Use of 3D organoids as a model to study idiopathic form of Parkinsons disease

Organoids are becoming particularly popular in modeling diseases that are difficult to reproduce in animals, due to anatomical differences in the structure of a given organ. Thus, they are a bridge between the in vitro and in vivo models. Human midbrain is one of the structures that is currently being intensively reproduced in organoids for modeling Parkinson’s disease (PD). Thanks to three-dimensional (3D) architecture and the use of induced pluripotent stem cells (iPSCs) differentiation into organoids, it has been possible to recapitulate a complicated network of dopaminergic neurons. In this work, we present the first organoid model for an idiopathic form of PD. iPSCs were generated from peripheral blood mononuclear cells of healthy volunteers and patients with the idiopathic form of PD by transduction with Sendai viral vector. iPSCs were differentiated into a large multicellular organoid-like structure. The mature organoids displayed expression of neuronal early and late markers. Interestingly, we observed statistical differences in the expression levels of LIM homeobox transcription factor alpha (early) and tyrosine hydroxylase (late) markers between organoids from PD patient and healthy volunteer. The obtained results show immense potential for the application of 3D human organoids in studying the neurodegenerative disease and modeling cellular interactions within the human brain

Carbon fibers as a new type of scaffold for midbrain organoid development

The combination of induced pluripotent stem cell (iPSC) technology and 3D cell culture creates a unique possibility for the generation of organoids that mimic human organs in in vitro cultures. The use of iPS cells in organoid cultures enables the differentiation of cells into dopaminergic neurons, also found in the human midbrain. However, long-lasting organoid cultures often cause necrosis within organoids. In this work, we present carbon fibers (CFs) for medical use as a new type of scaffold for organoid culture, comparing them to a previously tested copolymer poly-(lactic-co-glycolic acid) (PLGA) scaffold. We verified the physicochemical properties of CF scaffolds compared to PLGA in improving the efficiency of iPSC differentiation within organoids. The physicochemical properties of carbon scaffolds such as porosity, microstructure, or stability in the cellular environment make them a convenient material for creating in vitro organoid models. Through screening several genes expressed during the differentiation of organoids at crucial brain stages of development, we found that there is a correlation between PITX3, one of the key regulators of terminal differentiation, and the survival of midbrain dopaminergic (mDA) neurons and tyrosine hydroxylase (TH) gene expression. This makes organoids formed on carbon scaffolds an improved model containing mDA neurons convenient for studying midbrain-associated neurodegenerative diseases such as Parkinson’s disease

Origin of the induced pluripotent stem cells affects their differentiation into dopaminergic neurons

Neuronal differentiation of human induced pluripotent stem (iPS) cells, both in 2D models and 3D systems in vitro, allows for the study of disease pathomechanisms and the development of novel therapies. To verify if the origin of donor cells used for reprogramming to iPS cells can influence the differentiation abilities of iPS cells, peripheral blood mononuclear cells (PBMC) and keratinocytes were reprogrammed to iPS cells using the Sendai viral vector and were subsequently checked for pluripotency markers and the ability to form teratomas in vivo. Then, iPS cells were differentiated into dopaminergic neurons in 2D and 3D cultures. Both PBMC and keratinocyte-derived iPS cells were similarly reprogrammed to iPS cells, but they displayed differences in gene expression profiles and in teratoma compositions in vivo. During 3D organoid formation, the origin of iPS cells affected the levels of FOXA2 and LMX1A only in the first stages of neural differentiation, whereas in the 2D model, differences were detected at the levels of both early and late neural markers FOXA2, LMX1A, NURR1, TUBB and TH. To conclude, the origin of iPS cells may significantly affect iPS differentiation abilities in teratomas, as well as exerting effects on 2D differentiation into dopaminergic neurons and the early stages of 3D midbrain organoid formation

Chalcogen-Varied Imidazolone Derivatives as Antibiotic Resistance Breakers in Staphylococcus aureus Strains

In this study, a search for new therapeutic agents that may improve the antibacterial activity of conventional antibiotics and help to successfully overcome methicillin-resistant Staphylococcus aureus (MRSA) infections has been conducted. The purpose of this work was to extend the scope of our preliminary studies and to evaluate the adjuvant potency of new derivatives in a set of S. aureus clinical isolates. The study confirmed the high efficacy of piperazine derivatives of 5-arylideneimidazol-4-one (7–9) tested previously, and it enabled the authors to identify even more efficient modulators of bacterial resistance among new analogs. The greatest capacity to enhance oxacillin activity was determined for 1-benzhydrylpiperazine 5-spirofluorenehydantoin derivative (13) which, at concentrations as low as 0.0625 mM, restores the effectiveness of β-lactam antibiotics against MRSA strains. In silico studies showed that the probable mechanism of action of 13 is related to the binding of the molecule with the allosteric site of PBP2a. Interestingly, thiazole derivatives tested were shown to act as both oxacillin and erythromycin conjugators in S. aureus isolates, suggesting a complex mode of action (i.e., influence on the Msr(A) efflux pump). This high enhancer activity indicates the high potential of imidazolones to become commercially available antibiotic adjuvants

L'influence de l'hypoxie sur l'expression de podoplanine dans les fibroblastes associés au cancer (CAF) et son rôle dans la progression du cancer du sein

Tumor is a pathologic tissue including cancer cells, a modified extracellular matrix, endothelial cells, blood and lymphatic vessels, immune and inflammatory cells and activated fibroblasts called cancer-associated fibroblasts (CAFs). Podoplanin (PDPN), mucin-type transmembrane glycoprotein is expressed in tumor cells and CAFs, helps metastasis. Its role in metastaticprocess has been demonstrated for breast cancer cells into lymph nodes. Here we show that PDPN modulates the CCL21/CCR7 chemokine/receptor axis in a hypoxia-dependent manner. Cancer progression depends on the tumour stroma in which CAFs differ from normal fibroblasts. CAFs promote tumour growth, recruitment of endothelial progenitor cells and angiogenesis. In breast cancer up to 80% of fibroblasts display the CAF phenotype. Here a PDPN expressing model of CAFs made it possible to demonstrate the involvement of CCL21/CCR7 axis in the tumor cell-to-CAF recognition through podoplanin binding of CCL21. PDPN positive CAFs secrete microRNAs, which control gene expression at post-transcriptional level and influence cancer cells. MiR-21 is a key regulator of the oncogenic process, through its downstream target proteins among which the tumor suppressor, PTEN. We analyzed the effect of miR-21, but also oncogenic and hypoxia dependent miRs: miR-210 and miR-29b, on PDPN expression in fibroblasts in conditions mimicking the intra tumor microenvironment, i.e. in hypoxia. This points to crucial differences as compared to normoxia. Moreover we uncover the effect of podoplanin on angiogenesis by endothelial cells colocalizing with CAFs expressing podoplanin and on the expression of most prominent proangiogenic factors.Le tissu tumoral comprend, outre les cellules cancéreuses, une matrice extra-cellulaire modifiée, les cellules endothéliales des vaisseaux sanguins et lymphatiques, immunes et inflammatoires et des fibroblastes actives associés au cancer (CAFs). La podoplanine (PDPN), glycoprotéine transmembranaire de type mucine, y est exprimée dans les cellules cancéreuses et les CAFs. Elleaidea la métastase comme montré dans le carcinome mammaire envahissant les ganglions lymphatiques. Ce travail montre que PDPN module l’interaction chimiokine/récepteur de l’axe CCL21/CCR7 et dépend de l’hypoxie. La progression tumorale est aidée par le stroma ou les CAFs ont des propriétés particulières par rapport aux fibroblastes normaux. Ils promeuvent la croissance tumorale, le recrutement des précurseurs endothéliaux et l’angiogenèse. Dans le carcinome mammaire, 80% des fibroblastes ont un phénotype CAF. Un modèle de CAFs exprimant la PDPN a permis de démontrer l’implication de CCL21/CCR7 dans la reconnaissance entre cellules tumorales et CAFs via la liaison CCL21/PDPN. Les CAFsPDPN+ secrètent des microARNs qui contrôlent des gènes des cellules cancéreuses. MiR-21 est un régulateur oncogène fondamental, par son action sur le suppresseur de tumeur PTEN. Nous avons analysé l’effet de miR-21, ainsi que de miR-210 et miR-29b sur PDPN dans les fibroblastes en hypoxie pour mimer le microenvironnement intratumoral et mis en évidence les différences biologiques comparativement à la normoxie ainsi que l’effet de la podoplanine sur l’angiogenèse par les cellules endothéliales en colocalization avec les CAFs exprimant la podoplanine et sur l’expression des facteurs proangiogéniques

The influence of hypoxia on podoplanin expression in cancer-associated fibroblasts (CAF) and its role in the progression of breast cancer

Le tissu tumoral comprend, outre les cellules cancéreuses, une matrice extra-cellulaire modifiée, les cellules endothéliales des vaisseaux sanguins et lymphatiques, immunes et inflammatoires et des fibroblastes actives associés au cancer (CAFs). La podoplanine (PDPN), glycoprotéine transmembranaire de type mucine, y est exprimée dans les cellules cancéreuses et les CAFs. Elleaidea la métastase comme montré dans le carcinome mammaire envahissant les ganglions lymphatiques. Ce travail montre que PDPN module l’interaction chimiokine/récepteur de l’axe CCL21/CCR7 et dépend de l’hypoxie. La progression tumorale est aidée par le stroma ou les CAFs ont des propriétés particulières par rapport aux fibroblastes normaux. Ils promeuvent la croissance tumorale, le recrutement des précurseurs endothéliaux et l’angiogenèse. Dans le carcinome mammaire, 80% des fibroblastes ont un phénotype CAF. Un modèle de CAFs exprimant la PDPN a permis de démontrer l’implication de CCL21/CCR7 dans la reconnaissance entre cellules tumorales et CAFs via la liaison CCL21/PDPN. Les CAFsPDPN+ secrètent des microARNs qui contrôlent des gènes des cellules cancéreuses. MiR-21 est un régulateur oncogène fondamental, par son action sur le suppresseur de tumeur PTEN. Nous avons analysé l’effet de miR-21, ainsi que de miR-210 et miR-29b sur PDPN dans les fibroblastes en hypoxie pour mimer le microenvironnement intratumoral et mis en évidence les différences biologiques comparativement à la normoxie ainsi que l’effet de la podoplanine sur l’angiogenèse par les cellules endothéliales en colocalization avec les CAFs exprimant la podoplanine et sur l’expression des facteurs proangiogéniques.Tumor is a pathologic tissue including cancer cells, a modified extracellular matrix, endothelial cells, blood and lymphatic vessels, immune and inflammatory cells and activated fibroblasts called cancer-associated fibroblasts (CAFs). Podoplanin (PDPN), mucin-type transmembrane glycoprotein is expressed in tumor cells and CAFs, helps metastasis. Its role in metastaticprocess has been demonstrated for breast cancer cells into lymph nodes. Here we show that PDPN modulates the CCL21/CCR7 chemokine/receptor axis in a hypoxia-dependent manner. Cancer progression depends on the tumour stroma in which CAFs differ from normal fibroblasts. CAFs promote tumour growth, recruitment of endothelial progenitor cells and angiogenesis. In breast cancer up to 80% of fibroblasts display the CAF phenotype. Here a PDPN expressing model of CAFs made it possible to demonstrate the involvement of CCL21/CCR7 axis in the tumor cell-to-CAF recognition through podoplanin binding of CCL21. PDPN positive CAFs secrete microRNAs, which control gene expression at post-transcriptional level and influence cancer cells. MiR-21 is a key regulator of the oncogenic process, through its downstream target proteins among which the tumor suppressor, PTEN. We analyzed the effect of miR-21, but also oncogenic and hypoxia dependent miRs: miR-210 and miR-29b, on PDPN expression in fibroblasts in conditions mimicking the intra tumor microenvironment, i.e. in hypoxia. This points to crucial differences as compared to normoxia. Moreover we uncover the effect of podoplanin on angiogenesis by endothelial cells colocalizing with CAFs expressing podoplanin and on the expression of most prominent proangiogenic factors

Rola interakcji CCL21 - CCR7 w specyficznej adhezji komórek NK do śródbłonkowych komórek HPLNEC.B3

Tworzenie naczyń krwionośnych wewnątrz guza może być kontrolowane na różne sposoby. Szczególny nacisk kładzie się na badanie strategii umożliwiających hamowanie neowaskularyzacji poprzez blokowanie aktywacji i proliferacji komórek śródbłonka. Jednym z naturalnych mechanizmów obronnych organizmu jest działanie komórek naturalni zabójcy (NK). Poprzednio w naszym laboratorium wykazano, że zastymulowane komórki NK są zdolne do eliminacji komórek śródbłonkowych obecnych w guzie. Przedstawiono również wyniki sugerujące, że w patologicznych warunkach komórki śródbłonka zaangażowane w tworzenie naczyń krwionośnych wewnątrz guza mogą być rozpoznane jako „niebezpieczne” i niszczone przez komórki NK. W niniejszej pracy skupiliśmy się na badaniu podłoża specyficznej adhezji komórek NK do komórek HPLNEC.B3, w szczególności roli oddziaływania chemokiny CCL21 z jej receptorem CCR7. Jako modelu do badań użyliśmy komórek NK aktywowanych IL-2, które jak pokazano są zdolne do rozpoznawania, zabijania i adhezji do komórek śródbłonka. Ponadto użyliśmy komórek HPLNEC.B3 pochodzących z peryferyjnych węzłów chłonnych ponieważ stanowią one miejsce docelowe dla przerzutów wielu nowotworów, w szczególności czerniaka. Zaobserwowaliśmy, że CCL21 efektywnie zwiększa adhezję komórek NK. Inkubacja komórek NK z przeciwciałem neutralizującym receptor CCR7 obniża adhezję co sugeruje, że ten receptor jest zaangażowany w proces adhezji. Co więcej, po indukowanej temperaturą agregacji receptora na powierzchni komórek NK (capping) zaobserwowano wzrost poziomu adhezji komórek NK do śródbłonka. Udział GAG został przedstawiony poprzez hamujący efekt siarczanu chondroityny E efektywnie zmniejszającego poziom adhezji. Jako modelu naśladującego mikrośrodowisko wewnątrz guza użyliśmy komórek hodowanych w hipoksji. Zaobserwowaliśmy, że w środowisku o znacznie obniżonym poziomie tlenu poziom adhezji komórek NK jest niższy, jak również poziom produkowanej przez same komórki śródbłonka chemokiny spada. Jednakże różnica między podstawową adhezją komórek NK a tą indukowaną CCL21 jest większa w hipoksji w porównaniu do normoksji. Zaprezentowanie szczególnych zdolności aktywowanych komórek NK, które w specyficznych warunkach są zdolne do rozpoznawania i niszczenia komórek śródbłonka in vitro pozwala przypuszczać, że in vivo komórki te mogłyby uczestniczyć w inhibicji unaczynienia guza. Również znajomość mechanizmu zachodzenia adhezji mogłaby pomóc w zwiększeniu tych zdolności.Tumor angiogenesis may be controlled by several mechanisms. Here, strategies that could block tumor neovascularization, endothelial cell (EC) activation and proliferation were tested. Natural killer (NK) cells play an important role among the natural protection mechanisms. It has been previously shown in our laboratory, that in pathological conditions, ECs involved in tumor angiogenesis may be recognized as “dangerous” cells and then killed by NK cells. The present study was focused on the background of specific adhesion of NK cells to HPLNEC.B3 cells. The role of CCL21-CCR7 interaction in the specific adhesion of NK cells to HPLNEC.B3 endothelial cells was considered. After IL-2 activation NK cells were shown to be able to recognize, adhere to and kill endothelial cells. HPLNEC.B3 that are endothelial cells derived from peripheral lymph nodes, were chosen because they are the place of metastasis of many types of cancers, especially melanoma. We observed that CCL21 chemokine potently increases the NK adhesion. Treatment with neutralizing antibody for receptor of this chemokine, CCR7, decreased adhesion to the basal level indicating the involvement of this receptor. Moreover induction of capping of CCR7 receptor by temperature also increase level of attached NK cells. The involvement of GAGs (glycosaminoglycans) has been demonstrated by the inhibitory effect of chondroitin sulfate E which efficiently caused decrease in adhesion level. As a model of tumor microenvironment we used hypoxic HPLNEC.B3 cells. Our observations indicates that under hypoxic condition level of attached NK cells is lower as well as amount of produced CCL21 protein. However, the differences between basal adhesion and this induced by CCL21 is higher under hypoxic conditions in comparison to normoxic conditions. The demonstration that, under specific conditions stimulated NK cells are able to recognize and kill ECs in vitro allows hypothesizing that in vivo NK cells may participate in tumor angiogenesis inhibition and the knowledge of the mechanism of this reaction may help to enhance their cytotoxic action

Preliminary studies of antimicrobial activity of new synthesized hybrids of 2-thiohydantoin and 2-quinolone derivatives activated with blue light

Thiohydantoin and quinolone derivatives have attracted researchers’ attention because of a broad spectrum of their medical applications. The aim of our research was to synthesize and analyze the antimicrobial properties of novel 2-thiohydantoin and 2-quinolone derivatives. For this purpose, two series of hybrid compounds were synthesized. Both series consisted of 2-thiohydantoin core and 2-quinolone derivative ring, however one of them was enriched with an acetic acid group at N3 atom in 2-thiohydantoin core. Antibacterial properties of these compounds were examined against bacteria: Staphylococcus aureus, Bacillus subtilis, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae. The antimicrobial assay was carried out using a serial dilution method to obtain the MIC. The influence of blue light irradiation on the tested compounds was investigated. The relative yield of singlet oxygen ((1)O(2)*, (1)Δ(g)) generation upon excitation with 420 nm was determined by a comparative method, employing perinaphthenone (PN) as a standard. Antimicrobial properties were also investigated after blue light irradiation of the suspensions of the hybrids and bacteria placed in microtitrate plates. Preliminary results confirmed that some of the hybrid compounds showed bacteriostatic activity to the reference Gram-positive bacterial strains and a few of them were bacteriostatic towards Gram-negative bacteria, as well. Blue light activation enhanced bacteriostatic effect of the tested compounds