Screening the mammalian extracellular proteome for regulators of embryonic human stem cell pluripotency - PubMed (original) (raw)
. 2010 Feb 23;107(8):3552-7.
doi: 10.1073/pnas.0914019107. Epub 2010 Feb 2.
Lori L Jennings, Mark Knuth, Anthony P Orth, Heath E Klock, Weija Ou, Julie Feuerhelm, Mitchell V Hull, Eric Koesema, Yuping Wang, Jia Zhang, Chunlei Wu, Charles Y Cho, Andrew I Su, Serge Batalov, Hong Chen, Kristen Johnson, Bryan Laffitte, Deborah G Nguyen, Evan Y Snyder, Peter G Schultz, Jennifer L Harris, Scott A Lesley
Affiliations
- PMID: 20133595
- PMCID: PMC2840467
- DOI: 10.1073/pnas.0914019107
Screening the mammalian extracellular proteome for regulators of embryonic human stem cell pluripotency
Rodolfo Gonzalez et al. Proc Natl Acad Sci U S A. 2010.
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 genome-wide 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 long-term pluripotent growth of human embryonic stem cells without bFGF or TGFbeta/Activin/Nodal ligand supplementation. Our results further indicate that activation of the pigment epithelium-derived factor receptor-Erk1/2 signaling pathway by the pigment epithelium-derived 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.
Conflict of interest statement
The authors declare no conflict of interest.
Figures
Fig. 1.
Flowchart of target selection through protein production. An initial bioinformatic evaluation identified 4,642 predicted extracellular targets. Filtering for biomedical relevance reduced this set to 1,206, 529 of which were selected for production based on template availability. A total of 1,174 clones were processed, yielding a final protein collection of 806 purified proteins (69%), which met threshold criteria.
Fig. 2.
Human ESC self-renewal assay. (A) Percentage of OCT4 positive hESCs after treatment with 806 different proteins. (B–M) Representative images showing hESC-H9 cell morphology and OCT4 and NANOG expression after growing hESC-H9 cells in either bFGF, TEMD1, EBI3, or PEDF for five passages in UM medium. Values are the mean ± SD for three measurements. (Scale bar, 100 μm.)
Fig. 3.
Analysis of hESC cell pluripotency after long-term culture (17 passages) with PEDF. (A–C) H9 cells cultured in vitro were induced to form embryoid bodies, and the derived cells were stained with different differentiation markers: endoderm (AFP and Sox17), mesoderm (SMA), or ectoderm (MAP2). (Scale bar, 100 μm). (D and E) H&E images of teratomas that formed in SCID mice after injection of 1 × 104 hESCs (H9 or H1 cells) that were grown with PEDF for 17 passages. (Scale bar, 50 μm.)
Fig. 4.
Expression and effects of PEDF receptor knockdown in hESCs. (A and B) Undifferentiated hESC-H9 cells grown with UM medium plus PEDF (100 ng/mL) were fixed and stained for the PEDF receptor. (C) Western blot analysis of PEDFR protein expression 72 hours after hESC-H9 cells grown in UM medium plus 100 ng/mL PEDF were treated with control or with PEDFR shRNA lenti-particles A or B. (D–I) Analysis of OCT4 expression 72 hours hESC-H9 and H1 cells were treated with control or PEDFR shRNA lenti-particles A or B. Values are the mean ± SD for three measurements. (Scale bar, 100 μm.)
Fig. 5.
Activation of the ERK1/2 signaling pathway by PEDF supports hESC self-renewal and pluripotency. (A) hES-H9 cells were serum starved for 16 hours and then treated with PEDF (100 ng/mL) or PEDF (100 ng/mL) plus ERK1/2 inhibitor PD98059 (10 μM) for 15 minutes. Cytosolic extracts (30 μg) were subjected to immunoblotting with anti-phospho ERK Ab (pERK; Top) or with anti-general ERK Ab (ERK; Bottom). (B) Analysis of stem cell–associated protein expression 7 days after hESCs were treated with PEDF (100 ng/mL) or PEDF (100 ng/mL) plus PD98059 (10 μM) in UM medium. (C–H) Immunofluorescence analysis of OCT4 expression in hES-H1 and H9 cells treated with PEDF (100 ng/mL), PEDF (100 ng/mL) plus DMSO, or PEDF (100 ng/mL) plus PD98059 (10 μM) in UM medium for 7 days. Values are the mean ± SD for three measurements. (Scale bar, 100 μm.)
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