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Quantitative analysis of human pluripotency and neural specification by in-depth (phospho)proteomic profiling

Posted on August 2nd, 2018 by claire.malley@nih.gov
Images
Figure 1. Controlled Neural Induction and Schematic Diagram of the (Phospho)Proteomic Workflow to Compare Human Pluripotency and Neural Multipotency
Figure 2. Comparative Analysis of (Phospho)Proteomes of Pluripotent hESCs and Multipotent hNSCs
Figure 3. S/T Kinases and Their Predicted Substrate Motifs
Figure 4. Mapping TGF-β and WNT pathways in hESCs and hNSCs

Ilyas Singeç1, Andrew M. Crain1, Junjie Hou2, Brian T.D. Tobe1, Maria Talantova1, Alicia A. Winquist1, Kutbuddin S. Doctor3, Jennifer Choy1, Xiayu Huang3, Esther La Monaca4, David M. Horn5, Dieter A. Wolf2, Stuart A. Lipton1, Gustavo J. Gutierrez1, Laurence M. Brill2, Evan Y. Snyder1

  1. Center for Stem Cells and Regenerative Medicine, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA 92037, USA
  2. Proteomics Facility, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA 92037, USA
  3. Informatics and Data Management, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA 92037, USA
  4. Department of Biology, Vrije Universiteit Brussel, 1050 Brussels, Belgium
  5. Thermo Fisher Scientific Inc., San Jose, CA 95134, USA

Highlights

  • 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

Abstract

Controlled differentiation of human embryonic stem cell (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 proteomicand phosphoproteomic profiles of hESCs and their specified multipotent neural stem cellderivatives (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.