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

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[.]

Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy

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PLURIPOTENT STEM CELL THERAPIES

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

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 fi bers, 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 diffi cult 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,

fl ow 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-defi ned 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 effi ciency of different myogenic

differentiation treatments

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

1 Molecular Medicine, Mayo Clinic, Rochester, MN;

2 Endocrinology, Mayo Clinic, Rochester, MN.

Derivation of diabetes-specifi c 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 immunodefi cient mice Of note, removal of the primary tumor by

unilateral nephrectomy resulted in recurrent and metastatic tumor

development

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 modifi ed 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-TGF-iPSC-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

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

1 Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany; 2 Department of Pediatric Pneumology, Allergology and Neonatology, Hannover Medical School, Hannover, Germany; 3 Institute 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 signifi cant 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-defi ciency (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 PAP-iPSCs 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

PLURIPOTENT STEM CELL THERAPIES

In summary, we generated murine Csf2rb-defi cient 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

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

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 9 (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 signifi cant apoptosis due

to spontaneous dimerization and did not interfere with their

self-renewal 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<0.01) The induction of iC9-iPSC cell death by CID exposure was

completely inhibited by the pan-caspase inhibitor, qVD-Oph (% live

cells; 93.62±0.97%), suggesting that this cytotoxic effect was caused

by the enforced dimerization of caspase 9 by CID We subsequently

generated mesenchymal stromal cells from iC9-iPSC (iPSC-MSC),

by culturing the iPSC with serum-free medium containing the TGFβ

pathway inhibitor, SB-431542 Flow cytometric analysis showed

these iPSC-MSCs expressed MSC phenotypic markers (positive

for CD44, CD73, CD90, CD105 and negative for CD14, CD19,

CD34, CD45, HLA-DR), and were also capable of adipocytic and

osteoblastic differentiation in appropriate Finally, iPS-MSCs carrying

iC9 consistently and rapidly underwent robust apoptosis by the

administration of CID (% of live cells; 3.37% in the presence of CID

vs 61.03% in the absence of CID)

In conclusion, we were able to genetically modify iPSC to express

iC9 as a safety switch, without altering the self-renewal properties

or the pluripotency of the cells iC9-iPSC could be efficiently

eliminated in the presence of CID either as primitive cells or following

differentiation into MSC Hence the iC9 suicide gene may allow the

clinical benefi ts of hiPSC therapy while providing an additional safety

mechanism to control adverse events

Disease-Specifi c Induced Pluripotent Stem Cells

Yohei Sato,1,2 Hiroshi Kobayashi,1,2 Takashi Higuchi,1 Hiroyuki Ida,1,2 Susumu Minamisawa,3 Yoshikatsu Eto,4 Takumi Era,5

Shigemi Kimura,6 Toya Ohashi.1,2

1 Gene Therapy, Jikei University School of Medicine, Tokyo, Japan; 2 Pediatrics, Jikei University School of Medicine, Tokyo, Japan; 3 Cell Physiology, Jikei University School of Medicine, Tokyo, Japan; 4 Advanced Clinical Research Center, Institute

of Neurological Disorders, Kanagawa, Japan; 5 Department of Cell Modulation, Kumamoto University, Kumamoto, Japan;

6 Department of Child Development, Kumamoto University, Kumamoto, Japan.

Pompe disease is an inherited neuromuscular disorder caused by

a genetic defi ciency of acid-alpha-glucosidase (GAA) Based on the clinical phenotype, Pompe disease is classifi ed as infantile or late-onset Hypertrophic cardiomyopathy is usually high in infantile Pompe disease In late-onset Pompe disease, cardiovascular complications such as cardiac hypertrophy and arrhythmia are clinically present; however, cardiac involvement is less frequent and milder than that

in the infantile form Patient/disease-specifi c induced pluripotent stem cells (iPSCs) have been used for disease modeling, drug screening, and cell therapy Huang et al have reported that infantile Pompe disease iPSCs can be differentiated into cardiomyocyte-like cells (Hum Mol Genet 2011) We attempted to generate late-onset Pompe disease iPSCs, which could differentiate into cardiomyocytes, for disease modeling We also attempted to generate gene-corrected Pompe disease iPSCs by using lentiviral gene transfer

GAA was cloned into cDNA-expressing third-generation lentiviral vector (CS2-EF1α-GAA) To assess the transfection effi cacy, Venus,

a YFP variant protein, was also cloned into the vector (CS2-EF1α-Venus) Then, we transfected lentiviral vectors containing GAA and Venus into control iPSCs (TkDA3-4, healthy donor) at MOI

10, 50, and 100 to estimate the optimized titer for gene correction Expressions of GAA and Venus in iPSCs were examined in a dose-dependent manner

Pompe disease iPSCs (HPS0175, 0176, 0177) were generated from

fi broblasts reprogrammed by the Sendai Virus acquired from late-onset Pompe disease patients Control iPSCs (HPS0223) were also generated from healthy donors by using the Sendai Virus Healthy control and Pompe disease iPSCs showed similar pluripotency characteristics, which were observed using immunohistochemistry and RT-PCR

Robust cardiomyocyte differentiation was induced according

to the directed differentiation protocol using the GSK-3 and Wnt inhibitors Beating cardiomyocytes were observed 10 days after the differentiation of healthy control (HPS0223) and Pompe disease iPSCs (HPS0175) Cardiomyocytes derived from Pompe disease iPSCs demonstrated disease-specifi c hallmarks such as high glycogen accumulation and lysosomal enlargement

In late-onset Pompe disease iPSCs, glycogen accumulation and lysosomal enlargement are sustained even after cardiomyocyte differentiation Our results are compatible with those previously published, showing that cardiac hypertrophy sometimes accompanies late-onset Pompe disease Pathological changes in differentiated cardiomyocytes might elucidate the cardiovascular complications

of the late-onset Pompe disease We would like to generate gene-corrected iPSCs and differentiate into cardiomyocyte to evaluate the effi cacy of gene transfer