Cell Transplantation 19(9) Abstracts

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Cell Transplantation, Vol. 19, pp. 1063–1071, 2010
 0963-6897/10 $90.00 + .00
DOI: 10.3727/096368910X498278
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Injectable VEGF Hydrogels Produce Near Complete Neurological and Anatomical Protection Following Cerebral Ischemia in Rats

Dwaine F. Emerich,* Eduardo Silva,† Omar Ali,* David Mooney,‡ William Bell,* Seong Jin Yu,§ Yuji Kaneko,§ and Cesar Borlongan§

*InCytu, Inc., Lincoln, RI, USA
†Wyss Institute for Biologically Inspired Engineering, Cambridge, MA, USA
‡School of Engineering and Applied Sciences, Harvard University, Cambridge, MA, USA
§Department of Neurosurgery and Brain Repair, University of South Florida, Tampa, FL, USA

Vascular endothelial growth factor (VEGF) is a potent proangiogenic peptide and its administration has been considered as a potential neuroprotective strategy following cerebral stroke. Because VEGF has a short half-life and limited access to the brain parenchyma following systemic administration, approaches are being developed to deliver it directly to the site of infarction. In the present study, VEGF was incorporated into a sustained release hydrogel delivery system to examine its potential benefits in a rat model of cerebral ischemia. The hydrogel loaded with VEGF (1 μg) was stereotaxically injected into the striatum of adult rats 15 min prior to a 1-h occlusion of the middle cerebral artery. Two days after surgery, animals were tested for motor function using the elevated bias swing test (EBST) and Bederson neurological battery. Control animals received either stroke alone, stroke plus injections of a blank gel, or a single bolus injection of VEGF (1 μg). Behavioral testing confirmed that the MCA occlusion resulted in significant deficits in the the EBST and Bederson tests. In contrast, the performance of animals receiving VEGF gels was significantly improved relative to controls, with only modest impairments observed. Cerebral infarction analyzed using 2,3,5-triphenyl-tetrazolium chloride staining confirmed that the VEGF gels significantly and potently reduced the lesion volume. No neurological or histological benefits were conferred by either blank gel or bolus VEGF injections. These data demonstrate that VEGF, delivered from a hydrogel directly to the brain, can induce significant functional and structural protection from ischemic damage in a rat model of stroke.

Key words: Alginate; Stroke; Vascular endothelial growth factor (VEGF); Hydrogel; Neuroprotection; Transplantation

Address correspondence to Dwaine F. Emerich, Ph.D., InCytu, Inc., 701 George Washington Highway, Lincoln, RI 02865, USA. Tel: 401-499-6662; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation
, Vol. 19, pp. 1073–1084, 2010
0963-6897/10 $90.00 + .00
DOI: 10.3727/096368910X503415
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Changes in Host Blood Factors and Brain Glia Accompanying the Functional Recovery After Systemic Administration of Bone Marrow Stem Cells in Ischemic Stroke Rats

Ming Yang,* Xiaotao Wei,† Jing Li,† Lynn A. Heine,‡ Robert Rosenwasser,§ and Lorraine Iacovitti*†

*Department of Neurology, Thomas Jefferson University, Philadelphia, PA, USA
†Farber Institute for Neurosciences, Thomas Jefferson University, Philadelphia, PA, USA
‡Department of Surgery, Thomas Jefferson University, Philadelphia, PA, USA
§Department of Neurological Surgery, Thomas Jefferson University, Philadelphia, PA, USA

In this study, we examined the effects of systemic administration of rat or human bone marrow stromal stem cells (MSC) at early and later times following middle cerebral artery occlusion (MCAO) on blood cytokines/growth factors, brain glia, and motor behavior in rats. Rats were tail vein injected with rat (r) and human (h) MSCs at 1 or 7 days post-MCAO. In some rats (N = 4) MSCs isolated from transgenic GFP rats were used to track the migration of cells peripherally and centrally at 2.5 and 28 days. Motor behavior was assessed using the modified Neurological Severity Score/climbing test at various time points before and after MCAO and transplantation. Prior to sacrifice at 1, 7, or 28 days post-MCAO, blood serum was collected for cytokine array analysis. Brains were analyzed for markers of activated microglia (CD11) and reactive astrocytes (GFAP). Administration of either allogeneic (rMSCs) or xenogeneic (hMSCs) stem cells produced a significant recovery of motor behavior after MCAO, with cells delivered at 1 day having greater effect than those at 7 days. Correlated with recovery was an amplification in activated microglia, reactive astrocytes, and new blood vessels in the infarct region, resulting in greater preservation in brain integrity. Concomitantly, expression of blood cytokines/chemokines (IL-13, MMP2, MIP) and growth factors/receptors (VEGF, neuropilin, EPOR, TROY, NGFR, RAGE) were modified following MSC administration. Because only rare GFP-labeled MSCs were observed in the brain, these effects did not depend on the central incorporation of stem cells. The early systemic administration of allogeneic or xenogeneic MSCs soon after experimental stroke produces a structural/functional recovery in the brain which is correlated with an increase in activated brain glia and changes in circulating cytokines and growth factors. Stem cells therefore induce an important neuroprotective and/or regenerative response in the host organism.

Key words: Bone marrow transplantation; Middle cerebral artery occlusion; Stroke; Inflammatory factors; Microglia; Reactive astrocytes


Cell Transplantation, Vol. 19, pp. 1085–1101, 2010
0963-6897/10 $90.00 + .00
DOI: 10.3727/096368910X505468
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Ectopic Dopaminergic Progenitor Cells From
En1+/Otx2lacZ Transgenic Mice Survive and Functionally Reinnervate the Striatum Following Transplantation in a Rat Model of Parkinson’s Disease

Christina Hackl,*1 Anna Papazoglou,*1 Claudia Ganser,* Alexander Klein,* Nilima Prakash,† Wolfgang Wurst,† and Guido Nikkhah*

*Department of Stereotactic and Functional Neurosurgery, Laboratory of Molecular Neurosurgery, Neurocenter, University Hospital Freiburg, Freiburg, Germany
†Helmholtz Centre Munich, German Research Centre for Environmental Health (GmbH) and Technical University Munich, Institute of Developmental Genetics, Munich/Neuherberg, Germany

Cell-based therapies for Parkinson’s disease (PD) using neural stem cells to replace the lost dopamine neurons is currently an intense area of research. In this study we have evaluated the restorative potential of ectopic dopaminergic (DA) neurons derived from the rostral hindbrain (RH) of En1+/Otx2lacZ transgenic mice. The genetic modification of the DA progenitor domain in the En1+/Otx2lacZ mice is a gain of function, resulting in the enlargement of the area containing DA neurons, as well as an increase in their absolute number in the midbrain/hindbrain region. Amphetamine-induced rotation performed after cell transplantation into the unilaterally 6-hydroxydopamine-lesioned rat striatum revealed that animals with transgenic RH-derived DA grafts exhibited functional recovery similar to transgenic and wild-type ventral mesencephalon (VM)-derived DA grafts. Morphological analyses revealed equivalent numbers of surviving DA neurons from both homotopic VM- and ectopic RH-derived grafts from transgenic donors with low numbers of surviving serotonergic (5-HT) neurons. Conversely, grafts derived from wild-type donors contained predominantly surviving DA neurons or 5-HT neurons when they were prepared from the VM or RH, respectively. The study demonstrates the pattern of survival and functional potential of ectopic DA neurons derived from the RH of En1+/Otx2lacZ transgenic mice and that cell transplantation is an important neurobiological tool to characterize newly generated DA neural stem cells in vivo.

Key words: Midbrain/hindbrain boundary; Orthodenticle homolog 2 (Otx2); Neuron cell transplantation; DA progenitor domain; Ventral mesencephalon; Nucleus raphe

1
These authors provided equal contribution to this work.
Address correspondence to Guido Nikkhah, M.D., Ph.D., Department of Stereotactic and Functional Neurosurgery, Laboratory of Molecular Neurosurgery, Neurocenter, University Hospital Freiburg, Breisacher Str. 64, D-79106 Freiburg, Germany. Tel: +49-761-270-5063; Fax: +49-761-270-5010; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation
, Vol. 19, pp. 1103–1122, 2010
0963-6897/10 $90.00 + .00
DOI: 10.3727/096368910X503406
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Analysis of Dosing Regimen and Reproducibility of Intraspinal Grafting of Human Spinal Stem Cells in Immunosuppressed Minipigs

Dusan Usvald,*1 Peter Vodicka,*1 Jana Hlucilova,*1 Radek Prochazka,* Jan Motlik,* Karolina Kuchorova,‡§ Karl Johe,† Silvia Marsala,‡ Miriam Scadeng,¶ Osamu Kakinohana,‡ Roman Navarro,‡ Marian Santa,# Michael P. Hefferan,‡ Tony L. Yaksh,‡ and Martin Marsala‡§

*Institute of Animal Physiology and Genetics, Czech Academy of Sciences, Libechov, Czech Republic
†Neuralstem, Inc., Rockville, MD, USA
‡Anesthesiology Research Laboratory, University of California, San Diego, La Jolla, CA, USA
§Institute of Neurobiology, Slovak Academy of Sciences, Kosice, Slovakia
¶UCSD Center for Functional MRI, University of California, San Diego, La Jolla, CA, USA
#Faculty of Health, Department of Emergency Medicine, University of Presov, Presov, Slovakia

In recent studies using a rat aortic balloon occlusion model, we have demonstrated that spinal grafting of rat or human neuronal precursors or human postmitotic hNT neurons leads to progressive amelioration of spasticity and rigidity and corresponding improvement in ambulatory function. In the present study, we characterized the optimal dosing regimen and safety profile of human spinal stem cells (HSSC) when grafted into the lumbar spinal cord segments of naive immunosuppressed minipigs. Gottingen-Minnesota minipigs (18–23 kg) were anesthetized with halothane, mounted into a spine-immobilization apparatus, and received five bilateral injections of HSSC delivered in 2, 4, 6, 8, or 10 μl of media targeted into L2–L5 central gray matter (lamina VII). The total number of delivered cells ranged between 2,500 and 100,000 per injection. Animals were immunosuppressed with Prograf® for the duration of study. After cell grafting, ambulatory function was monitored daily using a Tarlov’s score. Sensory functions were assessed by mechanically evoked skin twitch test. Animals survived for 6–7 weeks. Three days before sacrifice animals received daily injections of bromodeoxyuridine (100 mg/kg; IV) and were then transcardially perfused with 4% paraformaldehyde. Th12–L6 spinal column was then dissected; the spinal cord was removed and scanned with MRI. Lumbar transverse spinal cord sections were then cut and stained with a combination of human-specific (hNUMA, hMOC, hNSE, hSYN) or nonspecific (DCX, MAP2, GABA, CHAT) antibodies. The total number of surviving cells was estimated using stereological quantification. During the first 12–24 h after cell grafting, a modest motor weakness was observed in three of eight animals but was no longer present at 4 days to 7 weeks. No sensory dysfunction was seen at any time point. Postmortem MRI scans revealed the presence of the individual grafts in the targeted spinal cord areas. Histological examination of spinal cord sections revealed the presence of hNUMA-immunoreactive grafted cells distributed between the base of the dorsal horn and the ventral horn. In all grafts intense hMOC, DCX, and hSYN immunoreactivity in grafted cells was seen. In addition, a rich axodendritic network of DCX-positive processes was identified extending 300–700 μm from the grafts. On average, 45% of hNUMA-positive neurons were GABA immunoreactive. Stereological analysis of hNUMA-positive cells showed an average of 2.5- to 3-fold increase in number of surviving cells compared with the number of injected cells. Analysis of spinal structural morphology showed that in animals injected with more than 50,000 cells/injection or volumes of injectate higher than 6 μl/injection there was tissue expansion and disruption of the local axodendritic network. Based on these data the safe total number of injected cells and volume of injectate were determined to be 30,000 cells delivered in ≤6 μl of media. These data demonstrate that highly reproducible delivery of a potential cell therapeutic candidate into spinal parenchyma can be achieved across a wide range of cell doses by direct intraspinal injections. The resulting grafts uniformly showed robust cell survival and progressive neuronal maturation.

Key words: Immunosuppression; Spinal stem cell; Spinal cord; Grafting; Survival; Dose response

1
These authors provided equal contribution to this work.
Address correspondence to Martin Marsala, M.D., Anesthesiology Research Laboratory-0695, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0695, USA. Tel: 858-822-3805; Fax: 858-822-3249; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation
, Vol. 19, pp. 1123–1132, 2010
0963-6897/10 $90.00 + .00
DOI: 10.3727/096368910X516664
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Suspension Culture of Mammalian Cells Using Thermosensitive Microcarrier That Allows Cell Detachment Without Proteolytic Enzyme Treatment

Hee Seok Yang,*1 Oju Jeon,†1 Suk Ho Bhang,‡ Soo-Hong Lee,§ and Byung-Soo Kim‡

*Department of Bioengineering, Hanyang University, Seoul, Republic of Korea
†Department of Chemical Engineering, Hanyang University, Seoul, Republic of Korea
‡School of Chemical and Biological Engineering, Seoul National University, Seoul, Republic of Korea
§Department of Biomedical Sciences, CHA Stem Cell Institute, Pochon CHA University, Seoul, Republic of Korea

Microcarriers are used to expand anchorage-dependent cells in large-scale suspension bioreactors. Proteolytic enzyme treatment is necessary to detach cells cultured on microcarriers for cell harvest or scale-up, but the enzyme treatment damages the cells and extracellular matrices and complicates the culture process. Here, we fabricated thermosensitive microcarriers from which cells can be detached by temperature change without proteolytic enzyme treatment. A thermosensitive polymer, poly-N-isopropylacrylamide (pNIPAAm), was incorporated on the surface of Cytodex-3® microcarriers. pNIPAAm-grafted microcarriers allowed human bone marrow-derived mesenchymal stem cells (hBMMSCs) to adhere, spread, and grow successfully on the microcarriers as nongrafted microcarriers did. By dropping temperature below 32°C, more than 82.5% of hBMMSCs were detached from pNIPAAm-grafted microcarriers. The trypsin treatment for cell detachment induced apoptosis and death of some of the detached cells, but cell detachment from pNIPAAm-grafted microcarriers by temperature change significantly reduced the apoptosis and cell death. pNIPAAm-grafted microcarriers can significantly reduce cell extracellular matrix damage in the cell detachment process and simplify the cell detachment process by avoiding proteolytic enzyme treatment. pNIPAAm-grafted microcarriers would be valuable to a variety of potential fields demanding a large amount of cells without cell damage, such as cell therapy, tissue engineering, and other biological and clinical applications.

Key words: Cell culture; Microcarrier; Thermosensitive

1
These authors provided equal contribution.
Address correspondence to Byung-Soo Kim, School of Chemical and Biological Engineering, Seoul National University, San 56-1, Sillim-dong, Gwanak-gu, Seoul 151-744, Republic of Korea. Tel: +82 2 880 1509; Fax: +82 2 888 1604; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation
, Vol. 19, pp. 1133–1142, 2010
0963-6897/10 $90.00 + .00
DOI: 10.3727/096368910X505486
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Effectiveness of a Web-Based Automated Cell Distribution System

Joyce C. Niland, Tracey Stiller, James Cravens, Janice Sowinski, John Kaddis, and Dajun Qian

Department of Information Sciences, City of Hope National Medical Center, Duarte, CA, USA

In recent years, industries have turned to the field of operations research to help improve the efficiency of production and distribution processes. Largely absent is the application of this methodology to biological materials, such as the complex and costly procedure of human pancreas procurement and islet isolation. Pancreatic islets are used for basic science research and in a promising form of cell replacement therapy for a subset of patients afflicted with severe type 1 diabetes mellitus. Having an accurate and reliable system for cell distribution is therefore crucial. The Islet Cell Resource Center Consortium was formed in 2001 as the first and largest cooperative group of islet production and distribution facilities in the world. We previously reported on the development of a Matching Algorithm for Islet Distribution (MAID), an automated web-based tool used to optimize the distribution of human pancreatic islets by matching investigator requests to islet characteristics. This article presents an assessment of that algorithm and compares it to the manual distribution process used prior to MAID. A comparison was done using an investigator’s ratio of the number of islets received divided by the number requested pre- and post-MAID. Although the supply of islets increased between the pre- versus post-MAID period, the median received-to-requested ratio remained around 60% due to an increase in demand post-MAID. A significantly smaller variation in the received-to-requested ratio was achieved in the post- versus pre-MAID period. In particular, the undesirable outcome of providing users with more islets than requested, ranging up to four times their request, was greatly reduced through the algorithm. In conclusion, this analysis demonstrates, for the first time, the effectiveness of using an automated web-based cell distribution system to facilitate efficient and consistent delivery of human pancreatic islets by enhancing the islet matching process.

Key words: Human islets; Algorithm; Automated cell distribution system; Islet Cell Resources (ICR); Islet equivalents (IEQs)

Address correspondence to Joyce C. Niland, Ph.D., Chair, Department of Information Sciences, City of Hope National Medical Center, 1500 East Duarte Road, Duarte, CA 91010-3000, USA. Tel: 626-256-4673, ext. 63032; Fax: 626-301-8802; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation
, Vol. 19, pp. 1143–1155, 2010
0963-6897/10 $90.00 + .00
DOI: 10.3727/096368910X504487
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

In Vitro and In Vivo Analysis of Endothelial Progenitor Cells From Cryopreserved Umbilical Cord Blood: Are We Ready for Clinical Application?

Valérie Vanneaux,*† Fida El-Ayoubi,† Catherine Delmau,† Catherine Driancourt,† Séverine Lecourt,* Aurore Grelier,* Audrey Cras,* Wendy Cuccuini,‡ Jean Soulier,‡ Jean-Jacques Lataillade,† Marie-Caroline Lebousse-Kerdiles,† Jean François Oury,§ Olivier Sibony,§ Jean-Pierre Marolleau,¶ Marc Benbunan,* Georges Uzan,† and Jérôme Larghero*

*Assistance Publique-Hôpitaux de Paris, Hôpital Saint Louis, Unité de Thérapie Cellulaire, Université Paris Diderot, INSERM Unit UMRS940, Paris, France
†INSERM Unit U972, IFR69, “Les cellules souches: de leurs niches à leurs applications thérapeutiques,” Hôpital Paul Brousse, Villejuif, France
‡Assistance Publique-Hôpitaux de Paris, Hôpital Saint Louis, Laboratoire Central d’Hématologie, Paris, France
§Assistance Publique-Hôpitaux de Paris, Hôpital Robert Debré, Service de Gynécologie-Obstetrique, Université Paris Diderot, Paris, France
¶Département d’He´matologie Clinique, Hôpital d’Amiens, Amiens, France

Umbilical cord blood (CB) represents a main source of circulating endothelial progenitor cells (cEPCs). In view of their clinical use, in either the autologous or allogeneic setting, cEPCs should likely be expanded from CB kept frozen in CB banks. In this study, we compared the expansion, functional features, senescence pattern over culture, and in vivo angiogenic potential of cEPCs isolated from fresh or cryopreserved CB (cryoCB). cEPCs could be isolated in only 59% of cryoCB compared to 94% for fresh CB, while CB units were matched in terms of initial volume, nucleated and CD34+ cell number. Moreover, the number of endothelial colony-forming cells was significantly decreased when using cryoCB. Once cEPCs culture was established, the proliferation, migration, tube formation, and acetylated-LDL uptake potentials were similar in both groups. In addition, cEPCs derived from cryoCB displayed the same senescence status and telomeres length as that of cEPCs derived from fresh CB. Karyotypic aberrations were found in cells obtained from both fresh and cryoCB. In vivo, in a hind limb ischemia murine model, cEPCs from fresh and cryoCB were equally efficient to induce neovascularization. Thus, cEPCs isolated from cryoCB exhibited similar properties to those of fresh CB in vitro and in vivo. However, the low frequency of cEPCs colony formation after cryopreservation shed light on the need for specific freezing conditions adapted to cEPCs in view of their future clinical use.

Key words: Endothelial progenitor cells (EPCs); Cord blood (CB); Regenerative medicine

Address correspondence to Pr Járôme Larghero, Cell Therapy Unit, Saint Louis Hospital, 1, avenue Claude Vellefaux, 75475 Paris cedex 10, France. Tel: 33 1 42 49 47 50; Fax: 33 1 42 49 47 55; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation
, Vol. 19, pp. 1157–1168, 2010
0963-6897/10 $90.00 + .00
DOI: 10.3727/096368910X504496
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Transplantation of Human Amnion Epithelial Cells Reduces Hepatic Fibrosis in Immunocompetent CCl
4-Treated Mice

Ursula Manuelpillai,* Jorge Tchongue,† Dinushka Lourensz,† Vijesh Vaghjiani,* Chrishan S. Samuel,‡§ Alison Liu,† Elizabeth D. Williams,¶ and William Sievert†#

*Centre for Reproduction & Development, Monash Institute of Medical Research, Monash University, Clayton, Victoria, Australia
†Centre for Inflammatory Diseases, Department of Medicine, Monash University, Clayton, Victoria, Australia
‡Howard Florey Institute, University of Melbourne, Melbourne, Victoria, Australia
§Department of Biochemistry and Molecular Biology, University of Melbourne, Melbourne, Victoria, Australia
¶Centre for Cancer Research, Monash Institute of Medical Research, Monash University, Clayton, Victoria, Australia
#Gastroenterology and Hepatology Unit, Monash Medical Centre, Clayton, Victoria, Australia

Chronic liver injury and inflammation lead to hepatic fibrosis, cirrhosis, and liver failure. Embryonic and mesenchymal stem cells have been shown to reduce experimental liver fibrosis but have potential limitations, including the formation of dysplastic precursors, tumors, and profibrogenic cells. Other stem-like cells may reduce hepatic inflammation and fibrosis without tumor and profibrogenic cell formation. To test this hypothesis we transplanted human amnion epithelial cells (hAEC), isolated from term delivered placenta, into immunocompetent C57/BL6 mice at week 2 of a 4-week regimen of carbon tetrachloride (CCl4) exposure to induce liver fibrosis. Two weeks following hAEC infusion, intact cells expressing the human-specific markers inner mitochondrial membrane protein and human leukocyte antigen-G were found in mouse liver without evidence of host rejection of the transplanted cells. Human albumin, known to be produced by hAEC, was detected in sera of hAEC-treated mice. Human DNA was detected in mouse liver and also spleen, lungs, and heart of some animals. Following hAEC transplantation, CCl4-treated animals showed decreased serum ALT levels and reduced hepatocyte apoptosis, compared to controls. hAEC-treated mouse liver had lower TNF-α and IL-6 protein levels and higher IL-10 compared to animals given CCl4 alone. Compared to CCl4 controls, hAEC-treated mice showed fewer activated collagen-producing hepatic stellate cells and less fibrosis area and collagen content. Reduced hepatic TGF-β levels in conjunction with a twofold increase in the active form of the collagen-degrading enzyme matrix metalloproteinase-2 in hAEC-treated mice compared to CCl4 controls may account for the reduction in fibrosis. hAEC transplantation into immunocompetent mice leads to cell engraftment, reduced hepatocyte apoptosis, and decreased hepatic inflammation and fibrosis.

Key words: Amnion epithelial cells; Hepatic inflammation; Liver fibrosis; Cellular therapy; Xenotransplantation

Address correspondence to Dr. Ursula Manuelpillai, Monash Institute of Medical Research, Monash University, Clayton, VIC 3168, Australia. Tel: +61 3 9594 7012; Fax: +61 3 9594 7416; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation
, Vol. 19, pp. 1169–1180, 2010
0963-6897/10 $90.00 + .00
DOI: 10.3727/096368910X503398
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Genetic Modification of Donor Hepatocytes Improves Therapeutic Efficacy for Hemophilia B in Mice

Yao-Ming Wu,*1 Chung-Yang Kao,†1 Yu-Jen Huang,* I-Shing Yu,† Hsuan-Shu Lee,‡ Hong-Shiee Lai,* Po-Huang Lee,* Chia-Ni Lin,† and Shu-Wha Lin†§

*Department of Surgery, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan
†Department of Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University College of Medicine, Taipei, Taiwan
‡Department of Internal Medicine, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan
§Department of Laboratory Medicine, National Taiwan University Hospital, Taipei, Taiwan

Hepatocyte transplantation (Tx) holds promise for curing genetic liver diseases. However, a limited number of donor hepatocytes can be transplanted into the host liver. Recipient preconditioning and donor cell engineering are under investigation to improve cell engraftment. In theory, genetically engineered cells secreting therapeutic proteins with superior function could compensate for poor engraftment efficiency. We have generated a bioengineered human coagulation factor IX (FIX) with augmented specific activity (named FIXTriple). The aim of this study was to evaluate therapeutic efficacy of cell therapy using hemophilia B (HB) as a disease model by transplanting FIX-Triple-secreting hepatocytes. The donor hepatocytes were isolated from FIX-Triple knock-in (KI) or FIX-WT (wild-type) KI mice and transplanted intrasplenically into FIX knock-out (KO) mice. FIX-Triple KI recipients exhibited fourfold higher plasma FIX clotting activity than FIX-WT KI recipients. By repeated Txs, the clotting activity of FIX-Triple KI recipients even increased to more than 10% of normal mouse plasma. The engraftment and FIX production efficiencies of transplanted cells were equivalent between the FIX-WT KI and FIX-Triple KI donors. A hemostatic function assay showed that FIX-Triple KI recipients with repeated Txs had more enhanced clot kinetics and a greater maximum rate of thrombus generation than those with a single Tx. Moreover, FIX inhibitors in these recipients rarely developed. In conclusion, hepatocyte Tx with genetically engineered donor cells is an effective therapeutic strategy for HB.

Key words: Hepatocyte transplantation; Genetically engineered donor cells; Bioengineered coagulation factor IX; Hemophilia B

1
These two authors provided equal contribution to this work.
Address correspondence to Dr. Shu-Wha Lin, Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, No.7, Chung-San S. Road, Taipei 100, Taiwan, R.O.C. Tel: (886) 2 23123456, ext. 66927; Fax: (886) 2 23817083; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 19, pp. 1181–1193, 2010
0963-6897/10 $90.00 + .00
DOI: 10.3727/096368910X504469
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Efficient Genetic Modification of Cynomolgus Monkey Embryonic Stem Cells With Lentiviral Vectors

Weiqiang Li,*†1 Chang Liu,*†1 Jie Qin,*† Li Zhang,‡ Rui Chen,*† Jing Chen,*† Xinbing Yu,*† Guifu Wu,§ Bruce T. Lahn,*†‡ Yongshui Fu,¶ and Andy Peng Xiang*†#**

*Center for Stem Cell Biology and Tissue Engineering, Sun Yat-sen University, Guangzhou, Guangdong, P.R. China
†The Key Laboratory for Stem Cells and Tissue Engineering, Ministry of Education, Guangzhou, P.R. China
‡Department of Human Genetics and Howard Hughes Medical Institute, University of Chicago, Chicago, IL, USA
§Division of Cardiology, First Affiliated Hospital, Sun Yat-sen University, Key Laboratory on Assisted Circulation, Ministry of Health, Guangzhou, P.R. China
¶Guangzhou Blood Center, Guangzhou, P.R. China
#Department of Biochemistry, Zhongshan Medical School, Sun Yat-sen University, Guangzhou, Guangdong, P.R. China
**State Key Laboratory of Ophthalmology, Sun Yat-sen University, Guangzhou, P.R. China

Embryonic stem (ES) cells have the ability to undergo indefinite self-renewal in vitro and give rise during development to derivatives of all three primary germ layers (ectoderm, endoderm, and mesoderm), which make them a highly prized reagent in cell and gene therapy. Efficient introduction of various genes of interest into primate ES cells has proven to be difficult. Here, we demonstrated that the self-inactivating HIV-1-based lentiviral vectors constructed by MultiSite gateway technology are efficient tools for the transduction of cynomolgus monkey (Macaca fasicularis) ES (cmES) cells. After antibiotic selection, all of the transduced cells can stably express the reporter gene (humanized Renilla GFP or dTomato) while maintaining their stem cell properties, including continuous expression of stem cell markers, alkaline phosphatase (AKP), OCT-4, SSEA-4, and TRA-1-60, formation of embryoid bodies in vitro and teratomas in vivo containing derivatives of three embryonic germ layers. This approach will provide a useful tool for both gene function studies and in vivo cell tracking of stem cells.

Key words: Primate embryonic stem cells; Lentivirus; Transduction; Green fluorescent protein

1
These authors provided equal contribution to this work.
Address correspondence to Andy Peng Xiang, Center for Stem Cell Biology and Tissue Engineering, Sun Yat-sen University, 74# Zhongshan Road 2, Guangzhou, Guangdong, 510080, P.R. China. Tel: +86-20-87335822; Fax: +86-20-87335858; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it or Yongshui Fu, Guangzhou Blood Center, Luyuan Road 31#, Guangzhou, 510095, P.R. China. Tel: +86-20-83593187; Fax: +86-20- 83575958; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation
, Vol. 19, pp. 1195–1208, 2010
0963-6897/10 $90.00 + .00
DOI: 10.3727/096368910X504478
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Deficiency of Either P-Glycoprotein or Breast Cancer Resistance Protein Protect Against Acute Kidney Injury

Miriam Huls,* Joost P. H. Schoeber,* Catherine M. Verfaillie,† Aernout Luttun,‡ Fernando Ulloa-Montoya,† Aswin L. Menke,§ Lars R. van Bolderen,¶ Rob M. Woestenenk,§ Gerard F. M. Merkx,# Jack F. M. Wetzels,** Frans G. M. Russel,* and Rosalinde Masereeuw*

*Department of Pharmacology and Toxicology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands
†Interdepartmental Stem Cell Institute, Katholieke Universiteit Leuven, Leuven, Belgium
‡Centre for Molecular and Vascular Biology, Katholieke Universiteit Leuven, Leuven, Belgium
§Central Hematology Laboratory, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands
¶Department of Radiation Oncology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands
#Department of Human Genetics, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands
**Department of Nephrology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands

The kidney has a high capacity to regenerate after ischemic injury via several mechanisms, one of which involves bone marrow-derived (stem) cells. The ATP binding cassette transporters, P-glycoprotein and breast cancer resistance protein, are determinants for the enriched stem and progenitor cell fraction in bone marrow. Because they are upregulated after acute kidney injury, we hypothesized that both efflux pumps may play a role in protecting against renal injury. Surprisingly, transporter-deficient mice were protected against ischemia-induced renal injury. To further study this, bone marrow from irradiated wild-type mice was reconstituted by bone marrow from wild-type, P-glycoprotein- or breast cancer resistance protein-deficient mice. Four weeks later, kidney injury was induced and its function evaluated. Significantly more bone marrow-derived cells were detected in kidneys grafted with transporter-deficient bone marrow. A gender mismatch study suggested that cell fusion of resident tubular cells with bone marrow cells was unlikely. Renal function analyses indicated an absence of renal damage following ischemia-reperfusion in animals transplanted with transporter-deficient bone marrow. When wild-type bone marrow was transplanted in breast cancer resistance protein-deficient mice this protection is lost. Furthermore, we demonstrate that transporter-deficient bone marrow contained significantly more monocytes, granulocytes, and early outgrowth endothelial progenitor cells.

Key words: Bone marrow transplantation; Ischemia-reperfusion injury; Renal function; Side population; ABC transporter

Address correspondence to Rosalinde Masereeuw, Ph.D., Department of Pharmacology and Toxicology (149), Radboud University Nijmegen Medical Centre, Nijmegen Centre for Molecular Life Sciences, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. Tel: +31 243613730; Fax: +31 243614214; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation
, Vol. 19, pp. 1209–1213, 2010
0963-6897/10 $90.00 + .00
DOI: 10.3727/096368910X504441
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Brief Communication

The Porcine Circovirus Type 1 in Porcine Kidney 15 Cell Line Is Not Transferred to Mice Lymphoid Cells After Xenoimplantation Into the Peritoneal Cavity

P. Hernández Jáuregui,* M. Anaya Ruiz,* F. Romero Pastrana,* G. Delgado López,* A. Pimentel Morales,* E. Tena Betancourt,† and E. Gómez Conde‡

*Centro de Investigación Biomédica de Oriente, Instituto Mexicano del Seguro Social (IMSS), Puebla, México
†Servicio de Cirugía Experimental y Bioterio, Coordinación de Investigación en Salud del IMSS, México D.F, México
‡Unidad Médica de Alta Especialidad CMN MAC IMSS, Puebla, México

The porcine circovirus type 1 (PCV1) has been identified within lymphoid tissues of experimental infected pigs and suggested to induce an immunosuppressive stage in pigs. The virus does not induce a cytophatic effect in the pig-derived cell line PK-15. Because PCV1 is prevalent in many pig cells and tissues, the risk of inducing a viral xenozoonosis by PCV1 was raised for the xenoimplantation of pig cells into human hosts. The present work evaluated if PCV1 is able to replicate in mice tissues after xenoimplantation of PCV1-infected pig cells. Active growing PK-15 cells harboring PCV1 with or without microencapsulation in sodium alginate were implanted into the peritoneal cavity of mice. After 1 month postimplantation in mice, peritoneal macrophages, spleen, and lymph nodes were harvested and analyzed with the polymerase chain reaction technique (PCR). No evidence of circovirus type 1 DNA was detected within the mice tissues.

Key words: Xenoimplantation; PK-15 cells; Porcine circovirus type 1 (PCV1); Mice

Address correspondence to Pablo Hernández Jáuregui, D.V.M., Ph.D., Centro de Investigación Biomédica de Oriente, Instituto Mexicano del Seguro Social (IMSS), Km 4.5 Carretera Atlixco Metepec, C.P. 74360, Puebla, México. E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it