ABSTRACT
REST (RE1 silencing transcription factor), also known as NRSF (neuron-restrictive silencer factor), is a well-known transcriptional repressor of neural genes in non-neural tissues and stem cells. Dysregulation of REST activity is thought to play a role in diverse diseases including epilepsy, cancer, Down's syndrome and Huntington's disease. The role of REST/NRSF in control of human embryonic stem cell (hESC) fate has never been examined. To evaluate the role of REST in hESCs we developed an inducible REST knockdown system and examined both growth and differentiation over short and long term culture. Interestingly, we have found that altering REST levels in multiple hESC lines does not result in loss of self-renewal but instead leads to increased survival. During differentiation, REST knockdown resulted in increased MAPK/ERK and WNT signaling and increased expression of mesendoderm differentiation markers. Therefore we have uncovered a new role for REST in regulation of growth and early differentiation decisions in human embryonic stem cells.
Subject(s)
Cell Differentiation , Gene Knockdown Techniques , Human Embryonic Stem Cells/metabolism , MAP Kinase Signaling System , Repressor Proteins/metabolism , Cell Line , Cell Survival , Humans , Repressor Proteins/geneticsABSTRACT
Supramolecular nanosubstrate-mediated delivery (SNSMD) leverages the power of molecular self-assembly and a nanostructured substrate platform for the low toxicity, highly efficient co-delivery of biological factors encapsulated in a nanovector. Human fibroblasts are successfully reprogrammed into induced pluripotent stems and transdifferentiated into induced neuronal-like cells.
Subject(s)
Cell Transdifferentiation , Cellular Reprogramming Techniques/methods , Cellular Reprogramming , Cell Line , Cell Transdifferentiation/genetics , Cellular Reprogramming/genetics , Fibroblasts/cytology , Fibroblasts/metabolism , Gene Transfer Techniques , Genetic Vectors , Humans , Nanocapsules , Nanotechnology , Polymorphism, Single NucleotideABSTRACT
High-content screening (HCS) permits simultaneous observation and analysis of multiple cellular variables including cell morphology, survival, and differentiation in live cells at the single-cell level, at the level of the culture well, and across the entire culture. By combining high-throughput technologies such as robotics, chemical libraries, and automated high-resolution microscopy, scientists are able to evaluate a much broader array of experimental conditions than can be studied using conventional cell biological techniques that study fewer parameters at any one time. Thus, HCS assays provide a means to vastly improve our basic understanding of stem cell biology. We have developed a HCS assay that allows the study of the effects of hundreds of small molecules in parallel. The protocol described in this chapter was developed to assess the effects of small molecules on the survival, proliferation, and expression of pluripotent markers following single-cell dissociation of human embryonic stem cells, but can be applied to the study of other types of stem cells including induced pluripotent stem cells. A detailed protocol for the setup of HCS assays and the parameters used to identify chemical modifiers of survival in human pluripotent stem cells, as well as secondary assays used to validate the small-molecule "hits" obtained during the high-content screen, are described.
