369 Doxycyclin Inducible Expression of Human Cytidine Deaminase (hCDD) in Hematopoietic Cells Mediates Protection Against Cytosine Arabinoside Ara C In Vitro and In Vivo Molecular Therapy Volume 19, S[.]
STEM CELL THERAPIES II failure In addition to being an alternate source for HSCs, iPS cells also have the advantage of excellent ex vivo viability, making them amenable to both Mpl gene addition and targeted gene correction We are testing these approaches in the Mpl-/- mice, a disease model which demonstrates both a platelet and stem cell defect Using retroviral vectors expressing Oct 3/4, Sox 2, and Klf-4 we reprogrammed Mpl/fibroblasts into iPS cells In our initial studies the Mpl-/- iPS cells were corrected by transduction with lentiviral vectors expressing a codon-optimized Mpl transgene driven by engineered versions of the Mpl promoter To induce hematopoietic differentiation the corrected iPS cells were first cultured under conditions to generate embryonic bodies and then transduced with a retroviral vector expressing HOXB4-IRES-GFP In some studies murine ES cells were similarly differentiated by HOXB4 overexpression The resultant HSC-like cells were then used to rescue lethally irradiated Mpl-/- recipients Our results show engraftment of repopulating cells that produce progenitor marrow cells which express c-Kit, CD41, and to a lesser extent CD45 In the peripheral blood of these mice almost all of the RBCs and some CD45+ WBCs were shown to be derived from engrafted HSC-like differentiated iPSCs or ESCs Although engraftment appeared robust at early time points most mice developed severe anemia and died by weeks post-transplantation We are now optimizing conditions for generating durable engraftment of HSC-like cells differentiated from ESCs and corrected iPSCs We also plan to evaluate different methods for correcting the Mpl-/- iPSCs including testing different versions of the Mpl promoter to drive expression of a codon-optimized Mpl transgene and the generation of iPSCs that have undergone gene correction by homologous recombination 367 Targeting NF-kB Enhances the Regenerative Potential of Muscle Stem Cells through Multiple Mechanisms Jonathan D Proto,1,2 Aiping Lu,1 Kayla Imbrogno,1 Ying Tang,1 Paul D Robbins,3 Bing Wang,1 Johnny Huard.1 Orthopedic Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA; 2Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA; 3Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA Multiple groups have demonstrated that members of the nuclear factor kappa B (NF-kB) protein family are able to negatively regulate myogenesis Until now, little has been found to suggest a regulatory role for NF-kB in myogenic cells beyond differentiation and growth NF-kB contains five subunits, two of which, a p50/p65 heterodimer, are thought to mediate the suppression of myogenesis In this investigation, we examined the consequences of NF-kB blockade on the regenerative phenotype of muscle-derived stem cells (MDSCs) isolated from the gastrocnemius of p65 haploinsuffient mice (p65+/-) and their wild type littermates (p65+/+) Results thus far have found that p65+/- MDSCs have enhanced myogenic potential in vitro and regenerative capacity in vivo, in accordance with the reports of others studying myoblasts We also found p65+/- MDSCs to display increases in cell proliferation under normal growth conditions as well as enhanced survival under oxidative stress However, as a pleiotropic transcription factor, growth factors and cytokines are among NF-kB’s target genes We investigated the impact of p65 reduction on MDSC growth factor expression and found that p65+/- MDSCs express higher levels of hepatocyte growth factor Our preliminary data also demonstrates that MDSCs secrete anti-inflammatory factors capable of limiting the expression of inflammatory cytokines in activated macrophages in vitro We have further tested this hypothesis in vivo using a cardiotoxin injury model and found that delivery of p65+/MDSCs post-injury results in engraftments that are associated with reduced inflammation and fiber necrosis compared to p65+/+ MDSC engraftments in injured skeletal muscle Taken collectively, our results suggest that NF-kB/p65 reduction enhances MDSC-mediated muscle Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy regeneration through multiple mechanisms Therefore, NF-kB may be a potential therapeutic target to improve muscle pathology in diseases of degeneration via enhancing not only the myogenic potential of MDSCs, but their anti-inflammatory properties as well 368 Recombinase Strategies To Make and Modify iPS Cells Marisa Karow,1 Christopher L Chavez,1 Alfonso P Farruggio,1 Jonathan M Gesinger,1 Joseph C Wu,1 Yanru Chen-Tsai,1 Michele P Calos.1 Stanford University School of Medicine, Stanford, CA Induced pluripotent stem cells (iPSC) have revolutionized the stem cell field iPSC are most often produced by using retroviruses, but the resulting cells are ill-suited for clinical application Several alternative strategies to make iPSC have been developed, but none encompass within them a simple methodology to facilitate the precise insertion of a therapeutic gene for gene therapy approaches Here we report practical strategies to create and genetically modify murine iPSC that rely on plasmid DNA and the sequential application of three site-specific recombinases PhiC31 integrase was used to insert the reprogramming cassette at preferred locations in the genome, producing iPSC demonstrated to be pluripotent by a full range of in vitro and in vivo criteria, including teratoma formation and generation of chimeric mice The reprogramming plasmid includes the target attP site for a second site-specific integrase, which can be utilized for precise insertion of a therapeutic gene into the target site Cre recombinase can be employed for clean excision of the entire reprogramming cassette and most plasmid sequences We demonstrated the efficient operation of this type of strategy in accessible cell types, including fibroblasts and adipose stem cells Furthermore, to illustrate a clinically relevant example, we generated iPSC from the A/J limb girdle muscular dystrophy mouse model, introduced the therapeutic dysferlin gene, and deleted the reprogramming genes This simple strategy produces pluripotent cells that are corrected for a genetic defect and have the potential to be used in a clinical setting 369 Doxycyclin-Inducible Expression of Human Cytidine Deaminase (hCDD) in Hematopoietic Cells Mediates Protection Against Cytosine-Arabinoside Ara-C In Vitro and In Vivo Nico Lachmann,1 Nils Pfaff,1 Sebastian Brennig,1 Tobias Cantz,2 Axel Schambach,3 Christopher Baum,3 Thomas Moritz.1 REBIRTH Cluster of Excellence, RG Reprogramming, Hannover Medical School, Hannover, Germany; 2REBIRTH Cluster of Excellence, JRG Stem Cell Biology, Hannover Medical School, Hannover, Germany; 3REBIRTH Cluster of Excellence, Department of Experimental Hematology, Hannover Medical School, Hannover, Germany Hematopoietic stem cells (HSCs) genetically modified to overexpress drug resistance genes have been advocated to overcome chemotherapy induced myelosuppression Recently, we demonstrated that overexpression of hCDD from a constitutive spleen focus forming virus (SFFV)-derived promoter protects hematopoietic cells from Ara-C toxicity in vitro and in vivo However, these studies also indicated substantial lymphotoxicity by high level constitutive CDD expression (Rattmann et al Blood, 2006) To circumvent this problem, we now have established a doxycyclin (Dox)-inducible (TET-ON) CDD-expression system and have evaluated this system in murine in vitro and in vivo protection assays In vitro CDD-mediated Ara-C resistance was evaluated in 32D cells as well as primary Linclonogenic progenitor cells In these studies cells were co-transduced with two lentiviral constructs expressing CDD (TET-CDD) and the reverse transactivator protein (rtTA3) In 32D cells Dox-inducible S143 STEM CELL THERAPIES II CDD expression was observed within 24h and protected cells from Ara-C concentrations of up to 5000 nM (applied for 48hrs), whereas control- or untransduced cells died at a 25-fold lower dose After Dox withdrawal transgene expression remained detectable for at least three days Similar protection was observed in primary lin- murine cells and progenitor cell derived colonies were protected from Ara-C doses of 300 to 600 nM while untransduced control cells did not yield colony growth at doses of 50 nM Ara-C or higher In vivo studies were performed by transplanting C57Bl/6 mice with Lin-cells from Rosa26-M2rtTA mice previously transduced with the TET.CDD or a control SIN lentiviral vector Transgene expression was induced by Dox administration starting four weeks post transplantation In this model Dox administration induced stable transgene expression in peripheral blood B-, T- and myeloid cells peaking 15 days after start of administration and remaining detectable for 21 days after Dox withdrawal No significant lymphotoxicity was detected in these studies Moreover, the TET.CDD vector conveyed significant protection against Ara-C (500 mg/kg, d1-4, i.v.) to the hematopoietic system as measured by granulocyte counts (0.26 +/-0.25 versus 0.8 +/0, p=0.02) and platelet counts (584 +/-159 versus 883 +/-194, p=0.02) on day after CTX application Therefore, taken together, our data demonstrate efficient Dox-inducible hCDD expression in 32D and primary murine bone marrow cells in vitro as well as in our murine in vivo bone marrow transplant/ gene transfer model Most importantly, in the latter model Dox-inducible CDD expression not only allowed for significant protection from Ara-C induced myelotoxicity but also abrogated the lymphotoxicity observed previously with high and constitutive hCDD expression 370 Enhanced Cell Survival, Differentiation and Down-Regulation of Hypoxia-Related Genes by Increased Oxygen Supply Dmitriy Sheyn,1,2 Shimon Benjamin,1 Shiran Ben David,2 Ilan Kallai,1 Yoram Zilberman,1 Anthony Oh,2 Gadi Pelled,1,2 Dan Gazit,1,2 Zulma Gazit.1,2 Hebrew University of Jerusalem, Jerusalem, Israel; 2Cedars-Sinai Medical Center, Los Angeles Introduction: Osteogenesis of mesenchymal stem cells (MSCs) is highly dependent on oxygen supply Reduction of oxygen availability within the scaffold limits osteogenic differentiation We have shown that perfluorotributylamine (PFTBA), a synthetic oxygen carrier, enhances MSC-based bone formation in vivo In this study we aim to explore the mechanism of this phenomenon We hypothesize that a transient increase in oxygen levels generated by PFTBA will affect MSC survival, proliferation and differentiation, thus affecting bone formation and metabolism in vitro and in vivo To test this hypothesis, we incorporated PFTBA into MSC-loaded alginate beads and investigated the effect on oxygen availability, cell survival, osteogenesis and the expression of hypoxia-related genes Materials and Methods: MSCs overexpressing the BMP2 gene were encapsulated in alginate beads that had been supplemented with 10% (w/v) PFTBA A control group consisted of similar cell-loaded beads containing PBS Monitoring of oxygen levels was performed using a needle-type oxygen microsensor S144 -A To evaluate cell growth, cells were retrieved from the beads, counted and stained with PI; then cell death was analyzed using FACS The effect of PFTBA on MSC osteogenesis was measured using an ALP assay Gene expression was evaluated using qRTPCR To verify that the MSCs maintained their osteogenic potential in vivo, alginate beads with or without PFTBA were implanted subcutaneously in C3H/HeN mice The implants were harvested after or weeks and analyzed using µCT Results: Oxygen measurements showed that supplementation of PFTBA significantly increased the available oxygen level during a 96-hour period [figure1B] PFTBAcontaining beads displayed an elevation in cell viability, which was stably preserved throughout weeks of culture [figure1C] Cell death analysis showed that beads with PFTBA contained a significantly lower ratio of dead cells throughout the experiment In addition, cells in the PBS group expressed significantly more hypoxia-related genes such as VEGF, DDIT3 and PKG1 Moreover, we found that PFTBA supplementation led to an increase in the osteogenic differentiation of MSCs based on ALP expression in vitro Similar result was obtained in vivo where the implantation of MSCs in alginate beads resulted in higher bone volume in the PFTBA group, as shown by µCT Conclusions: Our results indicate that there is an oxygen shortage during the first days after cell encapsulation in alginate beads and that PFTBA supplementation increases the local oxygen level in the vicinity of the cells Consequently, cell survival and osteogenesis are increased and hypoxia-related genes are downregulated These results indicate that oxygen supply might accelerate MSC-based bone regeneration 371 In Vitro Reprogramming of Murine Gall Bladder Cells to beta-Like Cells Raymond D Hickey,1 Kathryn M Schubert,1 Jessie S Coleman,1 Feorillo Galivo,1 Eric Lagasse,2 Markus Grompe.1 Oregon Stem Cell Center, OHSU, Portland, OR; 2McGowan Institute, Pittsburgh, PA Current cell therapies for type I diabetes are restricted to transplantation of donor islets, but success has been hindered by both immune rejection of transplanted cells and by lack of donor islets available In theory, a more suitable cell therapy approach would involve an autologous source of cells that could be expanded and reprogrammed in vitro to beta cells, which could then be transplanted back to the patient without the need of long-term immune suppression We hypothesized that gall bladder cells (GBCs) could fulfill these requirements, as removal of the gall bladder is a minimally invasive surgery with no adverse effects Additionally, the gall bladder and pancreas are developmentally related, and therefore we reasoned that these cells would be more readily reprogrammable to beta Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy ... rejection of transplanted cells and by lack of donor islets available In theory, a more suitable cell therapy approach would involve an autologous source of cells that could be expanded and reprogrammed... transgene expression remained detectable for at least three days Similar protection was observed in primary lin- murine cells and progenitor cell derived colonies were protected from Ara- C doses of. .. untransduced control cells did not yield colony growth at doses of 50 nM Ara- C or higher In vivo studies were performed by transplanting C5 7Bl/6 mice with Lin -cells from Rosa26-M2rtTA mice previously