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Keywords:

  • Human iPS cells;
  • Adenoviral vector;
  • DNA integration;
  • Human ES cells;
  • Parkinson's disease;
  • Dopamine neurons

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES
  10. Supporting Information

Mouse and human fibroblasts have been transformed into induced pluripotent stem (iPS) cells by retroviral transduction or plasmid transfection with four genes. Unfortunately, viral and plasmid DNA incorporation into chromosomes can lead to disruption of gene transcription and malignant transformation. Tumor formation has been found in offspring of mice generated from blastocysts made mosaic with iPS cells. To proceed with iPS cells for human therapy, reprogramming should be done with transient gene expression. Recently, adenoviral vectors have been used to produce mouse iPS cells without viral integration. Here, we report the successful creation of human iPS cells from embryonic fibroblasts using adenoviral vectors expressing c-Myc, Klf4, Oct4, and Sox2. After screening 12 colonies, three stable iPS cell lines were established. Each cell line showed human embryonic stem cell morphology and surface markers. Southern blots and polymerase chain reaction demonstrated that there was no viral DNA integration into iPS cells. Fingerprinting and karyotype analysis confirmed that these iPS cell lines are derived from the parent human fibroblasts. The three human iPS cell lines can differentiate to all three germ layers in vitro, including dopaminergic neurons. After s.c. injection into nonobese diabetic–severe combined immunodeficient mice, each human iPS line produced teratomas within 5 weeks postimplantation. We conclude that adenoviral vectors can reprogram human fibroblasts to pluripotent stem cells for use in individualized cell therapy without the risk for viral or oncogene incorporation. STEM CELLS 2009;27:2667–2674


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES
  10. Supporting Information

Stem cell research offers great promise for understanding basic mechanisms of human development and differentiation, as well as the hope for new treatments for diseases such as diabetes, Parkinson's disease, and myocardial infarction. However, human embryonic stem (ES) cell research also raises ethical and political controversies. The groundbreaking work of Shinya Yamanaka has shown that adult mouse and human fibroblasts can be transformed into induced pluripotent stem (iPS) cells by retroviral transduction with only four genes encoding the transcription factors c-Myc, Klf4, Oct4, and Sox2 [1, 2]. This work was confirmed by others in murine and human systems [3–5]. The reprogramming of somatic cells to iPS cells avoids the ethical problems specific to ES cell research. Reprogramming fibroblasts to stem cells makes it possible to create unique pluripotent stem cells to treat individual patients and study human disease.

A shortcoming of the initial reprogramming method was the use of a retrovirus or lentivirus to transduce fibroblasts. These viruses are integrated into host chromosomes where they can cause insertional mutagenesis, interfere with gene transcription, and induce malignant transformation [6]. The average retroviral infection leads to 10-20 retroviral integration sites in human iPS cell lines [2, 7]. In the first report of germline-competent mouse iPS cells, 20% of chimeric mice developed tumors that were attributable to the reactivation of the c-Myc proviral transgene that had integrated into the host cell genome [8]. This result was corroborated by a report from another group describing cancer-related deaths in 18 of 36 iPS chimeric mice [9]. Higher rates of malignancies are not usually observed in chimeric mice derived from ES cells; therefore, the tumorigenicity of iPS cells constitutes a major safety concern.

To proceed with iPS cells for human therapy, reprogramming should be done with nonintegrating strategies. Several groups have sought alternatives to retroviral gene integration. Yamanaka's group transfected plasmids without using virus to generate mouse iPS cells [10]. Two recent papers have shown that mouse and human iPS cells can be produced by piggyBac transposition with four genes in a single plasmid, thus significantly improving induction efficiency [11, 12]. Another method has used Cre-recombinase-excisable viruses to reprogram mouse cells [13, 14]. Thomson's laboratory has described production of human iPS cells using nonintegrating episomal vectors [15]. After removal of the episome, iPS cells were free of vector and transgene sequences. Ding's laboratory reported the generation of mouse iPS cells by the insertion of transducing proteins into the cytoplasm of fibroblasts in the presence of valproic acid [16].

Replication-defective adenoviral vectors have proven useful for gene insertion into cells without integration into chromosomal DNA [17, 18]. Gene expression can persist for days, which should provide sufficient time to reprogram fibroblasts to pluripotent cells. Stadtfeld and colleagues have used adenoviral vectors to generate mouse iPS cells from liver cells and fibroblasts without viral integration [19].

In this report, we successfully produced iPS cells from human embryonic fibroblasts using adenoviral vectors expressing c-Myc, Klf4, Oct4, and Sox2. The human iPS cell lines express ES cell-specific markers and can be differentiated into all three germ layers in vitro and in vivo. We demonstrate that human iPS cells generated with adenovirus are free of any viral or transgene integration. Because our laboratory has a long interest in cell therapy for Parkinson's disease, we show that the human iPS cells can be differentiated into dopamine neurons.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES
  10. Supporting Information

Construction of Adenovirus

We used the Ad-Easy system (The Johns Hopkins Oncology Center, Baltimore, MD, http://www.coloncancer.org/adeasy.htm) to create adenoviruses expressing human Oct4, Sox2, Klf4 and c-Myc. Four expressed sequence tag clones containing full coding sequences were obtained from Open Biosystems (Huntsville, AL, http://www.openbiosystems.com). A 1.4-kb fragment of human Oct4 cDNA was cloned into NotI-XbaI sites of pShuttle-cytomegalovirus (CMV) vector [18, 20]. Similarly, cDNA fragments for human Sox2 (1.5 kb), Klf4 (2.0 kb), and c-Myc (1.5 kb) were cloned into XhoI-HindIII sites of pShuttle-CMV vector. Sequences were verified by restriction digestion. Homologous recombinations were performed in bacterial AdEasy-1 cells. The adenoviruses were produced in HEK293 cells and purified to high titer, as we and others have described [18, 20-22]. Viral titer was determined by plaque assays and is expressed as plaque-forming units per μl (pfu/μl).

Culture of Human Embryonic Fibroblast IMR90 Cells

Human embryonic fibroblast IMR90 cells were purchased from the American Type Culture Collection (Manassas, VA, http://www.atcc.org; Catalog No. CCL-186). IMR90 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) containing 10% fetal bovine serum (Invitrogen) and 1% penicillin–streptomycin–L-glutamine (Invitrogen). IMR90 cells were passaged using 0.05% trypsin-EDTA (Invitrogen) and replated at 1:5 dilutions.

Adenovirus Transduction of IMR90 Cells

IMR90 cells were plated at a density of 5 × 104 to 5 × 106 cells per 100-mm tissue culture plate. The next day (day 2), IMR90 cells were about 40% confluent. The cells were incubated with medium containing four adenoviruses (Ad-cMyc, Ad-Klf4, Ad-Oct4, and Ad-Sox2) plus Ad-green fluorescent protein (GFP), each at a multiplicity of infection of 50-500 pfu/cell. Twenty-four hours later, adenoviruses were removed and replaced with normal IMR90 culture medium. On day 4, IMR90 cells had grown to near confluence. The cells were transduced with the same five adenoviruses at the same concentrations as on day 2. Twenty-four hours later, adenoviruses were removed and replaced with normal IMR90 culture medium. On day 7, IMR90 cells were dissociated using 0.05% trypsin-EDTA and plated onto mouse embryonic fibroblast feeder layers (PMEF) (Millipore, Billerica, MA, http://www.millipore. com) at a concentration of 1 × 106 cells per 100-mm plate. From day 8 onward, the culture medium was changed to human ES cell medium consisting of DMEM/F12, 20% Knockout Serum Replacement, nonessential amino acids, sodium pyruvate, 1% penicillin–streptomycin–L-glutamine, 0.1 mM β-mercaptoethanol, and 4 ng/ml basic fibroblast growth factor (bFGF). The medium was changed every other day.

iPS Cell Generation

By days 25-30, a total of 26 colonies showing ES cell-like morphology emerged under various conditions (Table 1). Twelve colonies with promising morphology were selected with a P-20 pipette tip. These colonies were briefly dissociated in 0.05% trypsin-EDTA and plated on mouse embryonic fibroblasts in individual wells of a 96-well plate in human ES cell medium. The colonies were further expanded in 24-well plate and six-well plate cultures. Finally, three iPS clones were established. The doubling time of iPS cells was about 36 hours.

Table 1. Summary of human iPS cell induction by adenovirus
  1. IRM90 cells were transduced with adenovirus MOI of 50-500 pfu/cell. Higher MOIs resulted in higher percentages of GFP+ cells when analyzed at day 7. ES cell-like colonies appeared after transduction with adenovirus at 150-300 pfu/cell. Definitive human iPS cell lines were established with adenovirus at 200-250 pfu/cell. The low efficiency of human iPS cell formation (about 0.0002%) may be the result of a low percentage of cells coexpressing all four genes as well as gene dilution from ongoing cell division. Because IMR90 cells were continuously dividing, adenoviral vectors were diluted after each cell division. Successful iPS induction required exposure to adenovirus at day 2 and day 4 after culture of IRM90 cells.

  2. Abbreviations: ES, embryonic stem; GFP, green fluorescent protein; iPS, induced pluripotent stem; MOI, multiplicity of infection.

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Immunofluorescent Staining for ES Cell Markers and Three Germ Layers

Human iPS cell colonies and differentiated cells were fixed in 4% paraformaldehyde for 20 minutes. The cells were washed three times with phosphate-buffered saline (PBS) and incubated in blocking buffer (PBS, 5% normal goat serum, 0.1% Triton X-100) for 30 minutes. Cells were incubated overnight at 4°C in the following primary antibody solutions (from Chemicon, Temecula, CA, http://www.chemicon.com, unless otherwise noted): rabbit anti-Oct4 (1:200), rabbit anti-Nanog (1:200), rat anti-stage-specific embryonic antigen (SSEA)-3 (1:100), mouse anti-SSEA-4 (1:100), mouse anti-TRA-1-60 (1:100), mouse anti-TRA-1-81 (1:100), mouse anti-smooth muscle actin (SMA) (1:200), mouse anti-α-fetoprotein (AFP) (1:200), rabbit anti-Nestin (1:200), mouse anti-microtubule-associated protein (MAP)2 (1:200), mouse anti-βIII-tubulin (TuJ1; 1:200; Promega, Madison, WI, http://www.promega.com), rabbit anti-tyrosine hydroxylase (TH) (1:150, Pel-Freez, Rogers, AK, http://www.invitrogen.com). After three washes with PBS, the cells were incubated with fluorescein isothiocyanate-conjugated secondary antibodies for 1 hour. Fluorescent staining was examined with a Nikon Diaphot fluorescent microscope (Nikon Instruments, Inc., Melville, NY, http://www.nikoninstruments.com), and images were taken by SPOT digital camera (SPOT Imaging Solutions, a division of Diagnostic Instruments, Inc., Sterling Heights, MI, http://www.diaginc.com). For alkaline phosphatase (ALP) staining, iPS cells were fixed with 4% paraformaldehyde for 2 minutes, followed by a 15-minute incubation with staining solution (Alkaline Phosphatase Detection Kit; Millipore).

Reverse Transcription-Polymerase Chain Reaction Analysis

Total RNA was purified from human iPS cells using the RNeasy Kit (Qiagen, Hilden, Germany, http://www1.qiagen.com). Reverse transcription-polymerase chain reaction (RT-PCR) was performed according to established methods [23] using an RT-PCR kit (Invitrogen) with the primers listed in supplemental online Table 1. Primers overlapping the coding region and 3′-untranslated region were used for endogenous mRNA. Primers covering only the coding region were used for adenovirus-mediated mRNA. Total RNA from human ES cells (BG01V) was used as a positive control. For adenovirus-mediated transcription, total RNA from HEK293 cells infected with each adenovirus was used as a positive control.

Bisulfite Sequencing

Genomic DNA was purified from human iPS cells using the DNeasy Kit (Qiagen). The conversion of unmethylated cytosines to uracil was carried out as described in the EZ DNA Methylation-Gold Kit (Zymo Research, Orange, CA, http://www.zymoresearch.com). One microgram genomic DNA was treated in each reaction, and 2 μl of elution was used for each PCR reaction. The primers GAG GCT GGA GCA GAA GGA TTG CTT TGG and CCC CCC TGG CCC ATC ACC TCC ACC ACC TGG were used to amplify a genomic DNA fragment (460 bp) in the human Oct4 promoter using the following conditions (Herculase II Fusion DNA Polymerase system; Stratagene, La Jolla, CA, http://www.stratagene.com): 1× Herculase buffer, 2 mM MgCl2, 0.2 mM deoxynucleotide triphosphate (dNTP), 0.25 μM primers, 100 ng genomic DNA, and 1 μl Herculase DNA polymerase. The cycling conditions were 95°C for 5 minutes, followed by 35 cycles of 95°C for 30 seconds, 59°C for 30 seconds, 72°C for 1 minute, and finally 72°C for 10 minutes. The resultant PCR products were cloned into pCR2.1 vector (Invitrogen) and sequenced.

Karyotyping and DNA Fingerprinting

The human iPS cells were cultured in six-well plates as described above. Cells at 80%–90% confluence were mitotic arrested with colcemid (0.05% μg/ml) for 3 hours and harvested using 0.05% trypsin-EDTA. Hypotonization was performed with 0.075 M KCl, and a 3:1 mixture of methanol and glacial acidic acid was used for cell fixation. Slides were dropped and aged for 8 hours at 57°C. Giemsa/trypsin/Leishman banding was performed following a standard protocol, with 2.5-minute incubations in 0.6% trypsin and Leishman's stain. Imaging and karyotyping were performed using BandView software (Applied Spectral Imaging, Vista, CA, http://www.spectral-imaging.com). To confirm that human iPS clones were derived from IMR90, short tandem repeat (STR) analysis was performed in the DNA Sequencing Core at the University of Colorado – Denver.

PCR Analysis for Viral Gene Integration

Human iPS cells were cultured in six-well plates as described above. Cells were dissociated using 0.05% trypsin-EDTA. After centrifugation at 200g for 5 minutes, cell pellets were suspended in 200 μl PBS with 20 μl proteinase K (10 mg/ml) and 4 μl RNaseA (100 mg/ml). Total genomic DNA was purified using the DNeasy Blood and Tissue Kit according to provided protocols (Qiagen). The DNA concentration was measured by absorbance at 260 nm. For genomic DNA PCR, we used the Herculase II Fusion DNA Polymerase system (Stratagene) with the following setup: 1× Herculase buffer, 2 mM MgCl2, 0.2 mM dNTP, 0.25 μM primers, 100 ng genomic DNA, and 1 μl Herculase DNA polymerase. The cycling conditions were 95°C for 5 minutes, followed by 35 cycles of 95°C for 30 seconds, 55°C for 30 seconds, 72°C for 1 minute, and finally 72°C for 10 minutes. The primers are listed in supplemental online Table 2. We used 1 pg of adenoviral vector as a positive control. We analyzed 15 μl PCR products in 1% agarose gel.

Southern Blotting

The probes for Southern blots were generated by random-primed DNA labeling with digoxigenin (DIG)-2′-deoxyuridine 5′-triphosphate (Roche, Basel, Switzerland, http://www.roche-applied-science.com). DNA fragments for Oct4, Klf4, c-Myc, and Sox2 (1.5-2.0 kb) were purified from pCMV-Shuttle vector after digestion with NotI-XbaI or XhoI-HindIII. The pAd-Klf4 adenoviral plasmid DNA was digested with BamHI-HindIII, which in theory would generate 9 DNA fragments (2, 2.8, 2.9, 3.2, 3.5, 4.5, 4.7, 5.3, and 6.0 kb). All adenoviral fragments were purified and used for producing probes for whole adenoviral vector blots.

Genomic DNA was purified from iPS cells, IMR90cells, and HEK293 cells with the DNeasy kit (Qiagen). Purified genomic DNA (5 μg) was digested with BamHI overnight. As a positive control for whole vector blotting, pAd-Klf4 adenoviral plasmid DNA was digested with BamHI-HindIII. As a positive control for cDNA blots, pAd-Oct4 adenoviral plasmid DNA was digested with NotI-XbaI, whereas pAd-Klf4, pAd-cMyc, and pAd-Sox2 adenoviral plasmid DNA was digested with XhoI-HindIII.

Digested DNA fragments were separated on 0.8% agarose gels and transferred to a nylon membrane (Amersham Biosciences, Piscataway, NJ, http://www.amersham.com). The membrane was incubated with DIG-labeled DNA probes in DIG Easy Hyb buffer (Roche) at 42°C overnight with constant agitation. After washing, ALP-conjugated anti-DIG antibody (1:10,000; Roche) was added to the membrane. Chemiluminescent signals were detected using x-ray film after incubation with CDP-Star (Roche).

In Vitro Differentiation

Human iPS cells were dissociated using 0.05% trypsin-EDTA and were replated on noncoated Petri dishes at 1 × 105 cells per 35-mm dish. The medium was the same as human ES cell medium, but without bFGF. The medium was changed every 2 days. After 7 days in floating culture, embryoid bodies formed. We then transferred embryoid bodies to 0.1% gelatin-coated tissue culture dishes (12-well) using the same medium. The medium was changed every other day once embryoid bodies were attached to the dish. Differentiated cells were fixed after 8 days in adherent culture.

Dopamine Neuron Differentiation

Mouse PA6 stromal cells were cultured in DMEM with 10% fetal calf serum and 1% penicillin–streptomycin–L-glutamine. The day before iPS cell differentiation, PA6 cells were dissociated using 0.05% trypsin-EDTA and seeded on 0.1% gelatin-coated 12-well plates at 1 × 105 cells/well [24–26]. Human iPS cells were dissociated using 0.05% trypsin-EDTA and seeded on the PA6 cell layer at 1,000 iPS cells/well. The medium was ES cell medium without bFGF and was changed every 2 days. The iPS cells were differentiated on PA6 cells for 14-16 days. After differentiation, cells were fixed and stained with antibodies for βIII-tubulin (TuJ1), MAP2, and TH [23].

Teratoma Formation

To examine whether human iPS cells can form teratomas in vivo, we s.c. injected 2 × 106 iPS cells in 0.1 ml PBS to 8-week-old nonobese diabetic severe combined immunodeficient (NOD/SCID) (National Cancer Institute, Bethesda, MD, http://www.cancer.gov) mice. Two mice were injected for each iPS clone. For a control, IMR90 cells (2 × 106 cells in 0.1 ml PBS) were also injected into two mice. After 5-6 weeks, tumors were dissected and fixed in 4% paraformaldehyde. Samples were embedded in paraffin and processed with hematoxylin and eosin staining in the Pathology Laboratory at the University of Colorado Hospital.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES
  10. Supporting Information

We created four adenoviruses expressing Oct4, Sox2, c-Myc, and Klf4 under control of the CMV promoter. In an initial experiment, human fetal fibroblasts, IMR90, were plated at a density of 50,000, 500,000, or 5,000,000 cells per 100-mm dish. On day 2, the four adenoviruses plus Ad-GFP were added at a concentration of 250 pfu/cell. On day 7, about 45%–55% of the fibroblasts were GFP+ (Table 1). The fibroblasts were dissociated and replated on mouse embryonic fibroblasts and cultured with human ES cell medium. No ES cell-like colonies were formed under these conditions. Because adenoviruses lose their expression after cell division, we decided to repeat adenovirus treatment on day 4.

In a new set of experiments, the IMR90 cells were plated at a density of 500,000 cells per 100-mm dish. On day 2, the four adenoviruses plus Ad-GFP were added at a concentration of 50-500 pfu/cell. On day 4, adenovirus exposure was repeated at the same concentrations. On day 7, IMR90 cells were replated on mouse embryonic fibroblasts in human ES cell medium (Fig. 1A-1C). On days 25-30, ES cell-like colonies emerged (Fig. 1D). All colonies had lost intrinsic Ad-GFP expression, as expected for transient expression of the adenoviral vectors (Fig. 1E). We picked up a subset of iPS colonies showing good ES cell-like morphology, and excluding those colonies with apparent differentiated cells, dark centers, or colonies that appeared too large or too small in size. The ES cell-like colonies were expanded, and stable iPS cell lines were established (Table 1). A third series of iPS cell induction experiments was performed using 5 × 104 to 5 × 106 cells per 100-mm dish with adenovirus infections on day 2 and day 4. The results confirmed that human iPS cells can be generated by repeated adenovirus infections at a concentration of 250 pfu/cell with 500,000 cells per 100-mm dish, as shown in Table 1. Of 12 ES cell-like colonies, three independent human iPS cell lines were established. The iPS cell induction efficiency is low, about 0.0002% (one iPS cell clone per 500,000 starting fibroblasts).

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Figure 1. Generation of human induced pluripotent stem (iPS) cells with adenoviral vectors from IMR90 human fetal fibroblasts. (A): Timeline of experimental design. IMR90 cells were transduced with adenovirus (Ad) on day 2 and day 4. On day 7, IMR90 cells were dissociated and seeded on mouse embryonic fibroblasts. Colonies appeared at days 25-30 in embryonic stem (ES) cell culture medium. (B): Phase contrast photomicrograph of IMR90 fibroblasts on day 7. (C): Green fluorescent protein (GFP) expression in transduced IMR90 cells on day 7. (D, E): An embryonic stem (ES) cell-like colony emerged at day 30, as shown by phase contrast (black arrows in (D)). Fluorescence microscopy of the same colony (white arrows in (E)) was GFP, whereas some surrounding IMR90 cells remained GFP+. (F): Expanded iPS colonies show typical ES cell morphology by phase contrast microscopy. (G): Human iPS cells were positive for alkaline phosphatase (ALP) staining. (H-M): Human iPS cells expressed six human ES cell markers, including Oct4 (H), Nanog (I), stage-specific embryonic antigen (SSEA)-3 (J), SSEA-4 (K), TRA-1-60 (L), and TRA-1-81 (M). Bar length: 200 μm (B, C), 2 mm (D–G), 1 mm (H--M). (O): Reverse transcription-polymerase chain reaction analysis of mRNA expression in the three human iPS cell lines (iPS1, iPS6, and iPS9). Positive controls were the human ES cell line BG01V and HEK293 cells infected with each adenovirus. (P): Oct4 promoter methylation analysis in three human iPS cell lines. In the Oct4 promoter region, 10 cytosine guanine dinucleotides (CpGs) were examined after bisulfite treatment. Each horizontal line presents one DNA sequencing result, with a total of eight sequences analyzed for each iPS cell line. Filled circles indicate methylated CpGs, open circles indicate unmethylated CpGs. Results show that all three iPS lines had little methylation compared with the original IMR90 fibroblasts.

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As shown in Figure 1F, human iPS cells demonstrated morphology typical of human ES cells and were positive for ALP staining (Fig. 1G). Human iPS cells expressed ES cell markers such as Oct4, Nanog, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81 (Fig. 1H–1M). Results of RT-PCR analysis showed that all three iPS clones expressed mRNA for endogenous Oct4, Sox2, Klf4, c-Myc, and Nanog (Fig. 1O). By contrast, we could not detect any adenoviral vector-derived mRNA for Oct4, Sox2, Klf4, or c-Myc (Fig. 1O). We analyzed the methylation status of cytosine guanine dinucleotides (CpGs) in the Oct4 promoter in the three iPS clones. Results showed that CpGs were highly unmethylated in all three clones, compared with the highly methylated CpGs in parent IMR90 cells (Fig. 1P). This finding indicates that the Oct4 promoter was activated in the iPS cells.

STR analysis of three iPS clones revealed identical results as with IMR90 cells, indicating that all three iPS cells were derived from parent IMR90 cells (Table 2). Cytogenetic analysis demonstrated that each human iPS cell line had the same karyotype (46, XX) as the parent IMR90 cells (an example is shown in Fig. 2). All three iPS cell lines were diploid, in contrast to previous findings that adenovirus-generated mouse iPS cells were tetraploid in three of 13 iPS cell lines [19].

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Figure 2. Karyotype analysis using Giemsa/trypsin/Leishman banding of human induced pluripotent stem cells derived from IMR90 fibroblasts. Ten metaphase spreads were analyzed and an additional 21 cells were counted. All cells had a normal female karyotype (46, XX). An example from iPS-1 line is shown.

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Table 2. Short tandem repeat analysis of human induced pluripotent stem (iPS) cell clones generated from IMR90 cells
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To check for viral integration into genomic DNA, we performed Southern blotting and PCR analysis. Genomic DNA from iPS cells, IMR90 cells, and HEK293 cells was digested with BamHI. To detect viral DNA integration, adenoviral Klf4 plasmid DNA (pAd-Klf4) was digested with BamHI-HindIII, and all fragments were purified for probe labeling. To detect any transgene integration, the whole cDNA fragments of Oct4, Klf4, c-Myc, and Sox2 (1.5-2.0 kb) were used for probe labeling. In all five Southern blots, there were no viral DNA or transgene-specific bands in any of the three iPS cell lines (Fig. 3A–3E). In genomic DNA PCR analysis, all three iPS cell lines amplified a 650-bp human engrailed one (En1) promoter fragment, the same size as from positive controls using the human En1 promoter plasmid (Fig. 4B). The amplification of a randomly chosen DNA fragment helps demonstrate that genomic DNA from the three iPS cell lines is intact. By contrast, all seven PCR reactions were negative for adenoviral gene integration in all three human iPS cell lines (Fig. 4B). These results demonstrate that there was no adenoviral integration in iPS cells generated by adenoviral vectors.

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Figure 3. Southern blots show no viral DNA integration in human induced pluripotent stem (iPS) cells generated by adenoviral vectors. Genomic DNAs from iPS cells, IMR90 cells, and HEK293 cells were digested with BamHI. DNA fragments of Oct4, Klf4, c-Myc, and Sox2 (1.5-2.0 kb) were used for digoxigenin probe labeling. (A): Digestion of adenoviral Klf4 plasmid DNA (pAd-Klf4) with BamHI-HindIII resulted in nine pieces of fragments that were purified for probe labeling and positive controls (equivalent to 0.2, 1, and 5 integrations per genome). We found two bands (open arrows) in all three iPS cells as well as in IMR90 and HEK293 cells, indicating they were nonspecific bands. In positive controls, pAd-Klf4 showed bands in the range of 2-6 kb, as expected (solid arrows in (A)). In HEK293 cells, two strong bands at 5-6 kb were seen, reflecting adenoviral E1 integration fragments, whereas two weak bands at 3-4 kb appear to be nonspecific (lane 6 in (A)). (B–E): Positive controls were loaded in amounts of 0.2-5 integrations per genome after adenoviral Oct4 plasmid DNA (pAd-Oct4) was digested with NotI-XbaI (B), whereas adenoviral Klf4, c-Myc, and Sox2 plasmid DNA (pAd-Klf4, pAd-cMyc, and pAd-Sox2, respectively) was digested with XhoI-HindIII (C–E). In each of the four cDNA blots (B-E), all three human iPS cell lines showed the same bands as in IMR90 and HEK293 cells, indicating that these bands were either endogenous genes or nonspecific bands (open arrows in (B–E)). In positive controls, single bands at the expected size were seen (solid arrows in (B–E)).

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Figure 4. Polymerase chain reaction (PCR) analysis of viral gene integration in human induced pluripotent stem (iPS) cells. (A): Schematic drawing of adenoviral vector. Four genes, c-Myc (C), Klf4 (K), Oct4 (O), and Sox2 (S), were cloned into the multiple cloning site downstream from the cytomegalovirus (CMV) promoter. Restriction sites in the adenoviral backbone were labeled. Arrowheads show primer pairs used for detecting gene integration in genomic DNA. To detect integration of any of the four genes, the forward primer originated in the CMV promoter and the reverse primer originated in the inserted gene. Three primer pairs, B1, B2, and B3, were used to detect adenoviral backbone fragments. (B): Representative PCR gel images from each primer pair. One picogram of adenoviral vector was used for positive (+) controls. Water was used for negative (−) controls. A fragment corresponding to a portion of the human engrailed one (En1) promoter was used as an internal control.

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We differentiated human iPS cells to test their pluripotency. The iPS cells formed embryoid bodies after 7 days in suspension cultures (Fig. 5A). Embryoid bodies were allowed to differentiate further in adherent cultures. After 8 days in adherent culture, neural cells appeared (Fig. 5B). Immunofluorescent staining showed that human iPS cells can differentiate into all three germ layers—mesoderm (SMA, Fig. 5C), endoderm (AFP, Fig. 5D), and ectoderm (Nestin, Fig. 5E).

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Figure 5. In vitro and in vivo differentiation of human induced pluripotent stem (iPS) cells. (A): Embryoid bodies were formed after 7 days in floating culture and then replated. (B): After 8 days of adherent culture, embryoid bodies differentiated into many cell types, including neuroepithelial cells, as judged by morphology. (C–E): Immunostaining shows that embryoid bodies can differentiate into all three germ layers—mesoderm (smooth muscle actin [SMA] in (C)), endoderm (α-fetoprotein [AFP] in (D)), and ectoderm (Nestin in (E)). (F–H): After plating on PA6 cells, human iPS cells differentiated into microtubule-associated protein 2 (MAP2)+(F) and βIII-tubulin-positive neurons (TuJ1, (G)) with long neurites. Some neurons were positive for the dopaminergic marker tyrosine hydroxylase (TH) (H). (I–L) All three human iPS cell lines formed teratomas 5-6 weeks after s.c. injection into nonobese diabetic severe combined immunodeficient mice. Various tissues of the three germ layers were detected by hematoxylin and eosin staining, including neural tissue (I), gut-like epithelium (J), cartilage (K), and muscle (L). Bar length: 5 mm (A), 1 mm (B), 200 μm (C–H), 100 μm (I–L).

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To produce dopamine neurons, iPS cells were plated on PA6 cells for 2 weeks [24–26]. Immunostaining revealed a large number of neurons positive for MAP2 and βIII-tubulin/TuJ1 (Fig. 5F, 5G). Subsequent immunostaining for TH, the rate-limiting enzyme in dopamine synthesis, showed clusters of cells with morphology compatible with dopamine neurons (Fig. 5H). Optimization of midbrain dopamine neuron production from iPS cells is now being pursued in our laboratories.

To examine the differentiation potential of human iPS cells in vivo, we injected 2 × 106 iPS cells into NOD/SCID mice s.c.. After 5-6 weeks, large teratomas formed from all three iPS clones, weighing about 1 g by 6 weeks. Histology revealed a wide range of tissue types including neural tissue (Fig. 5I), gut-like epithelium (Fig. 5J), cartilage (Fig. 5K), and muscle (Fig. 5L).

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES
  10. Supporting Information

In this paper, we believe that we are the first to report that an adenoviral vector can reprogram human embryonic fibroblasts to iPS cells. These iPS cells show features typical of human ES cells, including surface markers and highly unmethylated Oct4 promoter. We have shown that adenovirus-generated human iPS cells are pluripotent and can be differentiated into all three germ layers in vitro and in vivo. Most important, we have demonstrated that human iPS cells generated by adenovirus are free from viral DNA integration into host chromosomes. Therefore, iPS cells created by the adenovirus method eliminate the risk for malignant transformation associated with retrovirus or plasmid transfection methods. Because we would like to use iPS cells to develop cells for transplant into patients with Parkinson's disease, we have shown that iPS cells can be differentiated into cells with markers compatible with dopamine neurons, as we have described with human ES cells [23, 26]. Refinement of the differentiation and purification protocols could yield cells appropriate for human therapeutic trials.

We have found that the optimal adenoviral concentration for making human iPS cells is 200-250 pfu/cell in a fibroblast density of 5 × 105 cells/65 cm2 with infections applied at day 2 and day 4. The overall efficiency for generating human iPS cells is quite low, about 0.0002%, similar to that reported for adenoviral induction of mouse iPS cells [19]. The low efficiency of human iPS cell formation may be a result of a low percentage of cells coexpressing all four genes and dilution of the viral titer with progressive cell replication. Because IMR90 cells were continuously dividing, adenoviral vectors were lost after each cell division. To improve induction efficiency, we are developing an adenoviral vector expressing all four genes in a single vector, as demonstrated by retroviral and plasmid transfection methods [10-12, 27, 28].

We have not found any tetraploid human iPS cells in our adenovirus experiments. In mice, using an adenovirus-based iPS cell induction method, Stadtfeld et al. [19] have found tetraploid iPS cell clones that were derived from mouse fetal liver cells (one of nine clones) and mouse adult hepatocytes (two of three clones), but not from mouse fibroblasts. Others have shown that 53% of adult liver cells are tetraploid, whereas nearly all fetal hepatocytes are diploid [29, 30]. Because the frequency of tetraploidy in mouse iPS cells matched the ratio of tetraploidy in fetal and adult liver cells, it is most likely that tetraploid mouse iPS cells were derived from starting tetraploid liver cells and not from cell fusion induced by adenovirus.

With improved induction efficiency, our adenoviral method should be useful for reprogramming fibroblasts derived from adults and even elderly people. We plan to use these vectors to generate iPS cells from patients with genetic forms of Parkinson's disease and other neurodegenerative disorders. These integration-free iPS cells will be valuable for studying disease mechanisms, drug screening, and toxicity testing.

In summary, we successfully generated human iPS cells using adenoviral vectors without viral gene integration into the genome. The adenoviral method can create patient-specific iPS cells that can be converted into specific cell types for modeling disease and for treating individual patients without the risk for chromosomal disruption.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES
  10. Supporting Information

The study was supported by the Leopold Korn and Michael Korn Professorship in Parkinson's Disease at the University of Colorado School of Medicine and the Edward D. and Anna Mitchell Family Foundation. Wenbo Zhou, Ph.D., is a Coleman Faculty Fellow at the University of Colorado. We thank Bette K. DeMasters, M.D., for assistance with histology of the iPS cell teratomas.

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  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES
  10. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES
  10. Supporting Information

Additional supporting information available online.

FilenameFormatSizeDescription
STEM_201_sm_SuppTab1.doc41KSupplemental Table 1 Primers used for RT-PCR Analysis.
STEM_201_sm_SuppTab2.doc40KSupplemental Table 2 Primers used for genomic PCR Analysis.

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