Abstract
Several methods allow reprogramming of differentiated somatic cells to embryonic stem cell-like cells. However, the process of reprogramming remains inefficient and the underlying molecular mechanisms are poorly understood. Here, we report the optimization of somatic cell fusion with embryonic stem cells in order to provide an efficient, quantitative assay to screen for factors that facilitate reprogramming. Following optimization, we achieved a reprogramming efficiency 15–590 fold higher than previous protocols. This allowed observation of cellular events during the reprogramming process. Moreover, we demonstrate that overexpression of the Spalt transcription factor, Sall4, which was previously identified as a regulator of embryonic stem cell pluripotency and early mouse development, can enhance reprogramming. The reprogramming activity of Sall4 is independent of an N-terminal domain implicated in recruiting the nucleosome remodeling and deacetylase corepressor complex, a global transcriptional repressor. These results indicate that improvements in reprogramming assays, including fusion assays, may allow the systematic identification and molecular characterization of enhancers of somatic cell reprogramming.
Highlights
The developmental programs of somatic cells are characterized by remarkably stable patterns of gene expression and repression
Optimization of an Efficient Reprogramming Assay Several different protocols have been developed to reprogram somatic cells via cell fusion with embryonic stem cells (ESCs), with protocol efficiencies typically less than 0.001% [12,20]
Such low efficiencies lead to technical difficulties in screening for positive regulators of somatic cell reprogramming
Summary
The developmental programs of somatic cells are characterized by remarkably stable patterns of gene expression and repression. Nuclear reprogramming was first demonstrated as an integral part of mammalian development; following fusion of the egg and sperm, the fused gametic nucleus must be reprogrammed, through a series of changes that include DNA demethylation and chromatin remodeling, to that of an embryonic cell if development is to be successful [5,6,9] In methods such as somatic cell nuclear transfer (SCNT), the nucleus of a somatic cell is transferred to an enucleated oocyte for reprogramming to an embryonic cell state, through the use of the endogenous machinery [3,10,11]. In all methods, the efficiency of reprogramming is very low, suggesting that additional components of the reprogramming pathways remain to be identified
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