Efficient Differentiation of Embryonic Stem Cells into Mesodermal Precursors by BMP, Retinoic Acid and Notch Signalling

The ability to direct differentiation of mouse embryonic stem (ES) cells into specific lineages not only provides new insights into the pathways that regulate lineage selection but also has translational applications, for example in drug discovery. We set out to develop a method of differentiating ES cells into mesodermal cells at high efficiency without first having to induce embryoid body formation. ES cells were plated on a feeder layer of PA6 cells, which have membrane-associated stromal-derived inducing activity (SDIA), the molecular basis of which is currently unknown. Stimulation of ES/PA6 co-cultures with Bone Morphogenetic Protein 4 (BMP4) both favoured self-renewal of ES cells and induced differentiation into a Desmin and Nestin double positive cell population. Combined stimulation with BMP4 and all-trans Retinoic Acid (RA) inhibited self-renewal and resulted in 90% of cells expressing Desmin and Nestin. Quantitative reverse transcription-polymerase chain reaction (qPCR) analysis confirmed that the cells were of mesodermal origin and expressed markers of mesenchymal and smooth muscle cells. BMP4 activation of a MAD-homolog (Smad)-dependent reporter in undifferentiated ES cells was attenuated by co-stimulation with RA and co-culture with PA6 cells. The Notch ligand Jag1 was expressed in PA6 cells and inhibition of Notch signalling blocked the differentiation inducing activity of PA6 cells. Our data suggest that mesodermal differentiation is regulated by the level of Smad activity as a result of inputs from BMP4, RA and the Notch pathway

MYC Induces a Hybrid Energetics Program Early in Cell Reprogramming

Cell reprogramming is thought to be associated with a full metabolic switch from an oxidative- to a glycolytic-based metabolism. However, neither the dynamics nor the factors controlling this metabolic switch are fully understood. By using cellular, biochemical, protein array, metabolomic, and respirometry analyses, we found that c-MYC establishes a robust bivalent energetics program early in cell reprogramming. Cells prone to undergo reprogramming exhibit high mitochondrial membrane potential and display a hybrid metabolism. We conclude that MYC proteins orchestrate a rewiring of somatic cell metabolism early in cell reprogramming, whereby somatic cells acquire the phenotypic plasticity necessary for their transition to pluripotency in response to either intrinsic or external cues.Torres and colleagues describe an MYC-dependent remodeling of mitochondrial dynamics and metabolism during the first stage of cell reprogramming. Endogenous MYC activity was found to be necessary for cell reprogramming, likely by establishing a hybrid metabolic state characterized by elevated glycolytic flux and a somatic oxidative metabolic rate. MYC polarized mitochondria by increasing ATPIF1 and labeled cells prone to cell reprogramming.MINECO/FEDER, UE (J.T.); grant SAF2013-41945-R MINECO and Fundación Ramón ArecesPeer Reviewe

Effect of BMP4 on differentiation of ES cells cultured on PA6 feeder cells.

<p>ES cells were induced to differentiate on PA6 cells as indicated and analysed by immunofluorescence microscopy (<b>A, B</b> and <b>C</b>) or flow cytometry (<b>D</b>). The immunofluorescence images show expression of NCam and Oct4 (<b>A</b>), Desmin and Oct4 (<b>B</b>) or Keratin 14 (<b>C</b>). Quantification of the immunofluorescence experiments from (<b>A</b>) and (<b>B</b>) is shown in the lower bar diagrams. Colonies were counted as single positive for NCam (NCam+), Oct4 (Oct4+) or Double positive (DP) in (<b>A</b>); and single positive for Desmin (Des+), Oct4 (Oct4+) or double positive (DP) in (<b>B</b>). Data are represented as the average ± Standard Error of the Mean (SEM) of 3 independent differentiation experiments (***, <i>p</i><0.001, n = 3). (<b>D</b>) ES cells were induced to differentiate for 7 days with BMP4 and RA on CSFE-labelled PA6 and Desmin expression was assessed by flow cytometry (right hand dot plots). Side (SS) and forward scatter (FS) profiles are shown in the left hand dot plot. IC: isotype control antibody. Scale bars, 50 µm.</p

Mesenchymal gene expression signature of differentiated ES.

<p>(<b>A</b>) Marker expression profiling of ES cell-derived mesodermal precursors at the 3^rd passage showing downregulation of markers for undifferentiated cells and upregulated expression of mesodermal markers. (<b>B</b>) Expression of markers for mesenchymal stem (MSC) and smooth muscle (SMC) cells. Gene expression was normalised to undifferentiated ES cells and represented in log(2) scale. Data are the average ± SEM of 3 independent differentiation experiments. ES, undifferentiated ES cells; HSC, haematopoietic stem cells; Ect, ectoderm; Mes, mesoderm; End, endoderm; Cdm, cardiomyocyte; Skm, skeletal muscle.</p

SDIA combined with BMP4 and RA treatment induces differentiation of ES cells into mesodermal precursors.

<p>(<b>A</b>) Total RNA was extracted after 9 days of ES cell culture under the conditions indicated. Expression of markers for the three germ layers was assessed by qPCR and represented as relative gene expression normalised to undifferentiated ES cells. Gene expression analyses showed robust induction of mesodermal markers by combined stimulation with BMP4 and RA. (<b>B–D</b>) ES cells were induced to differentiate on PA6 cells in serum-free medium supplemented with BMP4+Retinoic Acid (RA) for 9 days and expression of Desmin, Nestin and Gata4 was analysed by immunofluorescence staining (<b>B</b>, <b>C</b>). Images are three dimensional projections of the mean fluorescence intensity of z-stacks and show co-expression of Desmin with Nestin and Gata4. (<b>D</b>) ES cells were differentiated as in (<b>C</b>) for 7 days and expression of Gata4 analysed by qPCR as in (<b>A</b>). Undiff, undifferentiated ES cells; d7, ES cells differentiated for 7 days in the presence of BMP4 and RA; PA6, PA6 feeder cells as a negative control. Data in (<b>A</b>) and (<b>D</b>) are represented as the average ± SEM of 3 independent differentiation experiments conducted in triplicate (n = 3). Scale bars, 50 µm.</p

Repression of Smad-dependent transcription by RA and SDIA.

<p>(<b>A</b>) Expression of Notch ligands and receptors in PA6, determined by qPCR, relative to their values in undifferentiated ES cells. Data are represented in log(2) scale as the average ± SEM of 3 independent RNA extractions conducted in triplicate. (<b>B</b>) ES cells were induced to differentiate on PA6 cells in serum-free medium in the presence of DMSO (as vehicle control) or a γ-Secretase Inhibitor (γSI) and expression of the neroectodermal markers <i>Sox1</i> and <i>Nestin</i> was analysed by qPCR. Data are represented as the average ± SEM of a representative experiment conducted in triplicate. (<b>C</b>) ES cells were induced to differentiate on PA6 cells in serum-free medium supplemented with BMP4 and RA in the presence of DMSO (as vehicle control) or γ-Secretase Inhibitor (γSI). Representative micrographs show morphology of differentiated colonies (phase contrast; left hand panels) and expression of Desmin (green immunofluorescence; middle panels). DAPI was used as nuclear counterstaining (blue; right hand panels). (<b>D</b>) Percentage of colonies positive for Desmin, represented as the average ± SEM of 3 independent differentiation experiments. (<b>E</b>) Reporter assays of Smad-dependent transcription in ES cells plated either on gelatine (black bars) or mitomycin C-treated PA6 cells (white bars) and stimulated as indicated. Data were normalised to unstimulated (control) ES cells plated on gelatine and represented as the average ± SEM of 3 independent experiments conducted in triplicate. (*) <i>p</i>-value<0.05, n = 3. (<b>F</b>) Schematic illustrating crosstalk between the BMP4-, RA- and SDIA/Jag1-activated pathways in controlling ES cell differentiation. Scale bar in (<b>C</b>), 50 µm.</p

Differentiation dynamics of ES cells by BMP4+RA.

<p>(<b>A</b>) Schematic of the differentiation protocol. (<b>B</b>) Cells were recorded by time-lapse microscopy and cultured as indicated. Phase contrast images of the same field are shown. Arrowhead shows colony of ES cells that differentiated into cells of mesenchymal appearance. (<b>C</b>) Phase-contrast images of ES cell-derived mesodermal progenitors cultured for 7 days in NSC medium (left panel, scale bar 100 µm) or at the 3^rd passage in NSC medium (right panel, scale bar 50 µm).</p