express therapeutic genes. Fetal tissues could provide a third source of astrocytes for transplantation. These approaches are limited by the use of potentially hazardous viral vectors, the need for multiple surgical procedures or large amounts of fetal tissue, and the restricted proliferative capacity of differentiated neural cell types. Embryonic stem cells could provide an alternative source of transgenic astrocytes that circumvents these difficulties. These cells are totipotent cells obtained from the inner cell mass of the embryo while in the blastocyst stage. 20
Ronald J. Benveniste, Gordon Keller and Isabelle Germano
Yasushi Takagi, Masaki Nishimura, Asuka Morizane, Jun Takahashi, Kazuhiko Nozaki, Junya Hayashi and Nobuo Hashimoto
damage. 4, 13 Embryonic stem cells can be expanded to large numbers while maintaining their potential to differentiate into various somatic cell types of the three germ layers, and the in vitro differentiation of ESCs thus can provide donor cells for transplantation therapies. Indeed, ESCs have been found to differentiate in vitro into many clinically relevant cell types, including hematopoietic cells, cardiomyocytes, insulin-secreting cells, neurons, and glia. 4, 13 Following transplantation into the central nervous system, ESC-derived neural precursors have been
Mahmud Uzzaman, Ronald J. Benveniste, Gordon Keller and Isabelle M. Germano
For gene therapy strategies currently in clinical trials, viral vectors are used to deliver transgenes directly to normal and tumor cells within the central nervous system (CNS). The use of viral vectors is limited by several factors. The aim of this study was to assess whether embryonic stem cell (ESC)–derived astrocytes expressing a doxycycline-inducible transgene can be used as a vector for gene therapy.
The authors generated a pure population of ESC-derived astrocytes carrying a transgene, tumor necrosis factor–related apoptosis-inducing ligand (TRAIL), inserted in the chromosome under the control of a highly regulated doxycycline-inducible expression system. Fully differentiated ESC-derived astrocytes were stereotactically transplanted in the mouse brain, and then cell migration and transgene expression were studied.
The ESC-derived astrocytes started to migrate from the transplant site 48 hours after the procedure. They were found to have migrated throughout the brain tissue by 6 weeks. Transplanted ESC-derived astrocytes expressed the TRAIL transgene after doxycycline induction throughout the duration of the experiment. Teratoma formation was not observed in long-term experiments (12 weeks).
These data show that ESC-derived astrocytes can be used as delivery vectors for CNS tumors. This technique might have a major impact on the treatment of patients with malignant gliomas and a wide spectrum of other neurological diseases.
Sunit Das, Michael Bonaguidi, Kenji Muro and John A. Kessler
E mbryonic stem cells are pluripotent cells derived from the inner cell mass of the early mammalian embryo. Pluripotency distinguishes ES cells from multipotent progenitor cells (also known as adult stem cells), which persist throughout life; these cells are capable of giving rise to a tissue-specific, limited number of cell types. The term “embryonic stem cell” was first introduced in 1981 to distinguish embryo-derived from teratocarcinoma-derived pluripotent cells and was stated to define a cell with the following essential characteristics: 1) derivation
Daniel J. Guillaume and Su-Chun Zhang
-specific manner. 42 These cells differentiate into myelin-producing oligodendrocytes when placed into the dysmyelinated shiverer mouse brain, 58 but appropriately become neurons or glia when grafted into areas of injured brain or spinal cord, 55 suggesting that the fate of these cells is influenced by their environment. Because neural stem/ progenitor cells have relatively restricted differentiation potential compared to cells of lower lineage stages, they appear to have a lower risk of tumor formation. Embryonic Stem Cells Embryonic stem cells possess several features
Isabelle M. Germano, Mahmud Uzzaman, Ronald J. Benveniste, Milana Zaurova and Gordon Keller
to previously published protocols. 6 The Ainv-18 embryonic stem cells (gift from Michael Kyba) constitutively express a doxycycline-binding transcriptional activator fusion protein and include a doxycycline-responsive promoter upstream of a Lox cloning site. 22 Genes cloned into the Lox site by using Cre-assisted recombination 31 are then expressed when the cells are exposed to doxycycline. The ES cells were directed to differentiate into astrocytes by using protocols recently developed in our laboratory. 6 The A172 human glioma cell lines were used to test
Sudhakar Vadivelu, Marina M. Platik, Luke Choi, Molly L. Lacy, Aarti R. Shah, Yun Qu, Terrence F. Holekamp, Daniel Becker, David I. Gottlieb, Jeffrey M. Gidday and John W. McDonald
GLUT-1 was also observed at a distance of 1.5 to 2 mm from the transplant epicenter. Confocal examination revealed that the ESC-derived endothelial cells had aligned with host endothelial cells ( Fig. 3G–K ) to form chimeric microvasculature. 17, 32 Fig. 3. Embryonic stem cell—derived vascular cells survive and create chimeric microvessels. A: Transmission electron micrograph of the basal lamina (arrowheads) demonstrating a GFP-labeled endothelial cell derived from a transplanted ESC. Arrows indicate DAB precipitate. Images of multichannel
Do-Hun Lee, Eun Young Kim, Sepill Park, Ji Hoon Phi, Seung-Ki Kim, Byung-Kyu Cho, Jinho Lim and Kyu-Chang Wang
clinical trials. 2 , 13 , 20 Embryonic stem cells are pluripotent cells isolated from the inner cell mass of blastocysts. 19 , 23 Their characteristics of proliferating indefinitely and differentiating into enormously diverse cell types have attracted much attention from clinicians facing increasing numbers of patients who suffer from various degenerative disorders. Recently, much progress has been made in the application of stem cell therapy to the treatment of disorders such as chronic heart failure, stroke, brain tumor, and Parkinson disease. 5 , 10 Nonetheless
Sudhakar Vadivelu, Daniel Becker and John W. McDonald
To identify and evaluate stem cell–derived oligodendrocytes obtained for cell transplantation therapies, the authors developed a novel model to examine single, adult oligodendrocytes in situ.
Green fluorescent protein–expressing, mouse embryonic stem cells (ESCs) were neural induced and additionally staged in an oligosphere preparatory step for high-yield derivation of oligodendrocyte progenitors. These transplantable, induced progenitors were injected into postnatal Day 2 rat pups, in which spinal cord sections were then examined at 3 and 9 weeks posttransplantation.
Transplanted oligosphere ESCs survived and integrated anatomically into postnatal and adult white matter, generating targeted regions of chimeric spinal cord. A simple model for identifying adult oligodendrocytes in situ is presented, which is suitable for use in further studies examining functional myelination and derivation of oligodendrocytes from genetically engineered ESC lines, including human ESCs. Results from the model presented here demonstrate a unique method for examining transplantable oligodendrocyte progenitors derived from ESCs for repair of white matter disease.
Christopher Thomas Scott
research. The Globalization of Embryonic Stem Cell Research It has been a decade since James Thomson's isolation of hESCs. Yet restrictions on hESC research raise questions about how individual nations will fare in an increasingly fractured legal and political landscape. Studies conducted at Stanford University and the University of Michigan reveal an “innovation gap,” whereby the peer-reviewed literature publication rate for research using hESC lines is increasing in offshore laboratories, but is decreasing in US laboratories. 17 Tracking which lines go where