Small molecule screening with laser cytometry can be used to identify pro-survival molecules in human embryonic stem cells

• Published in: PloS one Vol. 8; no. 1; p. e54948
• Main Authors: Sherman, Sean P, Pyle, April D
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 29.01.2013; Public Library of Science (PLoS)
• Subjects: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine - analogs & derivatives; 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine - pharmacology; Algorithms; Animals; Apoptosis; Biology; Cell culture; Cell cycle; Cell fate; Cell survival; Cell Survival - drug effects; Chemistry; Cloning; Cytometry; Dissociation; DNA - metabolism; Drug Evaluation, Preclinical - economics; Drug Evaluation, Preclinical - methods; Drug screening; Embryo cells; Embryonic stem cells; Embryonic Stem Cells - cytology; Embryonic Stem Cells - drug effects; Embryonic Stem Cells - metabolism; Embryos; Genetic engineering; Green Fluorescent Proteins - metabolism; High-Throughput Screening Assays; Humans; Kinases; Laser Scanning Cytometry - economics; Laser Scanning Cytometry - methods; Lasers; Medical research; Mice; Molecular biology; Octamer Transcription Factor-3 - metabolism; Pluripotency; Propidium - metabolism; Protein Kinase Inhibitors - pharmacology; Proteins; Regenerative medicine; rho-Associated Kinases - antagonists & inhibitors; Screening; Signaling; Small Molecule Libraries - pharmacology; Spectrometry, Fluorescence; Staining and Labeling; Stem cells; Survival; Therapy; Time Factors; Toxicity; Toxicity testing; Los Angeles California; United States > US; California
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0054948

Differentiated cells from human embryonic stem cells (hESCs) provide an unlimited source of cells for use in regenerative medicine. The recent derivation of human induced pluripotent cells (hiPSCs) provides a potential supply of pluripotent cells that avoid immune rejection and could provide patient-tailored therapy. In addition, the use of pluripotent cells for drug screening could enable routine toxicity testing and evaluation of underlying disease mechanisms. However, prior to establishment of patient specific cells for cell therapy it is important to understand the basic regulation of cell fate decisions in hESCs. One critical issue that hinders the use of these cells is the fact that hESCs survive poorly upon dissociation, which limits genetic manipulation because of poor cloning efficiency of individual hESCs, and hampers production of large-scale culture of hESCs. To address the problems associated with poor growth in culture and our lack of understanding of what regulates hESC signaling, we successfully developed a screening platform that allows for large scale screening for small molecules that regulate survival. In this work we developed the first large scale platform for hESC screening using laser scanning cytometry and were able to validate this platform by identifying the pro-survival molecule HA-1077. These small molecules provide targets for both improving our basic understanding of hESC survival as well as a tool to improve our ability to expand and genetically manipulate hESCs for use in regenerative applications.