BACKGROUND AND OBJECTIVES: Use of somatic cells as a feeder layer to maintain the embryonic stem cells (ESCs) in undifferentiated state limits the stem cell research design, since experimental data may result from a combined ESCs and feeder cell response to various stimuli. Therefore, present study was designed to evaluate the developmental competence of the buffalo ESCs over different homogenous feeders and compare with various extracellular matrices using different concentrations of LIF. METHODS AND RESULTS: Inner cell masses (ICMs) of in vitro hatched blastocysts were cultured onto homologous feeders viz. fetal fibroblast, granulosa and oviductal cell feeder layers and synthetic matrices viz. fibronectin, collagen type I and matrigel in culture medium. Developmental efficiency was found higher for ESCs cultured on fetal fibroblast and granulosa layers (83.33%) followed by fibronectin (77.78%) at 30 ng LIF. Oviductal feeder was found to be the least efficient feeder showing only 11.11% undifferentiated primary ESC colonies at 30 ng LIF. However, neither feeder layer nor synthetic matrix could support the development of primary colonies at 10 ng LIF. Expression of SSEA- 4, TRA-1-60 and Oct-4 were found positive in ESC colonies from all the feeders and synthetic matrices with 20 ng and 30 ng LIF. CONCLUSIONS: Fetal fibroblast and granulosa cell while, amongst synthetic matrices, fibronectin were found to be equally efficient to support the growth and maintenance of ESCs pluripotency with 30 ng LIF. This well-defined culture conditions may provide an animal model for culturing human embryonic stem cells in the xeno-free or feeder-free conditions for future clinical applications.
BACKGROUND AND OBJECTIVES: Use of somatic cells as a feeder layer to maintain the embryonic stem cells (ESCs) in undifferentiated state limits the stem cell research design, since experimental data may result from a combined ESCs and feeder cell response to various stimuli. Therefore, present study was designed to evaluate the developmental competence of the buffalo ESCs over different homogenous feeders and compare with various extracellular matrices using different concentrations of LIF. METHODS AND RESULTS: Inner cell masses (ICMs) of in vitro hatched blastocysts were cultured onto homologous feeders viz. fetal fibroblast, granulosa and oviductal cell feeder layers and synthetic matrices viz. fibronectin, collagen type I and matrigel in culture medium. Developmental efficiency was found higher for ESCs cultured on fetal fibroblast and granulosa layers (83.33%) followed by fibronectin (77.78%) at 30 ng LIF. Oviductal feeder was found to be the least efficient feeder showing only 11.11% undifferentiated primary ESC colonies at 30 ng LIF. However, neither feeder layer nor synthetic matrix could support the development of primary colonies at 10 ng LIF. Expression of SSEA- 4, TRA-1-60 and Oct-4 were found positive in ESC colonies from all the feeders and synthetic matrices with 20 ng and 30 ng LIF. CONCLUSIONS: Fetal fibroblast and granulosa cell while, amongst synthetic matrices, fibronectin were found to be equally efficient to support the growth and maintenance of ESCs pluripotency with 30 ng LIF. This well-defined culture conditions may provide an animal model for culturing human embryonic stem cells in the xeno-free or feeder-free conditions for future clinical applications.
Authors: J A Thomson; J Itskovitz-Eldor; S S Shapiro; M A Waknitz; J J Swiergiel; V S Marshall; J M Jones Journal: Science Date: 1998-11-06 Impact factor: 47.728