Identificación, caracterización y aislamiento de células madre somáticas en los miomas humanos

En los últimos años la Medicina y la Biología han experimentado varias revoluciones. La más reciente es la denominada Medicina Regenerativa (Robey, 2000), basada principalmente en el conocimiento y potencial uso terapéutico de las células madre con la finalidad de regenerar tejidos u órganos dañados y, de este modo, poder tratar enfermedades crónico-degenerativas hasta ahora incurables, así como poder profundizar en los mecanismos que regulan el cáncer. Las células madre o troncales son células indiferenciadas con capacidad de auto-renovación y que pueden, en circunstancias determinadas, generar otras células diferenciadas de igual o distinta estirpe celular. Dependiendo de su procedencia se distinguen, clásicamente, tres tipos diferentes de células madre: embrionarias, primordiales y adultas, aunque nuestro grupo se va a centrar en estas últimas. Se ha propuesto que tras su formación fetal, tejidos y órganos mantienen una población restringida de células indiferenciadas en estadio quiescente denominadas células madre adultas. Al dividirse, las células madre adultas generan un tipo de célula intermedia parcialmente diferenciada y con capacidad de división, que dará lugar a células diferenciadas propias de ese órgano (Robey, 2000). Se caracterizan por su auto-renovación o capacidad para generar copias idénticas de sí mismas durante períodos prolongados de tiempo y por su capacidad para dar lugar a células o tejidos propios de la hoja embrionaria a la que pertenece su linaje. Se han aislado células madre adultas humanas en muchos tejidos como neural (Gage, 2000), hígado (Forbes y cols. 2002), páncreas (Bonner-Weir, S. y Sharma, A. 2002), órganos hematopoyéticos (Bonnet, D. 2002), músculo cardíaco (Hughes SM, Blau HM. 1990), músculo esquelético (Seale, P. y Rudnicki, M.A. 2000), epidermis (Watt, F.M. 1988),epitelio gastrointestinal (Marshman, E., Booth, C., y Potten, C. S. 2002),bazo (Otto, W.R. 2002),endometrio (Cervelló y cols,2006) y más recientemente en miometrio (Ono y cols, 2007). Por otro lado, el tejido miometrial humano, debido a su enorme desarrollo durante el embarazo (hasta en 20 veces su tamaño), requiere de la existencia de una población de células madre adultas funcionalmente relevantes y muy activas. Estas han sido identificadas aisladas y caracterizadas en los últimos años (Ono y cols., 2007). Dicha población celular debe permanecer indiferenciada en sus nichos, susceptible de ser inducida durante un posible embarazo. Además, podría disponer de mecanismos de autocontrol para evitar patologías derivadas de su proliferación benigna incontrolada como es la formación de miomas. Basándonos en estas premisas, la presente tesis doctoral tiene por objeto la identificación, caracterización y aislamiento de la población de células candidatas a ser las células madre somáticas formadoras de miomas. En la actualidad, una de cada cuatro mujeres desarrolla miomas, siendo la patología ginecológica más frecuente que causa dolor, alteraciones menstruales e infertilidad. Su origen es desconocido y aunque sabemos que es hormono-dependiente solo es tratable mediante cirugía. Los resultados de este estudio son un fuerte argumento para apoyar la implicación de las células de la Side Population (SP) aisladas de miomas humanos, como supuestas células iniciadoras del tumor. Estas células tienen una elevada capacidad de proliferación in vitro bajo condiciones de hipoxia, un posible origen mesenquimal con la capacidad de diferenciarse in vitro en adipocitos y osteocitos, así como la capacidad para dar lugar a un tejido similar a un mioma in vivo (modelo animal). Sin embargo, las limitaciones de este modelo hacen indispensable la búsqueda de marcadores específicos basados en las características de la SP, como se sugiere en el análisis genómico. Por otro lado, el establecimiento de las dos líneas celulares generadas LeioSP1-2, podría proporcionar un excelente modelo in vitro e in vivo para ampliar conocimientos acerca de la fisiología de los miomas y apoyar aún más la idea del origen de los mismos como una enfermedad derivada a partir de la división anómala de éstas células madre adultas y no de cualquier tipo celular del miometrio.Human uterine fibroids or leiomyomas are the most common benign tumor of the female genital tract affecting 25-30% of women worldwide. They are estrogen-dependent tumors within the myometrium composed by high extracellular matrix content. Typical leiomyoma symptoms, such as pain, menstrual disorders and infertility (1), tend to increase with age and tumor size, constituting the main cause of hysterectomy (2) since no effective long-term medical treatment is available. Despite its high prevalence, knowledge about the cells initiating these uterine tumors is scarce (3), although their origin is associated with disorders in the function, proliferation and fate of a subset of myometrial cells (4, 5). A large body of evidence points to its clonal origin (6) and high proliferation features under hypoxic conditions (7, 8). The cancer stem cells (CSC) or cell initiators of tumors (CIT) hypothesis suggests that tumor cells are organized hierarchically, similarly to normal tissues, with a small self-renewing population of stem cells (SC) generating a large population of proliferative cells that differ from the rest. Thus, they are initiated through a small subset of tumor cells that are ultimately responsible for their formation and growth. CSC/CIT have been identified in a variety of solid tumors, including breast (9), prostate (10), colorectal (11), pancreatic (12), brain (13, 14), and ovarian cancer (15-19). Side population (SP) cells have been successfully used as a tool to identify somatic SC in different organs and tissues, such as bone marrow (20,21), mammary gland (22), skin (23), lung (24), dental pulp (25), endometrium (26-28) and uterine myometrium (29), and also in tumors such as ovarian cancer (18). However, accumulated evidences also demonstrate that tumor progression could result from the interaction between CSC/CIT and their microenvironment (30), specifically with the stromal compartment (31, 32). In this context, our aim was to isolate and characterize the SP population from human leiomyomas, analyzing its clonogenic activity under hypoxic conditions and establishing two MyoSP cell lines (MyoSP1 and MyoSP2). Here we report in vitro and in vivo assays demonstrating the ability of these cell lines to differentiate into mesenchymal lineages cell types, and to form a tissues-like leiomyoma in an animal model

Updated approaches for management of uterine fibroids.

Uterine anatomy and uterine fibroids (UFs) characteristics have been classically considered as almost a unique issue in gynecology and reproductive medicine. Nowadays, the management of UF pathology is undergoing an important evolution, with the patient’s quality of life being the most important aspect to consider. Accordingly, surgical techniques and aggressive treatments are reserved for only those cases with heavy symptomatology, while the clinical diagnostic based on size and number of UFs remains in a second plane in these situations. Moreover, the development of several noninvasive surgical techniques, especially the appearance of ulipristal acetate as a medical etiological treatment, has substantially changed the clinical indications. As a consequence, after almost 2 decades without relevant updates, it has been necessary to update the protocols for the management of UFs in the Spanish Society of Gynecology and Obstetrics twice. Accordingly, we believe that it is necessary to translate our experience to protocolize the medical care for patients with UFs, incorporating these new therapeutic options, and selecting the best treatment for them. We highlight the importance of achieving the patient’s goals and decisions by improving the clinical diagnosis for these type of pathologies, allowing enhanced personalized treatments, as well as the reduction of potential risks and unnecessary surgeries.post-print366 K

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

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

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

Molecular and Cellular Insights into the Development of Uterine Fibroids

Uterine leiomyomas represent the most common benign gynecologic tumor. These hormone-dependent smooth-muscle formations occur with an estimated prevalence of ~70% among women of reproductive age and cause symptoms including pain, abnormal uterine bleeding, infertility, and recurrent abortion. Despite the prevalence and public health impact of uterine leiomyomas, available treatments remain limited. Among the potential causes of leiomyomas, early hormonal exposure during periods of development may result in developmental reprogramming via epigenetic changes that persist in adulthood, leading to disease onset or progression. Recent developments in unbiased high-throughput sequencing technology enable powerful approaches to detect driver mutations, yielding new insights into the genomic instability of leiomyomas. Current data also suggest that each leiomyoma originates from the clonal expansion of a single transformed somatic stem cell of the myometrium. In this review, we propose an integrated cellular and molecular view of the origins of leiomyomas, as well as paradigm-shifting studies that will lead to better understanding and the future development of non-surgical treatments for these highly frequent tumors

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

Magnetic nanoparticles as a new approach to improve the efficacy of gene therapy against differentiated human uterine fibroid cells and tumor-initiating stem cells

Photograph of a scene at a Tulsa refinery and tank farm

Integrative Genomic and Transcriptomic Profiling Reveals a Differential Molecular Signature in Uterine Leiomyoma versus Leiomyosarcoma

The absence of standardized molecular profiling to differentiate uterine leiomyosarcomas versus leiomyomas represents a current diagnostic challenge. In this study, we aimed to search for a differential molecular signature for these myometrial tumors based on artificial intelligence. For this purpose, differential exome and transcriptome-wide research was performed on histologically confirmed leiomyomas (n = 52) and leiomyosarcomas (n = 44) to elucidate differences between and within these two entities. We identified a significantly higher tumor mutation burden in leiomyosarcomas vs. leiomyomas in terms of somatic single-nucleotide variants (171,863 vs. 81,152), indels (9491 vs. 4098), and copy number variants (8390 vs. 5376). Further, we discovered alterations in specific copy number variant regions that affect the expression of some tumor suppressor genes. A transcriptomic analysis revealed 489 differentially expressed genes between these two conditions, as well as structural rearrangements targeting ATRX and RAD51B. These results allowed us to develop a machine learning approach based on 19 differentially expressed genes that differentiate both tumor types with high sensitivity and specificity. Our findings provide a novel molecular signature for the diagnosis of leiomyoma and leiomyosarcoma, which could be helpful to complement the current morphological and immunohistochemical diagnosis and may lay the foundation for the future evaluation of malignancy risk

Immunophenotyping of the XY donor-derived cells in the endometria of transplanted patients.

<p>(A) XY donor-derived cells (indicated by arrows, orange and green signals) in the absence of the hematopoietic marker CD45+ (blue signal) in the recipients' endometria. Lower panel: spleen as a positive control for CD45 (blue). (B) Co-localization of XY donor-derived cells (indicated by arrows) with epithelial marker CD9 (blue) in recipients' endometria indicate the coexistence of XY+CD9+ cells in epithelial glands. Lower panel: epithelial endometrium as a positive control for CD9 (blue). (C) Co-localization of XY donor-derived cells (indicated by arrows) with stromal marker Vm (blue) in recipients' endometria, indicated by the overlapping of the XY+Vm+ expression in the stromal fraction. Lower panel: stromal endometrium as a positive control for Vm (blue). All the images were acquired using a 63×/1.7× oil immersion objective with a confocal microscope. Note that DAPI signals are shown in gray in all the images.</p