Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright  The American Society of Gene Therapy S132 STEM CELLS identification/isolation of HSCs. However, CD52 is expressed on some cells with colony-forming and NOD/SCID-engrafting capacities, both at the mRNA and membrane protein level. It is not clear how to reconcile the expression of CD52 of HSPCs with the considerable existing laboratory and clinical information that CD52 has not been found to cause myelosuppression, when used for marrow purging and for in vivo treatment of bone marrow transplant patients. 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. 346. The ACE System, a Mammalian Artificial Chromosome Engineering Technology: Delivery and Transgene Expression in Human Adult & Embryonic Stem Cells Sandra L. Vanderbyl, 1 Brent Sullenbarger, 2 Neil MacDonald, 1 Melanie Golembiewski, 2 Carl F. Perez, 1 Harry C. Ledebur, Jr., 1 Larry C. Lasky. 2,3 1 Preclinical Programs, Chromos Molecular Systems, Inc., Burnaby, BC, Canada; 2 Pathology and Internal Medicine, The Ohio State University, Columbus, OH; 3 Center for Stem Cell and Regenerative Medicine, Cleveland, OH. The ACE System is a potent biological engineering system consisting of a mammalian artificial chromosome engineered with multiple site-specific integration sites, expression-optimized shuttle vectors to specifically transfer genes, and a proprietary integrase to catalyze specific incorporation of a payload onto the ACE. ACEs are promising gene delivery vehicles for gene-based cell therapies as they are stably maintained, non-integrating, autonomously replicating, and are easily isolated to high purities by flow sorting. We published the first reports of the transfer and stable transgene expression of a mammalian artificial chromosome into hMSCs and hHSCs (Stem Cells 22:324-33, 2004; Exp Hematol 33:1470-1476, 2005). We will update our progress, including an hMSCs enrichment strategy resulting in stable EPO transgene expression, more than 50 days post EPO-ACE transfer, at levels of 100-200 IU/cell/day. We also report the first successful ACE transfer into human embryonic stem cells (hESCs). We quantified the delivery of IdUrd- labeled ACEs to hESCs, via flow cytometry at 24-48 hours post- transfection, a screening technique that utilizes a FITC-conjugated anti-BrdUrd B44 clone antibody that binds to the IdUrd-DNA adduct. We detected IdUrd-labeled ACEs in 13% of the cells 24-48 hours post-transfer. Gene expression studies are underway. The combination of ACEs and the multipotency of adult & embryonic stem cells represent a unique approach for the study of stem cell fate/biology and for the development of novel gene-based cell therapies. 347. Expression of the N-Formyl Peptide Receptor in Bone Marrow-Derived Human Mesenchymal Stem Cells Anand Viswanathan, 1 Richard G. Painter, 1 Nicholas A. Lanson, Jr., 1 Guoshun Wang. 1 1 Departments of Medicine and Genetics, Gene Therapy Program, Louisiana State University Health Sciences Center, New Orleans, LA. Human mesenchymal stem cells (hMSCs) from adult bone marrow can differentiate into a variety of different cell types including airway epithelial cells, suggesting the theraputic potential of stem cells in tissue injury repair. However, approaches for the efficient recruitment of hMSCs to specific sites have not been established. To this end, we have investigated whether hMSCs express the N-formyl peptide receptor (FPR), which is typically expressed in polymorphonuclear leukocytes and is responsible for the neutrophil chemotactic response. Reverse transcription PCR using FPR specific primers resulted in a positive amplification of this mRNA. Sequencing of the amplicon proved 100% homology to the published FPR sequence. Immunofluorescence staining using a monoclonal antibody against human FPR and flow cytometric analyses demonstrated that hMSCs express FPR at the protein level. Actin polymerization after formylated peptide stimulation is one of the basic cellular responses indicative of the FPR function. We incubated hMSCs with 100nM of N-formyl methionyl leucyl phenylalanine (fMLP). Staining with phalloidin-conjugated fluorescein and flow cytometric analyses showed significant F-actin polymerization in hMSCs. Also, hMSCs were attracted toward fMLP gradients in Boyden chambers and binding of flourescein-labeled formyl-Nle-Leu-Phe-Nle-Tyr-Lys displayed the existence and functionality of the receptor in hMSCs. Thus, we conclude that hMSCs express functional formyl peptide receptors on their surface membrane and are capable of chemotaxis toward their ligand. The expression of the N-formyl peptide receptor on hMSCs, cells not normally associated with immune responses, could serve as a mechanism of directing stem cells to the sites of inflammation. Therefore, endogenous expression or over-expression of the receptor within hMSCs could be manipulated in a manner to target these cells to specific inflammatory sites, such as the lungs in cystic fibrosis. Funded in part by CFF and NIH. We thank Drs. Darwin J. Prockop and Bruce Bunnell at Tulane University for providing human hMSCs, technical support and advice. 348. Kruppel-Like Factor 4 Regulates Proliferation of Human and Mouse Hematopoietic Stem-Progenitor Cells, but Is Not Essential for Mouse Hematopoietic Repopulation Jonathan K. Alder, 1 Robert W. Georgantas, 1 Richard L. Hildreth, 1 Curt I. Civin. 1 1 Oncology, Johns Hopkins University School of Medicine, Baltimore, MD. Quiescence is a key characteristic of hematopoietic stem cells (HSCs), postulated to prevent premature exhaustion of the stem cell pool and to protect HSCs from insults such as proliferative stress, irradiation, and chemotoxins. Similarly, quiescence is also thought to play a role in chemoprotection of “cancer stem cells” postulated to give rise to various hematologic malignancies. Understanding of pathways that regulate the cell cycle may contribute to our ability to genetically modify and expand HSC. Several Kruppel-like factor family members, including KLF1, KLF2, KLF3, and KLF6 have been shown to have pivotal roles in hematopoiesis. Experiments in zebrafish have suggested that KLF4 may also play a role in hematopoiesis. Here we found that enforced expression of KLF4 in hematopoietic cells induced cell cycle arrest without triggering apoptosis. Based on the high levels of expression of KLF4 in mouse and human hematopoietic stem-progenitor cells (HSPCs) and that KLF4 has previously been shown to regulate p21 cip1/waf1 , we hypothesized that KLF4 regulates proliferation of these cells through regulation of p21 cip1/waf1 . We next examined hematopoiesis of KLF4 -/- mice to test whether lack of KLF4 led to HSPC expansion or led to other hematopoietic abnormalities. However, KLF4 -/- fetal liver cells had normal numbers of all mature lineages and provided normal radioprotection, similar to wild type controls. Furthermore, in long-term competitive repopulation assays, KLF4 -/- mouse HSPCs demonstrated hematopoietic potency