In mice, the entry of germ cells into meiosis crucially depends on the expression of stimulated by retinoic acid gene 8 (Stra8). Stra8 is expressed specifically in pre-meiotic germ cells of females and males, at fetal and postnatal stages, respectively, but the mechanistic details of its spatiotemporal regulation are yet to be defined. In particular, there has been considerable debate regarding whether retinoic acid is required, in vivo, to initiate Stra8 expression in the mouse fetal ovary. We show that the distinctive anterior-to-posterior pattern of Stra8 initiation, characteristic of germ cells in the fetal ovary, is faithfully recapitulated when 2.9 kb of the Stra8 promoter is used to drive eGFP expression. Using in vitro transfection assays of cutdown and mutant constructs, we identified two functional retinoic acid responsive elements (RAREs) within this 2.9 kb regulatory element. We also show that the transcription factor DMRT1 enhances Stra8 expression, but only in the presence of RA and the most proximal RARE. Finally, we used CRISPR/Cas9-mediated targeted mutation studies to demonstrate that both RAREs are required for optimal Stra8 expression levels in vivo.

• MeSH
• adaptorové proteiny signální transdukční genetika   metabolismus   MeSH
• CRISPR-Cas systémy genetika   MeSH
• meióza MeSH
• mutageneze MeSH
• myši inbrední C57BL MeSH
• myši transgenní MeSH
• myši MeSH
• ovarium cytologie   metabolismus   MeSH
• plod cytologie   metabolismus   MeSH
• promotorové oblasti (genetika) MeSH
• regulace genové exprese účinky léků   MeSH
• regulační oblasti nukleových kyselin genetika   MeSH
• retinoidní X receptory genetika   metabolismus   MeSH
• transkripční faktory genetika   metabolismus   farmakologie   MeSH
• tretinoin farmakologie   MeSH
• vazebná místa MeSH
• vývoj plodu genetika   MeSH
• zárodečné buňky cytologie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Histological, immunohistochemical and molecular examination of bioptic samples of 30 normal adult auricular cartilages and small samples from 6 ear cartilages from aborted foetuses was performed. The adult cartilage was the tissue with minimal proliferative activity, which we were able to confirm with antibodies against Ki67 in contrast to a high proliferative activity in the auricular cartilage of foetal tissues. It may therefore be presumed that the process of foetal tissue maturation is undoubtedly associated with a significant reduction in proliferative activity. The mature lamella of the adult auricular cartilage has a histological tri-lamellar structure. There are a great number of elastic fibres in the intercellular matrix of the central zone, which are conversely present in only small amounts in both peripheral layers. While the external layer of the concave surface of the cartilage contains a fewer number of oval elements, the external layer of the convex side is composed of numerous fusiform chondrocytes. Antibodies against various subtypes of S-100 protein showed that auricular chondrocyte activity is modified depending on the configuration of individual distinct zones (isoforms A1, A6, B2 and P were positive in all layers, isoforms A2 and A2 in peripheral zones). The most active cells metabolically are most likely chondrocytes in both external layers adjacent to the perichondrium. We have also demonstrated α-smooth muscle actin (SMA)-positive chondrocytes in both peripheral layers of the auricular cartilage adjacent to the perichondrium. In addition, we found definite differences in the distribution of actin-positive cells depending on the external shape of the pinna. The majority of these fusiform cells were localised primarily in the areas of great curvature of the pinna, especially the convex side, as mentioned above. On the basis of these unique structural features we assume that the ear cartilage may embody an example of the socalled intelligent biological material, which has its internal structure made in such a way as to more easily develop and yet still maintain all the shape characteristics of the human auricle. The knowledge of these specific structural characteristics is important especially for use of auricular cartilage in auricular reconstruction.

• MeSH
• dospělí MeSH
• imunohistochemie MeSH
• lidé MeSH
• plod cytologie   MeSH
• posmrtné změny MeSH
• proteiny S100 metabolismus   MeSH
• ušní chrupavka cytologie   embryologie   MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Human induced pluripotent stem cell line was generated from commercially available primary human prostate fibroblasts HPrF derived from a fetus, aged 18-24 weeks of gestation. The fibroblast cell line was reprogrammed with Yamanaka factors (OCT4, SOX2, c-MYC, KLF4) using CytoTune™-iPS 2.0 Sendai Reprogramming Kit. Pluripotency of the derived transgene-free iPS cell line was confirmed both in vitro by detecting the expression of factors of pluripotency on a single-cell level, and in vivo using teratoma formation assay. This iPS cell line will be a useful tool for studying both normal prostate development and prostate cancer disease.

• MeSH
• fibroblasty * cytologie   metabolismus   MeSH
• indukované pluripotentní kmenové buňky * cytologie   metabolismus   MeSH
• lidé MeSH
• plod * cytologie   embryologie   MeSH
• přeprogramování buněk MeSH
• prostata * cytologie   embryologie   MeSH
• techniky buněčného přeprogramování * MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The fetus is thought to be protected from exposure to foreign antigens, yet CD45RO+ T cells reside in the fetal intestine. Here we combined functional assays with mass cytometry, single-cell RNA sequencing and high-throughput T cell antigen receptor (TCR) sequencing to characterize the CD4+ T cell compartment in the human fetal intestine. We identified 22 CD4+ T cell clusters, including naive-like, regulatory-like and memory-like subpopulations, which were confirmed and further characterized at the transcriptional level. Memory-like CD4+ T cells had high expression of Ki-67, indicative of cell division, and CD5, a surrogate marker of TCR avidity, and produced the cytokines IFN-γ and IL-2. Pathway analysis revealed a differentiation trajectory associated with cellular activation and proinflammatory effector functions, and TCR repertoire analysis indicated clonal expansions, distinct repertoire characteristics and interconnections between subpopulations of memory-like CD4+ T cells. Imaging mass cytometry indicated that memory-like CD4+ T cells colocalized with antigen-presenting cells. Collectively, these results provide evidence for the generation of memory-like CD4+ T cells in the human fetal intestine that is consistent with exposure to foreign antigens.

• MeSH
• antigen Ki-67 genetika   imunologie   metabolismus   MeSH
• antigen prezentující buňky cytologie   imunologie   metabolismus   MeSH
• antigeny CD5 genetika   imunologie   metabolismus   MeSH
• CD4-pozitivní T-lymfocyty cytologie   imunologie   metabolismus   MeSH
• imunofenotypizace MeSH
• imunologická paměť genetika   imunologie   MeSH
• kultivované buňky MeSH
• lidé MeSH
• plod cytologie   imunologie   metabolismus   MeSH
• průtoková cytometrie MeSH
• stanovení celkové genové exprese metody   MeSH
• střeva cytologie   embryologie   imunologie   MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• vývojová regulace genové exprese imunologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

INTRODUCTION: Magnetic resonance (MR) imaging is suitable for noninvasive long-term tracking. We labeled human induced pluripotent stem cell-derived neural precursors (iPSC-NPs) with two types of iron-based nanoparticles, silica-coated cobalt zinc ferrite nanoparticles (CZF) and poly-l-lysine-coated iron oxide superparamagnetic nanoparticles (PLL-coated γ-Fe2O3) and studied their effect on proliferation and neuronal differentiation. MATERIALS AND METHODS: We investigated the effect of these two contrast agents on neural precursor cell proliferation and differentiation capability. We further defined the intracellular localization and labeling efficiency and analyzed labeled cells by MR. RESULTS: Cell proliferation was not affected by PLL-coated γ-Fe2O3 but was slowed down in cells labeled with CZF. Labeling efficiency, iron content and relaxation rates measured by MR were lower in cells labeled with CZF when compared to PLL-coated γ-Fe2O3. Cytoplasmic localization of both types of nanoparticles was confirmed by transmission electron microscopy. Flow cytometry and immunocytochemical analysis of specific markers expressed during neuronal differentiation did not show any significant differences between unlabeled cells or cells labeled with both magnetic nanoparticles. CONCLUSION: Our results show that cells labeled with PLL-coated γ-Fe2O3 are suitable for MR detection, did not affect the differentiation potential of iPSC-NPs and are suitable for in vivo cell therapies in experimental models of central nervous system disorders.

• MeSH
• buněčná diferenciace * MeSH
• fibroblasty cytologie   MeSH
• imunoenzymatické techniky MeSH
• indukované pluripotentní kmenové buňky cytologie   MeSH
• kontrastní látky chemie   MeSH
• kultivované buňky MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• lidé MeSH
• lysin chemie   MeSH
• magnetická rezonanční tomografie metody   MeSH
• magnetické nanočástice chemie   MeSH
• neurony cytologie   MeSH
• plíce cytologie   MeSH
• plod cytologie   MeSH
• proliferace buněk MeSH
• průtoková cytometrie MeSH
• transmisní elektronová mikroskopie MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

Identifying the steps involved in striatal development is important both for understanding the striatum in health and disease, and for generating protocols to differentiate striatal neurons for regenerative medicine. The most prominent neuronal subtype in the adult striatum is the medium spiny projection neuron (MSN), which constitutes more than 85% of all striatal neurons and classically expresses DARPP-32. Through a microarray study of genes expressed in the whole ganglionic eminence (WGE: the developing striatum) in the mouse, we identified the gene encoding the transcription factor Forkhead box protein P1 (FoxP1) as the most highly up-regulated gene, thus providing unbiased evidence for the association of FoxP1 with MSN development. We also describe the expression of FoxP1 in the human fetal brain over equivalent gestational stages. FoxP1 expression persisted through into adulthood in the mouse brain, where it co-localised with all striatal DARPP-32 positive projection neurons and a small population of DARPP-32 negative cells. There was no co-localisation of FoxP1 with any interneuron markers. FoxP1 was detectable in primary fetal striatal cells following dissection, culture, and transplantation into the adult lesioned striatum, demonstrating its utility as an MSN marker for transplantation studies. Furthermore, DARPP-32 expression was absent from FoxP1 knock-out mouse WGE differentiated in vitro, suggesting that FoxP1 is important for the development of DARPP-32-positive MSNs. In summary, we show that FoxP1 labels MSN precursors prior to the expression of DARPP-32 during normal development, and in addition suggest that FoxP1 labels a sub-population of MSNs that are not co-labelled by DARPP-32. We demonstrate the utility of FoxP1 to label MSNs in vitro and following neural transplantation, and show that FoxP1 is required for DARPP-32 positive MSN differentiation in vitro.

• MeSH
• buněčná diferenciace fyziologie   MeSH
• corpus striatum * cytologie   embryologie   růst a vývoj   MeSH
• dopaminem a cAMP regulovaný fosfoprotein 32 metabolismus   MeSH
• embryo savčí MeSH
• forkhead transkripční faktory genetika   metabolismus   MeSH
• jaderné proteiny metabolismus   MeSH
• kultivované buňky MeSH
• myši knockoutované MeSH
• myši MeSH
• neparametrická statistika MeSH
• nervové kmenové buňky fyziologie   transplantace   MeSH
• neurony cytologie   metabolismus   MeSH
• novorozená zvířata MeSH
• plod cytologie   MeSH
• proteiny nervové tkáně metabolismus   MeSH
• represorové proteiny genetika   metabolismus   MeSH
• techniky in vitro MeSH
• transportní proteiny metabolismus   MeSH
• vývojová regulace genové exprese fyziologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

• MeSH
• chromozomální aberace MeSH
• genetická variace MeSH
• heterochromatin genetika   MeSH
• infertilita genetika   MeSH
• karyotypizace MeSH
• lidé MeSH
• lidské chromozomy, pár 9 genetika   MeSH
• mapování chromozomů MeSH
• plod cytologie   MeSH
• studie případů a kontrol MeSH
• těhotenství genetika   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• těhotenství genetika   MeSH
• ženské pohlaví MeSH
• Publikační typ
• abstrakty MeSH
• přednášky MeSH

Vývoj patologického fenotypu u pacientů s myotonickou dystrofií závisí značnou měrou na toxickém účinku transkriptu expandované repetice genu DMPK a jeho interakci s vazebnými proteiny. Vztah mezi CUGexp tohoto genu a vazebnými proteiny MBNL1 a MBNL2 byl dosud studován na tkáňových kulturách myoblastů a na transgenních zvířatech. V tomto sdělení podáváme první molekulárně genetickou analýzu fetálních tkání plodu z rodiny postižené myotonickou dystrofií prvního typu (MD1). Intrauterinní vývoj, zrání a diferenciace kosterního svalstva plodu (360 g, 22. týden) byly jen mírně opožděny. Paternálně zděděná expanze repetice CTG v genu DMPK (350 CTG) byla potvrzena molekulárně genetickou analýzou a následné histopatologické vyšetření plodu prokázalo znaky myotonické dystrofie. Tkáně získané autopsií (kosterní sval, jícen, žaludek a střevo) byly vyšetřeny histologickými i molekulárně genetickými (imunohistochemickou analýzou proteinů MBNL1 a MBNL2, a in situ hybridizací DMPK CUGexp) metodami. Intranukleární ložiska (foci) s CUG transkripty byla zjištěna v kosterním svalu, ve svalovině (muscularis externa) zažívací trubice, v cévní medii, endotelu a intramurálních nervových pleteních i v epitelu. Protein MBNL1 přitom s ložisky expandovaných transkriptů extenzivně kolokalizoval ve všech tkáních. Naproti tomu byl protein MBNL2 nalezen též v cytoplazmě. Přítomnost transkriptu DMPK s expandovanou repeticí CUG a proteinu MBNL1 v intranukleárních fokusech fetálních tkání MD1 pacientů může teoreticky znamenat sekvestraci proteinu MBNL1, a tak přispět ke generaci patologického fenotypu MD1.

Development of the pathological phenotype in patients with myotonic dystrophy (MD1) largely depends on toxic effects of expanded DMPK gene repeats (CUGexp) transcription and their interaction with binding proteins. The relationship between DMPK CUGexp, MBNL1 and MBNL2 has so far been studied on tissue cultures (myoblast cell lines) and transgenic animals. In this report, the first in situ molecular genetic analysis of fetal tissue from a family affected by MD1 is presented. Intrauterine development, maturation and differentiation of skeletal muscles of the fetus (360 g, 22nd week of pregnancy) were only slightly delayed. In the fetus, paternally inherited expansion of the CTG repeat in the DMPK gene (350 CTG) was confirmed by molecular analysis. Subsequent histopathological examination revealed signs of myotonic dystrophy. Fetal tissue obtained at autopsy (skeletal muscles, esophagus, stomach and intestines), were studied by histopathological, immunofluorescence (expression of MBNL1/MBNL2 proteins) and in situ hybridization (DMPK CUGexp) methods. Intranuclear CUGexp-containing foci were present in skeletal muscle fibers, muscularis externa of the esophagus, stomach and intestines, vascular smooth muscle, neurons and Schwann cells of intrinsic ganglionic plexuses, and epithelial cells. MBNL1 protein was largely co-localized with the CUGexp foci in all tissues examined. Contrarily, MBNL2 protein was also detected in the tissue cytoplasm. The presence of DMPK transcript with expanded CUG repeat and MBNL1 protein in the intranuclear foci of MD1 fetal tissues studied may theoretically result in sequestration of the protein and thus contribute to generation of the MD1 phenotype.

• Klíčová slova
• proteiny MBNL, fetální tkáň, DMPK mutace,
• MeSH
• fenotyp MeSH
• fluorescenční mikroskopie MeSH
• genetická transkripce genetika   MeSH
• histologické techniky MeSH
• hybridizace in situ fluorescenční MeSH
• imunohistochemie MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• mutační analýza DNA MeSH
• myotonická dystrofie * diagnóza   genetika   MeSH
• nemoci plodu MeSH
• otcové MeSH
• pitva MeSH
• plod * cytologie   patologie   MeSH
• polymerázová řetězová reakce MeSH
• protein-serin-threoninkinasy genetika   MeSH
• proteiny vázající RNA genetika   MeSH
• repetitivní sekvence nukleových kyselin MeSH
• ribonukleoproteiny genetika   MeSH
• rodokmen MeSH
• Southernův blotting MeSH
• zdraví rodiny MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH
• techniky in vitro MeSH

The resistance of transformed epithelial cells to a detachment-induced apoptosis (anoikis) can significantly affect their susceptibility to anticancer therapy. We showed that detachment of both fetal (FHC) and adenocarcinoma (HT-29) human colon epithelial cells resulted in the activation of the pro-survival Akt pathway, and significant changes in integrin-linked kinase (ILK) and focal adhesive kinase (FAK) phosphorylation. We demonstrated a detachment-induced and PI3K/Akt-mediated resistance to apoptotic effects of TRAIL, which was not associated with any changes in the cell surface TRAIL death receptor levels. Instead, a modulation of downstream intracellular signaling events was suggested to be involved. Our results may have important implications for optimization of new strategies in treatment of cancers at different stages of development.

• MeSH
• adenokarcinom enzymologie   patologie   MeSH
• aktivace enzymů účinky léků   MeSH
• anoikis účinky léků   MeSH
• buněčná adheze účinky léků   MeSH
• buněčná membrána účinky léků   metabolismus   MeSH
• buňky HT-29 MeSH
• chemorezistence účinky léků   MeSH
• epitelové buňky účinky léků   enzymologie   patologie   MeSH
• fokální adhezní tyrosinkinasy metabolismus   MeSH
• fosfatidylinositol-3-kinasy metabolismus   MeSH
• fosforylace účinky léků   MeSH
• lidé MeSH
• nádory tračníku enzymologie   patologie   MeSH
• plod cytologie   MeSH
• protein TRAIL farmakologie   MeSH
• protein-serin-threoninkinasy metabolismus   MeSH
• protoonkogenní proteiny c-akt metabolismus   MeSH
• receptory domény smrti metabolismus   MeSH
• signální transdukce účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Stable cell lines obtained by spontaneous immortalization might represent early stages of malignant transformation and be useful experimental models for studies of mechanisms of cancer development. The FHC (fetal human cells) cell line has been established from normal fetal colonic mucosa. Detailed characterization of this cell line and mechanism of spontaneously acquired immortality have not been described yet. Therefore, we characterized the FHC cell line in terms of its tumorigenicity, cytogenetics, and TP53 gene mutation analysis. FHC cells displayed capability for anchorage-independent growth in semisolid media in vitro and formed solid tumors after transplantation into SCID (severe combined immunodeficiency) mice. This tumorigenic phenotype was associated with hypotriploidy and chromosome number ranging from 66 to 69. Results of comparative genetic hybridization arrays showed that most chromosomes included regions of copy number gains or losses. Region 8q23 approximately 8q24.3 (containing, e.g., MYC proto-oncogene) was present in more than 20 copies per nucleus. Moreover, we identified mutation of TP53 gene in codon 273; triplet CGT coding Arg was changed to CAG coding His. Expression of Pro codon 72 polymorphic variant of p53 was also detected. Mutation of TP53 gene was associated with abolished induction of p21(Waf1/Cip1) and MDM-2 proteins and resistance to apoptosis after genotoxic treatment. Because of their origin from normal fetal colon and their relative resistance to the induction of apoptosis, FHC cells can be considered a valuable experimental model for various studies.

• MeSH
• apoptóza fyziologie   MeSH
• buněčná adheze fyziologie   MeSH
• buňky - růstové procesy fyziologie   MeSH
• cytogenetické vyšetření metody   MeSH
• fenotyp MeSH
• geny p53 MeSH
• HCT116 buňky MeSH
• hybridizace in situ fluorescenční MeSH
• karcinoembryonální antigen metabolismus   MeSH
• karyotypizace MeSH
• keratiny metabolismus   MeSH
• kolon cytologie   metabolismus   fyziologie   MeSH
• lidé MeSH
• mutační analýza DNA metody   MeSH
• myši SCID MeSH
• myši MeSH
• nádorová transformace buněk genetika   patologie   MeSH
• nádory tračníku genetika   patologie   MeSH
• plod cytologie   MeSH
• poškození DNA MeSH
• signální transdukce MeSH
• srovnávací genomová hybridizace MeSH
• transformované buněčné linie MeSH
• transplantace nádorů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH