High-content analysis (HCA) sits at the intersection of modern cell biology, high-resolution microscopy, advanced image processing, and analysis software. Constantly evolving cellular- imaging techniques and the ability to interrogate single cells can produce a wealth of information. Questions that could not be asked can now be answered.
Pharmaceutical and biotech industries have adopted HCA for target identification and validation, drug characterization, and predictive toxicity. HCA has surpassed high-throughput screening in measuring multiple biological pathways simultaneously or revealing off-target drug effects.
CHI’s upcoming “High Content Analysis” conference will cover the entire HCA gamut, from toxicity assessment and drug screening, to image analysis and data management, to pathway analysis and RNAi.
Stem cells hold great potential for regenerative medicine because of their ability to differentiate into all the cells of the body. However, the degree of pluripotency can vary and “a stem cell is not a stem cell is not a stem cell” says Paul Sammak, Ph.D., member of the McGowan Institute for Regenerative Medicine, The University of Pittsburgh. Dr. Sammak studies the paracrine and epigenetic control of trophectoderm differentiation in the early stages of embryonic development.
“By measuring lineage-specific transcription factor expression levels in single cells we identified culture conditions for the quantitative conversion from pluripotent to trophectoderm committed cells,” explains Dr. Sammak. “Treatment of the cells with a histone deacetylase inhibitor slowed differentiation in a dose-dependent manner. We believe that epigenetic regulators play a role in early blastocyst viability.
“A rapid, selective protocol for trophectoderm induction will allow us to perform further mechanistic studies on the role of environmental factors on human trophectoderm and blastocyst development,” he adds.
Dr. Sammak will present the results of a collaboration with Rami Mangoubi at the C. S. Draper Laboratories, which will demonstrate a live-cell quantitative method for following differentiation and changes in colony morphology using texture analysis.
“The combination of phenotypic measurements and HCA screening will allow us to better characterize stem cells for drug discovery or cell therapy.
“A major bottleneck in the clinical use of stem cells for regenerative medicine is an understanding of the precise differentiation conditions required for individual lines to make early cell-fate decisions,” notes Emanuel Nazareth, graduate student in the laboratory of Peter Zandstra, Institute of Biomaterials and Biomedical Engineering at the University of Toronto.
Cell distribution plays a major role in determining stem-cell fate, however, this distribution is difficult to measure or control. To address this, the group has developed a bioengineered cell-culture system that allows the control of colony size, spacing, and cell density.
Using high-content analysis they compared the response of four different stem-cell lines to six different growth factor cocktails chosen to induce rapid differentiation into either neurectoderm, primitive streak/mesendoderm, or extraembryonic lineages on two different substrates.
“By monitoring the single-cell protein expression of Oct4 and Sox2 we were able to discover and optimize conditions that resulted in the rapid induction of these cell fates,” explains Nazareth. Future work will not only refine the characterization of the effect of these ligands on cell-fate decisions in step-wise differentiation protocols but will also study the effects of various agonists and antagonists on these pathways.