BackgroundSmall membrane-permeable molecules are now widely used during maintenance and differentiation of embryonic stem cells of different species. In particular the glycogen synthase kinase 3 (GSK3) is an interesting target, since its chemical inhibition activates the Wnt/beta-catenin pathway. In the present comparative study four GSK3 inhibitors were characterized.MethodsCytotoxicity and potential to activate the Wnt/beta-catenin pathway were tested using the commonly used GSK3 inhibitors BIO, SB-216763, CHIR-99021, and CHIR-98014. Wnt/beta-catenin-dependent target genes were measured by quantitative PCR to confirm the Wnt-reporter assay and finally EC50-values were calculated.ResultsCHIR-99021 and SB-216763 had the lowest toxicities in mouse embryonic stem cells and CHIR-98014 and BIO the highest toxicities. Only CHIR-99021 and CHIR-98014 lead to a strong induction of the Wnt/beta-catenin pathway, whereas BIO and SB-216763 showed a minor or no increase in activation of the Wnt/beta-catenin pathway over the natural ligand Wnt3a. The data from the Wnt-reporter assay were confirmed by gene expression analysis of the TCF/LEF regulated gene T.ConclusionsOut of the four tested GSK3 inhibitors, only CHIR-99021 and CHIR-98014 proved to be potent pharmacological activators of the Wnt/beta-catenin signaling pathway. But only in the case of CHIR-99021 high potency was combined with very low toxicity.

PMM2-CDG, formerly known as congenital disorder of glycosylation-Ia (CDG-Ia), is caused by mutations in the gene encoding phosphomannomutase 2 (PMM2). This disease is the most frequent form of inherited CDG-diseases affecting protein N-glycosylation in human. PMM2-CDG is a multisystemic disease with severe psychomotor and mental retardation. In order to study the pathophysiology of PMM2-CDG in a human cell culture model, we generated induced pluripotent stem cells (iPSCs) from fibroblasts of a PMM2-CDG-patient (PMM2-iPSCs). Expression of pluripotency factors and in vitro differentiation into cell types of the three germ layers was unaffected in the analyzed clone PMM2-iPSC-C3 compared with nondiseased human pluripotent stem cells (hPSCs), revealing no broader influence of the PMM2 mutation on pluripotency in cell culture. Analysis of gene expression by deep-sequencing did not show obvious differences in the transcriptome between PMM2-iPSC-C3 and nondiseased hPSCs. By multiplexed capillary gel electrophoresis coupled to laser induced fluorescence detection (xCGE-LIF) we could show that PMM2-iPSC-C3 exhibit the common hPSC N-glycosylation pattern with high-mannose-type N-glycans as the predominant species. However, phosphomannomutase activity of PMM2-iPSC-C3 was 27% compared with control hPSCs and lectin staining revealed an overall reduced protein glycosylation. In addition, quantitative assessment of N-glycosylation by xCGE-LIF showed an up to 40% reduction of high-mannose-type N-glycans in PMM2-iPSC-C3, which was in concordance to the observed reduction of the Glc3Man9GlcNAc2 lipid-linked oligosaccharide compared with control hPSCs. Thus we could model the PMM2-CDG disease phenotype of hypoglycosylation with patient derived iPSCs in vitro. Knock-down of PMM2 by shRNA in PMM2-iPSC-C3 led to a residual activity of 5% and to a further reduction of the level of N-glycosylation. Taken together we have developed human stem cell-based cell culture models with stepwise reduced levels of N-glycosylation now enabling to study the role of N-glycosylation during early human development.

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.

The activation of the TGF-beta pathway by activin A directs ES cells into the definitive endoderm germ layer. However, there is evidence that activin A/TGF-beta is not solely responsible for differentiation into definitive endoderm. GSK3beta inhibition has recently been shown to generate definitive endoderm-like cells from human ES cells via activation of the canonical Wnt-pathway. The GSK3beta inhibitor CHIR-99021 has been reported to generate mesoderm from human iPS cells. Thus, the specific role of the GSK3beta inhibitor CHIR-99021 was analyzed during the differentiation of human ES cells and compared against a classic endoderm differentiation protocol. At high concentrations of CHIR-99021, the cells were directed towards mesodermal cell fates, while low concentrations permitted mesodermal and endodermal differentiation. Finally, the analyses revealed that GSK3beta inhibition rapidly directed human ES cells into a primitive streak-like cell type independently from the TGF-beta pathway with mesoderm and endoderm differentiation potential. Addition of low activin A concentrations effectively differentiated these primitive streak-like cells into definitive endoderm. Thus, the in vitro differentiation of human ES cells into definitive endoderm is initially independent from the activin A/TGF-beta pathway but requires high canonical Wnt-signaling activity.

Differentiation of pluripotent cells into endoderm-related cell types initially requires in vitro gastrulation into the definitive endoderm (DE). Most differentiation protocols are initiated from colonies of pluripotent cells complicating their adaption due to insufficiently defined starting conditions. The protocol described here was initiated from a defined cell number of dispersed single cells and tested on three different human embryonic stem cell lines and one human induced pluripotent stem cell line. Combined activation of ActivinA/Nodal signaling and GSK3 inhibition for the first 24 h, followed by ActivinA/Nodal signaling efficiently induced the DE state. Activation of ActivinA/Nodal signaling alone was not effective. Efficient GSK3 inhibition allowed the reduction of the ActivinA concentration during the entire protocol. A feeder-independent cultivation of pluripotent cells was preferred to achieve the high efficiency and robustness since feeder cells hindered the differentiation process. Additionally, inhibition of the phosphatidylinositol 3-kinase (PI3K) signaling pathway was not required, nonetheless yielding high cell numbers efficiently committed toward the DE. Finally, the endoderm generated could be differentiated further into PDX1-positive pan-pancreatic cells and NGN3-positive endocrine progenitors. Thus, this efficient and robust DE differentiation protocol is a step forward toward better reproducibility due to the well-defined conditions based on dispersed single cells from feeder-free-cultivated human pluripotent cells.