Stimulation of Activin A/Nodal signaling is insufficient to induce definitive endoderm formation of cord blood-derived unrestricted somatic stem cells

USSC were cultured for (1) three days with or without 100 ng/ml Activin A in either serum-free, low-serum or serum-containing media, (2) three days with or without 100 ng/ml Activin A in combination with 10 ng/ml FGF4 in pre-induction medium, or (3) four days with or without small molecules Induce Definitive Endoderm (IDE1, 100 nM; IDE2, 200 nM) in serum-free media. Formation of definitive endoderm was assessed using RT-PCR for gene markers of endoderm (Sox17, FOXA2 and TTF1) and lung epithelium (surfactant protein C; SPC) and cystic fibrosis transmembrane conductance regulator; CFTR). The differentiation capacity of Activin A treated USSC was also assessed.Activin A or IDE1/2 induced formation of Sox17+ definitive endoderm from hESC but not from USSC. Activin A treated USSC retained their capacity to form cells of the ectoderm (nerve), mesoderm (bone) and endoderm (lung). Activin A in combination with FGF4 did not induce formation of Sox17+ definitive endoderm from USSC. USSC express both Activin A receptor subunits at the mRNA and protein level, indicating that these cells are capable of binding Activin A.Stimulation of the Nodal signaling pathway with Activin A or IDE1/2 is insufficient to induce definitive endoderm formation from USSC, indicating that USSC differ in their stem cell potential from hESC.Unrestricted somatic stem cells (USSC) are a population of stem cells that can be isolated from umbilical cord blood at birth. USSC have been shown to form the following cell types: nerve (ectoderm); heart, cartilage, bone, fat and blood (mesoderm); and liver and lung (endoderm) [1,2]. Although USSC have been shown to form cells representative of all three germ line layers [1,3,4], it is unclear whether USSC have an identical or a restricted capacity when compared to human embryonic stem cells (hESC).Cord blood-derived USSC are an attractive source of cells for therapy, as unlike hESC, they can be obtained noninvasively at the time of birth, and have been shown not