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New approaches to the optimized maintenance of embryonic stem cells in culture

University of British Columbia

The realization of many stem cell-based therapies will rely on the development of methods for the expansion and controlled differentiation of stem cells and the development of assays to rapidly detect the results of culture manipulation. Mouse embryonic stem cells (ESC) provide a relatively abundant and high purity model system to investigate environmental cues that influence stem cell fate. The dynamics of loss of pluripotency following the removal of leukemia inhibitory factor (LIF) were investigated using three functional assays (chimeric mouse formation, embryoid body generation and colony forming ability). A rapid loss (>70%) of pluripotent cells was detected within 24 hours, with very low residual activity by all criteria within 72 hours. Surprisingly, functional endpoints of pluripotency correlated poorly with two commonly used markers, expression of Oct4 and SSEA-1. The embryoid body assay was then used in factorial and central composite design experiments to define optimized levels of ascorbic acid (AA), chondroitin sulphate and PD98059 (PD), three factors that enhance undifferentiated ESC maintenance. These experiments identified an unexpected negative interaction between AA and PD and allowed the development of a cocktail of these three factors that increased ESC yield 3-fold over untreated cultures. To identify more rapid endpoints for the detection of undifferentiated ESC, global gene expression profiling was performed on ESC induced to differentiate by removal of LIF, addition of retinoic acid and addition of DMSO. Gene expression profiles were determined using the Affymetrix platform and a new meta-analysis methodology was developed and applied to the resulting gene expression data sets. This analysis revealed that the expression of the stem cell factor receptor, c-kit, expressed on the cell surface, correlated with the frequency of undifferentiated ESC making it an ideal marker for further studies. Furthermore, the expression changes of seven genes - 103728_at, 8430410A17Rik, klf2, nr0b1, sox2, tcll and zfp42 - was able to predict undifferentiated ESC frequencies in both differentiation and maintenance cultures. These experiments should provide a useful framework for making further improvements in the culture of undifferentiated ESC.

Text

2011-01-26

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http://hdl.handle.net/2429/30875

Genetics

University of British Columbia

Graduate