Quantitative Analysis of Human Pluripotency and Neural Specification by In-Depth (Phospho)Proteomic Profiling

• Published in: Stem cell reports Vol. 7; no. 3; pp. 527 - 542
• Main Authors: Singec, Ilyas, Crain, Andrew M., Hou, Junjie, Tobe, Brian T.D., Talantova, Maria, Winquist, Alicia A., Doctor, Kutbuddin S., Choy, Jennifer, Huang, Xiayu, La Monaca, Esther, Horn, David M., Wolf, Dieter A., Lipton, Stuart A., Gutierrez, Gustavo J., Brill, Laurence M., Snyder, Evan Y.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 13.09.2016; Elsevier
• Subjects: Animals; Cell Differentiation - genetics; Cell Lineage - genetics; Cells, Cultured; Computational Biology - methods; Gene Expression Profiling; Gene Knockdown Techniques; Humans; Neurons - cytology; Neurons - metabolism; Phosphoproteins - metabolism; Pluripotent Stem Cells - cytology; Pluripotent Stem Cells - metabolism; Proteome; Proteomics - methods; Resource; Signal Transduction; Transcriptome
• ISSN: 2213-6711; 2213-6711
• DOI: 10.1016/j.stemcr.2016.07.019

Controlled differentiation of human embryonic stem cells (hESCs) can be utilized for precise analysis of cell type identities during early development. We established a highly efficient neural induction strategy and an improved analytical platform, and determined proteomic and phosphoproteomic profiles of hESCs and their specified multipotent neural stem cell derivatives (hNSCs). This quantitative dataset (nearly 13,000 proteins and 60,000 phosphorylation sites) provides unique molecular insights into pluripotency and neural lineage entry. Systems-level comparative analysis of proteins (e.g., transcription factors, epigenetic regulators, kinase families), phosphorylation sites, and numerous biological pathways allowed the identification of distinct signatures in pluripotent and multipotent cells. Furthermore, as predicted by the dataset, we functionally validated an autocrine/paracrine mechanism by demonstrating that the secreted protein midkine is a regulator of neural specification. This resource is freely available to the scientific community, including a searchable website, PluriProt. •Controlled neural induction produces pure cultures of PAX6+ neural stem cells•Most comprehensive (phospho)proteome mapping in pluripotent and multipotent cells•Prediction and validation of midkine as regulator of neural lineage commitment•Searchable and publicly available website presenting (phospho)proteomic dataset Snyder, Brill, Singec, and colleagues demonstrate detailed analysis of human pluripotency and controlled neural lineage entry by using quantitative label-free (phospho)proteomics. The accuracy of the large dataset (13,000 proteins; 60,000 non-redundant phosphorylation sites) allows precise characterization and comparison of pluripotent and multipotent “stemness.” Functional follow-up experiments validate that the understudied protein midkine controls neuralization of hESCs.