For the production and bio-banking of differentiated derivatives from human pluripotent stem cells (hPSCs) in large quantities for drug screening and cellular therapies, well-defined and robust procedures for differentiation and cryopreservation are required. Definitive endoderm (DE) gives rise to respiratory and digestive epithelium, as well as thyroid, thymus, liver, and pancreas. Here, we present a scalable, universal process for the generation of DE from human-induced pluripotent stem cells (hiPSCs) and embryonic stem cells (hESCs). Optimal control during the differentiation process was attained in chemically-defined and xeno-free suspension culture, and high flexibility of the workflow was achieved by the introduction of an efficient cryopreservation step at the end of DE differentiation. DE aggregates were capable of differentiating into hepatic-like, pancreatic, intestinal, and lung progenitor cells. Scale-up of the differentiation process using stirred-tank bioreactors enabled production of large quantities of DE aggregates. This process provides a useful advance for versatile applications of DE lineages, in particular for cell therapies and drug screening.

In vitro differentiation of human pluripotent stem cells (hPSCs) into definitive endoderm (DE) represents a key step towards somatic cells of lung, liver and pancreas. For future clinical applications, mass production of differentiated cells at chemically defined conditions and free of xenogeneic substances is envisioned. In this study we adapted our previously published two-dimensional (2D) DE induction protocol to three-dimensional (3D) static suspension culture in the absence of the xenogeneic extracellular matrix Matrigel. Next, fetal calf serum and bovine serum albumin present in the standard medium were replaced by a custom-made and xeno-free B-27. This yielded in a chemically defined and xenogeneic-free 3D culture protocol for differentiation of hPSCs into DE at efficiencies similar to standard 2D conditions. This novel protocol successfully worked with different hPSC lines including hESCs and hiPSCs maintained in two different stem cell media prior to differentiation. DE cells obtained by our novel BSA-free 3D protocol could be further differentiated into PDX1- or NKX6.1-expressing pancreatic progenitor cells. Notably, upon DE differentiation, we also identified a CXCR4⁺/NCAM⁺/EpCAM^low cell population with reduced DE marker gene expression. These CXCR4⁺/NCAM⁺/EpCAM^low cells emerge as a result of Wnt/beta-catenin hyperactivation via elevated CHIR-99021 concentrations and likely represent misspecified DE.

Growth factors are important regulators during organ development. For many vertebrates (but not humans) it is known how they contribute to the formation and expansion of PDX1-positive cells during pancreas organogenesis. Here, the effects of the fibroblast growth factors FGF2, FGF7, FGF10, and epidermal growth factor (EGF) on pancreas development in humans were assessed by using human pluripotent stem cells (hPSCs). During this, FGF2 was identified as a potent anti-pancreatic factor whereas FGF7, FGF10, and EGF increased the cell mass while retaining PDX1-positivity. FGF2 increased the expression of the anti-pancreatic factor sonic hedgehog (SHH) while suppressing PDX1 in a dose-dependent manner. Differentiating cells secreted SHH to the medium and we interrogated the cells’ secretome during differentiation to globally examine the composition of secreted signaling factors. Members of the TGF-beta-, Wnt-, and FGF-pathways were detected. FGF17 showed a suppressive anti-pancreatic effect comparable to FGF2. By inhibition of specific branches of FGF-receptor signaling, we allocated the SHH-induction by FGF2 to MEK/ERK-signaling and the anti-pancreatic effect of FGF2 to the receptor variant FGFR1c or 3c. Altogether, we report findings on the paracrine activity of differentiating hPSCs during generation of pancreatic progenitors. These observations suggest a different role for FGF2 in humans compared to animal models of pancreas organogenesis.

During the past decade, RNA‐guided Cas9 nuclease from microbial clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) has become a powerful tool for gene editing of human pluripotent stem cells (PSCs). Using paired CRISPR/Cas9 nickases (CRISPR/Cas9n) it is furthermore possible to reduce off‐target effects that may typically occur with traditional CRISPR/Cas9 systems while maintaining high on‐target efficiencies. With this technology and a well‐designed homology‐directed repair vector (HDR), we are now able to integrate transgenes into specific gene loci of PSCs in an allele conserving way. In this protocol we describe CRISPR/Cas9n design and homology directed repair vector design, transfection of human pluripotent stem cells and selection and expansion of generated cell clones. © 2020 The Authors.Basic Protocol 1: Repair template design and CRISPR/Cas9n constructionBasic Protocol 2: Transfection of human pluripotent stem cells by electroporationBasic Protocol 3: Genotyping of generated cell clones