Amenable epigenetic traits of dental pulp stem cells underlie high capability of xeno-free episomal reprogramming

Thekkeparambil Chandrabose et al. Stem Cell Research & Therapy (2018) 9:68 https://doi.org/10.1186/s13287-018-0796-2 RESEARCH Open Access Amenable epigenetic traits of dental pulp stem cells underlie high capability of xenofree episomal reprogramming Srijaya Thekkeparambil Chandrabose1†, Sandhya Sriram2†, Subha Subramanian2, Shanshan Cheng3, Wee Kiat Ong2,4, Steve Rozen3, Noor Hayaty Abu Kasim1* and Shigeki Sugii2,5* Abstract Background: While a shift towards non-viral and animal component-free methods of generating induced pluripotent stem (iPS) cells is preferred for safer clinical applications, there is still a shortage of reliable cell sources and protocols for efficient reprogramming. Methods: Here, we show a robust episomal and xeno-free reprogramming strategy for human iPS generation from dental pulp stem cells (DPSCs) which renders good efficiency (0.19%) over a short time frame (13–18 days). Results: The robustness of DPSCs as starting cells for iPS induction is found due to their exceptional inherent stemness properties, developmental origin from neural crest cells, specification for tissue commitment, and differentiation capability. To investigate the epigenetic basis for the high reprogramming efficiency of DPSCs, we performed genome-wide DNA methylation analysis and found that the epigenetic signature of DPSCs associated with pluripotent, developmental, and ecto-mesenchymal genes is relatively close to that of iPS and embryonic stem (ES) cells. Among these genes, it is found that overexpression of PAX9 and knockdown of HERV-FRD improved the efficiencies of iPS generation. Conclusion: In conclusion, our study provides underlying epigenetic mechanisms that establish a robust platform for efficient generation of iPS cells from DPSCs, facilitating industrial and clinical use of iPS technology for therapeutic needs. Keywords: Induced pluripotent stem cells, Dental pulp-derived mesenchymal stem cells, Stem cell therapeutics, Regenerative medicine, Xeno-free, Feeder-free, Episomal vector reprogramming Background The capability of human pluripotent stem cells to differentiate toward multilineage adult tissues provides ample cellular sources for cell therapy. Shinya Yamanaka and his group made the landmark discovery of reprogramming somatic cells by introducing transcription factors chosen from embryonic stem (ES) cells and transforming them into induced pluripotent stem (iPS) cells [1]. This exciting discovery paved the way for replacing the use of ethically and politically controversial ES cells in cell therapies. In * Correspondence: ; † Equal contributors 1 Department of Restorative Dentistry, Faculty of Dentistry, University of Malaya, 50603 Kuala Lumpur, Malaysia 2 Fat Metabolism and Stem Cell Group (FMSCG), Laboratory of Metabolic Medicine (LMM), Singapore Bioimaging Consortium (SBIC), Helios, Biopolis, A*STAR, Singapore 138667, Singapore Full list of author information is available at the end of the article vitro modelling of patients’ own cells mimicking human diseases became possible for studying the nature and complexity of genetic diseases and for application in drug discovery [2]. iPS technology can be used for elucidating the complexity of many human ailments, especially regarding genetic, degenerative, injured or age-associated conditions related to neurological systems, hepatic, cardiac, vision, bone disorders, wounding, autoimmune diseases, spinal cord injury, metabolic disorders, and certain types of cancers [3–5]. The initial attempts to induce reprogramming were undertaken with viral-mediated vectors for expressing the transcription factors; however, with time, multiple efforts were made to improve the reprogramming strategies to increase the efficiency of iPS generation and also to adhere to cell therapy practices. Attempts include improving the reprogramming strategies using synthetic © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Thekkeparambil Chandrabose et al. Stem Cell Research & Therapy (2018) 9:68 mRNAs, synthetic miRNAs, recombinant proteins, temperature-sensitive Sendai virus, and episomal plasmids [6, 7]. Currently, standard iPS culture systems are innately unstable and difficult for application in clinical therapies due to the presence of various xenogenic factors, such as feeder layers, growth factors, and extracellular matrix (ECM) components in culture media, which makes the quality control of cells difficult. Consequently, to improve the clinical utility of iPS cells, attempts were made to use feeder layers, serum, or ECM of human origin for supporting the iPS culture in a physiological niche [8–10]. However, the complication of limited sources of human materials and their laborious maintenance efforts hampered the possibility of testing these cells in human clinical trials. Hence, to facilitate the implementation of iPS cell-based therapies, xeno-free and feeder-free alternative materials and methods minimizing human materials were developed, but this development has been relatively slow due to the lower efficiency of reprogramming in most of the cell types tested [8, 11]. Significant efforts have been made at looking for an ideal starting cell population for reprogramming [12]. It is generally better to have more developmentally immature cells as the starting source as they exhibit inherently higher proliferation, differentiation, and regenerative properties. This reduces the risk of mutations, chromosomal damage, or accumulated epigenetic changes compared to older cells [7]. We chose dental pulp stem cells (DPSCs) as the starting material in our experiments as they are known for their outstanding biological characteristics [13]. DPSCs are immature mesenchymal stem cells (MSCs) placed in the same lineage of cells as the umbilical cord- and Wharton’s Jelly-derived stem cells [14]. DPSCs are a rich source of potent MSCs despite the small amount of tissue samples obtained [15]. They are highly multipotent, have immunomodulatory properties, and can be obtained relatively easily by tooth extraction procedures [16–19]. The isolation procedure of the DPSCs is simple and robust. Remarkably, DPSCs also support reprogramming and induction of pluripotency in a more refined manner, possibly due to their dual mesectodermal and neural crest origins and inherent expression of pluripotent factors including Oct-4, Nanog, c-Myc, Sox2, stage-specific embryonic antigens (SSEA-3, SSEA (...truncated)