Novel Hyperactive Transposons for Genetic Modification of Induced Pluripotent and Adult Stem Cells:...

Author contributions: E.B.: designed the experiments, characterized the hyperactive transposases by transfection into iPS and adult stem cells, conducted the G-banding analysis, analyzed the data and wrote part of the manuscript; J.M.: performed immunohistochemistry staining, microscopy, FIX expression analysis, analyzed the data and wrote part of the manuscript; A.A.-S.: performed the molecular biology experiments and analyzed the data; L. Ma.:designed the experiments and characterized the hyperactive transposases in myoblasts and MSC; M.Q.: designed and performed the Pax3 coaxed myogenic differentiation of iPS; L. Mátés.: designed and generated the hyperactive transposases and transposons; P.S.-B.: designed and performed the hepatic and cardiomyogenic differentiation of iPS; M.G.: designed and performed the neural and glial differentiation of iPS; B.Y.: performed the adipogenic and myogenic differentiation experiments from MSC and myoblasts, respectively. J.V.: conducted the cytogenetic analysis and the array comparative genomic hybridization (aCGH) analysis; M.Y.R.: contributed to the cardiac differentiation experiments of iPS; E.S.-K. : performed molecular biology and cell culture experiments; Z.I.: designed and supervised the generation of the hyperactive transposases and transposons, interpreted the data; C.V.: designed and supervised the hepatic, cardiomyogenic, neural and glial differentiation experiments, interpreted the data; M.S.: designed and supervised the Pax3 coaxed myogenic differentiation experiments, interpreted the data; Z.I.: designed and supervised the generation of the hyperactive transposases and transposons, interpreted the data; T.V.: designed and supervised the experiments, interpreted the data and wrote the manuscript; M.K.L.C.: designed, supervised and managed the experiments, interpreted the data, wrote the manuscript and coordinated the overall research effort.

Correspondence to: Marinee K.L. Chuah, PhD , e-mail: [email protected] , Phone: +(32) 16 330557; Fax: +(32) 16 345990, Vesalius Research Center, Flanders Institute of Biotechnology (VIB), University of Leuven, Faculty of Medicine, University Hospital Campus Gasthuisberg, Herestraat 49, 3000 Leuven, & Free University of Brussels (VUB), Faculty of Medicine, University Hospital Campus Jette, Laarbeeklaan 101, 1090 Brussel, Belgium; Thierry VandenDriessche, PhD, e-mail: [email protected] , Phone: +(32) 16 330558; Fax: +(32) 16 345990, Vesalius Research Center, Flanders Institute of Biotechnology (VIB), University of Leuven, Faculty of Medicine, University Hospital Campus Gasthuisberg, Herestraat 49, 3000 Leuven, & Free University of Brussels (VUB), Faculty of Medicine, University Hospital Campus Jette, Laarbeeklaan 101, 1090 Brussel, Belgium; Zsuzsanna Izsvák, PhD, e-mail: [email protected], Max Delbruck Center for Molecular Medicine, Robert Rossle Strasse, 10., 13092 Berlin, Germany, Phone: +(49) 30 9406-3510 or -2546; Fax: (49) 30 9406-2547 or -3382; # Corresponding authors, * equal contributions; Received March 15, 2010; accepted for publication August 05, 2010; available online without subscription through the open access option. ©AlphaMed Press 1066-5099/2010/$30.00/0 doi: stem.501

STEM CELLS®

EMBRYONIC STEM CELLS/INDUCED PLURIPOTENT STEM CELLS

Novel Hyperactive Transposons for Genetic Modification of Induced Pluripotent and Adult Stem Cells: A Non-Viral Paradigm for Coaxed Differentiation

Eyayu Belay1,2,*, Janka Mátrai1,2,*, Abel Acosta-Sanchez1,2, Ling Ma1, Mattia Quattrocelli4, Lajos Mátés3, Pau Sancho-Bru4, Martine Geraerts4, Bing Yang1, Vermeesch Joris5, Melvin Yesid Rincón1,2, Ermira Samara-Kuko1,2, Zoltán Ivics3,6, Catherine M. Verfaillie4, Maurilio Sampaolesi4,Zsuzsanna Izsvák3,6,#, Thierry VandenDriessche1,2,#, Marinee K. L. Chuah 1,2,#,

1 Flanders Institute for Biotechnology (VIB), Vesalius Research Center, University of Leuven, Leuven; 2 Free University of Brussels (VUB), Brussels, Belgium ; 3 Max Delbrück Center for Molecular Medicine, Berlin, Germany; 4 Stem Cell Institute, University of Leuven, Leuven, Belgium ; 5 Center for Human Genetics, University Hospital Gasthuisberg, Leuven, Belgium; 6 University of Debrecen, Debrecen, Hungary

Keywords Transposon Sleeping Beauty iPS myoblast stem cell mesenchymal stem cell muscle

ABSTRACTAdult stem cells and induced pluripotent stem cells (iPS) hold great promise for regenerative medicine. The development of robust non-viral approaches for stem cell gene transfer would facilitate functional studies and potential clinical applications. We have previously generated hyperactive transposases derived from Sleeping Beauty, using an in vitro molecular evolution and selection paradigm. We now demonstrate that these hyperactive transposases resulted in superior gene transfer efficiencies and expression in mesenchymal and muscle stem/progenitor cells, consistent with higher expression levels of therapeutically relevant proteins including coagulation factor IX. Their differentiation potential and karyotype was not affected. Moreover, stable transposition could also

be achieved in iPS which retained their ability to differentiate along neuronal, cardiac and hepatic lineages without causing cytogenetic abnormalities. Most importantly, transposon-mediated delivery of the myogenic PAX3 transcription factor into iPS coaxed their differentiation into MYOD+ myogenic progenitors and multinucleated myofibers, suggesting that PAX3 may serve as a myogenic “molecular switch” in iPS. Hence, this hyperactive transposon system represents an attractive non-viral gene transfer platform with broad implications for regenerative medicine, cell and gene therapy.

Transposons for stem cell and iPS engineering

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INTRODUCTION

There is a need to develop efficient and safe non-viral gene transfer approaches for genetic modification of adult stem cells. Unfortunately, non-viral vectors integrate very inefficiently into most primary cells and are rapidly diluted and/or degraded in a dividing stem cell population and its progeny, leading to only transient transgene expression. However, the use of non-viral gene delivery approaches in conjunction with the latest generation transposon technology may potentially overcome these limitations. Transposons derived from Sleeping Beauty (SB) are among the most promising for mammalian gene transfer [1, 2]. SB has been “resurrected” by molecular reconstruction from silent, ancestral transposons found in fish genomes which enabled transposition in mammalian cells [3]. However, transposition of these early-generation SB transposons was still relatively inefficient in most primary mammalian cells including stem cells [4-7]. To overcome this limitation, we have recently generated novel hyperactive transposases from SB using a high-throughput, in vitro molecular evolution and selection paradigm [8]. The particular hyperactive SB transposase SB100X, exhibited

100-fold enhancement of transposition in human cell lines as compared to the originally resurrected SB. Most importantly, SB100X also resulted in a significant enhancement of stable gene transfer efficiencies in CD34+ hematopoietic stem cells (HSC) [1, 8-12]. Hence, it is essential to also assess the performance of SB100X in other adult stem cell types, particularly induced pluripotent stem cells (iPS) and adult stem cells, including mesenchymal stem cells (MSC) and muscle stem/progenitor cells (myoblasts). These distinct stem/progenitor cells are relevant targets for regenerative medicine and gene/cell therapy of genetic and complex diseases, which underscores their clinical relevance.

iPS are promising adult stem cells for regenerative medicine and cell/gene therapy by virtue of their remarkable pluripotency, which resembles that of embryonic stem (ES) cells

[13-25]. They can be genetically corrected and be used as a source of cells capable of differentiating into disease-free transplantable cell types [19, 20, 26-29]. However, the differentiation pathways of iPS are not well understood and gene expression profiling and functional characterizations indicate that there are significant differences between iPS and ES cells [30]. Hence, it is important to demonstrate that paradigms of coaxed differentiation that were initially validated in ES cells can also be applied to iPS. The ability to coax the differentiation of iPS into clinically relevant, transplantable cell types is a key step towards possible clinical applications [31-33]. In particular, it is not known how to direct the differentiation of iPS into muscle progenitor cells, which has therapeutic implications for cell therapy of congenital degenerative muscle diseases, including Duchenne muscular dystrophy [34, 35].

The objectives of the present study therefore are: (i) to assess the performance of these novel hyperactive SB transposases in adult stem/progenitor cells, including MSC, myoblasts and iPS; (ii) to establish proof-of-concept that transposons could be used for coaxed differentiation of iPS. We therefore investigated the consequences of expressing PAX3 on myogenic differentiation [36-38], following transposon-mediated gene delivery in iPS and demonstrated that PAX3 may serve as a myogenic “molecular switch” in iPS that may replicate the normal myogenic developmental program.

METHODS

Characterization of hyperactive transposases in MSC and myoblasts 3.35 g SB transposon encoding GFP, FIX and NeoR, pT2-HB-CMV-GFP-Neo, pT2-HB-CMV-FIX-Neo and 1.68 g hyperactive SB transposases, SB11, SB16X, SB32X, SB80X,

SB100X and the inactive DDE SB (control) were nucleofected into MSC and myoblasts using Nucleofactor I (Lonza) followed by G418 selection (Supplementary Experimental Procedures). Transposon copy number,


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integration sites were determined by quantitative real-time PCR, excision product (EP) assay, splinkerette and LAM PCR followed by sequencing and BlastN analysis using the NCBI database (Supplementary Experimental Procedures and Supplementary Table 1). GFP expression was analyzed by fluorescent microscopy and FACS, FIX expression was assessed by Asserachrome FIX ELISA (Diagnostica Stago, USA). Adipogenic and myogenic differentiation was performed as described in the Supplementary Experimental Procedures. Karyotyping was performed by G-banding and by 1Mb–resolution array comparative genome hybridization (aCGH) (Supplementary Experimental Procedures).

Generation of the GFP+ and DsRed+ iPS Two different mouse iPS cell lines were used in this study. The iPS-Nanog-GFP(originally designated as iPS-MEF-Ng-20D-17) was provided by Dr. S. Yamanaka (Kyoto University, Japan) and expressed GFP from a

Nanog promoter 18 . The other iPS line (originally designated as O59 Oct4neomycin MEF) does not contain the Nanog-GFP construct and was obtained from Dr. R. Jaenisch (MIT, USA). iPS cells (106) were cultured on growth-arrested mouse embryonic fibroblasts (MEF) (106) in a 10cm dish in iPS medium (Supplementary Experimental

Procedures). iPS were nucleofected with 14 gpT2-HB-CMV-GFP-neo transposon

(Supplementary Figure S1A) and 6 g

SB100X or the inactive DDE mutant plasmid using program A-13 of the Nucleofector I (Lonza, Cologne Germany) and mouse ES nucleofection buffer. Transposition efficiency was assessed by FACS and excision assay (Supplementary Experimental Procedures). iPS-Nanog-GFP cells were nucleofected with

20 g pT2-HB-PGK-DsRed transposons and

10 g SB100X plasmids (Supplementary Figure S1A). Following successive subclonings individual colonies were picked from the transfected iPS-Nanog-GFP cells that were uniformly DsRed+. Transposition was confirmed using an excision product PCR-based assay and by sequencing the integration sites in the iPS-Nanog-GFP/DsRed+ cells. Karyotype analysis was performed based on G-

banding and aCGH (Supplementary Experimental Procedures).

Differentiation of iPS-Nanog-GFP/DsRed+

Cardiomyogenic differentiation. Transposed mouse iPS-Nanog-GFP/DsRed+ cells were plated at 20000 cells/cm2 in a low attachment plate (Corning) for EB induction using cardiomyogenic differentiation medium. After 10 days in culture, EB were plated on gelatin-coated plates for 4 days. Contracting cardiomyocytes were detected as of day 12 after EB induction (Supplementary Experimental Procedures). Hepatic differentiation: iPS-Nanog-GFP/DsRed+ cells were plated in 12 well plates at 2500 cells/cm2,pre-coated with 2% matrigel (Becton Dickinson) in 1ml iPS hepatic differentiation medium, 65% of which was changed every other day and completely at day 6 (definitive endoderm), at day 10 (hepatic endoderm) and at day 14 (bipotential hepatoblast). 10% FBS (Hyclone) was added on day 1-2, 2% FBS from day 3-6, and 0.5% FBS from day 7 to 28. The medium was supplemented with cytokines as described in the Supplementary Experimental Procedures. Neural and glial differentiation: Prior to differentiation, iPS-Nanog-GFP/DsRed+ cells were plated on 0.1% gelatin coated plates in mESC medium (Supplementary Experimental Procedures) to eliminate MEFs. The next day, cells were seeded in 0.1% gelatin coated 6-well plates in N2B27 medium at 10000 cells/cm2. 50% medium was changed every 2nd until day 7 (neural induction phase). At day 7, cells were replated in ultra-low attachment T75 flasks (Corning) in NSE medium (neural expansion phase). Fresh cytokines were added every second day until day 14. At day 14 the proliferating neurospheres were plated on 0.1% gelatin in NSE medium to obtain an adherent layer of radial-glial-like proliferating neural stem cells (NSCs). To obtain mature glial cells 50000 NSCs/cm2 were seeded in a 24 well plate coated with 0.1% gelatin in glial differentiation medium [39]. NS cells differentiate to GFAP positive astrocytes within 2 days after exposure to FCS. Under these conditions, there is minimal neural differentiation. For more details on neural and


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glial differentiation see the Supplementary Experimental Procedures. Coaxed differentiation of iPS using PAX3: iPS-Nanog-GFP/DsRed+ cells were nucleofected (program A-13) (Lonza) in mouse ES nucleofection

buffer with the pT2-HB-CMV- -globin-Pax3transposon (14 g) and SB100X or DDE

transposase (6 g). EBs derived using the hanging drop method were seeded on 24 or 6-well plates wells, coated with 1mg/ml calf skin collagen (Sigma), and kept 48h in iPS-medium without LIF, then switched to complete DMEM (Sigma) supplemented with 2% horse serum (Sigma) (Supplementary Experimental Procedures). In all the differentiation experiments, expression of lineage specific genes was assessed by quantitative RT-PCR [40], immunostaining [34] and subsequent microscopic analysis as described in the Supplementary Experimental Procedures.

RESULTS

Characterization of hyperactive transposases in adult stem cells An SB transposon encoding GFP and NeoR,pT2-HB-CMV-GFP-Neo, (Supplementary Figure S1A) was transfected into mesenchymal stem/progenitor cells (MSC) or muscle stem/progenitor cells (myoblasts) by nucleofection together with expression vectors encoding hyperactive SB transposases designated as SB16X, SB32X, SB80X, and SB100X (Supplementary Figure S1C). The denominator refers to the fold-increase in transposition efficiency relative to the originally rederived SB10 transposase [3] based on a marker mobilization assay in HeLa cells [8]. An early generation hyperactive SB11 transposase [4] and a catalytically

inactive DDE SB transposase were also included (Supplementary Figure S1B). Cytofluorimetric analysis 48 hr post-transfection revealed that GFP expression in transfected MSCs and myoblasts did not vary among any of the hyperactive constructs vs.

DDE control or SB11, based on the percentage of GFP+ cells and the mean fluorescent intensity. Typically, transfection efficiencies based on % GFP expression 48 hr post-nucleofection corresponded to 40 + 6 % in MSC and 38 + 6 % in myoblasts.

A significant increase in G418R/GFP+ MSC (Figure 1A) and myoblasts (Figure 1D) was obtained with those constructs encoding the hyperactive transposases (SB16X, SB32X, SB80X, SB100X), as compared to the early-generation SB11 transposase or the inactive

DDE transposase. The most robust increase (i.e. 10-fold; Student t-test, p < 0.001, n=3) was obtained with SB100X. This stable transgene expression resulted from bona fide transposition rather than transposition-independent genomic integration consistent with the canonical transposon integration sites (Supplementary Table 1). Transposon copy number in the transfected MSC and myoblasts was determined by quantitative real-time PCR yielding an average of 3-5 transposons/diploid cells (Supplementary Experimental Procedure and Figures 2A and 2C). The relative transposition efficiency in adult stem cells was further assessed using an excision product (EP) PCR-based assay [41, 42] (Figures 1B and 1E) that measures the accumulation of the donor plasmid from which the transposon has been excised. An average 6 to 10-fold increase in transposition efficiency could be obtained with SB100X compared to SB11, which correlated with the differential transgene expression between SB100X vs. SB11 (Figures 1A and 1D). To further confirm the superior transposition efficiency of SB100X and to validate its clinical relevance, we cotransfected MSC and myoblasts with a SB100X expression construct and a transposon encoding human coagulation factor IX (FIX), pT2-HB-CMV-FIX-Neo, (Supplementary Figure S1A) as a potential gene/cell therapy approach for hemophilia B. This resulted in robust and stable FIX expression levels, as measured by ELISA, which were significantly higher (Student t-test, p<0.001, n=3) than the levels obtained with SB11 (Figures 1C and 1F), indicating that long-term expression of high FIX levels in these transfected stem/progenitor cells was critically depending on efficient transposition with SB100X. We subsequently confirmed the functional FIX activity in the MSC transfected with SB100X and pT2-HB-CMV-FIX-Neo by directly assessing one of the key steps in the coagulation cascade, namely the conversion of factor X into factor Xa by activated FIX (FIXa). Functional FIX levels


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corresponding to 1.25 g FIX per 106 cells per 72 hrs could be obtained with SB100X, corresponding to 50% of the total amount of FIX produced by the transfected MSC (Figure 1C). In contrast, no functional FIX could be detected in MSC that were cotransfected with pT2-HB-CMV-FIX-Neo and the SB11 or

DDE expression plasmid. MSC and myoblasts transfected with SB100X retained their ability to undergo normal differentiation (Supplementary Experimental Procedure and Figures 2B and 2D) [43-45].

Molecular karyotyping by G-banding and by 1Mb–resolution array comparative genome hybridization (aCGH) revealed a normal karyotype in transfected MSC and myoblasts

similar to that of non-transfected or DDE-transfected controls (Figures 3A and 3B).

Characterization of hyperactive transposase (SB100X) in iPS Typically, nucleofection adversely affects cell viability but a condition was selected that was relatively well tolerated by the iPS cells resulting in 90% viability (Supplementary Methods). iPS were transfected with the pT2-HB-CMV-GFP-neo transposon (Supplementary Figure S1A) and an expression vector encoding the hyperactive transposase

SB100X or the inactive DDE mutant. Approximately 20% of the iPS cells were eGFP+ 48 h post-transfection. Subsequent expansion of the SB100X-transfected iPS resulted in the propagation of 5 % GFP+ iPS that stably expressed high levels of GFP, consistent with stable transposition (Figure 4A). Taking into account the actual percentage transfected iPS (i.e. 20% of the total iPS population), this corresponds to a net transposition efficiency of 25%. In contrast, iPS transfected with the catalytically inactive

DDE mutant did not yield any stably expressing GFP+ cells (Figure 4A). Similarly, the mean fluorescent intensity (MFI) of GFP in the SB100X transposed cells remained stable and high while in the cells nucleofected with

the inactive DDE construct GFP MFI decreases to the same level as in the non-nucleofected controls (Figure 4A). Transposed iPS that had undergone 20 successive passages continued to express high GFP levels for at

least 70 days after nucleofection (Supplementary Figure S3). This indicates that SB100X resulted in relatively robust stable transposition in iPS.

To confirm that the iPS that had undergone transposition remained pluripotent, we subsequently used iPS that stably expressed the GFP reporter gene from the Nanog promoter (i.e. iPS-Nanog-GFP) [18] which serves as a surrogate marker of pluripotency. iPS-Nanog-GFP cells were transfected with transposons encoding DsRed (i.e. pT2-HB-PGK-DsRed) (Supplementary Figure S1A) and an expression vector encoding the hyperactive transposase

SB100X or the inactive DDE mutant. An excision product PCR-based assay was performed confirming that the SB100X-transfected iPS-Nanog-GFP cells had

undergone transposition, whereas the DDE controls failed to yield any excision products (Figure 4B).

Individual colonies were picked from the transfected iPS-Nanog-GFP cells that were uniformly DsRed+ (Figure 4C). These purified iPS-Nanog-GFP/DsRed+ colonies stably expressed DsRed for several months under continuous in vitro expansion consistent with stable transgene expression following transposition. The integration sites of the iPS-Nanog-GFP/DsRed+ cells were sequenced, hereby confirming that they had undergone stable transposition (Supplementary Table 1). Most SB transposon integrations were not within genes but in intergenic regions. The iPS-Nanog-GFP/DsRed+colony used for the subsequent experiments had a normal karyotype based on G-banding and aCGH (Figures 3A and 3B). qPCR analysis confirmed that the selected iPS-Nanog-GFP/DsRed+ clone contained 1 copy/cell of the pT2-HB-PGK-DsRed transposon, which sufficed to obtain robust and sustained DsRed expression. Since the iPS-Nanog-GFP/DsRed+ colonies continued to express GFP from the Nanog promoter (yellow colonies) this suggests that the pluripotency of the transposed iPS-Nanog-GFP cells was maintained.

To independently confirm this iPS-Nanog-GFP/DsRed+ cells were induced to


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differentiate along mesodermal (cardiac), endodermal (hepatic) and ectodermal (neuronal) lineages. Induction of cardiomyogenic differentiation resulted in the emergence of cardiomyocyte-like cells exhibiting characteristic synchronous contractions (Supplementary Movie 1 and 2). Both DsRed and GFP were expressed in embryoid bodies, 3 and 6 days after induction of cardiomyogenic differentiation (Figure 5A). Contracting cardiomyocyte-like cells started to emerge 12 to 13 days after inducing cardiomyogenesis and express DsRed (Figure 5A, Supplementary Movie 1 and 2). This indicates that the PGK promoter used to drive DsRed in the transposon construct is not silenced upon cardiomyogenic differentiation. In contrast, GFP expression driven from the Nanog promoter declined upon induction of the cardiomyogenic differentiation program consistent with the loss of pluripotency in the differentiated iPS-Nanog-GFP/DsRed+

progeny. Conversely, those iPS-Nanog-GFP/DsRed+ derived cell clusters in which the Nanog promoter remained active were not contracting suggesting that some residual iPS-Nanog-GFP/DsRed+ cells had remained pluripotent. The cardiomyogenic phenotype of these iPS-Nanog-GFP/DsRed+ derived cells that had undergone transposition was confirmed by immunostaining and by quantitative RT-PCR using cardiac-specific markers (Figure 5). There was no significant

difference between SB100X versus DDE-transfected iPS-Nanog-GFP cells, indicating that transposition and/or expression of the hyperactive SB100X transposase did not adversely influence cardiomyogenic differentiation (Figure 5).

We have confirmed these findings on three independent iPS clones that were obtained after transfection of the murine iPS with the GFP-encoding transposon along with the SB100X construct. We demonstrated that these three iPS clones (clone # 1, #9, #12) continued to stably express GFP, during at least 70 days post-nucleofection (i.e. 20 passages), (Supplementary Figure S3A) and were capable of differentiating into embryoid bodies (EB). GFP expression was sustained in these iPS-derived EBs (Supplementary Figure S3B).

Moreover, their ability to undergo cardiomyogenic differentiation was maintained, consistent with the induction of expression of cardiac-specific markers (Supplementary Figure S3C).

The iPS-Nanog-GFP/DsRed+ cells were subsequently induced to differentiate into hepatic cells using conditions that mimic ES-cell differentiation into hepatocyte-like cells. The iPS-Nanog-GFP/DsRed+-derived hepatocyte-like cells exhibited a typically polygonal morphology and expressed hepatic markers (Figure 5C). There was no significant difference in hepatic differentiation potential in SB100X-transfected iPS-Nanog-GFP cells

compared to the inactive DDE mutant control (Figures 5C and 5D). Similarly, iPS-Nanog-GFP/DsRed+ cells that had undergone transposition retained their ability to differentiate into neurospheres and neural progenitor/stem cells (Figure 6A). Induction of neural and glial differentiation was maintained in the iPS-Nanog-GFP/DsRed+ cells consistent with characteristic morphological features and expression of lineage-specific markers as determined by immunostaining and qRT-PCR (Figures 6A and 6B).

To compare the expression pattern of these differentiation markers with that of normal tissues, we used primary cardiomyocytes, hepatocytes and brain cells, as reference cell types for cardiac, hepatic and neural differentiation. Post-natal (adult, neonatal) or pre-natal (embryonic, fetal) tissues were used, as indicated (Supplementary Figure S4). The qRT-PCR results show that expression pattern of these various lineage-specific markers in iPS cells, transfected with either SB100X or an inactive DDE SB mutant is not significantly different. In general, the expression pattern of differentiation markers closely resembles that of more immature reference cell types, whereas there are some notable differences, particularly compared to adult tissues (e.g. liver) (Supplementary Figure S4).

Coaxed myogenic differentiation of iPS by transposon-mediated Pax3 gene delivery It is particularly challenging to promote skeletal muscle differentiation from iPS. To


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better define the conditions necessary for myogenic differentiation of iPS and coax their differentiation into myogenic precursor cells we delivered a transposon encoding the myogenic transcription factor PAX3 (i.e. pT2-HB-CMV-Pax3) (Supplementary Figure S1A) along with SB100X transposase into the iPS-Nanog-GFP/DsRed+ cells. This resulted in 15% PAX3+ cells compared to < 5% in the

inactive DDE controls as determined by immunostaining (Figure 7A). At later time-points, PAX3 expression declined to the same

lower expression levels as the inactive DDEcontrols, suggesting transcriptional repression of the CMV promoter upon myogenic differentiation. PAX3 protein expression kinetics is consistent with the changes in Pax3 mRNA expression levels as determined by qRT-PCR (Figure 7C). Most importantly, induction of PAX3 expression following transfection with SB100X was associated with a significant induction of MYOD expression (Figures 7A and 7C). In contrast, there was no induction of MYOD expression in control iPS-Nanog-GFP/DsRed+ cells transfected with the

catalytically inactive DDE mutant yielding only <5% MYOD+ cells. Most importantly, induction of PAX3 expression following transposition with SB100X resulted in multinucleated myofiber-like cell clusters that expressed alpha myosin heavy chain

( MYHC), which were absent in the DDEcontrol (Figure 7B). GFP expression driven from the Nanog promoter in the iPS-Nanog-GFP/DsRed+-derived EB cells declined upon myogenic differentiation, consistent with the loss of pluripotency, whereas expression of DsRed driven from the PGK promoter in the pT2-HB-PGK-DsRed transposon was sustained (Figure 7D).

To further confirm that the loss of PAX3 expression was due to transcriptional silencing, and not a peculiarity of the iPS-Nanog-GFP/DsRed+ clone, we repeated the coaxed differentiation experiment using the Oct4neomycin MEF O59 iPS cells. The latter iPS cells were cotransfected with the pT2-HB-CMV- -globin-Pax3 transposon and the SB100X constructs. We confirmed by quantitative RT-PCR (Supplementary Figure S5) that in these iPS cells, PAX3 mRNA

expression also gradually decreases upon myogenic differentiation, consistent with the PAX3 expression pattern in iPS-Nanog-GFP/DsRed+ cells (Figure 7). We subsequently retrieved PAX3-transposon-genome junctions by LAM-PCR and mapped the integration sites in the mouse genome (Supplementary Table 2). This confirmed that the pT2-HB-CMV- -globin-Pax3 transposon DNA is stable integrated into the genome of these iPS-derived myogenic progenitors.

These results support the use of this novel transposon technology to enable coaxed differentiation of iPS and suggest that PAX3 may play a key role in promoting myogenic differentiation of iPS.

DISCUSSION

Stable genomic integration is an essential feature to enable long-lasting gene expression in dividing target cells, particularly in stem/progenitor cells and their differentiated progeny. Non-viral vectors are typically diluted and result in transient expression. Only a small fraction of the transfected cells contain integrated vectors (i.e. 1/105-1/106). To develop an efficient non-viral gene delivery paradigm for genetic modification of stem/progenitor cells, we had generated novel SB transposases by in vitro evolution and selection by hyperactivating mutations in both the DNA binding and the catalytic domain (Supplementary Figures S1B and S1C) [8]. We demonstrated that these novel hyperactive SB transposases consistently increased stable gene transfer efficiency in different clinically relevant adult stem/progenitor cells, particularly MSC, myoblasts and iPS. SB100X-mediated stable transposition was an obligate requirement to obtain efficient and sustained gene expression in these distinct stem cell populations. This accounted for sustained production of therapeutically relevant proteins (i.e. coagulation factor IX). The increased stable gene expression levels obtained with SB100X resulted from a substantial improvement in bona fide transposition, consistent with the retrieval of canonical transposon integration sites and the accumulation of transposon excision products.


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Moreover, mutational inactivation of the DDE-catalytic motif prevented stable gene expression.

Though MSC do not normally express FIX, our results indicate that they are capable to perform the essential post-translational modification

steps, including -carboxylation and propeptide cleavage, enabling production of biologically active FIX. Nevertheless, approximately 50% of the FIX produced by these genetically modified MSC is fully active, when the total amount of FIX produced is taken into account (Figure 1). This suggests that expression of very high levels of FIX in ectopic cell types, may possibly saturate some of these key post-translational modification steps, resulting in the production of inactive FIX molecules that are not fully processed, consistent with some of

our previous studies 45 .

Our results indicate that stable gene transfer and expression in stem cells is more efficient with SB100X than with early-generation SB transposases, in agreement with other studies [4, 6]. The increased transposition efficiency of SB100X is likely due to, at least in part, to improved folding properties resulting in increased transposase stability [8]. However, a more detailed structural analysis of the protein–DNA complex would be necessary to fully understand the hyperactive nature of SB100X. The performance of SB100X also compares favorably with that of other transposons. We and others have recently shown that SB100X is more efficient than piggyBac [1, 8, 10]. The transposase catalyzes the excision of the transposon from the donor plasmid and its subsequent integration into the target cell chromosome. Conversely, the transposase may potentially also catalyze the excision of these integrated transposon sequences from the host cell chromosomes. However, since the transposase-encoding construct is not endowed with any sequences that facilitate its own genomic integration, it will gradually be diluted in any dividing stem/progenitor cell population. Consequently, this would result in only transient expression of the transposase, hereby minimizing potential mobilization of any integrated transposons and local “hopping”.

We showed for the first time that this novel hyperactive transposase platform can be used to efficiently coax differentiation of iPS into the desired cell type in vitro by de novoexpression of lineage-specific transcription/differentiation factors in iPS. We demonstrated that transposon/SB100X-mediated delivery of the myogenic Pax3 gene induced PAX3 expression that results in coaxed myogenic differentiation. The emergence of long, cylindrical multi-nucleated

myofiber-like cell clusters expressing MYHC suggests that PAX3 not only initiates the myogenic program but also allows for the subsequent differentiation steps, including fusion of muscle progenitors. Hence, it is particularly encouraging that coaxed myogenic differentiation of iPS can replicate some of the critical steps in normal myogenic differentiation. This is consistent with the recent demonstration that (i) skeletal muscle differentiation of embryonic mesoangioblasts requires PAX3 activity [38]; (ii) that expression of PAX3 in embryoid bodies derived from ESC can give rise to functional skeletal muscle stem/progenitor cells [36, 37]; (iii) PAX3 activation promotes the differentiation of mesenchymal stem cells toward the myogenic lineage [46]. However, in these previous ESC-based studies, myogenic differentiation was incomplete since multi-nucleated myofibers could not be generated in vitro, in contrast to the current iPS study. This may be due to differences in PAX3 expression levels and kinetics or to intrinsic differences between iPS and ESC. The present study sets the stage to further improve the efficiency of coaxed differentiation of iPS into distinct cell types. Though the PAX3-transposon DNA is stably retained in the target cell genome of the myogenic precursors, consistent with the retrieval of bona fide transposition integration sites (Supplementary Table 2), PAX3 mRNA levels gradually declined (Figure 7 & Supplementary Figure 5). This suggests that the CMV promoter used to drive PAX3 expression, is repressed upon myogenic differentiation. In contrast, the PGK promoter remained active in both cardiomyogenic and myogenic precursors (Figures 5 & 7). This is consistent with previous studies indicating that


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viral promoters (like CMV) are more susceptible to transcriptional repression than promoters of tissue-specific or house-keeping

genes 47 . This may reflect epigenetic differences in promoter methylation and/or histone modifications.

The present study also contributed to refining the conditions that allow differentiation of genetically modified iPS that had undergone transposition into cardiomyogenic, hepatic, neuronal and myogenic lineages, consistent with the differentiation of ES cells into these distinct lineages. We demonstrated that it is possible to replicate in vitro at least some of the critical steps of normal differentiation during embryogenesis. For instance, the transposon-modified iPS-derived cardiomyogenic cells expressed the gap junction protein CNX43, which is essential for establishing normal cardiac architecture and ventricular conduction, consistent with their ability to undergo synchronous contractions. Similarly, genetically modified iPS were able to undergo hepatic differentiation. Finally, transposon-modified iPS gave rise to neurospheres and neural stem cells and exhibited characteristic neuronal or glial morphologic features. There was no significant difference in cardiomyogenic, hepatic or neuronal differentiation potential of iPS when transfected with SB100X compared to the

DDE inactive control (Figures 5 and 6), indicating that the hyperactive transposon technology did not adversely influence the intrinsic pluripotency of iPS. Similarly, transposon modification of MSC and myoblasts did not affect their stem/progenitor phenotype since adipogenic and myogenic differentiation were not impaired (Figures 2B and 2D). Though the cardiomyogenic, hepatic and neural cells derived from transposon-modified iPS share many characteristics with their normal cellular counterparts in the organism, they are not identical. In particular, expression of hepatic markers in iPS-derived hepatic cells or fetal hepatocytes is different from the expression pattern in fully mature adult hepatocytes. Hence, the current iPS differentiation protocols do not reliably replicate the final steps of differentiation towards more mature cellular phenotypes.

Additional studies are therefore required to further dissect the molecular signals required for terminal differentiation. In particular, further optimization of lineage-specific differentiation conditions of iPS is warranted to fully replicate the normal in vivo differentiation processes.

The use of non-viral vectors based on the hyperactive SB transposon technology may facilitate clinical implementation of ex vivo cell and gene therapy using stem/progenitor cells and iPS. Their use may reduce the risk of insertional oncogenesis since SB transposons do not exhibit an integration bias towards genes (Supplementary Table 1) [1, 8, 48] and since the intrinsic promoter/enhancer activity of the SB terminal repeat sequences is virtually

negligible, in contrast to the -retroviral LTR [49-52]. Consequently, they have a significantly improved safety profile compared

to -retroviral vectors [1, 2, 8]. aCGH and karyotypic analysis confirmed the absence of gross cytogenetic abnormalities in the transfected MSC and myoblast polyclonal populations and in the iPS-Nanog-GFP/DsRed+ clone. Hence, the SB100X transposon system does not seem to evoke any gross cytogenetic abnormalities, in contrast to

when the C31 integrase was used 53 .Nevertheless, the occurrence of cytogenetic abnormalities in less than 10% of the cells and/or deletions/duplications smaller than 1Mb cannot be excluded, since such changes would still be undetectable using aCGH. Moreover, the cases of insertional mutagenesis observed in the context of other randomly integrating vectors have shown that mutations occurring in less than 0.01% of the gene-modified cell population can potentially cause adverse events. Though the SB transposons have an

improved safety profile compared to -retroviral vectors, they can de facto act as insertional mutagens. Hence, it cannot be excluded that SB transposons may contribute to malignant transformation through random insertional oncogenesis, a feature that has actually been exploited to identify oncogenes

or tumor suppressor genes 2 . This risk varies depending on the strength of the internal/promoter enhancer, the transposition efficiency and copy number, the extent of local


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hopping and transposon mobilization and the target cell type. Hence, it is important to continue to improve their safety profile through the use of transcriptional insulators and/or by targeted integration using zinc finger domains

2 . Additional safety studies are therefore needed to further corroborate the safety of this transposon platform using sensitive

genotoxicity assays 52 54 55 .

CONCLUSION

In conclusion, this novel hyperactive transposon represents a promising emerging non-viral gene transfer platform technology that may pave the way towards novel applications in genomic research (i.e. functional genomics, insertional mutagenesis, enhancer/promoter trapping) and translational

studies including stem cell engineering, coaxed differentiation of iPS and gene/cell therapy.

ACKNOWLEDGMENTS

We thank A. Dalda for technical assistance. We acknowledge the financial support of EU FP6 (INTHER), EU FP7 (PERSIST), AFM, VIB, FWO (Flanders, Belgium) (G.0632.07 & G.0667.07 Odysseus award, FWO WOG), KULeuven SCIL Center of Excellence Award. MG was funded by FWO, PSB was funded by FWO and AGAURA (Beatriu de Pinós), MYR was funded through a COLCIENCIAS/LASPAU fellowship. We also wish to thank Dr. Yamanaka and Dr. Jaenisch for providing us with the different murine iPS clones.

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Figure 1. Relative stable transfection efficiency of human MSC (A) and myoblasts (D) transfected with the pT2HB-CMV-GFP-Neo transposon and constructs encoding different SB transposases. Inactive DDE was used as control. Pixel density of the area corresponding to GFP+ cells was quantified for each transposase, relative to SB100X (n=10 independent fields). GFP expression was assessed 5 weeks post-nucleofection. Transposition efficiency was assessed in MSC (B) and myoblasts (E) using an excision product (EP) PCR-assay (n=3). EP corresponds to a specific PCR-product of 316 bp. The standard mimics the EP from the transposition reaction and can be detected with the same PCR primers, yielding a fragment slightly different in size (245 bp). PCR products were separated on a 1% agarose gel and quantified by densitometric scanning. Each PCR band in

the DDE, SB11 and SB100X conditions corresponds to an independent transfection, which was repeated 3 times. FIX expression in MSC (C) and myoblasts (F) transfected with pT2-HB-CMV-FIX-Neo and constructs encoding SB100X, SB11 or DDE transposases at 24, 48 and 72 h. FIX production by the non-transposed, DDE, SB11 and SB100X transfected cells was assessed based on 3 independent transfections for each of the time-points tested.


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Figure 2. Real-time quantitative PCR specific on genomic DNA from MSC (A) and myoblasts (C) 5 weeks post-transfection. NeoR-specific primers/probe were used to detect stably integrated pT2-HB-CMV-GFP-Neo transposons. Threshold ( Rn) level in function of cycle number is shown. A reference standard composed of a known amount of serially diluted NeoR-containing transposon plasmid spiked into genomic DNA was used to establish linear [Ct]-log[C0] standard curves, with high correlation coefficients (R2>0.99). Consequently, transposon copy number could be determined by comparing the Ct value of the test sample with that obtained using the validated standard. Adipogenic differentiation of SB100X-transposed vs. non-transposed MSC using oil red O staining (B) (n=22 random fields) and differentiation of SB100X-transposed vs. non-transposed myoblasts into myotubes (D). Nuclei were stained with DAPI (blue) and immunostaining for desmin (red) confirmed the myogenic phenotype. Multinucleated myotubes are indicated (white circles). GFP fluorescence in SB100 vs. non-transposed controls is shown.


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Figure 3. Karyotyping by G-banding (A) and array comparative genome hybridisation (aCGH) at 1 Mb resolution (B) in a polyclonal population of MSC, myoblasts and a clone of iPS-Nanog-GFP/DsRed+ after transposition with SB100X relative to either non-transposed or DDE controls. The Y-axis of the aCGH (B) represents the log2 of the intensity ratios of the combined dye (Cy5 and Cy3) swap experiments of the SB100X-transposed vs. non-transposed control DNA for the three different cell types. Hybridization signals of those clones that exceeded twice the standard deviation (SD) in a dye-swap replicate experiment (red lines) are considered significant, whereas hybridization signals that fell within twice the SD are considered not statistically significantly different (green lines). In the X-axis, clones are ordered from the short-arm to the long-arm telomere of each chromosome, arranged from 1 to 22. In the iPS-Nanog-GFP/DsRed+ (lower right panel) the position of the centromere of each chromosome is indicated by a vertical black line. Non-transposed MSC and myoblasts or DDE-transposed iPS-Nanog-GFP cells yielded an identical karyotype as the SB100X transposed cells (data not shown).


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Figure 4. iPS transfected with the pT2HB-CMV-GFP-Neo transposon and SB100X or DDEconstructs. Kinetics of %GFP+ iPS and mean fluorescent intensity (MFI) (A), based on 3 independent transfections. Demonstration of transposition in iPS-Nanog-GFP/DsRed+ cells using an excision product (EP) PCR-based assay 3 days post-transfection. EP corresponds to a specific

PCR-product of 316 bp. Each PCR band in the DDE and SB100X conditions corresponds to an independent transfection, which was repeated 3 times. The standard mimics the EP from the transposition reaction and can be detected with the same PCR primers, yielding a fragment slightly different in size (245 bp). PCR products were separated on a 1% agarose gel and quantified by densitometric scanning (B) (n=3). Five successive subcloning of iPS-Nanog-GFP/DsRed+ colonies to obtain pure DsRed+ colonies showing only GFP, only DsRed and the combined GFP/DsRed fluorescence (C).


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Figure 5. Cardiomyogenic (A, B) and hepatic (C, D) differentiation of iPS-Nanog-GFP transfected

with the pT2-HB-PGK-DsRed transposon and SB100X or DDE constructs. Kinetics of GFP and DsRed expression in EBs and cardiomyogenic (CM) cells (A) using GFP (488nm) or DsRed filter (546nm) and overlay showing GFP+DsRed+ (orange/yellow) cells. Expression of cardiac (A, B) and hepatic markers (C, D) were assessed by immunostaining (A, C) and quantitative RT-PCR (B, D) (n=3). Ct values for each of the markers were determined based on 3 independent transfections for each of the time-points tested.


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Figure 6. Neural stem cell differentiation (A) and mature neural and glial differentiation (B) of

iPS-Nanog-GFP cells transfected with the pT2-HB-PGK-DsRed transposon and SB100X or DDE constructs. Expression of early neural stem cell and radial glial markers were assessed by quantitative RT-PCR (A) or immunostaining (B) (n=3). Ct values for each of the markers were determined based on 3 independent transfections for each of the time-points tested.


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Figure 7. Coaxed differentiation of iPS-Nanog-GFP/DsRed+ cells transfected with the pT2-HB-CMV- -globin-Pax3 transposon and SB100X or DDE constructs. PAX3, MYOD (A) and

MYHC (B) expression was assessed by immunostaining (A, B) or quantitative RT-PCR (C). Kinetics of GFP and DsRed expression in iPS-Nanog-GFP/DsRed+ cells undergoing myogenic differentiation (D) (n=3). Ct values for each of the markers were determined based on 3 independent transfections for each of the time-points tested.

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