Quantitative time-lapse analysis of pluripotency and trophectoderm differentiation at the single-cell level
Embryonic Stem Cells (ESCs) have the ability to change, or differentiate, into a diverse set of specific cell types (placental cells, neurons, muscle, etc.). Alternatively, they can also grow and divide to make more ESCs (self-renew). These abilities are the basis for their potential usefulness in medical applications. However, a population of ESCs usually does not differentiate into a single cell type. When apparently identical cells are placed together in the same culture dish, they typically generate a mixture of different cell types. This variability can arise from small variations in the local environment of each cell, from signals cells use to communicate with one another, or from intrinsic randomness (“stochasticity”) of the biochemical reactions that cells use to control differentiation. In order to manipulate ESCs it is crucial to understand the origins of this heterogeneity.
Recent work has made it clear that cellular decisions result not from the control of a single gene, but rather from the interaction of many genes and proteins. In the case of ESC development, some of the genes and interactions that control differentiation decisions are now known. However, it is unclear why they act differently in different cells. Traditional techniques average over large cell populations, making it difficult to address this issue. Because of the intrinsic variability in ESC differentiation it is crucial to study it in individual cells.
To this end, we will develop a microscopy-based system to simultaneously follow multiple genes in individual cells. We will create ESCs that contain three differently colored fluorescent protein labels that can be used to track the dynamics of important genes. We will record ‘movies’ of individual cells and their levels of gene expression as they grow and change into other cell types. Using this data in conjunction with mathematical models we will determine how the behavior of the system as a whole varies from cell to cell, how it responds to particular perturbations, and how it can be manipulated. More specifically, we will analyze the genetic system that allows ESCs to avoid differentiation (that is, to remain in the ESC state), as well as the system that allows them to change into placental cells.
In this effort, we will directly compare differentiation in mouse and human ESCs. This will shed light on the differences between the species and take advantage of their different pros and cons: mouse ESCs are easier to engineer, while some human ESCs are able to differentiate into placental cell types without genetic manipulation. The ability to use diverse human ESC lines will be crucial for this.
Together, the research proposed here will explore the fundamental basis for decision-making in individual cells. It will thus directly enable better strategies for controlling stem cell differentiation for therapeutic purposes.
Embryonic stem cells have enormous therapeutic potential for a wide variety of diseases. However, a fundamental problem that hinders their usefulness is that they typically give rise to a mixture of many different types of cells, rather than to the particular cell type that is most useful for a given application. The proposed research will analyze the genetic basis for this variability. It will thus play a pivotal role in facilitating the engineering of embryonic stem cells for therapeutic applications that will be of benefit to citizens of California and others.