events were noted during study period. Improvements in stroke scales (NIHSS, ESS, and EMS) and functional outcomes (mRS) from baseline to the end of the 12-month follow-up period were significantly greater in the PBSC than the control group. The fiber numbers asymmetry (FNA) scores based on diffusion tensor image (DTI) tractography were reduced in every PBSC-treated subject, but not in the control group. Reduction in the FNA scores correlated well with the improvement in NIHSS. Furthermore, a positive motor-evoked potential (MEP) response by transcranial magnetic stimulation (TMS) appeared in 9 of the 15 subjects in the PBSC group. This phase II study demonstrated that implantation of autologous CD34+ PBSC was safe, feasible, and effective in improving functional outcome.

Key words: Peripheral blood stem cells; Stroke; Immunosorting; CD34

Received November 20, 2013; final acceptance January 10, 2014. Online prepub date: January 29, 2014.
1These authors provided equal contribution to this work.
Address correspondence to Woei-Cherng Shyu, M.D., Ph.D., Center for Neuropsychiatry, Graduate Institute of Immunology, and Translational Medicine Research Center, China Medical University and Hospital, 2 Yuh-Der Road, Taichung, Taiwan 40447, Republic of China. Tel: +886-4-2205-2121, ext. 6034; Fax: +886-4-2208-0666; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 23, pp. 1613-1630, 2014
0963-6897/14 $90.00
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DOI: http://dx.doi.org/10.3727/096368914X676916
E-ISSN 1555-3892
Copyright ©
2014 Cognizant Comm. Corp.
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Repeated Administrations of Human Umbilical Cord Blood Cells Improve Disease Outcomes in a Mouse Model of Sanfilippo Syndrome Type III B

Alison E. Willing,*†‡1 Svitlana N. Garbuzova-Davis,*†‡§1 Olga Zayko,* Hiranya M. Derasari,* Ashley E. Rawls,* Chris R. James,* Ron F. Mervis,*† Cyndy D. Sanberg,¶ Nicole Kuzmin-Nichols,¶ and Paul R. Sanberg*†‡§#

*Center of Excellence for Aging and Brain Repair, Morsani College of Medicine, University of South Florida, Tampa, FL, USA
†Department of Neurosurgery and Brain Repair, Morsani College of Medicine, University of South Florida, Tampa, FL, USA
‡Department of Molecular Pharmacology and Physiology, Morsani College of Medicine, University of South Florida, Tampa, FL, USA
§Department of Pathology and Cell Biology, Morsani College of Medicine, University of South Florida, Tampa, FL, USA
Saneron CCEL Therapeutics, Inc., Tampa, FL, USA
#Department of Psychiatry, Morsani College of Medicine, University of South Florida, Tampa, FL, USA

Sanfilippo syndrome type III B (MPS III B) is an inherited disorder characterized by a deficiency of α-N-acetylglucosaminidase (Naglu) enzyme leading to accumulation of heparan sulfate in lysosomes and severe neurological deficits. We have previously shown that a single administration of human umbilical cord mononuclear cells (hUCB MNCs) into Naglu knockout mice decreased behavioral abnormalities and tissue pathology. In this study, we tested whether repeated doses of hUCB MNCs would be more beneficial than a single dose of cells. Naglu mice at 3 months of age were randomly assigned to either a Media-only group or one of three hUCB MNC treatment groups—single low dose (3 × 106
cells), single high dose (1.8 × 107 cells), or multiple doses (3 × 106 cells monthly for 6 months) delivered intravenously; cyclosporine was injected intraperitoneally to immune suppress the mice for the duration of the study. An additional control group of wild-type mice was also used. We measured anxiety in an open field test and cognition in an active avoidance test prior to treatment and then at monthly intervals for 6 months. hUCB MNCs restored normal anxiety-like behavior in these mice (p < 0.001). The repeated cell administrations also restored hippocampalcytoarchitecture, protected the dendritic tree, decreased GM3 ganglioside accumulation, and decreased microglial activation, particularly in the hippocampus and cortex. These data suggest that the neuroprotective effect of hUCB MNCs can be enhanced by repeated cell administrations.

Key words: Cord blood; Mononuclear cells; Naglu enzyme; Behavior; Hippocampus; Microglia; Heparan sulfate

Received August 16, 2013; final acceptance December 19, 2013. Online prepub date: December 30, 2013.
1These authors provided equal contribution to this work.
Address correspondence to Alison E. Willing, Ph.D., Center of Excellence for Aging and Brain Repair, Morsani College of Medicine, University of South Florida, 12901 Bruce B. Downs Blvd., MDC78, Tampa, FL 33612, USA. Tel: +1 813-974-7812; Fax: +1 813-974-3078; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 23, pp. 1631-1655, 2014
0963-6897/14 $90.00
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DOI: http://dx.doi.org/10.3727/096368914X685131
E-ISSN 1555-3892
Copyright ©
2014 Cognizant Comm. Corp.
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Functional Regeneration of Supraspinal Connections in a Patient With Transected Spinal Cord Following Transplantation of Bulbar OlfactoryEnsheathing Cells With Peripheral Nerve Bridging

Pawel Tabakow,* Geoffrey Raisman,† Wojciech Fortuna,*‡ Marcin Czyz,* Juliusz Huber,§ Daqing Li,† Pawel Szewczyk,¶ Stefan Okurowski,# Ryszard Miedzybrodzki,**†† Bogdan Czapiga,* BeataSalomon,‡‡ Agnieszka Halon,§§ Ying Li,† Joanna Lipiec,§ Aleksandra Kulczyk,§ and Wlodzimierz Jarmundowicz*

*Department of Neurosurgery, Wroclaw Medical University, Wroclaw, Poland
†Spinal Repair Unit, Department of Brain Repair and Rehabilitation, UCL Institute of Neurology, London, UK
Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland
§Department of Pathophysiology of Locomotor Organs, Karol Marcinkowski Medical University, Poznan, Poland
¶Department of General Radiology, Interventional Radiology and Neuroradiology, Wroclaw Medical University, Wroclaw, Poland
#Neurorehabilitation Center for Treatment of Spinal Cord Injuries AKSON, Wroclaw, Poland
**Bacteriophage Laboratory of the Ludwik Hirszfeld Institute of Immunology, and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland
††Department of Clinical Immunology of the Transplantation Institute, Medical University of Warsaw, Warsaw, Poland
‡‡University Clinical Hospital, Wroclaw, Poland
§§Department of Pathomorphology and Oncological Cytology, Wroclaw Medical University, Wroclaw, Poland

Treatment of patients sustaining a complete spinal cord injury remains an unsolved clinical problem because of the lack of spontaneous regeneration of injured central axons. A 38-year-old man sustained traumatic transaction of the thoracic spinal cord at upper vertebral level Th9. At 21 months after injury, the patient presented symptoms of a clinically complete spinal cord injury (American Spinal Injury Association class A-ASIA A). One of the patient’s olfactory bulbs was removed and used to derive a culture containing olfactory ensheathing cells and olfactory nerve fibroblasts. Following resection of the glial scar, the cultured cells were transplanted into the spinal cord stumps above and below the injury and the 8-mm gap bridged by four strips of autologous suralnerve. The patient underwent an intense pre- and postoperative neurorehabilitation program. No adverse effects were seen at 19 months postoperatively, and unexpectedly, the removal of the olfactory bulb did not lead to persistent unilateral anosmia. The patient improved from ASIA A to ASIA C. There was improved trunk stability, partial recovery of the voluntary movements of the lower extremities, and an increase of the muscle mass in the left thigh, as well as partial recovery of superficial and deep sensation. There was also some indication of improved visceral sensation and improved vascular autoregulation in the left lower limb. The pattern of recovery suggests functional regeneration of both efferent and afferent long-distance fibers. Imaging confirmed that the grafts had bridged the left side of the spinal cord, where the majority of the nerve grafts were implanted, and neurophysiological examinations confirmed the restitution of the integrity of the corticospinal tracts and the voluntary character of recorded muscle contractions. To our knowledge, this is the first clinical indication of the beneficial effects of transplanted autologous bulbar cells.

Key words: Paraplegia; Cell transplantation; Repair; Regeneration

Received February 18, 2014; final acceptance September 8, 2014. Online prepub date: October 21, 2014.
Address correspondence to Pawel Tabakow, M.D., Ph.D., Department of Neurosurgery, Wroclaw Medical University, Borowska str. 213, 50-556 Wroclaw, Poland. Tel: +48 606 137 846; Fax: +48 71 734 34 09; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 23, pp. 1657-1671, 2014
0963-6897/14 $90.00
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DOI: http://dx.doi.org/10.3727/096368913X674648
E-ISSN 1555-3892
Copyright ©
2014 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Transplantation of Enteric Neural Stem/Progenitor Cells Into the Irradiated Young Mouse Hippocampus

Ahmed M. Osman,* Kai Zhou,† Changlian Zhu,†‡ and Klas Blomgren*†§

*Karolinska Institute, Department of Women’s and Children’s Health, Karolinska University Hospital, Stockholm, Sweden
†Center for Brain Repair and Rehabilitation, Institute of Neuroscience and Physiology, University of Gothenburg, Gothenburg, Sweden
‡Department of Pediatrics, the Third Affiliated Hospital of Zhengzhou University, Zhengzhou, China
§Department of Pediatrics, The Queen Silvia Children’s Hospital, University of Gothenburg, Gothenburg, Sweden

Radiotherapy is an effective treatment for brain tumors but often results in cognitive deficits in survivors. Transplantation of embryonic or brain-derived neural stem/progenitor cells (BNSPCs) ameliorated cognitive impairment after irradiation (IR) in animal models. However, such an approach in patients requires a clinically relevant source of cells. We show for the first time the utilization of enteric neural stem/progenitor cells (ENSPCs) from the postnatal intestinal wall as a source of autologous cells for brain repair after injury caused by IR. Cells were isolated from the intestinal wall and propagated in vitro for 1 week. Differentiation assays showed that ENSPCs are multipotent and generated neurons, astrocytes, and myofibroblasts. To investigate whether ENSPCs can be used in vivo, postnatal day 9 mice were subjected to a single moderate irradiation dose (6 or 8 Gy). Twelve days later, mice received an intrahippocampal injection of syngeneic ENSPCs. Four weeks after  transplantation, 0.5% and 1% of grafted ENSPCs were detected in the dentate gyrus of sham and irradiated animals, respectively, and only 0.1% was detected after 16 weeks. Grafted ENSPCs remained undifferentiated but failed to restore IR-induced loss of BNSPCs and the subsequent impaired growth of the dentate gyrus. We observed microglia activation, astrogliosis, and loss of granule neurons associated with grafted ENSPC clusters. Transplantation of ENSPCs did not ameliorate IR-induced impaired learning and memory. In summary, whileautologous ENSPC grafting to the brain worked technically, even in the absence of immunosuppression, the protocols need to be modified to improve survival and integration.

Key words: Radiotherapy; Grafting; Neurogenesis; Pediatric oncology; Brain tumors; Late effects

Received July 25, 2013; final acceptance October 9, 2013. Online prepub date: October 22, 2013.
Address correspondence to Klas BlomgrenKarolinska Institute, Department of Women’s and Children’s Health, Karolinska University Hospital Q2:07, Stockholm 171 76, Sweden. Tel: +46 8 517 771 83; Fax: +46 8 517 717 74; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it