Month: May 2020

Similar reprogramming experiments were carried out in parallel with human keratinocytes, a somatic cell with a high reprogramming efficiency and fibroblasts, a cell type with Niraparib significantly lower reprogramming efficiency. Two serial spinfections of keratinocyte, fibroblast or astrocyte cultures resulted in over 90%, 50% and 40% of infected cells, respectively. Twelve days after the first infection, we started to observe the appearance of morphological hES-like colonies, coinciding with transgene silencing. However, we also detected partially reprogrammed colonies with non-hES morphological phenotypes, where transgene silencing, based on GFP expression, did not occur. Finally, eighteen days following the initial infection, we either fixed/stained or manually picked the hiPS cell colonies obtained for further culture and characterization. We observed a similar reprogramming efficiency for human astrocytes and keratinocytes, which was much higher when compared to fibroblasts. Similar reprogramming experiments performed using alkaline phosphatase staining to evaluate pluripotent colony formation showed comparable results. To better understand why astrocytes reprogram more efficiently than other cell types such as fibroblasts, we performed a real-time analysis of the expression of genes involved in different aspects of stem cell biology in the H9 hES cell line, astrocytes, keratinocytes and fibroblasts. Using a Pearson correlation as a distance measure between the different sets of values we observed that both, keratinocytes and astrocytes, are closer than fibroblasts to hES cells, which may contribute to the higher reprogramming efficiency of astrocytes. hES cells have a cell cycle signature structure characterized by a very shortG1phaseand ahighpercentage of cellsinS phase. We found that the characteristic stem cell cycle signature of hES cells is acquired in all the ASThiPS cell lines and differs greatly from the cell cycle profile observed in the cells of origin. Consequently, we observed a dramatic change in the expression profile of the cell cycle proteins involved in the G1/S transition in the ASThiPS cell lines to levels similar to those observed in hES cells. The induction of pluripotency by expression of Oct4, Sox2, Klf4 and c-Myc has been reported in human somatic cells of different origin. Recently, the generation of iPS cells from human neural stem cells has been achieved with only the expression of Oct4. These data suggest that the high similarity in the transcriptional program reported between these committed stem cells and hES cells helps to facilitate the reprogramming process. In our work, we demonstrated the ability to generate hiPS cells from a differentiated neural cell type. Furthermore, we showed that human astrocytes generated hiPS with a reprogramming efficiency similar to human keratinocytes, which is much higher than the efficiency observed for other cell types, such as fibroblasts.

The close packed structures emerge as model systems that approximate the network properties of self-organized molecular structures: They yield the local statistical averages and distributions similar to that of the self-assembled systems. Using these model networks as the basis, one may generate novel networks by introducing a few random links whereby the local properties are preserved while the desired global properties are approximated. The ultimate goal is to use both statistical and spectral characterization to design networks with desired properties and to determine the principles underlying organization of complex networks. The developmentally committed identity of somatic cells can be reverted to a pluripotent state through different reprogramming approaches. Among these methodologies, pluripotency is achieved by somatic cell nuclear transfer into enucleated unfertilized oocytes, cell fusion of differentiated cells with embryonic stem cells or treatment of differentiated cells with extracts derived from pluripotent cells. More recently, it has been reported that the induction of pluripotency in somatic cells can be achieved by the expression of defined transcription factors, including either the combination of Oct4, Sox2, Klf4 and cMyc, although the latter was found to be dispensable, or Oct4, Sox2, Nanog and Lin28. Induced pluripotent stem cells are epigenetically and functionally similar to ES cells, although BEZ235 studies comparing ES and iPS cells continue to more precisely examine the equivalence of these cell types. While the molecular mechanisms underlying the process of reprogramming remain obscure, recent reports indicate that classical hallmarks of malignancy such as inactivation of the p53 pathway or silencing of the ink4/arf locus leading to immortalization are shared between somatic cell reprogramming and cell transformation. In the last few years, rapid progress has been made towards improving the efficiency of iPS cell generation, development of integration-free strategies or substitution of some reprogramming factors with other proteins or chemical compounds. Though initial reports relied on the use of retroviral or lentiviral delivery systems to introduce the reprogramming transcription factors, induction of pluripotency can now be achieved with plasmid transfection, non-integrative episomal vectors, Piggy-Bac transposition, self-excisable vectors or by the delivery of reprogramming proteins. The universality of the process has been demonstrated by the generation of iPS cells from different species, as well as from different sources of somatic cells including fibroblasts, CD34+ cells, adipose cells, HUVEC cells, keratinocytes, neural stem cells or hepatocytes. Here, we report the generation of iPS cells from human astrocytes with a similar efficiency to keratinocytes, one of the human somatic cell types with the highest reported reprogramming efficiency to date. ASThiPS cells show a pluripotent gene expression signature.

We used the score values proposed by the manufacturer for microorganisms: values between 0.000 and 1.699 did not allow reliable cell identification; values between 1.700 and 1.999 allowed probable cell identification; scores higher than 2.0 were considered statistically significant and allowed the confident identification of different cell species. Finally, we used Multiexperiment Viewer version 4.3 software to perform hierarchical clustering with dendrogram representations of collected MALDI-TOF MS data. The mass values of peaks of the reference spectrum of each cell type were selected after treatment of these spectra by the FlexAnalysis software. In this report, we showed that a MALDI-TOF MS approach was able to identify intact immune cells. This method was rapid and easy to perform and did not require any additional components, in contrast to flow cytometry. In addition, the repertoire of analyzed molecules is different between MALDI-TOF MS and flow cytometry because MALDI-TOF MS is applicable to soluble molecules with a molecular weight ranging from 2 to 20 kDa, whereas flow cytometry detects surface markers or intracellular proteins through permeabilization procedures. Our MALDI-TOF MS approach extended to eukaryotic cells an approach previously used for bacterial identification. In this study, intact primary or cultured cells were washed in saline to eliminate contamination by components such as cytokines or albumin contained in FCS, and thawed samples were deposited on the MALDI target in which HCCA matrix was added. Clearly, spectra were constituted by a collection of peaks, and their masses and relative intensities varied according cell origin. Other attempts have been performed to analyze MALDI-TOF MS profiles of whole eukaryotic cells. A recent report shows that MALDI-TOF MS typing is efficient to Tubulin Acetylation Inducer HDAC inhibitor characterize 66 cell culture samples representing 34 species from insects to primates. Spectra of each cell type were composed of a variety of peaks with different masses and intensities, demonstrating the feasibility of our approach. However, as cell samples are treated by ethanol and formic acid/acetonitrile before assay, the two methods are not superimposable. Another report describes MALDI-TOF MS spectra from K562, BHK21 and GM15226 cell lines after lysis in 2,5-dihydroxybenzoic acid matrix solution. Again, obtained spectra show common peaks among the three cell types and specific peaks, demonstrating that MALDI-TOF MS performed on crude extracts of mammalian cell types may be useful to easily identify different cell types. In addition, we demonstrated that freezing cells at 280uC was sufficient to obtain high quality spectra, whereas cell lysis increased background noise in a non-interpretable way.

Dramatic increase in multinucleated cells, rounded cells with condensed ball-like nuclei, and cells undergoing cell death. Down-regulation experiments using RNA-interference have shown that Ncl is required for correct mitosis, given that absence of Ncl gave a prolonged cell cycle with misaligned chromosomes and defects in spindle organization. Similar experiments revealed that absence of Ncl forced cells into Tubacin growth arrest, accumulated in G2 phase, but also resulted in increased apoptosis and effects on the nucleus as well as defects in centrosome duplication. According to the above we reasoned that the found interaction between Ncl-P/Tpt1, which peaks during mitosis, plays a part in cell proliferation or cell cycle regulation. This is further supported by that we found the colocalization to decrease upon retinoic acid induced differentiation, since differentiated cells proliferate much slower than ES cells. We previously found an interaction between Tpt1 and Npm1 that also showed a cell cycle dependent pattern of interaction with a significant peak during mitosis. Ncl and Npm1 have previously been shown to be interaction partners. One could speculate if all three proteins may exist in a complex during mitosis, but we do not think this is the case. Npm1/Ncl was previously shown not to interact during prometaphase and metaphase, phases where we detect individual interactions of Ncl-P/Tpt1 and Tpt1/Npm1, suggesting that they are not part of a single complex. Remarkably, we found that Ncl-P also forms a complex with Oct4 and we observe this complex during interphase in both murine and human ES cells. Oct4 belongs to the POU transcription factor family. POU transcription factors were originally identified as DNA-binding proteins that are able to activate the transcription of genes containing an octameric sequence called the octameric motif, within the promoter or enhancer region. Oct4 has been proven to be essential for the identity of the pluripotential founder cell population in the mammalian embryo. Its endogenous expression is normally tightly restricted to embryonic stem and germ cells, and illegitimate Oct4 activation is also typical for embryonic carcinoma cells. The precise levels of Oct4 expression was reported to determine the fate of ES cells to either differentiate, dedifferentiate or self-renew. Oct4 has also been reported to be a potent nuclear reprogramming factor, where the Oct4 distribution and level in mouse clones showed consequences for pluripotency and the development of the somatic cell clones. With the newest method to reprogram somatic cell nuclei through retroviral introduction by different sets of transcription factors to create iPS cells, Oct4 has again been proven to be one of the necessary components for successful nuclear reprogramming.

OPN expression parallels the time course of macrophage infiltration into the infarct, a late event in the development of cerebral infarcts. This suggests that the upregulation of OPN is delayed until brain matrix remodeling is underway. While delayed expression of OPN in stroke has been reported, there are little data on its role in cerebral injury early during cerebral I-R. OPN can exist in two forms: secreted and intracellular. sOPN can engage various receptors on the cell surface, stimulating signal transduction pathways and cell adhesion. Certain of these receptors are upregulated following transient global cerebral I-R. Extensive post-translational modifications can modify the interaction of OPN with other proteins. OPN can be cleaved by thrombin, exposing a cryptic attachment motif that is capable of engaging additional integrins. iOPN, which lacks the signal sequence that targets the protein to secretory vesicles, possibly due to a down-stream alternative translational initiation signal, is expressed in Nutlin-3 548472-68-0 dendritic cells and macrophages of the immune system. In patients with advanced stages of Alzheimer’s disease, iOPN levels are increased in pyramidal neurons compared to normal human brain ; the authors suggested that iOPN may play a role in cell cycle progression, neuronal remyelination, and/or the formation of protein aggregates in Alzheimer’s Disease. The role of iOPN in the cellular response to stroke has not been studied. Here, we evaluated OPN expression early after cerebral I-R in three different areas of the brain. Next, we determined if the form of OPN we detected in cortical brain tissue was secreted or intracellular. Lastly, we investigated OPN expression in the presence of acetaminophen, a drug recently shown to reduce apoptosis and mitochondrial dysfunction in early cerebral I-R. For immunocytochemical analysis, sections were first washed in PBS. Endogenous hydrogen peroxidases were quenched using 0.3% H2O2 in PBS for 30 min. Sections were then blocked in 1% BSA for 1 h followed by incubation with 2A1 overnight at 4uC in a humidified chamber. The 2A1 antibody used in this study was generated in our laboratory; it binds to an epitope centrally located in the C-terminal thrombin cleavage fragment about 50 amino acids downstream of the RGD integrin-binding site. The epitope is not subject to post-translational modifications that can interfere with antibody-epitope recognition. A few sections were used as negative controls, where the primary antibody was omitted. Slides were then incubated with secondary HRPconjugated goat anti-mouse antibody for 1 h. Immunostaining was developed by the peroxidaseantiperoxidase procedure using 3-39-diaminobenzidine as the cosubstrate. Sections were counterstained with hematoxylin before mounting.