Improvement of Cell Survival During Human Pluripotent Stem Cell Definitive Endoderm Differentiation

Abstract

Definitive endoderm (DE) is a vital precursor for internal organs such as liver and pancreas. Efficient protocol to differentiate human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs) to DE is essential for regenerative medicine and for modeling diseases; yet, poor cell survival during DE differentiation remains unsolved. In this study, our use of B27 supplement in modified differentiation protocols has led to a substantial improvement. We used an SOX17-enhanced green fluorescent protein (eGFP) reporter hESC line to compare and modify established DE differentiation protocols. Both total live cell numbers and the percentages of eGFPpositive cells were used to assess differentiation efficiency. Among tested protocols, three modified protocols with serum-free B27 supplement were developed to generate a high number of DE cells. Massive cell death was avoided during DE differentiation and the percentage of DE cells remained high. When the resulting DE cells were further differentiated toward the pancreatic lineage, the expression of pancreatic-specific markers was significantly increased. Similar high DE differentiation efficiency was observed in H1 hESCs and iPSCs through the modified protocols. In B27 components, bovine serum albumin was found to facilitate DE differentiation and cell survival. Using our modified DE differentiation protocols, satisfactory quantities of quality DE can be produced as primary material for further endoderm lineage differentiation.

Chemically defined and xenogeneic-free differentiation of human pluripotent stem cells into definitive endoderm in 3D culture

“…Serum contains insulin-like growth factor (IGF) which inhibits DE differentiation of hPSCs by elevated PI3K signaling 12 . However, 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 . Thus, to improve cell survival, DE differentiation media commonly contain the xenogeneic supplements fetal calf serum (FCS), bovine serum albumin (BSA) or B-27 TM 16,18–20 .…”

“…However, 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 . Thus, to improve cell survival, DE differentiation media commonly contain the xenogeneic supplements fetal calf serum (FCS), bovine serum albumin (BSA) or B-27 TM 16,18–20 . B-27 TM is a serum-free media supplement composed of vitamins, antioxidants, lipids, steroids and trace elements but it also contains BSA 21 .…”

Human embryonic stem cell dispersion in electrospun PCL fiber scaffolds by coating with laminin‐521 and E‐cadherin‐Fc

“…The differentiation to definitive endoderm was accompanied with substantial cell death as reported by Refs. , yielding a heterogeneous cell population and dispersion at day 10. This protocol would need further optimization for instance by optimizing the medium components; however, this was outside of the scope of this article.…”

Development of Colonic Organoids Containing Enteric Nerves or Blood Vessels from Human Embryonic Stem Cells

“…The development of HCOs was performed in three distinct phrases including gendoderm and hindgut differentiation and colonic organoid development ( Figure 1 ). Endoderm differentiation was performed in reference to the previous study with some modifications [ 22 ]. On day 1, 200,000 human embryonic stem cells (hESCs) were seeded in a 24-well plate and treated with B27 (1:100) (Gibco, Grand Island, NY, USA, 12587-010), Activin A (100 ng/mL, Peprotech, Rock Hill, NJ, USA, 120-14E), and mesoderm differentiation compound CHIR-99021 (3 µM, Sigma-Aldrich, St. Louis, MO, USA, SML1046-25MG), in which B27 is the key component to improve cell survival in definitive endoderm.…”