Transcriptional analysis of early lineage commitment in human embryonic stem cells

Andrew L Laslett,Sam Wormald,Sean Grimmond,David Nikolic-Paterson,Adelia Lin,David Haylock,Brooke Gardiner,Lincon Stamp,Martin F Pera,Susan M Hawes

doi:10.1186/1471-213x-7-12

Abstract

BackgroundThe mechanisms responsible for the maintenance of pluripotency in human embryonic stem cells, and those that drive their commitment into particular differentiation lineages, are poorly understood. In fact, even our knowledge of the phenotype of hESC is limited, because the immunological and molecular criteria presently used to define this phenotype describe the properties of a heterogeneous population of cells.ResultsWe used a novel approach combining immunological and transcriptional analysis (immunotranscriptional profiling) to compare gene expression in hESC populations at very early stages of differentiation. Immunotranscriptional profiling enabled us to identify novel markers of stem cells and their differentiated progeny, as well as novel potential regulators of hESC commitment and differentiation. The data show clearly that genes associated with the pluripotent state are downregulated in a coordinated fashion, and that they are co-expressed with lineage specific transcription factors in a continuum during the early stages of stem cell differentiation.ConclusionThese findings, that show that maintenance of pluripotency and lineage commitment are dynamic, interactive processes in hESC cultures, have important practical implications for propagation and directed differentiation of these cells, and for the interpretation of mechanistic studies of hESC renewal and commitment. Since embryonic stem cells at defined stages of commitment can be isolated in large numbers by immunological means, they provide a powerful model for studying molecular genetics of stem cell commitment in the embryo.

Highlights

The mechanisms responsible for the maintenance of pluripotency in human embryonic stem cells, and those that drive their commitment into particular differentiation lineages, are poorly understood
Antibodies to epitopes expressed on the surface of primate pluripotent stem cells reveal heterogeneity in human embryonic stem cells (hESC) cultures In our investigations we used three monoclonal antibodies recognising canonical markers of hESC
The first was the monoclonal antibody GCTM-2, which recognises an epitope on the protein core of a high molecular weight pericellular matrix keratan sulphate/chondroitin sulphate proteoglycan [28]

Summary

Introduction

The mechanisms responsible for the maintenance of pluripotency in human embryonic stem cells, and those that drive their commitment into particular differentiation lineages, are poorly understood. The differentiation in vitro of hESC into a variety of tissue types of enormous potential significance to research and medicine, including neural tissue, blood, cardiac muscle, and many others, has been reported, and the first studies showing proof of principle of the application of hESC-derived neural cells in preclinical animal models of disease have recently been published [6,7]. While this record is impressive, very significant challenges remain ahead if hESC are going to fulfill their potential. The numerous studies of the hESC transcriptome and proteome, [8,9,10,11,12,13,14,15,16,17,18,19] which generally have compared hESC populations grown under conditions that support renewal to cultures undergoing overt differentiation, have produced a molecular blueprint of the pluripotent state, but this blueprint is limited in its resolution due to the inherent complexity of the cell populations under comparison

Methods

Results

Discussion

Conclusion