Screening the mammalian extracellular proteome for regulators of embryonic human stem cell pluripotency

Abstract

Approximately 3,500 mammalian genes are predicted to be secreted or single-pass transmembrane proteins. The function of the majority of these genes is still unknown, and a number of the encoded proteins might find use as new therapeutic agents themselves or as targets for small molecule or antibody drug development. To analyze the physiological activities of the extracellular proteome, we developed a large-scale, high-throughput protein expression, purification, and screening platform. For this study, the complete human extracellular proteome was analyzed and prioritized based on genomewide disease association studies to select 529 initial target genes. These genes were cloned into three expression vectors as native sequences and as N-terminal and C-terminal Fc fusions to create an initial collection of 806 purified secreted proteins. To determine its utility, this library was screened in an OCT4-based cellular assay to identify regulators of human embryonic stem-cell self-renewal. We found that the pigment epithelium-derived factor can promote longterm pluripotent growth of human embryonic stem cells without bFGF or TGFβ/Activin/Nodal ligand supplementation. Our results further indicate that activation of the pigment epithelium-derived factor receptor-Erk1/2 signaling pathway by the pigment epitheliumderived factor is sufficient to maintain the self-renewal of pluripotent human embryonic stem cells. These experiments illustrate the potential for discovering novel biological functions by directly screening protein diversity in cell-based phenotypic or reporter assays.secreted proteins | human embryonic stem cells | pigment epitheliumderived factor | automated protein expression | high throughput M any physiological processes are regulated by secreted proteins, making the extracellular proteome a rich source of molecules for understanding basic pathways involved in normal physiological function, development, and human disease. Although many examples of bioactive secreted proteins have been identified, recent technologic advances allow us to directly screen this proteomic diversity for new factors influencing important biological pathways. Efforts to survey the extracellular proteome using conditioned media from transiently transfected cell lines have shown that novel factors can be identified in a prospective screening approach. Lin et al. (1) evaluated ∼3,400 genes encompassing predicted secreted proteins or extracellular domains of single-pass transmembrane proteins. Each gene of interest was expressed by transient transfection using liposomal reagents in 293 T cells, and relative quantitation was determined by ELISA using an affinity tag. Conditioned media from this set was used to screen for biological activity in various assays. However, such screens are complicated by secondary metabolites, the release of cytoplasmic proteins by cell lysis, and variable and undefined levels of extracellular proteins. To overcome these challenges, we have created a collection of purified and physically characterized extracel...

Identification of molecules derived from human fibroblast feeder cells that support the proliferation of human embryonic stem cells

“…Previous studies have identified PEDF in a mouse and human fibroblast cell-conditioned medium, suggesting that this protein could also be a potential regulator of hESC. Finally, in a more recent study, it was firmly established that PEDF supports the self-renewal of pluripotent hESC, promoting long-term growth of pluripotent hESC in vitro without further bFGF or TGFβ/Activin/Nodal ligand supplementation [16].…”

Translational Profiles of Medullary Myofibroblasts during Kidney Fibrosis

“…The SerpinF1 gene produces the pigment epithelium-derived factor (PEDF), a secreted multifunctional protein that regulates stem cell fate. 28,29 In kidneys, PEDF serves as a marker for diabetic nephropathy, and exogenous PEDF ameliorates glomerular disease. 30,31 Thy1, a heavily glycosylated cell surface protein, regulates cell-cell and cell-matrix interactions, is expressed in certain fibroblast and mesenchymal cell populations, and plays roles in fibrosis.…”

Pigment Epithelium-derived Factor (PEDF) Prevents Retinal Cell Death via PEDF Receptor (PEDF-R)

“…The dependence of PEDF on PEDF-R has been reported for other biological activities, emphasizing the essential role of the PEDF⅐PEDF-R interaction. For example, human embryonic stem cells deficient in the PNPLA2 gene lacked the PEDF self-renewal activity (52); PNPLA2 knockdown in human melanoma cells resulted in the loss of the ability of PEDF to promote the mesenchymal-like phenotype that prevents metastasis (53); PEDF-mediated activation of PPAR␥ and repression of IL-8 required PEDF-R in human prostate cancer cells (54), and stimulation of PEDF-R triglyceride lipase activity in mouse muscle and liver is dependent on interactions with PEDF (55). We propose that the PEDF⅐PEDF-R interaction is critical for PEDF-mediated retina survival activity (Fig.…”