Abstract
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+/EpCAMlow cell population with reduced DE marker gene expression. These CXCR4+/NCAM+/EpCAMlow cells emerge as a result of Wnt/beta-catenin hyperactivation via elevated CHIR-99021 concentrations and likely represent misspecified DE.
Highlights
Human pluripotent stem cells possess an unlimited proliferative potential and can be differentiated into all somatic cell types
In a different approach efficient definitive endoderm (DE) differentiation was achieved from singularized human pluripotent stem cells (hPSCs) that initially formed spheroids in suspension culture without addition of exogenous extracellular matrices but the differentiation was performed in the presence of xenogeneic BSA24
Results hPSCs can be efficiently differentiated into the DE under 3D culture conditions
Summary
Human pluripotent stem cells (hPSCs) possess an unlimited proliferative potential and can be differentiated into all somatic cell types. PI3K signaling is required for self-renewal of hPSCs and the removal of serum at the beginning of DE differentiation in combination with activin A is associated with massive cell death[13,16,17]. To improve cell survival, DE differentiation media commonly contain the xenogeneic supplements fetal calf serum (FCS), bovine serum albumin (BSA) or B-27TM 16,18–20. In a different approach efficient DE differentiation was achieved from singularized hPSCs that initially formed spheroids in suspension culture without addition of exogenous extracellular matrices but the differentiation was performed in the presence of xenogeneic BSA24. We established static 3D conditions for DE differentiation of hPSCs in the absence of xenogeneic scaffolds and media supplements based on our previously published protocol[4]. These cells could be associated with a decreased expression of important DE marker genes suggesting that DE differentiation protocols should be optimized towards low NCAM-positivity
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