Modulation of the Pentose Phosphate Pathway Induces Endodermal Differentiation in Embryonic Stem Cells

Embryonic stem (ES) cells can differentiate in vitro into a variety of cell types. Efforts to produce endodermal cell derivatives, including lung, liver and pancreas, have been met with modest success. Understanding how the endoderm originates from ES cells is the first step to generate specific cell types for therapeutic purposes. Recently, it has been demonstrated that inhibition of Myc or mTOR induces endodermal differentiation. Both Myc and mTOR are known to be activators of the Pentose Phosphate Pathway (PPP). We found that, differentely from wild type (wt), ES cells unable to produce pentose sugars through PPP differentiate into endodermal precursors in cell culture conditions generally non-permissive to generate them. The same effect was observed when wt ES cells were differentiated in presence of chemical inhibitors of the PPP. These data highlight a new role for metabolism. Indeed, to our knowledge, it is the first time that modulation of a metaboli

Cardiomyocyte differentiation of embryonic stem cells on the surface of organic semiconductors

Purpose: Electrically active supports provide new horizons for bio-sensing and artificial organ design. Cell-based electrochemical biosensors can be used as bio-microactuators, applied to the biorobotics. Microchip-based bioassay systems can provide real-time cell analysis for preclinical drug design or for intelligent drug delivery devices. In regenerative medicine, electrically active supports can be used as bio-reactors to monitor cell activity, optimize the stem cell differentiation and control cell and tissue morphology. Biocompatibility and direct interaction of the electrically active surface with the cell surface is a critical aspect of this technology. Methods: In this work embryonic stem cells (AK7 ES) have been cultivated on the surface of thin films achieved through the evaporation of two aromatic compounds (T6 and PDI-8CN2) of particular interest for the fabrication of organic field-effect transistors (OFET). One of the potential advantages offered by the application of OFETs as bio-electronic supports is that they represent a powerful tool for the detection of bio-signals because their electrically active surface is an organic film. Results: The cell morphology on T6 and PDI-8CN2 surface shows to be similar to the usual cell appearance, as obtained when standard culture support (petri dish) are employed. Moreover, our experimental results demonstrate that stem cells can be lead to differentiation up to “beating” cardiomyocytes even on these electrically-active organic films. Conclusions: This investigation encourages the perspective to develop OFET-based biosensors in order to accurately characterize stem cells during the cardiac differentiation process and eventually increase their differentiation efficiency

Mechanism inducing endodermal cell differentiation.

<p>(A) RT-PCR of different lineage-specific marker genes in wt and <i>G6pdΔ</i> ES cells in presence of a lower oxygen concentration (5%) and in normal culture conditions (20%). (B) RT-PCR of different lineage-specific markers in differentiated wt E14 and <i>Pgd</i>+/− ES cells at 8, 10 and 13 days of neural differentiation. (C) Double immunostaining Sox17/βIII-tubulin/DAPI of cells at 10 days of differentiation showed areas of immunoreactive cells for Sox17 only in <i>Pgd</i>+/− ES cells. Scale bars, 75 µm. (D) qRT-PCR for Sox17 and GATA4 in wt and <i>G6pdΔ</i> ES cells at day 10 after treatment with D-(-)-ribose during neural differentiation. Values are means ± SD (n = 3). *<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001.</p

Endodermal induction in <i>G6pdΔ</i> ES cells.

<p>(A) Analysis of different markers in wt and <i>G6pdΔ</i> ES cells during neural differentiation. Expression profiles of undifferentiated ES cells (Oct4 and Nanog), neural precursors (Nestin), neurons (NF-L), astrocytes (GFAP), mesendodermal precursors (GATA4), endodermal precursors (Sox17), and cardiac precursors (Nkx2.5) markers were analyzed by RT-PCR. RNA was isolated from cells at different days of differentiation. Lane C, positive control, RNA isolated from 14dpc embryos. Amplified HPRT is shown as a positive control. (B) Double immunostaining Sox17/βIII-tubulin/DAPI of cells at 10 days of differentiation showed areas of immunoreactive cells for Sox17 only in <i>G6pdΔ</i> ES cells. Scale bars, 50 µm. (C) RT-PCR analysis of GATA4, Sox17, NF-L (neural marker), TH (dopaminergic neuron marker) and GAD65 (gabaergic neuron marker) on wt, two different <i>G6pdΔ</i> ES cell lines, and <i>G6pdΔ</i>^pG6pd during differentiation. Lane C, positive control, on RNA isolated from 14dpc embryos. Amplified HPRT is shown as a positive control. (D) Western blot analysis with anti-Cripto and anti-Actin antibodies performed on protein extracts from wt and <i>G6pdΔ</i> ES cells during neural differentiation. Actin was analyzed as loading control. Below each lane the relative quantities (RQ) with respect to related undifferentiated embryonic stem cells are indicated. (E) Western blot analysis with anti-phospho-Smad2 and anti-Actin antibodies performed on protein extracts from wt and <i>G6pdΔ</i> ES cells during neural differentiation. Actin was analyzed as loading control. Below each lane the relative quantities (RQ) with respect to related undifferentiated embryonic stem cells are indicated.</p