1049. Human Multipotential Stromal Cells (MSCs) Repeatedly Create Their Own Niches as They Are Expanded and Re-Expanded from Single Cells into Colonies

the dilTerentiation of embryoniccells to the dopaminergic neuronal phenotype.Embryonicstem (ES) cells can either maintain pluripotency or proliferateinto a varietyof pathways, thereby theoretically providing dilTerentiated cells for molecular and cellular studies and for cell-based therapies. However, it has proven very difficult to control the dilTerentiation processes well enough to obtain large numbersof phenotypically homogenous preparations ofcellssuchas pancreaticbeta cells, dopaminergicneuronsand cardiac muscles. A potentiallyusefulapproachto the in vitrohESdilTerentiation toward neuronal phenotypes has come from the description that co-culturing ES cell with the bone marrow stromal PA6 cell line induces a highly efficient differentiation toward the dopaminergic neuron phenotype neurons. We report the patterns of gcne expression of the human H9 ES cell during PA6-induced differentiation in vitro. using the AlTymetrix-based microarray platform.Aller two weeks of co-culture with PA6, we manually isolated the resulting roseuelike structures and further cultured them to produce neurospheres containing neuroectodermal cells that, in turn, differentiate into the neuronal phenotype.We have used RNA extracted from native H9, from spontaneously formed embryoid bodies and from PA6induced neuroectodermal cells and neurospheresto prepare probes for microarrayanalysis. We have confirmed the established stagespecific expression of a number of genes including oct3/4, nestin, N-CAM, SOX2, and PAX6 and have also identified and begun to characterizea numberof additional genes that regulatethis pathway. Identification of the roles of these genes in neuronal differentiation may become useful for a better understanding of these important developmental programs and alsofor the efficient in-vitro generation of differentiated neuronal cells.

1049. Human Multipotential Stromal Cells (MSCs) Repeatedly Create Their Own Niches as They Are Expanded and Re-Expanded from Single Cells into Colonies Joni H. Ylostalo,' Nikolay A. Bazhanov,' Darwin J. Prockop.' 'Center/or Gene Therapy, Tulane University Health Sciences Center. New Orleans, LA.

Human multipotential stromal cells (MSCs) arc plastic adherent cells derived from bone marrow and other tissues. When MSCs are plated in very lowdensitiesthey formcolonies,which showdistinct regions of inner very densely populated cells and outer cells that arc further apart (Figure IA). In the present study MSC colonies were examined in more detail by following the colony formation from a single cell and comparing the transcriptomes of the inner and outer regions of the colonies, MSCs from inner and outer regions of colonies were isolated using laser microdissection and pressurecatapulting(LMPC, Figure IB,C). TotalRNAwas isolated and amplified for microarray assays from 3 inner region (IN) and 3 outer region (OUT) samples. Microarray data analysis revealed a set of genes that could distinguish the IN from OUT samples. Manyof the genes up-regulated in INsamples wereassociated with "extracellular matrix" whilethegenes up-regulated inOUTsamples were associated with "cytoskeleton organization and biogenesis". The expressionof some of the identified genes and other important genes for MSC biology were confirmed with real-time RT-PCR using low density arrays (LDA). This analysis indicated a good correlation between microarrayand real-time PCR data with some genes showing much larger changes on real-time PCR (Figure ID, E). The presence of the protein products of selected up-regulated genes identified were confirmedwith immunocytochemistry. Cells from inside and outside regions of colonies showed similar growth kinetics and clonality,with only small differences. In this study we have shown that the transcriptomeof the cells derived from inside and outside regions of the colony are different, leading to distinct microenvironrnents, that can be re-created by expansion. S400

CLINICAL GENE THERAPY ORAL ABSTRACT SESSION

1050. Combined Modality Therapy of Locally Advanced Pancreas Cancer (LAPC) Integrating Radiation Inducible Gene Therapy with TNFerade™: Preliminary Results of a Randomized Phase 11/111 Trial Neil Senzer,'

'Cancer Research, MGlY Crowley Cancer Research Centers, Dallas, TX.

Background: TNFeradc is a replication-deficient adenovector containingthe humanTNF-lX gene regulatedby a chemo/radiationinduciblepromoter, Egr-I. The dose-selection phase of'this study in 50 patientswith non-resectableLAPCalso evaluatedthe safetyand earlyefficacyofTNFerade + chemoradiation. TheTNFeradeMTD, the dose used for the randomized phase of the trial, was determined to be 4 x 1011 PU.Witha follow-up(FU) rangeof I0.6-25.6months, the Kaplan-Meier meanand mediansurvivalprojections at the MTD were 11.2and 14.6months,respectively, with an 18-month survival rateof34%. 4/5 patientsreassessed assurgicallyresectable achieved pathologically negative margins and 4/11 patients remain alive at time of review. Methods:The amended phase 11/111 trial will enroll a total of 330 patients in a 2:I randomization; TNFerade + chemoradiotherapy (TNI') versus chcmo-radiothcrapy (SOC, standard of care) bothfollowed by gemcitabine/erlotinib maintenance until progression. The 5V,-week randomized treatmentcomponent consistsof continuousinfusion 5-FU(200mg/mvdayx 5 days/wk)and 50.4 Gy radiation/l.8 Gy/28 fractions with or without weekly intratumoral injectionsof 4 x 1011 PUTNFerade.TNFeradewas administeredby image-guided percutaneous transabdominal approach (PTA) in the first 5 I patients and, following safety review, by either PTAor via an ultrasound-guided endoscopic approach (EUS) in the currently accruing patients. Results: Fifty one patients have completed the planned safety assessment phase of the trial (PTA injections only) with 18 randomized to SOC and 33 to TNF. The documentation of 14 vascular events in 9 of33 TNI' patients and 7 events in 7 of 18 SOC patients did not suggest an increased risk in the TNI' arm. Kaplan-Meieranalysis shows a 42.5% absolute increase in overall survival withtheadditionofTNFerade.The l-year survivalis 70.5% TNF versus 28% SOC. Conclusion: These preliminaryresults of a prospectivelyrandomizedclinical assessmentof a replication-deficient adenovectorcontaining the human TNF-lX gene regulated by a chemo/radiation-induciblepromoterintegratedinto the combined modalitytherapyof LAPC suggesta trend in mediansurvival(using 75% confidence limits). The completed and reviewed run-in safety analysis confirms the safety data from prior phase I and II studies. These limited but provocative results support continued accrual as per protocol. Molecular Therapy Yofume 15. Supplement I, .\by 2007 Copyright © '111C American Societyo f Gene TIICr.lpr