Human embryonic stem cells (hESCs) exhibit apparently unlimited capacity for self-renewal and can be differentiated into cells of all 3 germ layers. In addition to providing an essentially unlimited stem cell supply for basic research, hESCs could potentially be used for generating cells and tissues for therapeutic applications, screens of drug and gene effects, toxicology studies and mass production of biomaterials.

Combinations of culture conditions, growth factors and small molecules are being optimized to promote the maintenance or the differentiation of hESCs toward specific fates. Thorough investigation of the potential interactions between factors used in these protocols requires multivariate experiments to which labour-intensive conventional hESC cultures are poorly suited. To facilitate the development of hESC high-throughput applications, we have derived a CA1 subline that exhibits high plating efficiency when dissociated as single cells. The adapted subline (CA1T) remained karyotypically normal and capable of differentiation toward the 3 germ layers in teratomas. The highly increased tolerance to single-cell dissociation of CA1T cells makes them particularly well suited for high-throughput screening applications. We have observed that an early drop in the proliferation rate of hESCs was a leading indicator of pluripotency maker loss, making cell proliferation assays a simple means to screen for early differentiation.

Using a microwell hESC proliferation assay, we have investigated the effects of common cell culture variables such as pH, optimized media component concentrations, as well as defined the toxic levels for different types of laser radiation and fluorescent dyes used for cell tracking. In addition, we have analyzed multiple protein candidates secreted by feeder cells to identify factors responsible for hESC maintenance. This simple screening platform exploits the rapid rate of expansion of undifferentiated hESCs and a nucleic acid quantification assay applicable to small test cultures. Within a few days our assay system allows the discrimination of subtle changes in media composition, and unlike more elaborate immunostaining-based detection methods requires only 2 pipeting steps and is amenable to monitoring with a fluorescence scanner or a plate reader.

The proliferative index of CA1T cells has provided a reliable surrogate readout for quantifying early changes in CA1 cell behaviour following exposure to differentiation stimuli. This CA1T-based assay system should therefore greatly facilitate future screening of hESCs for their responses to a wide range of conditions and thereby aid in the development of new reagents and knowledge of the pathways that control the biological properties of hESCs.

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