353. Biological Comparison of Mesenchymal Stem Cells Derived from Bone Marrow and Adipose Tissue

STEM CELLS 351. Inhibition of Thyroid Hormone Receptors in the Neural Stem Cells of Newborn and Adult Mice In Vivo: Consequences on Target Gene Expression 1

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Zahra Hassani, Gladys Alfama, Jean-Christophe François, Carinne Giovannangeli,2 Barbara A. Demeneix.1 1 Evolution des Régulations Endocriniennes, MNHN USM501/ CNRS UMR5166, Paris, France; 2Régulation et Dynamique des Génomes, MNHN USM503/CNRS UMR5153/ INSERMU565, Paris, France. The discovery of adult neural stem cells (NSC) in the mammalian brain a few years ago raised the possibility of new therapeutic perspectives for the treatment of genetic or inherited neurological disorders. However, little is known about the fundamental biology of this NSC population, and functional analysis of target genes has to be done to better understand the mechanisms underlying their proliferation or differentiation pathways. The tightly localised position of these neural stem cells in the subependymal area lining the lateral ventricles in the brain make them accessible to gene transfer by stereotaxic injections in the lateral ventricles. Knocking down the expression of a target gene by RNA interference (RNAi) in the NSC population of adult mice brains could lead to the determination of the function of a given protein in its in vivo cellular context. After having demonstrated that RNAi technology can be efficient in vivo by stereotaxic injection of small interfering RNAs (siRNAs) in the lateral ventricles of newborn mice (Hassani et al., 2005), we now address the question of whether small hairpin RNAs (shRNAs) could lead to the specific inhibition of a target gene in the same conditions. The advantage in the use of shRNAs is that we actually know that linear polyethylenimine (L-PEI) is able to specifically deliver plasmids to NSC and neural progenitors located in the subventricular zone of adult mice brains (Lemkine et al., 2002) after stereotaxic injections in the lateral ventricles. However, PEI is not an efficient vector for the delivery of siRNAs in the brain in vivo (Hassani et al., 2005). On this basis we injected plasmids carrying small hairpin RNAs complexed by L-PEI to the lateral ventricles of newborn mice brains in order to specifically inhibit the expression of a target gene. Our first observation was that shRNAs under the control of a H1- or a CMV-promoter are not efficient for the inhibition of the luciferase target gene in vivo. Surprisingly, we found that an hybrid CMV-H1 promoter directing the expression of the shRNA gave significantly better inhibition of the target gene than a H1- or CMV- promoter alone. As thyroid hormone tri-iodothyronin (T3) is necessary for proliferation of NSC in vivo (Lemkine et al., 2005), we sought to identify the genes involved in T3-mediated regulation of NSC proliferation. Two genes potentially implicated in NSC proliferation were tested and found to respond to T3 in the newborn mouse brain: CyclinD1 and Sox2. We found that cyclinD1 expression was significantly decreased after T3 treatment of hypothyroid animals, and that this repression involved Thyroid hormone Receptor alpha1 (TRa1). Sox2 is a transcription factor that is a major player in embryonic neurogenesis. Its expression is limited to NSC in the adult. Sox2 was found to be activated by T3 in newborn mice brains. We now aim to determine whether Sox2 is also regulated by T3 via TRa1 or via another TR, and we will assess this question by inhibiting TRa1 with specific shRNAs.

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352. CD34+ Hematopoietic Stem-Progenitor Cell MicroRNA Expression and Function: The miR Circuit Diagram of Differentiation Control Robert W. Georgantas,1 Richard L. Hildreth,1 John Alder,1 Carlo Croece,3 George A. Calin,3 Curt I. Civin.1,2 1 Divison of Immunology and Hematopoiesis, Kimmel Comp Cancer Center at Johns Hopkins, Baltimore, MD; 2Department of Pediatrics, Johns Hopkins School of Medicine, Baltimore, MD; 3 Department of Molecular Virology, Immunology, and Medical Genetics, Comprehensive Cancer Center at Ohio State Univ, Columbus, OH. Since lin-4, the founding member of the class, was described, microRNAs (miRs) have become a recently realized class of epigenetic elements which modify translation of mRNA to protein, and which also may result in gene silencing through chromatin remodeling. First discovered in C. elegans, miRs have been identified in numerous other organisms including drosophila, rat, mouse, and humans. To date, they have been shown to control cellular metabolism, apoptosis, differentiation and development. Even more importantly aberrant expression of miRs and deletion of miRs are highly associated with the development of various cancers. To better understand the role of miRs in normal hematopoiesis we have determined the microRNA expression profile of primary normal human PBSC and bone marrow CD34+ cells. We have combined this data with the extensive mRNA expression data obtained from CD34+ HSPC, CD34+/CD38-/Lin- HSC-enriched, and CD34+/ CD38+/Lin+ HPC-enriched populations in a previous study. (Cancer Research 64:4344) Combining these two datasets into one integrated database has allowed us to intricately examine the global interaction of HSPC mRNAs and microRNAs, and to also predict which miRs are involved with differentiation of the hematopoietic system. These findings offer promising new targets for the study of stem cell differentiation and targets for genetic therapies of hematopoietic stem cells. FINANCIAL DISCLOSURE of Curt I. Civin: The Johns Hopkins University holds patents on CD34 monoclonal antibodies and inventions related to stem cells. Dr. Civin is entitled to a share of the sales royalty received by the University under licensing agreements between the University, Becton Dickinson Corporation and Baxter HealthCare Corporation. The terms of this arrangement are being managed by the Johns Hopkins University in accordance with its conflict of interest policies.

353. Biological Comparison of Mesenchymal Stem Cells Derived from Bone Marrow and Adipose Tissue Reza Izadpanah,1 Cynthia Trygg,1 Christopher Kriedt,1 Bruce Bunnell.1,2 1 Division of Gene Therapy, Tulane National Primate Research Center, Covington, LA; 2Center for Gene Therapy, Tulane University Health Sciences Center, New Orleans, LA. The potential of adult mesenchymal stem cells (MSCs) to differentiate to diverse cell types in vitro and in vivo offers outstanding opportunities to use these cells as therapeutic agents. The biological characteristics of MSCs isolated from two distinct tissues, bone marrow and adipose tissue were evaluated in vitro and in vivo. MSCs derived from human and non-human primate (rhesus monkey) tissue sources were compared. The data indicate that MSCs isolated from rhesus bone marrow (rBMSCs) and human adipose tissue (hASCs) had more similar biologic properties than MSCs of

Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright  The American Society of Gene Therapy

STEM CELLS rhesus adipose tissue (rASCs) and human bone marrow MSCs (hBMSCs). Analyses of in vitro growth kinetics revealed shorter doubling time for rBMSCs and hASCs. rBMSCs and hASCs underwent significantly more population doublings than the other MSCs. MSCs from all sources showed a marked decrease in telomerase activity over extended culture; however they maintained their mean telomere length. All of the MSCs expressed embryonic stem cell markers, Oct-4, Rex-1 and Sox-2 for at least 10 passages. Early populations of MSCs types showed similar multilineage differentiation capability. However, only the rBMSCs and hASCs retain greater differentiation efficiency at higher passages. Overall in vitro characterization of MSCs from these two species and tissue sources revealed a high level of common biologic properties. The bone marrow and adipose tissue derived MSCs were then compared for the plasticity in vivo. In a time course study, the two types of MSCs were transplanted into the lateral ventricles of NIHIII immune deficient mice. The migration and engraftment of MSCs have been evaluated on 2, 4, and 6 weeks post engraftment. Our results indicated that while both cell types engrafted in multiple regions throughout the brain, the adipose tissue derived MSCs persisted in the brain for longer periods of time. The adipose tissue derived MSCs were detected up to six weeks post-transplantation. Since there is no ethical limitation on using adult stem cells, the results of this study suggest that adipose tissue and bone marrow will prove to be important sources of pluripotent stem cells.

354. Genetic Modification of Murine StromalDerived Cells (MSC) in Suspension Culture as a Model for the Rapid Generation of Transplantable Cytoreagents Yung-Wei Pan,1 Tammy Tien Luoh,1 Peter Kurre.1 Pediatrics & Cell and Developmental Biology, Oregon Health & Sience University, Portland, OR. 1

Bone marrow-derived mesenchymal stromal cells (MSC) can contribute to tissue repair after cardiovascular injury, and appear to have immunomodulatory activity in some experimental models. Convenient recovery, propagation, and extensive proliferation characteristics recommend them for a range of cellular therapies, some of which require their stable genetic modification. Others have previously demonstrated that HIV-derived lentiviral vectors efficiently transduce human mesenchymal cells (Zhang XY et al., Mol Ther. 2002 May;5:555-65). However, adherence and ongoing subculture of MSC risk differentiation and may increase phenotypic heterogeneity within the cell product. Our studies explored whether stromal-derived cells can be efficiently transduced without adherence and subculture requirements. Murine (m-) MSC were established from C57BL/6 animals and characterized to ascertain differentiation capacity along adipogenic and osteogenic phenotypes. Cells were transduced with 3rd generation lentiviral vectors expressing GFP from an elongation factor 1 alpha promoter and pseudotyped with vesicular stomatitis virus G (VSV-G) protein. We trypsinized and washed mMSC from subconfluent primary cultures and transduced them in suspension or adherent after replating with subsequent determination of gene transfer, fluorescence intensity, and proviral copy numbers. Results demonstrated that gene transfer in suspension is sensitive to changes in vector- and fibronectin concentration, and routinely exceeded transfer to mMSC exposed in standard adherent culture. We were able to transduce up to 58% of cells after a single 1 hour incubation with vector at multiplicity of infection (MOI) of 10. Consistent with studies in murine fibroblasts, fluorescence intensity tended to increase with MOI. This was mirrored by a rapid rise in proviral copy number (up to 16 copies at MOI 10), determined by real-time PCR. We conducted experiments in mMSC (5th passage) immediately upon thawing, again showing gene transfer in over 50% of target cells. We further confirmed gene transfer rates Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright  The American Society of Gene Therapy

of up to 35% in mMSC progenitors from day 14 initiation culture commonly termed CFU fibroblast (CFU-F)- after a single vector exposure (MOI 10) of murine whole bone marrow immediately upon harvest. Gene transfer and CFU-F clonogenicity after transduction in suspension decreased at MOI greater than 3, suggesting VSV-G Env protein toxicity, previously described by others. In conclusion, our studies demonstrated rapid (1 hour), efficient HIV-lentiviral transduction of mMSC in suspension and evaluated important parameters governing the efficiency of gene transfer under these conditions. We suggest that this protocol will be useful in the context of cellular therapy using MSC, other stromal-derived, or endothelial cells as cytoreagents for the production of secreted proteins - as recently described (Bigger BW et al., Gene Ther. 2006 Jan;13(2):117-26.) for coagulation factor IX released from primary hematopoietic cells.

355. A Platform for Gene Delivery to Embryonic Stem Cells Using HSV April M. Sunyog,1 Darren Wolfe,2 James Wechuck,2,3 David Krisky,2,4 Ying Jiang,2 Joseph Glorioso.2 1 Molecular Virology and Microbiology, University of Pittsburgh, Pittsburgh, PA; 2Molecular Genetics and Biochemistry, University of Pittsburgh, Pittsburgh, PA; 3Bioengineering, University of Pittsburgh, Pittsburgh, PA; 4Pathology, University of Pittsburgh, Pittsburgh, PA. Embryonic development is a complex process involving integrated pathways directing the ordered proliferation, migration, and differentiation of stem cells into a developing embryo. Murine embryonic stem (mES) lines are immortal and homogeneous, and provide a relevant model of embryogenesis via embryoid body formation. An efficient gene delivery system for mES cells will facilitate investigations into the molecular events of embryonic differentiation. Here we describe the engineering and characterization of a replication-defective Herpes simplex virus-1 (HSV-1) vector, JDββ, as a platform gene delivery vehicle. In this vector, the immediate early (IE, α) genes ICP4 and ICP22, and one full genomic joint element have been deleted. IE genes ICP27 and ICP0, whose low-level expression is necessary for transgene expression, have been converted into early (E, β) genes in order to constrain their expression to ICP4-complementing cells, effectively eliminating their expression in normal cells. JDββ does not express toxic viral genes in non-complementing cells including mES cells. JDββ contains a green fluorescent protein (GFP) transgene under the control of the HCMV IE promoter and transduces mES cells with high efficiency. Transduction with JDββ, even at elevated MOI, does not alter subsequent embryoid body formation nor the expression of endogenous germ layer markers during embryoid body formation, as determined by quantitative-PCR analysis. JDββ will be further engineered to express developmentally relevant genes individually or in combination. This vector may be a useful tool for the experimental stem cell and regenerative medicine fields due to the attributes of non-toxicity, large transgene capacity, robust transgene expression, and non-integrating genome.

356. Engineering of Cell Surface Sialic Acids for Polymeric Gene Delivery Yihua Loo,1,2 S.-Gopalan Sampathkumar,1 Prabhani U. Atukorale,1 Kevin J. Yarema,1 Kam W. Leong.2 1 Biomedical Engineering, Johns Hopkins University, Baltimore, MD; 2Biomedical Engineering, Duke University, Durham, NC. Uptake of plasmid vectors through the plasma membrane of a cell is a one of the barriers to efficient non-viral gene delivery. To facilitate uptake, we explored the use of ‘oligosaccharide engineering’ S135