|ognizant Communication Corporation|
The Regenerative Medicine Journal
VOLUME 17, NUMBER 7, 2008
Cell Transplantation, Vol. 17, pp. 723-734, 2008
0963-6897/08 $90.00 + 00
Copyright © 2008 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.
Antisense RNA Sequences Modulating the Ataxin-1 Message: Molecular Model of Gene Therapy for Spinocerebellar Ataxia Type 1, a Dominant-Acting Unstable Trinucleotide Repeat Disease
Youxin Gao,1 Tao Zu,1 Walter C. Low,2 Harry T. Orr,1,3 and R. Scott McIvor1,3
1Institute of Human Genetics, Department of Genetics, Cell
Biology and Development, University of Minnesota, Minneapolis, MN, USA
2Department of Neurosurgery, University of Minnesota, Minneapolis, MN, USA
3Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN, USA
Spinocerebellar ataxia type 1 (SCA1) is a dominant inherited disease caused by expanded trinucleotide repeats resulting in an increased polyglutamine tract in the gene product. As a potential therapeutic approach for SCA1, we tested antisense RNAs targeting two regions of the ataxin-1 message. Single-stranded regions around the translational initiation site and the intron 8 splice donor site of the ataxin-1 message were identified by computer-assisted RNA secondary structure prediction. Plasmids were generated to contain a 254-bp antisense sequence spanning the translation initiation site (pLasBDini) or a 317-bp sequence spanning the intron 8 splice donor site (pLasBDei) of the ataxin-1 message. These plasmids were transfected into Chinese hamster ovary cells engineered to express either expanded or unexpanded ataxin-1 message and protein. Reduced levels of mutant ataxin-1 message (82 CAG repeats), wild-type ataxin-1 message (30 CAG repeats), and ataxin-1 protein were observed by Northern and Western blot analyses in pLasBDini-transfected clones. pLasBDei-transfected 293 cells exhibited a shift in ataxin-1 message to a size several kilobases longer than that of the natural message. Reverse transcriptase/polymerase chain reaction assays demonstrated the retention of message spanning the intron 8 splice acceptor and the inability to amplify sequences between exons 8 and 9, implying that normal splicing of intron 8 had been interrupted. We conclude that antisense RNAs were effective in reducing or modifying ataxin-1 messages in transfected cells, and may be an effective genetic strategy for therapy of SCA1 and similar dominant-acting neurological disorders.
Key words: Cerebellum; Ataxia; Spinocerebellar ataxia type 1; Gene therapy
Address correspondence to R. Scott McIvor, Ph.D., Institute of Human Genetics, MMC 206, 420 Delaware St. S.E., University of Minnesota, Minneapolis, MN 55455, USA. Tel: (612) 626-1497; Fax: (612) 626-1435; E-mail: email@example.com
Adult Spinal Cord Stem/Progenitor Cells Transplanted as Neurospheres Preferentially Differentiate Into Oligodendrocytes in the Adult Rat Spinal Cord
Andrea J. Mothe, Iris Kulbatski, Ann Parr, Michael Mohareb, and Charles H. Tator
Toronto Western Research Institute and Krembil Neuroscience Centre, University of Toronto, Toronto, ON, Canada
Neural stem/progenitor cells (NSPCs) capable of generating new neurons and glia reside in the adult mammalian spinal cord. Transplantation of NSPCs has therapeutic potential for spinal cord injury, although there is limited information on the ability of these cells to survive and differentiate in vivo. Neurospheres cultured from the periventricular region of the adult spinal cord contain NSPCs that are self-renewing and multipotent. We examined the survival, proliferation, migration, and differentiation of adult spinal cord NSPCs generated from green fluorescent protein (GFP) transgenic rats and transplanted into the intact spinal cord. The grafted GFP-expressing cells survived for at least 6 weeks in vivo and migrated from the injection site along the rostro-caudal axis of the spinal cord. Transplanted cells transiently proliferated following transplantation and approximately 17% of the GFP-positive cells were apoptotic at 1 day. Also, better survival was seen with NSPCs transplanted as neurospheres in comparison to NSPCs transplanted as dissociated cells. By 1 week posttransplantation, grafted cells primarily expressed an oligodendrocytic phenotype and only 2% differentiated into astrocytes. Approximately 75% versus 38% of the grafted cells differentiated into oligodendrocytes after transplantation into spinal white versus gray matter, respectively. This is the first report to examine the time course of cell survival, proliferation, apoptosis, and phenotypic differentiation of transplanted NSPSs in the spinal cord. This is also the first report to examine the differences between transplanted NSPCs grafted as neurospheres or dissociated cells, and to compare the differentiation potential after transplantation into spinal cord white versus gray matter.
Key words: Adult neural stem/progenitor cells; Neurospheres; Survival; Proliferation; Apoptosis; Differentiation; Oligodendrocytes
Address correspondence to Andrea Mothe, Toronto Western Research Institute, McL 12-423, 399 Bathurst Street, Toronto, ON, M5T 2S8, Canada. Tel: 416-603-5032; E-mail: firstname.lastname@example.org
Lesion-Induced Increase in Survival and Migration of Human Neural Progenitor Cells Releasing GDNF
Soshana Behrstock,*# Allison D. Ebert,* Sandra Klein, Melanie Schmitt, Jeannette M. Moore, and Clive N. Svendsen
The Waisman Center, University of Wisconsin Madison, Madison, WI, USA
The use of human neural progenitor cells (hNPC) has been proposed to provide neuronal replacement or astrocytes delivering growth factors for brain disorders such as Parkinson's and Huntington's disease. Success in such studies likely requires migration from the site of transplantation and integration into host tissue in the face of ongoing damage. In the current study, hNPC modified to release glial cell line-derived neurotrophic factor (hNPCGDNF) were transplanted into either intact or lesioned animals. GDNF release itself had no effect on the survival, migration, or differentiation of the cells. The most robust migration and survival was found using a direct lesion of striatum (Huntington's model) with indirect lesions of the dopamine system (Parkinson's model) or intact animals showing successively less migration and survival. No lesion affected differentiation patterns. We conclude that the type of brain injury dictates migration and integration of hNPC, which has important consequences when considering transplantation of these cells as a therapy for neurodegenerative diseases.
Key words: Parkinson's disease; Huntington's disease; Striatum; Stem cell transplants
*These two authors contributed equally to this work.
#Current address: Promega Corporation, 2800 Woods Hollow Lane, Madison, WI, USA.
Address correspondence to Clive N. Svendsen, T611 Waisman Center, University of Wisconsin Madison, 1500 Highland Avenue, Madison, WI 53705-2280, USA. Tel: (608) 265-8668; Fax: (608) 263-5267; E-mail: email@example.com
Ascorbic Acid Increases the Number of Dopamine Neurons In Vitro and in Transplants to the 6-OHDA-Lesioned Rat Brain
V. Bagga,1 S. B. Dunnett,1 and R. A. Fricker-Gates2
1Brain Repair Group, School of Biosciences, Cardiff University,
Cardiff, Wales, UK
2Schools of Medicine and Life Sciences, and Institute for Science and Technology in Medicine, Keele University, Keele, Staffordshire, UK
The inadequate survival of dopamine neurons following intracerebral transplantation is in part attributed to the generation of reactive oxygen species and subsequent oxidative stress. To address this, we investigated whether the antioxidant ascorbic acid (vitamin C) had any effect on the yields of dopamine neurons derived from E14 rat ventral mesencephalic cells in vitro and in grafts. Following in vitro differentiation in medium containing ascorbic acid at concentrations ranging from 20 to 100 mM, significantly more neurons were immunopositive for the marker of mesencephalic dopamine neurons, tyrosine hydroxylase (TH), when compared to standard differentiation conditions containing no ascorbic acid. Mesencephalic cell suspensions supplemented with 100 mM ascorbic acid were also transplanted into unilateral 6-OHDA-lesioned rats and behavioral rotation was assessed at 2, 4, and 6 weeks posttransplantation. Grafts pretreated with ascorbic acid contained significantly more surviving dopamine neurons compared to nontreated grafts. However, no significant difference in rotation score was observed, with both groups showing a reversal and overcompensation of rotational bias. In addition, no evidence of neurogenesis of nigral dopamine neurons was observed in transplant groups. While the increased number of dopamine neurons observed in our study following ascorbic acid treatment may reflect a selective survival effect, our in vitro results suggest that ascorbic acid may act to increase the number dopamine neurons, both in culture and following transplantation, by stimulating dopaminergic differentiation of neural precursors from the fetal ventral mesencephalon.
Key words: Ascorbic acid; Dopaminergic neurons; Neuronal survival; Parkinson's disease; Transplantation; Oxidative stress
Address correspondence to R. A. Fricker-Gates, Schools of Medicine and Life Sciences, Huxley Building, Keele University, Keele, Staffordshire. ST5 5BG, UK. Tel: (0)1782 733874; Fax: (0)1782 733516; E-mail: firstname.lastname@example.org
Preliminary Survival Studies on Autologous Cultured Skin Fibroblasts Transplantation by Injection
Yuming Zhao,1* Jiaqi Wang,2* Xiaoqing Yan,1 Dan Li,1 and Jun Xu1
1Department of the Plastic and Reconstructive Surgery, Clinical
Division of Surgery, Chinese PLA (People's Liberation Army) General Hospital,
2Department of the Aesthetic & Plastic Surgery on the Face and Neck, The Plastic Surgery Hospital, Beijing, China
In the correction of aesthetic impairments on the face, dermal, and superficial subcutaneous defects, adequately safe implant material is required. Cultured autologous skin fibroblasts, as a protein repair system, create a living injectable system that has been utilized effectively to treat rhytids, depressed scars, subcutaneous atrophy, acne irregularities, and laser wounds. To evaluate the new method, we have investigated the survival and collagen secretion of autologous transplanted fibroblasts. In this study, rabbit fibroblasts were cultured and expanded. Cells (8 × 107/ml) were injected into the superficial and deep dermal junction of the dorsal ears. Two rabbits were injected independently with labeled [3H]TdR fibroblasts; similarly, eight rabbits were given unlabeled transplanted cells in the right ear and vehicle in the left. Each site was injected three times with the same amount of cells every 2 weeks. The grafts were evaluated for 5 months. After explantation, the samples were collected from the injected sites and stained with autoradiography, H&E, and sirius red, respectively. According to the histological observations, the [3H]TdR-labeled cells survived and large amounts of embryo fibroblasts were found in the experimental subgroup of the labeled cell group. The depth of dermis was significantly different between the experimental subgroup (701.3 ± 31.5 mm) and the control subgroup (638.3 ± 23.9 mm) of the unlabeled group (p < 0.01). There was also a significant difference of collagen III between the experimental subgroup (2.63 ± 1.41 cm2) and the control subgroup (1.05 ± 0.90 cm2) (p < 0.05). There was no significant difference of collagen I between the experimental subgroup (56.25 ± 14.41 cm2) and the control subgroup (55.41 ± 16.59 cm2) (p > 0.05). The results obtained demonstrate that the distinction of the depth of dermis should be interpreted by the increase of collagen III, instead of collagen I, which is produced by the transplanted fibroblasts. The investigation indicated that transplanted autologous skin fibroblasts could provide a potential and effective approach to treat minor facial tissue deficiencies.
Key words: Fibroblasts; [3H]TdR; Autoradiography; Cell transplantation; Collagen; Implant material
*These two authors contributed equally to this report.
Address correspondence to Jun Xu, M.D., Department of the Plastic and Reconstructive Surgery, Clinical Division of Surgery, Chinese PLA (People's Liberation Army) General Hospital, 28 Fuxing Road, Beijing, 100853 China. Tel: +86-10-66875550; Fax: +86-10-66938150; E-mail: email@example.com
The Effect of Isolation Methods and the Use of Different Enzymes on Islet Yield and In Vivo Function
Omaima M. Sabek,1 Patricia Cowan,2 Daniel W. Fraga,1 and A. Osama Gaber1
1Department of Surgery, The Methodist Hospital, Houston,
2Department of Acute & Chronic Care, University of Tennessee, Memphis, TN, USA
The ability to isolate high-yield pure and viable islets from human cadaver pancreas donors is dependent on donor factor as well as isolation factors. The aim of this study was to examine factors influencing islets recovery and in vivo function with an emphasis on donor and isolation methods as well as to compare the effectiveness of Liberase, widely used in clinical islet isolation, with Serva for the isolation of pure functional islets. The results of 123 islet isolations using Liberase for digestion were compared with those of 113 isolations with Serva. Islet equivalents per gram of tissue were similar between Liberase and Serva (3620 ± 1858 vs. 4132 ± 2104, p < 0.2) as well as the percent purity (75 ± 16 vs. 74 ± 15, p < 0.9). In vivo function of islets from 71 isolations (Liberase = 45, Serva = 26) were further tested by transplantation into NOD-SCID mice following short-term culture (<6 days, n = 71). Our data show that both Liberase- and Serva-isolated islets showed similar function results following short-term culture. These data demonstrate that there is no difference in islet yield, purity, and function between the two enzymes. However, when these 71 isolations were analyzed for in vivo function with emphasis on donor factors, cold ischemia time (12.0 ± 5.3 vs. 15.0 ± 5.7, p < 0.04), islet integrity (1.6 ± 0.7 vs. 1.3 ± 0.5, p < 0.05), and female gender were the only factors that correlated with in vivo function. We also compared the mechanical-shaking method for islets isolation with hand-shaking methods. Our results show that although there is no different in islet yield, purity, and integrity between different enzymes using the same method, hand-shaking method yields more islets with better integrity than mechanical-shaking method.
Key words: Islets; Yield; Viability; Enzyme; Isolation factor
Address correspondence to Omaima M. Sabek, Ph.D., Department of Surgery, The Methodist Hospital, 6550 Fannin Street, Smith Tower 1661, Houston, TX 77030, USA. Tel: 713-441-6309; Fax: 713-790-3085; E-mail: OMSabek@tmhs.org
Protection of Human Pancreatic Islets Using a Lentiviral Vector Expressing Two Genes: cFLIP and GFP
Elizabeth S. Fenjves,1 M. Sofia Ochoa,1 Sirlene Cechin,1 Carlota Gay-Rabinstein,1 Ingrid Pérez-Alvarez,1 Hirohito Ichii,1 Armando Mendez,1 Camillo Ricordi,1 and Michael A. Curran2
1Diabetes Research Institute, University of Miami School
of Medicine, Miami, FL, USA
2Memorial Sloan Kettering Cancer Center, New York, NY, USA
Pancreatic islet transplantation can provide insulin independence to diabetic patients. However, apoptosis of islets often leads to early graft failure. Genetic engineering with protective gene(s) can improve the viability of these cells. Here we show successful transduction of human islets with a feline immunodeficiency virus (FIV) vector expressing both a cytoprotective (cFLIP) gene and the green fluorescent protein (GFP). Despite using low virus titers to maximize safety, transduced islets expressed both genes, resulting in improved b-cell metabolic activity and viability. Although only ~10% of total islet cells were transduced, the significant viability advantages suggest a "barrier" effect in which protecting the periphery of the islet shields the core. These results provide the first demonstration that a lentiviral vector can express two genes in islets. Furthermore, the engineered islets are resistant to a variety of apoptotic stimuli, suggesting the potential of this approach in enhancing the viability of transplanted cells.
Key words: Diabetes; Pancreatic islet transplantation; Feline immunodeficiency virus (FIV); Gene therapy; cFLIP
Address correspondence to Michael A. Curran, Memorial Sloan Kettering Cancer Center, 415 East 68th Street, ZHC 1545, Box 470, New York, NY 10021, USA. Tel: 646-888-2348; Fax: 646-422-0470; E-mail: firstname.lastname@example.org
Rescue of Fertility in Homozygous Mice for the Urokinase Plasminogen Activator Transgene by the Transplantation of Mouse Hepatocytes
Nicolas M. Brezillon,1,2,3 Lucie DaSilva,1,2,3 David L'Hôte,4,5 Florence Bernex,6,7 Julie Piquet,2 Nadine Binart,1 Serban Morosan,8,9 and Dina Kremsdorf1,2,3
1INSERM, U845, 75015 Paris, France
2Université Paris Descartes, facultéé de médecine René Descartes, CHU Necker, 75015 Paris, France
3Institut Pasteur, Département de Virologie, 75015 Paris, France
4INSERM U567/CNRS UMR8104, Université Paris Descartes, faculté de médecine René Descartes, CHU Cochin, 75014 Paris, France
5INRA, UMR 1061, Université de Limoges, 87000 Limoges, France
6INRA, UMR 955, 94704 Maison-Alfort, France
7Ecole Nationale Vétérinaire d'Alfort, Service d'Anatomopathologie, 94704 Maisons-Alfort, France
8Université Pierre et Marie Curie, Centre d'expérimentation fonctionnelle, 75005 Paris, France
9Facultatea de Medicina Veterinara, 700490 Iasi, Romania
Development of the urokinase plasminogen activator/SCID (uPA/SCID) transgenic mouse model has opened new perspectives for the study of different biological mechanisms such as liver regeneration, stem cell differentiation, and human hepatic pathogens. We observed that homozygous uPA/SCID mice (uPA+/+/SCID) had a small offspring, indicating a fertility defect. The goal of this study was thus to rescue the fertility of homozygous uPA mice. A deregulation of ovarian function with an absence of corpus luteum was observed in female uPA+/+/SCID mice. In male uPA+/+/SCID mice, a decrease of the weight of the testes, epididymis, seminal vesicle, and prostate was measured. This was associated with an absence of seminal and prostatic secretions and a reduction in testicular sperm production. We hypothesized that the infertility of mice was the consequence of uPA-induced liver injury. Thus, in order to rescue liver function, hepatocytes from mice negative for the uPA transgene were transplanted into uPA+/+/SCID mice. Thirty days after cell transplantation, the livers of transplanted uPA+/+/SCID mice were totally repopulated and presented a normal morphology. Furthermore, transplantation restored normal body weight, life span, and reproductive organ function. In conclusion, we demonstrated that the transplantation of uPA+/+/SCID mice with healthy hepatocytes was sufficient to rescue the reproductive capacity of female and male uPA homozygous animals, highlighting the importance of normal liver function to reproductive capability.
Key words: Hepatocyte transplantation; Fertility; uPA/SCID mice; Cell therapy
Address correspondence to Dina Kremsdorf, INSERM U845, CHU Necker, 156, rue de Vaugirard, Paris 75015, France. Tel: +33-1-40615343; Fax: +33-1-40615581; E-mail: email@example.com
Vitrification Successfully Preserves Hepatocyte Spheroids
R. Magalhães,1 X. W. Wang,2 S. S. Gouk,1 K. H. Lee,1 C. M. Ten,3 H. Yu,3,4 and L. L. Kuleshova1
1Low Temperature Preservation Unit, National University Medical
Institutes Yong Loo Lin School of Medicine, National University of Singapore,
2Department of General Surgery, Xiang Ya Hospital, Central South University, Changsha Hunan, PR China
3Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
4Institute of Bioengineering and Nanotechnology, Agency for Science, Technology and Research, Singapore
This is the first report on low-temperature preservation of self-assembled cell aggregates by vitrification, which is both a time- and cost-effective technology. We developed an effective protocol for vitrification (ice-free cryopreservation) of hepatocyte spheroids that employs rapid stepwise exposure to cryoprotectants (10.5 min) at room temperature and direct immersion into liquid nitrogen (-196°C). For this, three vitrification solutions (VS) were formulated and their effects on vitrified-warmed spheroids were examined. Cryopreservation using ethylene glycol (EG)-sucrose VS showed excellent preservation capability whereby highly preserved cell viability and integrity of vitrified spheroids were observed, through confocal and scanning electron microscopy imaging, when compared to untreated control. The metabolic functions of EG-sucrose VS-cryopreserved spheroids, as assessed by urea production and albumin secretion, were not significantly different from those of control within the same day of observation. In both the vitrification and control groups, albumin secretion was consistently high, ranging from 47.57 ± 14.39 to 70.38 ± 11.29 mg/106 cells and from 56.84 ± 14.48 to 71.79 ± 16.65 mg/106 cells, respectively, and urea production gradually increased through the culture period. The efficacy of vitrification procedure in preserving the functional ability of hepatocyte spheroids was not improved by introduction of a second penetrating cryoprotectant, 1,2-propanediol (PD). Spheroids cryopreserved with EG-PD-sucrose VS showed maintained cell viability; however, in continuous culture, levels of both metabolic functions were lower than those cryopreserved with EG-sucrose VS. EG-PD VS, in which nonpenetrating cryoprotectant (sucrose) was excluded, provided poor protection to spheroids during cryopreservation. This study demonstrated that sucrose plays an important role in the effective vitrification of self-assembled cell aggregates. In a broad view, the excellent results obtained suggest that the developed vitrification strategy, which is an alternative to freezing, may be effectively used as a platform technology in the field of cell transplantation.
Key words: Hepatocyte spheroids; Vitrification; Preservation; Cryoprotectants
Address correspondence to L. L. Kuleshova, Ph.D., Low Temperature Preservation Unit, National University Medical Institutes Yong Loo Lin School of Medicine, National University of Singapore, Block MD11 603-01B-11, 10 Medical Drive, Singapore 117597. Tel: +65 6516 3359; Fax: +65 6773 5461; E-mail: firstname.lastname@example.org or email@example.com
Recipient Immune Repertoire and Engraftment Site Influence the Immune Pathway Effecting Acute Hepatocellular Allograft Rejection
Phillip H. Horne,1 Keri E. Lunsford,1 Jon P. Walker,2 Mitchel A. Koester,3 and Ginny L. Bumgardner3
1Integrated Biomedical Science Graduate Program, College
of Medicine, The Ohio State University Medical Center, Columbus, OH, USA
2Division of Digestive Diseases, Department of Internal Medicine, The Ohio State University Medical Center, Columbus, OH, USA
3Department of Surgery, Comprehensive Transplant Center, The Ohio State University Medical Center, Columbus, OH, USA
As novel acute allograft rejection mechanisms are being discovered, determining the conditions that promote or subvert these distinct rejection pathways is important to interpret the clinical relevance of these pathways for specific recipient groups as well as specific tissue and organ transplants. We have employed a versatile hepatocellular allograft model to analyze how the host immune repertoire and immune locale influences the phenotype of the rejection pathway. In addition, we investigated how peripheral monitoring of cellular and humoral immune parameters correlates with the activity of a specific rejection pathway. Complete MHC mismatched hepatocellular allografts were transplanted into immune competent CD4-deficient, CD8-deficient, or C57BL/6 hosts to focus on CD8-dependent, CD4-dependent, or combined CD4 and CD8-dependent alloimmunity, respectively. Hepatocellular allografts were transplanted to the liver or kidney subcapsular space to investigate the influence of the immune locale on each rejection pathway. The generation of donorreactive DTH, alloantibody, and allospecific cytotoxicity was measured to assess both cellular and humoral immunity. Graft-infiltrating lymphocytes were phenotyped and enumerated in each recipient group. In the presence of CD8+ T cells, cytolytic cellular activity is the dominant mechanism of graft destruction and is amplified in the presence of CD4+ T cells. The absence of CD8+ T cells (CD8 KO) results in potent humoral immunity as reflected by high levels of cytotoxic alloantibody and graft rejection with similar kinetics. Transplant to the liver compared to the kidney site is distinguished by more rapid kinetics of rejection and alloimmunity, which is predominately cell mediated rather than a mix of both humoral and cell-mediated immunity. These studies define several rejection mechanisms occurring in distinct immune conditions, highlighting the plasticity of acute allograft rejection responses and the need to design specific monitoring strategies for these pathways to allow dynamic immune assessment of clinical transplant recipients and targeted immunotherapies.
Key words: Cell transplantation; Cytotoxicity; Alloantibody; Delayed type hypersensitivity (DTH); Engraftment site
Address correspondence to Ginny L. Bumgardner, M.D., Ph.D., F.A.C.S.,Department of Surgery, Division of Transplant, The Ohio State University Medical Center, 1654 Upham Drive, 373 Means Hall, Columbus, OH 43210-1250, USA. Tel: 614-293-6177; Fax: 614-293-4541; E-mail: firstname.lastname@example.org
Xenotransplantation of Human Umbilical Cord Blood Mononuclear Cells to Rats With D-Galactosamine-Induced Hepatitis
Ana I. Álvarez-Mercado,1,2 María J. Sáez-Lara,1,2 María V. García-Mediavilla,3 Sonia Sánchez-Campos,3 Francisco Abadía,4 María Cabello-Donayre,1,2 Ángel Gil,1,2 Javier González-Gallego,3 and Luis Fontana1,2
1Department of Biochemistry and Molecular Biology II, School
of Pharmacy, Campus de Cartuja s/n, 18071 Granada, Spain
2Institute of Nutrition and Food Technology, Biomedical Research Center, Parque Tecnológico Ciencias de la Salud, Avda. del Conocimiento s/n, 18100 Armilla, Granada, Spain
3Centro de Investigación Biomédica en Red de Enferemedades Hepáticas y Digestivas (CIBEREHD), and Institute of Biomedicine, University of León, Campus de Vegazana s/n, 24071 León, Spain
4Department of Cell Biology, School of Sciences, University of Granada, Campus de Fuentenueva s/n, 18071 Granada, Spain
Cord blood is an attractive cell source in regenerative medicine and represents an alternative to bone marrow. The aim of this study was to investigate whether human umbilical cord blood mononuclear (HUCBM) cells might be valuable in hepatic regenerative medicine. HUCBM cells differentiated in vitro into hepatocytes, as suggested by expression of albumin, cytokeratin-18, glutamine synthetase, a-fetoprotein, and cytochrome P450 3A4 at both mRNA and protein levels in a time-dependent fashion. In contrast, the hematopoietic phenotype was gradually lost, as demonstrated by disappearance of CD45 expression. The regenerative potential of HUCBM cells was tested by using a human-to-rat xenotransplant model in which HUCBM cells were intraportally injected into rats with D-galactosamine-induced hepatitis. Liver histology and biochemical markers of hepatic damage were determined. Presence of human cells was detected in blood and liver of both control and D-galactosamine-treated animals. Cell transplantation produced an improvement in both the histological damage and liver function, as demonstrated by plasma values of alkaline phosphatase, <gk>g-glutamyl transferase, lactate dehydrogenase, and total and direct bilirubins. Results obtained suggest that HUCBM cells are capable of hepatic engraftment in this human-to-rat xenotransplant model and that transplantation of HUCBM cells may be a suitable therapy for liver disease.
Key words: D-Galactosamine; Experimental hepatitis; Human hematopoietic mononuclear cells; Umbilical cord blood; Xenotransplantation
Address correspondence to Luis Fontana, Ph.D., Departamento de Bioquímica y Biología Molecular II, Facultad de Farmacia, Campus de Cartuja s/n, 18071 Granada, Spain. Tel: 34 958 242335; Fax: 34 958 248960; E-mail: email@example.com
Determination of Viability of Human Cartilage Allografts by a Rapid and Quantitative Method Not Requiring Cartilage Digestion
Carmen López,1 Nuria Ajenjo,1* Maria J. Muñoz-Alonso,1# Pilar Farde,1 J. León,1 and J. Gómez-Cimiano2
1Departmento de Biología Molecular, Instituto de Biomedicina
y Biotecnologia, CSIC, Universidad de Cantabria, Santander, Spain
2Servicio de Artroscopias, Hospital Universitario Marqués de Valdecilla, Santander, Spain
Fresh osteochondral allograft transplantation is increasingly used for the treatment of cartilage pathologies of the knee. It is believed that transplantation success depends on the presence of viable chondrocytes in the graft, but methods to evaluate graft viability require the isolation of chondrocytes by enzymatic digestion of the cartilage and/or the use of radioactive precursors. We have adapted the well-known cell viability assay based on the reduction of tetrazolium derivatives to evaluate cartilage viability. We took advantage from the histological properties of cartilage tissue and the fact that some tetrazolium derivatives (e.g., WST-1, XTT) give soluble reduction products that can permeate the hyaline cartilage matrix. We have validated this assay in human cartilage explants from arthrotomy interventions and deceased donors, measuring the reduced product in the explant supernatant. Using this method we have compared the performance of several culture media in cartilage viability. From those tested, DMEM supplemented with fetal bovine serum results in higher viability of the cartilage and the explants remain viable at least 15 days in culture at 37°C. Cartilage cells continued expressing chondrocyte-specific genes, suggesting the maintenance of chondrogenic phenotype. The described method offers a quantitative and convenient method to measure the viability of human cartilage grafts.
Key words: Chondrocyte; Osteochondral graft; Transplantation; Tetrazolium; Viability
*Present address: Centro Nacional de Investigaciones Oncológicas,
#Present address: Instituto de Investigaciones Biomédicas, CSIC, Madrid, Spain.
Address correspondence to Javier León, Facultad de Medicina, 39011 Santander, Spain. Tel: 34-942-201952; Fax: 34-942.201945; E-mail: firstname.lastname@example.org
Effects of Direct Transplantation of Multipotent Mesenchymal Stromal/Stem Cells Into the Demyelinated Spinal Cord
D. P. J. Hunt,1 K. A. Irvine,2 D. J. Webber,1 D. A. S. Compston,1 W. F. Blakemore,2 and S. Chandran1
1Cambridge Centre for Brain Repair, Department of Clinical
Neuroscience, Forvie Site, Addenbrookes Hospital, University of Cambridge,
2Department of Veterinary Medicine, University of Cambridge, Cambridge, UK
The adult bone marrow contains a population of multipotent mesenchymal stromal cells (MSCs), defined by plastic adherence, expression of stromal cell surface markers, and differentiation into mesenchymal lineages. There has been much interest in the possible therapeutic use of MSCs in the treatment of demyelinating diseases of the central nervous system. One therapeutic possibility is that these cells may be able to remyelinate when directly injected into the demyelinated spinal cord. Here we examine the effects of direct transplantation of green fluorescent protein (GFP)-labeled MSCs into a model of focal spinal cord demyelination induced by ethidium bromide. We demonstrate that direct intralesional injection of undifferentiated MSCs does not lead to remyelination. Furthermore, we report that transplanted MSCs migrate into areas of normal tissue, deposit collagen, and are associated with axonal damage. These findings support the need for further experimental evaluation of the safety and efficacy of direct parenchymal injection of MSCs into demyelinated lesions and highlight an important issue regarding potential clinical consequences of culture heterogeneity of MSCs between centers.
Key words: Bone marrow; Mesenchymal stromal cell; Remyelination; Demyelination; Multiple sclerosis
Address correspondence to Dr. David Hunt, Cambridge Centre for Brain
Repair, Forvie Site, Robinson Way, University of Cambridge, Cambridge CB2
2PY, UK. Tel: 01223 331160; Fax: 01223 331174; E-mail: email@example.com