MDM4 enables efficient human iPS cell generation from PBMCs using synthetic RNAs

MDM4 enables efficient human iPS cell generation from PBMCs using synthetic RNAs | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article MDM4 enables efficient human iPS cell generation from PBMCs using synthetic RNAs Masato Nakagawa, Mizuho Nogi, Hatsuki Doi, Hirohisa Ohno, Megumi Mochizuki, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6250001/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 08 Sep, 2025 Read the published version in Scientific Reports → Version 1 posted 15 You are reading this latest preprint version Abstract If iPS cells can be established easily and efficiently using freshly collected blood cells, it will enhance regenerative and personalized medicine. While there have been reports of iPS derivation from blood-derived endothelial progenitor cells using RNA, none have been documented from peripheral blood-derived mononuclear cells (PBMCs). In this study, we established a method to generate iPS cells from PBMCs using synthetic RNAs and found that MDM4, which suppresses p53, improved reprogramming efficiency. Biological sciences/Stem cells/Reprogramming Biological sciences/Stem cells/Pluripotent stem cells/Induced pluripotent stem cells Figures Figure 1 Figure 2 Main Text Recently, the development of regenerative medicine and cell therapy using iPS cells has gained significant momentum. Additionally, valiant efforts to elucidate disease states and discover new therapeutic drugs with patient-derived iPS cells are evident 1–5 . More recently, attempts have been made to create individual iPS cells tailored for personalized medicine. Consequently, there is an increasing demand for more efficient production of high-quality iPS cells. Human iPS cells were first established from skin-derived fibroblasts (human dermal fibroblasts, HDFs) on feeder cells using retroviruses 6 . The method was stable enough to produce iPS cells, making it suitable for research. However, many challenges had to be addressed when considering future applications for iPS cell-based cell therapy and regenerative medicine. The world's first clinical-grade iPS cells were produced from human peripheral blood-derived mononuclear cells (PBMCs) using plasmid vectors under feeder-free culture conditions 7 . Since there is a risk that plasmid vectors may integrate into the genomic DNA of iPS cells generated via this method, an approach avoiding the insertion of exogenous genes has become necessary. Currently, the most efficient method for generating iPS cells involves using Sendai virus 8 . However, it is crucial to consider the biosafety level requirements and restrictions on vector modification as potential limitations when utilizing Sendai virus vectors. Meanwhile, researchers have reported that synthetic RNA, including messenger RNA (mRNA) and microRNA (miRNA), can be used to create iPS cells 9 , 10 . The transfected RNA does not remain in the cells, and the ability to synthesize any RNA with the desired sequence in vitro represents a significant advantage. Although synthetic RNA has been utilized to generate iPS cells from human fibroblasts and blood-derived endothelial progenitor cells, their generation from PBMCs has yet to be reported 11 . In this study, we produced iPS cells from PBMCs using synthetic RNA and found that MDM4, which suppresses the function of p53, significantly increased the reprogramming efficiency. A protocol for reprogramming human dermal fibroblasts (HDFs) using synthetic RNA was validated with a previously described method 11 . We primarily utilized the StemRNA 3rd Gen Reprogramming Kit (ReproCELL, Japan) for this experiment. The number of synthetic RNA transfections varied from four to one, and the minimum number of transfections necessary to produce iPS cells was assessed by immunostaining on day 9. During the same experiment, we also evaluated the effects of tumor suppressor gene TP53 and its regulatory genes (e.g., MDM2 and MDM4 , also known as MDMX ) on reprogramming efficiency. Human dermal fibroblasts were cultured for four days and harvested, with the required number of cells collected. The synthetic RNA for reprogramming was mixed with the transfection reagent, combined with the cell suspension, and with iMatrix-511, a substrate for iPS cell culture, before being seeded onto a culture plate. StemFit AK03 without bFGF was the reprogramming medium until colonies appeared (up to day 7). Immunostaining was conducted on day 9, with the number of TRA-1-60-positive colonies counted using ImageJ, and the reprogramming efficiency was calculated based on the number of HDFs seeded on day 0 (Fig. 1b and 1c). When mCherry mRNA was added to the reprogramming kit, colonies appeared to fill the entire well after four transfections. Colonies were still observed even when we reduced the number of transfections to two. However, no colonies appeared after one transfection. Since previous reports indicated that suppressing p53 functions increases reprogramming efficiency 12 , we investigated whether it could enhance RNA-mediated reprogramming. Human dermal fibroblasts were transfected with the reprogramming kit alongside p53 wild-type (WT), dominant-negative p53 (R175H 13 ), MDM2, or MDM4, which are known to inhibit p53 14 . p53 WT decreased the reprogramming efficiency, while p53 R175H increased it. MDM2 and MDM4 did not affect the reprogramming efficiency of HDFs (Fig. 1b, 1c, and Supplementary Table 1). We established multiple iPS cell clones from HDFs using synthetic RNA. Morphological, karyotypic, and gene expression analyses were conducted on MN388#4. The results were consistent with those of previously reported iPS cells (Fig. 1d and 1e). Next, we examined whether we could produce iPS cells from PBMCs using synthetic RNA. While the method and frequency of RNA transfection were identical to those used for reprogramming HDF, the culture medium used was optimized for PBMCs (Fig. 2a). iPS cell-like colonies of sufficient size emerged approximately 14 days after the initial RNA transfection, at which point immunostaining was conducted. When reprogramming PBMCs, only a limited number of colonies appeared after adding mCherry mRNA to the reprogramming kit, but the quantity increased slightly with the introduction of p53 R175H. By contrast to HDF, MDM4 mRNA significantly enhanced the efficiency of PBMC reprogramming (Fig. 2b and 2c). MDM4 serine 367 phosphorylation is known to lead to degradation via the ubiquitin pathway. The mutant S367A (MDM4-SA), with serine replaced by alanine, has been reported to be resistant to degradation 15 . We thus investigated the impact of this mutant on PBMC reprogramming (Fig. 2d, 2e, and 2f). Among the three samples of PBMCs presented in Fig. 2d, MDM4-S367A yielded the highest number of TRA-1-60-positive colonies. Notably, in the case of PBMC lot 2, which exhibited poor reprogramming efficiency, almost no colonies were observed except when MDM4-S367A was involved (Fig. 2e, 2f, and 2d, along with Supplementary Table 2). We also assessed the effect of the MDM4-S367D (MDM4-SD) mutant, which mimics the phosphorylated state, but its reprogramming ability was comparable to that of MDM4-WT. iPS cell clones established using synthetic RNA from PBMCs exhibited normal colony morphology and karyotype (Fig. 2g) with similar gene expression as a well-established iPS cell line (Fig. 2h). In this study, we successfully used synthetic RNA to generate iPS cells from HDFs and PBMCs isolated from human blood. Notably, our work revealed the critical inclusion of MDM4, which inhibits p53, with other reprogramming factors when utilizing blood cells. The initial step in establishing a synthetic RNA-based reprogramming protocol involved mixing all cells, culture medium, synthetic RNA, and coating substrate, then seeding them together during the initial transfection. This approach is believed to enhance efficiency compared to introducing RNA into adherent cells, as RNA can be delivered from the entire cell surface. Suppressing p53 functions is known to increase reprogramming efficiency 12 , 16-18 . As such, dominant-negative mutants of p53 and siRNA targeting p53 are routinely employed. Once again, the dominant-negative mutant of p53 had the most significant effect when human dermal fibroblasts (HDFs) were reprogrammed using synthetic RNA. MDM4, as well as MDM2, is well known to suppress the function of p53. However, both MDM2 and MDM4 had little effect on HDF reprogramming using synthetic RNA. By contrast, MDM4 exhibited the strongest effect when reprogramming PBMCs, indicating that the reprogramming mechanism with synthetic RNA varies slightly between HDFs and PBMCs. MDM4 may serve additional roles in PBMC reprogramming beyond suppressing p53 function. We aim to clarify this in future research and develop more efficient reprogramming methods. The results of this study enabled the production of iPS cells from PBMCs isolated from blood. This development will significantly contribute to cell therapy and regenerative medicine using iPS cells. Research and development of personalized medicine utilizing individual iPS cells has gained momentum recently. Because blood samples can be acquired with relative ease and minimal invasion, our findings will prove invaluable to further advance iPS cell-based personalized medicine. Separating PBMCs from a blood sample takes approximately an hour, and mRNA transfection is safe due to its short half-life and lack of genomic integration 19-22 . Thus, PBMCs represent a promising cell resource for generating iPS cells with mRNA for clinical applications. The immediate availability of blood samples for iPS cell establishment after collection offers a notable advantage. Methods Synthesis of mRNA in vitro mCherry, d2EGFP, p53 WT, p53 R175H, MDM2 WT, MDM4 WT, MDM4 S367A, and MDM4 S367D cDNAs were amplified by PCR and subcloned into plasmid vectors (T7 promoter) for in vitro RNA synthesis. We used a kit (NIPPON GENE, CUGA 7 in vitro Transcription kit, 307-13531) to synthesize mRNA and followed the attached protocol. CleanCap Reagent AG (3'OMe) (TriLink, N-7413-1) was used as a capping reagent. The Monarch RNA Cleanup Kit (NEW ENGLAND Biolabs, T2040S) was used to purify the synthesized RNA, and a Qubit 4 Fluorometer was used to measure its concentration and purity (Invitrogen, Q33238). HDF reprogramming Frozen HDF stocks (Cell Applications, INC., CA10605f) were thawed and cultured in DMEM (Nacalai Tesque, 08459-64) containing 10% FBS (SIGMA, F7524) for 4 days. Cells were harvested with trypsin (Gibco, 25200056), and the required number of cells were prepared in StemFit AK03N medium (AJINOMOTO, SF010-002) without bFGF (StemFit AK03N (-)) at a concentration of 5.0 x 10 5 cells/mL (day 0). The StemRNA 3rd Gen Reprogramming Kit (REPROCELL, 00–0076) was used for reprogramming. The transduction method followed the protocol attached to the kit. For reprogramming, 5.0 x 10 4 cells were seeded in one well of a 24-well plate. A total of 200 ng of mRNA encoding OSKMNL (0.76 uL), EKB (0.67 uL), and the additional factors synthesized in our lab (0.13 uL, 100 ng/mL), and microRNAs (0.16 uL) was mixed with 0.72 uL of Lipofectamine MessengerMAX Transfection Reagent (Invitrogen, LMRNA001) as the RNA transfection solution (RNAs and MessengerMAX were diluted with Opti-MEM medium (Invitrogen, 31985062)). Cell suspension (100 uL), RNA transfection solution, iMatrix-511 (1.3 uL) (MATRIXOME, 892 − 011), and StemFit AK03N (-) medium were mixed (total volume was 800 uL) and seeded into a well immediately. From day 1 to day 3, synthetic RNA was introduced as a medium and transfection solution mixture. The medium was changed to StemFit AK03N (-) on day 4 and StemFit AK03N containing bFGF (StemFit AK03N (+)) on day 7. Cells were immunostained with an anti-TRA-1-60 antibody or passaged on day 9. PBMC reprogramming Frozen PBMC stocks (Cellular Technology Ltd, CTL-UP1) were thawed and cultured in StemSpan SFEM II (STEMCELL Technologies, ST-09605) supplemented with six cytokines (20 ng/mL IL-3 (090-05761), 50 ng/mL IL-6 (098-06041), 10 ng/mL TPO (207-17581), 20 ng/mL Flt3L (061-05391), 50 ng/mL SCF (197-15511), and 10 ng/mL G-CSF (072-06101) (Fujifilm Wako Chemicals)) (SS6F medium) for 4 days. Cells were harvested after trypsinization, and the required number of cells was prepared in SS6F medium at a concentration of 1.0 x 10 6 cells/mL (day 0). The StemRNA 3rd Gen Reprogramming Kit was used for reprogramming. For reprogramming, 1.0 x 10 5 cells were seeded in one well of a 24-well plate. A total of 400 ng of mRNA encoding OSKMNL (1.48 uL), EKB (1.33 uL), and the additional factors synthesized in our lab (0.3 uL, 100 ng/mL), and microRNAs (0.31 uL) was mixed with 1.44 uL of Lipofectamine MessengerMAX Transfection Reagent as the RNA transfection solution (RNAs and MessengerMAX were diluted with Opti-MEM medium). Cell suspension (100 uL), RNA transfection solution, iMatrix-511 (2.5 uL), and SS6F medium were mixed (total volume was 800 uL) and seeded into a well immediately. From day 1 to 3, synthetic RNA was introduced as an SS6F medium and transfection solution mixture. The medium was changed to a mixture of SS6F medium and StemFit AK03N (+) (mix ratio 3:2) on day 4 and StemFit AK03N (+) on day 7. Cells were immunostained with an anti-TRA-1-60 antibody or passaged on day 14. Immunostaining and colony counting Reprogrammed cells were fixed with a 4% paraformaldehyde phosphate buffer solution (Fujifilm Wako Chemicals, 163-20145) for 10 min at RT. Fixed cells were stained with an anti-TRA-1-60 antibody (BD, 560071) followed by an anti-mouse Alexa488 antibody (Invitrogen, A21042). Fluorescent images of whole wells were acquired using a microscope system (Keyence, BZ-710), and colony numbers were measured using ImageJ software. iPS cell culture Cultures for maintaining pluripotency or differentiating iPS cells were performed according to previous methods 7 , 23 . Human iPS cell lines were cultured on 0.5 µg/cm 2 iMatrix-511 (892 − 011/012, Matrixome) in StemFit AK03N containing bFGF (Ajinomoto) for 7 days. TrypLE Select Enzyme (12563011, Gibco) was used to detach and dissociate cells. Cell numbers were counted using Countess 3 (Invitrogen). After mixing cells, culture medium, iMatrix-511, and Y-27632 (Rock inhibitor, 10 µM, 18188-04, Nacalai) in a single tube, the mixture was seeded onto a plate. Human iPS cells were cultured as low-density single cells by plating 2.08 ×10 3 live cells/cm 2 . When culturing and differentiating hiPS cells without bFGF, StemFit AK03N without bFGF (solution C not included) was used when cells were collected as single cells. Quantification of gene expression Total RNA was purified, and RT-qPCR was performed as described previously 7 . GAPDH , NANOG , NR2F2 , FOXP2 , and HEY1 expression was detected using TaqMan Gene Expression Assays (Invitrogen, Assay ID of GAPDH: Hs99999905_m1; NANOG: Hs02387400_g1; NR2F2: Hs00819630_m1; FOXP2: Hs00362818_m1; HEY1: Hs01114113_m1). Relative gene expression levels were calculated using the ΔΔCt method, normalizing to GAPDH. Karyotyping Karyotype analysis using the G-banding method was outsourced (Special Reference Laboratories (SRL), Japan). Statistics GraphPad Prism (GraphPad Software Inc.) was used for statistics. Data are presented as means ± standard deviation (SD). Unless specified otherwise, no statistically significant differences were detected. Human involvement in this study We confirm that our study did not involve direct interaction with human subjects. The frozen PBMC stocks (Cellular Technology Ltd, CTL-UP1) used in the study were obtained commercially, and we did not collect blood samples from human participants. Additionally, the human dermal fibroblasts (HDFs) used in the study were also sourced from commercial providers. Therefore, this study does not fall under the category of human subject research that requires ethical approval. Declarations Data availability The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request. Acknowledgments This study was supported by the Core Center for iPS Cell Research, AMED (Japan) (24bm1323001h0002, 25bm1323001h0003), the iPS Cell Research Fund (Japan), and the World Premier International Research Center Initiative (WPI), MEXT (Japan). Author contributions M. Nakagawa conceived and drafted this manuscript. M. Nogi conducted reprogramming experiments using HDFs, while M. Nakagawa handled those with PBMCs. H.O., M.N., K.H., and H.S. developed the mRNA synthesis method. Competing interests The authors declare that they have no competing interests. References Nonaka, H. et al. Induced pluripotent stem cell-based assays recapture multiple properties of human astrocytes. J Cell Mol Med 28 , e18214 (2024). Otsuka, Y. et al. Phototoxicity avoidance is a potential therapeutic approach for retinal dystrophy caused by EYS dysfunction. JCI Insight 9 , e174179 (2024). Yano, H. et al. Human iPSC-derived CD4+ Treg-like cells engineered with chimeric antigen receptors control GvHD in a xenograft model. Cell Stem Cell 31 , 795-802.e6 (2024). Paul, S. K. et al. Retrotransposons in Werner syndrome-derived macrophages trigger type I interferon-dependent inflammation in an atherosclerosis model. Nat Commun 15 , 4772 (2024). Okamoto, H. et al. Defective flow space limits the scaling up of turbulence bioreactors for platelet generation. Commun Eng 3 , 1–12 (2024). Takahashi, K. et al. Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors. Cell 131 , 861–872 (2007). Nakagawa, M. et al. A novel efficient feeder-free culture system for the derivation of human induced pluripotent stem cells. Sci Rep 4 , 3594 (2014). Ye, H. & Wang, Q. Efficient Generation of Non-Integration and Feeder-Free Induced Pluripotent Stem Cells from Human Peripheral Blood Cells by Sendai Virus. Cell Physiol Biochem 50 , 1318–1331 (2018). Kogut, I. et al. High-efficiency RNA-based reprogramming of human primary fibroblasts. Nat Commun 9 , 745 (2018). McGrath, P. S., Diette, N., Kogut, I. & Bilousova, G. RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells. J Vis Exp (2018) doi:10.3791/58687. Poleganov, M. A. et al. Efficient Reprogramming of Human Fibroblasts and Blood-Derived Endothelial Progenitor Cells Using Nonmodified RNA for Reprogramming and Immune Evasion. Human Gene Therapy 26 , 751–766 (2015). Hong, H. et al. Suppression of induced pluripotent stem cell generation by the p53-p21 pathway. Nature 460 , 1132–1135 (2009). Boettcher, S. et al. A dominant-negative effect drives selection of TP53 missense mutations in myeloid malignancies. Science 365 , 599–604 (2019). Joerger, A. C. & Fersht, A. R. The p53 Pathway: Origins, Inactivation in Cancer, and Emerging Therapeutic Approaches. Annu Rev Biochem 85 , 375–404 (2016). Okamoto, K. et al. DNA damage-induced phosphorylation of MdmX at serine 367 activates p53 by targeting MdmX for Mdm2-dependent degradation. Mol Cell Biol 25 , 9608–9620 (2005). Marión, R. M. et al. A p53-mediated DNA damage response limits reprogramming to ensure iPS cell genomic integrity. Nature 460 , 1149–1153 (2009). Kawamura, T. et al. Linking the p53 tumour suppressor pathway to somatic cell reprogramming. Nature 460 , 1140–1144 (2009). Li, H. et al. The Ink4/Arf locus is a barrier for iPS cell reprogramming. Nature 460 , 1136–1139 (2009). Tani, H. & Akimitsu, N. Genome-wide technology for determining RNA stability in mammalian cells: historical perspective and recent advantages based on modified nucleotide labeling. RNA Biol 9 , 1233–1238 (2012). Sharova, L. V. et al. Database for mRNA Half-Life of 19 977 Genes Obtained by DNA Microarray Analysis of Pluripotent and Differentiating Mouse Embryonic Stem Cells. DNA Res 16 , 45–58 (2009). Schwanhäusser, B. et al. Global quantification of mammalian gene expression control. Nature 473 , 337–342 (2011). Goodarzi, H. et al. Systematic discovery of structural elements governing stability of mammalian messenger RNAs. Nature 485 , 264–268 (2012). Cheng, Y.-S. et al. Simultaneous binding of bFGF to both FGFR and integrin maintains properties of primed human induced pluripotent stem cells. Regen Ther 25 , 113–127 (2024). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 08 Sep, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 23 Jun, 2025 Reviews received at journal 16 Jun, 2025 Reviews received at journal 26 May, 2025 Reviews received at journal 22 May, 2025 Reviewers agreed at journal 19 May, 2025 Reviewers agreed at journal 18 May, 2025 Reviewers agreed at journal 18 May, 2025 Reviewers agreed at journal 17 May, 2025 Reviews received at journal 17 Apr, 2025 Reviewers agreed at journal 02 Apr, 2025 Reviewers invited by journal 28 Mar, 2025 Editor assigned by journal 28 Mar, 2025 Editor invited by journal 27 Mar, 2025 Submission checks completed at journal 25 Mar, 2025 First submitted to journal 25 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6250001","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":439399341,"identity":"e50c8d35-ef4b-4c48-a1ef-93ecaf4d8835","order_by":0,"name":"Masato Nakagawa","email":"data:image/png;base64,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","orcid":"","institution":"Center for iPS Cell Research and Application (CiRA), Kyoto University","correspondingAuthor":true,"prefix":"","firstName":"Masato","middleName":"","lastName":"Nakagawa","suffix":""},{"id":439399342,"identity":"d4d20d3c-22da-44ef-bacf-16e63fa4e91d","order_by":1,"name":"Mizuho Nogi","email":"","orcid":"","institution":"Center for iPS Cell Research and Application (CiRA), Kyoto University","correspondingAuthor":false,"prefix":"","firstName":"Mizuho","middleName":"","lastName":"Nogi","suffix":""},{"id":439399343,"identity":"a9967b7f-867a-4391-8463-b86bfc86aa64","order_by":2,"name":"Hatsuki Doi","email":"","orcid":"","institution":"Premium Research Institute for Human Metaverse Medicine (WPI-PRIMe), Osaka University","correspondingAuthor":false,"prefix":"","firstName":"Hatsuki","middleName":"","lastName":"Doi","suffix":""},{"id":439399344,"identity":"c2bea585-0b2a-48f5-bebf-63b534f1026d","order_by":3,"name":"Hirohisa Ohno","email":"","orcid":"","institution":"Center for iPS Cell Research and Application (CiRA), Kyoto University","correspondingAuthor":false,"prefix":"","firstName":"Hirohisa","middleName":"","lastName":"Ohno","suffix":""},{"id":439399346,"identity":"fc046169-b3e0-46d1-a7b5-a4ddf4e8b18d","order_by":4,"name":"Megumi Mochizuki","email":"","orcid":"","institution":"Center for iPS Cell Research and Application (CiRA), Kyoto University","correspondingAuthor":false,"prefix":"","firstName":"Megumi","middleName":"","lastName":"Mochizuki","suffix":""},{"id":439399348,"identity":"900872d7-0c4e-486d-990b-53f7bcc45da5","order_by":5,"name":"Karin Hayashi","email":"","orcid":"","institution":"Center for iPS Cell Research and Application (CiRA), Kyoto University","correspondingAuthor":false,"prefix":"","firstName":"Karin","middleName":"","lastName":"Hayashi","suffix":""},{"id":439399350,"identity":"eab6286d-afbe-49bd-9657-b38632724bbb","order_by":6,"name":"Hirohide Saito","email":"","orcid":"","institution":"Center for iPS Cell Research and Application (CiRA), Kyoto University","correspondingAuthor":false,"prefix":"","firstName":"Hirohide","middleName":"","lastName":"Saito","suffix":""}],"badges":[],"createdAt":"2025-03-18 06:38:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6250001/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6250001/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-16446-y","type":"published","date":"2025-09-08T15:56:53+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":80747902,"identity":"4b1e29d1-a61f-4d5a-a517-92f3f363f929","added_by":"auto","created_at":"2025-04-16 15:42:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":620252,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGeneration of iPS cells from HDFs using synthetic RNA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea. Schematic diagram of HDF reprogramming experiments using synthetic RNA. On day 0 (d0), HDFs were harvested and seeded together with the required number of cells mixed with media (StemFit AK03N without bFGF), coating substrate (iMatrix-511), and synthetic RNA transfection solution. The transfection of synthetic RNA from day 1 to day 3 was performed by replacing the old medium with a fresh medium containing the newly prepared synthetic RNA transfection solution. The medium was changed to StemFit AK03N containing bFGF on day 7, with TRA-1-60 immunostaining performed on day 9.\u003c/p\u003e\n\u003cp\u003eb. Representative images of TRA-1-60 immunostaining. The number of transfections is shown at the top as Trf×1-4 (n = 3). Factors introduced with reprogramming factors (i.e., OCT3/4, SOX2, KLF4, c-MYC, NANOG, and LIN28A) (OSKMNL) are shown on the left.\u003c/p\u003e\n\u003cp\u003ec. Impact by the number of transfections and additional factors on reprogramming efficiency. The number of TRA-1-60-positive colonies was determined by ImageJ from the immunostaining data shown in Fig. 1b, with the reprogramming efficiency calculated from the number of HDFs used for reprogramming on day 0. Mean (SD) for n = 3 independent experiments; ∗∗p \u0026lt; 0.01, ∗p \u0026lt; 0.05, analyzed by one-way ANOVA with Tukey's test.\u003c/p\u003e\n\u003cp\u003ed. Establishment of HDF-derived iPS cells using synthetic RNA. Phase-contrast image of colonies of clone MN388#4 (top) and karyotyping results (bottom).\u003c/p\u003e\n\u003cp\u003ee. Gene expression analysis in undifferentiated and differentiated states. RNA was extracted from cells cultured in a medium with bFGF (undifferentiated state) or without bFGF (differentiated state), with gene expression confirmed by real-time quantitative PCR using TaqMan probes (n = 3).\u003c/p\u003e","description":"","filename":"figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6250001/v1/790d46232abde6bc9aaccdc2.png"},{"id":80746903,"identity":"885d4d42-1b60-4142-99b6-430f4e62d113","added_by":"auto","created_at":"2025-04-16 15:34:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":623948,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGeneration of iPS cells from PMBCs using synthetic RNA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea. Schematic diagram of PBMC reprogramming experiments using synthetic RNA. On day -4 (d-4), frozen stocks were thawed and incubated in the PBMC culture medium (SS6F). On day 0 (d0), PBMCs were harvested and seeded together with the required number of cells mixed with media (SS6F), coating substrate (iMatrix-511), and synthetic RNA transfection solution. The transfection of synthetic RNA from day 1 to day 3 was performed by replacing the old medium with a fresh medium containing the newly prepared synthetic RNA transfection solution. The medium was changed to a mix of SS6F and StemFit AK03N containing bFGF on day 4 and StemFit AK03N containing bFGF on day 7, with TRA-1-60 immunostaining performed on day 14.\u003c/p\u003e\n\u003cp\u003eb and c. Effect of dominant-negative p53 and MDMs on PBMC reprogramming. The number of TRA-1-60-positive colonies was determined by ImageJ from the immunostaining data (b), with the reprogramming efficiency calculated from the number of PBMCs used for reprogramming on day 0 (n = 4). Relative reprogramming efficiencies were normalized to PBMCs transfected with \u003cem\u003emCherry\u003c/em\u003e mRNA as the additional factor (c). Mean (SD) for n = 4 independent experiments; ∗∗∗p \u0026lt; 0.001, ∗∗p \u0026lt; 0.01, ∗p \u0026lt; 0.05, analyzed by one-way ANOVA with Tukey's test.\u003c/p\u003e\n\u003cp\u003ed. Representative images of TRA-1-60 immunostaining. Eight lots of PBMCs were used in this experiment (n = 8). This figure shows immunostaining results from three lots of PBMCs (PBMC1-3, lot numbers are listed in Supplementary Table 2). Factors introduced together with OSKMNL reprogramming factors are shown on the left.\u003c/p\u003e\n\u003cp\u003ee and f. Effect of MDM4s on reprogramming efficiency. The number of TRA-1-60-positive colonies was determined by ImageJ from the immunostaining data shown in Fig. 2d (e), with the reprogramming efficiency calculated from the number of PBMCs used for reprogramming on day 0 (f). Mean (SD) for n = 8 independent experiments; ∗∗p \u0026lt; 0.01, ∗p \u0026lt; 0.05, analyzed by one-way ANOVA with Tukey's test.\u003c/p\u003e\n\u003cp\u003eMDM4-WT: MDM4 wild-type, MDM4-SA: MDM4 S367A, MDM4-SD: MDM4 S367D.\u003c/p\u003e\n\u003cp\u003eg. Establishment of PBMC-derived iPS cells using synthetic RNA. Phase-contrast image of colonies of clones MN245C13 and MN333#4 (top) and karyotyping results (bottom).\u003c/p\u003e\n\u003cp\u003ee. Gene expression analysis in undifferentiated and differentiated states. RNA was extracted from cells cultured in a medium with bFGF (undifferentiated state) or without bFGF (differentiated state), with gene expression confirmed by real-time quantitative PCR using TaqMan probes (n = 3).\u003c/p\u003e","description":"","filename":"figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6250001/v1/2314214ca0a721ce82e3f235.png"},{"id":91359021,"identity":"1a1c2954-4571-41ba-97e6-f76f5ed8756a","added_by":"auto","created_at":"2025-09-15 16:04:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1651037,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6250001/v1/a57c7cd4-0b36-4a54-bb5b-64a2e60524da.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"MDM4 enables efficient human iPS cell generation from PBMCs using synthetic RNAs","fulltext":[{"header":"Main Text","content":"\u003cp\u003eRecently, the development of regenerative medicine and cell therapy using iPS cells has gained significant momentum. Additionally, valiant efforts to elucidate disease states and discover new therapeutic drugs with patient-derived iPS cells are evident\u003csup\u003e1\u0026ndash;5\u003c/sup\u003e. More recently, attempts have been made to create individual iPS cells tailored for personalized medicine. Consequently, there is an increasing demand for more efficient production of high-quality iPS cells.\u003c/p\u003e\n\u003cp\u003eHuman iPS cells were first established from skin-derived fibroblasts (human dermal fibroblasts, HDFs) on feeder cells using retroviruses\u003csup\u003e6\u003c/sup\u003e. The method was stable enough to produce iPS cells, making it suitable for research. However, many challenges had to be addressed when considering future applications for iPS cell-based cell therapy and regenerative medicine. The world\u0026apos;s first clinical-grade iPS cells were produced from human peripheral blood-derived mononuclear cells (PBMCs) using plasmid vectors under feeder-free culture conditions\u003csup\u003e7\u003c/sup\u003e. Since there is a risk that plasmid vectors may integrate into the genomic DNA of iPS cells generated via this method, an approach avoiding the insertion of exogenous genes has become necessary. Currently, the most efficient method for generating iPS cells involves using Sendai virus\u003csup\u003e8\u003c/sup\u003e. However, it is crucial to consider the biosafety level requirements and restrictions on vector modification as potential limitations when utilizing Sendai virus vectors.\u003c/p\u003e\n\u003cp\u003eMeanwhile, researchers have reported that synthetic RNA, including messenger RNA (mRNA) and microRNA (miRNA), can be used to create iPS cells\u003csup\u003e9\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e10\u003c/sup\u003e. The transfected RNA does not remain in the cells, and the ability to synthesize any RNA with the desired sequence in vitro represents a significant advantage. Although synthetic RNA has been utilized to generate iPS cells from human fibroblasts and blood-derived endothelial progenitor cells, their generation from PBMCs has yet to be reported\u003csup\u003e11\u003c/sup\u003e. In this study, we produced iPS cells from PBMCs using synthetic RNA and found that MDM4, which suppresses the function of p53, significantly increased the reprogramming efficiency.\u003c/p\u003e\n\u003cp\u003eA protocol for reprogramming human dermal fibroblasts (HDFs) using synthetic RNA was validated with a previously described method\u003csup\u003e11\u003c/sup\u003e. We primarily utilized the StemRNA 3rd Gen Reprogramming Kit (ReproCELL, Japan) for this experiment. The number of synthetic RNA transfections varied from four to one, and the minimum number of transfections necessary to produce iPS cells was assessed by immunostaining on day 9. During the same experiment, we also evaluated the effects of tumor suppressor gene \u003cem\u003eTP53\u003c/em\u003e and its regulatory genes (e.g., \u003cem\u003eMDM2\u003c/em\u003e and \u003cem\u003eMDM4\u003c/em\u003e, also known as \u003cem\u003eMDMX\u003c/em\u003e) on reprogramming efficiency. Human dermal fibroblasts were cultured for four days and harvested, with the required number of cells collected. The synthetic RNA for reprogramming was mixed with the transfection reagent, combined with the cell suspension, and with iMatrix-511, a substrate for iPS cell culture, before being seeded onto a culture plate. StemFit AK03 without bFGF was the reprogramming medium until colonies appeared (up to day 7). Immunostaining was conducted on day 9, with the number of TRA-1-60-positive colonies counted using ImageJ, and the reprogramming efficiency was calculated based on the number of HDFs seeded on day 0 (Fig. 1b and 1c). When \u003cem\u003emCherry\u003c/em\u003e mRNA was added to the reprogramming kit, colonies appeared to fill the entire well after four transfections. Colonies were still observed even when we reduced the number of transfections to two. However, no colonies appeared after one transfection. Since previous reports indicated that suppressing p53 functions increases reprogramming efficiency\u003csup\u003e12\u003c/sup\u003e, we investigated whether it could enhance RNA-mediated reprogramming. Human dermal fibroblasts were transfected with the reprogramming kit alongside p53 wild-type (WT), dominant-negative p53 (R175H\u003csup\u003e13\u003c/sup\u003e), MDM2, or MDM4, which are known to inhibit p53\u003csup\u003e14\u003c/sup\u003e. p53 WT decreased the reprogramming efficiency, while p53 R175H increased it. MDM2 and MDM4 did not affect the reprogramming efficiency of HDFs (Fig. 1b, 1c, and Supplementary Table 1). We established multiple iPS cell clones from HDFs using synthetic RNA. Morphological, karyotypic, and gene expression analyses were conducted on MN388#4. The results were consistent with those of previously reported iPS cells (Fig. 1d and 1e).\u003c/p\u003e\n\u003cp\u003eNext, we examined whether we could produce iPS cells from PBMCs using synthetic RNA. While the method and frequency of RNA transfection were identical to those used for reprogramming HDF, the culture medium used was optimized for PBMCs (Fig. 2a). iPS cell-like colonies of sufficient size emerged approximately 14 days after the initial RNA transfection, at which point immunostaining was conducted. When reprogramming PBMCs, only a limited number of colonies appeared after adding \u003cem\u003emCherry\u003c/em\u003e mRNA to the reprogramming kit, but the quantity increased slightly with the introduction of p53 R175H. By contrast to HDF, \u003cem\u003eMDM4\u003c/em\u003e mRNA significantly enhanced the efficiency of PBMC reprogramming (Fig. 2b and 2c). MDM4 serine 367 phosphorylation is known to lead to degradation via the ubiquitin pathway. The mutant S367A (MDM4-SA), with serine replaced by alanine, has been reported to be resistant to degradation\u003csup\u003e15\u003c/sup\u003e. We thus investigated the impact of this mutant on PBMC reprogramming (Fig. 2d, 2e, and 2f). Among the three samples of PBMCs presented in Fig. 2d, MDM4-S367A yielded the highest number of TRA-1-60-positive colonies. Notably, in the case of PBMC lot 2, which exhibited poor reprogramming efficiency, almost no colonies were observed except when MDM4-S367A was involved (Fig. 2e, 2f, and 2d, along with Supplementary Table 2). We also assessed the effect of the MDM4-S367D (MDM4-SD) mutant, which mimics the phosphorylated state, but its reprogramming ability was comparable to that of MDM4-WT. iPS cell clones established using synthetic RNA from PBMCs exhibited normal colony morphology and karyotype (Fig. 2g) with similar gene expression as a well-established iPS cell line (Fig. 2h).\u003c/p\u003e\n\u003cp\u003eIn this study, we successfully used synthetic RNA to generate iPS cells from HDFs and PBMCs isolated from human blood. Notably, our work revealed the critical inclusion of MDM4, which inhibits p53, with other reprogramming factors when utilizing blood cells. The initial step in establishing a synthetic RNA-based reprogramming protocol involved mixing all cells, culture medium, synthetic RNA, and coating substrate, then seeding them together during the initial transfection. This approach is believed to enhance efficiency compared to introducing RNA into adherent cells, as RNA can be delivered from the entire cell surface.\u003c/p\u003e\n\u003cp\u003eSuppressing p53 functions is known to increase reprogramming efficiency\u003csup\u003e12\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e16-18\u003c/sup\u003e. As such, dominant-negative mutants of p53 and siRNA targeting p53 are routinely employed. Once again, the dominant-negative mutant of p53 had the most significant effect when human dermal fibroblasts (HDFs) were reprogrammed using synthetic RNA. MDM4, as well as MDM2, is well known to suppress the function of p53. However, both MDM2 and MDM4 had little effect on HDF reprogramming using synthetic RNA. By contrast, MDM4 exhibited the strongest effect when reprogramming PBMCs, indicating that the reprogramming mechanism with synthetic RNA varies slightly between HDFs and PBMCs. MDM4 may serve additional roles in PBMC reprogramming beyond suppressing p53 function. We aim to clarify this in future research and develop more efficient reprogramming methods.\u003c/p\u003e\n\u003cp\u003eThe results of this study enabled the production of iPS cells from PBMCs isolated from blood. This development will significantly contribute to cell therapy and regenerative medicine using iPS cells. Research and development of personalized medicine utilizing individual iPS cells has gained momentum recently. Because blood samples can be acquired with relative ease and minimal invasion, our findings will prove invaluable to further advance iPS cell-based personalized medicine. Separating PBMCs from a blood sample takes approximately an hour, and mRNA transfection is safe due to its short half-life and lack of genomic integration\u003csup\u003e19-22\u003c/sup\u003e. Thus, PBMCs represent a promising cell resource for generating iPS cells with mRNA for clinical applications. The immediate availability of blood samples for iPS cell establishment after collection offers a notable advantage.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cb\u003eSynthesis of mRNA in vitro\u003c/b\u003e\u003c/p\u003e\u003cp\u003emCherry, d2EGFP, p53 WT, p53 R175H, MDM2 WT, MDM4 WT, MDM4 S367A, and MDM4 S367D cDNAs were amplified by PCR and subcloned into plasmid vectors (T7 promoter) for in vitro RNA synthesis. We used a kit (NIPPON GENE, CUGA 7 in vitro Transcription kit, 307-13531) to synthesize mRNA and followed the attached protocol. CleanCap Reagent AG (3'OMe) (TriLink, N-7413-1) was used as a capping reagent. The Monarch RNA Cleanup Kit (NEW ENGLAND Biolabs, T2040S) was used to purify the synthesized RNA, and a Qubit 4 Fluorometer was used to measure its concentration and purity (Invitrogen, Q33238).\u003c/p\u003e\u003cp\u003e\u003cb\u003eHDF reprogramming\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFrozen HDF stocks (Cell Applications, INC., CA10605f) were thawed and cultured in DMEM (Nacalai Tesque, 08459-64) containing 10% FBS (SIGMA, F7524) for 4 days. Cells were harvested with trypsin (Gibco, 25200056), and the required number of cells were prepared in StemFit AK03N medium (AJINOMOTO, SF010-002) without bFGF (StemFit AK03N (-)) at a concentration of 5.0 x 10\u003csup\u003e5\u003c/sup\u003e cells/mL (day 0). The StemRNA 3rd Gen Reprogramming Kit (REPROCELL, 00\u0026ndash;0076) was used for reprogramming. The transduction method followed the protocol attached to the kit. For reprogramming, 5.0 x 10\u003csup\u003e4\u003c/sup\u003e cells were seeded in one well of a 24-well plate. A total of 200 ng of mRNA encoding OSKMNL (0.76 uL), EKB (0.67 uL), and the additional factors synthesized in our lab (0.13 uL, 100 ng/mL), and microRNAs (0.16 uL) was mixed with 0.72 uL of Lipofectamine MessengerMAX Transfection Reagent (Invitrogen, LMRNA001) as the RNA transfection solution (RNAs and MessengerMAX were diluted with Opti-MEM medium (Invitrogen, 31985062)). Cell suspension (100 uL), RNA transfection solution, iMatrix-511 (1.3 uL) (MATRIXOME, 892\u0026thinsp;\u0026minus;\u0026thinsp;011), and StemFit AK03N (-) medium were mixed (total volume was 800 uL) and seeded into a well immediately. From day 1 to day 3, synthetic RNA was introduced as a medium and transfection solution mixture. The medium was changed to StemFit AK03N (-) on day 4 and StemFit AK03N containing bFGF (StemFit AK03N (+)) on day 7. Cells were immunostained with an anti-TRA-1-60 antibody or passaged on day 9.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePBMC reprogramming\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFrozen PBMC stocks (Cellular Technology Ltd, CTL-UP1) were thawed and cultured in StemSpan SFEM II (STEMCELL Technologies, ST-09605) supplemented with six cytokines (20 ng/mL IL-3 (090-05761), 50 ng/mL IL-6 (098-06041), 10 ng/mL TPO (207-17581), 20 ng/mL Flt3L (061-05391), 50 ng/mL SCF (197-15511), and 10 ng/mL G-CSF (072-06101) (Fujifilm Wako Chemicals)) (SS6F medium) for 4 days. Cells were harvested after trypsinization, and the required number of cells was prepared in SS6F medium at a concentration of 1.0 x 10\u003csup\u003e6\u003c/sup\u003e cells/mL (day 0). The StemRNA 3rd Gen Reprogramming Kit was used for reprogramming. For reprogramming, 1.0 x 10\u003csup\u003e5\u003c/sup\u003e cells were seeded in one well of a 24-well plate. A total of 400 ng of mRNA encoding OSKMNL (1.48 uL), EKB (1.33 uL), and the additional factors synthesized in our lab (0.3 uL, 100 ng/mL), and microRNAs (0.31 uL) was mixed with 1.44 uL of Lipofectamine MessengerMAX Transfection Reagent as the RNA transfection solution (RNAs and MessengerMAX were diluted with Opti-MEM medium). Cell suspension (100 uL), RNA transfection solution, iMatrix-511 (2.5 uL), and SS6F medium were mixed (total volume was 800 uL) and seeded into a well immediately. From day 1 to 3, synthetic RNA was introduced as an SS6F medium and transfection solution mixture. The medium was changed to a mixture of SS6F medium and StemFit AK03N (+) (mix ratio 3:2) on day 4 and StemFit AK03N (+) on day 7. Cells were immunostained with an anti-TRA-1-60 antibody or passaged on day 14.\u003c/p\u003e\u003cp\u003e\u003cb\u003eImmunostaining and colony counting\u003c/b\u003e\u003c/p\u003e\u003cp\u003eReprogrammed cells were fixed with a 4% paraformaldehyde phosphate buffer solution (Fujifilm Wako Chemicals, 163-20145) for 10 min at RT. Fixed cells were stained with an anti-TRA-1-60 antibody (BD, 560071) followed by an anti-mouse Alexa488 antibody (Invitrogen, A21042). Fluorescent images of whole wells were acquired using a microscope system (Keyence, BZ-710), and colony numbers were measured using ImageJ software.\u003c/p\u003e\u003cp\u003e\u003cb\u003eiPS cell culture\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCultures for maintaining pluripotency or differentiating iPS cells were performed according to previous methods\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Human iPS cell lines were cultured on 0.5 \u0026micro;g/cm\u003csup\u003e2\u003c/sup\u003e iMatrix-511 (892\u0026thinsp;\u0026minus;\u0026thinsp;011/012, Matrixome) in StemFit AK03N containing bFGF (Ajinomoto) for 7 days. TrypLE Select Enzyme (12563011, Gibco) was used to detach and dissociate cells. Cell numbers were counted using Countess 3 (Invitrogen). After mixing cells, culture medium, iMatrix-511, and Y-27632 (Rock inhibitor, 10 \u0026micro;M, 18188-04, Nacalai) in a single tube, the mixture was seeded onto a plate. Human iPS cells were cultured as low-density single cells by plating 2.08 \u0026times;10\u003csup\u003e3\u003c/sup\u003e live cells/cm\u003csup\u003e2\u003c/sup\u003e. When culturing and differentiating hiPS cells without bFGF, StemFit AK03N without bFGF (solution C not included) was used when cells were collected as single cells.\u003c/p\u003e\u003cp\u003e\u003cb\u003eQuantification of gene expression\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTotal RNA was purified, and RT-qPCR was performed as described previously\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eGAPDH\u003c/em\u003e, \u003cem\u003eNANOG\u003c/em\u003e, \u003cem\u003eNR2F2\u003c/em\u003e, \u003cem\u003eFOXP2\u003c/em\u003e, and \u003cem\u003eHEY1\u003c/em\u003e expression was detected using TaqMan Gene Expression Assays (Invitrogen, Assay ID of GAPDH: Hs99999905_m1; NANOG: Hs02387400_g1; NR2F2: Hs00819630_m1; FOXP2: Hs00362818_m1; HEY1: Hs01114113_m1). Relative gene expression levels were calculated using the ΔΔCt method, normalizing to GAPDH.\u003c/p\u003e\u003cp\u003e\u003cb\u003eKaryotyping\u003c/b\u003e\u003c/p\u003e\u003cp\u003eKaryotype analysis using the G-banding method was outsourced (Special Reference Laboratories (SRL), Japan).\u003c/p\u003e\u003cp\u003e\u003cb\u003eStatistics\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGraphPad Prism (GraphPad Software Inc.) was used for statistics. Data are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Unless specified otherwise, no statistically significant differences were detected.\u003c/p\u003e\u003cp\u003e\u003cb\u003eHuman involvement in this study\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe confirm that our study did not involve direct interaction with human subjects. The frozen PBMC stocks (Cellular Technology Ltd, CTL-UP1) used in the study were obtained commercially, and we did not collect blood samples from human participants. Additionally, the human dermal fibroblasts (HDFs) used in the study were also sourced from commercial providers. Therefore, this study does not fall under the category of human subject research that requires ethical approval.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eData availability\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003eAcknowledgments\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Core Center for iPS Cell Research, AMED (Japan) (24bm1323001h0002, 25bm1323001h0003), the iPS Cell Research Fund (Japan), and the World Premier International Research Center Initiative (WPI), MEXT (Japan).\u003c/p\u003e\n\u003cp\u003eAuthor contributions\u003c/p\u003e\n\u003cp\u003eM. Nakagawa conceived and drafted this manuscript. M. Nogi conducted reprogramming experiments using HDFs, while M. Nakagawa handled those with PBMCs. H.O., M.N., K.H., and H.S. developed the mRNA synthesis method.\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eNonaka, H. \u003cem\u003eet al.\u003c/em\u003e Induced pluripotent stem cell-based assays recapture multiple properties of human astrocytes. \u003cem\u003eJ Cell Mol Med\u003c/em\u003e \u003cstrong\u003e28\u003c/strong\u003e, e18214 (2024).\u003c/li\u003e\n\u003cli\u003eOtsuka, Y. \u003cem\u003eet al.\u003c/em\u003e Phototoxicity avoidance is a potential therapeutic approach for retinal dystrophy caused by EYS dysfunction. \u003cem\u003eJCI Insight\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, e174179 (2024).\u003c/li\u003e\n\u003cli\u003eYano, H. \u003cem\u003eet al.\u003c/em\u003e Human iPSC-derived CD4+ Treg-like cells engineered with chimeric antigen receptors control GvHD in a xenograft model. \u003cem\u003eCell Stem Cell\u003c/em\u003e \u003cstrong\u003e31\u003c/strong\u003e, 795-802.e6 (2024).\u003c/li\u003e\n\u003cli\u003ePaul, S. K. \u003cem\u003eet al.\u003c/em\u003e Retrotransposons in Werner syndrome-derived macrophages trigger type I interferon-dependent inflammation in an atherosclerosis model. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 4772 (2024).\u003c/li\u003e\n\u003cli\u003eOkamoto, H. \u003cem\u003eet al.\u003c/em\u003e Defective flow space limits the scaling up of turbulence bioreactors for platelet generation. \u003cem\u003eCommun Eng\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, 1\u0026ndash;12 (2024).\u003c/li\u003e\n\u003cli\u003eTakahashi, K. \u003cem\u003eet al.\u003c/em\u003e Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e131\u003c/strong\u003e, 861\u0026ndash;872 (2007).\u003c/li\u003e\n\u003cli\u003eNakagawa, M. \u003cem\u003eet al.\u003c/em\u003e A novel efficient feeder-free culture system for the derivation of human induced pluripotent stem cells. \u003cem\u003eSci Rep\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, 3594 (2014).\u003c/li\u003e\n\u003cli\u003eYe, H. \u0026amp; Wang, Q. Efficient Generation of Non-Integration and Feeder-Free Induced Pluripotent Stem Cells from Human Peripheral Blood Cells by Sendai Virus. \u003cem\u003eCell Physiol Biochem\u003c/em\u003e \u003cstrong\u003e50\u003c/strong\u003e, 1318\u0026ndash;1331 (2018).\u003c/li\u003e\n\u003cli\u003eKogut, I. \u003cem\u003eet al.\u003c/em\u003e High-efficiency RNA-based reprogramming of human primary fibroblasts. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 745 (2018).\u003c/li\u003e\n\u003cli\u003eMcGrath, P. S., Diette, N., Kogut, I. \u0026amp; Bilousova, G. RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells. \u003cem\u003eJ Vis Exp\u003c/em\u003e (2018) doi:10.3791/58687.\u003c/li\u003e\n\u003cli\u003ePoleganov, M. A. \u003cem\u003eet al.\u003c/em\u003e Efficient Reprogramming of Human Fibroblasts and Blood-Derived Endothelial Progenitor Cells Using Nonmodified RNA for Reprogramming and Immune Evasion. \u003cem\u003eHuman Gene Therapy\u003c/em\u003e \u003cstrong\u003e26\u003c/strong\u003e, 751\u0026ndash;766 (2015).\u003c/li\u003e\n\u003cli\u003eHong, H. \u003cem\u003eet al.\u003c/em\u003e Suppression of induced pluripotent stem cell generation by the p53-p21 pathway. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e460\u003c/strong\u003e, 1132\u0026ndash;1135 (2009).\u003c/li\u003e\n\u003cli\u003eBoettcher, S. \u003cem\u003eet al.\u003c/em\u003e A dominant-negative effect drives selection of TP53 missense mutations in myeloid malignancies. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e365\u003c/strong\u003e, 599\u0026ndash;604 (2019).\u003c/li\u003e\n\u003cli\u003eJoerger, A. C. \u0026amp; Fersht, A. R. The p53 Pathway: Origins, Inactivation in Cancer, and Emerging Therapeutic Approaches. \u003cem\u003eAnnu Rev Biochem\u003c/em\u003e \u003cstrong\u003e85\u003c/strong\u003e, 375\u0026ndash;404 (2016).\u003c/li\u003e\n\u003cli\u003eOkamoto, K. \u003cem\u003eet al.\u003c/em\u003e DNA damage-induced phosphorylation of MdmX at serine 367 activates p53 by targeting MdmX for Mdm2-dependent degradation. \u003cem\u003eMol Cell Biol\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 9608\u0026ndash;9620 (2005).\u003c/li\u003e\n\u003cli\u003eMari\u0026oacute;n, R. M. \u003cem\u003eet al.\u003c/em\u003e A p53-mediated DNA damage response limits reprogramming to ensure iPS cell genomic integrity. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e460\u003c/strong\u003e, 1149\u0026ndash;1153 (2009).\u003c/li\u003e\n\u003cli\u003eKawamura, T. \u003cem\u003eet al.\u003c/em\u003e Linking the p53 tumour suppressor pathway to somatic cell reprogramming. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e460\u003c/strong\u003e, 1140\u0026ndash;1144 (2009).\u003c/li\u003e\n\u003cli\u003eLi, H. \u003cem\u003eet al.\u003c/em\u003e The Ink4/Arf locus is a barrier for iPS cell reprogramming. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e460\u003c/strong\u003e, 1136\u0026ndash;1139 (2009).\u003c/li\u003e\n\u003cli\u003eTani, H. \u0026amp; Akimitsu, N. Genome-wide technology for determining RNA stability in mammalian cells: historical perspective and recent advantages based on modified nucleotide labeling. \u003cem\u003eRNA Biol\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 1233\u0026ndash;1238 (2012).\u003c/li\u003e\n\u003cli\u003eSharova, L. V. \u003cem\u003eet al.\u003c/em\u003e Database for mRNA Half-Life of 19 977 Genes Obtained by DNA Microarray Analysis of Pluripotent and Differentiating Mouse Embryonic Stem Cells. \u003cem\u003eDNA Res\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e, 45\u0026ndash;58 (2009).\u003c/li\u003e\n\u003cli\u003eSchwanh\u0026auml;usser, B. \u003cem\u003eet al.\u003c/em\u003e Global quantification of mammalian gene expression control. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e473\u003c/strong\u003e, 337\u0026ndash;342 (2011).\u003c/li\u003e\n\u003cli\u003eGoodarzi, H. \u003cem\u003eet al.\u003c/em\u003e Systematic discovery of structural elements governing stability of mammalian messenger RNAs. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e485\u003c/strong\u003e, 264\u0026ndash;268 (2012).\u003c/li\u003e\n\u003cli\u003eCheng, Y.-S. \u003cem\u003eet al.\u003c/em\u003e Simultaneous binding of bFGF to both FGFR and integrin maintains properties of primed human induced pluripotent stem cells. \u003cem\u003eRegen Ther\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 113\u0026ndash;127 (2024).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6250001/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6250001/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIf iPS cells can be established easily and efficiently using freshly collected blood cells, it will enhance regenerative and personalized medicine. While there have been reports of iPS derivation from blood-derived endothelial progenitor cells using RNA, none have been documented from peripheral blood-derived mononuclear cells (PBMCs). In this study, we established a method to generate iPS cells from PBMCs using synthetic RNAs and found that MDM4, which suppresses p53, improved reprogramming efficiency.\u003c/p\u003e","manuscriptTitle":"MDM4 enables efficient human iPS cell generation from PBMCs using synthetic RNAs","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-16 15:34:26","doi":"10.21203/rs.3.rs-6250001/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-23T07:52:59+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-17T01:33:17+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-26T09:17:31+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-23T00:01:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"129022293821437930557622017908560537040","date":"2025-05-19T06:22:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"94181725696951980468672076550581846383","date":"2025-05-19T03:54:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"45449885328104961088715645878582685846","date":"2025-05-19T02:35:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"169156525618377236546921575411014364480","date":"2025-05-17T07:51:02+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-17T15:55:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"326028725024608128117942466512203963591","date":"2025-04-02T10:31:01+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-28T09:17:50+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-28T09:07:44+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-03-27T06:06:26+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-25T09:02:49+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-03-25T09:01:43+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"821f7642-6a08-4858-8369-b433c1d90ae0","owner":[],"postedDate":"April 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":46776030,"name":"Biological sciences/Stem cells/Reprogramming"},{"id":46776031,"name":"Biological sciences/Stem cells/Pluripotent stem cells/Induced pluripotent stem cells"}],"tags":[],"updatedAt":"2025-09-15T16:00:17+00:00","versionOfRecord":{"articleIdentity":"rs-6250001","link":"https://doi.org/10.1038/s41598-025-16446-y","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-09-08 15:56:53","publishedOnDateReadable":"September 8th, 2025"},"versionCreatedAt":"2025-04-16 15:34:26","video":"","vorDoi":"10.1038/s41598-025-16446-y","vorDoiUrl":"https://doi.org/10.1038/s41598-025-16446-y","workflowStages":[]},"version":"v1","identity":"rs-6250001","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6250001","identity":"rs-6250001","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}