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Meeting Report The Promises and Challenges of Regenerative Medicine, October 20–22 2004, Kobe, Japan John Morser, Fiona M. McDonald Regenerative Medicine, Nihon Schering K.K. Research Center, Kobe, Japan Abstract This report provides a brief summary of information presented at a workshop on regenerative medicine held in Kobe, Japan, on October 20–22, 2004. A major focus of the workshop was the identification and characterization of adult and embryonic stem cells, including approaches to manipulate
these—in terms both of maintaining stemness and of driving differentiation toward a desired phenotype—and current developments toward their therapeutic use in regenerative medicine. Stem Cells 2005;23:707–709
Meeting Report
potential for rapid and complete self-renewal, making them a very useful model organism for studying the fundamental processes of regeneration. Agata’s group has looked at genes associated with different aspects of the regenerative process, using dsRNA gene knockdown, with particular emphasis on the neural system. They have identified several genes whose knockdown results in disturbances of regeneration such as DjotxA (absence of visual neurons), DjwntA (ectoptic eye formation in the posterior area of the head), clone #721HH (late formation of ectopic eyes in the posterior body), Djnetrin, or cadherin (disturbances in network formation). They have identified a further gene that they have named noudarake (brains everywhere) (Djndk). Djndk encodes for a protein similar to the fibroblast growth factor (FGF) receptor but without a kinase domain, and expression of Djndk in Xenopus oocytes neutralizes the effects of FGF. Although Djndk is expressed only in the head region, its knockdown results in development of ectopic brains all along the body. These findings have led them to propose a model in which, under normal conditions, trapping or capture of brain activators in the head region prevents these from inducing ectopic brain formation in other areas of the body. In the same session, Shin-Ichi Nishikawa (Riken CDB) described the use of the hair follicle melanocyte system to study
An Ernst Schering Research Foundation (ESRF) (Berlin, http:// www.schering-fg.de) workshop, jointly organized with the Riken Center for Developmental Biology (Riken CDB) (Kobe, Japan, http://www.cdb.riken.jp/en/index.html), entitled “The Promises and Challenges of Regenerative Medicine” was held in Kobe on October 20–22, 2004. This workshop, which was number 54 in the series of ESRF workshops, coincided with the official opening of the Nihon Schering Research Center in Kobe, which will concentrate on preclinical research in the area of regenerative medicine. The purpose of the workshop was to bring together a group of invited preclinical and clinical scientists with experts in various fields relevant to regenerative medicine to encourage discussion and generate new ideas. The opening lecture, an overview of perspectives in regenerative medicine, was given by Ronald McKay (National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD), who is also the 2004 winner of the Ernst Schering prize. In session I (Evolution, development, and regeneration), Kiyokazu Agata (Riken CDB) reported on his group’s studies using Planaria as a model organism. These flatworms have a high proportion of pluripotent stem cells and show an unlimited
Correspondence: Fiona McDonald, Ph.D., Regenerative Medicine, Nihon Schering K.K. Research Center, Kobe 650-0047, Japan. Telephone: 81-78-304-7226; Fax: 81-78-304 7242; e-mail:
[email protected] Received November 12, 2004; accepted for publication February 1, 2005. ©AlphaMed Press 1066-5099/2005/$12.00/0 doi: 10.1634/stemcells.2004-0315
Stem Cells 2005;23:707–709 www.StemCells.com
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stem cell–niche interactions. In this system, the stem cell and amplifying compartments are spatially separated, and so melanocyte stem cells here interact only with their environmental niche and not with their progeny (as is the case in the gut or testes). The environment of the niche is essential to maintain stem cell survival in the hibernating state, and the FOXO pathway seems to be involved in this effect. Using single-cell reverse transcription– polymerase chain reaction, they have compared gene expression in melanocyte stem cells and activated melanocytes to identify specific stemness genes and find differences in expression of genes such as SOX10 and Bcl2 (which is required for melanocyte survival but is absent in melanocyte stem cells). There seems to be a general suppression of gene expression in stem cells compared with activated melanocytes, with the exception of ribosomal proteins, which are expressed similarly in both. In session II (Embryonic and adult stem cells), Norio Nakatsuji (Department of Development and Differentiation, Kyoto University, Kyoto, Japan) and Alan Trounson (Monash Institute of Reproduction and Development, Monash University, Victoria, Australia) described the regulations and guidelines governing the generation and use of human embryonic stem (ES) cell lines in Japan and Australia, as well as the current status of characterization and availability of such cell lines. The Kyoto group has also generated nonhuman primate (cynomolgus monkey) ES cell lines and shown that these can be differentiated into, for example, dopaminergic neurons or retinal pigmented epithelial cells. These monkey ES cells can be transfected with yellow fluorescent protein, allowing them to be visualized after differentiation and transplantation. Alan Trounson also described specialized ES cell lines such as those that are trisomic for chromosome 13, triploid Fanconi’s anemia, and Fragile X repeat expansion, which have been generated and can be used to investigate genomic and epigenetic effects for these diseases. Neurospheres generated from human ES cells can be differentiated to neurons or to a glial lineage, and differentiation to astrocytes also occurs on transplantation of human ES cells into rodent brain. Trounson showed that it is also possible to differentiate human ES cells to cardiomyocytes. In discussion, the issue was raised of the possibility of contamination of human ES cells by the murine feeder layers used in their culture. Also in this session, Hideki Taniguchi (Department of Regenerative Medicine, Yokohama City University, Yokohama, Japan) described studies with hepatic and pancreatic stem cells. Single hepatic cells could be identified that formed clonal colonies, with the recloned progeny showing increased albumin and urea production and developing bile duct-like structures. Transplantation of hepatic stem cells from enhanced green fluorescent protein (EGFP) transgenic mice into mice with either liver or bile duct injury resulted in development of EGFP expressing hepatic or bile duct structures. Colony-forming cells isolated from mouse pancreas were found to be mostly cMet+ cKit– CD45 –Ter11a – and to
Regenerative Medicine Workshop express cytokeratin 19. Growing pancreatic stem cells in 3D culture gave rise to islet-like structures. Sessions III and IV (Regeneration in specific indications) concentrated mainly on current or future regenerative approaches for important organ systems. Ruby Ghadially (Department of Dermatology, University of California, San Francisco) described approaches to identify human skin stem cells, with particular focus on the identification of interfollicular epidermal stem cells. Her data suggest that the density of stem cells in the interfollicular epidermis is lower than had been expected (at less than 0.01%). Shigeru Kinoshita (Department of Ophthalmology, Kyoto Prefectural University, Kyoto, Japan) described the characterization of corneal epithelial cells using gene expression analysis. His group has used transplantation of corneal epithelial cell layers grown on amniotic membrane to treat severe corneal injury, using autologous cells for cases with unilateral injury and donor cells for bilateral injury, from which no autologous cells can be obtained. As an alternative to the allogenic transplants for bilateral injury, which require both local and systemic immunosuppression, clinical studies on the use of autologous grafts of noncorneal mucosal layers (generated from oral mucosal epithelium) are ongoing and seem promising. Luis Parada (University of Texas Southwestern Medical Center at Dallas) presented the case for glioblastoma being a stem cell disease. Neurofibromatosis 1–deficient (NF1– /–) patients show a high incidence of astrocytoma, which can progress to intractable glioblastoma multiforme. Conditional knockout of NF1 in the peripheral nervous system of the mouse gave rise to peripheral neurofibroma associated with loss of p53. These tumors showed a very good match (histologically and in terms of molecular markers) to human neurofibroma. Conditional knockout of NF1 in the mouse central nervous system results in 100% incidence of optic glioma. When these animals also carried a p53 mutation, there was a 100% incidence of glioblastoma. In all of the animals, nestin, a marker of neural stem cells, was present in some cells in the region of the lateral ventricles before tumor appearance. In a few animals that had pure tumor because the tumor had invaginated into the ventricles, the tumor contained markers for oligodendrocytes, astroglia, and neurons, implying a stem cell origin. Hiromitsu Nakauchi (Center for Experimental Medicine, Tokyo University, Tokyo) described the role of Polycomb proteins, in particular Bmi-1, in maintenance of cellular memory in hematopoietic stem cells. Bmi-1 knockout mice show a progressive loss of bone marrow hematopoietic stem cells and a failure of long-term marrow repopulation (also by fetal liver cells). Stem cells are present but cannot make large colonies (growth stops after about 8 days in culture), and there is a decrease in the number of mixed colonies, with those that do form being mostly macrophage/neutrophil lineages. Bmi-1– /– CD34 – c-Kit Sca-1 lineage marker (CD34 –KSL) cells showed defective selfrenewal and accelerated differentiation, mostly to macrophages.
Morser, McDonald Re-expression of Bmi-1 can rescue the knockout phenotype and restore the differentiation pattern. Overexpression of HoxB4 or other members of the Polycomb gene family did not show this effect. Bmi-1 seems also to be of importance for other stem cells because defects are apparent in other organ systems. For example, Bmi-1 knockouts show decreased spermatogenesis with increasing age. Yoshiki Sasai (Riken CDB) described investigations into directed neuronal differentiation from uncommitted Xenopus embryonic cells by use of PA6 cells as a feeder layer to provide what he described as stromal-derived inducing activity (SDIA). This drives differentiation of the embryonic cells to a mainly midbrain dopaminergic neuronal phenotype. Differentiation toward sensory-type neurons can be driven by late treatment of the PA6 feeder SDIA-producing cells with BMP4, whereas Wnt blockade can promote differentiation toward rostral neuronal cell types. The factors associated with SDIA activity of PA6 cells have not yet been identified. The SDIA seems to accumulate at the surface of the PA6 cells but is not present in conditioned medium. However, conditioned extracellular matrix can also induce neuronal differentiation, as can coculture on a transwell filter. Exposing primate ES cells to SDIA gives rise to tyrosine hydroxylase–positive dopaminergic neurons, which, when transplanted into a primate model of Parkinson’s disease, result in improvements in motor activity and local dopaminergic function as assessed by positron emission tomography. Takayuki Asahara (Riken CDB) reported on the characterization of endothelial precursor cells (EPCs). Their transplantation into the myocardium of nude rats with acute myocardial infarction resulted in improved myocardial function, decreased fibrosis, and increased capillary density. The isolation of EPCs has resulted in the definition of a new mechanism, vasculogenesis, which is in contrast to angiogenesis. Vasculogenesis is defined as de novo vessel formation by in situ incorporation, differentiation, migration, and/or proliferation of bone marrow–derived EPCs. More recently, the source of EPCs has been widened to include other sources than bone marrow such as in situ tissue-resident EPCs. The final lecture of the workshop was given by Doris Taylor (Center for Cardiovascular Repair, University of Minnesota, Minneapolis). She described the effects of transplantation of autologous skeletal muscle myoblasts into scarred myocardium in
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a rabbit model of acute myocardial infarction. The myoblasts did engraft in the scar tissue, but not in large numbers. The transplant seemed to improve myocardial contractility, although the mechanism underlying this effect is not known. A phase II/III clinical trial in myocardial infarction/heart failure is ongoing. Clinical studies have also been performed using bone marrow–derived stem cells. In this case, the patients who did best were those whose endothelial precursor cells could migrate in vitro. She also described approaches to atherosclerosis prevention using bone marrow–derived mononuclear cells. The plaque burden in apolipoprotein E – /– mice fed a high-fat diet was reduced by administration of bone marrow mononuclear cells from young wild-type mice. The treatment did not reduce lipid levels but did decrease inflammation, as assessed by measurement of systemic interleukin-6. In a rabbit model of high-fat diet combined with balloon injury, neointima formation was again reduced by administration of bone marrow mononuclear cells, in this case also associated with reductions in inflammatory cytokines. The meeting provided a good overview of state-of-the-art stem cell basic research, and in several areas, regenerative approaches using stem cells show therapeutic potential. There was general consensus that significant progress has been made in our ability to identify and characterize stem cells in various organ systems and to manipulate these both in vitro and in vivo to increase their regenerative capacity. Particularly encouraging were the reports that it is becoming increasingly possible to direct differentiation of stem cells toward the desired phenotype and those showing organ repair, with the formation of complex structures after administration of a single precursor cell population. However, it was also clear that our understanding of the processes underlying organ repair is still insufficient to explain the functional effects seen in some of the models presented, particularly in cases in which the precursor cells are obtained from a source other than the target organ. Also, the question of the potential consequences of the use of feeder layers from nonhuman species was raised. A further workshop entitled “Stem Cells in Reproduction and the Brain” is planned for September 2005 in Kobe, Japan. The full proceedings of the workshop will be published by Springer Verlag (Nishikawa S-I, Morser J, eds. Ernst Schering Research Foundation Workshop, Vol. 54, The Promises and Challenges of Regenerative Medicine).
