Temporal analysis of neural differentiation using quantitative proteomics

The ability to derive neural progenitors, differentiated neurons and glial cells from human embryonic stem cells (hESCs) with high efficiency holds promise for a number of clinical applications. However, investigating the temporal events is crucial for defining the underlying mechanisms that drive this process of differentiation along different lineages. We carried out quantitative proteomic profiling using a multiplexed approach capable of analyzing eight different samples simultaneously to monitor the temporal dynamics of protein abundance as human embryonic stem cells differentiate into motor neurons or astrocytes. With this approach, a catalog of ∼1200 proteins along with their relative quantitative expression patterns was generated. The differential expression of the large majority of these proteins has not previously been reported or studied in the context of neural differentiation. As expected, two of the widely used markers of pluripotency, alkaline phosphatase (ALPL) and LIN28, were found to be downregulated during differentiation, while S-100 and tenascin C were upregulated in astrocytes. Neurofilament 3 protein, doublecortin and CAM kinase-like 1 and nestin proteins were upregulated during motor neuron differentiation. We identified a number of proteins whose expression was largely confined to specific cell types, embryonic stem cells, embryoid bodies and differentiating motor neurons. For example, glycogen phosphorylase (PYGL) and fatty acid binding protein 5 (FABP5) were enriched in ESCs, while beta spectrin (SPTBN5) was highly expressed in embryoid bodies. Karyopherin, heat shock 27 kDa protein 1 and cellular retinoic acid binding protein 2 (CRABP2) were upregulated in differentiating motor neurons but were downregulated in mature motor neurons. We validated some of the novel markers of the differentiation process using immunoblotting and immunocytochemical labeling. To our knowledge, this is the first large-scale temporal proteomic profiling of human stem cell differentiation into neural cell types highlighting proteins with limited or undefined roles in neural fate.