Differentiation of extraembryonic endoderm stem cell lines and parietal endoderm into visceral endoderm: the art of XEN cells

The extraembryonic endoderm of mammals is essential for nutritive support of the foetus and patterning of the early embryo. Visceral and parietal endoderm are major subtypes of this lineage with the former exhibiting most, if not all, of the embryonic patterning properties. Extraembryonic endoderm (XEN) cell lines derived from the primitive endoderm of mouse blastocysts represent a cell culture model of this lineage, but are biased towards parietal endoderm in culture and in chimaeras. Here, I further characterise XEN cells and show that these cell lines exhibit high levels of heterogeneity. In an effort for XEN cells to adopt visceral endoderm character different aspects of the in vivo environment were mimicked. I found that BMP4 and laminin promote a mesenchymal-to-epithelial transition of XEN cells with upregulation of epithelial markers and downregulation of mesenchymal markers. Gene expression analysis showed the differentiated XEN cells most resembled extraembryonic visceral endoderm. Correspondingly, inhibition of Erk and BMP signalling drives XEN cells toward parietal endoderm fate. Finally, I show that BMP4 treatment of freshly isolated parietal endoderm from Reichert’s membrane promotes its visceral endoderm differentiation. This suggests that parietal endoderm is still developmentally plastic and can be transdifferentiated to a visceral endoderm in response to BMP. Generation of visceral endoderm from XEN cells uncovers the true potential of these blastocyst-derived cells and is a significant step towards modelling early developmental events ex vivo

Virus-free induction of pluripotency and subsequent excision of reprogramming factors

Reprogramming of somatic cells to pluripotency, thereby creating induced pluripotent stem (iPS) cells, promises to transform regenerative medicine. Most instances of direct reprogramming have been achieved by forced expression of defined factors using multiple viral vectors1-7. However, such iPS cells contain a large number of viral vector integrations1,8, any one of which could cause unpredictable genetic dysfunction. While c-Myc is dispensable for reprogramming9,10, complete elimination of the other exogenous factors is also desired since ectopic expression of either Oct4 or Klf4 can induce dysplasia11,12. Two transient transfection reprogramming methods have been published to address this issue13,14. However, the efficiency of either approach is extremely low, and neither has thus far been applied successfully to human cells. Here we show that non-viral transfection of a single multiprotein expression vector, which comprises the coding sequences of c-Myc​,​ Klf4​,​ Oct4 and Sox2 linked with 2A peptides, can reprogram both mouse and human fibroblasts. Moreover, the transgene can be removed once reprogramming has been achieved. iPS cells produced with this non-viral vector show robust expression of pluripotency markers, indicating a reprogrammed state confirmed functionally by in vitro differentiation assays and formation of adult chimeric mice. When the single vector reprogramming system was combined with a piggyBac transposon15,16 we succeeded in establishing reprogrammed human cell lines from embryonic fibroblasts with robust expression of pluripotency markers. This system minimizes genome modification in iPS cells and enables complete elimination of exogenous reprogramming factors, efficiently providing iPS cells that are applicable to regenerative medicine, drug screening and the establishment of disease models

Differentiation of extraembryonic endoderm stem cell lines and parietal endoderm into visceral endoderm : the art of XEN cells

The extraembryonic endoderm of mammals is essential for nutritive support of the foetus and patterning of the early embryo. Visceral and parietal endoderm are major subtypes of this lineage with the former exhibiting most, if not all, of the embryonic patterning properties. Extraembryonic endoderm (XEN) cell lines derived from the primitive endoderm of mouse blastocysts represent a cell culture model of this lineage, but are biased towards parietal endoderm in culture and in chimaeras. Here, I further characterise XEN cells and show that these cell lines exhibit high levels of heterogeneity. In an effort for XEN cells to adopt visceral endoderm character different aspects of the in vivo environment were mimicked. I found that BMP4 and laminin promote a mesenchymal-to-epithelial transition of XEN cells with upregulation of epithelial markers and downregulation of mesenchymal markers. Gene expression analysis showed the differentiated XEN cells most resembled extraembryonic visceral endoderm. Correspondingly, inhibition of Erk and BMP signalling drives XEN cells toward parietal endoderm fate. Finally, I show that BMP4 treatment of freshly isolated parietal endoderm from Reichert’s membrane promotes its visceral endoderm differentiation. This suggests that parietal endoderm is still developmentally plastic and can be transdifferentiated to a visceral endoderm in response to BMP. Generation of visceral endoderm from XEN cells uncovers the true potential of these blastocyst-derived cells and is a significant step towards modelling early developmental events ex vivo.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Adenovirus dodecahedron, as a drug delivery vector.

Stability studies show that Dds can be conveniently stored and transported, and can potentially be used for therapeutic purposes under various climates. Successful BLM delivery by Ad Dds demonstrates that the use of virus like particle (VLP) results in significantly improved drug bioavailability. These experiments open new vistas for delivery of non-permeant labile drugs

BMP signaling induces visceral endoderm differentiation of XEN cells and parietal endoderm

The extraembryonic endoderm of mammals is essential for nutritive support of the fetus and patterning of the early embryo. Visceral and parietal endoderm are major subtypes of this lineage with the former exhibiting most, if not all, of the embryonic patterning properties. Extraembryonic endoderm (XEN) cell lines derived from the primitive endoderm of mouse blastocysts represent a cell culture model of this lineage, but are biased towards parietal endoderm in culture and in chimeras. In an effort to promote XEN cells to adopt visceral endoderm character we have mimicked different aspects of the in vivo environment. We found that BMP signaling promoted a mesenchymal-to-epithelial transition of XEN cells with up-regulation of E-cadherin and down-regulation of vimentin. Gene expression analysis showed the differentiated XEN cells most resembled extraembryonic visceral endoderm (exVE), a subtype of VE covering the extraembryonic ectoderm in the early embryo, and during gastrulation it combines with extraembryonic mesoderm to form the definitive yolk sac. We found that laminin, a major component of the extracellular matrix in the early embryo, synergised with BMP to promote highly efficient conversion of XEN cells to exVE. Inhibition of BMP signaling with the chemical inhibitor, Dorsomorphin, prevented this conversion suggesting that Smad1/5/8 activity is critical for exVE induction of XEN cells. Finally, we show that applying our new culture conditions to freshly isolated parietal endoderm (PE) from Reichert\u27s membrane promoted VE differentiation showing that the PE is developmentally plastic and can be reprogrammed to a VE state in response to BMP. Generation of visceral endoderm from XEN cells uncovers the true potential of these blastocyst-derived cells and is a significant step towards modelling early developmental events ex vivo. © 2011 Elsevier Inc

BMP signaling induces visceral endoderm differentiation of XEN cells and parietal endoderm

The extraembryonic endoderm of mammals is essential for nutritive support of the fetus and patterning of the early embryo. Visceral and parietal endoderm are major subtypes of this lineage with the former exhibiting most, if not all, of the embryonic patterning properties. Extraembryonic endoderm (XEN) cell lines derived from the primitive endoderm of mouse blastocysts represent a cell culture model of this lineage, but are biased towards parietal endoderm in culture and in chimeras. In an effort to promote XEN cells to adopt visceral endoderm character we have mimicked different aspects of the in vivo environment. We found that BMP signaling promoted a mesenchymal-to-epithelial transition of XEN cells with up-regulation of E-cadherin and down-regulation of vimentin. Gene expression analysis showed the differentiated XEN cells most resembled extraembryonic visceral endoderm (exVE), a subtype of VE covering the extraembryonic ectoderm in the early embryo, and during gastrulation it combines with extraembryonic mesoderm to form the definitive yolk sac. We found that laminin, a major component of the extracellular matrix in the early embryo, synergised with BMP to promote highly efficient conversion of XEN cells to exVE. Inhibition of BMP signaling with the chemical inhibitor, Dorsomorphin, prevented this conversion suggesting that Smad1/5/8 activity is critical for exVE induction of XEN cells. Finally, we show that applying our new culture conditions to freshly isolated parietal endoderm (PE) from Reichert\u27s membrane promoted VE differentiation showing that the PE is developmentally plastic and can be reprogrammed to a VE state in response to BMP. Generation of visceral endoderm from XEN cells uncovers the true potential of these blastocyst-derived cells and is a significant step towards modelling early developmental events ex vivo. © 2011 Elsevier Inc

Dd stability under different conditions of temperature, pH and ionic strength, analyzed by native gel electrophoresis.

<p>Purified Dds were dialyzed overnight at 4°C (A, B) or for 24 h at 37°C (C) against different buffers at the indicated pH. (A) Effect of pH on Dd solubility. Dd was dialyzed against the following 50 mM buffers: MES, pH 6; Hepes, pH 7; Tris, pH 8 and CAPS, pH 9. Left panel: samples were prepared in duplicates and after dialysis the second batch was centrifuged at 13000 rpm for 30 min. The first four lanes contain the dialyzed samples; the next lanes contain the supernatants after centrifugation. Right panel: samples were dialyzed against the same buffers as described for the left panel and also against citric acid, pH 4, and acetic acid, pH 5, but all buffers contained 150 mM NaCl. Samples were prepared in duplicates and one batch was kept at 4°C while the second one was incubated for 20 min at 30°C. Soluble proteins contained in the supernatants obtained by 30 min centrifugation at 13000 rpm were applied on agarose gel. (B) Dd stability in carbonate buffer. Carbonate buffer (100 mM) was prepared at the indicated pH and used for Dds dialysis. Some samples were incubated at 37°C for 20 min. All samples were centrifuged as above and the supernatants were electrophoresed on native agarose gel. First and last lanes, control Dd and Pb preparations, respectively. (C) Effect of ionic conditions on Dd thermal stability. Dds were dialyzed for 24 h at 37°C against the purification buffer containing NaCl or (NH₄)₂SO₄ (AS) at indicated concentrations or against PBS, pH 7.5. Non-treated samples (T for total) or supernatants after centrifugation at 13000 rpm for 30 min (S) were analyzed on the agarose gel. The first two lanes contain control Dd while the last lane contains Pb preparations, all in purification buffer.</p

Purification of recombinant Ad3 DB expressed in the baculovirus system.

<p>(A) Dodecahedra initially purified on sucrose density gradient were fractionated on a Q-Sepharose column in 20 mM Tris buffer, pH 7.5, using NaCl gradient. (B) Analysis of purified Dds. Left panel - negative stain electron microscopy (EM) of Dds purified on sucrose density gradient. Middle and right panels - non-denaturing agarose gel electrophoresis of fractions recovered from the Q-Sepharose column with detection with ethidium bromide (EtBr, middle panel) followed by Commassie Brilliant Blue (CBB) staining (right panel). (C) Negative stain EM showing free pentameric bases recovered in peak 1 (left panel) and complete dodecahedra in peak 2 (right panel) of the Q-Sepharose column (P1 and P2 in A). Scale bar equals 100 nm. (D) Flow cytometry analysis of HeLa cells transduced with Dd (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005569#s4" target="_blank">Material and Methods</a>). Sucrose density gradient-purified Dds – purple curve, Q-Sepharose-purified Dds - green curve. Blue curve shows the antibody background in the absence of Dd.</p

Dd stability upon lyophilization, inside HeLa cells and in human serum.

<p>(A) Purified Dds were dialyzed overnight at 4°C against water or 150 mM (NH₄)₂SO₄ in water. Mannitol (0.4%) and sucrose (0.4%) were added to samples marked „Cryoprotect. +”. Dd samples were frozen at −80°C, dried in speed-vac or lyophilized (marked Lyoph. +). Dry samples were reconstituted in the starting volume of water. All preparations were centrifuged for 30 min at 13000 rpm and the supernatants were applied onto native agarose gel. (B) Stability of Dd after application to HeLa cells. Purified Dds (2 µg in 100 µl) were applied to 2×10⁴ portions of HeLa cells. After indicated periods of penetration cell lysates were analyzed on SDS-PAGE (left panel) or on native agarose gel (two right panels). Control Dd samples contained 30 ng protein, while control Pb sample contained 10 ng protein. (C) Stability of Dd upon incubation in human serum. Samples of Dd concentrated by ultrafiltration in Microcon unit (Millipore) (5 µg each) were incubated in human serum (HS) at 4°C for 2 h (lane 4) and at 37°C for 15 min or for 2 h (lanes 5 and 6, respectively). Samples were resolved by native agarose gel electrophoresis and analyzed by Western blot performed with anti-Dd serum. The upper part shows CBB stained gel with proteins remaining after transfer, and the lower part the developed Western blot. Lanes 1 and 7 show Dd non treated or incubated for 2 h at 37°C, respectively, in the absence of serum. Lanes 2 and 3 show human serum after 2 h incubation at 4 and 37°C, respectively. Dd samples incubated with the serum are denoted in bold.</p

Effect of Dd and Dd-BLM conjugate on HeLa cells.

<p>(A) Flow cytometry analysis of Dd (green curve) and Dd-BLM (pink curve) cell entry. Cells were treated with appropriate vector for 1 h at 37°C as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005569#s4" target="_blank">Material and Methods</a>. The blue curve shows the antibody background in the absence of Dd. (B) Cells were treated with 1 µg Dd or Dd-BLM for indicated times and analyzed with anti-Dd serum (in red) by confocal microscopy, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005569#s4" target="_blank">Material and Methods</a>. Nuclei were stained blue with DAPI. Last row shows the 50 h-treatment without nuclear staining. Scale bar equals 20 µm.</p