Production and characterization of a panel of anti-mouse placenta-specific protein 1 (plac1) monoclonal antibodies.

Placenta-Specific Protein 1 (PLAC1) is essential for normal placental and embryonic development. It is widely expressed in various types of cancer cells. We produced a panel of anti-mouse plac1 monoclonal antibodies (mAbs) with different applications. Two recombinant proteins were produced containing either the extracellular domain (ED) plus tetanus toxin P2, P30, pan-DR epitope (PADRE), and KDEL3 (main plac1) or ED plus KDEL3 (control plac1). Recombinant proteins were used for immunization and screening. Positive clones were selected by ELISA and flow cytometry. Purified mAbs were tested by ELISA, WB, flow cytometry, immunohistochemistry (IHC), and immunofluorescent (IF). A combination of bioinformatics tools was used to predict the target epitope(s) of the mAbs. Eight anti-mouse plac1 mAbs (all IgG1/κ1) were generated, all reacting with high affinity in ELISA. Seven clones recognized plac1 in both reduced and non-reduced Western blots, while one only recognized the non-reduced form. Cross-inhibition ELISA revealed that all mAbs recognized overlapping epitopes with a shared motif except for 5C9. Four clones reacted with the native antigen in flow cytometry, but none were functional in IF or IHC staining. The produced multifunctional mAbs can be used to investigate different aspects of PLAC1 biology in reproduction and cancer.

SARS-CoV-2 nucleocapsid: Biological functions and implication for disease diagnosis and vaccine design.

The coronavirus disease 2019 (COVID-19) pandemic is transmitted by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has affected millions of people all around the world, leading to more than 6.5 million deaths. The nucleocapsid (N) phosphoprotein plays important roles in modulating viral replication and transcription, virus-infected cell cycle progression, apoptosis, and regulation of host innate immunity. As an immunodominant protein, N protein induces strong humoral and cellular immune responses in COVID-19 patients, making it a key marker for studying N-specific B cell and T cell responses and the development of diagnostic serological assays and efficient vaccines. In this review, we focus on the structural and functional features and the kinetic and epitope mapping of B cell and T cell responses against SARS-CoV-2 N protein to extend our understanding on the development of sensitive and specific diagnostic immunological tests and effective vaccines.

Diagnostic performance of a novel antigen-capture ELISA for the detection of SARS-CoV-2.

BACKGROUND AND AIMS: The coronavirus disease 2019 (COVID-19) pandemic is a serious health problem worldwide. Early virus detection is essential for disease control and management. Viral antigen detection by ELISA is a cost-effective, rapid, and accurate antigen diagnostic assay which could facilitate early viral detection. METHOD: An antigen-capture sandwich ELISA was developed using novel nucleocapsid (NP)-specific mouse monoclonal antibodies (MAbs). The clinical performance of the assay was assessed using 403 positive and 150 negative respiratory samples collected during different SARS-CoV-2 variants outbreaks in Iran. RESULTS: The limit of detection of our ELISA assay was found to be 43.3 pg/ml for recombinant NP. The overall sensitivity and specificity of this assay were 70.72% (95% CI: 66.01-75.12) and 100% (95% CI: 97.57-100), respectively, regardless of Ct values and SARS-CoV-2 variants. There was no significant difference in our assay sensitivity for the detection of Omicron subvariants compared to Delta variant. Assay sensitivity for the BA.5 Omicron subvariant was calculated as 91.89% (95% CI: 85.17-96.23) for samples with Ct values < 25 and 82.70% (95% CI: 75.19-88.71) for samples with Ct values < 30. CONCLUSION: Our newly developed ELISA method is reasonably sensitive and highly specific for detection of SARS-CoV-2 regardless of the variants and subvariants of the virus.

Epitope mapping of neutralising anti-SARS-CoV-2 monoclonal antibodies: Implications for immunotherapy and vaccine design.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease 2019 (COVID-19) pandemic. This disease has currently affected more than 346 million people and resulted in more than 5.5 million deaths in many countries. Neutralising monoclonal antibodies (MAbs) against the SARS-CoV-2 virus could serve as prophylactic/therapeutic agents in COVID-19 infection by providing passive protection against the virus in individuals. Until now, no Food and Drug Administration/European Medicines Agency-approved neutralising MAb against SARS-CoV-2 virus exists in the market, though a number of MAbs have been authorised for emergency use. Therefore, there is an urgent need for development of efficient anti-SARS-CoV-2 neutralising MAbs for use in the clinic. Moreover, neutralising anti-SARS-CoV-2 MAbs could be used as beneficial tools for designing epitope-based vaccines against the virus. Given that the target epitope of a MAb is a crucial feature influencing its neutralising potency, target epitopes of neutralising anti-SARS-CoV-2 MAbs already reported in the literature and reactivity of these MAbs with SARS-CoV-2 variants are reviewed herein.

Inhibition of transcription factor T-cell factor 3 (TCF3) using the oligodeoxynucleotide strategy increases embryonic stem cell stemness: possible application in regenerative medicine.

The transcription factor T-cell factor 3 (TCF3), one component of the Wnt pathway, is known as a cell-intrinsic inhibitor of many pluripotency genes in embryonic stem cells (ESCs) that influences the balance between pluripotency and differentiation. In this study, the effects of inhibition of TCF3 transcription factor on the stemness of mouse ESCs (mESCs) were investigated using the decoy oligodeoxynucleotides (ODNs) strategy. The TCF3 decoy and its scramble ODNs were designed and synthesized. The interaction specificity of the TCF3 decoy with the TCF3 transcription factor was evaluated by the electrophoretic mobility shift assay. Subcellular localization was carried out using fluorescence and confocal microscopy. Self-renewal and pluripotency of mESCs were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), cell cycle and apoptosis, alkaline phosphatase (ALP), embryoid body (EB) formation, and real-time assays. All experiments were performed in triplicate. The results showed that knockdown of TCF3 by decoy ODNs transfection in mESCs led to an increase in the cell proliferation, ALP enzyme activity, and master regulatory stemness genes and a decrease in the number and diameter of EBs. These results supported TCF3 as a potential target to maintain the pluripotency and self-renewal capacity of mESCs. Knockdown of the TCF3 transcription factor using decoy ODNs can be a promising method to maintain the stemness of stem cells in regenerative medicine and cell therapy researches.

Novel neutralizing mouse-human chimeric monoclonal antibodies against the SARS-CoV-2 receptor binding domain.

Introduction. Neutralizing antibodies have been widely used for the prophylaxis and treatment of COVID-19.Hypothesis. The major target for these neutralizing antibodies is the receptor-binding domain (RBD) of the viral spike protein.Aim. In the present study, we developed and characterized three neutralizing chimeric mouse-human mAbs for potential therapeutic purposes.Methodology. Light and heavy chain variable region genes of three mouse mAbs (m4E8, m3B6, and m1D1) were amplified and ligated to human Cγ1 and Cκ constant region genes by PCR. After cloning into a dual promoter mammalian expression vector, the final constructs were transiently expressed in DG-44 cells and the purified chimeric antibodies were characterized by ELISA and Western blotting. The neutralizing potency of the chimeric mAbs was determined by three different virus neutralization tests including sVNT, pVNT, and cVNT.Results. All three recombinant chimeric mAbs display human constant regions and are able to specifically bind to the RBD of SARS-CoV-2 with affinities comparable to the parental mAbs. Western blot analysis showed similar epitope specificity profiles for both the chimeric and the parental mouse mAbs. The results of virus neutralization tests (sVNT, pVNT, and cVNT) indicate that c4E8 had the most potent neutralizing activity with IC50 values of 1.772, 0.009, and 0.01 µg ml-1, respectively. All chimeric and mouse mAbs displayed a similar pattern of reactivity with the spike protein of the SARS-CoV-2 variants of concern (VOC) tested, including alpha, delta, and wild-type.Conclusion. The chimeric mAbs displayed neutralizing potency similar to the parental mouse mAbs and are potentially valuable tools for disease control.

Identification of immunodominant epitopes on nucleocapsid and spike proteins of the SARS-CoV-2 in Iranian COVID-19 patients.

Given the emergence of SARS-CoV-2 virus as a life-threatening pandemic, identification of immunodominant epitopes of the viral structural proteins, particularly the nucleocapsid (NP) protein and receptor-binding domain (RBD) of spike protein, is important to determine targets for immunotherapy and diagnosis. In this study, epitope screening was performed using a panel of overlapping peptides spanning the entire sequences of the RBD and NP proteins of SARS-CoV-2 in the sera from 66 COVID-19 patients and 23 healthy subjects by enzyme-linked immunosorbent assay (ELISA). Our results showed that while reactivity of patients' sera with reduced recombinant RBD protein was significantly lower than the native form of RBD (P < 0.001), no significant differences were observed for reactivity of patients' sera with reduced and non-reduced NP protein. Pepscan analysis revealed weak to moderate reactivity towards different RBD peptide pools, which was more focused on peptides encompassing amino acids (aa) 181-223 of RBD. NP peptides, however, displayed strong reactivity with a single peptide covering aa 151-170. These findings were confirmed by peptide depletion experiments using both ELISA and western blotting. Altogether, our data suggest involvement of mostly conformational disulfide bond-dependent immunodominant epitopes in RBD-specific antibody response, while the IgG response to NP is dominated by linear epitopes. Identification of dominant immunogenic epitopes in NP and RBD of SARS-CoV-2 could provide important information for the development of passive and active immunotherapy as well as diagnostic tools for the control of COVID-19 infection.

Epitope mapping of severe acute respiratory syndrome coronavirus 2 neutralizing receptor binding domain-specific monoclonal antibodies.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the outbreak led to the coronavirus disease 2019 (COVID-19) pandemic. Receptor binding domain (RBD) of spike (S) protein of SARS-CoV-2 is considered as a major target for immunotherapy and vaccine design. Here, we generated and characterized a panel of anti-RBD monoclonal antibodies (MAbs) isolated from eukaryotic recombinant RBD-immunized mice by hybridoma technology. Epitope mapping was performed using a panel of 20-mer overlapping peptides spanning the entire sequence of the RBD protein from wild-type (WT) Wuhan strain by enzyme-linked immunosorbent assay (ELISA). Several hybridomas showed reactivity toward restricted RBD peptide pools by Pepscan analysis, with more focus on peptides encompassing aa 76-110 and 136-155. However, our MAbs with potent neutralizing activity which block SARS-CoV-2 spike pseudovirus as well as the WT virus entry into angiotensin-converting enzyme-2 (ACE2) expressing HEK293T cells showed no reactivity against these peptides. These findings, largely supported by the Western blotting results suggest that the neutralizing MAbs recognize mainly conformational epitopes. Moreover, our neutralizing MAbs recognized the variants of concern (VOC) currently in circulation, including alpha, beta, gamma, and delta by ELISA, and neutralized alpha and omicron variants at different levels by conventional virus neutralization test (CVNT). While the neutralization of MAbs to the alpha variant showed no substantial difference as compared with the WT virus, their neutralizing activity was lower on omicron variant, suggesting the refractory effect of mutations in emerging variants against this group of neutralizing MAbs. Also, the binding reactivity of our MAbs to delta variant showed a modest decline by ELISA, implying that our MAbs are insensitive to the substitutions in the RBD of delta variant. Our data provide important information for understanding the immunogenicity of RBD, and the potential application of the novel neutralizing MAbs for passive immunotherapy of SARS-CoV-2 infection.

Does prior immunization with measles, mumps, and rubella vaccines contribute to the antibody response to COVID-19 antigens?

BACKGROUND: Incidence and severity of SARS-CoV2 infection are significantly lower in children and teenagers proposing that certain vaccines, routinely administered to neonates and children may provide cross-protection against this emerging infection. OBJECTIVE: To assess the cross-protection induced by prior measles, mumps and rubella (MMR) vaccinations against COVID-19. METHODS: The antibody responses to MMR and tetanus vaccines were determined in 53 patients affected with SARS-CoV2 infection and 52 age-matched healthy subjects. Serum levels of antibodies specific for NP and RBD of SARS-CoV2 were also determined in both groups of subjects with ELISA. RESULTS: Our results revealed significant differences in anti-NP (P<0.0001) and anti-RBD (P<0.0001) IgG levels between patients and healthy controls. While the levels of rubella- and mumps specific IgG were not different in the two groups of subjects, measles-specific IgG was significantly higher in patients (P<0.01). The serum titer of anti-tetanus antibody, however, was significantly lower in patients compared to healthy individuals (P<0.01). CONCLUSION: Our findings suggest that measles vaccination triggers those B cells cross-reactive with SARS-CoV2 antigens leading to the production of increased levels of measles-specific antibody.

Differential Antibody Response to SARS-CoV-2 Antigens in Recovered and Deceased Iranian COVID-19 Patients.

The coronavirus infectious disease 2019 (COVID-19), which is initiated by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has imposed critical challenges to global health. Understanding the kinetic of SARS-CoV-2-specific IgM and IgG responses in different subsets of COVID-19 patients is crucial to get insight into the humoral immune response elicited against the virus. We investigated IgM and IgG responses against SARS-CoV-2 nucleocapsid (N) and receptor-binding domain (RBD) of spike protein in two groups of recovered and deceased COVID-19 patients. The levels of IgM and IgG specific to N and RBD proteins were detected by ELISA. N- and RBD-specific IgM was higher in deceased patients in comparison with recovered patients, while there was no significant difference in N- and RBD-specific IgG between the two groups. A significant correlation was observed between IgG and IgM titers against RBD and N, in both groups of patients. These results argue against impaired antibody response in deceased COVID-19 patients.

Secretome of Aggregated Embryonic Stem Cell-Derived Mesenchymal Stem Cell Modulates the Release of Inflammatory Factors in Lipopolysaccharide-Induced Peripheral Blood Mononuclear Cells

Background: Bone marrow mesenchymal stem cells (BM-MSCs) have emerged as a potential therapy for various inflammatory diseases. Because of some limitations, several recent studies have suggested the use of embryonic stem cell-derived MSCs (ESC-MSCs) as an alternative for BM-MSCs. Some of the therapeutic effects of the ESC-MSCs are related to the secretion of a broad array of cytokines and growth factors, known as secretome. Harnessing this secretome for therapeutic applications requires the optimization of production of secretary molecules. It has been shown that aggregation of MSCs into 3D spheroids, as a preconditioning strategy, can enhance immunomodulatory potential of such cells. In this study, we investigated the effect of secretome derived from human ESC-MSCs (hESC-MSCs) spheroids on secretion of IL-1ß, IL-10, and tumor necrosis factor α (TNF-α) from lipopolysaccharide (LPS)-induced peripheral blood mononuclear cells (PBMCs). Methods: In the present study, after immunophenotyping and considering mesodermal differentiation of hESC-MSCs, the cells were non-adherently grown to prepare 3D aggregates, and then conditioned medium or secretome was extracted from the cultures. Afterwards, the anti-inflammatory effects of the secretome were assessed in an in vitro model of inflammation. Results: Results from this study showed that aggregate-prepared secretome from hESC-MSCs was able to significantly decrease the secretion of TNF-α (301.7 ± 5.906, p < 0.0001) and IL-1ß (485.2 ± 48.38, p < 0.001) from LPS-induced PBMCs as the indicators of inflammation, in comparison with adherent culture-prepared secretome (TNF-α: 166.6 ± 8.04, IL-1ß: 125.2 ± 2.73). Conclusion: Our study indicated that cell aggregation can be an appropriate strategy to increase immunomodulatory characteristics of hESC-MSCs.

Dual Effects of Cell Free Supernatants from Lactobacillus acidophilus and Lactobacillus rhamnosus GG in Regulation of MMP-9 by Up-Regulating TIMP-1 and Down-Regulating CD147 in PMADifferentiated THP-1 Cells.

OBJECTIVES: Recent studies have reported dysregulated expression of matrix metalloproteinases (MMPs), especially MMP-2, MMP-9, tissue inhibitor of metalloproteinase-1, -2 (TIMP-1, TIMP-2), and extracellular matrix metalloproteinase inducer (EMMPRIN/CD147) in activated macrophages of patients with inflammatory diseases. Therefore, MMP-2, MMP-9, and their regulators may represent a new target for treatment of inflammatory diseases. Probiotics, which are comprised of lactic acid bacteria, have the potential to modulate inflammatory responses. In this experimental study, we investigated the anti-inflammatory effects of cell-free supernatants (CFS) from Lactobacillus acidophilus (L. acidophilus) and L. rhamnosus GG (LGG) in phorbol myristate acetate (PMA)-differentiated THP-1 cells. MATERIALS AND METHODS: In this experimental study, PMA-differentiated THP-1 cells were treated with CFS from L. acidophilus, LGG and uninoculated bacterial growth media (as a control). The expression of MMP-2, MMP-9, TIMP-1, and TIMP-2 mRNAs were determined using real-time quantitative reverse transcription polymerase chain reaction (RTPCR). The levels of cellular surface expression of CD147 were assessed by flow cytometry, and the gelatinolytic activity of MMP-2 and MMP-9 were determined by zymography. RESULTS: Our results showed that CFS from both L. acidophilus and LGG significantly inhibited the gene expression of MMP-9 (P=0.0011 and P=0.0005, respectively), increased the expression of TIMP-1 (P<0.0001), decreased the cell surface expression of CD147 (P=0.0307 and P=0.0054, respectively), and inhibited the gelatinolytic activity of MMP-9 (P=0.0003 and P<0.0001, respectively) in PMA-differentiated THP-1 cells. Although, MMP-2 expression and activity and TIMP-2 expression remained unchanged. CONCLUSIONS: Our results indicate that CFS from L. acidophilus and LGG possess anti-inflammatory properties and can modulate the inflammatory response.

Anti-inflammatory Property of ß-D-Mannuronic Acid (M2000) on Expression and Activity of Matrix Metalloproteinase-2 and -9 through CD147 Molecule in Phorbol Myristate Acetate-differentiated THP-1 Cells.

The aim of this study was to evaluate the effects of M2000, a novel non-steroidal anti-inflammatory drug (NSAID) with immunosuppressive property and without gastro-nephrotoxicitic effects on matrix metalloproteinases (MMP)-2 and (MMP)-9 in phorbol myristate acetate (PMA)-differentiated THP-1 cells. Gene expression and activity of MMP-2 and MMP-9 are inhibited respectively by the tissue inhibitor of matrix metalloproteinase (TIMP)-2 and (TIMP)-1 and are induced by extracellular matrix metalloproteinase inducer (CD147/EMMPRIN). In this study, real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) was used to determine gene expression of MMP-2, MMP-9, TIMP-1, and TIMP-2. Flow cytometry and zymography were applied to determine cellular surface expression of CD147 and activity of MMP-2 and MMP-9, respectively. Our results showed that treatment of THP-1 cells with high concentration (25 µg/mL) of M2000 significantly decreased the cellular surface expression of CD147 (p<0.05) and the gene expression of MMP-2, MMP-9 and TIMP-1 (p<0.05), and inhibited the gelatinolytic activity of MMP-2 and MMP-9 (p<0.05). According to our results, M2000 can reduce inflammation through inhibition of the cellular surface expression of CD147 and decrease the gene expression and gelatinolytic activity of MMP-2 and MMP-9 in PMA-differentiated THP-1 cells.

The effect of Trimetazidine and Diazoxide on immunomodulatory activity of human embryonic stem cell-derived mesenchymal stem cell secretome.

Comprehensive proteome profiling of the factors secreted by mesenchymal stem cells (MSCs), referred to as secretome, revealed that it consists of cytokines, chemokines, growth factors, extracellular matrix proteins, and components of regeneration, vascularization, and hematopoiesis pathways. Harnessing this MSC secretome for therapeutic applications requires the optimization of production of secretary molecules. A variety of preconditioning methods have been introduced, which subject cells to stimulatory molecules to create the preferred response and stimulate persistent effects. Pharmacological preconditioning uses small molecules and drugs to increase survival of MSCs after transplantation or prolong release of effective secretary factors such as cytokines that improve immune system responses. In this study, we investigated the effect of secretome of human embryonic-derived mesenchymal stem cells (hESC-MSCs) preconditioned with Trimetazidine (TMZ) and Diazoxide (DZ) on immunomodulatory efficiency of these cells in LPS-induced peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from human peripheral blood and treated with concentrated hESC-MSC-derived conditioned medium and then, the secreted levels of IL-10, TNFα and IL-1ß were assessed by ELISA after induction with LPS. The results showed that TMZ and DZ-conditioned medium significantly enhanced immunomodulatory potential of hESC-MSCs by increasing the secretion of IL-10, TNFα and IL-1ß from LPS- induced PBMCs. We also found that hESC-MSCs did not secrete mentioned cytokines prior to or after the preconditioning with TMZ and DZ. In conclusion, our results implied that TMZ and DZ can be used to promote the immunomodulatory effects of hESC-MSC secretome. It is obvious that for applying of these findings in clinical demands, the potency of different pre-conditioned MSCs secretome on immune response needs to be more clarified.

Hypoxia Pre-Conditioned Embryonic Mesenchymal Stem Cell Secretome Reduces IL-10 Production by Peripheral Blood Mononuclear Cells.

BACKGROUND: Mesenchymal stem cells (MSCs) are important candidates for MSC-based cellular therapy. Current paradigm states that MSCs support local progenitor cells in damaged tissue through paracrine signaling. Therefore, study of paracrine effects and secretome of MSCs could lead to the appreciation of mechanisms and molecules associated with the therapeutic effects of these cells. This study analyzed anti-inflammatory and immune-modulatory effects of MSC secretomes derived from embryonic stem cells (ESCs) and bone marrow cells after hypoxia and normoxia preconditioning. METHODS: ESCs differentiated into MSCs and characterized by flow cytometry and differentiation into adipocytes and osteoblasts. The experimental groups consisted of individual groups of ESC-MSCs and BM-MSCs (bone marrow-derived mesenchymal stromal cells), which were preconditioned with either hypoxia or normoxia for 24, 48 and 72 h. After collecting the cell-free medium from each treatment, secretomes were concentrated by centrifugal filters. Using a peripheral blood mononuclear cell (PBMC) assay and ELISA, IL-10 concentration in PBMCs was evaluated after their incubation with different secretomes from preconditioned and non-preconditioned MSCs. RESULTS: A significant difference was observed between ESC-MSC normoxia and ESC-MSC hypoxia in IL-10 concentration, and normoxia secretomes increased IL-10 secretion from PBMCs. Moreover, the strongest IL-10 secretion from PBMCs could be detected after the stimulation by ESC-MSC conditioned secretomes, but not BM-MSC conditioned medium. CONCLUSIONS: Human hypoxia preconditioned ESC-MSC secretome indicated stronger immune-modulatory effects compared to BM-MSC conditioned medium. It could be suggested that induced MSCs confer less immune-modulatory effects but produce more inflammatory molecules such as tumor necrosis factor α, which needs further investigation.

Myc Decoy Oligodeoxynucleotide Inhibits Growth and Modulates Differentiation of Mouse Embryonic Stem Cells as a Model of Cancer Stem Cells.

BACKGROUND: Myc (c-Myc) alone activates the embryonic stem cell-like transcriptional module in both normal and transformed cells. Its dysregulation might lead to increased cancer stem cells (CSCs) population in some tumor cells. OBJECTIVE: In order to investigate the potential of Myc decoy oligodeoxynucleotides for differentiation therapy, mouse embryonic stem cells (mESCs) were used in this study as a model of CSCs. To our best of knowledge this is the first report outlining the application of Myc decoy in transcription factor decoy "TFD" strategy for inducing differentiation in mESCs. METHODS: A 20-mer double-stranded Myc transcription factor decoy and scrambled oligodeoxynucleotides (ODNs) were designed, analyzed by electrophoretic mobility shift (EMSA) assay and transfected into the mESCs under 2 inhibitors (2i) condition. Further investigations were carried out using fluorescence and confocal microscopy, cell proliferation and apoptosis analysis, alkaline phosphatase and embryoid body formation assay, real-time PCR and western blotting. RESULTS: EMSA data showed that Myc decoy ODNs bound specifically to c-Myc protein. They were found to be localized in both cytoplasm and nucleus of mESCs. Our results revealed the potential capability of Myc decoy ODNs to decrease cell viability by (16.1±2%), to increase the number of cells arrested in G0/G1 phases and apoptosis by (14.2±3.1%) and (12.1±3.2%), respectively regarding the controls. Myc decoy could also modulate differentiation in mESCs despite the presence of 2i/LIF in our medium the presence of 2i/LIF in our medium. CONCLUSION: The optimized Myc decoy ODNs approach might be considered as a promising alternative strategy for differentiation therapy investigations.