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Identification and functional analysis of genes regulating ES cell pluripotency Hsu, Lien

Abstract

Embryonic stem cells (ESC) are a pluripotent cell population with the capacity to undergo symmetrical self-renewing divisions, as well as to differentiate into cells of all three embryonic germ layers both in vitro under appropriate culture conditions and in vivo. Recent gene expression profiling studies have begun to identify the key genes defining the mouse ESC phenotype. In addition, comparisons of ESC gene expression profiles with those of differentiated cells, or various types of adult stem cells, have been done in an attempt to identify a set of critical ESC regulatory genes. However, these studies have been limited by a lack of related functional data for the ESC, correspondent to the expression analysis. In order to more accurately identify important regulatory genes a global gene expression profiling study on undifferentiated and early differentiated ESC was carried out at the BC Cancer Research Center (BCCRC) to identify those genes whose loss of expression correlates with loss of stem cell function. Further comparison of the BCCRC R1 ESC dataset with a similar R1 ESC data set generated through the Stem Cell Network (SCN) allowed us to identify candidate genes whose expression either significantly decreased in the first 18 hours following removal of leukemia inhibitory factor (LIF) or which continued decreasing over 72 hours. An additional comparison was carried out to identify over-lapping genes amongst 3 different ESC lines: R1, J1, V6.5 (all from SCN). Eight candidate genes, present in all data sets and fulfilling additional criteria, were identified through these analyses: Bc13, Klf5, Emp 1, Inhbb, Cxcl 1, Cbx2, Ankrd I, and 8430410Al7Riken. Their expression patterns were confirmed using quantitative real time PCR during ESC differentiation. Lenti-viral vectors for over-expression of these candidates were generated and used to obtain ESC populations showing increased expression. Functional studies, including embryoid body (EB) formation and colony forming cell (CFC) assays, as well as assessment of cell survival and proliferation rates, were done. Of note, Gbx2 and Inhbb were shown to partially rescue the differentiation potential of ESC following LIF removal, as well as to alter cell lineage differentiation and EB morphology. In addition, ESC over-expressing Inhbb or Cxcll showed differences in proliferation rate. Ankrd1 influenced the plating efficiency of ESC. This work lays the foundation for further studies into the molecular mechanisms that regulate ESC "sternness". Moreover, these studies may provide clues for the development of effective strategies to direct the differentiation of ESC into functional end cells of a specific type for use in cell replacement and cancer therapies.

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