ognizant Communication Corporation

The Regenerative Medicine Journal

VOLUME 19, NUMBER 5, 2010

Cell Transplantation, Vol. 19, pp. 509-523, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368909X485067
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Worldwide Survey of Published Procedures to Culture Human Embryonic Stem Cells

A. M. Fernandes,1* T. Meletti,1* R. Guimarães,2 M. P. Stelling,1 P. A. N. Marinho,1,3 A. S. Valladão,1 and S. K. Rehen1

1Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
2Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
3Programa de Engenharia Química/COPPE, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil

Since their derivation 11 years ago, human embryonic stem (hES) cells have become a powerful tool in both basic biomedical research and developmental biology. Their capacity for self-renewal and differentiation into any tissue type has also brought interest from fields such as cell therapy and drug screening. We conducted an extensive analysis of 750 papers (51% of the total published about hES cells between 1998 and 2008) to present a spectrum of hES cell research including culture protocols developed worldwide. This review may stimulate discussions about the importance of having unvarying methods to culture hES cells, in order to facilitate comparisons among data obtained by research groups elsewhere, especially concerning preclinical studies. Moreover, the description of the most widely used cell lines, reagents, and procedures adopted internationally will help newcomers on deciding the best strategies for starting their own studies. Finally, the results will contribute with the efforts of stem cell researchers on comparing the performance of different aspects related to hES cell culture methods.

Key words: Human embryonic stem (hES) cells; Cell culture protocols; Maintenance of hES cells; Peer review

Address correspondence to Stevens Rehen, Instituto de Ciências Biomédicas/UFRJ, Rua Rodolpho Paulo Rocco, 255-4º andar-CEDEPID-Hospital Universitário Clementino Fraga Fiho, Ilha do Fundão, Rio de Janeiro, RJ, Brazil. Tel: +55-21-25622931; E-mail: srehen@anato.ufrj.br

*These authors provided equal contribution.

Cell Transplantation, Vol. 19, pp. 525-536, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368910X491374
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

The Efficient Generation of Induced Pluripotent Stem (iPS) Cells From Adult Mouse Adipose Tissue-Derived and Neural Stem Cells

Pollyanna A. Tat, Huseyin Sumer, Karen L. Jones, Kyle Upton, and Paul J. Verma

Centre for Reproduction and Development, Monash Institute of Medical Research, Melbourne, Victoria, Australia

Ectopic expression of key reprogramming transgenes in somatic cells enables them to adopt the characteristics of pluripotency. Such cells have been termed induced pluripotent stem (iPS) cells and have revolutionized the field of somatic cell reprogramming, as the need for embryonic material is obviated. One of the issues facing both the clinical translation of iPS cell technology and the efficient derivation of iPS cell lines in the research laboratory is choosing the most appropriate somatic cell type for induction. In this study, we demonstrate the direct reprogramming of a defined population of neural stem cells (NSCs) derived from the subventricular zone (SVZ) and adipose tissue-derived cells (ADCs) from adult mice using retroviral transduction of the Yamanaka factors Oct4, Sox2, Klf4, and c-Myc, and compared the results obtained with a mouse embryonic fibroblast (mEF) control. We isolated mEFs, NSCs, and ADCs from transgenic mice, which possess a GFP transgene under control of the Oct4 promoter, and validated GFP expression as an indicator of reprogramming. While transduction efficiencies were not significantly different among the different cell types (mEFs 68.70 ± 2.62%, ADCs 70.61 ± 15.4%, NSCs, 68.72 ± 3%, p = 0.97), the number of GFP-positive colonies and hence the number of reprogramming events was significantly higher for both NSCs (13.50 ± 4.10 colonies, 0.13 ± 0.06%) and ADCs (118.20 ± 38.28 colonies, 1.14 ± 0.77%) when compared with the mEF control (3.17 ± 0.29 colonies, 0.03 ± 0.005%). ADCs were most amenable to reprogramming with an 8- and 38-fold greater reprogramming efficiency than NSCs and mEFs, respectively. Both NSC iPS and ADC iPS cells were demonstrated to express markers of pluripotency and could differentiate to the three germ layers, both in vitro and in vivo, to cells representative of the three germ lineages. Our findings confirm that ADCs are an ideal candidate as a readily accessible somatic cell type for high efficiency establishment of iPS cell lines.

Key words: Induced pluripotent stem (iPS) cells; Reprogramming; Adipose-derived cells; Neural stem cells

Address correspondence to Paul J. Verma, Monash Institute of Medical Research, 27-31 Wright Street, Clayton 3172, VIC, Australia. Tel: +61 3 9594 7000; Fax: +61 3 9594 7416; E-mail: Paul.Verma@med.monash.edu.au

Cell Transplantation, Vol. 19, pp. 537-548, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368910X498269
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Distribution, Differentiation, and Survival of Intravenously Administered Neural Stem Cells in a Rat Model of Amyotrophic Lateral Sclerosis

Dinko Mitrecic,1,2 Charles Nicaise,1 Srecko Gajovic,2 and Roland Pochet1

1Laboratory of Histology, Neuroanatomy and Neuropathology, Université Libre de Bruxelles, Brussels, Belgium
2Laboratory for Neurogenetics and Developmental Genetics, Croatian Institute for Brain Research, University of Zagreb, Zagreb, Croatia

The transplantation of neural stem cells (NSCs) is a challenging therapeutic strategy for the treatment of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). To provide insight into the potential of the intravenous delivery of NSCs, we evaluated the delivery of NSCs marked with green fluorescent protein to the central nervous system (CNS) via intravenous tail vein injections in an ALS model. The injected cell fates were followed 1, 3, and 7 days after transplantation. The highest efficiency of cell delivery to the CNS was found in symptomatic ALS (up to 13%), moderate in presymptomatic ALS (up to 6%), and the lowest in wild-type animals (up to 0.3%). NSCs injected into ALS animals preferentially colonized the motor cortex, hippocampus, and spinal cord, and their differentiation was characterized by a decrease of nestin expression and the appearance of MAP2-, GFAP-, O4-, and CD68-positive cells. Tumor necrosis factor (TNF) administration increased the CNS delivery of transplanted cells in wild-type and presymptomatic, but not ALS symptomatic animals. Moreover, a TNF-related increase in NSC differentiation and survival was detected. Apoptosis was detected as the main cause of the loss of transplanted cells and it was influenced by TNF. Although 3 days after TNF treatment cell death was accelerated, TNF slowed down apoptosis after 7 days. This study provides elementary facts about the process occurring after NSCs leave the blood stream and enter the nervous tissue affected by inflammation/degeneration, which should help facilitate the planning of future bench-to-bedside translational projects.'

Key words: Amyotrophic lateral sclerosis (ALS); Neural stem cells; Cell transplantation therapy; Tumor necrosis factor (TNF); Rat

Address correspondence to Dinko Mitrecic, Laboratory of Histology, Neuroanatomy and Neuropathology, 808 route de Lennik ; B-1070 Brussels, Belgium. Tel: 32 2 5556471; Fax: 32 2 5556285; E-mail: dominic@mef.hr

Cell Transplantation, Vol. 19, pp. 549-564, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368910X491383
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Transplant of GABAergic Precursors Restores Hippocampal Inhibitory Function in a Mouse Model of Seizure Susceptibility

I. Zipancic,1,2* M. E. Calcagnotto,1* M. Piquer-Gil,1 L. E. Mello,3 and M.  Álvarez-Dolado1

1Department of Cell Therapy and Regenerative Medicine, Andalusian Center for Molecular Biology and Regenerative Medicine (CABIMER), Seville, Spain
2Centro de Investigación Príncipe Felipe (CIPF), Valencia, Spain
3Department of Physiology, Universidade Federal de São Paulo (UNIFESP), São Paulo, Brazil

Defects in GABAergic function can cause epilepsy. In the last years, cell-based therapies have attempted to correct these defects with disparate success on animal models of epilepsy. Recently, we demonstrated that medial ganglionic eminence (MGE)-derived cells grafted into the neonatal normal brain migrate and differentiate into functional mature GABAergic interneurons. These cells are able to modulate the local level of GABA-mediated synaptic inhibition, which suggests their suitability for cell-based therapies. However, it is unclear whether they can integrate in the host circuitry and rescue the loss of inhibition in pathological conditions. Thus, as proof of principle, we grafted MGE-derived cells into a mouse model of seizure susceptibility caused by specific elimination of GABAergic interneuron subpopulations in the mouse hippocampus after injection of the neurotoxic saporin conjugated to substance P (SSP-Sap). This ablation was associated with significant decrease in inhibitory postsynaptic currents (IPSC) on CA1 pyramidal cells and increased seizure susceptibility induced by pentylenetetrazol (PTZ). Grafting of GFP+ MGE-derived cells in SSP-Sap-treated mice repopulates the hippocampal ablated zone with cells expressing molecular markers of mature interneurons. Interestingly, IPSC kinetics on CA1 pyramidal cells of ablated hippocampus significantly increased after transplantation, reaching levels similar to the normal mice. More importantly, this was associated with reduction in seizure severity and decrease in postseizure mortality induced by PTZ. Our data show that MGE-derived cells fulfill most of the requirements for an appropriate cell-based therapy, and indicate their suitability for neurological conditions where a modulation of synaptic inhibition is needed, such as epilepsy.

Key words: Cell therapy; GABA; Epilepsy; Interneuron; Medial ganglionic eminence (MGE); Saporin

Address correspondence to Manuel Álvarez-Dolado, Centro de Biología Molecular y Medicina Regenerativa (CABIMER), Av. Americo Vespucio s/n-41092 Seville, Spain. Tel: +34 954-467829; E-mail: manuel.alvarez@cabimer.es

*These authors provided equal contribution.

Cell Transplantation, Vol. 19, pp. 565-572, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368910X509077
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Optimizing Orthotopic Cell Transplantation in the Mouse Adrenal Gland

Cibele C. Cardoso,1,2 Stefan R. Bornstein,1 and Peter J. Hornsby2

1Department of Internal Medicine III, University Medical Center, University of Dresden, Dresden, Germany
2Department of Physiology, and Sam and Ann Barshop Institute for Longevity and Aging Studies, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA

Orthotopic cell transplantation models are important for a complete understanding of cell-cell interactions as well as tumor biology. In published studies of orthotopic transplantation in the mouse adrenal gland, human neuroblastoma cells have been shown to invade and occupy the adrenal, but in these investigations a true orthotopic model was not established. Here we show an orthotopic model in which transplanted cells are retained within the adrenal gland by formation of a fibrin clot. To establish an appropriate technique, we used brightly fluorescent 10 mm polystyrene microspheres injected into the mouse adrenal gland. In the absence of fibrinogen/thrombin for clot formation, much of the injected material was extruded to the outside of the gland. When the microspheres were injected in a fibrinogen/thrombin mixture, fluorescence was confined to the adrenal gland. As a model neoplastic cell originating from the cortex of the gland, we used a tumorigenic bovine adrenocortical cell line. When 3 x 105 cells were implanted orthotopically, by 16 days the cell mass had expanded and had invaded the cortex, whereas when 1 x 105 cells were used, tumor masses were much smaller. We therefore subsequently used 3 x 105 cells. When mice were sacrificed at different time points, we found that tumor growth resulting was progressive and that by 26 days cells there was extensive invasion into the cortex or almost complete replacement of the cortex with tumor cells. As a model neoplastic cell of neural crest origin, we used SK-N-AS human neuroblastoma cells. Orthotopic transplantation of 3 x 105 cells resulted in extensive invasion and destruction of the gland by 26 days. In summary, the present orthotopic model for intra-adrenal cell transplantation is valuable for investigation of growth of neoplastic cells of both cortical and medullary origin and should be useful for future studies of cortex-medulla interactions.

Key words: Adrenal gland; Orthotopic; Cortex; Medulla; Tumor; Neuroblastoma

Address correspondence to Peter J. Hornsby, Ph.D., Sam and Ann Barshop Institute for Longevity and Aging Studies, University of Texas Health Science Center, 15355 Lambda Drive, STCBM Bldg., San Antonio, TX 78245, USA. Tel: 210-562-5080; Fax: 281-582-3538; E-mail: Hornsby@uthscsa.edu

Cell Transplantation, Vol. 19, pp. 573-588, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368909X486048
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Bcl-2 Expression Enhances Myoblast Sheet Transplantation Therapy for Acute Myocardial Infarction

Katsukiyo Kitabayashi,1,2,3* Antti Siltanen,2* Tommi Pätilä,2,3 Muhammad Ali Asim Mahar,2 Ilkka Tikkanen,4 Jonna Koponen,5 Masamichi Ono,1 Yoshiki Sawa,1 Esko Kankuri,2,3 and Ari Harjula2,3

1Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Osaka, Japan
2Institute of Biomedicine, Pharmacology, University of Helsinki, Helsinki, Finland
3Department of Cardiothoracic Surgery, Helsinki University Meilahti Hospital, Helsinki, Finland
4Minerva Foundation Institute for Medical Research, Helsinki, Finland
5A.I. Virtanen Institute for Molecular Sciences, Department of Biotechnology and Molecular Medicine, University of Kuopio, Kuopio, Finland

Myoblast sheet transplantation is a promising novel treatment modality for heart failure after an ischemic insult. However, low supply of blood and nutrients may compromise sheet survival. The aim of this study was to investigate the effect of mitochondria-protective Bcl-2-modified myoblasts in cell sheet transplantation therapy. In the Bcl-2-expressing rat L6 myoblast sheets (L6-Bcl2), increased expression of myocyte markers and angiogenic mediators was evident compared to wild-type (L6-WT) sheets. The L6-Bcl2 sheets demonstrated significant resistance to apoptotic stimuli, and their differentiation capacity in vitro was increased. We evaluated the therapeutic effect of Bcl-2-modified myoblast sheets in a rat model of acute myocardial infarction (AMI). Sixty-four Wistar rats were divided into four groups. One group underwent AMI (n = 22), another AMI and L6-WT sheet transplantation (n = 17), and a third AMI and L6-Bcl2 sheet transplantation (n = 20). Five rats underwent a sham operation. Echocardiography was performed after 3, 10, and 28 days. Samples for histological analysis were collected at the end of the study. After AMI, the Bcl-2-expressing sheets survived longer on the infarcted myocardium, and significantly improved cardiac function. L6-Bcl2 sheet transplantation reduced myocardial fibrosis and increased vascular density in infarct and border areas. Moreover, the number of c-kit-positive and proliferating cells in the myocardium was increased in the L6-Bcl2 group. In conclusion, Bcl-2 prolongs survival of myoblast sheets, increases production of proangiogenic paracrine mediators, and enhances the therapeutic efficacy of cell sheet transplantation.

Key words: Apoptosis; Bcl-2; Cell sheet therapy; Myoblast; Myocardial infarction

Address correspondence to Antti Siltanen, Institute of Biomedicine, Pharmacology, Biomedicum, P.O. Box 63, FIN-00014 University of Helsinki, 00290 Helsinki, Finland. Tel: +358-9-191-25362; Fax: +358-9-191-25364; E-mail: antti.siltanen@helsinki.fi

*These authors provided equal contribution.

Cell Transplantation, Vol. 19, pp. 589-596, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368910X4863235
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Dystrophin Expression Following the Transplantation of Normal Muscle Precursor Cells Protects mdx Muscle From Contraction-Induced Damage

Joel Rousseau,1 Nicolas Dumont,2 Carl Lebel,1 Simon P. Quenneville,1 Claude H. Côté,2 Jérome Frenette,2 and Jacques P. Tremblay1

1Unité de recherche de recherche en Génétique Humaine, Centre de recherche de CHUL, CHUQ, Faculté de médecine, Université Laval, Sainte-Foy, Québec, Canada
2Unité CRML, Centre de recherche du CHUL, CHUQ, Faculté de médecine, Université Laval, Ste-Foy, QC, Canada

Duchenne muscular dystrophy (DMD) is the most frequent muscular dystrophy. Currently, there is no cure for the disease. The transplantation of muscle precursor cells (MPCs) is one of the possible treatments, because it can restore the expression of dystrophin in DMD muscles. In this study, we investigated the effects of myoblasts injected with cardiotoxin on the contractile properties and resistance to eccentric contractions of transplanted and nontransplanted muscles. We used the extensor digitorum longus (EDL) as a model for our study. We conclude that the sole presence of dystrophin in a high percentage of muscle fibers is not sufficient by itself to increase the absolute or the specific force in the EDL of transplanted mdx muscle. This lack of strength increase may be due to the extensive damage that was produced by the cardiotoxin, which was coinjected with the myoblasts. However, the dystrophin presence is sufficient to protect muscle from eccentric damage as indicated by the force drop results.

Key words: Duchenne muscular dystrophy; mdx; Muscle precursor cell transplantation; Muscle contraction

Address correspondence to Jacques P. Tremblay, Ph.D., Unité de recherche en Génétique Humaine, Centre de recherche du CHUL, 2705, Boulevard Laurier, Sainte-Foy, Québec, G1V 4G2, Canada. Tel: (418) 654-2186; Fax: (418) 654-2207; E-mail: Jacques-P.Tremblay@crchul.ulaval.ca

Cell Transplantation, Vol. 19, pp. 597-612, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368910X491806
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Adenosine A2A Agonist Administration Improves Islet Transplant Outcome: Evidence for the Role of Innate Immunity in Islet Graft Rejection

Preeti Chhabra,1 Kunjie Wang,2 Qiang Zeng,3 Mladen Jecmenica,1 Linda Langman,4 Joel Linden,5 Robert J. Ketchum,6,7 and Kenneth L. Brayman1

1Department of Surgery, University of Virginia, Charlottesville, VA, USA
2Department of Urology, West China Hospital, Sichuan University, Chengdu, China
3Department of Surgery, University of Pittsburgh, Pittsburgh, PA, USA
4Department of Cell Biology, University of Virginia, Charlottesville, VA, USA
5Departments of Medicine and Pharmacology, University of Virginia, Charlottesville, VA, USA
6Department of Structural Medicine, College of Osteopathic Medicine, Rocky Vista University, Parker, CO, USA
7Mountain West Research Foundation, Parker, CO, USA

Activation of adenosine A2A receptors inhibits inflammation in ischemia/reperfusion injury, and protects against cell damage at the injury site. Following transplantation 50% of islets die due to inflammation and apoptosis. This study investigated the effects of adenosine A2A receptor agonists (ATL146e and ATL313) on glucose-stimulated insulin secretion (GSIS) in vitro and transplanted murine syngeneic islet function in vivo. Compared to vehicle controls, ATL146e (100 nM) decreased insulin stimulation index [SI, (insulin)high glucose/(insulin)low glucose] (2.36 ± 0.22 vs. 3.75 ± 0.45; n = 9; p < 0.05). Coculture of islets with syngeneic leukocytes reduced SI (1.41 ± 0.17; p < 0.05), and this was restored by ATL treatment (2.57 ± 0.18; NS). Addition of a selective A2AAR antagonist abrogated ATL's protective effect, reducing SI (1.11 ± 0.42). ATL treatment of A2AAR+/+ islet/A2AAR-/- leukocyte cocultures failed to protect islet function (SI), implicating leukocytes as likely targets of A2AAR agonists. Diabetic recipient C57BL/6 mice (streptozotocin; 250 mg/kg, IP) received islet transplants to either the renal subcapsular or hepatic-intraportal site. Recipient mice receiving ATL therapy (ATL 146e or ATL313, 60 ng/kg/min, IP) achieved normoglycemia more rapidly than untreated recipients. Histological examination of grafts suggested reduced cellular necrosis, fibrosis, and lymphocyte infiltration in agonist-treated animals. Administration of adenosine A2A receptor agonists (ATL146e or ATL313) improves in vitro GSIS by an effect on leukocytes, and improves survival and functional engraftment of transplanted islets by inhibiting inflammatory islet damage in the peritransplant period, suggesting a potentially significant new strategy for reducing inflammatory islet loss in clinical transplantation.

Key words: Early islet loss; Adenosine receptor agonists in islet transplantation; Peritransplant islet graft survival; Innate immunity in early islet graft loss

Address correspondence to Kenneth L. Brayman, Department of Surgery, University of Virginia School of Medicine, P.O. Box 800709, Charlottesville, VA 22908-0709, USA. Tel: 434-924-9370; Fax: 434-924-5539; E-mail: klb9r@virginia.edu

Cell Transplantation, Vol. 19, pp. 613-628, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368910X486316
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Islet Isolation From Juvenile Porcine Pancreas After 24-h Hypothermic Machine Perfusion Preservation

Michael J. Taylor,1,3 Simona Baicu,1 Elizabeth Greene,1 Alma Vazquez,1 and John Brassil3

1Cell and Tissue Systems, N. Charleston, SC, USA
2Department of Surgery, Medical University of South Carolina, Charleston, SC, USA
3Organ Recovery Systems, Itasca, IL, USA

Pancreas procurement for islet isolation and transplantation is limited by concerns for the detrimental effects of postmortem ischemia. Hypothermic machine perfusion (HMP) preservation technology has had a major impact in circumventing ischemic injury in clinical kidney transplantation and is applied here to the preservation and procurement of viable islets after hypothermic perfusion preservation of porcine pancreata because pigs are now considered the donor species of choice for xenogeneic islet transplantation. Pancreases were surgically removed from young (<6 months) domestic Yorkshire pigs (25-32 kg), either before or after 30 min of warm ischemia time (WIT), and cannulated for perfusion. Each pancreas was assigned to one of six preservation treatment groups: fresh controls-processed immediately (cold ischemia <1 h) (G1, n = 7); static cold storage-flushed with cold UW-Viaspan and stored in UW-Viaspan at 2-4ºC for 24 h with no prior WIT (G2, n = 9); HMP perfused on a LifePort® machine at 4-6ºC and low pressure (10 mmHg) for 24 h with either KPS1 solution (G3, n = 7) or Unisol-UHK (G4, n = 7). Additional treatment groups to evaluate the effects of prior warm ischemia examined islet isolation after 30 min WIT in situ without (G5, n = 6) or with subsequent 24-h HMP with KPS1 (G6, n = 7). The pancreas was intraductally distended with Liberase PI enzyme and normothermically digested. The isolated islets were purified by a continuous density-gradient centrifugation. Perfusion-induced glandular edema was G3 = 138 ± 19%, G4 = 160 ± 16%, and G6 = 127 ± 22%. Islet yield (IEQ/g of pancreas) varied between the groups: G1 = 1,425 ± 610, G2 = 1,002 ± 262, G3 = 2,242 ± 449 (p < 0.05 vs. G2), G4 = 1,901 ± 420 (p < 0.05 vs. G2), G5 = 1,756 ± 329, and G6 = 1,396 ± 243. Islet stimulation indices were equivalent between the groups and similar to controls (G1). Insulin content (ng/IE) was different between the treatment groups with the highest insulin content in islets harvested from HMP pancreata. Dithizone staining for islets consistently showed more uniform digestion of the perfused organs, with greater separation of the tissue, less entrapped islets, and higher islet yield and purity. The salutary effects of HMP for 24 h were also manifest after 30-min prior warm ischemia. We conclude that 24 h of HMP is well tolerated, leading to moderate edema but no loss of function of the harvested islets. The edema appears to aid in enzymatic digestion, producing a greater yield and purity of islets compared with pancreas subjected to 24 h of static cold storage.

Key words: Hypothermic perfusion preservation; Cold storage; Pancreas preservation; Porcine pancreas preservation; Juvenile porcine pancreas; Porcine islets

Address correspondence to Michael J. Taylor, Ph.D., VP Research and Development, Cell and Tissue Systems, Inc., 2231 Technical Parkway, Suite A, N. Charleston, SC 29406, USA. Tel: (843) 722-6756; Fax: (843) 722-6657; E-mail: mtaylor@celltissuesystems.com

Cell Transplantation, Vol. 19, pp. 629-638, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368909X485058
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Monitoring of Intraportal Liver Cell Application in Children

Jochen Meyburg,1 Friederike Hoerster,1 Jan Schmidt,2 Johannes Poeschl,3 Georg F. Hoffmann,1 and Jens-Peter Schenk4

1Department of General Pediatrics, University Children's Hospital, Heidelberg, Germany
2Department of Visceral and Transplantation Surgery, University Hospital, Heidelberg, Germany
3Department of Neonatology, University Children's Hospital, Heidelberg, Germany
4Department of Diagnostic and Interventional Radiology, Division of Pediatric Radiology, University Hospital, Heidelberg, Germany

Despite recent advances and promising results in children, liver cell transplantation (LCT) should still be regarded as an experimental therapy. Several substantial complications are known from animal studies and individual patients. However, safety data on liver cell infusion in children are scarce. We used LCT in four children of different ages (3 weeks to 11 years, 3-40 kg) and underlying diseases [acute liver failure (n = 1), urea cycle disorders (n = 2), and Crigler-Najjar syndrome (n = 1)]. Vital parameters, portal vein flow (PVF), portal vein pressure (PVP), and liver enzymes were measured every 5 min during cell application and hourly thereafter between applications. An application protocol with discontinuation rules depending on changes in PVF and PVP was developed and successfully applied. Application was feasible in all children despite the catastrophic overall condition of the patient with acute liver failure. No application-related changes in vital parameters were found, and none of the children experienced clinical signs of portal vein thrombosis, pulmonary embolism, or anaphylactic reactions. Time courses for changes in PVF, PVP, and liver enzymes were obtained. Thorough monitoring of portal vein pressure and duplex sonography according to a defined protocol is likely to increase safety of cell application in pediatric LCT.

Key words: Hepatocyte transplantation; Urea cycle disorders; Acute liver failure; Portal vein pressure (PVP); Portal vein flow (PVF); Ornithine transcarbamylase deficiency; Carbamoylphosphate synthase deficiency; Citrullinemia

Address correspondence to Jochen Meyburg, M.D., University Children's Hospital, Im Neuenheimer Feld 430, D-69120 Heidelberg, Germany. Tel: +49 6221 5638428; Fax: +49 6221 565626; E-mail: jochen.meyburg@med.uni-heidelberg.de

Cell Transplantation, Vol. 19, pp. 639-643, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368910X491798
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Brief Communication
Transplantation of Cultured Autologous Melanocytes: Hope or Danger?

Rafal Czajkowski,1 Marta Pokrywczynska,1 Waldemar Placek,2 Barbara Zegarska,3 Tadeusz Tadrowski,2 and Tomasz Drewa1

1Department of Tissue Engineering, Nicolaus Copernicus University in Torun, Ludwik Rydygier Medical College in Bydgoszcz, Bydgoszcz, Poland
2Department of Dermatology, Nicolaus Copernicus University in Torun, Ludwik Rydygier Medical College in Bydgoszcz, Bydgoszcz, Poland
3Department of Cosmetology, Nicolaus Copernicus University in Torun, Ludwik Rydygier Medical College in Bydgoszcz, Bydgoszcz, Poland

Cultured human melanocytes are increasingly being used in the treatment of vitiligo. The growth media contain various types of mitogenic factors, both recombinant human (e.g., rhbFGF and rhSCF) and synthetic (e.g., TPA). High concentrations of mitogenic factors accelerate the cell cycle, and consequently may increase the risk of carcinogenesis of transplanted cells. Mutations of genes of the RAS/RAF/MEK/ERK signaling pathway are very often found in the early stages of the development of melanoma. TPA is considered to be an oncogenic factor, but so far there is no evidence to show that it is responsible for damage to the genetic material of cultured melanocytes. The aim of our study was to assess the risk of the development of mutations in selected genes of the RAS/RAF/MEK/ERK signaling pathway during the culturing of melanocytes in various growth media. Based on the results obtained, it can be concluded that TPA and high concentrations of other growth factors intensify the proliferation of melanocytes, without the risk of damage to the HRAS (exon 1 and 2), KRAS (exon 1 and 2), NRAS (exon 1 and 2), and BRAF (exon 11 and 15) genes. In order to assess the total safety of the transplantation of cultured melanocytes, it is necessary to carry out further studies on other signaling pathways as well as carry out biological tests on an animal model.

Key words: Melanoma; Melanomagenesis; Melanocyte culture; RAS/RAF/MEK/ERK pathway; Vitiligo

Address correspondence to Marta Pokrywczynska, M.Sc., Department of Tissue Engineering, Nicolaus Copernicus University in Torun, Ludwik Rydygier Medical College in Bydgoszcz, Karlowicza 24 street, 85-092 Bydgoszcz, Poland. Tel: 0048 52 585 37 37; Fax: 0048 52 585 37 42; Email: marthapok@interia.pl