Cell Transplantation 22(1) Abstracts

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Cell Transplantation, Vol. 22, pp. 1-14, 2013
0963-6897/13 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X653282
E-ISSN 1555-3892
Copyright © 2013 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Review

Cardiac Stem Cell Therapy: Stemness or Commitment?

Ashish Mehta and Winston Shim

Research and Development Unit, National Heart Centre Singapore, Singapore

Cardiac stem cell therapy to promote engraftment of de novo beating cardiac muscle cells in cardiomyopathies could potentially improve clinical outcomes for many patients with congestive heart failure. Clinical trials carried out over the last decade for cardiac regeneration have revealed inadequacy of current approaches in cell therapy. Chief among them is the choice of stem cells to achieve the desired outcomes. Initial enthusiasm of adult bone marrow stems cells for myocyte regeneration has largely been relegated to paracrine-driven, donor cell-independent, endogenous cardiac repair. However, true functional restoration in heart failure is likely to require considerable myocyte replacement. In order to match stem cell application to various clinical scenarios, we review the necessity to preprime stem cells towards cardiac fate before myocardial transplantation and if these differentiated stem cells could confer added advantage over current choice of undifferentiated stem cells. We explore differentiation ability of various stem cells to cardiac progenitors/cardiomyocytes and compare their applicability in providing targeted recovery in light of current clinical challenges of cell therapy.

Key words: Stem cells; Cardiomyocytes; Differentiation; Myocardial infarction; Regenerative medicine

Received October 8, 2011; final acceptance February 18, 2012. Online prepub date: August 27, 2012.
Address correspondence to Dr. Winston Shim, National Heart Centre Singapore, 17, Third Hospital Avenue, Mistri Wing, Singapore 168752. Tel: +65 64350752; Fax: +65 62263972; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 22, pp. 15-27, 2013
0963-6897/13 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X640466
E-ISSN 1555-3892
Copyright © 2013 Cognizant Comm. Corp.
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Survival, Integration, and Differentiation of Unrestricted Somatic Stem Cells in the Heart

Zhaoping Ding,* Sandra Burghoff,* Anja Buchheiser,† Gesine Kögler,† and Jürgen Schrader*

*Institut für Herz- und Kreislaufphysiologie, Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany
†Institute for Transplantation Diagnostics and Cell Therapeutics, Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany

Unrestricted somatic stem cells (USSCs) derived from human umbilical cord blood represent an attractive cell source to reconstitute the damaged heart. We have analyzed the cardiomyogenic potential and investigated the fate of USSCs after transplantation into rat heart in vivo. USSCs demonstrated cardiomyogenic differentiation properties characterized by the spontaneously beating activity and the robust expression of cardiac α-actinin and troponin T (cTnT) at protein and mRNA level after cocultivation with neonatal rat cardiomyocytes. To study the fate in vivo, eGFP+ USSCs were injected transcoronarily into immunosuppressed rats via a catheter-based technique. Nearly 80% USSCs were retained within the myocardium without altering cardiac hemodynamics. After 7 days, 20% of the transplanted cells survived in the host myocardium and showed elongated morphology with weak expression of cardiac-specific markers, while some eGFP+ USSCs were found to integrate into the vascular wall. After 21 days, only a small fraction of USSCs were found in the myocardium (0.13%); however, the remaining cells clearly exhibited a sarcomeric structure similar to mature cardiomyocytes. Identical results were also obtained in nude rats. In addition, we found some cells stained positively for activated caspase 3 paralleled by the massive infiltration of CD11b+ cells into the myocardium. In summary, USSCs can differentiate into beating cardiomyocytes by cocultivation in vitro. After coronary transplantation in vivo, however, long-term survival of differentiated USSCs was rather low despite a high initial fraction of trapped cells.

Key words: Stem cell; Unrestricted somatic stem cells (USSCs); Cardiogenesis; Transcoronary; Heart

Received March 4, 2011; final acceptance December 5, 2011. Online prepub date: April 17, 2012.
Address correspondence to Jürgen Schrader, M.D., Institut für Herz- und Kreislaufphysiologie, Heinrich-Heine-Universität Düsseldorf, Moorenstr. 5, 40225 Düsseldorf, Germany. Tel: +49 211 81 12670; Fax: +49 211 81 12672; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 22, pp. 19-40, 2013
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DOI: http://dx.doi.org/10.3727/096368912X654948
E-ISSN 1555-3892
Copyright © 2013 Cognizant Comm. Corp.
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ZNF281 Knockdown Induced Osteogenic Differentiation of Human Multipotent Stem Cells In Vivo and In Vitro

Kwang-Won Seo,*†‡1 Kyoung-Hwan Roh,*†1 Dilli Ram Bhandari,*† Sang-Bum Park,*† Seon-Kyung Lee,*† and Kyung-Sun Kang*†‡

*Adult Stem Cell Research Center, College of Veterinary Medicine, Seoul National University, Seoul, Republic of Korea
†Laboratory of Stem Cell and Tumor Biology, Department of Veterinary Public Health, College of Veterinary Medicine, Seoul National University, Seoul, Republic of Korea
‡Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul, Republic of Korea

ZNF281 is one of the core transcription factors in embryonic stem cells (ESCs) and has activation and repression roles in the transcription of ESC genes. A known target molecule of Zfp281 (the mouse homologue of ZNF281) is Nanog. However, NANOG is not expressed in most human multipotent stem cells (hMSCs). Here, we investigated the roles of ZNF281 with a gain- and loss-of-function study. The knockdown of ZNF281 in vivo and in vitro resulted in spontaneous osteochondrogenic differentiation and reduced the proliferation of hMSCs, as determined by cell morphology and molecular markers. When ZNF281-knockdown hMSCs were subcutaneously implanted into mice along with β-tricalcium phosphate (β-TCP), many cells were converted into osteoblasts within 4 weeks. In contrast, the overexpression of ZNF281 in hMSCs resulted in accelerated proliferation. The expression pattern of ZNF281 correlated well with the expression of β-CATENIN during differentiation and in the gain/loss-of-function study in hMSCs. The binding of ZNF281 to the promoter region of β-CATENIN was observed using a chromatin immunoprecipitation (ChIP) assay. In conclusion, we propose that ZNF281 plays an important role in the maintenance and osteogenic differentiation of stem cells via the transcriptional regulation of genes including β-CATENIN.

Key words: Zinc finger protein (ZNF281); b-CATENIN; WNT; Osteogenesis; Mesenchymal stem cells (MSC); Multipotency

Received January 12, 2011; final acceptance March 2, 2012. Online prepub date: September 7, 2012.
1These authors provided equal contribution to this work.
Address correspondence to Kyung-Sun Kang, D.V.M., Ph.D., Adult Stem Cell Research Center, College of Veterinary Medicine, Seoul National University, 151-742, Seoul, Republic of Korea. Tel: +82-2-880-1298; Fax: +82-2-876-7610; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 22, pp. 41-54, 2013
0963-6897/13 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X636984
E-ISSN 1555-3892
Copyright © 2013 Cognizant Comm. Corp.
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Interaction Between Adipose Tissue-Derived Mesenchymal Stem Cells and Regulatory T-Cells

Anja U. Engela, Carla C. Baan, Annemiek M. A. Peeters, Willem Weimar, and Martin J. Hoogduijn

Department of Internal Medicine, Transplantation Laboratory/Nephrology, Erasmus University Medical Center, Rotterdam, the Netherlands

Mesenchymal stem cells (MSCs) exhibit immunosuppressive capabilities, which have evoked interest in their application as cell therapy in transplant patients. So far it has been unclear whether allogeneic MSCs and host regulatory T-cells (Tregs) functionally influence each other. We investigated the interaction between both cell types using perirenal adipose tissue-derived MSCs (ASCs) from kidney donors and Tregs from blood bank donors or kidney recipients 6 months after transplantation. The immunomodulatory capacity of ASCs was not prejudiced by both Tregs from healthy donors and Tregs from graft recipients, indicating that ASCs were not targeted by the inhibitory effects of Tregs and vice versa. In addition, Tregs supported ASC function, as they did not alter the secretion of IFN-γ by immune cells and hence contributed to ASC activation and efficiency. ASCs exerted their suppressive role by expressing IDO, reducing levels of TNF-α, and by inducing the production of IL-10 in effector cells and Tregs. In conclusion, this study presents evidence that donor ASCs and acceptor Tregs do not impair each other’s function and therefore encourages the use of MSC therapy for the prevention of graft rejection in solid organ transplantation.

Key words: Mesenchymal stem cells (MSCs); Regulatory T-cells (Tregs); Immunosuppression; Transplantation

Received August 17, 2011; final acceptance January 26, 2012. Online prepub date: April 2, 2012.
Address correspondence to Anja U. Engela, P.O. Box 2040, Room Ee563a, 3000 CA Rotterdam, the Netherlands. Tel: +31(0)10-7035421; Fax: +31(0)10-7044718; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 22, pp. 55-64, 2013
0963-6897/13 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X640565
E-ISSN 1555-3892
Copyright © 2013 Cognizant Comm. Corp.
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Comparing the Functional Consequences of Human Stem Cell Transplantation in the Irradiated Rat Brain

Munjal M. Acharya, Lori-Ann Christie, Mary L. Lan, and Charles L. Limoli

Department of Radiation Oncology, University of California-Irvine, Irvine, CA, USA

Radiotherapy is a frontline treatment for the clinical management of CNS tumors. Although effective in eradicating tumor cells, radiotherapy also depletes neural stem and progenitor cells in the hippocampus that are important for neurogenesis and cognitive function. Consequently, the use of radiation to control primary and metastatic brain tumors often leads to debilitating and progressive cognitive decrements in surviving patients, representing a serious medical condition that, to date, has no satisfactory, long-term solutions. As a result, we have explored the use of stem cells as therapeutic agents to improve cognition after radiotherapy. Our past work has demonstrated the capability of cranially transplanted human embryonic (hESCs) and neural (hNSCs) stem cells to functionally restore cognition in rats 1 and 4 months after head-only irradiation. We have now expanded our cognitive analyses with hESCs and quantified both survival and differentiated fates of engrafted cells at 1 and 4 months after irradiation. Our findings indicate the capability of hESC transplantation to ameliorate radiation-induced cognitive dysfunction 1 month following cranial irradiation, using a hippocampal-dependent novel place recognition task. Irradiated animals not engrafted with stem cells experienced prolonged and significant cognitive dysfunction. Stereological estimates indicated that 35% and 17% of the transplanted hESCs survived at 1 and 4 months postgrafting, respectively. One month after irradiation and grafting, phenotypic analyses revealed that 26% and 31% of the hESCs differentiated into neurons and astrocytes, while at the 4-month time, neuronal and astrocytic differentiation was 7% and 46%, respectively. Comparison between present and past data with hESCs and hNSCs demonstrates equivalent cognitive restoration and a preference of hNSCs to commit to neuronal versus astrocytic lineages over extended engraftment times. Our data demonstrate the functional utility of human stem cell replacement strategies for ameliorating the adverse effects of cranial irradiation on cognition.

Key words: Human embryonic stem cells (hESCs); Human neural stem cells (hNSCs); Transplantation; Radiation; Cognition; Hippocampus; Spatial recognition memory

Received September 23, 2011; final acceptance February 15, 2012. Online prepub date: April 26, 2012.
Address correspondence to Prof. Charles L. Limoli, Department of Radiation Oncology, University of California-Irvine, Medical Sciences I, Room B-146B, Irvine, CA 92697-2695, USA. Tel: +1-949-824-3053; Fax: +1-949-824-3566; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 22, pp. 65-86, 2013
0963-6897/13 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X655037
E-ISSN 1555-3892
Copyright © 2013 Cognizant Comm. Corp.
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Electroacupuncture Promotes the Differentiation of Transplanted Bone Marrow Mesenchymal Stem Cells Overexpressing TrkC Into Neuron-Like Cells in Transected Spinal Cord of Rats

Ying Ding,* Qing Yan,* Jing-Wen Ruan,† Yan-Qing Zhang,* Wen-Jie Li,* Xiang Zeng,‡ Si-Fan Huang,* Yu-Jiao Zhang,* Jin-Lang Wu,§ Danny Fisher,¶ Hongxin Dong,¶ and Yuan-Shan Zeng*‡#

*Division of Neuroscience, Department of Histology and Embryology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China
†Department of Acupuncture of the First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China
‡Key Laboratory for Stem Cells and Tissue Engineering (Sun Yat-sen University), Ministry of Education, Guangzhou, China
§Department of Electron Microscope, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China
¶Department of Psychiatry and Behavioral Sciences, Northwestern University, Feinberg School of Medicine, Chicago, IL, USA
#Institute of Spinal Cord Injury, Sun Yat-sen University, Guangzhou, China

Our previous study indicated that electroacupuncture (EA) could increase neurotrophin-3 (NT-3) levels in the injured spinal cord, stimulate the differentiation of transplanted bone marrow mesenchymal stem cells (MSCs), and improve functional recovery in the injured spinal cord of rats. However, the number of neuron-like cells derived from the MSCs is limited. It is known that NT-3 promotes the survival and differentiation of neurons by preferentially binding to its receptor TrkC. In this study, we attempted to transplant TrkC gene-modified MSCs (TrkC-MSCs) into the spinal cord with transection to investigate whether EA treatment could promote NT-3 secretion in the injured spinal cord and to determine whether increased NT-3 could further enhance transplanted MSCs overexpressing TrkC to differentiate into neuron-like cells, resulting in increased axonal regeneration and functional improvement in the injured spinal cord. Our results showed that EA increased NT-3 levels; furthermore, it promoted neuron-phenotype differentiation, synaptogenesis, and myelin formation of transplanted TrkC-MSCs. In addition, TrkC-MSC transplantation combined with EA (the TrkC-MSCs + EA group) treatment promoted the growth of the descending BDA-labeled corticospinal tracts (CSTs) and 5-HT-positive axonal regeneration across the lesion site into the caudal cord. In addition, the conduction of cortical motorevoked potentials (MEPs) and hindlimb locomotor function increased as compared to controls (treated with the LacZ-MSCs, TrkC-MSCs, and LacZ-MSCs + EA groups). In the TrkC-MSCs + EA group, the injured spinal cord also showed upregulated expression of the proneurogenic factors laminin and GAP-43 and downregulated GFAP and chondroitin sulfate proteoglycans (CSPGs), major inhibitors of axonal growth. Together, our data suggest that TrkC-MSC transplantation combined with EA treatment spinal cord injury not only increased MSC survival and differentiation into neuron-like cells but also promoted CST regeneration across injured sites to the caudal cord and functional improvement, perhaps due to increase of NT-3 levels, upregulation of laminin and GAP-43, and downregulation of GFAP and CSPG proteins.

Key words: Transected spinal cord (TSC); Bone marrow mesenchymal stem cells (MSCs); Electroacupuncture; Tyrosine receptor kinase C (TrkC); Differentiation

Received September 25, 2011; final acceptance February 18, 2012. Online prepub date: September 21, 2012.
Address correspondence to Yuan-Shan Zeng, M.D., Ph.D., Department of Histology and Embryology, Zhongshan School of Medicine, Sun Yat-sen University, #74 Zhongshan 2nd Road, Guangzhou 510080, China. Tel: +86-20-87332698; Fax: +86-20-87332698; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it or Hongxin Dong, M.D., Ph.D., Department of Psychiatry and Behavioral Sciences, Feinberg School of Medicine, Northwestern University, 303 East Chicago Avenue, Chicago, IL 60611-3008, USA. Tel: 312-926-2323; Fax: 312-926-8080; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 22, pp. 87-97, 2013
0963-6897/13 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X653174
E-ISSN 1555-3892
Copyright © 2013 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

19F MRI Tracer Preserves In Vitro and In Vivo Properties of Hematopoietic Stem Cells

Brooke M. Helfer,* Anthony Balducci,* Zhina Sadeghi,† Charles O’Hanlon,* Adonis Hijaz,† Chris A. Flask,‡ and Amy Wesa*

*Celsense, Inc., Department of Research and Development, Pittsburgh, PA, USA
†Department of Urology, Case Western Reserve University, Cleveland, OH, USA
‡Departments of Radiology and Biomedical Engineering, Case Center for Imaging Research, Case Western Reserve University, Cleveland, OH, USA

Hematopoietic stem cells (HSCs) have numerous therapeutic applications including immune reconstitution, enzyme replacement, regenerative medicine, and immunomodulation. The trafficking and persistence of these cells after administration is a fundamental question for future therapeutic applications of HSCs. Here, we describe the safe and efficacious labeling of human CD34+ HSCs with a novel, self-delivering perfluorocarbon 19F magnetic resonance imaging (MRI) tracer, which has recently been authorized for use in a clinical trial to track therapeutic cells. While various imaging contrast agents have been used to track cellular therapeutics, the impact of this MRI tracer on HSC function has not previously been studied. Both human CD34+ and murine bone marrow (BM) HSCs were effectively labeled with the MRI tracer, with only a slight reduction in viability, relative to mock-labeled cells. In a pilot study, 19F MRI enabled the rapid evaluation of HSC delivery/retention following administration into a rat thigh muscle, revealing the dispersal of HSCs after injection, but not after surgical implantation. To investigate effects on cell functionality, labeled and unlabeled human HSCs were tested in in vitro colony forming unit (CFU) assays, which resulted in equal numbers of total CFU as well as individual CFU types, indicating that labeling did not alter multipotency. Cobblestone assay forming cell precursor frequency was also unaffected, providing additional evidence that stem cell function was preserved after labeling. In vivo tests of multipotency and reconstitution studies in mice with murine BM containing labeled HSCs resulted in normal development of CFU in the spleen, compared to unlabeled cells, and reconstitution of both lymphoid and myeloid compartments. The lack of interference in these complex biological processes provides strong evidence that the function and therapeutic potential of the HSCs are likely maintained after labeling. These data support the safety and efficacy of the MRI tracer for clinical tracking of human stem cells.

Key words: Cell tracking; Magnetic resonance imaging (MRI) tracer; Contrast agent; Preclinical safety; Stem cell therapy

Received April 20, 2011; final acceptance March 5, 2012. Online prepub date: August 2, 2012.
Address correspondence to Amy Wesa, 603 Stanwix St., Two Gateway Center, Suite 348, Pittsburgh, PA 15222, USA. Tel: +1 412-263-2870; Fax: +1 412-263-2878; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 22, pp. 99-117, 2013
0963-6897/13 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X638883
E-ISSN 1555-3892
Copyright © 2013 Cognizant Comm. Corp.
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Characterization, GFP Gene Nucleofection, and Allotransplantation in Injured Tendons of Ovine Amniotic Fluid-Derived Stem Cells

A. Colosimo,*1 V. Curini,*1 V. Russo,*A. Mauro,* N. Bernabò,*M. Marchisio,M. Alfonsi,§ A. Muttini,*M. Mattioli,* and B. Barboni*

*Department of Comparative Biomedical Sciences, University of Teramo, Teramo, Italy
StemTech Group, Chieti, Italy
‡Department of Biomorphology, University of Chieti, Chieti, Italy
§Department of Biomedical Sciences, University of Chieti, Chieti, Italy

Amniotic fluid has drawn increasing attention in the recent past as a cost-effective and accessible source of fetal stem cells. Amniotic fluid-derived mesenchymal stem cells (AFMSCs) that display high proliferation rate, large spectrum of differentiation potential, and immunosuppressive features are considered optimal candidates for allogeneic repair of mesenchymal damaged tissues. In this study, ovine AFMSCs (oAFMSCs) isolated from 3-month-old sheep fetuses were characterized for their proliferation rate, specific surface antigen and pluripotency marker expression, genomic stability, and mesenchymal lineage differentiation during their in vitro expansion (12 passages) and after nucleofection. The high proliferation rate of oAFMSCs gradually decreased during the first six subculture passages while the expression of surface molecules (CD29, CD58, CD166) and of pluripotency-associated markers (OCT4, TERT, NANOG, SOX2), the in vitro osteogenic differentiation potential, and a normal karyotype were maintained. Afterwards, oAFMSCs were nucleofected with a selectable plasmid coding for green fluorescent protein (GFP) using two different programs, U23 and C17, previously optimized for human mesenchymal stem cells. Transfection efficiencies were ~63% and ~37%, while cell recoveries were ~10% and ~22%, respectively. Nucleofected oAFMSCs expressing the GFP transgene conserved their pluripotency marker profile and retained a normal karyotype and the osteogenic differentiation ability. Seven single clones with a GFP expression ranging from 80% to 97% were then isolated and expanded over 1 month, thus providing stably transfected cells with long-term therapeutic potential. The in vivo behavior of GFP-labeled oAFMSCs was tested on a previously validated preclinical model of experimentally induced Achille’s tendon defect. The allotransplanted oAFMSCs were able to survive within the host tissue for 1 month enhancing the early phase of tendon healing as indicated by morphological and biomechanical results. Altogether these data suggest that genetically modified oAFMSCs might represent a valuable tool for in vivo preclinical studies in a highly valid translational model.

Key words: Amniotic fluid stem cells (AFMSCs); Sheep; Nucleofection; GFP reporter gene; Tendon healing

Received June 8, 2011; final acceptance January 28, 2012. Online prepub date: April 10, 2012.
1These authors provided equal contribution to this work.
Address correspondence to Alessia Colosimo, Ph.D., Department of Comparative Biomedical Sciences, University of Teramo, Piazza Aldo Moro 45, 64100 Teramo, Italy. Tel: +39-0861-266840; Fax: +39-0861-266929; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 22, pp. 119-131, 2013
0963-6897/13 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X638946
E-ISSN 1555-3892
Copyright © 2013 Cognizant Comm. Corp.
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Decrease of Global Methylation Improves Significantly Hepatic Differentiation of Ad-MSCs: Possible Future Application for Urea Detoxification

C. Seeliger,* M. Culmes,*† L. Schyschka,* X. Yan,*‡ G. Damm,* Z. Wang,*‡ J. Kleeff,§ W. E. Thasler,¶ J. Hengstler,# U. Stöckle,** S. Ehnert,** and A. K. Nüssler*,**

*Technical University Munich, MRI, Department of Trauma Surgery, Munich, Germany
†Technical University Munich, MRI, Department of Vascular Surgery, Munich, Germany
‡Union Hospital, Huazhong University of Science and Technology, Tongji Medical School, Wuhan, China
§Technical University Munich, MRI, Department of General Surgery, Munich, Germany
¶Ludwig Maximilians University Munich, Department of Surgery and Center for Liver Cell Research, Grosshadern Hospital, Munich, Germany
#Technical University Dortmund, IFADO, Dortmund, Germany
**Eberhard-Karls University Tubingen, Department of Trauma Surgery, Tubingen, Germany

Hepatocyte transplantation is considered to be an alternative to orthotopic liver transplantation. Cells can be used to bridge patients waiting for a donor organ, decrease mortality in acute liver failure, and support metabolic liver diseases. The limited availability of primary human hepatocytes for such applications has led to the generation of alternative hepatocyte-like cells from various adult stem or precursor cells. The aim of this study was to generate hepatocyte-like cells from adipose-derived mesenchymal stem cells (Ad-MSCs) for clinical applications, which are available “off the shelf.” Epigenetic changes in hepatocyte-like cells were induced by 5-azacytidine, which, in combination with other supplements, leads to significantly improved metabolic and enzymatic activities compared to nontreated cells. Cells with sufficient hepatic features were generated with a four-step protocol: 5-azacytidine (step 1); epidermal growth factor (step 2); fibroblast growth factor-4, dexamethasone, insulin-transferrin-sodium-selenite, and nicotinamide (step 3); and hepatocyte growth factor, dexamethasone, insulin-transferrin-sodium-selenite, and nicotinamide (step 4). Generated differentiated cells had higher phase I (CYP1A1/2, CYP2E1, CYP2B6, CYP3A4) and phase II activities compared to the undifferentiated cells. A strong expression of CYP3A7 and a weak expression of 3A4, as well as the important detoxification markers α-fetoprotein and albumin, could also be detected at the mRNA level. Importantly, urea metabolism (basal, NH4-stimulated, NH4- and ornithine-stimulated) was comparable to freshly isolated human hepatocytes, and unlike cryopreserved human hepatocytes, this activity was maintained after 6 months of cryopreservation. These findings suggest that these cells may be suitable for clinical application, especially for treatment of urea cycle disorders.

Key words: Differentiation; Adipose-derived mesenchymal stem cells (Ad-MSCs); Epigenetic; Urea metabolism; Cryopreservation

Received July 11, 2011; final acceptance February 3, 2012. Online prepub date: April 10, 2012.
Address correspondence to Prof. Dr. Andreas Nüssler, Eberhard Karls University Tubingen, Department of Trauma Surgery, Schnarrenbergstr. 95, 72076 Tubingen, Germany. Tel: +49-89-4140 6310; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 22, pp. 133-145, 2013
0963-6897/13 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X647162
E-ISSN 1555-3892
Copyright © 2013 Cognizant Comm. Corp.
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Insulin-Producing Cells From Adult Human Bone Marrow Mesenchymal Stem Cells Control Streptozotocin-Induced Diabetes in Nude Mice

Mahmoud M. Gabr,* Mahmoud M. Zakaria,* Ayman F. Refaie,* Amani M. Ismail,* Mona A. Abou-El-Mahasen,* Sylvia A. Ashamallah,* Sherry M. Khater,* Sawsan M. El-Halawani,* Rana Y. Ibrahim,* Gan Shu Uin,† Malgorzata Kloc,‡ Roy Y. Calne,†§ and Mohamed A. Ghoneim*

*Urology and Nephrology Center, Mansoura, Egypt
†Department of Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
‡The Methodist Hospital, Department of Surgery and The Methodist Hospital Research Institute, Houston, TX, USA
§Department of Surgery, Cambridge University, Cambridge, UK

Harvesting, expansion, and directed differentiation of human bone marrow-derived mesenchymal stem cells (BM-MSCs) could provide an autologous source of surrogate b-cells that would alleviate the limitations of availability and/or allogenic rejection following pancreatic or islet transplantation. Bone marrow cells were obtained from three adult type 2 diabetic volunteers and three nondiabetic donors. After 3 days in culture, adherent MSCs were expanded for two passages. At passage 3, differentiation was carried out in a three-staged procedure. Cells were cultured in a glucose-rich medium containing several activation and growth factors. Cells were evaluated in vitro by flow cytometry, immunolabeling, RT-PCR, and human insulin and c-peptide release in responses to increasing glucose concentrations. One thousand cell clusters were inserted under the renal capsule of diabetic nude mice followed by monitoring of their diabetic status. At the end of differentiation, ~5–10% of cells were immunofluorescent for insulin, c-peptide or glucagon; insulin, and c-peptide were coexpressed. Nanogold immunolabeling for electron microscopy demonstrated the presence of c-peptide in the rough endoplasmic reticulum. Insulin-producing cells (IPCs) expressed transcription factors and genes of pancreatic hormones similar to those expressed by pancreatic islets. There was a stepwise increase in human insulin and c-peptide release by IPCs in response to increasing glucose concentrations. Transplantation of IPCs into nude diabetic mice resulted in control of their diabetic status for 3 months. The sera of IPC-transplanted mice contained human insulin and c-peptide but negligible levels of mouse insulin. When the IPC-bearing kidneys were removed, rapid return of diabetic state was noted. BM-MSCs from diabetic and nondiabetic human subjects could be differentiated without genetic manipulation to form IPCs that, when transplanted, could maintain euglycemia in diabetic mice for 3 months. Optimization of the culture conditions are required to improve the yield of IPCs and their functional performance.

Key words: Human bone marrow mesenchymal stem cells (MSCs); Differentiation insulin; Soluble factors; Diabetes

Received April 5, 2011; final acceptance January 20, 2012. Online prepub date: June 15, 2012.
Address correspondence to Dr. Mohamed A. Ghoneim, M.D., M.D. (Hon.), FACS (Hon.), Urology and Nephrology Center, Mansoura-35516, Egypt. Tel: +20-50-2262226/2234545; Fax: +20-50-2235252; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 22, pp. 147-158, 2013
0963-6897/13 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X653057
E-ISSN 1555-3892
Copyright © 2013 Cognizant Comm. Corp.
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The Combined Expression of Pdx1 and MafA With Either Ngn3 or NeuroD Improves the Differentiation Efficiency of Mouse Embryonic Stem Cells Into Insulin-Producing Cells

Huiming Xu,* Kam Sze Tsang,† Juliana C. N. Chan,*‡§ Ping Yuan,§ Rongrong Fan,* Hideaki Kaneto,¶ and Gang Xu*‡§

*Department of Medicine and Therapeutics, The Chinese University of Hong Kong, The Prince of Wales Hospital, Shatin, Hong Kong SAR, China
†Department of Anatomical and Cellular Pathology, The Chinese University of Hong Kong, The Prince of Wales Hospital, Shatin, Hong Kong SAR, China
‡Hong Kong Institute of Diabetes and Obesity, The Chinese University of Hong Kong, The Prince of Wales Hospital, Shatin, Hong Kong SAR, China
§Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, The Prince of Wales Hospital, Shatin, Hong Kong SAR, China
¶Department of Metabolic Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, Japan

The use of pancreatic β-cells differentiated from embryonic stem (ES) cells or induced pluripotent stem (iPS) cells is a promising strategy in cell therapy. Pancreatic b-cell development is regulated by the sequential expression of a molecular network of transcription factors. In this experiment, we adopted a three-step differentiation protocol to differentiate mES (mouse ES) cells into insulin-secreting cells and overexpressed transcription factors by adenoviral vectors at various combinations at different time of differentiation. We found that the coexpression of Pdx1 and MafA with either Ngn3 or NeuroD, especially at the final stage of the three-step differentiation, significantly increased the differentiation efficiency. It also increased the glucose-stimulated insulin and C-peptide secretion in insulin-secreting cells derived from mES cells compared to the control green fluorescent protein (GFP) vector-transduced group. For the first time, we have demonstrated that the coexpression of Pdx1 and MafA during a specific time window of development can act synergistically with either Ngn3 or NeuroD to promote the differentiation of mES cells into insulin-secreting cells.

Key words: Transcription factors; Differentiation efficiency; Embryonic stem cells; Insulin-secreting cells

Received September 14, 2010; final acceptance November 30, 2011. Online prepub date: July 5, 2012.
Address correspondence to Dr. Gang Xu, Room 605, Li Ka Shing Institute of Health Science, The Chinese University of Hong Kong, The Prince of Wales Hospital, Hong Kong SAR, China. Tel: +85237636074; Fax: +85221446365; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 22, pp. 159-173, 2013
0963-6897/13 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X637028
E-ISSN 1555-3892
Copyright © 2013 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Significant Improvement in Islet Yield and Survival With Modified ET-Kyoto Solution: ET-Kyoto/Neutrophil Elastase Inhibitor

Tomohiko Machida,*1 Masahiro Tanemura,*†1 Yoshiaki Ohmura,* Tsukasa Tanida,* Hiroshi Wada,* Shogo Kobayashi,* Shigeru Marubashi,* Hidetoshi Eguchi,* Toshinori Ito,‡ Hiroaki Nagano,* Masaki Mori,* Yuichiro Doki,* and Yoshiki Sawa§

*Department of Gastroenterological Surgery, Osaka University Graduate School of Medicine, Osaka, Japan
†Department of Surgery and Institute for Clinical Research, National Hospital Organization Kure Medical Center, Hiroshima, Japan
‡Department of Complementary and Alternative Medicine, Osaka University Graduate School of Medicine, Osaka, Japan
§Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Osaka, Japan

Although islet transplantation can achieve insulin independence in patients with type 1 diabetes, sufficient number of islets derived from two or more donors is usually required to achieve normoglycemia. Activated neutrophils and neutrophil elastase (NE), which is released from these neutrophils, can directly cause injury in islet grafts. We hypothesized that inhibition of NE improves islet isolation and islet allograft survival. We tested our hypothesis by examining the effects of modified ET-Kyoto solution supplemented with sivelestat, a NE inhibitor (S-Kyoto solution), on islet yield and viability in islet isolation and the effect of intraperitoneally injected sivelestat on islet graft survival in a mouse allotransplant model. NE and proinflammatory cytokines such as tumor necrosis factor (TNF)-α and interleukin (IL)-6 increased markedly at the end of warm digestion during islet isolation and exhibited direct cytotoxic activity against the islets causing their apoptosis. The use of S-Kyoto solution significantly improved islet yield and viability. Furthermore, treatment with sivelestat resulted in significant prolongation of islet allograft survival in recipient mice. Furthermore, serum levels of IL-6 and TNF-α at 1 and 2 weeks posttransplantation were significantly higher in islet recipients than before transplantation. Our results indicated that NE released from activated neutrophils negatively affects islet survival and that its suppression both in vitro and in vivo improved islet yield and prolonged islet graft survival. The results suggest that inhibition of NE activity could be potentially useful in islet transplantation for patients with type 1 diabetes mellitus.

Key words: Type 1 diabetes; Islet transplantation; Neutrophil; Neutrophil elastase (NE)

Received June 17, 2011; final acceptance January 10, 2012. Online prepub date: April 2, 2012.
1These authors provided equal contribution to this work.
Address correspondence to Masahiro Tanemura, M.D., Ph.D., Department of Gastroenterological Surgery, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. Tel: +81-6-6879-3251; Fax: +81-6-6879-3259; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 22, pp. 175-187, 2013
0963-6897/13 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368912X653183
E-ISSN 1555-3892
Copyright © 2013 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

The Potential Use of Allogeneic Platelet-Rich Plasma for Large Bone Defect Treatment: Immunogenicity and Defect Healing Efficacy

Zhi-Yong Zhang,*1 Ai-Wen Huang,†1 Jun Jun Fan,*1 Kuanhai Wei,‡ Dan Jin,‡ Bin Chen,‡ Dan Li,* Long Bi,* Jun Wang,* and Guoxian Pei*

*Institute of Orthopaedics and Traumatology, Xijing Hospital, The Fourth Military Medical University, Xi’an, People’s Republic of China
†Department of Orthopaedics, the 309th Hospital of the PLA, Beijing, People’s Republic of China
‡Institute of Orthopaedics and Traumatology, Nanfang Hospital, Southern Medical University, Guangzhou, People’s Republic of China

Autologous platelet-rich plasma (PRP) has been extensively investigated for large bone defect treatment, but its clinical application is harassed by controversial outcome, due to highly variable PRP quality among patients. Alternatively, allogeneic PRP from well-characterized donors cannot only generate more consistent and reliable therapeutic effect but also avoid harvesting large quantities of blood, an additional health burdens to patients. However, the use of allogeneic PRP for bone defect treatment is generally less investigated, especially for its immunogenicity in such application. Here, we meticulously investigated the immunogenicity of allogeneic PRP and evaluated its healing efficacy for critical-sized defect treatment. Allogeneic PRP contained 4.1-fold and 2.7- to 4.9-fold higher amount of platelets and growth factors than whole blood, respectively. The intramuscular injection of allogeneic PRP to rabbits did not trigger severe and chronic immunoresponse, evidenced by little change in muscular tissue microstructure and CD4+/CD8+ T lymphocyte subpopulation in peripheral blood. The implantation of allogeneic PRP/deproteinized bone matrix (DPB) constructs (PRP + DPB) successfully bridged 1.5-cm segmental radial defects in rabbits, achieving similar healing capacity as autologous MSC/DPB constructs (MSC + DPB), with greater bone formation (1.1–1.5×, p < 0.05) and vascularization (1.3–1.6×, p < 0.05) than DPB alone, shown by histomorphometric analysis, bone mineral density measurement, and radionuclide bone imaging. Furthermore, the implantation of both allogeneic PRP- and autologous MSC-mediated DPB constructs (PRP + MSC + DPB) resulted in the most robust bone regeneration (1.2–2.1×, p < 0.05) and vascularization (1.3–2.0×, p < 0.05) than others (PRP + DPB, MSC + DPB, or DPB alone). This study has demonstrated the promising use of allogeneic PRP for bone defect treatment with negligible immunogenicity, great healing efficacy, potentially more consistent quality, and no additional health burden to patients; additionally, the synergetic enhancing effect found between allogeneic PRP and autologous MSCs may shed a light on developing new therapeutic strategies for large bone defect treatment.

Key words: Platelet-rich plasma (PRP); Mesenchymal stem cells (MSCs); Immunogenicity; Bone regeneration; Deproteinized bone matrix

Received October 9, 2011; final acceptance January 31, 2012. Online prepub date: August 2, 2012.
1These authors provided equal contribution to this work.
Address correspondence to Dr. Guoxian Pei, M.D., Ph.D., Professor and Chairman of Department of Orthopeadic Surgery, Xijing Hospital, The Fourth Military Medical University, 127 West Changle Road, Xi’an, Shaanxi, 710032, People’s Republic of China. Tel: +86-29-84775275; Fax: +86-29-83223558; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it