|ognizant Communication Corporation|
The Regenerative Medicine Journal
VOLUME 18, NUMBERS 10/11, 2009
Cell Transplantation, Vol. 18, pp. 1059-1067, 2009
0963-6897/09 $90.00 + 00
Copyright © 2009 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.
Protocols for Hematopoietic Stem Cell Expansion From Umbilical Cord Blood
Sonja Koestenbauer,1 Andreas Zisch,2 Gottfried Dohr,1 and Nicolas H. Zech2
1Institute for Cell Biology, Histology and Embryology, Centre
of Molecular Medicine, Medical University of Graz, 8010 Graz, Austria
2Department of Obstetrics, University Hospital Zurich, 8091 Zurich, Switzerland
The reconstitution of adult stem cells may be a promising source for the regeneration of damaged tissues and for the reconstitution of organ dysfunction. However, there are two major limitations to the use of such cells: they are rare, and only a few types exist that can easily be isolated without harming the patient. The best studied and most widely used stem cells are of the hematopoietic lineage. Pioneering work on hematopoietic stem cell (HSC) transplantation was done in the early 1970s by ED. Thomas and colleagues. Since then HSCs have been used in allogenic and autologous transplantation settings to reconstitute blood formation after high-dose chemotherapy for various blood disorders. The cells can be easily harvested from donors, but the cell number is limited, especially when the HSCs originate from umbilical cord blood (UCB). It would be desirable to set up an ex vivo strategy to expand HSCs in order to overcome the cell dose limit, whereby the expansion would favor cell proliferation over cell differentiation. This review provides an overview of the various existing HSC expansion strategies-focusing particularly on stem cells derived from UCB-of the parameters that might affect the outcome, and of the difficulties that may occur when trying to expand such cells.
Key words: Stem cells; Ex vivo expansion; Cord blood; Cell therapy
Address correspondence to Dr. Sonja Koestenbauer, Institute for Cell Biology, Histology and Embryology, Centre of Molecular Medicine, Medical University of Graz, Harrachgasse 21/7, 8010 Graz, Austria. Tel: 0043 (0) 316 385 72957; Fax: 0043 (0) 316 380 9625; E-mail: firstname.lastname@example.org or email@example.com
Deleterious Effect of Human Umbilical Cord Blood Mononuclear Cell Transplantation on Thioacetamide-Induced Chronic Liver Damage in Rats
Ana I. Álvarez-Mercado,1,2 María V. García-Mediavilla,3 Sonia Sánchez-Campos,3 Francisco Abadía,4 María J. Sáez-Lara,1,2 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, University of Granada, Campus de Cartuja s/n, 18071 Granada,
2Institute of Nutrition and Food Technology, Biomedical Research Center, Parque Tecnológico Ciencias de la Salud, Granada, Spain
3Centro de Investigación Biomédica en Red de Enfermedades 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
Our research group investigates whether human mononuclear cells isolated from umbilical cord blood (HUCBM cells) might be valuable in hepatic regenerative medicine. We recently demonstrated that HUCBM cell transplantation improves histological alterations and function of the liver in rats with acute liver damage induced by D-galactosamine. In the present study, HUCBM cells were transplanted into rats with thioacetamide (TAA)-induced liver cirrhosis, an experimental model that generates an intense fibrosis and mimics the histological and biochemical alterations found in the human disease. HUCBM transplantation had no effect on hepatic histology of cirrhotic animals. In contrast, analysis of plasma albumin and total bilirubin, liver damage markers, revealed a harmful effect of HUCBM cell transplantation in our experimental model of liver cirrhosis. Significantly higher plasma urea concentrations, marker of renal function, were observed in the cirrhotic and control rats intraportally injected with HUCBM cells than in those not receiving this therapy. Histological study revealed tubular and glomerular lesions in kidneys of cirrhotic animals transplanted with HUCBM cells. The glomeruli appeared ischemic, and the tubules showed a severe involvement that included peripheral asymmetric vacuolization and disappearance of the tubular lumen. Taken together, the histological and biochemical data suggest that the cirrhotic rats subjected to HUCBM cell therapy developed a hepatorenal syndrome.
Key words: Cell transplantation; Fibrosis; Human mononuclear cells; Liver cirrhosis; Thioacetamide; Umbilical cord blood
Address correspondence to Luis Fontana, Ph.D., Departamento de Bioquímica y Biología Molecular II, Facultad de Farmacia, University of Granada, Campus de Cartuja s/n, 18071 Granada, Spain. Tel: 34 958 242335; Fax: 34 958 248960; E-mail: firstname.lastname@example.org
Impaired Hepatocyte Regeneration in Acute Severe Hepatic Injury Enhances Effective Repopulation by Transplanted Hepatocytes
Chun-Hsien Yu,1,2 Hui-Ling Chen,3 Ya-Hui Chen,1,3,4 Ming-Fu Chang,5 Chin-Sung Chien,1,3,4 and Mei-Hwei Chang4
1Graduate Institute of Clinical Medicine, National Taiwan
University Hospital and National Taiwan University College of Medicine,
2Department of Pediatrics, Buddhist Tzu-Chi General Hospital, Taipei Branch, and Buddhist Tzu-Chi University College of Medicine, Hualien, Taiwan
3Hepatitis Research Center, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan
4Department of Pediatrics, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan
5Graduate Institute of Biochemistry and Molecular Biology, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan
Efficient repopulation by transplanted hepatocytes in the severely injured liver is essential for their clinical application in the treatment of acute hepatic failure. We studied here whether and how the transplanted hepatocytes are able to efficiently repopulate the toxin-induced acute injured liver. Male dipeptidyl peptidase IV-deficient F344 rats were randomized to receive retrorsine plus D-galactosamine (R+D-gal) treatment or D-galactosamine-alone (D-gal) to induce acute hepatic injury, and retrorsine-alone. In these models, retrorsine was used to inhibit the proliferation of endogenous hepatocytes while D-galactosamine induced acute hepatocyte damage. Wild-type hepatocytes (1 x 107/ml) were transplanted intraportally 24 h after D-galactosamine or saline injection. The kinetics of proliferation and repopulation of transplanted cells and the kinetics of cytokine response, hepatic stellate cell (HSC) activation, and matrix metalloproteinase (MMP2) expression were analyzed. We observed that early entry of transplanted hepatocytes into the hepatic plates and massive repopulation of the liver by transplanted hepatocytes occurred in acute hepatic injury induced by R+D-gal treatment but not by D-gal-alone or retrorsine-alone. The expressions of transforming growth factor-a and hepatocyte growth factor genes in the R+D-gal injured liver were significantly upregulated and prolonged up to 4 weeks after hepatocyte transplantation. The expression kinetics were parallel with the efficient proliferation and repopulation of transplanted hepatocytes. HSC was activated rapidly, markedly, and prolongedly up to 4 weeks after hepatocyte transplantation, when the expression of HGF gene and repopulation of transplanted hepatocytes were reduced afterward. Furthermore, the expression kinetics of MMP2 and its specific distribution in the host areas surrounding the expanding clusters of transplanted hepatocytes are consistent with those of activated HSC. Impaired hepatocyte regeneration after acute severe hepatic injury may initiate serial compensatory repair mechanisms that facilitate the extensive repopulation by transplanted hepatocytes that enter early the hepatic plates.
Key words: Retrorsine; D-Galactosamine; Regeneration; Growth factor; Liver failure
Address correspondence to Mei-Hwei Chang, M.D., Department of Pediatrics, National Taiwan University Hospital, 7 Chung-Shan S. Road, Taipei 100, Taiwan. Tel: +886-2-23123456, ext. 71701; Fax: +886-2-23114592; E-mail: email@example.com
Human Multipotent Mesenchymal Stromal Cells From Fetal Lung Expressing Pluripotent Markers and Differentiating Into Cell Types of Three Germ Layers
Cuiling Zheng,1 Shaoguang Yang,1 Zhenxing Guo,1,2 Wenbin Liao,1 Lei Zhang,1 Renchi Yang,1 and Zhong Chao Han1
1State Key Laboratory of Experimental Hematology, Institute
of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences
and Peking Union Medical College, Tianjin, P. R. China
2Department of Hematology/Oncology, First Hospital of Tsinghua University, Beijing, China
Multipotent mesenchymal stromal cells (MSCs) are a promising cell type for cell transplantation; however, their utilization remains limited until the availability of adequate alternative sources of MSCs and the thorough understanding of the biology of MSCs isolated from various sources are realized. Fetal lung has been identified as a rich source of MSCs. To explore the therapeutic potential of passaged fetal lung MSCs (FLMSCs), the present study evaluated their growth kinetics, telomere length, karyotype, immunophenotype, and the differentiation potential during in vitro expansion. FLMSCs could be easily amplified in vitro with no significant shorting of telomere length and had a normal karyotype. No significant differences between passage 5 or passage 25 were observed in the immunophenotype analysis using flow cytometry. Moreover, flow cytometry results provided the first demonstration, to our knowledge, that FLMSCs stably expressed pluripotent markers including Oct4, Nanog, Sox2, TRA-1-60, c-Myc, and SSEA-4 through 25 passages. In vitro differentiation studies as identified by confocal microscopy, flow cytometry, RT-PCR, and immunohistochemistry showed that FLMSCs had extended capacity of differentiating into mesodermal, ectodermal, and endodermal lineages, and that their potential for adipogenic, osteogenic, and chondrogenic differentiation may be maintained over 25 passages. Furthermore, osteogenic and chondrogenic differentiation was used as an indicator of their differentiation capability in vivo, as evidenced by ectopic bone and cartilage formation. In summary, these results suggest that FLMSCs are a primitive population and that their extensive in vitro expansion does not involve significant functional modification of the cells, including morphology, growth, karyotype, immunophenotype, and mesodermal differentiation potential. Hence, FLMSCs might constitute an attractive cell resource for cell transplantation to induce regeneration of damaged tissues/organs.
Key words: Multipotent mesenchymal stromal cells; Fetal lung; Pluripotent markers; Differentiation capacity
Address correspondence to Prof. Zhong Chao Han, State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, 288 Nanjing Road, Tianjin 300020, P. R. China. Tel: 86-22-27210717; Fax: 86-22-66211430; E-mail: firstname.lastname@example.org
Transcatheter Injection-Induced Changes in Human Bone Marrow-Derived Mesenchymal Stem Cells
Boon C. Heng,1 Steven H. Hsu,1 Catherine M. Cowan,1 Annie Liu,1 Julie Tai,1 Yen Chan,1 Warren Sherman,2 and Shubhayu Basu3
1Abbott Vascular, Santa Clara, CA, USA
2The Center for Interventional Vascular Therapy, Columbia University Medical Center, New York, NY, USA
3Global Cardiovascular Innovation Center, Cleveland, OH, USA
Human mesenchymal stem cells (hMSC) are being administered by direct intramyocardial (IM) injection into patients with myocardial dysfunction with an objective to improve clinical status. However, surprisingly little attention has been directed to qualifying hMSC functionality beyond simple viability. In particular, the transit of hMSCs through a small-caliber needle lumen, the final fluidic pathway for all IM injection devices, may be especially prone to inducing unwarranted effects on cell function. This study evaluated the changes in clonogenicity, gene expression, and cytokine secretion that may be induced in hMSC (20 million/ml) by injection through a 26-gauge Nitinol needle at two different flow rates compared to noninjected control samples. Results indicated that hMSC viability and colony forming unit (CFU) formation was not altered by changes in injection rate, although a trend toward lower titers was noted at the higher flow rate, for the specific batch of hMSCs studied. The gene expression and cytokine analysis data suggest that delivering a suspension of MSCs through narrow lumen needles may marginally alter certain gene expression programs, but that such in vitro effects are transient and not translated into measurable differences in protein production. Gene expression levels of four cytokines (bFGF, SDF-1, SCF, VEGF) were significantly different at 400 ml/min, and that of all cytokines were significantly different at 1600 ml/min when compared to controls (p < 0.05). These changes were less pronounced (statistically insignificant for most cases, p > 0.05) and, in certain instances directionally opposite, at 72 h. However, no differences in the amounts of secreted bFGF, EGF, or TGF-b were detectable at either of the two time points or flow rates. We infer that intramyocardial dministration by transcatheter techniques is unlikely to interfere with the machinery required for cell replication or secretion of regulatory and other growth factors, which are the mainstays of MSC contribution to cardiac tissue repair and regeneration.
Key words: Intramyocardial; Catheter; Delivery; Mesenchymal; Stem cells; Shear
Address correspondence to Shubhayu Basu, Ph.D., Director, Product Development, Global Cardiovascular Innovation Center, 10265 Carnegie Avenue, Cleveland, OH 44106, USA. E-mail: email@example.com or Warren Sherman, M.D., FACC, FSCAI, Director, Cardiac Cell-Based Endovascular Therapies, Center for Interventional Vascular Therapy, Columbia University Medical Center, 161 Ft. Washington Avenue IP-519, New York, NY 11032, USA. E-mail: firstname.lastname@example.org
Oscillating Pressure Treatment Upregulates Connexin43 Expression in Skeletal Myoblasts and Enhances Therapeutic Efficacy for Myocardial Infarction
Sae-Won Lee,1,3 Hyun-Jae Kang,1,2 Ji-Young Lee,1,3 Seock-Won Youn,1 Joo-Yun Won,1,3 Ji-Hyun Kim,1 Hyun-Chae Lee,1 Eun Ju Lee,1 Se-il Oh,1,2 Byung-Hee Oh,1,2 Young-Bae Park,1,2 and Hyo-Soo Kim1,2,3
1National Research Laboratory for Cardiovascular Stem Cells
and IRICT, Seoul National University Hospital, Seoul, Korea
2Department of Internal Medicine and Cardiovascular Center, Seoul National University Hospital, Seoul, Korea
3Ischemic/Hypoxic Disease Institute, Seoul National University College of Medicine, Seoul, Korea
Transplantation of autologous skeletal myoblasts (SMBs) is a potential therapeutic approach for myocardial infarction. However, their clinical efficacy and safety is still controversial. Electrical coupling through gap junction between SMBs and host myocardium is essential for synchronized contraction and electrical stability. Here, we investigated the effect of heart beat-simulating environment, oscillating pressure, on the expression of connexin43 in two types of SMBs from rat and mouse. We found that connexin43 is markedly decreased under ischemia-mimicking conditions such as serum starvation and hypoxia (1% O2) in rat primary cultured SMBs and mouse C2C12 SMB cell line. Interestingly, the decrease of connexin43 expression under serum starvation was attenuated by oscillating pressure. Oscillating pressure treatment increased the expression of connexin43 twofold through AP-1 stimulation, which was blocked by PD98059, ERK inhibitor. In coculture of cardiomyocytes and C2C12, pressure-treated C2C12 and cardiomyocytes were able to form functional gap junction, which was demonstrated by both calcein-AM dye transfer assay and measurement of simultaneous contraction. In rat myocardial infarction model, transplantation of SMBs pretreated with oscillating pressure resulted in lesser ventricular dilatation and better systolic function than transplantation of untreated SMBs and control group. These results suggested that application of oscillating pressure on SMBs before transplantation may be useful to promote therapeutic efficacy for myocardial infarction by enhancing gap junction formation between transplanted and host cells.
Key words: Skeletal myoblast; Gap junction; Ischemic heart; Serum starvation; Cardiomyocytes
Address correspondence to Hyo-Soo Kim, M.D., Ph.D., Department of Internal Medicine and Cardiovascular Center, Seoul National University Hospital, 28 Yongon-dong, Chongno-gu, Seoul 110-744, Korea. Tel: 82-2-2072-2226; Fax: 82-2-766-8904; E-mail: email@example.com
Sex of Muscle Stem Cells Does Not Influence Potency for Cardiac Cell Therapy
Lauren Drowley,1,4 Masaho Okada,2,4 Thomas R. Payne,4,5 Gregory P. Botta,4 Hideki Oshima,2,4 Bradley B. Keller,3,4 Kimimasa Tobita,3,4 and Johnny Huard1,2,4,5
1Department of Pathology, University of Pittsburgh, Pittsburgh,
2Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA, USA
3Developmental Biology, University of Pittsburgh, Pittsburgh, PA, USA
4Stem Cell Research Center, Children's Hospital of Pittsburgh of UPMC, Pittsburgh, PA, USA
5Cook Myosite, Inc., Pittsburgh PA, USA
We have previously shown that populations of skeletal muscle-derived stem cells (MDSCs) exhibit sex-based differences for skeletal muscle and bone repair, with female cells demonstrating superior engrafting abilities to males in skeletal muscle while male cells differentiating more robustly toward the osteogenic and chondrogenic lineages. In this study, we tested the hypothesis that the therapeutic capacity of MDSCs transplanted into myocardium is influenced by sex of donor MDSCs or recipient. Male and female MDSCs isolated from the skeletal muscle of 3-week-old mice were transplanted into recipient male or female dystrophin-deficient (mdx) hearts or into the hearts of male SCID mice following acute myocardial infarction. In the mdx model, no difference was seen in engraftment or blood vessel formation based on donor cell or recipient sex. In the infarction model, MDSC-transplanted hearts showed higher postinfarction angiogenesis, less myocardial scar formation, and improved cardiac function compared to vehicle controls. However, sex of donor MDSCs had no significant effects on engraftment, angiogenesis, and cardiac function. VEGF expression, a potent angiogenic factor, was similar between male and female MDSCs. Our results suggest that donor MDSC or recipient sex has no significant effect on the efficiency of MDSC-triggered myocardial engraftment or regeneration following cardiac injury. The ability of the MDSCs to improve cardiac regeneration and repair through promotion of angiogenesis without differentiation into the cardiac lineage may have contributed to the lack of sex difference observed in these models.
Key words: Stem cells; Cardiac; Cell transplantation; Sex
Address correspondence to Johnny Huard, 3343 Forbes Ave Suite 201, Pittsburgh, PA 15213, USA. Tel: 412-648-6798; Fax: 412-648-4066; Email: firstname.lastname@example.org
Amniotic Membrane Patching Promotes Ischemic Rat Heart Repair
Anna Cargnoni,1 Marco Di Marcello,2 Marino Campagnol,3 Claudia Nassuato,1 Alberto Albertini,4 and Ornella Parolini1
1Centro di Ricerca E. Menni, Fondazione Poliambulanza-Istituto
Ospedaliero, 25124 Brescia, Italy
2Ambulatorio Veterinario Cellatica, 25060 Cellatica, Brescia, Italy
3Facoltà di Medicina Veterinaria-Stabulario Centralizzato, 20133 Milano, Italy
4Istituto di Tecnologie Biomediche, CNR, 20090 Segrate-Milano, Italy
The amniotic membrane has long been applied for wound healing and treatment of ophthalmological disorders, even though the mechanisms underlying its actions remain to be clarified. Recently, cells derived from fetal membranes of human term placenta have raised strong interest in regenerative medicine for their stem cell potential and immunomodulatory features. Our study aimed to investigate the possible utility of amniotic membrane to limit postischemic cardiac injury. A fragment of human amniotic membrane was applied onto the left ventricle of rats that had undergone ischemia through left anterior descending coronary artery ligation. Echocardiographic assessment of morphological and functional cardiac parameters was then performed over a 3-month period. We demonstrated that application of an amniotic membrane fragment onto ischemic rat hearts could significantly reduce postischemic cardiac dysfunction. The amniotic membrane-treated rats showed higher preservation of cardiac dimensions and improved cardiac contractile function in terms of higher left ventricle ejection fraction, fractional shortening, and wall thickening. These improvements were apparent by day 7 after application of the amniotic membrane, persisted for at least 2 months, and occurred independently of cardiac injury severity. No engraftment of amniotic cells was detected into host cardiac tissues. Our results suggest that use of amniotic membrane may constitute a convenient vehicle for supplying cells that produce cardioprotective soluble factors, and reinforce the notion that this tissue constitutes a cell source with clinical potential that has yet to be completely revealed.
Key words: Amniotic membrane; Amniotic patch; Placenta; Myocardial infarction; Cardiac repair
Address correspondence to Ornella Parolini, Ph.D., Centro di Ricerca E. Menni, Fondazione Poliambulanza-Istituto Ospedaliero, Via Bissolati 57, I-25124 Brescia, Italy. Tel: ++390302455754; Fax: ++390302455704; E-mail: email@example.com or firstname.lastname@example.org
Local Delivery of Autologous Platelet in Collagen Matrix Simulated In Situ Articular Cartilage Repair
Yi Ying Qi,1,3 Xiao Chen,1 Yang Zi Jiang,1 Hong Xin Cai,2 Lin Lin Wang,1 Xing Hui Song,1 Xiao Hui Zou,4 and Hong Wei Ouyang1,2,3
1Center for Stem Cell and Tissue Engineering, School of Medicine,
Zhejiang University, Hangzhou, China
2Department of Orthopedadic Surgery, Sir Raw Raw Show Hospital, Zhejiang University, Hangzhou, China
3Institute of Cell Biology, School of Medicine, Zhejiang University, Hangzhou, China
4Women Hospital, School of Medicine, Zhejiang University, Hangzhou, China
Bone marrow released by microfracture or full-thickness cartilage defect can initiate the in situ cartilage repair. However, it can only repair small cartilage defects (<2 cm2). This study aimed to investigate whether autologous platelet-rich plasma (PRP) transplantation in collagen matrix can improve the in situ bone marrow-initiated cartilage repair. Full-thickness cartilage defects (diameter 4 mm, thickness 3 mm) in the patellar grooves of male New Zealand White rabbits were chosen as a model of in situ cartilage repair. They were treated with bilayer collagen scaffold (group II), PRP and bilayer collagen scaffold (group III), and untreated (group I), respectively (n = 11). The rabbits were sacrificed at 6 and 12 weeks after operation. The repaired tissues were processed for histology and for mechanical test. The results showed that at both 6 and 12 weeks, group III had the largest amounts of cartilage tissue, which restored a larger surface area of the cartilage defects. Moreover, group III had higher histological scores and more glycosaminoglycans (GAGs) content than those in the other two groups (p < 0.05). The Young's modulus of the repaired tissue in group II and group III was higher than that of group I (p < 0.05). Autologous PRP and bilayer collagen matrix stimulated the formation of cartilage tissues. The findings implicated that the combination of PRP with collagen matrix may repair larger cartilage defects that currently require complex autologous chondrocyte implantation (ACI) or osteochondral grafting.
Key words: Articular cartilage defect; Platelet-rich plasma (PRP); Collagen matrix; In situ cartilage repair
Address correspondence to Hong Wei Ouyang, Center for Stem Cell and Tissue Engineering, School of Medicine, Zhejiang University, 388 Yu Hang Tang Road, 310058 Hangzhou, China. Tel/Fax: +86-0571-88208262; E-mail: email@example.com
Transient Recovery in a Rat Model of Familial Amyotrophic Lateral Sclerosis After Transplantation of Motor Neurons Derived From Mouse Embryonic Stem Cells
Rodrigo López-González, Philip Kunckles, and Iván Velasco
Departamento de Neurociencias, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, México D.F. 04510, México
Embryonic stem (ES) cells can be induced to differentiate into motor neurons (MN). Animal models resembling MN degeneration and paralysis observed in familial amyotrophic lateral sclerosis (ALS) have been previously reported. In this work, we aimed to investigate whether transplanted MN could prevent motor deterioration in transgenic rats expressing a mutant form of human superoxide dismutase 1 (hSOD1G93A) associated with inherited ALS. Mouse ES cells were differentiated to neurons that express green fluorescent protein (GFP) under the promoter of the MN-specific gene hb9, as well as molecular markers indicative of MN identity. Cells were grafted into the lumbar spinal cord of adult wild-type (WT) or hSOD1G93A rats at 10 weeks of age, when transgenic animals are presymptomatic. Grafted cells with MN phenotype can survive for at least 1 week in hSOD1G93A animals. To quantitatively evaluate motor performance of WT and transgenic rats, we carried out weekly rotarod tests starting when the animals were 14 weeks old. Sham and grafted WT animals showed no decline in their ability to sustain themselves on the rotating rod. In contrast, sham hSOD1G93A rats decreased in motor performance from week 16 onwards, reaching paralysis by week 19 of age. In grafted transgenic animals, there was a significant improvement in rotarod competence at weeks 16 and 17 when compared to sham hSOD1G93A. However, in the following weeks, transplanted hSOD1G93A rats showed motor deterioration and eventually exhibited paralysis by week 19. At end-stage, we found only a few endogenous MN in sham and grafted hSOD1G93A rats by cresyl violet staining; no choline acetyl transferase-positive nor GFP-positive MN were present in grafted transgenic subjects. In contrast, WT rats analyzed at the same age possessed grafted GFP-positive MN in their spinal cords. These results strongly suggest that the transgenic hSOD1G93A environment is detrimental to grafted MN in the long term.
Key words: ES cells; Islet1; Choline acetyl transferase; Graft; Adult spinal cord; Paralysis
Address correspondence to Iván Velasco, Departamento de Neurociencias, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, AP 70-253, México D.F.-04510, México. Tel: 52-55-5622-5649; Fax: 52-55-5622-5607; E-mail: firstname.lastname@example.org
Compacted DNA Nanoparticle Gene Transfer of GDNF to the Rat Striatum Enhances the Survival of Grafted Fetal Dopamine Neurons
David M. Yurek,1 Anita M. Flectcher,1 Tomasz H. Kowalczyk,2 Linas Padegimas,2 and Mark J. Cooper2
1Department of Neurosurgery, University of Kentucky College
of Medicine, Lexington, KY, USA
2Copernicus Therapeutics, Inc., Cleveland, OH, USA
Previously it was established that infusion of glial cell line-derived neurotrophic factor (GDNF) protein into grafts of embryonic dopamine cells has a neurotrophic effect on the grafted cells. In this study we used a nonviral technique to transfer the gene encoding for GDNF to striatal cells. Plasmid DNA encoding for GDNF was compacted into DNA nanoparticles (DNPs) by 10 kDa polyethylene glycol (PEG)-substituted lysine 30-mers (CK30PEG10k) and then injected into the denervated striatum of rats with unilateral 6-hydroxydopamine lesions. Sham controls were injected with saline. One week later, experimental animals received either a ventral mesencephalic (VM) tissue chunk graft or a cell suspension VM graft implanted into the denervated striatum. Grafts were allowed to integrate for 4-6 weeks and during this period we monitored spontaneous and drug-induced motor activity. Using stereological cell counting we observed a 16-fold increase in the number of surviving TH+ cells within tissue chunk grafts placed into the striatum pretreated with pGDNF DNPs (14,923 ± 4,326) when compared to grafts placed into striatum pretreated with saline (955 ± 343). Similarly, we observed a sevenfold increase in the number of TH+ cells within cell suspension grafts placed into the striatum treated with pGDNF DNPs when compared to cell suspension grafts placed into the saline dosed striatum. Behaviorally, we observed significant improvement in rotational scores and in spontaneous forepaw usage of the affected forelimb in grafted animals receiving prior treatment with compacted pGDNF DNPs when compared to grafted animals receiving saline control pretreatment. Data analysis for protein, morphological, and behavioral measures suggests that compacted pGDNF DNPs injected into the striatum can result in transfected cells overexpressing GDNF protein at levels that provide neurotrophic support for grafted embryonic dopamine neurons.
Key words: Glial cell line-derived neurotrophic factor (GDNF); DNA nanoparticles; Gene transfer; Parkinson's disease
Address correspondence to David M. Yurek, Ph.D., Department of Neurosurgery, University of Kentucky College of Medicine, Health Sciences Research Building, Lexington, KY 40536-0305, USA. Tel: (859) 257-8219; Fax: (859) 323-6343; E-mail: David.Yurek@uky.edu
Cellular Epigenetic Modifications of Neural Stem Cell Differentiation
Rabindra P. Singh,1* Kevin Shiue,1* Dominic Schomberg,1* and Feng C. Zhou1,2,3
1Departments of Anatomy & Cell Biology, Indiana University
School of Medicine, Indianapolis, IN, USA
2Stark Neuroscience Research Institute, Indiana University School of Medicine, Indianapolis, IN, USA
3Department of Psychology, Indiana University Purdue University at Indianapolis, Indianapolis, IN, USA
Emerging information indicates that epigenetic modification (i.e., histone code and DNA methylation) may be integral to the maintenance and differentiation of neural stem cells (NSCs), but their actual involvement has not yet been illustrated. In this study, we demonstrated the dynamic nature of epigenetic marks during the differentiation of quiescent adult rat NSCs in neurospheres. A subpopulation of OCT4+ NSCs in the neurosphere contained histone marks, trimethylated histone 3 on lysine 27 (3me-H3K27), 2me-H3K4, and acetylated H4 (Ac-H4). A major decrease of these marks was found prior to or during differentiation, and was further diminished or reprogrammed in diverse subpopulations of migrated NSCs expressing nestin or b-III-tubulin. The DNA methylation mark 5-methyl-cytosine (5-MeC), and DNA methyltransferase (DNMT) 1 and 3a expression also correlated to the state of differentiation; they were highly present in undifferentiated NSCs but downregulated in migrated populations. In contrast, DNA methyl-CpG-binding protein (MBD1) was low in undifferentiated NSCs in neurospheres, but highly appeared in differentiating NSCs. Furthermore, we found an outward translocation of DNA methylation marker 5-MeC, DNMT1, DNMT3a, and MBD1 in NSCs as differentiation began and proceeded; 5-MeC from homogeneous nucleus to peripheral nucleus, and DMNT1a and 3a from nuclear to cytoplasm, indicating chromatin remodeling. Treatment with DNA methylation inhibitor, 5-aza-cytidine, altered DNA methylation and disrupted migration as indicated by a reduction of migrated neurons and differentiation. These results indicate that chromatin is dynamically remodeled when NSCs transform from the quiescent state to active growth, and that DNA methylation modification is essential for neural stem cell differentiation.
Key words: Neural progenitor cells; DNA methylation; Histone code; DNMT; MBD1; 5-aza-Cytidine
Address correspondence to Feng C. Zhou, Ph.D., Department of Anatomy & Cell Biology, Indiana University School of Medicine, 635 Barnhill Drive, MS 508, Indianapolis, IN 46202, USA. Tel: 317-274-7359; Fax: 317- 278-2040; E-mail: email@example.com
*Authors contributed equally.
Immune Cell Populations in Nonhuman Primate Islets
Lane C. K. Coffey,1,2 Dora M. Berman,1,3 Melissa A. Willman,1 and Norma S. Kenyon1,2,3,4
1Diabetes Research Institute, Miller School of Medicine,
University of Miami, Miami, FL, USA
2Department of Microbiology and Immunology, Miller School of Medicine, University of Miami, Miami, FL, USA
3Department of Surgery, Miller School of Medicine, University of Miami, Miami, FL, USA
4Department of Medicine, Miller School of Medicine, University of Miami, Miami, FL, USA
Islet transplantation is a promising cellular therapy for the treatment of type 1 diabetes (T1D). The immunogenicity of isolated islets has been of interest to the transplant community for many years, as upon transplantation, islets are damaged or destroyed through specific and nonspecific inflammatory and immune events. Antigen presenting cells (APC) are crucial intermediates in the generation of both innate and specific immune responses, and it has long been understood that some APC are resident in islets in situ, as well as after isolation. Our aim was to identify and characterize intraislet resident populations of APC and other immune cells in islets from nonhuman primates (Macaca fascicularis) in situ (pancreas biopsies obtained prerecovery) and after isolation using immunohistochemistry, confocal microscopy, and flow cytometry. The numbers of cells obtained in situ are similar to those in islets postisolation. Each isolated islet equivalent contains an average of 21.8 immune cells, 14.7 (67%) of which are APC. Many of these APC are dentritic cells and, surprisingly, 50% are B lymphocytes. The number of islet-resident immune cells increases with islet size, with greater numbers in large versus small islets (p < 0.001). The APC were localized around the exterior or spread evenly throughout the islets, with no definitive orientation identified. This knowledge will be useful to develop tailored modulation strategies to decrease immunogenicity, enhance engraftment, and ultimately prevent islet rejection.
Key words: Antigen presenting cells in islets; Lymphocytes in islets; Islet transplantation; Islet cell composition; Passenger leukocytes in islets; Dendritic cells in islets
Address correspondence to Norma S. Kenyon, Ph.D., Diabetes Research Institute, University of Miami, 1450 NW 10 Avenue (R-134), Miami, FL 33136, USA. Tel: (305) 243-5346; Fax: (305) 243-1042; E-mail: firstname.lastname@example.org
Quantitative Assessment of Islet Cell Products: Estimating the Accuracy of the Existing Protocol and Accounting for Islet Size Distribution
Peter Buchwald,1,2 Xiaojing Wang,1 Aisha Khan,1 Andres Bernal,3 Chris Fraker,1 Luca Inverardi,1 and Camillo Ricordi1
1Diabetes Research Institute, University of Miami Leonard
M. Miller School of Medicine, Miami, FL, USA
2Molecular and Cellular Pharmacology, University of Miami Leonard M. Miller School of Medicine, Miami, FL, USA
3BioRep Technologies, Miami, FL, USA
The ability to consistently and reliably assess the total number and the size distribution of isolated pancreatic islet cells from a small sample is of crucial relevance for the adequate characterization of islet cell preparations used for research or transplantation purposes. Here, data from a large number of isolations were used to establish a continuous probability density function describing the size distribution of human pancreatic islets. This function was then used to generate a polymeric microsphere mixture with a composition resembling those of isolated islets, which, in turn, was used to quantitatively assess the accuracy, reliability, and operator-dependent variability of the currently utilized manual standard procedure of quantification of islet cell preparation. Furthermore, on the basis of the best fit probability density function, which corresponds to a Weibull distribution, a slightly modified scale of islet equivalent number (IEQ) conversion factors is proposed that incorporates the size distribution of islets and accounts for the decreasing probability of finding larger islets within each size group. Compared to the current calculation method, these factors introduce a 4-8% downward correction of the total IEQ estimate, but they reflect a statistically more accurate contribution of differently sized islets.
Key words: Islets of Langerhans; Type 1 diabetes; Islet isolation; Frequency distribution; Probability density function; Weibull distribution
Address correspondence to Peter Buchwald, Diabetes Research Institute, University of Miami Leonard M. Miller School of Medicine, 1450 NW 10 Ave (R-134), Miami, FL 33136, USA. Tel: 305 243-9657; E-mail: email@example.com
Antiapoptotic Effect of Tacrolimus on Cytokine-Challenged Human Islets
José M. Balibrea del Castillo,1 M. Cruz García-Martín,2 Javier Arias-Díaz,1 Elena Giné,2 Elena Vara,2 and José L. Balibrea Cantero1
1Department of Surgery, Universidad Complutense, Madrid,
2Department of Biochemistry and Molecular Biology, Universidad Complutense, Madrid, Spain
Our goal was to investigate whether previously related antiapoptotic and anti-inflammatory effects of tacrolimus could be useful in protecting human islets cultured in the presence of several proinflammatory mediators. Human islets obtained from cadaveric donors after intraductal infusion with collagenase, mechanical digestion, and continuous Ficoll gradient purification were cultured in RPMI-1640 medium for 24 h. Escherichia coli lipopolysaccharide (10 mg/ml) or interleukin-1 (50 UI/ml) + g-IF (1000 UI/ml) and low-dose tacroliumus (5 ng/ml) were added. Homogenized samples (300 IE) from five different donors where assigned to four different experimental groups (control, treatment, cytokines, and cytokines + treatment). To evaluate islet damage and apoptotic response, nucleosome content, Bcl-2 protein levels, caspase-3, -8, and -9 levels, and insulin concentration were measured. Also, TNF-a and IL-6 levels where assessed as indicators of the inflammatory response. All proapoptotic markers, TNF-a, and IL-6 levels were augmented after both LPS and cytokine stimulation. Tacrolimus reduced significantly all of them and restored baseline values of nucleosome and caspase-9 in both experiments and Bcl-2 and caspase-3 when IL-1 + g-IF was added. Twenty-four-hour insulin concentration diminished when LPS or IL-1 + g-IF were present. Tacrolimus treatment restored insulin levels in both experiments. These results suggest that in vitro apoptotic events and media insulin concentration decrease after proinflammatory stimulation can be reverted using low-dose tacrolimus.
Key words: Islet transplantation; Apoptosis; Tacrolimus; Immunosuppression
Address correspondence to Dr. José M. Balibrea del Castillo, M.D., Ph.D., Avenida República Argentina, 227 Barcelona, Spain 08023. Tel: 34-609474822; E-mail: firstname.lastname@example.org
Long-Term Metabolic and Hormonal Effects of Exenatide on Islet Transplant Recipients With Allograft Dysfunction
Raquel N. Faradji,1,2* Tatiana Froud,1,3,4* Shari Messinger,1,5 Kathy Monroy,1 Antonello Pileggi,1,3 Davide Mineo,1,6 Thipaporn Tharavanij,1 Armando J. Mendez,1,2 Camillo Ricordi,1,3 and Rodolfo Alejandro1,2
1Clinical Islet Transplant Program, Diabetes Research Institute,
University of Miami Leonard M. Miller School of Medicine, Miami, FL, USA
2Department of Medicine, University of Miami Leonard M. Miller School of Medicine, Miami, FL, USA
3Department of Surgery, University of Miami Leonard M. Miller School of Medicine, Miami, FL, USA
4Department of Radiology, University of Miami Leonard M. Miller School of Medicine, Miami, FL, USA
5Department of Epidemiology, University of Miami Leonard M. Miller School of Medicine, Miami, FL, USA
6"Tor Vergata" University of Rome, Rome, Italy
The initial success of islet transplantation (ITx) is followed by graft dysfunction (GDF) and insulin reintroduction. Exenatide, a GLP-1 agonist, increases insulin and decreases glucagon secretion and has potential for b-cell regeneration. To improve functional islet mass, exenatide treatment was given to ITx recipients with GDF. The objective of this study was to assess metabolic and hormonal effects of exenatide in GDF. In this prospective, single-arm, nonrandomized study, 11 type 1 diabetes recipients of ITx with GDF had HbA1c, weight, insulin requirements, and 5-h mixed meal tolerance test (MMTT; with/without exenatide given before test) at baseline, 3, 6, and 12 months after initiating exenatide treatment. Baseline MMTT showed postprandial hyperglycemia and hyperglucagonemia. Daily exenatide treatment resulted in improved glucose, increased amylin/insulin ratio, and decreased proinsulin/insulin ratio as assessed by MMTT. Glucagon responses remained unchanged. Exenatide administration 1 h before MMTT showed decreased glucagon and glucose at 0 min and attenuation in their postprandial rise. Time-to-peak glucose was delayed, followed by insulin, proinsulin, amylin, and C-peptide, indicating glucose-driven insulin secretion. Five subjects completed 12-month follow-up. Glucose and glucagon suppression responses after MMTT with exenatide were no longer observed. Retrospective 3-month analysis of these subjects revealed higher and sustained glucagon levels that did not suppress as profoundly with exenatide administration, associated with higher glucose levels and increased C-peptide responses. In conclusion, Exenatide suppresses the abnormal postprandial hyperglucagonemia and hyperglycemia observed in GDF. Changes in amylin and proinsulin secretion may reflect more efficient insulin processing. Different degrees of responsiveness to exenatide were identified. These may help guide the clinical management of ITx recipients.
Key words: Islet; Islet transplantation; Graft dysfunction; Exenatide; Metabolism; Diabetes; Glucagon; Insulin; Insulin independence
Address correspondence to Rodolfo Alejandro, M.D., Diabetes Research Institute, 1450 NW 10th Avenue (R-134), Miami, FL 33136, USA. Tel: (305) 243-5324; Fax: (305) 243-1058; E-mail: email@example.com
*These two authors contributed equally to this work.