ognizant Communication Corporation

The Regenerative Medicine Journal

VOLUME 19, NUMBER 4, 2010

Cell Transplantation, Vol. 19, pp. 369-386, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368909X484266
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Monocyte Derivatives Promote Angiogenesis and Myocyte Survival in a Model of Myocardial Infarction

M. Bouchentouf,1 P. Paradis,1 K. A. Forner,1 J. Cuerquis,1 M. N. Boivin,1 J. Zheng,1 M. R. Boulassel,2 J. P. Routy,2 E. L. Schiffrin,1 and J. Galipeau1

1Lady Davis Institute for Medical Research, Jewish General Hospital, McGill University, Montreal, Quebec, Canada
2Royal Victoria Hospital, Division of Hematology, McGill University Health Center, Montreal, Quebec, Canada

In this study, we have investigated the hypothesis that previously reported beneficial effect of peripheral blood mononuclear cells cultured under angiogenic conditions on cardiovascular function following ischemia is not limited to EPCs but also to monocytes contained therein. We first purified and analyzed the phenotype and secretome of human and murine blood monocytes cultured under angiogenic conditions (named MDs for monocyte derivatives) and tested their effect in a mouse model of myocardial infarction (MI). FACS analysis of MDs shows that these cells express mature endothelial cell markers and that their proliferative capacity is virtually absent, consistent with their end-differentiated monocytic ontogeny. MDs secreted significant levels of HGF, IGF-1, MCP-1, and sTNFR-1 relative to their monocyte precursors. MDs were unable to form vascular networks in vitro when cultured on matrix coated flasks. Treatment of murine HL-1 cardiomyocyte cell line with MD-conditioned medium reduced their death induced by TNF-a, staurosporine, and oxidative stress, and this effect was dependent upon MD-derived sTNFR-1, HGF, and IGF-1. We further demonstrate that MD secretome promoted endothelial cell proliferation and capacity to form vessels in vitro and this was dependent upon MD-derived MCP-1, HGF, and IGF-1. Echocardiography analysis showed that MD myocardial implantation improved left ventricle fractional shortening of mouse hearts following MI and was associated with reduced myocardial fibrosis and enhancement of angiogenesis. Transplanted MDs and their secretome participate in preserving functional myocardium after ischemic insult
and attenuate pathological remodeling.

Key words: Myocardial infarction; Monocytes; Transplantation; Growth factors; Angiogenesis; Apoptosis; Cardiac remodeling

Address correspondence to Jacques Galipeau, M.D., FRCPC, Associate Professor of Medicine, Sir Mortimer B. Davis Jewish General Hospital, Lady Davis Institute for Medical Research, McGill University, 3755 Cote Ste-Catherine Road, Montreal, Quebec, Canada H3T 1E2. Tel: +1 (514) 340 8214; Fax: +1 (514) 340 8281; E-mail: jacques.galipeau@mcgill.ca

Cell Transplantation, Vol. 19, pp. 387-398, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368909X484257
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Transplantation of Long-term Cultured Porcine Islets in the Rat: Prolonged Graft Survival and Recipient Growth on Reduced Immunosuppression

Josephine K. R. A. Rijkelijkhuizen, Annemiek Töns, Onno T. Terpstra, and Eelco Bouwman

Department of Surgery, Leiden University Medical Centre, Leiden, The Netherlands

To evaluate whether further improvement in porcine islet xenotransplantation is feasible, a number of questions were addressed. Earlier we showed significant improvement in the nude mouse of the porcine islets by selection through long-term culture. Now these islets were tested in the stringent pig-to-rat model. Islets were isolated from adult pigs, cultured for 1.5-3 weeks and transplanted to rats. Possible rejection mechanisms were assessed by interference of the cellular response with cyclosporine A (CsA), blocking macrophages with gadolinium chloride (GdCl), and suppressing the humoral response with cyclophosphamide. Modifications in graft size and condition were analyzed. Untreated control recipients showed primary nonfunction (PNF). CsA treatment could fully overcome PNF and resulted in graft survival from 10 to over 134 days. Rejection was the main cause of function loss. Although rejection could not be prevented by intensifying the induction therapy, increased maintenance immunosuppression effectively blocked rejection, albeit at the expense of toxicity. Blocking the humoral response was ineffective; all grafts showed PNF. In contrast, depletion of macrophages fully prevented PNF. Combination of GdCl and CsA gave no additional effect, and grafts were rejected between 57 and 162 days. Generally, graft survivals were similar to those reported in the literature; however, long-term cultured islets required much less maintenance immunosuppression. Cessation of graft function was not always due to rejection; in some cases "islet exhaustion" was found, possibly caused by discrepancy between the graft size and the rapidly growing recipient. Neither the presence of damaged islet tissue in the graft nor the size of the graft exerted any influence on graft survival. On rejection, no real infiltration of the graft was seen; destruction gradually processed from the outside. The good functional capability of the cultured islets was illustrated by disappearance of the clinical symptoms and increase in body weight, which almost doubled in the long-term survivors.

Key words: Adult porcine islets; Rat; Survival; Xenotransplantation

Address correspondence to Eelco Bouwman, Department of Surgery D6-36, Leiden University Medical Centre, PO Box 9600, 2300 RC Leiden, The Netherlands. Tel: +31 (0)71 5262987; Fax: +31 (0)71 5266746; E-mail: e.bouwman@lumc.nl

Cell Transplantation, Vol. 19, pp. 399-408, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368909X481782
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Continuous Delivery of Stromal Cell-Derived Factor-1 From Alginate Scaffolds Accelerates Wound Healing

Sina Y. Rabbany,1,2* Joseph Pastore,3* Masaya Yamamoto,2,4 Tim Miller,3 Shahin Rafii,2,5 Rahul Aras,3 and Marc Penn3,6

1Bioengineering Program, Hofstra University, Hempstead, NY, USA
2Department of Genetic Medicine, Howard Hughes Medical Institute, Weill Cornell Medical College, New York, NY, USA
3Juventas Therapeutics, Inc., Cleveland, OH, USA
4Department of Biomaterials, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan
5Howard Hughes Medical Institute, Weill Cornell Medical College, New York, NY, USA
6Department of Stem Cell Biology and Regenerative Medicine, Cleveland Clinic, Cleveland, OH, USA

Proper wound diagnosis and management is an increasingly important clinical challenge and is a large and growing unmet need. Pressure ulcers, hard-to-heal wounds, and problematic surgical incisions are emerging at increasing frequencies. At present, the wound-healing industry is experiencing a paradigm shift towards innovative treatments that exploit nanotechnology, biomaterials, and biologics. Our study utilized an alginate hydrogel patch to deliver stromal cell-derived factor-1 (SDF-1), a naturally occurring chemokine that is rapidly overexpressed in response to tissue injury, to assess the potential effects SDF-1 therapy on wound closure rates and scar formation. Alginate patches were loaded with either purified recombinant human SDF-1 protein or plasmid expressing SDF-1 and the kinetics of SDF-1 release were measured both in vitro and in vivo in mice. Our studies demonstrate that although SDF-1 plasmid- and protein-loaded patches were able to release therapeutic product over hours to days, SDF-1 protein was released faster (in vivo Kd 0.55 days) than SDF-1 plasmid (in vivo Kd 3.67 days). We hypothesized that chronic SDF-1 delivery would be more effective in accelerating the rate of dermal wound closure in Yorkshire pigs with acute surgical wounds, a model that closely mimics human wound healing. Wounds treated with SDF-1 protein (n = 10) and plasmid (n = 6) loaded patches healed faster than sham (n = 4) or control (n = 4). At day 9, SDF-1-treated wounds significantly accelerated wound closure (55.0 ± 14.3% healed) compared to nontreated controls (8.2 ± 6.0%, p < 0.05). Furthermore, 38% of SDF-1-treated wounds were fully healed at day 9 (vs. none in controls) with very little evidence of scarring. These data suggest that patch-mediated SDF-1 delivery may ultimately provide a novel therapy for accelerating healing and reducing scarring in clinical wounds.

Key words: Dermal delivery; Tissue remodeling; Neovascularization; Tissue scarring; Hydrogels

Address correspondence to Marc Penn, Department of Stem Cell Biology and Regenerative Medicine, Cleveland Clinic, Cleveland, OH, 44195, USA. Tel: (216) 444-6697; E-mail: pennm@ccf.org or Sina Y. Rabbany, Department of Genetic Medicine, Weill Cornell Medical College, New York, NY 10021, USA. Tel: (212) 746-7014; E-mail: sir2007@med.cornell.edu

*These two authors contributed equally.

Cell Transplantation, Vol. 19, pp. 409-418, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368909X484707
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Human Fetal Liver-Derived Stem Cell Transplantation as Supportive Modality in the Management of End-Stage Decompensated Liver Cirrhosis

Aleem A. Khan,1* Mahaboob V. Shaik,1* N. Parveen,1 A. Rajendraprasad,1 Mohammed A. Aleem,1 M. Aejaz Habeeb,1 G. Srinivas,2 T. Avinash Raj,2 Santosh K. Tiwari,1 K. Kumaresan,3 J. Venkateswarlu,1 Gopal Pande,2 and C. M. Habibullah1

1Centre for Liver Research and Diagnostics, Deccan College of Medical Sciences, Kanchanbagh, Hyderabad, Andhra Pradesh, India
2Center for Cellular and Molecular Biology, Uppal Road, Hyderabad, Andhra Pradesh, India
3KK Scan Centre, Somajiguda, Hyderabad, Andhra Pradesh, India

Liver transplantation is the only existing modality for treating decompensated liver cirrhosis. Several factors, such as nonavailability of donors, combined with operative risks, complications associated with rejection, usage of immunosuppressive agents, and cost intensiveness, make this strategy available to only a few people. With a tremendous upsurge in the mortality rate of patients with liver disorders worldwide, there is a need to search for an alternative therapeutic tool that can combat the above limitations and serve as a supportive therapy in the management of liver diseases. Cell therapy using human fetal liver-derived stem cells can provide great potential to conservatively manage end-stage liver diseases. Therefore, the present investigation aimed to study and prove the safety and efficacy of human fetal liver-derived stem cell transplantation in patients with end-stage liver cirrhosis. Twenty-five patients with liver cirrhosis of different etiologies were infused with human fetal liver-derived stem cells (EpCAM+ve) labeled with Tc-HMPAO through hepatic artery. Our high throughput analysis using flow cytometry, RT-PCR, and cellular characterization exemplifies fetal liver cells with their high proliferation rate could be the best source for rejuvenating the diseased liver. Further, no episodes related to hepatic encephalopathy recurred in any of the subjects following hepatic stem cell transplantation. There was marked clinical improvement observed in terms of all clinical and biochemical parameters. Further, there was decrease in mean MELD score (p < 0.01) observed in 6 months follow-up in all patients. Therapy using human fetal liver stem/progenitor cells offers a potentially supportive modality to organ transplantation in the management of liver diseases.

Key words: Liver cirrhosis; Encephalopathy; Liver transplantation; Epithelial cell adhesion molecule; HLA

Address correspondence to Dr. C. M. Habibullah, Director, Laboratory of Stem cell Research and Regenerative Medicine, Centre for Liver Research and Diagnostics, Deccan College of Medical Sciences, Kanchanbagh, Hyderabad 500 058, Andhra Pradesh, India. Tel/Fax: +91-40-24342954; E-mail: cmhabib@rediffmail.com

*Equal contribution.

Cell Transplantation, Vol. 19, pp. 419-429, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368910X498250
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Efficient and Rapid Labeling of Transplanted Cell Populations With Superparamagnetic Iron Oxide Nanoparticles Using Cell Surface Chemical Biotinylation for In Vivo Monitoring by MRI

Po-Wah So,1,2 Tammy Kalber,3 David Hunt,4 Michael Farquharson,5 Alia Al-Ebraheem,5 Harold G. Parkes,4 Rolf Simon,6 and Jimmy D. Bell3

1Preclinical Imaging Unit, Institute of Psychiatry, King's College London, London, UK
2Biological Imaging Centre, Imaging Sciences Department, Hammersmith Hospital Campus, Imperial College London, London, UK
3Metabolic and Molecular Imaging Group, MRC Clinical Sciences Centre, Hammersmith Hospital Campus, Imperial College London, London, UK
4Department of Neuroinflammation, Institute of Neurology, University College London, London, UK
5The Department of Medical Physics and Applied Radiation Sciences, McMaster University, Hamilton, Ontario, Canada
6Forschungszentrum Karlsruhe GmbH in der Helmholtz-Gemeinschaft Institut für Synchrotron, Eggenstein-Leopoldshafen, Germany

Determination of the dynamics of specific cell populations in vivo is essential for the development of cell-based therapies. For cell tracking by magnetic resonance imaging (MRI), cells need to internalize, or be surface labeled with a MRI contrast agent, such as superparamagnetic iron oxide nanoparticles (SPIOs): SPIOs give rise to signal loss by gradient-echo and T2-weighted MRI techniques. In this study, cancer cells were chemically tagged with biotin and then magnetically labeled with anti-biotin SPIOs. No significant detrimental effects on cell viability or death were observed following cell biotinylation. SPIO-labeled cells exhibited signal loss compared to non-SPIO-labeled cells by MRI in vitro. Consistent with the in vitro MRI data, signal attenuation was observed in vivo from SPIO-labeled cells injected into the muscle of the hind legs, or implanted subcutaneously into the flanks of mice, correlating with iron detection by histochemical and X-ray fluorescence (XRF) methods. To further validate this approach, human mesenchymal stem cells (hMSCs) were also employed. Chemical biotinylation and SPIO labeling of hMSCs were confirmed by fluorescence microscopy and flow cytometry. The procedure did not affect proliferation and multipotentiality, or lead to increased cell death. The SPIO-labeled hMSCs were shown to exhibit MRI signal reduction in vitro and was detectable in an in vivo model. In this study, we demonstrate a rapid, robust, and generic methodology that may be a useful and practical adjuvant to existing methods of cell labeling for in vivo monitoring by MRI. Further, we have shown the first application of XRF to provide iron maps to validate MRI data in SPIO-labeled cell tracking studies.

Key words: Magnetic resonance imaging (MRI); Cell labeling; Chemical biotinylation; Superparamagnetic iron oxide nanoparticles (SPIOs)

Address correspondence to Dr. Po-Wah So, Preclinical Imaging Unit, Institute of Psychiatry, King's College London, James Black Centre, 125, Coldharbour Lane, London, SE5 9NU. UK. Tel: +44(0)20 7848 5451; E-mail: po-wah.so@kcl.ac.uk

Cell Transplantation, Vol. 19, pp. 431-441, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368909X484699
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

In Vivo MRI Stem Cell Tracking Requires Balancing of Detection Limit and Cell Viability

K. Nohroudi,1 S. Arnhold,2 T. Berhorn,1 K. Addicks,1 M. Hoehn,3 and U. Himmelreich3*

1Department of Anatomy I, University of Cologne, Cologne, Germany
2Department of Veterinary Anatomy, University of Giessen, Giessen, Germany
3Max Planck Institute for Neurological Research, Cologne, Germany

Cell-based therapy using adult mesenchymal stem cells (MSCs) has already been the subject of clinical trials, but for further development and optimization the distribution and integration of the engrafted cells into host tissues have to be monitored. Today, for this purpose magnetic resonance imaging (MRI) is the most suitable technique, and micron-sized iron oxide particles (MPIOs) used for labeling are favorable due to their low detection limit. However, constitutional data concerning labeling efficiency, cell viability, and function are lacking. We demonstrate that cell viability and migratory potential of bone marrow mesenchymal stromal cells (BMSCs) are negatively correlated with incorporated MPIOs, presumably due to interference with the actin cytoskeleton. Nevertheless, labeling of BMSCs with low amounts of MPIOs results in maintained cellular function and sufficient contrast for in vivo observation of single cells by MRI in a rat glioma model. Conclusively, though careful titration is indicated, MPIOs are a promising tool for in vivo cell tracking and evaluation of cell-based therapies.

Key words: Bone marrow mesenchymal stromal cells (BMSCs); Magnet resonance imaging (MRI); Electron microscopy; Micron-sized iron oxide particles (MPIOs); Cell migration

Address correspondence to Klaus Nohroudi, Department of Anatomy I, University of Cologne, Joseph-Stelzmann-Straße 9, D-50931 Köln, Germany. Tel: +49-(0)221-478-5438; Fax: +49-(0)221-478-6711; E-mail: klaus.nohroudi@uk-koeln.de

*Current address: Biomedical NMR unit, Katholieke Universiteit, 3000 Leuven, Belgium.

Cell Transplantation, Vol. 19, pp. 443-451, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368909X484680
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Bilirubin Promotes De Novo Generation of T Regulatory Cells

Fredy Rocuts,1* Xinyu Zhang,1* Jun Yan,2 Yinan Yue,1 Michael Thomas,1 Fritz H. Bach,1 Eva Czismadia,1 and Hongjun Wang1

1Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA
2Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA

We have previously demonstrated that bilirubin administration to the recipient induces tolerance towards islet cell transplants across a complete MHC mismatch in a mouse model. Here we assess the mechanisms of such protection. Bilirubin treatment of recipients improved function of islet allografts by suppressing expressions of proinflammatory and proapoptotic genes in those islets and by increasing Foxp3+ T regulatory (Treg) cells at the site of transplanted islets at various days after transplantation. No prolongation of graft survival was observed in recipients treated with bilirubin when CD4+CD25+ T cells were predepleted from those recipients, indicating that Treg cells are necessary for the protective effect of bilirubin. Adoptive transfer of Treg cells from tolerant mice into Rag1-/- recipients resulted in long-term acceptance of skin allografts in an alloantigen-specific manner, suggesting that Treg cells are sufficient to induce tolerance. In addition, bilirubin treatment promoted de novo generation of Treg cells in Rag1-/- recipients. Thus, bilirubin treatment to the recipients prolongs islet allograft survival via a Treg-dependent manner in which CD4+CD25+ Treg cells are both necessary and sufficient for tolerance induction and graft acceptance. Bilirubin treatment promotes de novo generation of Treg cells that might account for the protective effects of bilirubin given to recipients.

Key words: Islet transplantation; Bilirubin; T regulatory cells (Tregs); Allograft survival

Address correspondence to Hongjun Wang, Ph.D., Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Ave., SL427, Boston, MA 02215, USA. Tel: 617-667-2856; Fax: 617-667-0892; E-mail: hwang3@bidmc.harvard.edu

*These two authors contributed equally to this work.

Cell Transplantation, Vol. 19, pp. 453-469, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368909X485049
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Humanized Culture Medium for Clinical Expansion of Human Erythroblasts

Giovanni Migliaccio,1,2 Massimo Sanchez,2 Francesca Masiello,2 Valentina Tirelli,2 Lilian Varricchio,1 Carolyn Whitsett,1 and Anna Rita Migliaccio1,3

1Division of Hematology and Oncology, Tisch Cancer Institute, New York, NY, USA
2Cell Biology and Neuroscience, Istituto Superiore Sanità, Rome, Italy
3Hematology, Oncology and Molecular Medicine, Istituto Superiore Sanità, Rome, Italy

Ex vivo-generated erythroblasts represent alternative transfusion products. However, inclusion of bovine components in media used for their growth precludes clinical use, highlighting the importance of developing culture media based on pharmaceutical grade reagents. In addition, because adult blood generates ex vivo lower numbers of erythroblasts than cord blood, cord blood has been proposed as the source of choice for ex vivo erythroblast production. To clarify the potential of adult blood to generate erythroblasts ex vivo, experiments were designed to identify growth factors [stem cell factor (SCF), interleukin-3 (IL-3), erythropoietin (EPO), and/or thrombopoietin (TPO)] and the optimal concentration and addition schedule of hormones (dexamethasone and estradiol) sustaining maximal erythroid amplification from adult blood mononuclear cells (MNC) using media with serum previously defined as human erythroid massive amplification culture (HEMAser). Adult MNC stimulated with SCF and IL-3 in combination with EPO generated a 6-12-fold increase in erythroid cells while TPO was ineffective. Dexamethasone and estradiol (both at 10-6 M) exerted partially overlapping but nonredundant functions. Dexamethasone was indispensable in the first 10 days of culture while estradiol was required from day 10 on. The growth factor and hormone combinations identified in HEMASser were then used to formulate a media composed of dialyzed pharmaceutical grade human albumin, human albumin-lipid liposomes, and iron-saturated recombinant human tranferrin (HEMAdef). HEMAdef sustained erythroid amplification as efficiently as HEMAser for cord blood MNC and 10-fold higher than HEMAser for adult blood MNC. In fact, the numbers of erythroblasts generated in HEMAdef by adult MNC were similar to those generated by cord blood MNC. In conclusion, this study identifies growth factors, hormone combinations, and human protein-based media that allow similar levels of ex vivo erythroid expansion from adult and cord blood MNC, paving the way to evaluate adult blood as a source of ex vivo-expanded erythroblasts for transfusion.

Key words: Erythroblasts; Human erythroid massive amplification (HEMA) culture; Growth factors; Human albumin; Transfusion

Address correspondence to Anna Rita Migliaccio, Tisch Cancer Institute, Mount Sinai School of Medicine, One Gustave L Levy Place, Box 1079, New York, NY 10029, USA. Tel: (212) 241-6974; Fax: (212) 876-5276; E-mail: annarita.migliaccio@mssm.edu

Cell Transplantation, Vol. 19, pp. 471-486, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368909X484707
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Analysis of In Vitro and In Vivo Characteristics of Human Embryonic Stem Cell-Derived Neural Precursors

Nataliya Kozubenko,1,2 Karolina Turnovcova,1,2 Miroslava Kapcalova,1,2 Olena Butenko,1 Miroslava Anderova,1,2 Vendula Rusnakova,3 Mikael Kubista,3,4 Ales Hampl,1,2,5 Pavla Jendelova,1,2 and Eva Sykova1,2

1Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Prague, Czech Republic
2Department of Neuroscience and Center for Cell Therapy and Tissue Repair, Charles University, Second Medical Faculty, Prague, Czech Republic
3Institute of Biotechnology, Academy of Sciences of the Czech Republic, Prague, Czech Republic
4TATAA Biocenter, Lundberg Laboratory, Goteborg, Sweden
5Faculty of Medicine, Masaryk University, Brno, Czech Republic

During the last decade, much progress has been made in developing protocols for the differentiation of human embryonic stem cells (hESCs) into a neural phenotype. The appropriate agent for cell therapy is neural precursors (NPs). Here, we demonstrate the derivation of highly enriched and expandable populations of proliferating NPs from the CCTL14 line of hESCs. These NPs could differentiate in vitro into functionally active neurons, as confirmed by immunohistochemical staining and electrophysiological analysis. Neural cells differentiated in vitro from hESCs exhibit broad cellular heterogeneity with respect to developmental stage and lineage specification. To analyze the population of the derived NPs, we used fluorescence-activated cell sorting (FACS) and characterized the expression of several pluripotent and neural markers, such as Nanog, SSEA-4, SSEA-1, TRA-1-60, CD24, CD133, CD56 (NCAM), b-III-tubulin, NF70, nestin, CD271 (NGFR), CD29, CD73, and CD105 during long-term propagation. The analyzed cells were used for transplantation into the injured rodent brain; the tumorigenicity of the transplanted cells was apparently eliminated following long-term culture. These results complete the characterization of the CCTL14 line of hESCs and provide a framework for developing cell selection strategies for neural cell-based therapies.

Key words: Human embryonic stem cells (hESCs); Neural differentiation; Fluorescent-activated cell sorting; RT-PCR; Transplantation

Address correspondence to Prof. Eva Sykova, Dr.Sc., Institute of Experimental Medicine, ASCR, Videnska 1083, 142 20 Prague 4, Czech Republic. Tel: +420 241 062 230; Fax: +420 241 062 782; E-mail: sykova@biomed.cas.cz

Cell Transplantation, Vol. 19, pp. 487-503, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368909X486057
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Targeted Disruption of Outer Limiting Membrane Junctional Proteins (Crb1 and ZO-1) Increases Integration of Transplanted Photoreceptor Precursors Into the Adult Wild-Type and Degenerating Retina

R. A. Pearson,1 A. C. Barber,1 E. L. West,1 R. E. MacLaren,2* Y. Duran,1 J. W. Bainbridge,1 J. C. Sowden,3 and R. R. Ali14

1Department of Genetics, University College London Institute of Ophthalmology, London, UK
2Vitreoretinal Service, Moorfields Eye Hospital, London, UK
3Developmental Biology Unit, University College London Institute of Child Health, London, UK
4Molecular Immunology Unit, University College London Institute of Child Health, London, UK

Diseases culminating in photoreceptor loss are a major cause of untreatable blindness. Transplantation of rod photoreceptors is feasible, provided donor cells are at an appropriate stage of development when transplanted. Nevertheless, the proportion of cells that integrate into the recipient outer nuclear layer (ONL) is low. The outer limiting membrane (OLM), formed by adherens junctions between Mu¨ller glia and photoreceptors, may impede transplanted cells from migrating into the recipient ONL. Adaptor proteins such as Crumbs homologue 1 (Crb1) and zona occludins (ZO-1) are essential for localization of the OLM adherens junctions. We investigated whether targeted disruption of these proteins enhances donor cell integration. Transplantation of rod precursors in wild-type mice achieved 949 ± 141 integrated cells. By contrast, integration is significantly higher when rod precursors are transplanted into Crb1rd8/rd8 mice, a model of retinitis pigmentosa and Lebers congenital amaurosis that lacks functional CRB1 protein and displays disruption of the OLM (7,819 ± 1,297; maximum 15,721 cells). We next used small interfering (si)RNA to transiently reduce the expression of ZO-1 and generate a reversible disruption of the OLM. ZO-1 knockdown resulted in similar, significantly improved, integration of transplanted cells in wild-type mice (7,037 ± 1,293; maximum 11,965 cells). Finally, as the OLM remains largely intact in many retinal disorders, we tested whether transient ZO-1 knockdown increased integration in a model of retinitis pigmentosa, the rho-/- mouse; donor cell integration was significantly increased from 313 ± 58 cells without treatment to 919 ± 198 cells after ZO-1 knockdown. This study shows that targeted disruption of OLM junctional proteins enhances integration in the wild-type and degenerating retina and may be a useful approach for developing photoreceptor transplantation strategies.

Key words: Stem cell; Migration; Transplantation; Degeneration; Müller glia

Address correspondence to Rachael A. Pearson or Robin R. Ali, Department of Genetics, University College London Institute of Ophthalmology, 11-43 Bath Street, London, EC1V 9EL, UK. Tel: +44 (0)20 76084022; Fax: +44 (0)20 7608 6863; E-mail: rachael.pearson@ucl.ac.uk or r.ali@ucl.ac.uk

*Current address: Nuffield Laboratory of Ophthalmology, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, UK.