Identificación, caracterización y aislamiento de células madre somáticas del endometrio murino y humano.

RESUMEN El presente trabajo de investigación tiene como objetivo la identificación de células madre somáticas en el endometrio murino y humano. En la mayoría de los tejidos de individuos adultos, existen pequeñas poblaciones de células indiferenciadas que poseen la capacidad de diferenciarse a tipos celulares maduros, dividiéndose a su vez para mantener el balance necesario de células progenitoras indiferenciadas. Estas células reciben el nombre de células madre somáticas y se caracterizan por ser células indiferenciadas presentes en tejidos u órganos diferenciados; siendo además las encargadas del mantenimiento, regeneración y renovación celular en el lugar dónde residen. Por otro lado, el endometrio es un tejido altamente dinámico con capacidad regenerativa y proliferadora, renovándose mensualmente de novo y así sucesivamente durante cada ciclo menstrual y durante toda la vida reproductiva de la mujer. En este contexto, la hipótesis de partida de este trabajo ha sido que en el endometrio murino y humano debe de existir una población de células madre somáticas responsables de su alta capacidad regenerativa. Este trabajo se ha desarrollado basándose en dos modelos: el murino y el humano. Se elaboró un modelo animal mediante el cual se demuestra la existencia de células madre somáticas en el endometrio del ratón. A los ratones neonatos (3 días de edad) se les admministró 5-Bromo-2-desoxiuridina (BrdU), tras esto los animales fueron sacrificados en diferentes momentos de su vida, entre ellos día 7 (control de la técnica), día 14 (momento del destete), día 21 (madurez sexual) y días 49 en adelante (vida adulta). La localización de las células que retuvieron el marcaje se realizó mediante inmunohistoquímica y además las células que presentaron tinción se co-localizaron con marcadores típicos de células madre como Oct-4 (marcador de células madre embrionarias) y c-Kit (marcador de células madre hematopoyéticas). Los estudios murinos permitieron concluir tanto a nivel proteico como molecular la existencia de una población de células madre somáticas en el endometrio del ratón, encontrándose estas localizadas en la región basal del estroma endometrial. Del mismo modo, en el estudio se realizó el análisis de muestras endometriales humanas para determinar la existencia y presencia de células madre somáticas en este tejido. La técnica de detección de esta población celular se realizó mediante el método del Hoescht y la identificación de la Side Population (SP) por citometría de flujo con capacidad de separación celular. Esta técnica permitió aislar la población SP desde el endometrio humano correspondiente a las fracciones epiteliales y estromales de este tejido. Dicha población fue caracterizada a nivel molecular y presentó marcadores de indiferenciación típicos tal como Oct-4 y c-Kit. Además las células SP aisladas presentaron actividad telomerasa, actividad típica de células inmortales e indiferenciadas. El porcentaje de células SP en el endometrio humano permaneció constante en los rangos de edades estudiadas, sugiriendo así que esta población permanecería estable a lo largo de la vida de la mujer. Ela análisis fenotípico determinó el posible origen mesenquimal de las células aisladas ya que presentaron el marcador de células mesenquimales CD90. Estos avances en la identificación y caracterización de células madre somáticas en el endometrio humano y murino ofrecen un nuevo punto de vista para el estudio de la endometriosis y el cáncer de endometrio; ya que es posible que las células madre somáticas endometriales jueguen un papel fundamental en la fisiopatología de determinadas enfermedades ginecológicas como las anteriormente citadas. __________________________________________________________________________________________________Nowadays the identification, characterization and isolation of somatic stem cells in the majority of the tissues/organs have been one of the major challenges carried out by the cellular biology. The endometrium in menstruating and non-menstruating species has cycles of growth, differentiation and regeneration during the reproductive life. In this regard, the concept that endometrial stem cells are responsible for the regenerative capacity of this tissue was proposed many years ago. This study is based in two models: murine and human. Although the mice do not menstruate, in the murine endometrium occurs phenomenon of growth, differentiation, regression and reabsorption. For this reason, in the reproductive and regenerative medicine, the mouse is a well- established animal model for research studies based on the existence of the presence of somatic stem cells in this tissue. A reliable method for identifying the putative endometrial somatic stem cells in murine endometrium is based on the use of 5-brome-2-deoxyuridine (BrdU). Moreover, uteri from BrdU-treated mice were analysed by inmuno-histochemistry in an attempt to co-localize LRCs with undifferentiated markers like c-Kit and Oct-4 The Hoechst method and the isolation of the Side Population (SP) are one of the techniques applied to isolate human somatic stem cells from different tissues. In this study we demonstrate the existence of SP cells in the human endometrium. Furthermore, the SP cells isolated expressed typical undifferentiated markers such as c-KIT and OCT-4 at mRNA levels, and they have an intermediate profile of telomerase activity between human embryonic stem cells line and differentiated cells. The phenotypic analysis of this SP population is suggestive of a putative mesenchymal origin (CD90+)

Comparison of different sources of platelet-rich plasma as treatment option for infertility-causing endometrial pathologies

Objective. To study the effect of human plasma from different sources such as umbilical cord blood and adult blood platelet-rich plasma (PRP) on the regeneration of endometrial damage. Design. Composition analysis, in vitro approaches and a pre-clinical murine model using plasma to promote endometrial regeneration. Setting. Hospital and university laboratories. Patients/Animals. Adult plasma from 4 Atrophy patients and one fertile woman, commercial umbilical cord plasma and uterine-damaged NOD/SCID mice model were used. Intervention(s). Endometrial stromal cells from primary culture and an endometrial stem cell line were cultured in vitro and uterine-damaged NOD/SCID mice were treated with plasma samples from several origins. Main Outcome Measure(s). All plasma samples contain molecules with a high potential for regeneration (SCF, PDGFBB, THBS1, VWF). Furthermore, the highest increase in in vitro proliferation and migration rate was found when endometrial stromal cells were treated with umbilical cord plasma, adult PRP also revealed a significant increment. In the mouse model, a higher expression of Ki67 and Hoxa10 in the endometrium was detected after applying adult PRP and the proteomic analysis revealed a specific protein expression profile depending on the treatment. The damaged uterine tissue showed more pro22 regenerative markers after applying umbilical cord plasma (Stat5a, Uba3, Thy1) in comparison to the other treatments (non-activated umbilical cord plasma, activated adult PRP and not treatment). Conclusion. Human PRP possesses regeneration properties usable for endometrial pathologies. Besides that, these regenerative effects seem to be more apparent when the source of obtaining is umbilical cord blood

Stem cell paracrine actions in tissue regeneration and its potential therapeutic effect in human endometrium: a retrospective study.

Objective: Determining genetic and paracrine mechanisms behind endometrial regeneration in Asherman's Syndrome and Endometrial Atrophy (AS/EA) patients after autologous CD133+ bone marrow-derived stem cells (CD133+BMDSCs) transplantation. Design: Retrospective study using human endometrial biopsies and mouse models. Setting: Fundación-IVI, IIS-La Fe, Valencia, Spain. Samples: Endometrial biopsies collected before and after CD133+BMDSCs therapy, from 8 women with AS/EA (NCT02144987). And uterus from 5 mice, with only left horns receiving CD133+BMDSCs therapy. Methods: In human samples, hematoxylin and eosin (H&E) staining, RNA arrays, PCR validation and neutrophil elastase (NE) immunohistochemistry (IHQ). In mouse samples, PCR validation and protein immunoarrays

Bone Marrow-Derived Cells from Male Donors Do Not Contribute to the Endometrial Side Population of the Recipient

Accumulated evidence demonstrates the existence of bone marrow-derived cells origin in the endometria of women undergoing bone marrow transplantation (BMT). In these reports, cells of a bone marrow (BM) origin are able to differentiate into endometrial cells, although their contribution to endometrial regeneration is not yet clear. We have previously demonstrated the functional relevance of side population (SP) cells as the endogenous source of somatic stem cells (SSC) in the human endometrium. The present work aims to understand the presence and contribution of bone marrow-derived cells to the endometrium and the endometrial SP population of women who received BMT from male donors. Five female recipients with spontaneous or induced menstruations were selected and their endometrium was examined for the contribution of XY donor-derived cells using fluorescent in situ hybridization (FISH), telomapping and SP method investigation. We confirm the presence of XY donor-derived cells in the recipient endometrium ranging from 1.7% to 2.62%. We also identify 0.45–0.85% of the donor-derived cells in the epithelial compartment displaying CD9 marker, and 1.0–1.83% of the Vimentin-positive XY donor-derived cells in the stromal compartment. Although the percentage of endometrial SP cells decreased, possibly being due to chemotherapy applied to these patients, they were not formed by XY donor-derived cells, donor BM cells were not associated with the stem cell (SC) niches assessed by telomapping technique, and engraftment percentages were very low with no correlation between time from transplant and engraftment efficiency, suggesting random terminal differentiation. In conclusion, XY donor-derived cells of a BM origin may be considered a limited exogenous source of transdifferentiated endometrial cells rather than a cyclic source of BM donor-derived stem cells

Reconstruction of Endometrium from Human Endometrial Side Population Cell Lines

Endometrial regeneration is mediated, at least in part, by the existence of a specialized somatic stem cell (SSC) population recently identified by several groups using the side population (SP) technique. We previously demonstrated that endometrial SP displays genotypic, phenotypic and the functional capability to develop human endometrium after subcutaneous injection in NOD-SCID mice. We have now established seven human endometrial SP (hESP) cell lines (ICE 1–7): four from the epithelial and three from the stromal fraction, respectively. SP cell lines were generated under hypoxic conditions based on their cloning efficiency ability, cultured for 12–15 passages (20 weeks) and cryopreserved. Cell lines displayed normal 46XX karyotype, intermediate telomerase activity pattern and expressed mRNAs encoding proteins that are considered characteristic of undifferentiated cells (Oct-4, GDF3, DNMT3B, Nanog, GABR3) and those of mesodermal origin (WT1, Cardiac Actin, Enolase, Globin, REN). Phenotype analysis corroborated their epithelial (CD9+) or stromal (vimentin+) cell origin and mesenchymal (CD90+, CD73+ and CD45−) attributes. Markers considered characteristic of ectoderm or endoderm were not detected. Cells did not express either estrogen receptor alpha (ERα) or progesterone receptor (PR). The hESP cell lines were able to differentiate in vitro into adipocytes and osteocytes, which confirmed their mesenchymal origin. Finally, we demonstrated their ability to generate human endometrium when transplanted beneath the renal capsule of NOD-SCID mice. These findings confirm that SP cells exhibit key features of human endometrial SSC and open up new possibilities for the understanding of gynecological disorders such as endometriosis or Asherman syndrome. Our cell lines can be a valuable model to investigate new targets for endometrium proliferation in endometriosis

Off-label use of rituximab for systemic lupus erythematosus in Europe

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Human Endometrial Side Population Cells Exhibit Genotypic, Phenotypic and Functional Features of Somatic Stem Cells

During reproductive life, the human endometrium undergoes around 480 cycles of growth, breakdown and regeneration should pregnancy not be achieved. This outstanding regenerative capacity is the basis for women's cycling and its dysfunction may be involved in the etiology of pathological disorders. Therefore, the human endometrial tissue must rely on a remarkable endometrial somatic stem cells (SSC) population. Here we explore the hypothesis that human endometrial side population (SP) cells correspond to somatic stem cells. We isolated, identified and characterized the SP corresponding to the stromal and epithelial compartments using endometrial SP genes signature, immunophenotyping and characteristic telomerase pattern. We analyzed the clonogenic activity of SP cells under hypoxic conditions and the differentiation capacity in vitro to adipogenic and osteogenic lineages. Finally, we demonstrated the functional capability of endometrial SP to develop human endometrium after subcutaneous injection in NOD-SCID mice. Briefly, SP cells of human endometrium from epithelial and stromal compartments display genotypic, phenotypic and functional features of SSC

Poètiques de resistència/resiliència

La brutal realitat de la pandèmia i el que en deriva ens força –ens obliga– a replantejar els principis i fonaments de l’art en una societat que desapareix per moments: més que líquida, descobrim ara una societat volatilitzada, críptica, desesperada. La desigualtat que creix de manera desmesurada i el canvi climàtic, que ja es mesura en catàstrofes. Les violències, directes o estructurals, derivades de diferències mal enteses. Cada vegada més descosits, ens cal emparar-nos en valors positius i transformadors. Com a noves persones creadores, els i les artistes novells han de presentar percepcions del món que els hi ha tocat habitar –viure– amb llenguatges que adrecin aquestes problemàtiques. Amb propostes artístiques que deixin de banda prejudicis impostats, han de buscar el diàleg amb el públic per generar confluències i complicitats

Overexpression of the truncated form of high mobility group a proteins (HMGA2) in human myometrial cells induces leiomyoma-like tissue formation

The pathogenesis of uterine leiomyomas, the most common benign tumor in women, is still unknown. This lack of basic knowledge limits the development of novel non-invasive therapies. Our group has previously demonstrated that leiomyoma side population (SP) cells are present in tumor lesions and act like putative tumor-initiating stemcells in human leiomyoma. Moreover, accumulated evidence demonstrates that these benign tumors of mesenchymal origin are characterized by rearrangements of the High Mobility Group A proteins (HMGA). In this work, we tested the hypothesis that leiomyoma development may be due to overexpression of HMGA2 (encoding high mobility group AT-hook2) in myometrial stem cells using in vitro and in vivo approaches. Our work demonstrates that the truncated/short form of HMGA2 induces myometrial cell transformation toward putative tumor-initiating leiomyoma cells and opens up new possibilities to understand the origin of leiomyomas and the development of new therapeutic approaches.Fil: Mas, Aymara. Universidad de Valencia; EspañaFil: Cervelló, Irene. Universidad de Valencia; EspañaFil: Fernández Alvarez, Ana Julia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Faus, Amparo. Universidad de Valencia; EspañaFil: Díaz, Ana. Universidad de Valencia; EspañaFil: Burgués, Octavio. Universidad de Valencia; EspañaFil: Casado, Marta. University of Stanford; Estados UnidosFil: Simón, Carlos. Universidad de Valencia; Españ

Future Challenges and Opportunities of Extracellular Matrix Hydrogels in Female Reproductive Medicine

Bioengineering and reproductive medicine have progressed shoulder to shoulder for several decades. A key point of overlap is the development and clinical translation of technologies to support reproductive health, e.g., scaffold-free constructs, polymeric scaffolds, bioprinting or microfluidics, and hydrogels. Hydrogels are the focus of intense study, and those that are derived from the extracellular matrix (ECM) of reproductive tissues and organs are emerging as promising new players given their results in pre-clinical models. This literature review addresses the recent advances in the use of organ-specific ECM hydrogels in reproductive medicine, considering the entire female reproductive tract. We discuss in-depth papers describing the development of ECM hydrogels, their use in in vitro models, and their in vivo application in preclinical studies. We also summarize the functions of hydrogels, including as grafts, carriers for cell transplantation, or drug depots, and present the potential and possible scope for use of ECM hydrogels in the near future based on recent scientific advances