Cell Transplantation 21(4) Abstracts

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Cell Transplantation, Vol. 21, pp. 625–632, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X623899
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Impact of Tissue Volume and Purification on Clinical Autologous Islet Transplantation for the Treatment of Chronic Pancreatitis

Shinichi Matsumoto,* Morihito Takita,* Masayuki Shimoda,† Koji Sugimoto,* Takeshi Itoh,* Daisuke Chujo,‡ Jeffery A. SoRelle,§ Yoshiko Tamura,¶ Ana M. Rahman,¶ Nicholas Onaca,¶ Bashoo Naziruddin,¶ and Marlon F. Levy¶

*Baylor All Saints Islet Cell Laboratory, Fort Worth, TX, USA
†Baylor University Medical Center at Dallas, Dallas, TX, USA
‡Baylor Institute for Immunology Research, Dallas, TX, USA
§Institute of Biomedical Studies, Baylor University, Waco, TX, USA
¶Baylor Simmons Transplant Institute, Dallas, TX, USA

Autologous islet transplantation after total pancreatectomy is an excellent treatment for painful chronic pancreatitis. Traditionally, islets have been isolated without purification; however, purification is applied when the tissue volume is large. Nevertheless, the impact of tissue volume and islet purification on clinical outcomes of autologous islet transplantation has not been well examined. We analyzed 27 cases of autologous islet transplantation performed from October 2006 to January 2011. After examining the relationship between tissue volume and portal pressure at various time points, we compared islet characteristics and clinical outcomes between cases with complications (complication group) and without (noncomplication group), as well as cases with purification (purification group) and without (nonpurification group). Tissue volume significantly correlated with maximum (R = 0.61), final (R = 0.53), and delta (i.e., difference between base and maximum; R = 0.71) portal pressure. The complication group had a significantly higher body mass index, tissue volume, islet yield, and portal pressure (maximum, final, delta), suggesting that complications were associated with high tissue volume and high portal pressure. Only one of four patients (25%) in the complication group became insulin free, whereas 11 of 23 patients (49%) in the noncomplication group became insulin free with smaller islet yields. The purification group had a higher islet yield and insulin independence rate but had similar final tissue volume, portal pressure, and complication rates compared with the nonpurification group. In conclusion, high tissue volume was associated with high portal pressure and complications in autologous islet transplantation. Islet purification effectively reduced tissue volume and had no negative impact on islet characteristics. Therefore, islet purification can reduce the risk of complications and may improve clinical outcome for autologous islet transplantation when tissue volume is large.

Key words: Autologous islet transplantation; Tissue volume; Purification; Portal thrombosis; Portal pressure

Received February 15, 2011; final acceptance April 28, 2011. Online prepub date: February 2, 2012.
Address correspondence to Shinichi Matsumoto, M.D., Ph.D., Baylor All Saints Islet Cell Laboratory, 1400 8th Avenue, Fort Worth, TX 76104, USA. Tel: +1-817-922-2570; Fax: +1-817-922-4645; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it or Bashoo Naziruddin, Ph.D., Baylor Simmons Transplant Institute, 3410 Worth Street Suite 950, Dallas, TX 75246, USA. Tel: 214-820-2662; Fax: 214-820-7142; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 633–648, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X576027
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Anti-Inflammatory Properties of Exenatide in Human Pancreatic Islets

S. R. Cechin,* I. Pérez-Álvarez,* E. Fenjves,* R. D. Molano,* A. Pileggi,* P-O. Berggren,*† C. Ricordi,*† and R. L. Pastori*

*Diabetes Research Institute, University of Miami Leonard M. Miller School of Medicine, Miami, FL, USA
†The Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska Institutet, Stockholm, Sweden

Exenatide is an analog of the incretin hormone glucagon-like peptide (GLP-1) that is used for the treatment of T2D for their metabolic effects. In addition to its insulinotropic effects, exenatide increases functional islet mass and improves their survival. Improved outcomes have been reported in recent clinical islet transplantation trials for the treatment of type 1 diabetes. The purpose of this study was to investigate whether exenatide has anti-inflammatory properties in human islets. Exenatide treatment improved islet function, significantly reduced content of inflammation-related molecules (tissue factor, IFN-γ, IL-17, IL-1β, and IL-2) and caspase 3 activation, whereas increased phosphorylation of ERK1/2, STAT3, and Akt in vitro. Immunostaining showed expression of GLP-1R in β-cells but not in α-cells. IL-1β  colocalized with GLP-1R in β-cells. Induction of serine proteinase inhibitor 9 (PI-9) was detected after exposure of human islets to exenatide in vitro and after transplantation into immunodeficient mice. GLP-1 induced PI-9 expression in vitro but to a lower extent than exenatide. This effect was partially blocked by the antagonist exendin-9 in vitro. As assessed by immunostaining PI-9 is mostly expressed in β-cells but not in α-cells. In conclusion, we describe anti-inflammatory and cytoprotective properties of exenatide in human islets. Exenatide-mediated PI-9 expression, the only known granzyme B inhibitor, unveils potential immunoregulatory properties.

Key words: Human pancreatic islets; Glucagon-like peptide-1; Exenatide; Inflammation; Cytokines; Proteinase inhibitor 9 (PI-9); Nude mice; Islet transplantation

Received December 1, 2010; final acceptance February 15, 2011. Online prepub date: June 7, 2011.
Address correspondence to Ricardo L. Pastori, Ph.D., Diabetes Research Institute, University of Miami Leonard M. Miller School of Medicine, 1450 NW 10th Avenue, Miami, FL 33136, USA. Tel: +1 (305) 243-5349; Fax: +1 (305) 243-4404; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 649–655, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X623826
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

The Effects of Digestion Enzymes on Islet Viability and Cellular Composition

Itzia Iglesias,* Luis Valiente,* Keh-Dong Shiang,† Hirohito Ichii,‡ Fouad Kandeel,* and Ismail H. Al-Abdullah*

*Southern California Islet Cell Resources Center, Department of Diabetes, Endocrinology and Metabolism, City of Hope National Medical Center and Beckman Research Institute, Duarte, CA, USA
†Division of Biostatistics, Department of Information Sciences, City of Hope National Medical Center and Beckman Research Institute, Duarte, CA, USA
‡Department of Surgery, University of California, Irvine, CA, USA

The choice of enzyme blend is critical for successful islet isolation. Islet yield, viability, integrity, and function are important factors that influence the outcome of islet transplantation. Liberase HI has been used as a standard enzyme for pancreas digestion and has successfully produced islets that reversed diabetes. However, the replacement of Liberase HI with collagenase NB1 has significantly influenced the process outcome, both in quality and quantity of the isolated islets. The assessment of islet cells by Flow Cytometry (FC) has been reported to be useful for evaluating islet quality. The aim of this study was to assess the isolation outcomes and islet quality when comparing human islet cell processed with Liberase HI and NB1. A total of 66 islet isolations, 46 processed using Liberase HI and 20 using Serva NB1, were retrospectively analyzed. Islet yield, function in vitro, islet cell viability by FC, as well as isolation-related factors were compared. There was no significant difference in donor characteristics such as age and height; however, body mass index (BMI) in the Liberase HI group was significantly higher. There was also no significant difference in prepurification, postisolation, or postculture IEQ or percent recovery between the two groups. Flow data showed Liberase HI preparations had a significantly higher percent of live cells (DAPI) and NG+/TMRE+ when compared to NB1. Stimulation Indices (SI) for Liberase HI (n = 45) showed 3.17 and NB1 (n = 18) 2.71 (p = NS). The results of Annexin V/DAPI staining for live, apoptotic, and necrotic cells were 50.7 ± 2.24%, 14.4 ± 1.02%, and 27.8 ± 1.92% for Liberase HI versus 48.1 ± 1.93%, 12.3 ± 0.92%, and 33.9 ± 2.28% for NB1. Islets isolated using Liberase HI showed higher viable β cells by NG/TMRE staining and decreased necrosis by Annexin V/DAPI staining. FC assessment may be useful for determining the choice of digestion enzyme to maximize viable islets.

Key words: Islet; Assessment; Digestion enzymes; Collagenase; Ductal cells; Apoptosis

Received January 14, 2011; final acceptance April 12, 2011. Online prepub date: January 10, 2012.
Address correspondence to Ismail H. Al-Abdullah, Ph.D., Department of Diabetes, Endocrinology and Metabolism, City of Hope National Medical Center and Beckman Research Institute, 1500 E. Duarte Road, Duarte, CA 91010, USA. Tel: 626-256-4673, ext. 60109; Fax: 626-301-8136; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it .


Cell Transplantation, Vol. 21, pp. 657–669, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X593136
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Perfluorocarbon Emulsions Prevent Hypoxia of Pancreatic β-Cells

E. Maillard,*† M. T. Juszczak,† A. Langlois,* C. Kleiss,* M. C. Sencier,* W. Bietiger,* M. Sanchez-Dominguez,‡ M. P. Krafft,‡ P. R. V. Johnson,† M. Pinget,*§ and S. Sigrist*

*Centre Européen d’Etude du Diabète (CeeD), Strasbourg, France
†Nuffield Department of Surgical Sciences, University of Oxford, Oxford, UK
‡Institut Charles Sadron, Strasbourg, France
§University de Strasbourg (UdS), Strasbourg, France

As oxygen carriers, perfluorocarbon emulsions might be useful to decrease hypoxia of pancreatic islets before transplantation. However, their hydrophobicity prevents their homogenisation in culture medium. To increase the surface of contact between islets and Perfluorooctyl bromide (PFOB), and consequently oxygen delivery, we tested effect of a PFOB emulsion in culture medium on β-cell lines and rat pancreatic islets. RINm5F β-cell line or pancreatic rat islets were incubated for 3 days in the presence of PFOB emulsion in media (3.5% w/v). Preoxygenation of the medium was performed before culture. Cell viability was assessed by apoptotic markers (Bax and Bcl-2) and by staining (fluoresceine diacetate and propidium iodide). β-Cell functionality was determined by insulin release during a glucose stimulation test and. Hypoxia markers, HIF-1α and VEGF, were studied at days 1 and 3 using RT-PCR, Western blotting, and ELISA. PFOB emulsions preserved viability and functionality of RINm5F cells with a decrease of HIF-1α and VEGF expression. Islets viability was preserved during 3 days of culture. Secretion of VEGF was higher in untreated control (0.09 ± 0.041 μg VEGF/mg total protein) than in PFOB emulsion incubated islets (0.02 ± 0.19 μg VEGF/mg total protein, n = 4, p < 0.05) at day 1. At day 3, VEGF secretion was increased as compared to day 1 in control (0.23 ± 0.04 μg VEGF/mg total protein) but it was imbalance by the presence of PFOB emulsion (0.09 ± 0.03 μg VEGF/mg total protein, n = 5, p < 0.05). While insulin secretion was maintained in response to a glucose stimulation test until day 3 when islets were incubated in the presence of PFOB emulsion preoxygenated (0.81 ± 0.16 at day 1 vs. 0.75 ± 0.24 at day 3), the ability to secrete insulin in the presence of high glucose concentration was lost in islets controls (0.51 ± 0.18 at day 1 vs. 0.21 ± 0.13 at day 3). Atmospheric oxygen delivery by PFOB emulsion might be sufficient to decrease islets hypoxia. However, to improve islets functionality, overoxygenation is needed. Finally, maintenance of islet viability and functionality for several days after isolation could improve the outcome of islets transplantation.

Key words: Oxygen; Hypoxia; Hyperoxia; Perfluorocarbon; Islet; Insulin; Apoptosis; VEGF

Received August 10, 2010; final acceptance April 15, 2011. Online prepub date: September 22, 2011.
Address correspondence to Séverine Sigrist, Ph.D., Centre Européen d’Etude du Diabète, Responsable de laboratoire, Boulevard René Leriche, 67200 Strasborg, France. Tel: 03 90 20 12 12; Fax: 06 08 74 90 90; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 671–677, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X600975
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Normal Hepatocyte Transplantation Delays the Emergence of Chemically Induced Preneoplastic Nodules in Rat Liver

Maria Paola Serra, Silvia Doratiotto, Fabio Marongiu, and Ezio Laconi

Department of Biomedical Sciences, Unit of Experimental Medicine, University of Cagliari, Cagliari, Italy

Cancer often arises in a background of chronic tissue damage. It is also increasingly appreciated that such an injured tissue microenvironment might foster the selective emergence of altered cells, leading to neoplasia. Accordingly, reversal of chronic tissue damage could represent a potential strategy to counteract neoplastic disease. In these studies, we aim to investigate whether transplantation of normal cells in the context of an injured, neoplastic-prone microenvironment might impact on the evolution of the carcinogenic process. A rat model of chemically induced hepatocarcinogenesis was used. Animals were given a single dose of diethylnitrosamine (DENA), followed by two injections of retrorsine (RS), a pyrrolizidine alkaloid that imposes a persistent block on hepatocyte cell cycle. At the end of this protocol, rats were either given no further treatment or injected, via the portal circulation, with 4 million normal hepatocytes isolated from a syngenic donor. After 3 months, rats given DENA+RS alone displayed numerous discrete nodular lesions (up to 30 per liver), ranging 1 to 3 mm in size. On the other hand, in animals receiving DENA+RS and transplantation, donor hepatocytes were able to repopulate over 50% of the host liver, as expected. Most importantly, both the number and the size of hepatocyte nodules were greatly reduced in these animals (percent nodular area was 1.8 ± 0.3, down from a control value of 8.5 ± 2.8). The above data indicate that strategies aimed at reestablishing a normal tissue microenvironment might be relevant to the management of neoplastic disease.

Key words: Cell transplantation; Tumor microenvironment; Cell competition; Liver repopulation; Liver carcinogenesis

Received July 26, 2010; final acceptance April 25, 2011. Online prepub date: September 22, 2011.
Address correspondence to Dr. Ezio Laconi, M.D., Ph.D., Dipartimento di Scienze Biomediche, Unità di Medicina Sperimentale, Via Porcell 4, III piano, Università di Cagliari, 09124, Cagliari, Italy. Tel: +39 070 675 8342; Fax: +39 070 662574; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 679–691, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X612440
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Can Magnetic Targeting of Magnetically Labeled Circulating Cells Optimize Intramyocardial Cell Retention?

Aurélie Chaudeurge,*† Claire Wilhelm,‡§ Annabel Chen-Tournoux,*† Patrick Farahmand,*¶# Valérie Bellamy,*† Gwennhael Autret,¶#** Christine Ménager,†† Albert Hagège,*¶# Jerome Larghéro,‡‡§§ Florence Gazeau,‡§ Olivier Clément,¶#** and Philippe Menasché*¶#

*INSERM U633, Laboratory of Surgical Research, Paris, France
†Assistance Publique-Hôpitaux de Paris, Ecole de Chirurgie, Paris, France
‡Laboratoire Matière et Systèmes Complexes MSC, CNRS UMR 7057, Paris, France
§Université Paris-Diderot, Paris, France
¶Assistance Publique-Hôpitaux de Paris, Hôpital Européen Georges Pompidou, Department of Cardiovascular Surgery, Paris, France
#Université Paris Descartes, Sorbonne Paris Cité, Paris, France
**INSERM, U970, Paris Cardiovascular Research Center-PARCC, Paris, France
††Univ Paris 06-CNRS-ESPCI Laboratoire PECSA UMR7195, Paris, France
‡Assistance Publique-Hôpitaux de Paris, Hôpital St. Louis, Laboratory of Cell Therapy, Paris, France
§University Paris Diderot, Paris, France

Therapeutic intracavitary stem cell infusion currently suffers from poor myocardial homing. We examined whether cardiac cell retention could be enhanced by magnetic targeting of endothelial progenitor cells (EPCs) loaded with iron oxide nanoparticles. EPCs were magnetically labeled with citrate-coated iron oxide nanoparticles. Cell proliferation, migration, and CXCR4 chemokine receptor expression were assessed in different labeling conditions and no adverse effects of the magnetic label were observed. The magnetophoretic mobility of labeled EPCs was determined in vitro, with the same magnet as that subsequently used in vivo. Coronary artery occlusion was induced for 30 min in 36 rats (31 survivors), followed by 20 min of reperfusion. The rats were randomized to receive, during brief aortic cross-clamping, direct intraventricular injection of culture medium (n = 7) or magnetically labeled EPCs (n = 24), with (n = 14) or without (n = 10) subcutaneous insertion of a magnet over the chest cavity (n = 14). The hearts were explanted 24 h later and engrafted cells were visualized by magnetic resonance imaging (MRI) of the heart at 1.5 T. Their abundance in the myocardium was also analyzed semiquantitatively by immunofluorescence, and quantitatively by real-time polymerase chain reaction (RT-PCR).Although differences in cell retention between groups failed to be statistically significant using RT-PCR quantification, due to the variability of the animal model, immunostaining showed that the average number of engrafted EPCs was significantly ten times higher with than without magnetic targeting. There was thus a consistent trend favoring the magnet-treated hearts, thereby suggesting magnetic targeting as a potentially new mean of enhancing myocardial homing of intravascularly delivered stem cells. Magnetic targeting has the potential to enhance myocardial retention of intravascularly delivered endothelial progenitor cells.

Key words: Myocardial infarction; Endothelial progenitor cells (EPCs); Magnetic targeting; Homing

Received August 20, 2010; final acceptance April 10, 2011. Online prepub date: November 11, 2011.
Address correspondence to Philippe Menasché, INSERM U633, Laboratory of Surgical Research, Paris, France. Tel: +1 (305) 243-5349; Fax: +1 (305) 243-4404; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 693–705, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X623844
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Combined Mesenchymal Stem Cell Sheets and rhBMP-2-Releasing Calcium Sulfate–rhBMP-2 Scaffolds for Segmental Bone Tissue Engineering

Yiying Qi,*1 Yulu Wang,†1 Weiqi Yan,* Hang Li,* Zhongli Shi,* and Zhijun Pan*

*Department of Orthopedic Surgery, the Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China
†Department of Orthopedic Surgery, the First Affiliated Hospital, Baotou Medical School, Baotou, China

Repair of segmental bone defects remains a major challenge for orthopedic surgeons. This study aimed to investigate whether recombinant human bone morphogenetic protein-2 (rhBMP-2)-loaded calcium sulfate (CS) combined with mesenchymal stem cell (MSC) sheets could accelerate bone regeneration in ulnar segmental defects of rabbits. In vitro, the osteogenic differentiation of MSCs cultured on rhBMP-2-loaded CS was investigated. Forty complete 1.2-cm bone defects were treated with CS (group A), rhBMP-2-loaded CS (group B), MSC sheet-wrapped CS (group C), and MSC sheet-wrapped rhBMP-2-loaded CS (group D). At 4 and 8 weeks after implantation, the samples were treated by X-ray, microcomputed tomography, and histological observation. The rhBMP-2 could be released from the rhBMP-2-loaded CS scaffolds and maintain its bioactivity. The alkaline phosphatase (ALP) of MSCs cultured on rhBMP-2-loaded CS was significantly higher than that of CS at both 7 and 14 days (p < 0.05). The defects treated with MSC sheet-wrapped rhBMP-2-loaded CS showed significantly higher scores by X-ray analysis and more bone formation determined by both histology and microcomputed tomography than the other three groups at both 4 and 8 weeks after implantation (p < 0.05). No significant difference in X-ray score and bone formation was found between groups B and C, both significantly higher than group A (p < 0.05). The results suggested that MSC sheet-wrapped rhBMP-2-loaded CS may be an effective approach to promote the repair of segmental bone defects and has great potential for repairing large segmental bone defects in clinic.

Key words: Mesenchymal stem cells (MSCs); Bone regeneration; Osteogenic differentiation; Recombinant human bone morphogenetic protein (rhBMP-2); Calcium sulfate

Received December 19, 2010; final acceptance April 25, 2011. Online prepub date: January 10, 2012.
1These authors provided equal contribution to this work and were regarded as co-first author.
Address correspondence to Zhijun Pan, Department of Orthopaedics, the Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310009, China. Tel/Fax: +86-571-87783515; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it or Weiqi Yan, Department of Orthopedic Surgery, the Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310009, China. Tel/Fax: +86-571-87784603; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 707–721, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X582769
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
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Local Transplantation of Granulocyte Colony-Stimulating Factor-Mobilized Human Peripheral Blood Mononuclear Cells for Unhealing Bone Fractures

Tomoaki Fukui,*† Tomoyuki Matsumoto,*† Yutaka Mifune,*† Taro Shoji,*† Tomoya Kuroda,*† Yohei Kawakami,*† Atsuhiko Kawamoto,* Masaaki Ii,* Shin Kawamata,‡ Masahiro Kurosaka,† Takayuki Asahara,*§ and Ryosuke Kuroda†

*Group of Vascular Regeneration, Institute of Biomedical Research and Innovation, Kobe, Hyogo, Japan
†Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, Kobe, Hyogo, Japan
‡Stem Cell Bank Research Group, Institute of Biomedical Research and Innovation, Kobe, Hyogo, Japan
§Department of Regenerative Medicine Science, Tokai University School of Medicine, Isehara, Kanagawa, Japan

We previously reported the therapeutic potential of human peripheral blood (hPB) CD34+ cells for bone fracture healing via vasculogenesis/angiogenesis and osteogenesis. Transplantation of not only hPB CD34+ cells but also hPB total mononuclear cells (MNCs) has shown their therapeutic efficiency for enhancing ischemic neovascularization. Compared with transplantation of purified hPB CD34+ cells, transplantation of hPB MNCs is more attractive due to its simple method of cell isolation and inexpensive cost performance in the clinical setting. Thus, in this report, we attempted to test a hypothesis that granulocyte colony-stimulating factor-mobilized (GM) hPB MNC transplantation could also contribute to fracture healing via vasculogenesis/angiogenesis and osteogenesis. Nude rats with unhealing fractures received local administration of the following materials with atelocollagen: 1 × 107 GM hPB MNCs (Hi group), 1 × 106 GM hPB MNCs (Lo group), or PBS (PBS group). Immunohistochemistry and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated human cell-derived vasculogenesis and osteogenesis in the Hi and Lo groups, but not in the PBS group at week 1. Intrinsic angiogenesis and osteogenesis assessed by rat capillary, osteoblast density, and real-time RT-PCR analysis was significantly enhanced in the Hi group compared to the other groups. Blood flow assessment by laser doppler perfusion imaging showed a significantly higher blood flow ratio at week 1 in the Hi group compared with the other groups. Morphological fracture healing was radiographically and histologically confirmed in about 30% of animals in the Hi group at week 8, whereas all animals in the other groups resulted in nonunion. Local transplantation of GM hPB MNCs contributes to fracture healing via vasculogenesis/angiogenesis and osteogenesis.

Key words: Mononuclear cells; Fracture healing; Osteogenesis; Vasculogenesis; CD34+ cells

Received October 13, 2009; final acceptance April 22, 2011. Online prepub date: September 16, 2011.
Address correspondence to Tomoyuki Matsumoto, M.D., Ph.D., Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, Hyogo 650-0017 Japan. Tel: +81-78-382-5985; Fax: +81-78-351-6944; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 723–737, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X586783
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
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Assessment of Neuroprotective Effects of Human Umbilical Cord Blood Mononuclear Cell Subpopulations In Vitro and In Vivo

Johannes Boltze,*†1 Doreen M. Reich,*1 Susann Hau,*‡ Klaus G. Reymann,§ Maria Strassburger,§ Donald Lobsien,¶ Daniel-Christoph Wagner,* Manja Kamprad,# and Tobias Stahl**

*Fraunhofer Institute for Cell Therapy and Immunology, Leipzig, Germany
†Translational Centre for Regenerative Medicine, University of Leipzig, Leipzig, Germany
‡VITA34 AG, Leipzig, Germany
§Leibniz Institute for Neurobiology, Project Group Neuropharmacology, Magdeburg, Germany
¶Department for Neuroradiology, University of Leipzig, Leipzig, Germany
#Institute of Clinical Immunology and Transfusion Medicine, University of Leipzig, Leipzig, Germany
**Faculty of Veterinary Medicine, Department of Anatomy, Histology and Embryology, University of Leipzig, Leipzig, Germany

Experimental transplantation of human umbilical cord blood (hUCB) mononuclear cells (MNCs) in rodent stroke models revealed the therapeutic potential of these cells. However, effective cells within the heterogeneous MNC population and their modes of action are still under discussion. MNCs and MNC fractions enriched (CD34+) or depleted (CD34) for CD34-expressing stem/progenitor cells were isolated from hUCB. Cells were transplanted intravenously following middle cerebral artery occlusion in spontaneously hypertensive rats and directly or indirectly cocultivated with hippocampal slices previously subjected to oxygen and glucose deprivation. Application of saline solution or a human T-cell line served as controls. In vivo, MNCs, CD34+ and CD34 cells reduced neurofunctional deficits and diminished lesion volume as determined by magnetic resonance imaging. MNCs were superior to other fractions. However, human cells could not be identified in brain tissue 29 days after stroke induction. Following direct application on postischemic hippocampal slices, MNCs reduced neural damage throughout a 3-day observation period. CD34+ cells provided transient protection for 2 days. The CD34 fraction, in contrast to in vivo results, failed to reduce neural damage. Direct cocultivation of MNCs was superior to indirect cocultivation of equal cell numbers. Indirect application of up to 10-fold MNC concentrations enhanced neuroprotection to a level comparable to direct cocultivation. After direct application, MNCs migrated into the slices. Flow cytometric analysis of migrated cells revealed that the CD34+ cells within MNCs were preferably attracted by damaged hippocampal tissue. Our study suggests that MNCs provide the most prominent neuroprotective effect, with CD34+ cells seeming to be particularly involved in the protective action of MNCs. CD34+ cells preferentially home to neural tissue in vitro, but are not superior concerning the overall effect, implying that there is another, still undiscovered, protective cell population. Furthermore, MNCs did not survive in the ischemic brain for longer periods without immunosuppression.

Key words: Stroke; Human umbilical cord blood (hUCB); Cell transplantation; Magnetic resonance imaging; Cell therapy; Neuroprotection

Received June 30, 2010; final acceptance April 27, 2011. Online prepub date: September 16, 2011.
1These authors provided equal contribution to this work.
Address correspondence to Johannes Boltze, M.D., Fraunhofer-Institute for Cell Therapy and Immunology, Perlickstr.1, D-04103 Leipzig, Germany. Tel: +49-341-97-25814; Fax: +49-341-97-25829; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 739–747, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X612459
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
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Neural Stem/Progenitor Cells Transplanted to the Hypoglossal Nucleus Integrates With the Host CNS in Adult Rats and Promotes Motor Neuron Survival

Michael Fagerlund,1 Cynthia Pérez Estrada,1 Nasren Jaff, Mikael Svensson, and Lou Brundin

Department of Clinical Neuroscience and Departments of Neurosurgery and Neurology, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden

Transplantation of neural stem cells and the mobilization of endogenous neuronal precursors in the adult brain have been proposed as therapeutic strategies for central nervous system disorders and injuries. The aim of the present study was to investigate the possible survival and integration of grafted neural progenitor cells (NPCs) from the subventricular zone (SVZ) in a hypoglossal nerve avulsion model with substantial neuronal loss. Adult neural progenitor cells (NPCs) from the subventricular zone (SVZ) were cultured from inbred transgenic eGFP Lewis rats and transplanted to the hypoglossal nucleus of inbred Lewis rat from the same family but that were not carrying the eGFP strain after avulsion of the hypoglossal nerve. Grafted cells survived in the host more than 3 months and differentiated into neurons [βIII tubulin (Tuj-1 staining)] with fine axon- and dendrite-like processes as well as astrocytes (GFAP) and oligodendrocytes (O4) with typical morphology. Staining for synaptic structures (synaptophysin and bassoon) indicated integration of differentiated cells from the graft with the host CNS. Furthermore, transplantation of NPCs increased the number of surviving motoneurons in the hypoglossal nucleus after nerve avulsion that, if untreated, result in substantial neuronal death. The NPCs used in this study expressed VEGF in vitro as well as in vivo following transplantation that may mediate the rescue effect of the axotomized motoneurons.

Key words: Brain stem; Neural progenitor cells; Nerve injury; Motor neurons; Transplantation; Integration

Received August 30, 2010; final acceptance April 15, 2011. Online prepub date: December 13, 2011.
1These authors provided equal contribution to this work.
Address correspondence to Mikael Svensson, Department of Neurosurgery, Karolinska University Hospital, SE-17176 Stockholm, Sweden. Fax: +46-8-5177-1778; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 749–762, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X586774
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

The Colayer Method as an Efficient Way to Genetically Modify Mesencephalic Progenitor Cells Transplanted Into 6-OHDA Rat Model of Parkinson’s Disease

Andreas Ratzka,*1 Ieva Kalve,*†1 Meltem Özer,*† André Nobre,*† Maike Wesemann,* Julia Jungnickel,* Christiane Köster-Patzlaff,* Olga Baron,*† and Claudia Grothe*†

*Institute of Neuroanatomy, Hannover Medical School, Hannover, Germany
†Center for Systems Neuroscience (ZSN), Hannover, Germany

Exogenous cell replacement represents a potent treatment option for Parkinson’s disease. However, the low survival rate of transplanted dopaminergic neurons (DA) calls for methodological improvements. Here we evaluated a method to combine transient genetic modification of neuronal progenitor cells with an optimized cell culture protocol prior to intrastriatal transplantation into 6-hydroxydopamine (6-OHDA) unilateral lesioned rats. Plasmid-based delivery of brain-derived neurotrophic factor (BDNF) increases the number of DA neurons, identified by tyrosine hydroxylase immunoreactivity (TH-ir), by 25% in vitro, compared to enhanced green fluorescence protein (EGFP)-transfected controls. However, the nucleofection itself, especially the cell detachment and reseeding procedure, decreases the TH-ir neuron number to 40% compared with nontransfected control cultures. To circumvent this drawback we established the colayer method, which contains a mix of nucleofected cells reseeded on top of an adherent sister culture in a ratio 1:3. In this setup TH-ir neuron number remains high and could be further increased by 25% after BDNF transfection. Comparison of both cell culture procedures (standard and colayer) after intrastriatal transplantation revealed a similar DA neuron survival as seen in vitro. Two weeks after grafting TH-ir neuron number was strongly reduced in animals receiving the standard EGFP-transfected cells (271 ± 62) compared to 1,723 ± 199 TH-ir neurons in the colayer group. In contrast to the in vitro results, no differences in the number of grafted TH-ir neurons were observed between BDNF, EGFP, and nontransfected colayer groups, neither 2 nor 13 weeks after transplantation. Likewise, amphetamine and apomorphine-induced rotational behavior improved similarly over time in all groups. Nevertheless, the colayer protocol provides an efficient way for neurotrophic factor release by transplanted progenitor cells and will help to study the effects of candidate factors on survival and integration of transplanted DA neurons.

Key words: Parkinson’s disease (PD); Dopaminergic (DA) neurons; Cell replacement; Brain-derived neurotrophic factor (BDNF); Nucleofection

Received January 19, 2011; final acceptance April 21, 2011. Online prepub date: September 16, 2011.
1These authors provided equal contribution to this work.
Address correspondence to Prof. Dr. rer. nat. Claudia Grothe, Institute of Neuroanatomy, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. Tel: +49-511-532-2896; Fax: +49-511-532-2880; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 763–771, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X623907
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Fates of Murine Pluripotent Stem Cell-Derived Neural Progenitors Following Transplantation Into Mouse Cochleae

Koji Nishimura, Takayuki Nakagawa, Tatsunori Sakamoto, and Juichi Ito

Department of Otolaryngology, Head and Neck Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan

This study evaluated the tumorigenesis risk of induced pluripotent stem (iPS) cells after transplantation into the cochlea. One mouse embryonic stem (ES) cell line and three mouse iPS cell lines, one derived from adult mouse tail-tip fibroblasts (TTFs) and two from mouse embryonic fibroblasts (MEFs), were neutrally induced by stromal cell-inducing activity. Before transplantation, the efficiency of neural induction and the proportion of residual undifferentiated cells were evaluated using immunocytochemistry, and no significant differences were observed in the ratios of colonies expressing βIII tubulin, nestin, or octamer (Oct)3/4. Four weeks after transplantation into the cochleae of neonatal mice, the number of surviving transplants of TTFderived iPS cells generated by retroviral infection was significantly higher than those of MEF-derived iPS cells generated by plasmid transfection. Teratoma formation was identified in one of five cochleae transplanted with TTF-derived iPS cells. However, no significant differences were found in the cell proliferation activity or the extent of differentiation into mature neurons among the cell lines. These findings emphasize the necessity of selecting appropriate iPS cell lines and developing methods to eliminate undifferentiated cells after neural induction, in order to establish safe iPS cell-based therapy for the inner ear.

Key words: Cell therapy; Hearing loss; Inner ear; Pluripotent stem cell; Teratoma

Received November 25, 2010; final acceptance April 27, 2011. Online prepub date: February 2, 2012.
Address correspondence to Takayuki Nakagawa, M.D., Ph.D., Department of Otolaryngology, Head and Neck Surgery, Graduate School of Medicine, Kyoto University, Kawahara-cho 54, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. Tel: +81-75-751-3346; Fax: +81-75-751-7225; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it