Brief Report: Impaired Cell Reprogramming in Nonhomologous End Joining Deficient Cells

Authors

  • F. Javier Molina-Estevez,

    1. Division of Hematopoietic Innovative Therapies (HIT), Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT)/Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBER-ER), Madrid, Spain
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  • M. Luz Lozano,

    1. Division of Hematopoietic Innovative Therapies (HIT), Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT)/Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBER-ER), Madrid, Spain
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  • Susana Navarro,

    1. Division of Hematopoietic Innovative Therapies (HIT), Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT)/Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBER-ER), Madrid, Spain
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  • Yaima Torres,

    1. Department of Regenerative Cardiology, Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III, Madrid, Spain
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  • Ivana Grabundzija,

    1. Department of Mobile DNA, Max Delbrück Center for Molecular Medicine, Berlin, Germany
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  • Zoltan Ivics,

    1. Division of Medical Biotechnology, Paul Ehrlich Institute, Langen, Germany
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  • Enrique Samper,

    1. Department of Regenerative Cardiology, Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III, Madrid, Spain
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  • Juan A. Bueren,

    1. Division of Hematopoietic Innovative Therapies (HIT), Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT)/Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBER-ER), Madrid, Spain
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  • Guillermo Guenechea

    Corresponding author
    1. Division of Hematopoietic Innovative Therapies (HIT), Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT)/Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBER-ER), Madrid, Spain
    • Division of Hematopoietic Innovative Therapies (HIT), CIEMAT/CIBERER, Edificio 70, Av. Complutense 40, 28040 Madrid, Spain===

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    • Telephone: +34-914962530; Fax: +34-913466484


  • Author contributions: F.J.M.-E.: conception and design, collection and/or assembly of data, data analysis and interpretation, and manuscript writing; M.L.L.: conception and design, collection and/or assembly of data, and data analysis and interpretation; S.N.: conception and design, collection and/or assembly of data, and data analysis and interpretation; Y.T.: collection and/or assembly of data and data analysis and interpretation; I.G.: provision of study material or patients; Z.I.: provision of study material or patients and manuscript writing; E.S.: collection and/or assembly of data and data analysis and interpretation; J.A.B.: conception and design, data analysis and interpretation, manuscript writing, and final approval of the manuscript; G.G.: conception and design, data analysis and interpretation, manuscript writing, and final approval of the manuscript.

Abstract

Although there is an increasing interest in defining the role of DNA damage response mechanisms in cell reprogramming, the relevance of proteins participating in nonhomologous end joining (NHEJ), a major mechanism of DNA double-strand breaks repair, in this process remains to be investigated. Herein, we present data related to the reprogramming of primary mouse embryonic fibroblasts (MEF) from severe combined immunodeficient (Scid) mice defective in DNA-PKcs, a key protein for NHEJ. Reduced numbers of induced pluripotent stem cell (iPSC) colonies were generated from Scid cells using reprogramming lentiviral vectors (LV), being the reprogramming efficiency fourfold to sevenfold lower than that observed in wt cells. Moreover, these Scid iPSC-like clones were prematurely lost or differentiated spontaneously. While the Scid mutation neither reduce the proliferation rate nor the transduction efficacy of fibroblasts transduced with reprogramming LV, both the expression of SA-β-Gal and of P16/INK4a senescence markers were highly increased in Scid versus wt MEFs during the reprogramming process, accounting for the reduced reprogramming efficacy of Scid MEFs. The use of improved Sleeping Beauty transposon/transposase systems allowed us, however, to isolate DNA-PKcs-deficient iPSCs which preserved their parental genotype and hypersensitivity to ionizing radiation. This new disease-specific iPSC model would be useful to understand the physiological consequences of the DNA-PKcs mutation during development and would help to improve current cell and gene therapy strategies for the disease. STEM Cells 2013;31:1726–1730

Introduction

In 2006, Shinya Yamanaka and colleagues stirred up the stem cell research field by presenting a breakthrough method to reprogram adult somatic cells into induced pluripotent stem cells (iPSCs) by retroviral-mediated transfer of a limited number of transcription factor-encoding genes, namely, Oct3/4(Pou5f1), Klf4, Sox2, and cMyc [1].

Aiming to discern the biochemical pathways required for the efficient and safe reprogramming of adult somatic cells into iPSCs, recent studies have shown the relevance of telomere homeostasis [2–4], signal transducers, and DNA repair proteins [4–7] in cell reprogramming. These results have shown that a significant DNA stress—reflected by an increased number of DNA double strand breaks (DSBs)—is produced during the reprogramming process. Additionally, these studies showed the relevance of functional DNA repair pathways in cell reprogramming. Strikingly, although nonhomologous end joining (NHEJ) is a major pathway of DNA DSB repair [8, 9], the role of NHEJ in cell reprogramming has not been studied so far.

Taking into account that the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) plays a key role in NHEJ [10, 11], we have investigated the impact that the knockout of Prkdcscid (encoding for DNA-PKcs) has in the efficacy of cell reprogramming.

Materials and Methods

Primary mouse embryo fibroblast (MEFs) at E13.5 stage were isolated in-house from severe combined immunodeficient (Scid) BALB/c JHan Hsd-Prkdcscid and isogenic wt mice obtained from Harlan Laboratories (www.harlan.com, Barcelona, Spain). Passage 2 MEFs were either transduced or transfected using newly developed reprogramming lentiviral vectors (LV) [12, 13] or Sleeping Beauty transposon (SB) [14, 15] in conjunction with hyperactive transposase mutant SB100X [14] and then cultured for iPSC derivation. Further details can be found in Supporting Information Material and Methods.

Results and Discussion

To investigate the relevance of NHEJ in cell reprogramming, in the first set of experiments we investigated the generation of alkaline phosphatase positive (AP+) iPSC-like clones after transduction of Scid and wt MEFs with the STEMCCA RedLight LV that carries the reprogramming genes Oct3, Klf4, Sox2, and mCherry marker [12, 13] (Supporting Information Fig. 1A). While wt MEFs generated an average of 30.6 ± 17.5 iPSC-like colonies per 105 cells 17 days after transduction, the number of AP-positive Scid iPSC-like clones was decreased by threefold (10.98 ± 6.78 iPSC-like colonies per 105 cells) (Fig. 1A). Moreover, whereas several wt iPSCs could be maintained in the long-term and showed the characteristic properties of iPSCs (Supporting Information Fig. 1B, 1C, 1D), all Scid clones degenerated during the first passages, evidencing the relevance of NHEJ in the generation and/or expansion of iPSCs.

Figure 1.

Characterization of wt and Scid MEFs transduced with reprogramming LVs. (A): Reprogramming efficiency was determined as AP positive colonies count. 105 MEFs were transduced with the STEMCCA RedLight vector and grown under ES conditions. Cells were fixed and scored 17 days post-transduction. (B): To quantify telomere length, telomeric and centromeric probes were assayed on MEFs metaphases from either mock or STEMCCA-RedLight LV-transduced cultures 5 days after transduction. ***, p < .001; t test. (C): Cell proliferation was followed on mock (light bars) and transduced (dark bars) cultures upon cell reprogramming counting the cells after trypan blue staining. *, p < .05. (D): Transduction efficacy was measured by fluorescence activated cell sorting at the different times showed (left). Mean percentage of mCherry fluorescence in Scid and wt MEFs transduced with STEMCCA RedLight LV at different MOIs are represented in the right panel. (E): Expression levels of p53, p21/CIP1, p15/INK4b, and p16/INK4a were determined by quantitative polymerase chain reaction in mock (−) and transduced cells (LV) 10 days after transduction. Gapdh-normalized expression values were relativized to wt mock samples for comparison purposes. (F): SA-β-Gal staining was performed on Scid and wt samples 10 days after transduction with STEMCCA RedLight vector. At least 200 cells per well were counted in different fields of six-well plates, and the increase of the SA-β-Gal positive fraction was calculated using parallel mock cultures as reference. Abbreviations: AP, alkaline phosphatase; MEFs, mouse embryonic fibroblasts; MOI, multiplicity of infection; LV, lentiviral vector; Scid, severe combined immunodeficiency; SA-β-Gal, senescence associated β-galactosidase; wt, wild type.

Only cells with telomeres over a threshold length and with a significant proliferation ability can be efficiently reprogrammed [2, 3]. Therefore, to investigate whether any of these properties were affected in Scid cells, we measured by quantitative fluorescence in situ hibrization (q-FISH) the telomere length of wt and Scid MEFs prior and also after transduction with reprogramming LV. As shown in Figure 1B, the telomeres' length observed in all Scid groups was significantly longer when compared with their wt counterparts. These results mimic the observations in fresh samples, which showed increased telomere lengths in heterozygous and homozygous Scid MEFs with respect to wt MEFs (Supporting Information Fig. 2A, 2B). Although the role of DNA-PK in telomere elongation and capping has been a subject of discussion [16, 17], our results are consistent with previous observations [17, 18] and discard the hypothesis that eroded telomeres could account for the reprogramming deficiency observed in Scid MEFs.

When the cell expansion rate was determined in untransduced wt and Scid primary MEFs, no significant differences between them were noted (threefold to fourfold expansion after 7 days of culture in either wt or Scid MEFs; Fig. 1C light bars). In all instances, good viabilities above 90% were routinely observed (not shown). Noteworthy, transduction with the STEMCCA-RedLight LV increased the expansion rate both in wt and Scid MEFs, although differences were only significant in Scid MEFs after 7 days of transduction (Fig. 1C). These results discarded that a defective proliferation rate was accounting for the impaired reprogramming of Prkdcscid MEFs.

The Scid mutation may confer a restricted LV-transduction to MEFs [19, 20], hence leading to a specific defective reprogramming of Scid cells. To investigate whether a reduced transduction efficacy was accounting for the restricted ability of Prkdcscid MEFs for being reprogrammed, wt and Scid MEFs were transduced with multiplicities of infection (MOIs) of 5 and 10 transduction units (TUs) per cell using the STEMCCA-RedLight LV. Thereafter, the percentage of mCherry-positive cells was determined at 3 and 7 days post-transduction. As shown in Figure 1D, similar and MOI-dependent percentages of mCherry positive cells were observed in wt and Scid MEFs both at 3 and 7 days post-transduction, indicating that a reduced transduction efficacy of Scid MEFs with the reprogramming LV was not accounting for the restricted reprogramming of these cells.

Next, to explain whether the genetic stress induced by cell reprogramming mediated a differential apoptotic or senescent response in Scid MEFs, the expression of key regulators controlling these cell functions was determined 10 days after transduction of wt and Scid MEFs with the STEMCCA-RedLight LV. A very weak expression of p53, p21, or p15/Ink4b was observed both in wt and Scid MEFs, either transduced or not. In contrast to this observation, a marked increase in p16/INK4a, incompatible with cell reprogramming [2], was specifically observed in Scid cells transduced with the reprogramming vector (Fig. 1E). This observation was consistent with senescence associated β-galactosidase staining (SA-β-Gal; Fig. 1F), where a tendency of increased staining was consistently observed in reprogramming Scid cells.

In a further attempt to generate Prkdcscid iPSCs, cells were either transduced with the STEMCCA-Myc LV (Supporting Information Fig. 1A) or nucleofected with the reprogramming T2/OSKM transposon and the hyperactive Sleeping Beauty transposase (SB100X) [14, 21] (Supporting Information Fig. 3A). Compared to the number of AP+ colonies generated with the STEMCCA-RedLight LV (Fig. 1A), AP+ colonies generated with the STEMCCA-Myc LV (98 ± 8 AP+ colonies per 105 cells and 12 ± 1 per 105 cells; wt and Scid, respectively) and, more markedly, with the T2/OSKM SB (313 ± 83 per 105 cells and 117 ± 7 AP+ colonies per 105 cells; wt and Scid cells, respectively) were clearly increased (Fig. 2A). To discard that a differential integration efficiency of these two vectors accounted for differences in cell reprogramming, vector copy numbers were calculated in the LV- and SB-generated iPSC clones. In all instances, values of 1–2 integrations per cell were found (data not shown). Telomere quantification also showed similar results in either the LV- or SB-treated MEFs (Supporting Information Fig. 3B) and were highly consistent with data obtained in Figure 1B.

Figure 2.

Characterization of wt and Scid MEFs transfected with reprogramming transposons. (A): Reprogramming efficiency was determined as AP positive colonies count. 105 MEFs were transduced with the STEMCCA cMyc vector or nucleofected with the T2/OSKM reprogramming vector and the SB100X transposase and grown under ES conditions. Cells were fixed and scored 17 days post-transduction. (B): Expression levels of p53, p21/CIP1, p15/INK4b, and p16/INK4a were determined by quantitative polymerase chain reaction (PCR) in mock (−) and transposon ransfected cells (Tn) 10 days after transduction. Gapdh-normalized expression values were relativized to wt mock samples for comparison purposes. (C): All clones derived were positive for SSEA-1 surface marker analyzed by flow cytometry. Characteristic histograms from wt and Scid Tn-iPS clones are shown. (D): We demonstrate expression of pluripotency markers. ×100 magnified photographs show immunodetection of Nanog, SSEA1, and Oct3/4 in Scid and wt Tn-iPS in the colonies. (E): To confirm endogenous expression of embryonic stem cell (ESC) factors, RNA extracts from Tn-iPS grown on feeder-free cultures were processed for quantitative PCR (qPCR). Expression levels of Nanog, c-Myc, and Sox2 from wt and Scid Tn-iPS are shown referred to mouse ESC J1 levels. (F): Karyotype of the Tn-iPS clones generated was determined on fixed metaphases from feeder-free cultures. (G): Diagnostic AluI restriction of the Scid locus matched expected genotype for the Scid and wt parental MEFs and derived Tn-iPS. (H): Radiation sensitivity of primary MEFs and Tn-iPSs from Scid and wt backgrounds. Cells were exposed to x-rays, and survival fraction was assessed in CFU-F and AP+ iPSC colonies, respectively. Abbreviations: AP, alkaline phosphatase; CFU-F, colony forming units-fibroblast; iPSC, induced pluripotent stem cell; MEF, mouse embryonic fibroblast; Scid, severe combined immunodeficiency; Tn, transposon transfected cells; wt, wild type.

Further analyses performed in SB-transfected cells showed that, in contrast to data previously obtained with STEMCCA-RedLight (Fig. 1E), a very weak expression of p16/INK4a was now observed in Scid cells (Fig. 2B). Consistent with this observation, similar SA-β-Gal staining was observed in SB-transfected wt and Scid MEFs (Supporting Information Fig. 3C). Additional experiments showed that differences in the induction of SA-β-Gal+ cells by the reprogramming STEMCCA-RedLight and the T2/OSKM were highly significant (p < .001; n = 3). The more limited senescence induced by the T2/OSKM SB transposon on Scid cells may thus account for the increased reprogramming efficacy of this construct on Scid MEFs.

While Prkdcscid iPSC colonies could be generated from Scid MEFs transduced with the STEMCCA-Myc LV, the higher reprogramming efficacy of the transposon approach, particularly in Scid cells (Fig. 2A), allowed us to generate and propagate in vitro several Prkdcscid transposon-mediated iPSC clones (Tn-iPSC). These clones maintained the characteristic iPSC morphology (Supporting Information Fig. 3D), expressed the expected markers of pluripotency (Fig. 2C–2E), generated teratomas with tissue structures from the three germ layers (Supporting Information Fig. 3E), and some of them retained a normal karyotype (Fig. 2F). The presence of the Scid mutation in Prkdcscid Tn-iPSCs was confirmed by means of PCR assays (Fig. 2G). Additionally, we demonstrated that, as with parental Prkdcscid MEFs, Prkdcscid Tn-iPSCs were also hypersensitive to ionizing radiation as expected for cells deficient in NHEJ [22–24] (Fig. 2H).

Conclusion

Taking together, our results demonstrate that Scid cells lacking fully functional DNA-PKcs and thus with an impaired NHEJ ability, are characterized by a defective cell reprogramming. We also demonstrate that this reprogramming barrier can be surpassed using the efficient reprogramming SB transposon system, which facilitated the generation of Prkdcscid Tn-iPSC clones that retained their characteristic radiation hypersensitivity of NHEJ cells. These novel findings will lead to further research on the interaction of NHEJ proteins and delivery systems available for cell reprogramming. Besides, Prkdc-/- iPSCs should constitute an excellent model to investigate molecular and developmental defects associated to NHEJ deficiency in pluripotent stem cells.

Acknowledgements

We thank Gustavo Mostoslavsky for kindly providing pSTEMCCA c-Myc and pSTEMCCA RedLight plasmids and to Rosa Maria Yañez for her unvaluable advice on senescence and aging; José C. Segovia, Oscar Quintana, Victoria Moleiro, Juan Camilo Estrada, Elena Almarza, Diego León, Israel Orman, Raquel Chinchón, Laura Cerrato, Aurora de la Cal, Sergio Losada and Angelines Acevedo for collaboration in experimental work; Miguel Angel Martín, Jesús Martínez, and Edilia de Almeida for careful maintenance of the animals; Fundación Marcelino Botín for promoting translational research at the Division of Hematopoietic Innovative Therapies (HIT) of the CIEMAT/CIBERER. This work was supported by the following grants: European Commision FP7 Program (PERSIST; Ref 222878), Spanish Ministry of Economy and Competitiveness (International Cooperation on Stem Cell Research Plan E; Ref PLE 2009/0100; SAF 2009-07164), Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III (RETICS-RD06/0010/0015 and PI08/0701), Dirección General de Investigación de la Comunidad de Madrid (CellCAM; Ref S2010/BMD-2420), EU FP7 InduStem (grant number 230675), Fundació la Marató de TV3 (Ref 464/C/2012), Hungarian stem cell project TÁMOP-4.2.2-08/1-2008-0015 and the Bundesministerium für Bildung und Forschung (ReGene, grant number 01GN1003A).

Disclosure of Potential Conflicts of Interest

The authors indicate no potential conflicts of interest.

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