Project description:WNT signaling inhibition at an intermediate stage of differentiation is a key requirement for cardiac induction of human ES cells, suggesting that endogenous WNT activity interferes with cardiomyocyte formation. Two downstream genes of endogenous WNT signaling, MSX1 and CDX2, were induced during cardiac differentiation to reveal whether they may account for the global negative effects played by endogenous WNT signaling in this process.
Project description:Efficient directed cardiac induction of human embryonic stem cells requires an intermediate inhibition of WNT signaling, shortly after the initial induction of mesoderm. To reveal the significance of the WNT inhibition step during differentiation, two differential time-course analyses were performed - both comparing WNT-inhibited and non-WNT-inhibited samples: In the first time-course, the WNT inhibitor IWP-2 was either added or omitted on days 2 and 3, and in the second time-course, IWP-2 was either added or omitted on days 1 and 2.
Project description:A simultaneous stimulation of the Activin / FGF, BMP, and WNT pathways is required for promoting most efficient mesoderm induction in human embryonic stem cells, as well as for subsequent differentiation into cardiomyocytes. To reveal the contributions of three of these signaling pathways to mesoderm formation and cardiac induction, comparative differentiation time-courses were recorded, varying the combinations of signaling factors administered to the cells during the first day of differentiation: FGF (F) + BMP (B) + WNT (W) treatment during the first 24 hours, or FGF + BMP, or BMP + WNT, or FGF + WNT
Project description:Background: Ion channels are key determinants for the function of excitable cells but little is known about their role and involvement during cardiac development. Earlier work identified Ca2+-activated potassium channels of small and intermediate conductance (SKCas) as important regulators of neural stem cell fate. Here, we have investigated their impact on the differentiation of pluripotent cells towards the cardiac lineage. Methods and Results: We have applied the SKCa-activator EBIO on embryonic stem cells and identified this particular ion channel family as a new critical target involved in the generation of cardiac pacemaker-like cells: SKCa-activation led to rapid remodeling of the actin cytoskeleton, inhibition of proliferation, induction of differentiation and diminished teratoma formation. Time-restricted SKCa-activation induced cardiac mesoderm and commitment to the cardiac lineage as shown by gene regulation, protein and functional electrophysiological studies. In addition, the differentiation into cardiomyocytes was modulated in a qualitative fashion, resulting in a strong enrichment of pacemaker-like cells. This was accompanied by induction of the sino-atrial gene program and in parallel by a loss of the chamber-specific myocardium. In addition, SKCa activity induced activation of the Ras-Mek-Erk signaling cascade, a signaling pathway involved in the EBIO-induced effects. Conclusions: SKCa-activation drives the fate of pluripotent cells towards the cardiac lineage and preferentially into pacemaker-like cardiomyocytes. This provides a novel strategy for the enrichment of cardiomyocytes and in particular, the generation of a specific subtype of cardiomyocytes, pacemaker-like cells, without genetic modification. Untreated ES cells in three independent experiments: - Untreated control ES cells sample 1 (Con_1) - Untreated control ES cells sample 2 (Con_2) - Untreated control ES cells sample 3 (Con_3) EBIO-treated ES cells in three independent experiments: - EBIO-treated ES cells sample 1 (EBIO_1) - EBIO-treated ES cells sample 2 (EBIO_2) - EBIO-treated ES cells sample 3 (EBIO_3) Untreated differentiated ES cells in two independent experiments: - Untreated control differentiated ES cells sample 1 (Con_day5+10_1) - Untreated control differentiated ES cells sample 2 (Con_day5+10_2) EBIO-treated differentiated ES cells in two independent experiments: - EBIO-treated differentiated ES cells sample 1 (EBIO_day5+10_1) - EBIO-treated differentiated ES cells sample 2 (EBIO_day5+10_2)
Project description:This study compares cardiac induction time-courses using (i) wild-type hESCs subjected to a standard directed differentiation protocol, (ii) EOMES knockout hESCs subjected to the same protocol, and (iii) EOMES KO / TET-ON hESCs subjected to a TET-ON protocol.
Project description:ES cells differentiated in the presence of the Wnt inhibitor DKK1 fail to express the transcription factor Snail and undergo EMT. We generated an ES cell line, A2.snail, that induced Snail expression upon addition of doxycycline addition. Microarrays were used to gain a global picture of ES cell differentiation at early timepoints after Snail was expressed during Wnt inhibition.
Project description:We performed whole transcriptome RNA sequencing experiments of neural differentiation time courses starting from human ES cells (EGFP-H1) and mouse EGFP-EpiS cells. Cells were sampled at high temporal frequency using an automated Tecan Tissue Culture robotics system to sample mouse cells every 4 mins and human cells every 10 mins for the first 10 hours of neural differentiation.
Project description:In vitro cardiac differentiation can be readily achieved in human induced pluripotent stem cells (hiPSCs) via temporal modulation of Wnt signalling. However, its differentiation trajectory and the in vivo counterpart of intermediate progenitor populations has yet to be elucidated. This study aims to capture the most diverse amount of distinct cell populations during in vitro cardiac differentiation by performing single cell RNA-sequencing at day 2, 4, 5 and 9 cells undergoing in vitro cardiac differentiation. Our study has revealed in vitro cardiac differentiation is analogous with in vivo second heart field cell specification.
