|ognizant Communication Corporation|
The Regenerative Medicine Journal
VOLUME 18, NUMBER 4, 2009
Cell Transplantation, Vol. 18, pp. 381-389, 2009
0963-6897/09 $90.00 + 00
Copyright © 2009 Cognizant Comm. Corp.
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ERas Is Expressed in Primate Embryonic Stem Cells But Not Related to Tumorigenesis
Yujiro Tanaka,1,5* Tamako Ikeda,1* Yukiko Kishi,1 Shigeo Masuda,1 Hiroaki Shibata,1,3 Kengo Takeuchi,4 Makoto Komura,5 Tadashi Iwanaka,5 Shin-ichi Muramatsu,2 Yasushi Kondo,6 Kazutoshi Takahashi,7 Shinya Yamanaka,7 and Yutaka Hanazono1
1Division of Regenerative Medicine, Center for Molecular
Medicine, Jichi Medical University, Tochigi, Japan
2Division of Neurology, Department of Internal Medicine, Jichi Medical University, Tochigi, Japan
3Tsukuba Primate Research Center, National Institute of Biomedical Innovation, Ibaraki, Japan
4Department of Pathology, Cancer Institute Hospital, Tokyo, Japan
5Department of Pediatric Surgery, Graduate School of Medicine, University of Tokyo, Tokyo, Japan
6Mitsubishi Tanabe Pharma, Osaka, Japan
7Center for iPS Cell Research and Application, Institute for Integrated Cell-Material Sciences, Kyoto University, Kyoto, Japan
The ERas gene promotes the proliferation of and formation of teratomas by mouse embryonic stem (ES) cells. However, its human orthologue is not expressed in human ES cells. This implies that the behavior of transplanted mouse ES cells would not accurately reflect the behavior of transplanted human ES cells and that the use of nonhuman primate models might be more appropriate to demonstrate the safety of human ES cell-based therapies. However, the expression of the ERas gene has not been examined in nonhuman primate ES cells. In this study, we cloned the cynomolgus homologue and showed that the ERas gene is expressed in cynomolgus ES cells. Notably, it is also expressed in cynomolgus ES cell-derived differentiated progeny as well as cynomolgus adult tissues. The ERas protein is detectable in various cynomolgus tissues as assessed by immunohistochemisty. Cynomolgus ES cell-derived teratoma cells, which also expressed the ERas gene at higher levels than the undifferentiated cynomolgus ES cells, did not develop tumors in NOD/Shi-scid, IL-2Rgnull (NOG) mice. Even when the ERas gene was overexpressed in cynomolgus stromal cells, only the plating efficiency was improved and the proliferation was not promoted. Thus, it is unlikely that ERas contributes to the tumorigenicity of cynomolgus cells. Therefore, cynomolgus ES cells are more similar to human than mouse ES cells despite that ERas is expressed in cynomolgus and mouse ES cells but not in human ES cells.
Key words: Embryonic stem cell; ERas; Cynomolgus monkey; Tumorigenesis
Address correspondence to Yutaka Hanazono, M.D., Ph.D., Professor, Division of Regenerative Medicine, Center for Molecular Medicine, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke, Tochigi 329-0498, Japan. Tel: +81-285-58-7451; Fax: +81-285-44-5205; E-mail: firstname.lastname@example.org
Neurotrophism of Bone Marrow Stromal Cells to Embryonic Stem Cells: Noncontact Induction and Transplantation to a Mouse Ischemic Stroke Model
Tao Yang,1,2 Kam Sze Tsang,1,2 Wai Sang Poon,2,3 and Ho Keung Ng1,2
1Li Ka Shing Institute of Health Sciences, The Chinese University
of Hong Kong, Hong Kong
2Department of Anatomical and Cellular Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Hong Kong
3Department of Surgery, The Chinese University of Hong Kong, Prince of Wales Hospital, Hong Kong
Embryonic stem (ES) cell-derived cell products may serve as a source of cells for regenerative medicine. Currently available technologies for the induction of ES cells into neural lineage cells require extended culturing in vitro and complex procedural manipulations, with variable yields of heterogeneous cells, which have hindered the prospective use of cell derivatives for treatment of ischemic stroke. We established a simple and efficient method to derive mouse ES cells into neural lineage cells using an 8-day coculture with the bone marrow stromal cells MS5, followed by a 6-day propagation culture and a 4-day selection culture. The protocol generated a relatively high yield of neural lineage cells without any mesodermal and endodermal lineage commitment. In in vivo study, these derived cells could improve the cognitive function of ischemic stroke mice. Three weeks after transplantation, migration of implanted cells to lesioned areas was noted. It was also evident of a normalization of pyramidal neuron density and morphology in hippocampal CA1 region. One (1/17) episode of teratoma development was noted. Data suggested that MS5 cells may exert a neurotrophic effect to enhance neural differentiation of ES cells and MS5-induced ES cell-derived cells appeared to be applicable to cell therapy for ischemic stroke.
Key words: Embryonic stem cell; Stromal cell; Coculture; Differentiation; Transplantation; Ischemic stroke
Address correspondence to Dr. Kam Sze Tsang, Department of Anatomical and Cellular Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong. Tel: (852) 2632 1157; Fax: (852) 2632 4552; E-mail: email@example.com
Transplantation of Allogeneic and Xenogeneic Placenta-Derived Cells Reduces Bleomycin-Induced Lung Fibrosis
Anna Cargnoni,1 Lucia Gibelli,1 Alessandra Tosini,1 Patrizia Bonassi Signoroni,1 Claudia Nassuato,1 Davide Arienti,2 Guerino Lombardi,2 Alberto Albertini,3 Georg S. Wengler,1 and Ornella Parolini1
1Centro di Ricerca E. Menni, Fondazione Poliambulanza-Istituto
Ospedaliero, 25124 Brescia, Italy
2Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna, Reparto del Benessere animale, IZSLER, 25124 Brescia, Italy
3Istituto di Tecnologie Biomediche, CNR, 20090 Segrate-Milano, Italy
Fetal membranes (amnion and chorion) have recently raised significant attention as potential sources of stem cells. We have recently demonstrated that cells derived from human term placenta show stem cell phenotype, high plasticity, and display low immunogenicity both in vitro and in vivo. Moreover, placenta-derived cells, after xenotransplantation, are able to engraft in solid organs including the lung. On these bases, we studied the effects of fetal membrane-derived cells on a mouse model of bleomycin-induced lung fibrosis. Fetal membrane-derived cells were infused 15 min after intratracheal bleomycin instillation. Different delivery routes were used: intraperitoneal or intratracheal for both xenogeneic and allogeneic cells, and intravenous for allogeneic cells. The effects of the transplanted cells on bleomycin-induced inflammatory and fibrotic processes were then scored and compared between transplanted and control animals at different time points. By PCR and immunohistochemistry analyses, we demonstrated the presence of transplanted cells 3, 7, 9, and 14 days after transplantation. Concomitantly, we observed a clear decrease in neutrophil infiltration and a significant reduction in the severity of bleomycin-induced lung fibrosis in mice treated with placenta-derived cells, irrespective of the source (allogeneic or xenogeneic) or delivery route. Our findings constitute further evidence in support of the hypothesis that placenta-derived cells could be useful for clinical application, and warrant further studies toward the use of these cells for the repair of tissue damage associated with inflammatory and fibrotic degeneration.
Key words: Fetal membrane-derived cells; Placenta; Amnion; Cell transplantation; Lung fibrosis; Inflammation
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: firstname.lastname@example.org or email@example.com
DAPI Diffusion After Intravitreal Injection of Mesenchymal Stem Cells in the Injured Retina of Rats
Paula Castanheira,1 Leonardo Torquetti Torquetti,2 Débora Rodrigues Soares Magalhãs,1 Marcio B. Nehemy,2 and Alfredo M. Goes1
1Department of Biochemistry and Immunology, Biological Sciences
Institute, Federal University of Minas Gerais, Belo Horizonte, Brazil
2Department of Ophthalmology, Medicine Faculty, São Geraldo Eye Hospital, Federal University of Minas Gerais, Belo Horizonte, Brazil
To evaluate DAPI (4´,6-diamidino-2-phenylindole) as a nuclear tracer of stem cell migration and incorporation it was observed the pattern of retinal integration and differentiation of mesenchymal stem cells (MSCs) injected into the vitreous cavity of rat eyes with retinal injury. For this purpose adult rat retinas were submitted to laser damage followed by transplantation of DAPI-labeled BM-MSCs grafts and double-labeled DAPI and quantum dot-labeled BM-MSCs. To assess a possible DAPI diffusion as well as the integration and differentiation of DAPI-labeled BM-MSCs in laser-injured retina, host retinas were evaluated 8 weeks after injury/transplantation. It was demonstrated that, 8 weeks after the transplant, most of the retinal cells in all neural retinal presented nuclear DAPI labeling, specifically in the outer nuclear layer (ONL), inner nuclear layer (INL), and ganglion cell layer (GCL). Meanwhile, at this point, most of the double-labeled BM-MSCs (DAPI and quantum dot) remained in the vitreous cavity and no retinal cells presented the quantum dot marker. Based on these evidences we concluded that DAPI diffused to adjacent retinal cells while the nanocrystals remained labeling only the transplanted BM-MSCs. Therefore, DAPI is not a useful marker for stem cells in vivo tracing experiments because the DAPI released from dying cells in moment of the transplant are taken up by host cells in the tissue.
Key words: Mesenchymal stem cells; Stem cell-based therapy; Retinal damage; Retinal regeneration; DAPI; Quantum dot
Address correspondence to Alfredo M. Goes, Department of Biochemistry and Immunology, Biological Sciences Institute, Universidade Federal de Minas Gerais, Ave. Antonio Carlos, 6627 ICB, Q4-167 zip code 31.270-901, Belo Horizonte, Minas Gerais, Brazil. Tel: 55 (31) 34092632; E-mail: firstname.lastname@example.org
A Novel Strategy Incorporated the Power of Mesenchymal Stem Cells to Allografts for Segmental Bone Tissue Engineering
Xiao Hui Zou,1* Hong Xin Cai,2* Zi Yin,3 Xiao Chen,3 Yang Zi Jiang,3 Hu Hu,4 and Hong Wei Ouyang2,3
1Women Hospital, School of Medicine, Zhejiang University,
2Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, China
3Center for Stem Cell and Tissue Engineering, School of Medicine, Zhejiang University, China
4Department of Pathology and Pathophysiology, School of Medicine, Zhejiang University, China
Mesenchymal stem cells (MSCs) hold great promise for bone regeneration. However, the power of mesenchymal stem cells has not been applied to structural bone allografts in clinical practice. This study designed a new strategy to enhance the efficiency of allografts for segmental bone regeneration. Isolated MSCs were cultured to form a cell sheet. The MSC sheet was then wrapped onto structural allografts. The assembled structures were cultured in vitro to evaluate the differentiation potential of MSC sheet. The assembled structures were implanted subcutaneously into nude mice as well as into the segmental radius defect of rabbits to investigate the efficiency of MSC sheets to repopulate allografts for bone repair. MSC sheets, upon assembling on bone grafts, showed similar differentiation properties to the in situ periosteum in vitro. After implantation the MSC sheets accelerated the repopulation of bone grafts in nude mice. Moreover, MSC sheets induced thicker cortical bone formation and more efficient graft-to-bone end fusion at the segmental bone defects in rabbits. This study thus presented a novel, more efficient, and practical strategy for large weight-bearing bone reconstruction by using MSC sheets to deliver large number of MSCs to repopulate the bone allografts.
Key words: Mesenchymal stem cells; Bone allografts; Tissue engineering
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: 0086-0571-88208262; E-mail email@example.com
*Authors have equal contribution to this article.
Bone Regeneration and Neovascularization Processes in a Pellet Culture System for Periosteal Cells
Mari Akiyama and Masaaki Nakamura
Department of Biomaterials, Osaka Dental University, Osaka, Japan
Reliable bone regeneration can be achieved with a pellet culture system using bovine periosteal cells. However, bone regeneration and neovascularization processes in this system have remained unclear. The present study aimed to clarify the extracellular environment and neovascularization process. To detect components of the extracellular matrix secreted by cells and to identify the conditions necessary for bone regeneration in the body, Western blotting and in vivo tests in nude mice were performed. Cells were cultured with or without ascorbic acid and culture supernatant was precipitated. Western blotting showed that culture supernatant contained collagen type I, procollagen type I, and procollagen type I C-terminus when cells were cultured with ascorbic acid. Cells cultured with ascorbic acid formed partial bony tissues at 2 weeks after grafting to nude mice, while bone formation was missing without ascorbic acid. Immunostaining was performed using species-specific vascular endothelial cell markers to ascertain whether vascular endothelial cells were bovine or murine (nude mouse). Immunohistological methods showed vascular endothelial cells in osseous tissue formed in the subcutaneous tissue of nude mice were murine. Extracellular matrix synthesis in vitro and host blood flow in vivo are essential for bone regeneration.
Key words: Bovine periosteal cells; Collagen type I; Procollagen type I; Western blotting; Species-specific vascular endothelial cell markers
Address correspondence to Mari Akiyama, Department of Biomaterials, Osaka Dental University, Hirakata-shi, Osaka 573-1121, Japan. Tel: +81-72-864-3056; Fax: +81-72-864-3156; E-mail: firstname.lastname@example.org
Clinical Use of Fructosamine in Islet Transplantation
Thipaporn Tharavanij,1,2 Tatiana Froud,1,3 Cristiane B. Leitao,1 David A. Baidal,1 Charlotte N. Paz-Pabon,1 Messinger Shari,1,4 Pablo Cure,1 Karina Bernetti,1 Camillo Ricordi,1,3 and Rodolfo Alejandro1,4
1Diabetes Research Institute, University of Miami, Miami,
2Department of Medicine, Thammasat University, Pratumthani, Thailand
3Department of Surgery, University of Miami, Miami, FL, USA
4Department of Medicine, University of Miami, Miami, FL, USA
Many islet transplant recipients have medical conditions that could interfere with the accuracy of HbA1c measurements (e.g., anemia/dapsone use). Fructosamine is less prone to have clinical interferences and reflects glucose control in a shorter period of time than HbA1c. This study aimed to validate fructosamine use in islet transplant subjects and to evaluate its effectiveness as a predictor for islet graft dysfunction. Thirty-three islet transplant recipients who had concomitant fructosamine and HbA1c data available were retrospectively analyzed. HbA1c, fructosamine, mean capillary blood glucose, and islet graft function (fasting Cpeptide/ glucose ratio) were assessed. There was a significant and positive association between fructosamine and HbA1c (p < 0.0001). Both variables were also positively associated with mean overall and fasting capillary glucose. Neither fructosamine nor HbA1c was shown by ROC analysis to significantly discriminate between periods with and without subsequent graft dysfunction. HbA1c >6% was predictive of this outcome 1 month in advance (OR 2.95, p = 0.003). However, although significantly associated with graft dysfunction, use of this cutoff as a predictor of dysfunction has poor sensitivity (50%) and specificity (77.6%). Fructosamine above the normal range (>270 mmol/L Quest Diagnostics) was also predictive of ensuing dysfunction (OR 2.47, p = 0.03); however, it had similarly poor sensitivity (62%) and specificity (64%). Fructosamine can be used as an alternative to HbA1c for glycemic assessment in islet transplant recipients in situations with HbA1c assay interference. Neither HbA1c nor fructosamine are good predictors of islet graft dysfunction.
Key words: Fructosamine; HbA1C; Islet transplantation; Diabetes; Graft dysfunction
Address correspondence to Rodolfo Alejandro, M.D., Diabetes Research Institute, University of Miami, 1450 NW 10th Avenue (R-134), Miami, FL 33136, USA. Tel: (305) 243-5324; Fax: (305) 243-1058; E-mail: email@example.com
Reversible Regulation of Cell Cycle-Related Genes by Epigallocatechin Gallate for Hibernation of Neonatal Human Tarsal Fibroblasts
Jung Yoon Bae,1 Jun Kanamune,1 Dong-Wook Han,1,2 Kazuaki Matsumura,1 and Suong-Hyu Hyon1
1Department of Medical Simulation Engineering, Research Center
for Nano Medical Engineering, Institute for Frontier Medical Sciences,
Kyoto University, Kyoto 606-8507, Japan
2Department of Nanomedical Engineering, College of Nanoscience & Nanotechnology, Pusan National University, Pusan 609-735, Korea
We investigated the hibernation effect of epigallocatechin-3-O-gallate (EGCG) on neonatal human tarsal fibroblasts (nHTFs) by analyzing the expression of cell cycle-related genes. EGCG application to culture media moderately inhibited the growth of nHTFs, and the removal of EGCG from culture media led to complete recovery of cell growth. EGCG resulted in a slight decrease in the cell population of the S and G2/M phases of cell cycle with concomitant increase in that of the G0/G1 phase, but this cell cycle profile was restored to the initial level after EGCG removal. The expression of cyclin D1 (CCND1), CCNE2, CCN-dependent kinase 6 (CDK6), and CDK2 was restored, whereas that of CCNA, CCNB1, and CDK1 was irreversibly attenuated. The expression of a substantial number of genes analyzed by cDNA microarray was affected by EGCG application, and these affected expression levels were restored to the normal levels after EGCG removal. We also found the incorporation of FITC-EGCG into the cytosol of nHTFs and its further nuclear translocation, which might lead to the regulation of the exogenous signals directed to genes for cellular responses including proliferation and cell cycle progression. These results suggest that EGCG temporarily affects not only genes related to the cell cycle but also various other cellular functions.
Key words: Epigallocatechin-3-O-gallate; Neonatal human tarsal fibroblasts; Hibernation; Cell cycle; cDNA microarray
Address correspondence to Suong-Hyu Hyon, Ph.D., Department of Medical Simulation Engineering, Research Center for Nano Medical Engineering, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. Tel: 81-75-751-4109; Fax: 81-75-751-4141; E-mail: firstname.lastname@example.org
A Simple Breeding Protocol for the Procurement of Accurately Staged Rat Donor Embryos for Neural Transplantation
U. M. Weyrauch, E. M. Torres, A. L. Baird, and S. B. Dunnett
Department of Biosciences, Cardiff University, Cardiff, UK
Obtaining accurately staged rat embryos can be difficult because of the variety of breeding protocols employed and because precise staging cannot be confirmed until excision of the embryos from the dam. The detection of estrus, pairing of animals, and confirmation of pregnancies is generally left to commercial suppliers, as in-house breeding can be laborious and unpredictable. Here we describe a simple, reliable in-house breeding protocol for the generation of accurately staged embryos as assessed by measurements of average crown to rump length (CRL).
Key words: Rat breeding; Cell transplantation; Estrus cycle; Embryo staging; Donor age
Address correspondence to Ulrike M. Weyrauch, Cardiff University, Biosciences Building, Museum Avenue, Cardiff CF10 3US, UK. Tel: 0044-(0)2920874684; Fax: 0044-(0)2920876749; E-mail: email@example.com
Naive Rat Amnion-Derived Cell Transplantation Improved Left Ventricular Function and Reduced Myocardial Scar of Postinfarcted Heart
Kazuro L. Fujimoto,1 Toshio Miki,1 Li J. Liu,2 Ryotaro Hashizume,1 Stephen C. Strom,3 William R. Wagner,1,4 Bradley B. Keller,2,4 and Kimimasa Tobita2,4
1Department of Surgery, McGowan Institute for Regenerative
Medicine, University of Pittsburgh, Pittsburgh, PA, USA
2Department of Pediatrics, McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA, USA
3Department of Pathology, McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA, USA
4Department of Bioengineering, McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA, USA
Stem cells contained in the amniotic membrane may be useful for cellular repair of the damaged heart. Previously, we showed that amnion-derived cells (ADCs) express embryonic stem cell surface markers and pluripotent stem cell-specific transcription factor genes. These ADCs also possess the potential for mesoderm (cardiac) lineage differentiation. In the present study we investigated whether untreated naive ADC transplantation into the injured left ventricular (LV) myocardium is beneficial as a cell-based cardiac repair strategy in a rat model. ADCs were isolated from Lewis rat embryonic day 14 amniotic membranes. FACS analysis revealed that freshly isolated ADCs contained stage-specific embryonic antigen-1 (SSEA-1), Oct-4-positive cells, and mesenchymal stromal cells, while hematopoietic stem cell marker positive cells were absent. Reverse transcription-PCR revealed that naive ADCs expressed cardiac and vascular specific genes. We injected freshly isolated ADCs (2 x 106 cells suspended in PBS, ADC group) into acutely infarcted LV myocardium produced by proximal left coronary ligation. PBS was injected in postinfarction controls (PBS group). Cardiac function was assessed at 2 and 6 weeks after injection. ADC treatment attenuated LV dilatation and sustained LV contractile function at 2 and 6 weeks in comparison to PBS controls (p < 0.05, ANOVA). LV peak systolic pressure and maximum dP/dt of ADC-treated heart were higher and LV end-diastolic pressure and negative dP/dt were lower than in PBS controls (p < 0.05). Histological assessment revealed that infarcted myocardium of the ADC-treated group had less fibrosis, thicker ventricular walls, and increased capillary density (p < 0.05). The fate of injected ADCs was confirmed using ADCs derived from EGFP(+) transgenic rats. Immunohistochemistry at 6 weeks revealed that EGFP(+) cells colocalized with von Willebrand factor, <gk>a-smooth muscle actin, or cardiac troponin-I. Our results suggest that naive ADCs are a potential cell source for cellular cardiomyoplasty.
Key words: Amnion; Stem cell; Rat; Cardiac function; Acute myocardial infarction
Address correspondence to Toshio Miki, M.D., Ph.D., Department of
Surgery, McGowan Institute for Regenerative Medicine, University of Pittsburgh,
3025 East Carson Street, Suite 238, Pittsburgh, PA 15203, USA. Tel: +1-412-383-7033;
Fax: +1-412-383-9460; E-mail: firstname.lastname@example.org