OBJECTIVES: To characterize basal differentiation tendencies of a human embryonic stem (hES) cell line, KCL-002. MATERIALS AND METHODS: In vitro specification and differentiation of hES cells were carried out using embryoid body (EB) cultures and tests of pluripotency and in vivo differentiation were performed by teratoma assays in SCID mice. Real-time PCR, immunohistochemistry, flow cytometry and histological analyses were used to identify expression of genes and proteins associated with the ectodermal, endodermal and mesodermal germ layers. RESULTS: Undifferentiated KCL-002 cells expressed characteristic markers of pluripotent stem cells such as Nanog, Sox-2, Oct-4 and TRA 1-60. When differentiated in vitro as EB cultures, expression of pluripotency, endodermal and ectodermal markers decreased rapidly. In contrast, mesodermal and mesenchymal markers such as VEGFR-2, α-actin and vimentin increased during EB differentiation as shown by qPCR, immunostaining and flow cytometric analyses. Teratoma formation in SCID mice demonstrated the potential to form all germ layers in vivo with a greater proportion of the tumours containing mesenchymal derivatives. CONCLUSIONS: The data presented suggest that the KCL-002 hES cell line is pluripotent and harbours a bias in basal differentiation tendencies towards mesodermal and mesenchymal lineage cells. Characterizing innate differentiation propensities of hES cell lines is important for understanding heterogeneity between different cell lines and for further studies aimed at deriving specific lineages from hES cells.
OBJECTIVES: To characterize basal differentiation tendencies of a human embryonic stem (hES) cell line, KCL-002. MATERIALS AND METHODS: In vitro specification and differentiation of hES cells were carried out using embryoid body (EB) cultures and tests of pluripotency and in vivo differentiation were performed by teratoma assays in SCIDmice. Real-time PCR, immunohistochemistry, flow cytometry and histological analyses were used to identify expression of genes and proteins associated with the ectodermal, endodermal and mesodermal germ layers. RESULTS: Undifferentiated KCL-002 cells expressed characteristic markers of pluripotent stem cells such as Nanog, Sox-2, Oct-4 and TRA 1-60. When differentiated in vitro as EB cultures, expression of pluripotency, endodermal and ectodermal markers decreased rapidly. In contrast, mesodermal and mesenchymal markers such as VEGFR-2, α-actin and vimentin increased during EB differentiation as shown by qPCR, immunostaining and flow cytometric analyses. Teratoma formation in SCIDmice demonstrated the potential to form all germ layers in vivo with a greater proportion of the tumours containing mesenchymal derivatives. CONCLUSIONS: The data presented suggest that the KCL-002 hES cell line is pluripotent and harbours a bias in basal differentiation tendencies towards mesodermal and mesenchymal lineage cells. Characterizing innate differentiation propensities of hES cell lines is important for understanding heterogeneity between different cell lines and for further studies aimed at deriving specific lineages from hES cells.
Authors: Kevin A D'Amour; Alan D Agulnick; Susan Eliazer; Olivia G Kelly; Evert Kroon; Emmanuel E Baetge Journal: Nat Biotechnol Date: 2005-10-28 Impact factor: 54.908
Authors: J Itskovitz-Eldor; M Schuldiner; D Karsenti; A Eden; O Yanuka; M Amit; H Soreq; N Benvenisty Journal: Mol Med Date: 2000-02 Impact factor: 6.354
Authors: Justin G Lees; Sue Anne Lim; Tristan Croll; Georgia Williams; Sylvia Lui; Justin Cooper-White; Leon R McQuade; Bagyalakshmi Mathiyalagan; Bernard E Tuch Journal: Regen Med Date: 2007-05 Impact factor: 3.806
Authors: Michael J Abeyta; Amander T Clark; Ryan T Rodriguez; Megan S Bodnar; Renee A Reijo Pera; Meri T Firpo Journal: Hum Mol Genet Date: 2004-01-28 Impact factor: 6.150