210 IPSC Derived Hematopoietic Progenitor Cells Generated From a Murine Model of Hereditary Pulmonary Alveolar Proteinosis Recapitulate the GM CSF Dependent Functional Defects of the Disease Molecular[.]
PLURIPOTENT STEM CELL THERAPIES 208 CD106 Defines a Population of Myogenic Progenitor-Like Cells Derived from Induced Pluripotent Stem Cells Jonathan M Geisinger,1 Christopher B Bjornson,1 Chunli Zhao,1 Tawny L Neal,1 Michele P Calos.1 Genetics, Stanford University School of Medicine, Stanford, CA Cell-based therapies are attractive for treating degenerative muscle disorders, such as Duchenne muscular dystrophy and limb girdle muscular dystrophy 2B and 2D, due to their potential to not only repair damaged muscle fibers, but also to provide a source of progenitor cells However, cell-based therapies depend on being able to obtain a large enough number of the engraftable cell type, be it myoblasts or satellite cells Induced pluripotent stem cells (iPSCs) can be used to overcome this barrier because they can generate essentially limitless numbers of cells and can differentiate into many different cell types There exist a number of muscle differentiation protocols for obtaining myogenic cells from differentiated iPSC cultures, but it remains somewhat unclear as to what actual cell type the resulting myogenic cells represent This ambiguity makes it more difficult to compare different protocols Thus, we sought to identify potentially myogenic cells in cultures of differentiated iPSCs based on developmental myogenesis and adult satellite cell biology In this work, we utilize an embryoid body formation-based differentiation protocol with mdx mouse iPSCs to generate cultures from which we isolated CD106+ Sca-1- cells via FACS, which we refer to as myogenic progenitor-like (MPL) cells This combination of markers has previously been used to isolate murine satellite cells from muscle These iPSC-derived MPL cells express satellite cells markers, as demonstrated by qRT-PCR, flow cytometric analysis, western blotting, and immunocytochemistry Upon prolonged culture, these MPL cells express markers of mature muscle, such as myogenin Additionally, we observed that MPL cells were consistently ~11% of the living sorted cells These data characterize a previously unappreciated, well-defined population of myogenic progenitor-like cells derived from differentiated iPS cells that are potentially useful for personalized and allogenic cell therapy approaches in muscle, lay the groundwork for identifying a similar population of cells in differentiated human iPS cultures, and provides a way to measure the efficiency of different myogenic differentiation treatments 209 Lentivector-Mediated c-MYC Expression Plays a Key Role in Consistent Teratoma Formation upon Transplantation of iPSC-Derived Progenitor Cells Yasuhiro Ikeda,1 Moustafa El-Khatib,1 Ohmine Seiga,1 Egon Jacobus,1 Jason Tonne,1 Yogish Kudva.2 Molecular Medicine, Mayo Clinic, Rochester, MN; Endocrinology, Mayo Clinic, Rochester, MN Derivation of diabetes-specific induced pluripotent stem cells (iPSCs) and their differentiation into functional β-cells can provide the foundation for new diagnostic and therapeutic applications A major concern regarding the use of iPSC-derived cells is the risk of teratoma formation upon transplantation The primary source of teratomas is residual undifferentiated pluripotent cells The use of integrating reprogramming vectors can also increase the risk of tumorigenicity of iPSC progeny, due to insertional mutagenesis or sustained expression of the reprogramming factors Indeed, we have experienced consistent teratoma formation upon transplantation of lenti-iPSC-derived pancreatic progenitor cells into a kidney capsule of immunodeficient mice Of note, removal of the primary tumor by unilateral nephrectomy resulted in recurrent and metastatic tumor development S80 Here, we assessed the mechanism of teratoma formation upon transplantation of iPSC progeny The use of pancreatic progenitor cells from transgene-free (TGF) iPSCs and a modified differentiation protocol, which involved enzymatic dissociation at each step-wise differentiation stage to eliminate residual pluripotent cells, resulted in teratoma-free regeneration of human islets in vivo The use of TGF-iPSCs or the enzymatic dissociation protocol alone reduced the incidence, but did not prevent tumor formation We then tested the incidence of teratoma formation using a TGF-iPSC line, super-infected by lentiviral vectors expressing the four reprogramming factors (4F), c-MYC alone or a marker GFP gene Although transplantation of Lenti-GFP-TGF-iPSC or control TGF-iPSC progeny did not result in tumors, mouse recipients of Lenti-c-MYC- or Lenti-4F-TGFiPSC-derived cells rapidly developed teratoma/solid tumors Thus, the use of both TGF-iPSCs and enzymatic dissociation in iPSC differentiation steps can achieve teratoma-free islet regeneration Our data also indicate that c-MYC expression through an integrated lenti-reprogramming vector plays a crucial role in teratoma formation upon transplantation of iPSC progeny 210 IPSC-Derived Hematopoietic Progenitor Cells Generated From a Murine Model of Hereditary Pulmonary Alveolar Proteinosis Recapitulate the GM-CSF Dependent Functional Defects of the Disease Adele Mucci,1 Nico Lachmann,1 Christine Happle,2 Mania Ackermann,1 Silke Glage,3 Gesine Hansen,2 Thomas Moritz.1 Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany; 2Department of Pediatric Pneumology, Allergology and Neonatology, Hannover Medical School, Hannover, Germany; 3Institute of Pathology, Hannover Medical School, Hannover, Germany Hereditary pulmonary alveolar proteinosis (herPAP) is an extremely rare lung disease resulting from the inability of alveolar macrophages to clear the alveolar spaces from surfactant phospholipids This defect stems from homozygous or compound mutations within the genes encoding the α- or b-chain of the GM-CSF receptor, CSF2RA or CSF2RB, respectively Thus far only a symptomatic treatment is available that consists of intensive antibiotic therapy and repetitive whole lung lavage The invasive nature of the latter approach as well as its assotiation with significant cardiovascular morbidity emphasizes the necessity of alternative therapies Hence, we here investigate the suitability of a gene therapy approach based on hematopoietic cells derived from induced pluripotent stem cells (iPSC) Studies were performed in a murine model for Csf2rb-deficiency (Csf2rb-/- mice) iPSCs were generated from lin- bone marrow cells of Csf2rb-/- mice utilizing lentiviral overexpression of the standard Yamanaka-factors OSKM The resulting PAP-iPSCs displayed all major pluripotency criteria including SSEA-1 expression, alkaline phosphatase activity, endogenous Sox2, Oct4, Klf4, Nanog reactivation, as well as the capacity to differentiate into the three germ layers as assessed by teratoma formation Following an eight-day embryoid-body based differentiation protocol, the PAP-iPSCs gave rise to CD41+ hematopoietic progenitor cells (HPCs) that were capable to differentiate into granulocyte-, monocyte-, and erythrocyte-containing colonies comparable to HPCs derived from control iPSCs However, upon differentiation with GM-CSF, PAP-iPSCs - in contrast to control iPSCs - were unable to form GM-type colonies, recapitulating the defect found in primary lin- bone marrow cells of Csf2rb-/- mice Furthermore, the obtained CD41+ HPCs form both control and PAPiPSCs were differentiated into macrophage-like cells in the presence of M-CSF iPSC-derived macrophages expressed CD45, CD11b and F4/80, exhibited typical chemokine secretion, and activated the transcription factor STAT5 in response to IL-3 and GM-CSF in a similar manner to bone marrow-derived macrophages Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy PLURIPOTENT STEM CELL THERAPIES In summary, we generated murine Csf2rb-deficient iPSC lines, which upon hematopoietic differentiation recapitulated GM-CSF dependent functional defects characteristic of PAP These cells – upon genetic correction - appear as a promising source to test future cell and gene therapy strategies 211 Inducible Caspase 9-Safety Switch for Therapies Using Induced Pluripotent Stem Cells Shigeki Yagyu,1 Valentina Hoyos,1 Francesca Del Bufalo,1 Maksim Mamonkin,1 Wade Tao,1 Carlos A Ramos,1 Malcolm K Brenner.1 Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX Human induced pluripotent stem cells (hiPSC) could be an unlimited source of material for autologous cell therapy and tissue engineering, since the cells can proliferate extensively in vitro and differentiate into multiple lineages Unfortunately, undifferentiated hiPSC can cause teratomas after transplantation, so that complete removal of these tumorigenic cells is essential if hiPSC-based cell therapy is to be safe in clinical practice To control tumorigenic hiPSC, we have stably transduced these cells with a lentiviral vector carrying an inducible caspase (iC9) suicide gene (iC9-iPSC) The gene product can be dimerized by administration of a small molecule chemical inducer of dimerization (CID) that triggers apoptosis through the intrinsic (mitochondrial) pathway The transfer and expression of iC9 to hiPSC did not induce significant apoptosis due to spontaneous dimerization and did not interfere with their selfrenewal and pluripotency Following exposure to 10nM of CID, the iC9-iPSC rapidly apoptosed (residual live cells 24hrs after addition of CID = 0.75%±0.12% vs 82.79%±0.41% in the absence of CID, p