Differences in definitive endoderm induction approaches using growth factors and small molecules

Abstract

Definitive endoderm (DE) is the first stage of human pluripotent stem cell (hPSC) differentiation into hepatocyte-like cells. Developing human liver cell models for pharmaceutical applications is highly demanding. Due to the vast number of existing protocols to generate DE cells from hPSCs, we aimed to compare the specificity and efficiency of selected published differentiation conditions. We differentiated two hPSC lines (induced PSC and embryonic stem cell) to DE cells on Matrigel matrix using growth factors (Activin A and Wnt-3a) and small molecules (sodium butyrate and IDE 1) in different combinations. By studying dynamic changes during 6 days in cell morphology and the expression of markers for pluripotency, DE, and other germ layer lineages, we found that Activin A is essential for DE differentiation, while Wnt-3a and sodium butyrate are dispensable. Although sodium butyrate exerted rapid DE differentiation kinetics, it caused massive cell death and could not generate sufficient cells for further differentiation and applications. We further discover that IDE 1 could not induce DE as reported previously. Hereby, we compared different conditions for DE induction and found an effective six day-protocol to obtain DE cells for the further differentiation and applications.

Analysis of differentially expressed genes among human hair follicle–derived iPSCs, induced hepatocyte-like cells, and primary hepatocytes

“…Quantitative analysis indicated that using B27 could effectively induce the differentiation of hiPSCs into endoderm cells [ 22 ]. A few studies suggested that activin A could be used in the process of forming endoderm rows [ 23 , 24 ]. Hay et al [ 25 ] used activin A combined with Wnt3a to induce liver endoderm cells of relatively consistent form and could play the function of hepatocytes more efficiently than other cytokine combinations.…”

Robust parameter design of human induced pluripotent stem cell differentiation protocols defines lineage-specific induction of anterior-posterior gut tube endodermal cells

“…Upon selecting these reagents and their dose ranges, the routine immature hepatocyte (IH) induction protocol in our laboratory 8,29 and the studies assessing the reagent efficacy in dose-dependent manner were taken into considered to enlarge the experimental space (Table S3). [30][31][32][33][34][35][36][37] By utilizing the L 18 array, the scale of the experiments is considered to be 1/243 that of the traditional brute-force approach since only 18 experimental conditions are necessary for the entire screening instead of the 4374 (= 2 1 × 3 7 ) conditions that would otherwise be required for all combinations of chosen cell signaling modifiers and their doses.…”

“…[30][31][32][33][34][35][36][37] By utilizing the L 18 array, the scale of the experiments is considered to be 1/243 that of the traditional brute-force approach since only 18 experimental conditions are necessary for the entire screening…”

Single-cell qPCR demonstrates that Repsox treatment changes cell fate from endoderm to neuroectoderm and disrupts epithelial-mesenchymal transition

“…By systematically optimizing the differentiation protocol, Loh et al were able to differentiate hPSCs into > 98% pure SOX17-expressing DE cells within 48 h [2, 3]. In vertebrate embryos and during hPSC differentiation, activation of TGF-β/activin/nodal signaling by activin A is imperative for DE specification [4].…”