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Contents

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. References

The aim of this study was to further clarify the mechanisms involved in inducing pluripotency using canine foetal fibroblast cells. The two pluripotency-related transcription factors, OCT4 and SOX2, coupled to a fluorescent reporter gene were transduced, individually or in combination, using a lentiviral system. Stable transgenic cell lineages were obtained and canine cells showed to be highly responsive to the integration and expression of human SOX2 and OCT4, also depending on the amount of virus used for incubation. Such positive results are essential for the establishment of pluripotency induction through the incorporation of known transcription factors into the genome of somatic cells.


Introduction

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. References

The use of human embryonic stem cells (ESCs) in clinical trials remains controversial because of ethical concerns over the harvesting of human embryos for the isolation of ESCs, and also because of the risks associated with immune rejection of heterologous transplanted ESCs. Consequently, the use of induced pluripotent stem cells (iPSCs) using a set of transcription factors, such as OCT3/4, SOX2, KLF4 and c-MYC (Yamanaka factors), into differentiated somatic cells may help to overcome the immune rejection problem (Takahashi and Yamanaka 2006).

Induced pluripotent stem cells are similar to ESCs in morphology, proliferation and pluripotency. Successful generation of iPSCs has been reported for humans (Takahashi and Yamanaka 2006) and other species (Takahashi et al. 2007; Telugo et al. 2010 including the dog (Luo et al. 2011; Whitworth et al. 2012). Although the use of iPSCs in basic research is moving forward, their use as a therapeutic tool remains a challenge, mostly because of the lack of appropriate animal models for testing their efficacy and safety (Luo et al. 2011).

For a long time, the dog has served as a valuable model for human diseases; thus, approximately 58% of dog genetic diseases resemble specific human disorders caused by mutations in the same gene (Luo et al. 2011) and – more practically – the lifespan of dogs is considerably longer than that of rodents, thus allowing more adequate long-term studies in disease processes and therapeutics conducted in the rodent models (Whitworth et al. 2012).

Direct reprogramming generates iPSCs with the molecular profile and developmental potential of ESCs. However, several studies have suggested that small differences in gene expression or chromatin modifications yield iPS cell lines with reduced pluripotency when compared with ESCs (Carey et al. 2011). To harness the full potential of iPSC technology, it is important to understand the mechanisms involved. Therefore, the aim of this study was to obtain further information on the cellular mechanisms underlying induced pluripotency in dog cells for the future establishment of iPS cells that can be used for cell therapy and other purposes.

Materials and Methods

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. References

The study protocol was approved by the research ethics committee (2377/2011) of the Faculty of Animal Science and Food Engineering, University of Sao Paulo, Brazil.

Preparation of canine foetal fibroblasts

Foetal fibroblasts were obtained from one foetus at approximately 30 days of gestation.

Following ovariohysterectomy, the uterus from a pregnant animal was removed and immediately taken to the laboratory as previously described (Miglino et al. 2006; Martins et al. 2011) . After aseptic removal of organs and of the head, the foetus was washed in PBS-L (without calcium or magnesium) supplemented with antibiotics (1% penicillin–streptomycin; Invitrogen, Carlsbad, CA, USA) and minced. Fragments of approximately 1 mm were incubated with collagenase IV (40 mg/ml; Sigma, St. Louis, MO, USA) for 3 h at 38.5°C. After incubation, cells were washed and in vitro cultured in Dulbecco's modified Eagle medium (DMEM) (Gibco, Carlsbad, CA, USA, Invitrogen) supplemented with 10% foetal bovine serum and 1% penicillin–streptomycin (Gibco, Invitrogen). Culture media were changed every 3 or 4 days until the cells were subconfluent. The foetal fibroblasts were maintained in culture or frozen and stored at −80°C for further experiments.

Lentiviral production and transduction of canine fibroblasts

Plasmids contain the transcription factors human (h) OCT4-vexGFP and SOX2-mCitrine, each of which linked to a fluorescent reporter (Papapetrou et al. 2009). Lentiviral production consisted of the lipofection of 293FT cells (Invitrogen) with Lipofectamine 2000 reagent (Invitrogen) following manufacturer's suggestions. Supernatant was recovered at 48 and 72 h after transfection, filtered and concentrated by ultracentrifugation (28 000 × g for 2 h); supernatant was removed by pouring the tube and the remaining media culture was pipetted and used in the transductions. Stable transgenic cell lineages were produced by transducing 105 canine fibroblasts cells plated the day before with 100 μl or 250 μl for SOX2, 100 μl for OCT4 and a mix of the two factors (50 μl for SOX2 and 100 μl for OCT4) of the concentrated lentivirus produced with each of the transcription factors supplemented with 6 μg/ml polibrene (Sigma).

Induced pluripotent stem cell culture

Induced pluripotent stem cells were maintained on mitomycin-treated feeder layers (MEFs) with iPSC medium, which consisted of Dulbecco's modified Eagle's medium/F-12 (Gibco) supplemented with 20% (v/v) knockout serum (Gibco), minimal essential medium (MEM) non-essential amino acid solution (Sigma), glutamine (Invitrogen), 0.055 μm β-mercaptoethanol, 5 ng/ml human bFGF (Invitrogen) and 1000 U human LIF (Millipore, Billerica, MA, USA).

Flow cytometry

Percentage of cells expressing the fluorescent reporter was analysed by flow cytometry (FACSAria; BD Biosciences, San Jose, CA, USA), and positive cells were sorted and in vitro cultured or cryopreserved for further studies. To confirm the stability of SOX2-mCitrine and OCT4-vexGFP transgenic canine fibroblasts, assays were performed with qPCR and confocal microscopy.

Confocal microscopy

Live cells were submitted to confocal microscopy equipped with a fluorescence filter spectrum (Leica TSC SP5 AOBS; Leica Microsystems, Wetzlar, Germany) allowing the analysis of OCT4-vexGFP (excitation laser 405 nm, emission 535 nm) and Sox-mCitrine (excitation laser 514 nm, emission 529 nm). The pictures were analysed by software LAS-AF (Leica Application Suite and Advanced Fluorescence; Leica Microsystems) with modules Live Data, FRAP, FRET AB and SE.

Polymerase chain reaction

Total RNA (from purified samples) was isolated by the method of TRIzol Reagent (Invitrogen). Quantification of RNA was on a UV spectrophotometer at 260 nm. The cDNA High Capacity Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA) was used for cDNA synthesis; this kit uses random hexamers for the conversion of total RNA into cDNA.

Gene expression was assessed by quantitative PCR (Step One Real Time PCR Systems; Life Technologies, Carlsbad, CA, USA). The reactions were performed using a commercial assay system (SYBR® Green PCR Master Mix; Life Technologies); target genes were OCT4 and SOX2. Glyceraldehyde 3-phosphate dehydrogenase gene (GAPDH) served as a housekeeping gene. The sequences of the primers used are shown in Table 1. Reaction conditions consisted of 40 cycles with annealing temperature of 60°C. Quantification of OCT4 and SOX2 was by normalizing the signals with GAPDH signals using the inline image method (Livak and Schmittgen 2001).

Table 1. Sequences of the primers used for qPCR
GeneSequences (5′–3′)
OCT4_FWDCAGGCCCGAAAGAGAAAGC
OCT4_REVCGGGCACTGCAGGAACA
SOX2_FWDTGCGAGCGCTGCACAT
SOX2_REVTCATGAGCGTCTTGGTTTTCC
GAPDH_FWDAAGGCCATCACCATCTTCCA
GAPDH_REVCCACTACATACTCAGCACCAGCAT

Results

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. References

Flow cytometry

Stable cell lineages expressing the pluripotency-related transcription factors SOX2-mCitrine, OCT4-vexGFP and SOX2-mCitrine + OCT4-vexGFP were produced. The percentage of positive cells was calculated using non-transduced cells as controls. The results obtained and those after incubation with different volumes of the virus-containing supernatant are shown in Table 2. Regarding SOX2-positive cells, however not significantly different, fibroblasts transduced with a smaller amount of virus (100 μl) showed a higher percentage of positive cells compared with those that were transduced with 250 μl of supernatant (96.3% vs 90.2% of positive cells).

Table 2. Flow cytometry analysis of stable SOX2-mCitrine and OCT4-vexGFP transgenic canine fibroblasts
PlasmidsPositive cells, %
SOX2-mCitrine (100 μl)96.3
SOX2-mCitrine (250 μl)90.2
OCT4-vexGFP (100 μl)91.4
SOX2-mCitrine (50 μl) + OCT4-vexGFP (100 μl)73.2 + 92.8

Confocal microscopy

Confocal analysis enabled the visualization of the fluorescent protein reporters of both constructs (OCT4-vexGFP and SOX2-mCitrine), also in the double transduced cells (OCT4-vexGFP + SOX2-mCitrine). Both fluorescent proteins were equally distributed throughout the cell cytoplasm (Fig. 1).

image

Figure 1. Micrographs of canine foetal fibroblasts expressing the fluorescent protein reporters. a and a′: OCT4-vexGFP and vexGFP merged with bright field, b and b′: SOX2-mCitrine and merged; c, c′ and c″: OCT4-vex, SOX2-mCitrine and double positive, respectively

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Gene expression analysis

First results obtained on day 6 after transduction show the expression of both transcription factors in transduced cells, while controls were negative (Fig. 2).

image

Figure 2. Relative quantification of genes OCT4 (a) and SOX2 (b) using qPCR

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Discussion

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. References

Herein, we report the production of genetically modified canine foetal fibroblast cell lines, expressing different human transcription factors related to pluripotency. Such cell lines are important for the development of IPSC.

The percentage of cells positively transduced was estimated via the fluorescent reporter gene expression and flow cytometry. Canine cells were shown to be highly responsive to the integration and expression of human SOX2 and OCT4 apparently exceeding the responsiveness seen in other species (Huangfu et al. 2008).

Fluorescent microscopy analysis showed that the distribution of reporter proteins was similar for both transcription factors and that protein localization is not restricted to a particular cell region. The characterization of such positive results is essential for the establishment of induction of pluripotency through the incorporation of known transcription factors into the genome of somatic cells.

OCT4 and SOX2 expression in transduced cells showed a high exogenous expression of each targeted gene in single and double transfected cells. However, no interaction between OCT4 and SOX2 was observed, at least until 6 days after transduction. As day 6 after genetic modification is too early to allow for a possible and proper reprogramming of the endogenous genome, gene expression observed in this study must be due to the expression of the ‘transgene’. According to Hammachi et al. (2012), OCT4 is an essential regulator of pluripotency in vivo and in vitro in ESCs, as well as a key mediator of the reprogramming of somatic cells into iPSCs, and its interaction with SOX2 may be essential for such purpose (Rizzino 2009).

As already known, species other than humans and mice are refractory to the establishment of true ESCs. Therefore, optimization and characterization of direct reprogramming in domestic animals such as the dog are extremely helpful and may contribute in clarifying the mechanisms involved in cellular reprogramming pathways.

Acknowledgements

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. References

This study was supported by grants from the State of São Paulo Research Foundation (FAPESP grant 2012/01060-4); Gonçalves, NJN and Bressan, FF were supported by FAPESP graduate fellowships 2011/22915-5 and 2009/11631-6, respectively.

Conflicts of interest

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. References

None of the authors have any conflicts of interest to declare.

References

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. References
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