351 In Vitro Induction of the Hematopoietic Progenitor/Stem Cells from Human ES Cells Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy S136 STEM CEL[.]
STEM CELL THERAPIES I 350 Identification and Characterization of Chondrogenic Progenitor Cells in Adult Skeletal Muscle Guangheng Li, Bo Zheng, Laura B Meszaros, Joseph B Vella, Karin A Corsi, Arvydas Usas, Tomoyuki Matsumoto, Johnny Huard Stem Cell Research Center, Children’s Hospital of Pittsburgh, Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh Introduction: When ectotopic bone formation is induced in skeletal muscle by the addition of bone morphogenic protein (BMP), a chondrogenic phase is typically observed This suggests that there exists chondrogenic cells in skeletal muscle Previously, multipotent cells have been isolated from skeletal muscle vasculature (e.g pericytes, myoendothelial cells) In the present study, we sought to identify these chondrogenic cells and to determine if endothelial cells participate in chondrogenesis METHODS: Transgenic mice with LacZ expressing endothelial cells (FVB/N-Tg(TIE2-lacZ)182Sato/J) were induced to generate ectopic bone in skeletal muscle by injection of AAV-BMP4 Muscle derived cells (MDCs) were then isolated from Fischer rats and sorted by flow cytometry (FACS) and evaluated for their chondrogenic and myogenic potential using immunostaining (desmin, vimentin and MyoD) RESULTS: Endothelial cells (blue) in skeletal muscle vasculature were not observed to participate in the chondrogenic phase Legend: (a) Surface marker profile of freshly isolated MDCs (b) Phenotypic characterization of MDCs (c) Chondrogenic assay demonstrates positive chondrogenic differentiation of MDCs (d, e) Typical chondrocytes which occupy lacunae can be observed in the MDCs pellet DISCUSSION: Despite the isolation of multipotent cells from muscle vasculature our results suggest that endothelial cells not participate in the chondrogenic phase of ectopic bone formation However, chondrogenic cells have previously been isolated from rat MDCs, particularly the fibrogenic cells residing in the muscle fascia (FDCs) (ORS 2007 abstract) In the present study, FACS sorting and subsequent characterization of MDCs demonstrated that MDCs contain at least two populations of cells, one that is chondrogenic and one that is myogenic These chondrogenic cells are the likely candidate that participates in the chondrogenic phase of ectopic bone formation 351 In Vitro Induction of the Hematopoietic Progenitor/Stem Cells from Human ES Cells Legend: a AAV-BMP4 virus was injected into the gluteofemoral muscle pocket of a FVB/N-Tg (TIE2-lacZ)182Sato/J mouse b X-gal staining showed lacZ positive endothelial cells stained in blue c lacZ positive cells were not observed in the newly formed cartilage tissue at 17 days following injection of AAV-BMP4 virus d No lacZ positive cells were observed with higher magnification MDCs contain myogenic cells as well as chondrogenic cells that may arise from peri- and endomysium S136 Ryo Kurita, Rui Kageyama, Yoshie Miura, Takafumi Hiramoto, Tomoko Yokoo, Michiyo Okada, Atsushi Takahashi, Hiroyuki Inoue, Kenzaburo Tani Molecular and Clinical Genetics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan The human embryonic stem (ES) cells are considered to differentiate to three lineage cells in vitro and in vivo as mouse ES cells However, the best condition to induce these cells into the target cells/tissues and the safety of these differentiated cells/tissues remains unknown We have recently reported that the lentivirally transduced ES cells of common marmoset, small nonhuman primate, differentiated efficiently into hematopoietic progenitor cells in vitro without the requirement of stromal cells In this study, we examined whether the lentivirally transduction of tal1/scl induced human ES cells into hematopoietic progenitor cells in vitro, too First of all, we determined the optimal culture condition to induce multilineage hematopoietic progenitor cells from human ES cells of hES-1 cells based on the expressions of Brachyury, Flk1 and CD34 Then we established four human ES cell lines lentivirally expressing tal1/ scl gene The expression of hematopoietic cell markers including CD34, CD235a and CD133 was significantly increased in the embryoid bodies derived from tal1/scl-expressing ES cells cultured in the optimal growth factor condition The number of hematopoietic Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy STEM CELL THERAPIES I progenitor cells increased in two tal1/scl-expressing ES cell lines determined by colony-forming assay In vivo hematopoiesis of the tal1/scl-expressing ES cells in immune deficient mice is now under investigation Our results suggested that the combination of growth factor and tal1/scl gene transduction is highly effective to induce the hematopoietic stem cells from human ES cells at least in vitro 352 Gene Delivery to Human Mesenchymal Stem Cells Using Helper Virus-Free Packaged HSV-1 Amplicons Yasser M El-Sherbini, Mark Stevenson, Leonard W Seymour, Richard W Wade-Martins.2 Clinical Pharmacology, University of Oxford, Oxford, United Kingdom; 2Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom 1 Bone marrow mesenchymal stem cells (MSCs) have recently received much attention as a promising cellular delivery vehicle due to the ease of genetic manipulation and in vitro culturing The multipotency of MSCs also makes them promising candidates for regenerative medicine applications In the present study we evaluate the efficiency of genetically modifying MSCs using large carrying capacity (152 Kb) herpes simplex type (HSV-1) based amplicons In the present study we have used four amplicons, pHSVCMVGL, pHSVEF1GL, pHSVPUBGL and pHSVCA9GL pHSVCMVGL is a DNA plasmid containing the HSV-1 packaging signal (pac) and HSV-1 origin of replication (oriS); it also expresses enhanced green fluorescence protein (EGFP) under the control of the HSV-1 immediate early promoter (IE4/5) and luciferase under the control of the immediate early cytomegalovirus (CMV) promoter pHSVEF1GL, pHSVPUBGL and pHSVCA9GL have been modified from the pHSVCMVGL where the CMV promoter driving luciferase is replaced by the human elongation factor promoter (EF1), Polyubiquitin promoter and Carbonic anhydrase hypoxia specific promoter (CA9) respectively The amplicons successfully transduced MSCs with a transduction efficiency of 6% at MOI 10; however the efficiency of transduction was increased up to 30% at the same MOI by applying a centrifugation force during the transduction The ability of the vector to deliver a functional gene was also proven by strong expression from the EGFP and luciferase reporter genes within cells post transduction The levels of luciferase expression driven by the four promoters varied accordingly EF1, PUB and CA9 was nearly one tenth the expression from CMV However the EF1 promoter showed as much as twice the expression from PUB On the other hand CA9 hypoxic promoter didn’t show any sign of upregulation when hypoxic condition (5% oxgen) was applied comparable to normoxic conditions The transduction with HSV-1 amplicons altered neither the morphological features nor the phenotypic surface markers of the transduced MSCs as shown by fluorescence microscopy and flow cytometry respectively The transduction also did not affect the differentiation plasticity of the engineered cells as we were able to differentiate the transduced cells into adipocytes This study demonstrates the possibilities of using extrachromosomal vectors with large carrying capacity to modify MSCs, thereby avoiding deleterious affect associated with integrating vectors Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy 353 Novel Neural Stem Cell-Mediated Tumor Selective Gene Therapy for Breast Cancer Metastases Donghong Zhao,1 Joseph Najbauer,1 Elizabeth Garcia,1 Marianne Z Metz,1 Carlotta A Glackin,1 Seung U Kim,2 Karen S Aboody.1 City of Hope National Medical Center and Beckman Research Institute, Duarte, CA; 2University of British Columbia, Vancouver, BC, Canada Metastases to multiple organs are the primary cause of mortality in breast cancer patients It is estimated that ∼80% of women who die as a result of breast cancer have lymph node, lung, bone or brain metastases Current therapies cannot effectively eradicate breast cancer after metastasis to distant organs, with a poorer prognosis if bone and brain metastases are present In addition, chemotherapy doses are limited by toxicity to normal tissues Neural stem cells (NSCs) have the potential to overcome obstacles that limit current chemotherapy strategies by targeting therapeutic agents specifically to primary and metastatic breast tumor sites—thus, increasing the therapeutic index of a given drug In effect, NSCs offer a platform for tumor-localized chemotherapy production, thereby decreasing associated toxicities to normal tissues Here, we report a novel in vivo study that exploits the inherent tumor-tropism of NSCs (HB1.F3.CD, an established human NSC line) to effectively target breast tumor metastases in multiple organs, including liver, lung, lymph nodes, and femur This study was done in human breast cancer (MDA-MB-231, MCF-7) immunodeficient mouse models, employing Xenogen imaging and histochemical analysis to show formation of breast tumor metastases in multiple organs, including bone Our data demonstrate that NSCs localize to malignant cells throughout the body, with a greater preference for metastatic tumor sites in the liver, bone marrow, lung and lymph node, as compared to the primary mammary fat pad site, and with minimal NSC localization to normal tissues We also investigated the tropism of NSCs to various human breast cancer lines in vitro When we compared the migration of NSCs to different invasive breast cancer cell lines by Boyden chamber migration assays, we found that NSCs displayed a 3–4-fold increase in migration to highly invasive breast cancer cells (MDA-MB-231, MDA-MB-468, Hs578T) as compared to less invasive breast cancer cells (MCF-7, T47D, ZR75, SKBR3) These data showed that the potency of the NSC-tumor tropism strongly correlated with the invasiveness of the breast cancer cells in vitro To identify the cytokines responsible for attraction of NSCs to breast cancer cells, we screened 79 cytokines for their expression level in these invasive breast cancer cell lines Conditioned media were analyzed using a cytokine antibody array and demonstrated that IL-6 and IL-8 were highly expressed in the most invasive breast cancer cells Neutralizing antibodies against IL-6 inhibited 50% of the NSC migration to tumor-conditioned media, suggesting that IL-6 is an important cytokine mediating this selective, tumor-directed NSC migration, but that other factors are also involved This NSC-mediated therapeutic approach provides a novel tumor-localized strategy to eradicate breast tumor metastases and has significant clinical implications, especially for patients with advanced metastatic disease, who are refractory to currently available treatments S137 ...STEM CELL THERAPIES I progenitor cells increased in two tal1/scl-expressing ES cell lines determined by colony-forming assay In vivo hematopoiesis of the tal1/scl-expressing ES cells in immune... showed that the potency of the NSC-tumor tropism strongly correlated with the invasiveness of the breast cancer cells in vitro To identify the cytokines responsible for attraction of NSCs to breast... normal tissues We also investigated the tropism of NSCs to various human breast cancer lines in vitro When we compared the migration of NSCs to different invasive breast cancer cell lines by Boyden