251. PGE2 Increases Lentiviral Vector Transduction Efficiency of Human HSC

FETAL AND ADULT STEM CELLS 250. Rapidly Adhering Cells (RACs) Residing in Skeletal Muscle Contribute to Ectopic Adipose Cell Formation/Accumulation in Skeletal Muscle of Dystrophin/Utrophin-/- Mice

Jihee Sohn, Aiping Lu, Ying Tang, Bing Wang, Johnny Huard.1,2 1 Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA; 2Bioengineering and McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA. 1

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Ectopic fat formation/accumulation and infiltration in skeletal muscle is closely associated with several disorders including Duchenne muscular dystrophy (DMD), a degenerative muscle disorder. However, neither the origin of ectopic adipocytes, nor the mechanism of their formation in disease, is known. Based on a previously published preplate technique, we isolated two types of muscle derived cells; rapidly adhering cells (RACs) and slowly adhering cells (SACs) from skeletal muscle of C57BL (wt) and utrophin/dystrophin double knockout (dys-/-utro-/-, dKO) mice, which emulates the phenotype seen in DMD patients. Previously, we have shown that SACs are myogenic progenitor cells and are mainly involved in muscle fiber regeneration or degeneration. Here, we identify RACs as PDGFR + multipotent, mesenchymal progenitor cells and observe that dKO-RACs display increased adipogenic, osteogenic, and chondrogenic potentials compared to wt-RACs. We also show that, compared to other muscle-derived cell populations from dKO mice, RACs display significantly increased proliferation and adipogenic potentials both in vitro and in vivo. This study suggests that RACs may be the major contributor to ectopic fat cell formation within skeletal muscle in pathological conditions such as DMD. Therefore, targeting RACs to block adipogenesis in skeletal muscle may open new opportunities to treat muscle diseases.

251. PGE2 Increases Lentiviral Vector Transduction Efficiency of Human HSC

Garrett C. Heffner,1 Lauryn Christiansen,1 Francis J. Pierciey, Jr,1 Robert Kutner,1 Mitchell Finer,1 Gabor Veres.1 1 Stem Cell Biology, Bluebird Bio, Inc, Cambridge, MA. Hematopoietic stem cells are the most effective target cell population for gene therapy-based transplantation therapy of genetic diseases. Although lentiviral vectors has been demonstrated a better transduction efficiency than the previously used MLV based retroviral vectors and has a better safety profile based on their integration site preferences, further improvement in lentiviral vector transduction is necessary to successfully treat diseases requiring high level of gene expression and high percentage of corrected cells. In order to identify pathways and soluble factors involved with improved lentiviral transduction balanced with favorable safety and toxicity, gene expression profiling and small molecule screening was performed. We have identified PGE2 as being an important positive mediators of lentiviral transduction of CD34+ cells isolated from mobilized peripheral blood of adults. CD34+ cells transduced with lentiviral vector in the presence of cytokines and 10 uM PGE2 yielded vector copy number (VCN) approximately 2.5-fold elevated over CD34+ cells transduced in the absence of PGE2, and were associated with significant improvements in the percentage CD34+ cells transduced. Transduction of CD34+ cells in the presence of PGE2 had no negative effect on the frequency of colony-forming units in methylcellulose, or on the frequency of long-term culture initiating cells. Improvements in transduction were observed over a range of PGE2 concentrations. Finally, PGE2 was observed to improve transduction of prospectivelyisolated CD34+CD38- hematopoietic stem cells. These data suggest that PGE2-mediated improvements in lentiviral transduction of human CD34+ cells can result in consistently high transduction efficiency and provide benefit in gene therapy applications. S96

252. Effects of Cryopreservation and Donor Age on Cord Tissue and Adipose Tissue Use in Regenerative Medicine

David T. Harris,1 Mahmood S. Choudhery,1 Michael Badowski.1 Immunobiology, University of Arizona, Tucson, AZ.

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Mesenchymal stem cells (MSC) can be isolated from adult adipose tissue (AT) as well as neonatal cord tissue (CT). Both CT and AT can be utilized fresh or banked frozen for later applications. However, no study has been performed to examine these variables. Fresh and thawed CT and AT were expanded for 7-14 days to allow for outgrowth of MSCs. Expanded MSCs were analyzed by FACS. For differentiation, cells were grown in tissue specific differentiation media for 21 days and stained with tissue specific agents. In vitro assays for wound healing and angiogenesis were investigated by scratch and matrigel assays, respectively. FACS analysis showed no differences in expression of CD45, CD73, CD90 and CD105 between CT-MSC and AT-MSC, fresh or frozen. CT-MSC showed more proliferative potential than AT-MSC. Interestingly, when cultured in low numbers to determine colony forming units, CT-MSC showed less CFUs than AT-MSC. Cells from both sources (fresh and frozen) efficiently differentiated into adipose, bone, cartilage and neuronal structures as determined with histochemistry, immunoflourescence and real time RT-PCR. There was no difference in either the number or time of population doublings over the course of the experiment for fresh versus frozen tissues regardless of source. With the exception of osteocalcin, no significant difference was observed between fresh and frozen differentiated MSC. However, donor age had a significant influence on the differentiation capacity of in terms of proliferation, osteogenesis and chondrogenesis as measured by histochemistry, immunofluorescence and real-time qRT-PCR. In conclusion, MSC can easily be obtained from both tissues and it appears that adipose and cord tissues are suitable sources of stem cells for regenerative medicine, whether utilized fresh or previously banked frozen.

253. Amniotic Fluid Stem Cells for the Treatment of Hemophilia A

Chung-Jung Kuo,1 Saloomeh Mokhtari,1 Melisa Soland,1 Shay Soker,1 James Yoo,1 John Owen,2 Mary Ann Knovich,2 Anthony Atala,1 Graca Almeida-Porada,1 Christopher Porada.1 1 Wake Forest Institute for Regenerative Medicine, Winston-Salem, NC; 2Wake Forest University Health Sciences, Winston-Salem, NC. Hemophilia A (HA) is the most common inheritable coagulation deficiency, affecting 1 in 5000 boys. While current therapy for HA offers reliable prophylactic and therapeutic efficacy, it is prohibitively expensive, it does not cure the underlying disease, and it is unavailable to 75% of the world’s HA patients. In addition, 30% of HA patients develop antibodies to FVIII, complicating treatment and increasing morbidity/mortality. Novel therapies, such as gene therapy (GT) and/ or stem cell transplantation (SCT), can promise long-term or even permanent cure of HA following a single treatment. Since nearly 75% of HA patients are born into families with a history of HA, molecular diagnosis can be performed prenatally, making it possible to treat HA before birth. In previous studies, we reported that, after in utero transplantation (IUTx) into fetal sheep, transduced allogeneic bone marrow mesenchymal stem cells (BM-MSC) engraft at high levels within multiple organs, and durable immune tolerance is induced to the vector-encoded proteins. These findings suggest an in utero SCT/ GT strategy would be ideal for treating HA, since it could induce lifelong tolerance to FVIII, overcoming the immune-related hurdles currently hindering HA treatment. Recent studies have provided compelling evidence that MSC-like stem cells exist within the amniotic fluid (AFSC), raising the possibility that these cells could be harvested from the material collected for prenatal HA diagnosis, gene-corrected in vitro, and returned to the fetus to correct HA prior Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy