Anterior–Posterior Patterning of Definitive Endoderm Generated from Human Embryonic Stem Cells Depends on the Differential Signaling of Retinoic Acid, Wnt-, and BMP-Signaling

Abstract

As known from model organisms, such as frog, fish, mouse, and chicken, the anterior-posterior patterning of the definitive endoderm (DE) into distinct domains is controlled by a variety of signaling interactions between the DE and its surrounding mesoderm. This includes Wnt/FGFs and BMPs in the posterior half and all-trans-retinoic acid, TGF-β-ligands, Wnt-, and BMP-inhibitors in the anterior half of the DE sheet. However, it is currently unclear how these embryonic tissue interactions can be translated into a defined differentiation protocol for human embryonic stem cells. Activin A has been proposed to direct DE into a SOX2-positive foregut-like cell type. Due to the pleiotropic nature of SOX2 in pluripotency and developing cells of the foregut, we purified DE-cells by magnetic cell sorting and tested the effects of anteriorizing and posteriorizing factors on pure endoderm. We show in contrast to previous studies that the generation of the foregut marked by SOX2/FOXA2 double-positive cells does not depend on activin A/TGF-β-signaling but is mediated by the inhibition of Wnt- and BMP-signaling. Retinoic acid can posteriorize and at the same time dorsalize the foregut toward a PDX1-positive pancreatic duodenal cell type whereas active Wnt/beta-catenin signaling synergistically with FGF-2, BMP-4, and RA induces the formation of CDX2-positive posterior endoderm. Thus, these results provide new insights into the mechanisms behind cell specification of human DE derived from pluripotent stem cells. Stem Cells 2016;34:2635-2647.

Long Non-coding RNA Maternally Expressed 3 Increases the Expression of Neuron-Specific Genes by Targeting miR-128-3p in All-Trans Retinoic Acid-Induced Neurogenic Differentiation From Amniotic Epithelial Cells

“…All-trans retinoic acid (ATRA) is the active ingredient of Vitamin A and plays an important role in the development of the nervous system by providing trophic effects that support the proliferation, differentiation, and maintenance of neural cells. ATRA affects the differentiation of neural cells derived from stem cells, including embryonic stem cells, mesenchymal stem cells, induced pluripotent stem cells, and AECs (Mark et al, 2015;Samarut et al, 2015;Davenport et al, 2016).…”

Purification of Definitive Endoderm Generated from Pluripotent Stem Cells by Magnetic Cell Sorting

“…The differentiation protocol yielded an efficacy of 65% to 85% CXCR4-positive cells depending on the propensity of the PSC line used (Diekmann et al, 2015;Davenport et al, 2016). Typically, when seeding 100,000 cells per cm 2 one can expect a 4-to 8-fold increase in cell quantity compared to the initially seeded cell number.…”

“…During further embryonic development the endoderm gives rise to the linings of the gastrointestinal tract and various organs such as the thyroid, thymus, lungs, liver, and pancreas (Zorn and Wells, 2009). One aspect of regenerative medicine is the generation of somatic cells by differentiation from multipotent or pluripotent cells for cell replacement therapies of degenerative diseases (Naujok et al, 2011;Davenport et al, 2016). Pluripotent stem cells (PSCs)-such as embryonic stem cells (ESCs), epiblast stem cells (EpiSCs), or induced pluripotent stem cells (iPSCs)-represent a promising cell source for this purpose.…”

“…Nonetheless, the efficacy of the protocols currently used rarely touches 100% as the endoderm cells are contaminated with cells of non-endoderm origin or cells that retained pluripotency features since they did not adequately respond to the extrinsic stimuli (Davenport et al, 2016). These contaminants can cause serious problems: (1) The nonendoderm cells may negatively influence further differentiation by secretion of paracrine factors.…”

Stem Cell Therapy for Diabetes: Beta Cells versus Pancreatic Progenitors

“…A highly efficient definitive endoderm is induced by Activin/ Nodal signaling [13]. Activation of FGF and retinoid signaling, along with the inhibition of BMP and hedgehog signaling directs the endodermal cells to a pancreatic fate [14,15]. EGF and nicotinamide signaling enhance the generation of pancreatic progenitors and are crucial for the induction of NKX6.1 expression [16].…”