Characterization of antibodies to coagulation factor VIII

http://www.ester.ee/record=b4338686~S58*es

Assessment of the Potential of CDK2 Inhibitor NU6140 to Influence the Expression of Pluripotency Markers NANOG, OCT4, and SOX2 in 2102Ep and H9 Cells

As cyclin-dependent kinases (CDKs) regulate cell cycle progression and RNA transcription, CDKs are attractive targets for creating cancer cell treatments. In this study we investigated the effects of the small molecular agent NU6140 (inhibits CDK2 and cyclin A interaction) on human embryonic stem (hES) cells and embryonal carcinoma-derived (hEC) cells via the expression of transcription factors responsible for pluripotency. A multiparameter flow cytometric method was used to follow changes in the expression of NANOG, OCT4, and SOX2 together in single cells. Both hES and hEC cells responded to NU6140 treatment by induced apoptosis and a decreased expression of NANOG, OCT4, and SOX2 in surviving cells. A higher sensitivity to NU6140 application in hES than hEC cells was detected. NU6140 treatment arrested hES and hEC cells in the G2 phase and inhibited entry into the M phase as evidenced by no significant increase in histone 3 phosphorylation. When embryoid bodies (EBs) formed from NU6104 treated hES cells were compared to EBs from untreated hES cells differences in ectodermal, endodermal, and mesodermal lineages were found. The results of this study highlight the importance of CDK2 activity in maintaining pluripotency of hES and hEC cells and in differentiation of hES cells

Nocodazole Treatment Decreases Expression of Pluripotency Markers Nanog and Oct4 in Human Embryonic Stem Cells

Nocodazole is a known destabiliser of microtubule dynamics and arrests cell-cycle at the G2/M phase. In the context of the human embryonic stem cell (hESC) it is important to understand how this arrest influences the pluripotency of cells. Here we report for the first time the changes in the expression of transcription markers Nanog and Oct4 as well as SSEA-3 and SSEA-4 in human embryonic cells after their treatment with nocodazole. Multivariate permeabilised-cell flow cytometry was applied for characterising the expression of Nanog and Oct4 during different cell cycle phases. Among untreated hESC we detected Nanog-expressing cells, which also expressed Oct4, SSEA-3 and SSEA-4. We also found another population expressing SSEA-4, but without Nanog, Oct4 and SSEA-3 expression. Nocodazole treatment resulted in a decrease of cell population positive for all four markers Nanog, Oct4, SSEA-3, SSEA-4. Nocodazole-mediated cell-cycle arrest was accompanied by higher rate of apoptosis and upregulation of p53. Twenty-four hours after the release from nocodazole block, the cell cycle of hESC normalised, but no increase in the expression of transcription markers Nanog and Oct4 was detected. In addition, the presence of ROCK-2 inhibitor Y-27632 in the medium had no effect on increasing the expression of pluripotency markers Nanog and Oct4 or decreasing apoptosis or the level of p53. The expression of SSEA-3 and SSEA-4 increased in Nanog-positive cells after wash-out of nocodazole in the presence and in the absence of Y-27632. Our data show that in hESC nocodazole reversible blocks cell cycle, which is accompanied by irreversible loss of expression of pluripotency markers Nanog and Oct4

SOX2 Is Regulated Differently from NANOG and OCT4 in Human Embryonic Stem Cells during Early Differentiation Initiated with Sodium Butyrate

Transcription factors NANOG, OCT4, and SOX2 regulate self-renewal and pluripotency in human embryonic stem (hES) cells; however, their expression profiles during early differentiation of hES cells are unclear. In this study, we used multiparameter flow cytometric assay to detect all three transcription factors (NANOG, OCT4, and SOX2) simultaneously at single cell level and monitored the changes in their expression during early differentiation towards endodermal lineage (induced by sodium butyrate). We observed at least four distinct populations of hES cells, characterized by specific expression patterns of NANOG, OCT4, and SOX2 and differentiation markers. Our results show that a single cell can express both differentiation and pluripotency markers at the same time, indicating a gradual mode of developmental transition in these cells. Notably, distinct regulation of SOX2 during early differentiation events was detected, highlighting the potential importance of this transcription factor for self-renewal of hES cells during differentiation

The role of integrin β1 in the heterogeneity of human embryonic stem cells culture

The maintenance of the pluripotency of human embryonic stem (hES) cells requires special conditions for culturing. These conditions include specific growth factors containing media and extracellular matrix (ECM) or an appropriate substrate for adhesion. Interactions between the cells and ECM are mediated by integrins, which interact with the components of ECM in active conformation. This study focused on the characterisation of the role of integrin β1 in the adhesion, migration and differentiation of hES cells. Blocking integrin β1 abolished the adhesion of hES cells, decreasing their survival and pluripotency. This effect was in part rescued by the inhibition of RhoA signalling with Y-27632. The presence of Y-27632 increased the migration of hES cells and supported their differentiation into embryoid bodies. The differences in integrin β1 recycling in the phosphorylation of the myosin light chain and in the localisation of TSC2 were observed between the hES cells growing as a single-cell culture and in a colony. The hES cells at the centre and borders of the colony were found to have differences in their morphology, migration and signalling network activity. We concluded that the availability of integrin β1 was essential for the contraction, migration and differentiation ability of hES cells

Changes in Laminin Expression Pattern during Early Differentiation of Human Embryonic Stem Cells

<div><p>Laminin isoforms laminin-511 and -521 are expressed by human embryonic stem cells (hESC) and can be used as a growth matrix to culture these cells under pluripotent conditions. However, the expression of these laminins during the induction of hESC differentiation has not been studied in detail. Furthermore, the data regarding the expression pattern of laminin chains in differentiating hESC is scarce. In the current study we aimed to fill this gap and investigated the potential changes in laminin expression during early hESC differentiation induced by retinoic acid (RA). We found that laminin-511 but not -521 accumulates in the committed cells during early steps of hESC differentiation. We also performed a comprehensive analysis of the laminin chain repertoire and found that pluripotent hESC express a more diverse range of laminin chains than shown previously. In particular, we provide the evidence that in addition to α1, α5, β1, β2 and γ1 chains, hESC express α2, α3, β3, γ2 and γ3 chain proteins and mRNA. Additionally, we found that a variant of laminin α3 chain—145 kDa—accumulated in RA-treated hESC showing that these cells produce prevalently specifically modified version of α3 chain in early phase of differentiation.</p></div

hESC express a diverse range of laminin chains.

<p>(A) Western blot analysis of indicated laminin (LM) chains in hESC. The lysates of JEG-3 (α1 chain), A431 (α2, α3, β1-β3, γ1- γ3), A549 and JAR (α5) cells and human platelets (α4) were used as controls. See the text for a detailed explanation. (B) Flow cytometric analysis of laminin (LM) chains α1, α2, α3 and α5 in hESC on day 3 and 5 of RA treatment. Untreated hESC (mTeSR1) were used as controls. Average Fold Change based on Median Fluorescence Intensity (MFI) values was calculated in relation to corresponding control (mTeSR1) samples. Statistical significance with P-values less than 0.05 are labeled with “*”.</p

Laminin α5 chain expression increases in differentiating hESC.

<p>Flow cytometric analysis of RA-treated (red population) and control (mTeSR1, blue population) hESC stained with antibodies recognizing laminin (LM) α5 chain and SSEA-3 (A) or HAND1 (B).</p

The expression of laminin α5, β1, β2 and γ1 chains in differentiating RA-treated hESC.

<p>(A) Immunofluorescence analysis of laminin (LM) chains α5, β1, β2 and γ1 and OCT4 in RA-treated hESC. Laminin chains (red) and OCT4 (green) were detected with appropriate antibodies. Cell nuclei were labeled with DAPI (blue). Scale bar: 100 μm. (B) Multilayer confocal microscopy was used to visualize the LM β1 chain (red) localization and OCT4 (green) expression in RA-treated hESC. Cell nuclei were labeled with DAPI (blue). Scale bar: 20 μm. (C) Immunoprecipitation of laminin-511 and -521 from RA-treated hESC. The protein complexes were immunoprecipitated using laminin α5 chain-specific antibody. The laminin α5, β1, β2 and γ1 chains were detected by Western blot analysis using corresponding antibodies.</p

RA induces hESC differentiation and concomitant upregulation of HAND1 and GATA-4 expression.

<p>(A) Immunofluorescence analysis of OCT4 (green) in RA-treated hESC. Cell nuclei were labeled with DAPI (blue). Scale bar: 100 μm. (B) Flow cytometric analysis of OCT4, SSEA-3, NANOG, SOX-2, GATA-4 and HAND1 expression in RA-treated hESC on day 3 and 5. Untreated hESC (grown in mTeSR1) harvested at the identical time-points were used as controls. Average Fold Change based on Median Fluorescence Intensity (MFI) values was calculated in relation to corresponding control (mTeSR1) samples. Statistical significance with P-values less than 0.05 are labeled with “*” (C) Flow cytometric analysis of RA-treated and control (mTeSR1) hESC cells co-stained with antibodies recognizing OCT4 and GATA-4 or HAND1. Overlays of RA-treated (red) and control (mTeSR1, blue) hESC populations are presented. (D) Flow cytometric analysis of RA-treated and control (mTeSR1) hESC co-stained with GATA-4 and HAND1-specific antibodies. The percentages of cell populations in each quadrant are indicated on the density plots.</p