|ognizant Communication Corporation|
The Regenerative Medicine Journal
VOLUME 18, NUMBER 12, 2009
Cell Transplantation, Vol. 18, pp. 1261-1279, 2009
0963-6897/09 $90.00 + 00
Copyright © 2009 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.
Current Status of Stem Cell Therapy for Liver Diseases
Bruno Solano de Freitas Souza,1 Renata Campos Nogueira,1 Sheilla Andrade de Oliveira,2 Luiz Antonio Rodrigues de Freitas,1,3 Luiz Guilherme Costa Lyra,3,4 Ricardo Ribeiro dos Santos,1,4 Andre Castro Lyra,3,4 and Milena Botelho Pereira Soares1,4
1Centro de Pesquisas Gonçalo Moniz, Fundação
Oswaldo Cruz, Salvador, Bahia, Brazil
2Centro de Pesquisas Aggeu Magalhães, Fundação Oswaldo Cruz, Recife, Pernambuco, Brazil
3Universidade Federal da Bahia, Salvador, Bahia, Brazil
4Hospital São Rafael, Salvador, Bahia, Brazil
Liver failure is one of the main causes of death worldwide and is a growing health problem. Since the discovery of stem cell populations capable of differentiating into specialized cell types, including hepatocytes, the possibility of their utilization in the regeneration of the damaged liver has been a focus of intense investigation. A variety of cell types were tested both in vitro and in vivo, but the definition of a more suitable cell preparation for therapeutic use in each type of liver lesions is yet to be determined. Here we review the protocols described for differentiation of stem cells into hepatocytes, the results of cell therapy in animal models of liver diseases, as well as the available data of the clinical trials in patients with advanced chronic liver disease.
Key words: Liver diseases; Liver regeneration; Stem cells; Hepatocytes
Address correspondence to Milena Botelho Pereira Soares, Centro de Pesquisas Gonçalo Moniz, FIOCRUZ, Rua Waldemar Falcão, 121, Candeal, Salvador, BA, 40296-710, Brazil. Tel: +55 71 3176 2260; Fax: +55 71 3176 2272; E-mail: firstname.lastname@example.org
E-Cadherin Protects Primary Hepatocyte Spheroids From Cell Death by a Caspase-Independent Mechanism
Jennifer L. Luebke-Wheeler,1 Geir Nedredal,1 Le Yee,1 Bruce P. Amiot,2 and Scott L. Nyberg1
1Department of Surgery, Division of Transplant Surgery, Mayo
Clinic, Rochester MN, USA
2Brami Biomedical, Inc., Minneapolis, MN, USA
Cultivation of primary hepatocytes as spheroids creates an efficient three-dimensional model system for hepatic studies in vitro and as a cell source for a spheroid reservoir bioartificial liver. The mechanism of spheroid formation is poorly understood, as is an explanation for why normal, anchorage-dependent hepatocytes remain viable and do not undergo detachment-induced apoptosis, known as anoikis, when placed in suspension spheroid culture. The purpose of this study was to investigate the role of E-cadherin, a calcium-dependent cell adhesion molecule, in the formation and maintenance of hepatocyte spheroids. Hepatocyte spheroids were formed by a novel rocker technique and cultured in suspension for up to 24 h. The dependence of spheroid formation on E-cadherin and calcium was established using an E-cadherin blocking antibody and a calcium chelator. We found that inhibiting E-cadherin prevented cell-cell attachment and spheroid formation, and, surprisingly, E-cadherin inhibition led to hepatocyte death through a caspase-independent mechanism. In conclusion, E-cadherin is required for hepatocyte spheroid formation and may be responsible for protecting hepatocytes from a novel form of caspase-independent cell death.
Key words: Hepatocyte spheroids; E-Cadherin; Anoikis; Caspase-independent cell death
Address correspondence to Dr. Scott Nyberg, Mayo Clinic, 200 1st Street SW, Rochester, MN 55905, USA. Tel: (507) 266-9949; Fax: (507) 266-2810; E-mail: email@example.com
Functional Impact of Targeted Closed-Chest Transplantation of Bone Marrow Cells in Rats With Acute Myocardial Ischemia/Reperfusion Injury
Alexander Ghanem,1* Agnieszka Ziomka,2* Benjamin Krausgrill,2 Kerstin Schenk,2 Clemens Troatz,1 Tomas Miszalski-Jamka,3 Georg Nickenig,1 Klaus Tiemann,4# and Jochen Müller-Ehmsen2#
1Department of Medicine/Cardiology, University of Bonn, Bonn,
2Laboratory of Muscle Research and Molecular Cardiology, Department of Internal Medicine III, University Hospital of Cologne, Köln, Germany
3Department of Cardiology, University of Krakow, Krakow, Poland
4Department of Cardiology and Angiology, University Hospital Münster, Münster, Germany
Intramyocardial transplantation of bone marrow-derived stem cells is a potential therapeutic option after myocardial infarction (MI). Intramyocardial administration is invasive but allows efficient and targeted stem cell delivery. Aims of this study were validation of minimal-invasive, echo-guided closed-chest cell transplantation (CTx) of mononuclear (MNC) or mesenchymal stem cells (MSC) and quantification of systolic left ventricular function and assessment of contractile reserve with high-resolution reconstructive 3D-echocardiography (r3D-echo) 3 weeks after CTx. Female Fischer344 rats received syngeneic male MNC, MSC, or medium after myocardial ischemia and reperfusion via echo-guided percutaneous injection (open-chest for control). Left ventricular systolic function was measured and dysfunctional myocardium was quantified with r3D-echo. For investigation of contractile reserve and myocardial viability r3D-echo was additionally conducted during low-dose dobutamine 3 weeks after CTx. Cell persistence after echo-guided CTx was quantified via real-time PCR; scar size was measured histologically. Echo-guided percutaneous CTx was feasible in all animals (n = 30) without periprocedural complications. After 3 weeks, 1.4 ± 1.1% of transplanted MNC and 1.9 ± 1.2% of MSC were detected. These numbers were comparable to those after openchest intramyocardial injection of MNC (0.8 ± 1.1%; n = 8, p = 0.3). In r3D-echo no functional benefit was associated with CTx after MI and reperfusion. All groups (MNC, MSC, and controls) revealed a significant decrease of dysfunctional myocardium and similar contractile reserve during inotropic stimulation.In conclusion, percutaneous echo-guided closed-chest CTx promises to be an effective and safe approach for CTx in small-animal research. However, intramyocardial CTx of MNC or MSC had no influence on systolic function and contractile reserve after reperfused MI.
Key words: Closed-chest transplantation; Reconstructive 3D-echocardiography; Myocardial ischemia/reperfusion; Acute myocardial infarction
Address correspondence to Priv.-Doz. Dr. med. Jochen Müller-Ehmsen, Laboratory of Muscle Research and Molecular Cardiology, Department of Internal Medicine III, University Hospital of Cologne, Kerpener Str. 62, 50937 Köln, Germany. Tel: +49-221-478-32396; Fax: +49-221-478-32397; E-mail: firstname.lastname@example.org
*Drs. Ghanem and Ziomka contributed equally to this work.
#Drs. Tiemann and Müller-Ehmsen contributed equally to this work.
Refractory Angina Cell Therapy (ReACT) Involving Autologous Bone Marrow Cells in Patients Without Left Ventricular Dysfunction: A Possible Role for Monocytes
Nelson Americo Hossne, Jr.,1 Adriana Luckow Invitti,2,3 Enio Buffolo,1 Silvia Azevedo,2 José Salvador Rodrigues de Oliveira,4 Noedir Groppo Stolf,5 L. Eduardo Cruz,2,3 and Paul R. Sanberg6
1Cardiovascular Surgery Division, Surgery Department, Paulista
School of Medicine, Federal University of São Paulo, São
2Cryopraxis Criobiologia Ltda, Rio de Janeiro, Brazil
3Cellpraxis Bioengenharia, Rio de Janeiro, Brazil
4Hematology Division, Paulista School of Medicine, Federal University of São Paulo, São Paulo, Brazil
5Heart Institute, College of Medicine, University of São Paulo, São Paulo, Brazil
6Center of Excellence for Aging and Brain Repair, Department of Neurosurgery and Brain Repair, College of Medicine, and Office of Research and Innovation, University of South Florida, Tampa, FL, USA
Autologous bone marrow mononuclear cell (BMMC) transplantation has emerged as a potential therapeutic option for refractory angina patients. Previous studies have shown conflicting myocardium reperfusion results. The present study evaluated safety and efficacy of CellPraxis Refractory Angina Cell Therapy Protocol (ReACT), in which a specific BMMC formulation was administered as the sole therapy for these patients. The phase I/IIa noncontrolled, open label, clinical trial, involved eight patients with refractory angina and viable ischemic myocardium, without left ventricular dysfunction and who were not suitable for conventional myocardial revascularization. ReACT is a surgical procedure involving a single series of multiple injections (40-90 injections, 0.2 ml each) into ischemic areas of the left ventricle. Primary endpoints were Canadian Cardiovascular Society Angina Classification (CCSAC) improvement at 18 months follow-up and myocardium ischemic area reduction (assessed by scintigraphic analysis) at 12 months follow-up, in correlation with a specific BMMC formulation. Almost all patients presented progressive improvement in angina classification beginning 3 months (p = 0.008) postprocedure, which was sustained at 18 months follow-up (p = 0.004), as well as objective myocardium ischemic area reduction at 12 months (decrease of 84.4%, p < 0.004). A positive correlation was found between monocyte concentration and CCSAC improvement (r = -0.759, p < 0.05). Improvement in CCSAC, followed by correlated reduction in scintigraphic myocardium ischemic area, strongly suggests neoangiogenesis as the main stem cell action mechanism. The significant correlation between number of monocytes and improvement strongly supports a cell-related effect of ReACT. ReACT appeared safe and effective.
Key words: Monocytes; Angina; Cell therapy; Bone marrow mononuclear cell transplantation; Myocardia; Ischemia
Address correspondence to Nelson Americo Hossne Jr., Canário St., 943-Ap. 123-Moema, São Paulo, São Paulo, Brazil 04521-004. Tel: +55-11-8166-5050; Fax: +55-11-5052-0386; E-mail: email@example.com
Increased Apelin Following Bone Marrow Mononuclear Cell Transplantation Contributes to the Improvement of Cardiac Function in Patients With Severe Heart Failure
Lian Ru Gao,1 Ru Yi Xu,2 Ning Kun Zhang,1 Yu Chen,1 Zhi Guo Wang,1 Zhi Ming Zhu,1 Yu Xing Fei,1 Yi Cao,1 Hong Tao Xu,1 and Ye Yang1
1Department of Cardiology, Navy General Hospital, Beijing,
2Department of Geriatric Cardiology, Chinese PLA General Hospital, Beijing, China
We previously reported that intracoronary implantation of bone marrow mononuclear cells (BMMC) into ischemic hearts improved cardiac function after myocardial infarction. However, the mechanisms have not been elucidated. The present study investigates whether apelin, a newly described inotropic peptide with important cardiovascular regulatory properties, contributes to the functional improvement in patients with severe heart failure after cell transplantation. Forty consecutive patients with severe heart failure secondary to myocardial infarction were assigned to the BMMC therapy group or the standard medication group according to each patient's decision on a signed consent document. In 20 patients intracoronary cell infusion was performed, and another 20 patients were matched to receive standard medication as therapeutic controls. An additional 20 healthy subjects were designated as normal controls. Clinical manifestations, echocardiograms, and biochemical assays were recorded. Plasma apelin and brain natriuretic protein (BNP) levels were determined by enzyme immunoassay. Baseline levels of plasma apelin were significantly lower in all heart failure patients compared to normal subjects. In patients who underwent cell transplantation, apelin increased significantly from 3 to 21 days after operation, followed by significant improvement in cardiac function. In parallel, BNP varied inversely with the increase of apelin. In patients receiving standard medical treatment, apelin remained at a lower level. Our findings indicated that increased apelin levels following cell therapy may act as a paracrine mediator produced from BMMCs and play an important role in the treatment of heart failure through autocrine and paracrine mechanisms.
Key words: Cell transplantation; Apelin; Heart failure; Paracrine
Address correspondence to Dr. Lian Ru Gao, Department of Cardiology, Navy General Hospital, 6 Fucheng Road, Beijing 100037, China. Tel: 011-86-10-88180197; Fax: 011-86-10-68780127; E-mail: firstname.lastname@example.org
Sequential Hepatogenic Transdifferentiation of Adipose Tissue-Derived Stem Cells: Relevance of Different Extracellular Signaling Molecules, Transcription Factors Involved, and Expression of New Key Marker Genes
A. Bonora-Centelles,1,2,3 R. Jover,1,3,4 V. Mirabet,5 A. Lahoz,1,3 F. Carbonell,5 J. V. Castell,1,3,4 and M. J. Gómez-Lechón1,2,3
1Unidad de Hepatología Experimental, Centro de Investigación,
Hospital La Fe, Valencia, Spain
2Unidad de Terapia Celular Hepática, Hospital La Fe, Valencia, Spain
3CIBERehd, FIS, Spain
4Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad de Valencia, Valencia, Spain
5Centro de Transfusión de la Comunidad Valenciana, Generalidad Valenciana, Valencia, Spain
Adipose tissue contains a mesenchymal stem cell (MSC) population known as adipose-derived stem cells (ASCs) capable of differentiating into different cell types. Our aim was to induce hepatic transdifferentiation of ASCs by sequential exposure to several combinations of cytokines, growth factors, and hormones. The most efficient hepatogenic protocol includes fibroblastic growth factors (FGF) 2 and 4 and epidermal growth factor (EGF) (step 1), hepatocyte growth factor (HGF), FGF2, FGF4, and nicotinamide (Nic) (step 2), and oncostatin M (OSM), dexamethasone (Dex), and insulin-tranferrin-selenium (step 3). This protocol activated transcription factors [GATA6, Hex, CCAAT/enhancer binding protein a and b (CEBPa and b), peroxisome proliferator-activated receptor-g, coactivator 1 a (PGC1a), and hepatocyte nuclear factor 4 a (HNF4a)], which promoted a characteristic hepatic phenotype, as assessed by new informative markers for the step-by-step hepatic transdifferentiation of hMSC [early markers: albumin (ALB), a-2-macroglobuline (a2M), complement protein C3 (C3), and selenoprotein P1 (SEPP1); late markers: cytochrome P450 3A4 (CYP3A4), apolipoprotein E (APOE), acyl-CoA synthetase long-chain family member 1 (ACSL1), and angiotensin II receptor, type 1 (AGTR1)]. The loss of adipose adult stem cell phenotype was detected by losing expression of Thy1 and inhibitor of DNA binding 3 (Id3). The reexpression of phosphoenolpyruvate corboxykinase (PEPCK), apolipoprotein C3 (APOCIII), aldolase B (ALDOB), and cytochrome P450 1A2 (CYP1A2) was achieved by transduction with a recombinant adenovirus for HNF4a and finally hepatic functionality was also assessed by analyzing specific biochemical markers. We conclude that ASCs could represent an alternative tool in clinical therapy for liver dysfunction and regenerative medicine.
Key words: Adipose tissue; Mesenchymal stem cell; Hepatocyte differentiation; Transcription factors
Address correspondence to M. J. Gómez-Lechón, Unidad de Hepatología Experimental, Centro de Investigación, Hospital La Fe, Avda de Campanar 21, 46009-Valencia, Spain. Tel: +34 96 1973048; Fax: +34 96 1973018; E-mail: email@example.com
Use of Transduced Adipose Tissue Stromal Cells as Biologic Minipumps to Deliver Levodopa for the Treatment of Neuropathic Pain: Possibilities and Limitations
Nuria Cobacho, Ana Belén Serrano, Maria José Casarejos, Mari Angeles Mena, and Carlos Luis Paíno
Servicio de Neurobiología-Investigación, Hospital Ramón y Cajal, 28034 Madrid, Spain
Subarachnoidal grafting of monoamine-producing cells has been used with success to treat chronic pain in animal models. In the search for a source of autologous transplantable cells, capable of delivering neuroactive substances to the cerebrospinal fluid (CSF) to treat pain, we have tested adipose tissue-derived stromal cells (ADSCs) transduced to produce levodopa. Intrathecally grafted ADSCs survive for long term adhered to spinal cord and nerve root meninges. Cultured ADSCs were retrovirally transduced with tyrosine hydroxylase (TH) and/or GTP cyclohydroxylase 1 (GCH1) genes and stably expressed them for at least 6 weeks in culture. Singly transduced cultures did not produce measurable levodopa but doubly transduced or a mixture of singly transduced ADSCs were able to efficiently synthesize and release levodopa. When 0.5-1 x 106 THand GCH1-expressing ADSCs were intrathecally grafted in rats, elevated levels of levodopa and dopamine metabolites were found in CSF at 3 days, although at lower concentrations than expected. Unexpectedly, no levodopa was measurable in CSF at 6 days. In a rat model of neuropathic pain, intrathecal grafting of doubly transduced cells did not produce antiallodynic effects at 2 or 6 days, even when histological analysis revealed the presence of weak TH-immunoreactive subarachnoidal cell clusters. These results suggested that doubly transduced cells could indeed function as biological minipumps to enhance the dopaminergic neurotransmission at the spinal cord level but transgenes were rapidly silenced after intrathecal grafting. Transgene silencing was mimicked in culture by serum deprivation for 3 days. Serum addition at this point recovered transgene expression in just 6 h, as did, to a smaller degree, dbcAMP or histone deacetylase inhibitors. Transgene expression silencing in serum deprivation conditions was prevented by 5´-terminal IRES sequences. The present study does not discard the use of transduced cells as a strategy to treat chronic pain but shows that controlling transgene silencing in implanted cells needs to be achieved first.
Key words: Levodopa; Neuropathic pain; Intrathecal grafting; Analgesia; Transgene silencing; Rats
Address correspondence to Carlos L. Paíno, Servicio de Neurobiología-Investigación, Hospital Ramón y Cajal, Carretera de Colmenar km 9, 28034 Madrid, Spain. Tel: +34 913368385; Fax: +34 913369016; E-mail: firstname.lastname@example.org
Functional Recovery After the Transplantation of Neurally Differentiated Mesenchymal Stem Cells Derived From Bone Barrow in a Rat Model of Spinal Cord Injury
Sung-Rae Cho,1 Yong Rae Kim,2 Hoi-Sung Kang,3 Sun Hee Yim,1 Chang-il Park,1 Yoo Hong Min,4 Bae Hwan Lee,5 Ji Cheol Shin,1 and Jong-Baeck Lim3
1Department & Research Institute of Rehabilitation Medicine,
Yonsei University College of Medicine, Seoul, Korea
2Department of Rehabilitation Medicine, Pochun Joongmoon University College of Medicine, Seoul, Korea
3Department of Laboratory Medicine, Yonsei University College of Medicine, Seoul, Korea
4Department of Hematology, Yonsei University College of Medicine, Seoul, Korea
5Department of Physiology, Brain Research Institute and Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul, Korea
This study was designed to investigate functional recovery after the transplantation of mesenchymal stem cells (MSCs) or neurally differentiated MSCs (NMSCs) derived from bone marrow in a rat model of spinal cord injury (SCI). Sprague-Dawley rats were subjected to incomplete SCI using an NYU impactor to create a free drop contusion at the T9 level. The SCI rats were then classified into three groups; MSCs, NMSCs, and phosphate-buffered saline (PBS)-treated groups. The cells or PBS were administrated 1 week after SCI. Basso-Beattie-Bresnahan (BBB) locomotor rating scores were measured at 1-week intervals for 9 weeks. Somatosensory evoked potentials (SSEPs) and motor evoked potentials (MEPs) were also recorded 8 weeks after transplantation. While transplantation of MSCs led to a clear tendency of motor recovery, NMSC-treated rats had significantly improved BBB scores and showed significantly shortened initial latency, N1 latency, and P1 latency of the SSEPs compared to PBS controls. In addition, 5-bromo-2-deoxyuridine (BrdU)-prelabeled MSCs costained for BrdU and glial fibrillary acidic protein (GFAP) or myelin basic protein (MBP) were found rostrally and caudally 5 mm each from the epicenter of the necrotic cavity 4 weeks after transplantation. These results suggest that neurally differentiated cells might be an effective therapeutic source for functional recovery after SCI.
Key words: Spinal cord injury; Transplantation; Mesenchymal stem cells; Neural differentiation; Functional recovery
Address correspondence to Jong-Baeck Lim, M.D., Ph.D., Department of Laboratory Medicine, Yonsei University College of Medicine, 134 Shinchon-dong, Seodaemun-gu, Seoul, Korea 120-752. Tel: +82 2 2228-2447; Fax: +82 2 363-2794; E-mail: email@example.com
Intravenous Administration of 99mTc-HMPAO-Labeled Human Mesenchymal Stem Cells After Stroke: In Vivo Imaging and Biodistribution
Olivier Detante,1,2,3 Anaïck Moisan,2,4,5 Julien Dimastromatteo,2,5,6 Marie-Jeanne Richard,2,7,8,9 Laurent Riou,2,5 Emmanuelle Grillon,1,2 Emmanuel Barbier,1,2 Marie-Dominique Desruet,1,4,5 Florence De Fraipont,2,8,9 Christoph Segebarth,1,2 Assia Jaillard,1,2,10 Marc Hommel,2,3,11 Catherine Ghezzi,2,5 and Chantal Remy1,2
1INSERM, U836, Grenoble Cedex 9, France
2Université Joseph Fourier, Grenoble Cedex 9, France
3Unité Neuro-Vasculaire, Neurologie, Centre Hospitalier Universitaire de Grenoble, Grenoble Cedex 9, France
4Service de Biophysique et Médecine Nucléaire, Centre Hospitalier Universitaire de Grenoble, Grenoble Cedex 9, France
1Radiopharmaceutiques Biocliniques, INSERM, U877, Faculté de Médecine, La Tronche, France
6ERAS Labo, Saint Nazaire-les-Eymes, France
7Unité Mixte de Thérapie Cellulaire et Tissulaire, Centre Hospitalier Universitaire de Grenoble, Grenoble Cedex 9, France
8Unité de Cancérologie biologique et biothérapie, Centre Hospitalier Universitaire de Grenoble, Grenoble Cedex 9, France
9INSERM, U823, Institut Albert Bonniot, La Tronche Cedex, France
10Neuroradiologie/IRM, Centre Hospitalier Universitaire de Grenoble, Grenoble Cedex 9, France
11INSERM U003, Centre d'Investigation Clinique, Centre Hospitalier Universitaire, Grenoble Cedex 9, France
Human mesenchymal stem cells (hMSC) are a promising source for cell therapy after stroke. To deliver these cells, an IV injection appears safer than a local graft. We aimed to assess the whole-body biodistribution of IV-injected 99mTc-HMPAO-labeled hMSC in normal rats (n = 9) and following a right middle cerebral artery occlusion (MCAo, n = 9). Whole-body nuclear imaging, isolated organ counting (at 2 and 20 h after injection) and histology were performed. A higher activity was observed in the right damaged hemisphere of the MCAo group [6.5 ± 0.9 x 10-3 % of injected dose (ID)/g] than in the control group (3.6 ± 1.2 x 10-3 %ID/g), 20 h after injection. In MCAo rats, right hemisphere activity was higher than that observed in the contralateral hemisphere at 2 h after injection (11.6 ± 2.8 vs. 9.8 ± 1.7 x 10-3 %ID/g). Following an initial hMSC lung accumulation, there was a decrease in pulmonary activity from 2 to 20 h after injection in both groups. The spleen was the only organ in which activity increased between 2 and 20 h. The presence of hMSC was documented in the spleen, liver, lung, and brain following histology. IV-injected hMSC are transiently trapped in the lungs, can be sequestered in the spleen, and are predominantly eliminated by kidneys. After 20 h, more hMSC are found in the ischemic lesion than into the undamaged cerebral tissue. IV delivery of hMSC could be the initial route for a clinical trial of tolerance.
Key words: Human mesenchymal stem cell (hMSC); Stroke; Brain ischemia; Cell transplantation; Biodistribution; Nuclear imaging
Address correspondence to Olivier Detante, Grenoble Institut des Neurosciences, BP 170, 38042 Grenoble Cedex 9, France. Tel: 00.33.(0)4.56.52.05.88; Fax: 00.33.(0)4.56.52.05.98; E-mail: ODetante@chu-grenoble.fr
TNF, Pig CD86, and VCAM-1 Identified as Potential Targets for Intervention in Xenotransplantation of Pig Chondrocytes
Roberta Sommaggio, Rafael Máñez, and Cristina Costa
Institut d'Investigació Biomèdica de Bellvitge (IDIBELL), 08907 L'Hospitalet de Llobregat, Barcelona, Spain
Xenotransplantation of genetically engineered porcine chondrocytes may benefit many patients who suffer cartilage defects. In this work, we sought to elucidate the molecular bases of the cellular response to xenogeneic cartilage. To this end, we isolated pig costal chondrocytes (PCC) and conducted a series of functional studies. First, we determined by flow cytometry the cell surface expression of multiple immunoregulatory proteins in resting conditions or after treatment with human TNF-a, IL-1a, or IL-1b, which did not induce apoptosis. TNF-a and to a lesser extent IL-1a led to a marked upregulation of SLA I, VCAM-1, and ICAM-1 on PCC. SLA II and E-selectin remained undetectable in all the conditions assayed. Notably, CD86 was constitutively expressed at moderate levels, whereas CD80 and CD40 were barely detected. To assess their function, we next studied the interaction of PCC with human monoblastic U937 and Jurkat T cells. U937 cells adhered to resting and in a greater proportion to cytokine-stimulated PCC. Consistent with its expression pattern, pig VCAM-1 was key, mediating the increased adhesion after cytokine stimulation. We also conducted coculture experiments with U937 and PCC and measured the release of pig and human cytokines. Stimulated PCC secreted IL-6 and IL-8, whereas U937 secreted IL-8 in response to PCC. Finally, coculture of PCC with Jurkat in the presence of PHA led to a marked Jurkat activation as determined by the increase in IL-2 secretion. This process was dramatically reduced by blocking pig CD86. In summary, CD86 and VCAM-1 on pig chondrocytes may be important triggers of the xenogeneic cellular immune response. These molecules together with TNF could be considered potential targets for intervention in order to develop xenogeneic therapies for cartilage repair.
Key words: Xenotransplantation; Chondrocytes; Adhesion molecules; Costimulation; Cytokines
Address correspondence to Cristina Costa, Institut d'Investigació Biomèdica de Bellvitge (IDIBELL),Hospital Duran i Reynals, Gran Via km. 2,7, 08907 L'Hospitalet de Llobregat, Barcelona, Spain. Tel: 34-93-2607355; Fax: 34-93-2607426; E-mail: firstname.lastname@example.org
Secreted Products From the Porcine Choroid Plexus Accelerate the Healing of Cutaneous Wounds
C. G. Thanos,1,2 D. F. Emerich,3 B. E. Bintz,2 M. Goddard,1,2 J. Mills,4 R. Jensen,5 M. Lombardi,5 S. Hall,6 and K. Boekelheide6
1Department of Molecular Pharmacology, Physiology, and Biotechnology,
Brown University, Providence, RI, USA
2CytoSolv, Inc., Providence, RI, USA
3Glocester Institute of Regenerative Medicine, N. Scituate, RI, USA
4Kineteks, LLC, Warwick, RI, USA
5Harvard University Medical School, Cambridge, MA, USA
6Department of Pathology and Laboratory Medicine, Brown University, Providence, RI, USA
The choroid plexus (CP), located at the blood-brain interface, is partially responsible for maintaining the composition of cerebrospinal fluid. Epithelial cell clusters isolated from the CP secrete numerous biologically active molecules, and are neuroprotective when transplanted in animal models of Huntington's disease and stroke. The transcriptomic and proteomic profiles of CP may extend beyond CNS applications due to an abundance of trophic and regenerative factors, including vascular endothelial growth factor, transforming growth factor-b, and others. We used microarray to investigate the transcriptome of porcine CP epithelium, and then assessed the in vitro and in vivo regenerative capability of secreted CP products in cell monolayers and full-thickness cutaneous wounds. In vitro, CP reduced the void area of fibroblast and keratinocyte scratch cultures by 70% and 33%, respectively, compared to empty capsule controls, which reduced the area by only 35% and 6%, respectively. In vivo, after 10 days of topical application, CP conditioned medium lyophilate dispersed in antibiotic ointment produced a twofold improvement in incision tensile strength compared to ointment containing lyophilized control medium, and an increase in the regeneration of epidermal appendages from roughly 50-150 features per wound. Together, these data identify the CP as a source of secreted regenerative molecules to accelerate and improve the healing of superficial wounds and potentially other similar indications.
Key words: Choroid plexus; Wound healing; Regeneration; Topical; Growth factors
Address correspondence to Christopher G. Thanos, Ph.D., CytoSolv, Inc., 117 Chapman St., Suite 107, Providence, RI 02905, USA. Tel: 401-573-2001; Fax: 401-861-9777; E-mail: email@example.com