CDC2 mediates progestin initiated endometrial stromal cell proliferation: a PR signaling to gene expression independently of its binding to chromatin

Although non-genomic steroid receptor pathways have been studied over the past decade, little is known about the direct gene expression changes that take place as a consequence of their activation. Progesterone controls proliferation of rat endometrial stromal cells during the peri-implantation phase of pregnancy. We showed that picomolar concentration of progestin R5020 mimics this control in UIII endometrial stromal cells via ERK1-2 and AKT activation mediated by interaction of Progesterone Receptor (PR) with Estrogen Receptor beta (ERb) and without transcriptional activity of endogenous PR and ER. Here we identify early downstream targets of cytoplasmic PR signaling and their possible role in endometrial stromal cell proliferation. Microarray analysis of global gene expression changes in UIII cells treated for 45 min with progestin identified 97 up- and 341 down-regulated genes. The most over-represented molecular functions were transcription factors and regulatory factors associated with cell proliferation and cell cycle, a large fraction of which were repressors down-regulated by hormone. Further analysis verified that progestins regulate Ccnd1, JunD, Usf1, Gfi1, Cyr61, and Cdkn1b through PR-mediated activation of ligand-free ER, ERK1-2 or AKT, in the absence of genomic PR binding. ChIP experiments show that progestin promoted the interaction of USF1 with the proximal promoter of the Cdc2 gene. Usf1 knockdown abolished Cdc2 progestin-dependent transcriptional regulation and cell proliferation, which also blocked Cdc2 knockdown. We conclude that progestin-induced proliferation of endometrial stromal cells is mediated by ERK1-2 and AKT dependent early regulation of USF1, which directly induces Cdc2. To our knowledge, this is the first description of early target genes of progestin-activated classical PR via crosstalk with protein kinases and independently of hormone receptor binding to the genomic targets.Fil: Vallejo, Griselda. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: la Greca, Alejandro Damián. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Tarifa Reischle, Inti C.. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Mestre Citrinovitz, Ana Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Ballaré, Cecilia. Centro de Regulación Genómica; EspañaFil: Beato, Miguel. Centro de Regulación Genómica; España. Universitat Pompeu Fabra; EspañaFil: Saragüeta, Patricia Esther. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentin

Downregulation of E-cadherin in pluripotent stem cells triggers partial EMT

Epithelial to mesenchymal transition (EMT) is a critical cellular process that has been well characterized during embryonic development and cancer metastasis and it also is implicated in several physiological and pathological events including embryonic stem cell differentiation. During early stages of differentiation, human embryonic stem cells pass through EMT where deeper morphological, molecular and biochemical changes occur. Though initially considered as a decision between two states, EMT process is now regarded as a fluid transition where cells exist on a spectrum of intermediate states. In this work, using a CRISPR interference system in human embryonic stem cells, we describe a molecular characterization of the effects of downregulation of E-cadherin, one of the main initiation events of EMT, as a unique start signal. Our results suggest that the decrease and delocalization of E-cadherin causes an incomplete EMT where cells retain their undifferentiated state while expressing several characteristics of a mesenchymal-like phenotype. Namely, we found that E-cadherin downregulation induces SNAI1 and SNAI2 upregulation, promotes MALAT1 and LINC-ROR downregulation, modulates the expression of tight junction occludin 1 and gap junction connexin 43, increases human embryonic stem cells migratory capacity and delocalize β-catenin. Altogether, we believe our results provide a useful tool to model the molecular events of an unstable intermediate state and further identify multiple layers of molecular changes that occur during partial EMT.Fil: Aban, Cyntia. Fleni. Laboratorio de Investigación Aplicada a las Neurociencias; Argentina.Fil: Lombardi, Antonella. Fleni. Laboratorio de Investigación Aplicada a las Neurociencias; Argentina.Fil: Neiman, Gabriel. Fleni. Laboratorio de Investigación Aplicada a las Neurociencias; Argentina.Fil: Biani, María Celeste. Fleni. Laboratorio de Investigación Aplicada a las Neurociencias; Argentina.Fil: La Greca, Alejandro. Fleni. Laboratorio de Investigación Aplicada a las Neurociencias; Argentina.Fil: Waisman, Ariel. Fleni. Laboratorio de Investigación Aplicada a las Neurociencias; Argentina.Fil: Moro, Lucía Natalia. Fleni. Laboratorio de Investigación Aplicada a las Neurociencias; Argentina.Fil: Sevlever, Gustavo Emilio. Fleni. Laboratorio de Investigación Aplicada a las Neurociencias; Argentina.Fil: Miriuka, Santiago Gabriel. Fleni. Laboratorio de Investigación Aplicada a las Neurociencias; Argentina.Fil: Luzzani, Carlos. Fleni. Laboratorio de Investigación Aplicada a las Neurociencias; Argentina

188 MicroRNA characterization in equine induced pluripotent stem cells

Cell reprogramming has been well described in mouse and human cells. The expression of specific microRNAs has demonstrated to be essential for pluripotent maintenance and cell differentiation, but not much information is available in domestic species. A single microRNA can regulate the expression of hundreds of mRNA targets, a property given by a short sequence (called “seed”) in positions 2 to 8 from the 5′ end that is complementary to the 3′ untranslated region (UTR) tail of specific mRNAs. We aimed to generate horse induced pluripotent stem cells (iPSC), characterise them, and evaluate the expression of different microRNAs (miR-302a, b, c, d, miR-205, miR-145, miR-9, miR-96, miR-125b, and miR-296) in pluripotency and differentiation. Both cell states were evaluated (pluripotency and differentiation) in order to understand more deeply the complex network of transcriptional regulation in different contexts but with the same genomic background. Two equine iPSC lines (named L2 and L3) were characterised after the reprogramming of equine fibroblasts with the 4 human Yamanaka factors (OCT-4, SOX-2, c-MYC, KLF4). The pluripotency of both lines was assessed by phosphatase alkaline activity, expression of OCT-4, NANOG, and REX1 by RT-PCR, and by immunofluorescence of OCT-4, SOX-2, and c-MYC. In vitro differentiation to embryo bodies (EB) showed the capacity of the iPSC to differentiate into ectodermal, endodermal, and mesodermal phenotypes. MicroRNA expression was analysed by quantitative RT-PCR and resulted in higher expression of the miR-302 family, miR-9, and miR-96 in L2 and L3 v. fibroblasts (P ≤ 0.05), as previously shown in human pluripotent cells. Moreover, down-regulation of miR-145 and miR-205 was observed. After differentiation to EB, greater expression of miR-96 was observed in the EB compared with iPSC, and the expression of miR-205 was induced but only in the EB-L2. In addition, we performed in silico analysis of horse and human microRNAs. First, we compared the horse-miR-302/367 cluster with the human-miR-302/367 cluster and determined a 75% homology between them. Moreover, the seed region of the horse-miR-302 family resulted complementary to the 3′ UTR of horse cell cycle regulator genes CDK2, CYCLIN D1, and E2F1, and to the 3′ UTR of the RHOC gene, which is involved in the epithelial-mesenchymal transition. The miR-145 seed sequence was complementary to the 3′ UTR region of the OCT-4 and KLF-4 horse genes. With respect to miR-9 and miR-96, the seed sequence of these genes were complementary to the HES1 and PAX-6 genes. In all cases, the same gene targets were previously demonstrated in humans. In conclusion, we report the generation and characterization of equine iPSC and determined for the first time the expression of microRNAs in equine pluripotent cells. Moreover, several results led us to think that the horse microRNAs evaluated herein are highly conserved in sequence and function with respect to the human species. It will now be necessary to generate directed differentiations to derivatives of the 3 germ layers in order to strengthen our results. This is the first report to evaluate the expression and possible targets of microRNAs in pluripotent cells from domestic animals.Fil: Moro, Lucía Natalia. Fleni. Laboratorio de Investigación Aplicada a las Neurociencias; Argentina.Fil: Amín, Guadalupe. Fleni. Laboratorio de Investigación Aplicada a las Neurociencias; Argentina.Fil: Furmento, Verónica. Fleni. Laboratorio de Investigación Aplicada a las Neurociencias; Argentina.Fil: Waisman, Ariel. Fleni. Laboratorio de Investigación Aplicada a las Neurociencias; Argentina.Fil: Neiman, Gabriel. Fleni. Laboratorio de Investigación Aplicada a las Neurociencias; Argentina.Fil: La Greca, Alejandro. Fleni. Laboratorio de Investigación Aplicada a las Neurociencias; Argentina.Fil: Santín Velazque, Natalia Lucía. Fleni. Laboratorio de Investigación Aplicada a las Neurociencias; Argentina.Fil: Luzzani, Carlos. Fleni. Laboratorio de Investigación Aplicada a las Neurociencias; Argentina.Fil: Sevlever, Gustavo Emilio. Fleni. Laboratorio de Investigación Aplicada a las Neurociencias; Argentina.Fil: Vichera, Gabriel. Kheiron Biotech; Argentina.Fil: Miriuka, Santiago Gabriel. Fleni. Laboratorio de Investigación Aplicada a las Neurociencias; Argentina

Identification of the miRNAome of early mesoderm progenitor cells and cardiomyocytes derived from human pluripotent stem cells

MicroRNAs are small non-coding RNAs involved in post-transcriptional regulation of gene expression related to many cellular functions. We performed a small-RNAseq analysis of cardiac differentiation from pluripotent stem cells. Our analyses identified some new aspects about microRNA expression in this differentiation process. First, we described a dynamic expression profile of microRNAs where some of them are clustered according to their expression level. Second, we described the extensive network of isomiRs and ADAR modifications. Third, we identified the microRNAs families and clusters involved in the establishment of cardiac lineage and define the mirRNAome based on these groups. Finally, we were able to determine a more accurate miRNAome associated with cardiomyocytes by comparing the expressed microRNAs with other mature cells. MicroRNAs exert their effect in a complex and interconnected way, making necessary a global analysis to better understand their role. Our data expands the knowledge of microRNAs and their implications in cardiomyogenesis.Fil: Garate, Ximena. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: la Greca, Alejandro Damián. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Neiman, Gabriel. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bluguermann, Carolina. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Santín Velazque, Natalia Lucía. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Moro, Lucía Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Luzzani, Carlos Daniel. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Scassa, Maria Elida. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Sevlever, Gustavo. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Romorini, Leonardo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Miriuka, Santiago Gabriel. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Celldeath: A tool for detection of cell death in transmitted light microscopy images by deep learning-based visual recognition

Cell death experiments are routinely done in many labs around the world, these experiments are the backbone of many assays for drug development. Cell death detection is usually performed in many ways, and requires time and reagents. However, cell death is preceded by slight morphological changes in cell shape and texture. In this paper, we trained a neural network to classify cells undergoing cell death. We found that the network was able to highly predict cell death after one hour of exposure to camptothecin. Moreover, this prediction largely outperforms human ability. Finally, we provide a simple python tool that can broadly be used to detect cell death.Fil: la Greca, Alejandro Damián. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Pérez, Nelba. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Castañeda, Sheila Lucia. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Milone, Paula Melania. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Scarafia, Maria Agustina. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Möbbs, Alan Miqueas. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Waisman, Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Moro, Lucía Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Sevlever, Gustavo Emilio. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Luzzani, Carlos Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Miriuka, Santiago Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentin

Single cell transfection of human-induced pluripotent stem cells using a dropletbased microfluidic system

Microfluidic tools have recently made possible many advances in biological and biomedical research. Research in fields such as physics, engineering, chemistry and biology have combined to produce innovation in microfluidics which has positively impacted diverse areas such as nucleotide sequencing, functional genomics, single-cell studies, single molecules assays and biomedical diagnostics. Among these areas, regenerative medicine and stem cells have benefited from microfluidics since these tools have had a profound impact on their applications. In this study, we present a high-performance droplet-based system for transfecting individual human-induced pluripotent stem cells. We will demonstrate that this system has great efficiency in single cells and captured droplets, like other microfluidic methods but with lower cost. Moreover, this microfluidic approach can be associated with the PiggyBac transposase-based system to increase its transfection efficiency. Our results provide a starting point for subsequent applications in more complex transfection systems, single-cell differentiation interactions, cell subpopulations and cell therapy, among other potential applications.Fil: Pérez Sosa, Camilo José. Fundacion P/la Lucha C/enferm.neurologicas Infancia. Instituto de Neurociencias. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Ciudad Universitaria. Instituto de Neurociencias.; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Sanluis Verdes, Anahi. Fundacion P/la Lucha C/enferm.neurologicas Infancia. Instituto de Neurociencias. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Ciudad Universitaria. Instituto de Neurociencias.; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Waisman, Ariel. Fundacion P/la Lucha C/enferm.neurologicas Infancia. Instituto de Neurociencias. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Ciudad Universitaria. Instituto de Neurociencias.; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Lombardi, Antonella. Fundacion P/la Lucha C/enferm.neurologicas Infancia. Instituto de Neurociencias. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Ciudad Universitaria. Instituto de Neurociencias.; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Rosero, Gustavo. Universidad Tecnológica Nacional; ArgentinaFil: la Greca, Alejandro Damián. Fundacion P/la Lucha C/enferm.neurologicas Infancia. Instituto de Neurociencias. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Ciudad Universitaria. Instituto de Neurociencias.; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bhansali, Shekhar. Florida International University; Estados UnidosFil: Bourguignon, Natalia. Universidad Tecnológica Nacional; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Luzzani, Carlos Daniel. Fundacion P/la Lucha C/enferm.neurologicas Infancia. Instituto de Neurociencias. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Ciudad Universitaria. Instituto de Neurociencias.; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Perez, Maximiliano Sebastian. Universidad Tecnológica Nacional; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Miriuka, Santiago Gabriel. Universidad Tecnológica Nacional; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Lerner, Betiana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Tecnológica Nacional; Argentin

Extracellular vesicles from pluripotent stem cell-derived mesenchymal stem cells acquire a stromal modulatory proteomic pattern during differentiation

Mesenchymal stem/stromal cells (MSCs) obtained from pluripotent stem cells (PSCs) constitute an interesting alternative to classical MSCs in regenerative medicine. Among their many mechanisms of action, MSC extracellular vesicles (EVs) are a potential suitable substitute for MSCs in future cell-free-based therapeutic approaches. Unlike cells, EVs do not elicit acute immune rejection, and they can be produced in large quantities and stored until ready to use. Although the therapeutic potential of MSC EVs has already been proven, a thorough characterization of MSC EVs is lacking. In this work, we used a label-free liquid chromatography tandem mass spectrometry proteomic approach to identify the most abundant proteins in EVs that are secreted from MSCs derived from PSCs (PD-MSCs) and from their parental induced PSCs (iPSCs). Next, we compared both datasets and found that while iPSC EVs enclose proteins that modulate RNA and microRNA stability and protein sorting, PD-MSC EVs are rich in proteins that organize extracellular matrix, regulate locomotion, and influence cell–substrate adhesion. Moreover, compared to their respective cells, iPSCs and iPSC EVs share a greater proportion of proteins, while the PD-MSC proteome appears to be more specific. Correlation and principal component analysis consistently aggregate iPSCs and iPSC EVs but segregate PD-MSC and their EVs. Altogether, these findings suggest that during differentiation, compared with their parental iPSC EVs, PD-MSC EVs acquire a more specific set of proteins; arguably, this difference might confer their therapeutic properties.Facultad de Ciencias MédicasConsejo Nacional de Investigaciones Científicas y Técnica

HLA-G gene editing in tumor cell lines as a novel alternative in cancer immunotherapy

Cancer immunotherapies based mainly on the blockade of immune-checkpoint (IC) molecules by anti-IC antibodies offer new alternatives for treatment in oncological diseases. However, a considerable proportion of patients remain unresponsive to them. Hence, the development of novel clinical immunotherapeutic approaches and/or targets are crucial.W In this context, targeting the immune-checkpoint HLA-G/ILT2/ILT4 has caused great interest since it is abnormally expressed in several malignancies generating a tolerogenic microenvironment. Here, we used CRISPR/Cas9 gene editing to block the HLA-G expression in two tumor cell lines expressing HLA-G, including a renal cell carcinoma (RCC7) and a choriocarcinoma (JEG-3). Different sgRNA/Cas9 plasmids targeting HLA-G exon 1 and 2 were transfected in both cell lines. Downregulation of HLA-G was reached to different degrees, including complete silencing. Most importantly, HLA-G − cells triggered a higher in vitro response of immune cells with respect to HLA-G + wild type cells. Altogether, we demonstrated for the first time the HLA-G downregulation through gene editing. We propose this approach as a first step to develop novel clinical immunotherapeutic approaches in cancer.Fil: Palma, Maria Belen. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Departamento de Ciencias Morfológicas. Cátedra de Citología y Embriología A; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; ArgentinaFil: Tronik Le Roux, Diana. Saint-Louis Hospital; Francia. Universite de Paris 1 - Pantheon Sorbonne.; FranciaFil: Amin, Guadalupe. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Castañeda, Sheila Lucia. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Möbbs, Alan Miqueas. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Scarafia, Maria Agustina. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: la Greca, Alejandro Damián. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Daouya, Marina. Hôpital Saint-louis; Francia. Universite de Paris 1 - Pantheon Sorbonne.; FranciaFil: Poras, Isabelle. Hôpital Saint-louis; Francia. Universite de Paris 1 - Pantheon Sorbonne.; FranciaFil: Inda, Ana María. Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Departamento de Ciencias Morfológicas. Cátedra de Citología y Embriología A; Argentina. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas; ArgentinaFil: Moro, Lucía Natalia. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Carosella, Edgardo Delfino. Hôpital Saint-louis; Francia. Universite de Paris 1 - Pantheon Sorbonne.; FranciaFil: García, Marcela Nilda. Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Departamento de Ciencias Morfológicas. Cátedra de Citología y Embriología A; ArgentinaFil: Miriuka, Santiago Gabriel. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

HLA-G gene editing: a novel therapeutic alternative in cancer immunotherapy

Cancer immunotherapies based mainly on the blockade of immune-checkpoint (IC) molecules by anti-IC antibodies offer new alternatives for treatment in oncological diseases. However, a considerable proportion of patients remain unresponsive to them. Hence, the development of novel clinical immunotherapeutic approaches and/or targets are crucial. In this context, targeting the immune-checkpoint HLA-G/ILT2/ILT4 has caused great interest since it is abnormally expressed in several malignancies generating a tolerogenic microenvironment. Here, we used CRISPR/Cas9 gene editing to block the HLA-G expression in two tumor cell lines expressing HLA-G, including a renal cell carcinoma (RCC7) and a choriocarcinoma (JEG-3). Different sgRNA/Cas9 plasmids targeting HLA-G exon 1 and 2 were transfected in both cell lines. Downregulation of HLAG was reached to different degrees, including complete silencing. Most importantly, HLA-G – cells triggered a higher in vitro response of immune cells with respect to HLA-G + wild type cells. Altogether, we demonstrated for the first time the HLA-G downregulation through gene editing. We propose this approach as a first step to develop novel clinical immunotherapeutic approaches in cancer.Fil: Palma, María Belén. Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Cátedra de Citología, Histología y Embriología; Argentina. Fleni. Laboratorio de Investigación Aplicada a las Neurociencias; Argentina.Fil: Tronik-Le Roux, Diana. Saint-Louis Hospital; Francia. Université de Paris; Francia.Fil: Amín, Guadalupe. Fleni. Laboratorio de Investigación Aplicada a las Neurociencias; Argentina.Fil: Castañeda, Sheila. Fleni. Laboratorio de Investigación Aplicada a las Neurociencias; Argentina.Fil: Möbbs, Alan M. Fleni. Laboratorio de Investigación Aplicada a las Neurociencias; Argentina.Fil: Scarafia, María Agustina. Fleni. Laboratorio de Investigación Aplicada a las Neurociencias; Argentina.Fil: La Greca, Alejandro. Fleni. Laboratorio de Investigación Aplicada a las Neurociencias; Argentina.Fil: Daouya, Marina. Saint-Louis Hospital; Francia. Université de Paris; Francia.Fil: Poras, Isabelle. Saint-Louis Hospital; Francia. Université de Paris; Francia.Fil: Inda, Ana María. Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Cátedra de Citología, Histología y Embriología; Argentina. Comisión de Investigaciones Científicas; Argentina.Fil: Moro, Lucía Natalia. Fleni. Laboratorio de Investigación Aplicada a las Neurociencias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina.Fil: Carosella, Edgardo D. Saint-Louis Hospital; Francia. Université de Paris; Francia.Fil: García, Marcela N. Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Cátedra de Citología, Histología y Embriología; Argentina.Fil: Miriuka, Santiago G. Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Cátedra de Citología, Histología y Embriología; Argentina. Fleni. Laboratorio de Investigación Aplicada a las Neurociencias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Higher-order chromatin organization defines Progesterone Receptor and PAX2 binding to regulate estradiol-primed endometrial cancer gene expression

Estrogen (E2) and Progesterone (Pg), via their specific receptors (ER and PR respectively), are major determinants in the development and progression of endometrial malignancies. Here, we have studied how E2 and the synthetic progestin R5020 affect genomic functions in Ishikawa endometrial cancer cells. Using ChIPseq in cells exposed to the corresponding hormones, we identified cell specific binding sites for ER (ERbs) and PR (PRbs), which mostly correspond to independent sites but both adjacent to sites bound by PAX2. Analysis of long-range interactions by Hi-C showed enrichment of regions co-bound by PR and PAX2 inside TADs that contain differentially progestin-regulated genes. These regions, which we call “progestin control regions” (PgCRs), exhibit an open chromatin state prior to the exposure to the hormone. Our observations suggest that endometrial response to progestins in differentiated endometrial tumor cells results in part from binding of PR together with partner transcription factors to PgCRs, compartmentalizing hormone-independent open chromatin.Fil: la Greca, Alejandro Damián. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Bellora, Nicolás. Comision Nacional de Energia Atomica. Gerencia de Area de Aplicaciones de la Tecnologia Nuclear. Instituto de Tecnologias Nucleares Para la Salud.; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto de Investigaciones en Biodiversidad y Medioambiente. Universidad Nacional del Comahue. Centro Regional Universidad Bariloche. Instituto de Investigaciones en Biodiversidad y Medioambiente; ArgentinaFil: Le Dily, Francois. Centro de Regulación Genómica; España. Universitat Pompeu Fabra; EspañaFil: Jara, Rodrigo Agustin. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Quilez Oliete, Javier. Centro de Regulación Genómica; EspañaFil: Villanueva, José Luis. Centro de Regulación Genómica; EspañaFil: Vidal, Enrique. Centro de Regulación Genómica; EspañaFil: Merino, Gabriela Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Córdoba; ArgentinaFil: Fresno Rodríguez, Cristóbal. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Córdoba; ArgentinaFil: Tarifa Reischle, Inti Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Vallejo, Griselda. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Vicent, Guillermo P.. Centro de Regulación Genómica; EspañaFil: Fernandez, Elmer Andres. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Córdoba; ArgentinaFil: Beato, Miguel. Centro de Regulación Genómica; España. Universitat Pompeu Fabra; EspañaFil: Saragueta, Patricia Esther. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentin