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Molecular Mechanism of Systemic Delivery of Neural Precursor Cells to the Brain

Molecular Mechanism of Systemic Delivery of Neural Precursor Cells to the Brain: Assembly of Brain Endothelial Apical Cups and Control of Transmigration by CD44

Christine Rampona,b,c, Nicolas Weissa,b,c, Cyrille Debouxd,e, Nathalie Chaverota,b,c, Florence Millera,b,c, Delphine Buchetd,e, Hélène Tricoire-Leignela,b,c, Sylvie Cazaubona,b,c, Anne Baron-Van Evercoorend,e,f, Pierre-Olivier Courauda,b,c

Abstract

Systemically injected neural precursor cells (NPCs) were unexpectedly shown to reach the cerebral parenchyma and induce recovery in various diffuse brain pathologies, including animal models of multiple sclerosis. However, the molecular mechanisms supporting NPC migration across brain endothelium remain elusive. Brain endothelium constitutes the blood-brain barrier, which uniquely controls the access of drugs and trafficking of cells, including leukocytes, from the blood to the brain. Taking advantage of the availability of in vitro models of human and rat blood-brain barrier developed in our laboratory and validated by us and others, we show here that soluble hyaluronic acid, the major ligand of the adhesion molecule CD44, as well as anti-CD44 blocking antibodies, largely prevents NPC adhesion to and migration across brain endothelium in inflammatory conditions. We present further evidence that NPCs, surprisingly, induce the formation of apical cups at the surface of brain endothelial cells, enriched in CD44 and other adhesion molecules, thus hijacking the endothelial signaling recently shown to be involved in leukocyte extravasation. These results demonstrate the pivotal role of CD44 in the trans-endothelial migration of NPCs across brain endothelial cells: we propose that they may help design new strategies for the delivery of therapeutic NPCs to the brain by systemic administration.

 

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Neural Stem Cell Targeting of Glioma Is Dependent on Phosphoinositide 3-Kinase Signaling

Neural Stem Cell Targeting of Glioma Is Dependent on Phosphoinositide 3-Kinase Signaling

Stephen E. Kendalla, Joseph Najbauerb, Heather F. Johnstonc, Marianne Z. Metzb, Shan Lia,c, Marisa Bowersb, Elizabeth Garciab, Seung U. Kimd,e, Michael E. Barishc, Karen S. Aboodyb,c, Carlotta A. Glackina

Abstract

The utility of neural stem cells (NSCs) has extended beyond regenerative medicine to targeted gene delivery, as NSCs possess an inherent tropism to solid tumors, including invasive gliomas. However, for optimal clinical implementation, an understanding of the molecular events that regulate NSC tumor tropism is needed to ensure their safety and to maximize therapeutic efficacy. We show that human NSC lines responded to multiple tumor-derived growth factors and that hepatocyte growth factor (HGF) induced the strongest chemotactic response. Gliomatropism was critically dependent on c-Met signaling, as short hairpin RNA-mediated ablation of c-Met significantly attenuated the response. Furthermore, inhibition of Ras-phosphoinositide 3-kinase (PI3K) signaling impaired the migration of human neural stem cells (hNSCs) toward HGF and other growth factors. Migration toward tumor cells is a highly regulated process, in which multiple growth factor signals converge on Ras-PI3K, causing direct modification of the cytoskeleton. The signaling pathways that regulate hNSC migration are similar to those that promote unregulated glioma invasion, suggesting shared cellular mechanisms and responses.

 

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MSCs are Activated to Reduce Apoptosis in Part by Upregulation and STC-1

TISSUE-SPECIFIC STEM CELLS:

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Multipotent Stromal Cells (MSCs) are Activated to Reduce Apoptosis in Part by Upregulation and Secretion of Stanniocalcin-1 (STC-1)

Gregory J. Block 1, Shinya Ohkouchi 1, France Fung 1, Joshua Frenkel 1, Carl Gregory 1,
Radhika Pochampally 1, Gabriel DiMattia 2, Deborah E. Sullivan 3, Darwin J. Prockop 1*

1 Tulane Center for Gene Therapy, Tulane University Health Sciences Center, New Orleans, LA, 70112
2 London Regional Cancer Program and the Dept. of Oncology, Biochemistry, The University of Western Ontario
3 Tulane University, Department of Microbiology and Immunology, New Orleans LA, 70112
* To whom correspondence should be addressed. E-mail: dprocko@tulane.edu

Stem Cells Express, First published online December 18, 2008


Abstract

Multipotent stromal cells (MSCs) have been shown to reduce apoptosis in injured cells by secretion of paracrine factors, but these factors were not fully defined. We observed that co-culture of MSCs with previously UV irradiated fibroblasts reduced apoptosis of the irradiated cells, but fresh MSC conditioned media was unable reproduce the effect. Comparative Microarray analysis of MSCs grown in the presence or absence of UV irradiated fibroblasts demonstrated that the MSCs were activated by the apoptotic cells to increase synthesis and secretion of stanniocalcin-1 (STC-1), a peptide hormone that modulates mineral metabolism and has pleiotrophic effects that have not been fully characterized. We showed that STC-1 was required but not sufficient for reduction of apoptosis of UV-irradiated fibroblasts. In contrast, we demonstrated that MSC-derived STC-1 was both required and sufficient for reduction of apoptosis of lung cancer epithelial cells made apoptotic by incubation at low pH in hypoxia. Our data demonstrate that STC-1 mediates the anti-apoptotic effects of MSCs in two distinct models of apoptosis in vitro.

 

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Thrombopoietin Inhibits Murine Mast Cell Differentiation

Thrombopoietin Inhibits Murine Mast Cell Differentiation

Fabrizio Martelli, Barbara Ghinassi, Rodolfo Lorenzini, Alessandro M. Vannucchi, Rosa Alba Rana, Mitsuo Nishikawa, Sandra Partamian, Giovanni Migliaccio, Anna Rita Migliaccioa

ABSTRACT

We have recently shown that Mpl, the thrombopoietin receptor, is expressed on murine mast cells and on their precursors and that targeted deletion of the Mpl gene increases mast cell differentiation in mice. Here we report that treatment of mice with thrombopoietin or addition of this growth factor to bone marrow-derived mast cell cultures severely hampers the generation of mature cells from their precursors by inducing apoptosis. Analysis of the expression profiling of mast cells obtained in the presence of thrombopoietin suggests that thrombopoietin induces apoptosis of mast cells by reducing expression of the transcription factor Mitf and its target antiapoptotic gene Bcl2. STEM CELLS 2008;26:912–919

 

Disclosure of potential conflicts of interest is found at the end of this article.

 

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Efficient Differentiation of Functional Hepatocytes from Human Embryonic Stem Cells

Efficient Differentiation of Functional Hepatocytes from Human Embryonic Stem Cells

Sadhana Agarwal, Katherine L. Holton, Robert Lanza

ABSTRACT

Differentiation of human embryonic stem cells (hESCs) to specific functional cell types can be achieved using methods that mimic in vivo embryonic developmental programs. Current protocols for generating hepatocytes from hESCs are hampered by inefficient differentiation procedures that lead to low yields and large cellular heterogeneity. We report here a robust and highly efficient process for the generation of high-purity (70%) hepatocyte cultures from hESCs that parallels sequential hepatic development in vivo. Highly enriched populations of definitive endoderm were generated from hESCs and then induced to differentiate along the hepatic lineage by the sequential addition of inducing factors implicated in physiological hepatogenesis. The differentiation process was largely uniform, with cell cultures progressively expressing increasing numbers of hepatic lineage markers, including GATA4, HNF4 alpha, alpha-fetoprotein, CD26, albumin, alpha-1-antitrypsin, Cyp7A1, and Cyp3A4. The hepatocytes exhibited functional hepatic characteristics, such as glycogen storage, indocyanine green uptake and release, and albumin secretion. In a mouse model of acute liver injury, the hESC-derived definitive endoderm differentiated into hepatocytes and repopulated the damaged liver. The methodology described here represents a significant step toward the efficient generation of hepatocytes for use in regenerative medicine and drug discovery.

 

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Embryonic Stem Cells as a Platform for Analyzing Neural Gene Transcription

Embryonic Stem Cells as a Platform for Analyzing Neural Gene Transcription

Xiaodong Zhang, Scott A. Horrell, Deany Delaney, David I. Gottlieb

ABSTRACT

There is a need for improved methods to analyze transcriptional control of mammalian stem cell genes. We propose that embryonic stem cells (ESCs) will have broad utility as a model system, because they can be manipulated genetically and then differentiated into many cell types in vitro, avoiding the need to make mice. Results are presented demonstrating the utility of ESCs for analyzing cis-acting sequences using Olig2 as a model gene. Olig2 is a transcription factor that plays a key role in the development of a ventral compartment of the nervous system and the oligodendrocyte lineage. The functional role of an upstream region (USR) of the Olig2 gene was investigated in ESCs engineered at the undifferentiated stage and then differentiated into ventral neural cells with sonic hedgehog and retinoic acid. Deletion of the USR from the native gene via gene targeting eliminates expression in ventral neural cells differentiated in cell culture. The USR is also essential for regulated expression of an Olig2 transgene inserted at a defined foreign chromosomal site. A subregion of the USR has nonspecific promoter activity in transient transfection assays in cells that do not express Olig2. Taken together, the data demonstrate that the USR contains a promoter for the Olig2 gene and suggest that repression contributes to specific expression. The technology used in this study can be applied to a wide range of genes and cell types and will facilitate research on cis-acting DNA elements of mammalian genes.

 

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Comprehensive MicroRNA Profiling Reveals a Unique Human Embryonic Stem Cell Signature Dominated by a

Comprehensive MicroRNA Profiling Reveals a Unique Human Embryonic Stem Cell Signature Dominated by a Single Seed Sequence

Louise C. Laurenta,b, Jing Chenc, Igor Ulitskyd, Franz-Josef Muellerb,e, Christina Lua,b, Ron Shamird, Jian-Bing Fanc, Jeanne F. Loringb

ABSTRACT

Embryonic stem cells are unique among cultured cells in their ability to self-renew and differentiate into a wide diversity of cell types, suggesting that a specific molecular control network underlies these features. Human embryonic stem cells (hESCs) are known to have distinct mRNA expression, global DNA methylation, and chromatin profiles, but the involvement of high-level regulators, such as microRNAs (miRNA), in the hESC-specific molecular network is poorly understood. We report that global miRNA expression profiling of hESCs and a variety of stem cell and differentiated cell types using a novel microarray platform revealed a unique set of miRNAs differentially regulated in hESCs, including numerous miRNAs not previously linked to hESCs. These hESC-associated miRNAs were more likely to be located in large genomic clusters, and less likely to be located in introns of coding genes. hESCs had higher expression of oncogenic miRNAs and lower expression of tumor suppressor miRNAs than the other cell types. Many miRNAs upregulated in hESCs share a common consensus seed sequence, suggesting that there is cooperative regulation of a critical set of target miRNAs. We propose that miRNAs are coordinately controlled in hESCs, and are key regulators of pluripotence and differentiation.

 

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Applying a "Double-Feature" Promoter To Identify Cardiomyocytes Differentiated From HESC

Embryonic Stem Cells/Induced Pluripotent Stem Cells

 

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Applying a "Double-Feature" Promoter To Identify Cardiomyocytes Differentiated From Human Embryonic Stem Cells Following Transposon-Based Gene Delivery

Tamás I. Orbán, Ágota Apáti, Andrea Németh1, Nóra Varga1, Virág Krizsik1, Anita Schamberger1, Kornélia Szebényi1, Zsuzsa Erdei1, György Várady1, Éva Karászi1, László Homolya1, Katalin Német1, Elen Gócza2, Csaba Miskey3, Lajos Mátés3, Zoltán Ivics3, Zsuzsanna Izsvák3, Balázs Sarkadi1*

1Membrane Research Group of the Hungarian Academy of Sciences, Semmelweis University and National Blood Center, Budapest, Hungary
2Genetic Modification Program Group, Agricultural Biotechnology Center, Gödöllalt, Hungary
3Mobile DNA Group, Max-Delbrück Center for Molecular Medicine, Berlin, Germany

email: Balázs Sarkadi (sarkadi@biomembrane.hu)

*Correspondence to Balázs Sarkadi, Membrane Research Group of the Hungarian Academy of Sciences, Semmelweis University and National Blood Center, Dioszegi u. 64., Budapest, H-1113, Hungary

altAuthor contributions: T.I.O.: Conception and design; collection and assembly of data; data analysis and interpretation; manuscript writing; Á.A.: Conception and design; collection and assembly of data; data analysis and interpretation; manuscript writing; A.N.: Collection and assembly of data; N.V.: Collection and assembly of data; V.K.: Collection and assembly of data; A.S.: Collection and assembly of data; K.S.: Collection and assembly of data; Z.E.: Collection and assembly of data; G.V.: Collection and interpretation of data; É.K.: Collection of data; L.H.: Interpretation of data; K.N.: Collection and interpretation of data; E.G.: Interpretation of data; C.M.: Data analysis; L.M.: Interpretation of data; Z.Ivics Data analysis; manuscript writing; Z.Izsvák: Data analysis and interpretation of data; B.S.: Conception and design; financial support; collection and assembly of data; data analysis and interpretation; manuscript writing; final approval of manuscript.
altFirst published online in STEM CELLS Express February 20, 2009.
§Tamás I. Orbán and Ágota Apáti contributed equally to this work.
Phone/Fax: +36 1 372 4353

Funded by:

  • EU FP6-INTHER; Grant Number: LSHB-CT-2005018961
  • OTKA; Grant Number: AT 048986, NK72057, NKFP-1A-060/2004, ETT 405/2006
  • KKK grants

 


Keywords

Sleeping Beauty transposon • human embryonic stem cells • CAG promoter • altdouble-featurealt promoter • cardiomyocytes • lentiviral gene delivery

 


Abstract

Human embryonic stem (HuES) cells represent a new potential tool for cell- and gene-therapy applications. However, these approaches require the development of efficient, stable gene delivery, and proper progenitor cell and tissue separation methods. In HuES cell lines we have generated stable, EGFP-expressing clones using a transposon-based (Sleeping Beauty) system. This method yielded high percentage of transgene integration and expression. Similarly to a lentiviral expression system, both the undifferentiated state and the differentiation pattern of the HuES cells were preserved. By using the CAG promoter, in contrast to several other constitutive promoter sequences (such as CMV, EF1alt, or PGK), an exceptionally high EGFP expression was observed in differentiated cardiomyocytes. This phenomenon was independent of the transgene sequence, methods of gene delivery, copy number, and the integration sites. This altdouble-featurealt promoter behavior, that is providing a selectable marker for transgene expressing undifferentiated stem cells, and also specifically labeling differentiated cardiomyocytes, was assessed by transcriptional profiling. We found a positive correlation between CAG promoter-driven EGFP transcription and expression of cardiomyocyte-specific genes. Our experiments indicate an efficient applicability of transposon-based gene delivery into HuES cells, and provide a novel approach to identify differentiated tissues by exploiting a non-typical behavior of a constitutively active promoter, thereby avoiding invasive drug selection methods.

 

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Stem Cells and Diseases

The Promise of Stem Cells

Studying stem cells will help us understand how they transform into the dazzling array of specialized cells that make us what we are. Some of the most serious medical conditions, such as cancer and birth defects, are due to problems that occur somewhere in this process. A better understanding of normal cell development will allow us to understand and perhaps correct the errors that cause these medical conditions.

Another potential application of stem cells is making cells and tissues for medical therapies. Today, donated organs and tissues are often used to replace those that are diseased or destroyed. Unfortunately, the number of people needing a transplant far exceeds the number of organs available for transplantation. Pluripotent stem cells offer the possibility of a renewable source of replacement cells and tissues to treat a myriad of diseases, conditions, and disabilities including Parkinson's and Alzheimer's diseases, spinal cord injury, stroke, burns, heart disease, diabetes, osteoarthritis and rheumatoid arthritis.

Have Human Embryonic Stem Cells Successfully Treated Any Human Diseases?

Scientists have been able to do experiments with human embryonic stem cells (hESC) only since 1998, when a group led by Dr. James Thompson at the University of Wisconsin developed a technique to isolate and grow the cells. Moreover, Federal funds to support hESC research have been available since only August 9, 2001, when President Bush announced his decision on Federal funding for hESC research. Because many academic researchers rely on Federal funds to support their laboratories, they are just beginning to learn how to grow and use the cells. Thus, although hESC are thought to offer potential cures and therapies for many devastating diseases, research using them is still in its early stages.

Adult stem cells, such as blood-forming stem cells in bone marrow (called Hematopoietic Stem Cells, or HSCs), are currently the only type of stem cell commonly used to treat human diseases. Doctors have been transferring HSCs in bone marrow transplants for over 40 years. More advanced techniques of collecting, or "harvesting," HSCs are now used in order to treat leukemia, lymphoma and several inherited blood disorders.

The clinical potential of adult stem cells has also been demonstrated in the treatment of other human diseases that include diabetes and advanced kidney cancer. However, these newer uses have involved studies with a very limited number of patients.

 

Stem Cells 101

What are Stem Cell Lines?

Stem cell lines are stem cells that have been isolated from tissue or blood and held in liquid Culture Medium under conditions designed to support their growth and Proliferation. Under the correct conditions this proliferation enables substantial expansion of the cell numbers. Following expansion, the stem cell cultures can be harvested, divided into vials and preserved at ultra-low temperatures. This stock of frozen cells is called a cell bank and the freezing process is a crucial stage which enables the cell bank to be stored in a viable and stable state until required. The cells can be thawed and re-cultured for research or therapy. Holding the cells in suspended animation this way also enables extensive quality control and safety testing to be performed before the cells are approved for use. In some cases, such as embryonic stem cells the cultures appear to have the capacity to expand indefinitely, without changing. Such cell cultures are called stem cell lines.

 

Stem Cells 101

 

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