15 research outputs found
The origins of human embryonic stem cells : a biological conundrum
Human inner cell mass (ICM) cells isolated from in vitro fertilized blastocysts are the progenitor cells used to establish in vitro stable human embryonic stem cells (hESCs) which are pluripotent and self-renew indefinitely. This long-term perpetuation of hESCs in the undifferentiated state is thought to be an in vitro adaptation of the ICM cells. To investigate at the molecular level how hESCs acquired their unique properties, transcriptional profiles of isolated ICM cells and undifferentiated hESCs were compared. We identified 33 genes enriched in the ICM compared to the trophectoderm and hESCs. These genes are involved in signaling cascades (SEMA7A and MAP3K10), cell proliferation (CUZD1 and MS4A7) and chromatin remodeling (H1FOO and HRMT1L4). Furthermore, primordial germ cell-specific genes (SGCA and TEX11) were detected as expressed in the ICM cells and not hESCs. We propose that the transcriptional differences observed between ICM cells and hESCs might be accounted for by adaptive reprogramming events induced by the in vitro culture conditions which are distinct from that of in vitro fertilized blastocysts. hESCs are a distinct cell type lacking in the human embryo but, nonetheless, resemble the ICM in their ability to differentiate into cells representative of the endodermal, ectodermal and mesodermal cell lineages
Correction: Identification of a Potent Endothelium-Derived Angiogenic Factor.
El artĂculo original ha sido publicado: Jankowski V, Tölle M, Tran TNA, van der Giet M, Schuchardt M, et al. (2013) Identification of a Potent Endothelium-Derived Angiogenic Factor. PLoS ONE 8(7): e68575. doi:10.1371/journal.pone.006857
Morphological pictures of HUVEC/HUASMC co-cultures upon physiological FSS conditions.
<p>(<b>A</b>) Scanning electron microscopy picture of the homogenously colonized inside of a hollow fiber with confluently grown and characteristically cobblestone shaped human primary endothelial cells upon the application of low laminar FSS (0.1 N/m<sup>2</sup>) for 24 h (magnification: 1â¶500). (<b>B</b>) Scanning electron microscopy picture of HUASMCs on the hollow fiber outside with their typical cell cytoskeletal structure and morphology upon 0.1 N/m<sup>2</sup> applied for 24 h (magnification: 1â¶400). (<b>C</b>) Confocal microscopic immunolocalization of Cadherin-5 in co-cultivated HUVECs exposed to low laminar FSS (0.1 N/m<sup>2</sup>) over a period of five days (magnification: 1â¶400). (<b>D</b>)Cadherin-5 in HUVECs upon high laminar FSS (3 N/m<sup>2</sup>) (magnification: 1â¶400). (<b>E</b>) Confocal microscopic immunolocalization of α-smooth-muscle-actin in co-cultivated HUASMCs upon 3 N/m<sup>2</sup> luminally applied for a five day period (magnification: 1â¶400). (<b>F</b>) α-smooth-muscle-actin in co-cultivated HUASMCs upon high laminar FSS (3 N/m<sup>2</sup>) (magnification: 1â¶400).</p
Hoechst 33342 staining of human primary cell nuclei colonized onto polypropylene hollow fiber membranes.
<p>(<b>A</b>) Longitudinal section of the inside of a hollow fiber membrane confluently colonized with HUVECs and stained with Hoechst 33342 after 0.1 N/m<sup>2</sup> applied for 24 h (magnification: 1â¶100). (<b>B</b>) Longitudinal section of the outside of a HUASMC mono-culture module stimulated with low laminar FSS for 24 h (magnification: 1â¶100). (<b>C</b>) Cross-section of a polypropylene hollow fiber co-colonized with HUVECs and HUASMCs. In focus are HUVECs on the inside upon 0.1 N/m<sup>2</sup> applied for five days (magnification: 1â¶100). (<b>D</b>) Cross-section of a co-culture module showing HUASMCs on the outside of a hollow fiber after low laminar FSS stimulation for five days (magnification: 1â¶100).</p
Identification of a potent endothelium-derived angiogenic factor.
The secretion of angiogenic factors by vascular endothelial cells is one of the key mechanisms of angiogenesis. Here we report on the isolation of a new potent angiogenic factor, diuridine tetraphosphate (Up4U) from the secretome of human endothelial cells. The angiogenic effect of the endothelial secretome was partially reduced after incubation with alkaline phosphatase and abolished in the presence of suramin. In one fraction, purified to homogeneity by reversed phase and affinity chromatography, Up4U was identified by MALDI-LIFT-fragment-mass-spectrometry, enzymatic cleavage analysis and retention-time comparison. Beside a strong angiogenic effect on the yolk sac membrane and the developing rat embryo itself, Up4U increased the proliferation rate of endothelial cells and, in the presence of PDGF, of vascular smooth muscle cells. Up4U stimulated the migration rate of endothelial cells via P2Y2-receptors, increased the ability of endothelial cells to form capillary-like tubes and acts as a potent inducer of sprouting angiogenesis originating from gel-embedded EC spheroids. Endothelial cells released Up4U after stimulation with shear stress. Mean total plasma Up4U concentrations of healthy subjects (N=6) were sufficient to induce angiogenic and proliferative effects (1.34 ± 0.26 nmol L(-1)). In conclusion, Up4U is a novel strong human endothelium-derived angiogenic factor
Validation of the â<i>artificial artery</i>â as <i>in vitro</i> co-culture system with arterial functional characteristics mimicking <i>in vivo</i> conditions of the vasculature.
<p>Semi-quantitative RT-PCR analysis showing the total VWF mRNA expression in co-colonized HUVECs and HUASMCs. RNA of both cell types was isolated separately. The expression of VWF in 0.1 N/m<sup>2</sup> stimulated HUVECs was taken as reference. (<b>A</b>) MALDI mass spectrum from supernatants of the â<i>artificial arteryâ</i> after each day. Randomly selected peptides show constant molecular mass-signal intensities over a period of five days (abscissa: relative molecular mass m/z, zâ=â1; ordinate: relative intensity, arbitrary units). (<b>B</b>) MALDI mass spectrum of the supernatant of endothelial cells after stimulation with 3.0 N/m<sup>2</sup> (upper spectrum) and without stimulation (0.1 N/m<sup>2</sup>) (lower spectrum) (abscissa: relative molecular mass, m/z, zâ=â1; ordinate: relative intensity, arbitrary units). (<b>C</b>) Relative mass-signal intensities of Up<sub>4</sub>A in secretomes isolated from HUVECs, HUASMCs, and HUVEC/HUASMC co-cultures in the â<i>artificial artery</i>â after stimulating with 3 N/m<sup>2</sup> for five days. (<b>D</b>) Semi-quantitative RT-PCR analyses showing the total mRNA expression of KLF2, TIMP1, and CCND1 in co-colonized HUVECs exposed to 3 N/m<sup>2</sup> for five days. The expression of each gene in 0.1 N/m<sup>2</sup> stimulated HUVECs was taken as reference. (<b>E</b>) Semi-quantitative RT-PCR analysis showing the total EDN1 mRNA expression in co-colonized HUVECs (0.1 N/m<sup>2</sup> vs. 3 N/m<sup>2</sup>) and HUASMCs (0.1 N/m<sup>2</sup> vs. 3 N/m<sup>2</sup>). The expression of END1 in 0.1 N/m<sup>2</sup> stimulated HUVECs was taken as reference.</p
Figure 1
<p>(<b>A</b>) Angiogenic effects of endothelial secretome in the rat embryo chorioallantoic membrane. The primitive placenta and yolk sac of rat embryos cultured during organogenesis under negative control conditions (HBSS and bovine serum) (I). The corresponding vascular system is underdeveloped and could be improved by angiogenic factors like VEGF as positive control (II). More complex and structured blood vessels and red staining caused by red blood cells in the blood vessels (marked by arrows). Morphologic evaluation of angiogenic effect of the endothelial secretome (III), of the endothelial secretome after incubation with alkaline phosphate (IV), and of the endothelial secretome after incubation with alkaline phosphate in the presence of suramin (V). (<b>B</b>) MALDI-TOF-TOF mass spectrum of the fraction from the analytical reversed-phase chromatography. (<b>C</b>) Enhanced vascularisation of rat embryonic yolk sac membranes induced by increasing Up<sub>4</sub>U concentrations after 48 h of culture. Typical result out of 5 similar experiments. (D) Effect of increasing Up<sub>4</sub>U concentration on proliferation rate of human endothelial cells (nâ=â7). (<b>E</b>) Reversed phase chromatography of the fraction of human plasma containing the remaining nucleotides after exclusion of mononucleotides. (<b>F</b>) Up<sub>4</sub>U release of cultivated endothelial cells after stimulation by a cone-and-plate viscometer with shear stress of 3 N m<sup>-2</sup> (nâ=â11).</p
Figure 4
<p>(<b>A</b>)Effect of Up<sub>4</sub>U on phosphorylation of MAPK. Phosphorylation of MEK1, ERK1/2, Akt, and p38 measured by Luminexâą technique before (open bar) and after stimulation with Up<sub>4</sub>U for 10 min (filled bar). Ratio of phospho/total were normalized to protein content of the lysates and demonstrated as percent stimulation relative to control (*p<0.05; nâ=â5). (<b>B</b>)Effect of PD98059, U0126, SB2021902, and GSK on Up<sub>4</sub>U induced proliferation rate in vascular smooth muscle cells (<b>C</b>)Up<sub>4</sub>U amount of secretome from in-vivo/ex-vivo stimulated aortic rings. (*p<0.05; nâ=â4).</p
Molecular masses of Up<sub>4</sub>U fragments obtained by MALDI-TOF-TOF mass spectrometry (<b>Figure 1</b><b>.B)</b>.
<p>The first column shows the fragment masses measured by MALDI-TOF-TOF mass spectrometry; second column shows the fragments mass of Up<sub>4</sub>U isolated from the endothelial secretome; the third column the fragments mass of Up<sub>4</sub>U isolated from plasma; the fourth column shows the fragment masses calculated from their respective structures; the fifth column shows the fragments masses of synthesised Up<sub>4</sub>U. M<sup>+</sup>â=âprotonated parent ion; Ăâ=â uracil; Uâ=âuridine; pâ=âphosphate group, e.g. Up<sub>3</sub>â=â UTP; w/oâ=âwithout.</p
Figure 2
<p>(<b>A</b>)Effect of increasing Up<sub>4</sub>U concentrations on proliferation rate of vascular smooth muscle cells in the absence of PDGF (nâ=â6). (<b>B</b>)Effect of increasing Up<sub>4</sub>U concentrations on proliferation rate of vascular smooth muscle cells in the presence of PDGF (10<sup>â6</sup> mol L<sup>-1</sup> PDGF each; nâ=â3). (<b>C</b>)Effect of increasing UTP concentrations on proliferation rate of vascular smooth muscle cells in the presence of PDGF (10<sup>â6</sup> mol L<sup>-1</sup> PDGF each; nâ=â3). (<b>D</b>)Effect of increasing UDP concentrations on proliferation rate of vascular smooth muscle cells in the presence of PDGF (10<sup>â6</sup> mol L<sup>-1</sup> PDGF each; nâ=â3). (<b>E</b>)Effect of Up<sub>4</sub>U (10<sup>â7</sup> mol L<sup>-1</sup>) or ATPÎłS (10<sup>â7</sup> mol L<sup>-1</sup>) in the presence of PDGF (10<sup>â6</sup> mol L<sup>-1</sup> PDGF each; nâ=â3) and suramin (10<sup>â4</sup> mol L<sup>-1</sup>), PPADS (10<sup>â5</sup> mol L<sup>-1</sup>), MRS2179 (10<sup>â5</sup> mol L<sup>-1</sup>) or RBII (10<sup>â5</sup> mol L<sup>-1</sup>) on proliferation rate of vascular smooth muscle cells.</p