A Reliable and Efficient Protocol for Human Pluripotent Stem Cell Differentiation into the Definitive Endoderm Based on Dispersed Single Cells

Abstract

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.

Biphasic modulation of Wnt signaling supports efficient foregut endoderm formation from human pluripotent stem cells

“…Moreover, the use of our reporter cell line allowed us to track the canonical Wnt signaling activity after CHIR99021 administration. As an addition to previously published data (Toivonen et al, ; Loh et al, ; Diekmann et al, ; Siller et al, ), we were able to show a direct correlation between CHIR99021 administration and Wnt target gene expression.…”

Generation and Purification of Definitive Endoderm Cells Generated from Pluripotent Stem Cells

“…The optimal concentrations of CHIR-99021 and activin A should be determined for each human PSC line. The concentrations denoted here worked for all tested human PSC lines and induced high numbers of DE-committed cells (9). However, it might be worthwhile to define a lower threshold concentration especially for the recombinant protein activin A, which is expensive and puts a huge cost pressure on the laboratory.…”

“…PSCs can be differentiated into all adult cell types and consequently may be used in the future for potential treatments of a wide range of degenerative diseases (1). In vitro differentiation of PSCs into somatic cells of the lung (2), liver (3,4), and pancreas (5)(6)(7)(8) requires as a first step the generation of definitive endoderm cells (9,10). This step is rate limiting as the differentiation efficiency into a certain cell type of the aforementioned organs decreases significantly with each step from pluripotency towards somatic lineage selection.…”

“…This step is rate limiting as the differentiation efficiency into a certain cell type of the aforementioned organs decreases significantly with each step from pluripotency towards somatic lineage selection. The protocol described here uses a defined number of feeder-free cultured hPSC dispersed into single cells rather than colonies as starting material (9). Dispersed single cells can be differentiated efficiently into the definitive endoderm by a combined treatment with the small-molecule inhibitor of the GSK3, named CHIR-99021, activating the canonical Wnt-signaling pathway, and activin A, activating the Nodal/TGF-β pathway (9).…”

“…The protocol described here uses a defined number of feeder-free cultured hPSC dispersed into single cells rather than colonies as starting material (9). Dispersed single cells can be differentiated efficiently into the definitive endoderm by a combined treatment with the small-molecule inhibitor of the GSK3, named CHIR-99021, activating the canonical Wnt-signaling pathway, and activin A, activating the Nodal/TGF-β pathway (9). The protocol is independent from PI3K inhibition, which has been used in a number of other studies (8,11,12), thereby allowing a robust proliferation and good viability during differentiation (9).…”