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Muscular dystrophies (MDs) are chronic degenerative myopathies, whereof cardiac failure is still the most prominent cause of death. Cardiac cell therapies struggle with inefficient migration, engraftment and differentiation capacities of stem cells. We want to investigate whether epigenetic memory could be a tool to overcome these insufficiencies. We will derive canine cardiovascular progenitors (CPs) via factor-based- or direct reprogramming of isogenic skin fibroblast (SFs), cardiac fibroblasts (CFs) and cardiac mesoangioblasts (cMABs). We will ask whether the derived CPs express a bias towards cardiomyogenic differentiation or migration capacities. This will be examined by analysis of epigenetics, differentiation and migration. We will inject derived CPs in the systemic circulation of immunodeficient dystrophic mice to analyse migration and engraftment potential, and directly into the myocardium to analyse differentiation potential. As a proof of principle, we plan to inject golden retriever muscular dystrophy (GRMD) dogs by intra-coronary delivery and examine migration via multimodal imaging (MMI) and cardiac function by MRI/echocardiography. In summary, we will explore whether the epigenetic memory is a potential mechanism to boost cardiomyogenic and migration capacities of stem cells. In addition, this project can potentially contribute to refine cardiac cell therapies for not only MDs, but also in the broader context of cardiac failure.

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The pituitary gland takes, together with the hypothalamus, the main lead in coordinating the endocrine system to regulate vital physiological body processes like growth, puberty, reproduction and stress response. Because of this central role, malfunctioning of the pituitary can cause severe disorders like diabetes, cardiovascular disease, osteoporosis, infertility and psychological problems. Pituitary hormonal cell populations must therefore be maintained in a controlled and balanced manner. Previous studies revealed the existence of stem cells in the pituitary gland, mainly based on a side population (SP) and SOX2-expressing phenotype. Their role, however, remains uncertain. Lineage tracing showed that SOX2+ cells can give rise to all pituitary endocrine cell types during postnatal homeostasis, although only at a low degree. After damage in the gland, the pituitary stem cells are activated and appear involved in the subsequent regenerative process. Recently, it was found that the pituitary SP and SOX2+ stem cells are activated and expanded during tumor development and growth in the gland as occurring in the ‘dopamine receptor D2 knockout (Drd2-/-) mouse. Tumorigenesis is the most common pathology of the pituitary. However, underlying mechanisms remain elusive and current therapies are often unsatisfactory because of inoperability or therapy resistance.In the first part of our study, we investigated the role and significance of the pituitary stem cells in the postnatal gland by depleting the SOX2+ cells through diphtheria toxin (DT)-mediated ablation. In the second part, we further characterized the stem cells in the tumorous Drd2-/- pituitary in search of their role in development, progression and therapy resistance of pituitary tumors.Administration of DT to adult Sox2CreERT2/+;R26iDTR/+ mice (after tamoxifen-induced expression of DT receptor or DTR in SOX2+ cells) resulted in 80% obliteration of SOX2+ cells in the endocrine pituitary, coinciding with reduced pituisphere-forming activity. Counterintuitively for a stem cell population, the SOX2+ cell compartment did not repopulate. Considering the more active phenotype of the stem cells during early-postnatal pituitary maturation, SOX2+ cell ablation was also performed in 4- and 1-week-old animals. Ablation grade diminished with decreasing age and was accompanied by a proliferative reaction of the SOX2+ cells, suggesting a rescue attempt. Despite this activation, SOX2+ cells did also not recover. Finally, the major SOX2+ cell depletion in adult mice did not affect the homeostatic maintenance of pituitary hormonal cell populations, neither the cell (corticotrope) remodeling response to adrenalectomy challenge.To characterize the pituitary stem cell population during tumorigenesis, whole-genome expression analysis using RNA-sequencing (RNA-seq) was performed. Analysis revealed upregulation of different stemness factors and pathways including WNT, epithelial-mesenchymal transition (EMT), chemokine signaling and NOTCH in the SP of Drd2-/- pituitary. Transgenic lineage tracing of SOX2+ cells before and during pituitary tumor growth in the Drd2-/- mouse showed that the pituitary stem cells do not, or at least not to a substantial level, give rise to the tumor cells, suggesting rather an indirect, potentially paracrine role. Finally, higher resistance to the chemotherapeutic drug temozolomide was observed in the stem cell fraction of both mouse and human pituitary tumors as analyzed in vitro.In summary, our study shows that pituitary SOX2+ cells fail to regenerate after major depletion which does not affect adult endocrine cell homeostasis and remodeling. Thus, pituitary SOX2+ cells may constitute a copious stem cell reserve or may have other critical role(s) still to be clearly defined. In addition, pituitary stem cells appear activated during tumor formation in the (Drd2-/-) pituitary regarding their stemness molecular phenotype but are not directly linked to the tumor cells. This activation may imply a reaction against tumor development in the gland which may then indirectly affect tumor growth by paracrine signaling. Moreover, the stem cell fraction in pituitary tumors appears more resistant to chemotherapy; further characterization may lead to insights in the underlying mechanisms of therapy resistance and eventually to new therapeutic opportunities.In conclusion, our study adds new elements to the role of pituitary stem cells during homeostasis and tumorigenesis, but more research is clearly needed.

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In this project, we aim to use modern assessment techniques to study certain risk factors related to the development of pelvic floor prolapse in a clinical setting as well as in an experimental model. Pelvic floor prolapse is a bothersome condition when the vaginal wall or the uterus protrudes through the vaginal opening. It affects mainly women who were at least once pregnant and delivered vaginally. The project includes clinical part during which we tried to contribute to the discussion on risk factors related to the development of pelvic organ prolapse and other pelvic floor dysfunctions. More extensive, experimental part, aimed to further characterize a large ovine animal model for the development of pelvic organ prolapse and for vaginal surgery.The clinical study was conducted in Prague, Czech Republic, and included a large cohort of nulliparous women who delivered vaginally. One year after delivery one-third of women reported urinary incontinence, 13% had pelvic organ prolapse reaching the level of hymen or beyond and 3.3% reported some anorectal dysfunction mainly related to painful defecation. As a result of delivery, 18% of women sustained levator ani avulsion and 17% had levator hiatus ballooning. Both of which were tightly related to the current or future presence of pelvic organ prolapse and its recurrence after surgical correction. Demographic and obstetrical factors included age and body mass index increased the likely hood of urinary incontinence, age increases the risk for pelvic organ prolapse. Risk factors for levator ani avulsion included the forceps delivery, whereas epidural analgesia and perineal rupture grade I was decreasing.Experimentally we worked with the sheep as a large animal model. First, we characterized the pelvic floor of the virgin ewe and compared it to that of women. Second, we documented the effects of certain key life time events such as first delivery, menopause and under its replacement therapy. We identified many anatomical and structural similarities such as vaginal dimensions, the composition of the vaginal wall, attachments of levator ani muscle. Some anatomical structures present in women are not developed in sheep (i.e. the sacrospinous ligament, internal obturator muscle and obturator membrane) and their pelvic floor anatomy is adapted to their quadruped position and presence of the tail. We observed the effect of specific life span factors (first vaginal delivery, ovariectomy, hormonal replacement therapy) on active and passive biomechanical properties of the ovine vagina. Following the first vaginal delivery, ovine vagina became more spacious, its distal part was less stiff and smooth muscles generated lower contractile forces. Following artificially induced menopause vagina was narrower and its middle part becomes stiffer. Estradiol hormonal replacement returned the stiffness in within the range of the premenopausal animal. Histology showed only a limited amount of changes and had not sufficiently explained observed changes in biomechanics.The experimental work was dedicated to studying the effect of novel implants in the treatment of pelvic organ prolapse. In a comparative study, we used a bovine-derived acellular cross-linked collagen matrix (ACM) as an alternative to polypropylene flat meshes. Both types of implants were inserted in the ovine rectovaginal septum. After 6 months, ACMs showed more local graft-related complications and biomechanical properties comparable to polypropylene. Moreover, partial degradation of ACM had a negative impact on smooth muscle contractility. Due to theses observation, we concluded that ACM does not seem to have better biosafety profile than polypropylene.To proceed with last experimental study in sheep we firstly need to further explore the potential and feasibility of arm anchored implant. In a small study, we performed a trocar guided transvaginal insertion of an H-shaped implant self-tailored to fit ovine anatomy and dimensions. No serious complications were identified therefore we used the technique for bigger prospective study. Moreover, the surgical procedure was recorded for educational purposes.The subsequent study included previously described H-shaped mesh and flat mash, both fabricated from polymeric polyvinidylene fluoride loaded with iron particles that allow its visualization with magnetic resonance (MRI). In a longitudinal manner, we collected data documenting stable shape and position of implants. Initially, there was a drop in the effective surface area observed in both types of implants that remained stable until the end of the observational period. More detail analysis of thickness maps obtained from MRI data revealed two deformations patterns each of them specific for H-shape to flat mesh. Deformation of H-shaped implants was most probably related to distinct biomechanical properties of its central part and arms, whereas flat implants displayed heterogenic pattern most probably linked with pore aggregation caused with suturing. The polymer also showed a low rate of graft related complications, did not affect smooth muscle contractility yet increased the stiffness of augmented tissue.In general, this project has shown that pelvic organ prolapse is linked with maternal age and delivery-related injuries. Up to every eight women may have a symptomatic prolapse already 1 year after her first delivery. Moreover, those with muscle injury are in higher risk of POP development in the future and its recurrence after the primary surgical repair. To improve our knowledge on POP development and treatment we further explored the potential of a large ovine model for prolapse and vaginal surgery. We showed that many anatomical and morphological features and vaginal wall changes induced by specific life span factors (first delivery, artificial menopause, hormonal replacement) are to a certain extent similar to observations in women. We further used this model for testing novel implants and visualization techniques. We believe that the ovine model should be used in future research of pelvic organ prolapse pathophysiology and novel treatment techniques.