Human embryonic stem (ES) cells have the potential to form any cell type, but ironically, the first cell lineage to form during development still represents a surprising challenge. The first cell type to become specialized is an epithelial cell that later defines the boundary between the embryo and mother for the formation of the placenta. The placenta is the key organ that permits the blood of the mother to provide oxygen and nutrients to the fetus. It is composed of multiple cell types that are specialized for different functions but most of the fetal contributions are derived from the trophoblast cell lineage. Nearly 3% of human pregnancies are threatened by deficiencies of the function of the placenta to provide sufficient blood flow. This condition can result in dangerous increases in the mother’s blood pressure that threaten the health of both the mother and fetus. Studying the molecular details of the formation and function of the different cell types of the placenta is fundamentally medically important and biologically profound as placental development is a key process that helps defines the human species.
To fully utilize the potential of ES cells, we will start with an understanding of the homogeneity and possible bias in the differentiation fate of available human embryonic stem cell lines. We will characterize multiple human ES cell lines with regard to the types of proteins that form the internal cytoskeleton of the cells. These intermediate filament proteins are widely used for identifying cells of specific tissues. One of these may be characteristic of cells that generate the trophoblast lineage. We will confirm this by comparison with the simultaneous expression of a key determinant of trophoblast cells. To facilitate this analysis we will generate a human ES cell line with a colored marker protein when it changes to the trophoblast lineage. This line will permit the detection and purification of cells choosing this fate.
In mice a trophoblast stem cell has been isolated that is capable of self renewal and retains the capability of from different cell types of the placenta but not the embryo. In mice these cells have been experimentally generated by forced temporary expression of genes capable of triggering the specification of this lineage. However, this has not yet been successful with human ES cells. We propose to isolate trophoblast stem like cells by forcing the expression of genes in human embryonic stem cells that may be expressed at insufficient levels to trigger and maintain the trophoblast stem cell and by inhibiting the late stage differentiation of the same cells. The routine isolation of trophoblast cells from human ES cells will a valuable tool for identifying targets for modulation of placenta formation and function.
This research will contribute to the application of stem cell biology to the health of persons in California by providing additional screening criteria for different hESC lines. Until recently, the routine growth of hESC from single cells was challenging. The consequences of this are that differentiated cells, or cells selected for the ability to grow under non optimal conditions may accumulate in the populations of continuously cultivated hESC. Routine purification and expansion from single cells is standard microbiological practice for virus, bacteria and cell culture. Screens that can be applied to hESC lines to better characterize their homogeneity and monitor their quality are needed, particularly as new hESC lines are isolated and applications to patient use become imminent. The first part of this proposal will contribute to the quality control of hESC lines and provide tools for the identification of differentiated cells.
The second part of the proposal will attempt to generate a unique research resource for studying the formation and function of the multiple cell types that compose the placenta. Understanding the cellular and molecular basis of “poor placentation” has not received attention proportional to the frequency of occurrence during pregnancy because of the paucity of amenable experimental systems, the current keen competition for research support and the bias toward immediate disease application. The development of extraembryonic tissues, has historically not been the focus of most developmental biologists. Medical research has concentrated on tissues and cells from term patients. The routine isolation of multipotent trophoblast cells from hESCs would provide a new avenue of research that could identify new methods or strategies of improved maternal and fetal health.
SYNOPSIS: This project is focused on deriving trophoblast stem (TS) cells from hESC. A key feature of the trophoblast lineage is the expression of various intermediate filament (IF) gene products (e.g., keratins) and the PI has noted that hES cells already express some of these genes, in contrast to mESC, which do not express them until differentiation. The PI will use information from the mouse system to test the ability of defined regulatory pathways to stimulate enrichment of TS cells. In Aim 1, immunohistochemistry (IHC) will be used to describe the relative expression levels in a variety of hESC lines for IF genes (K7, K8, K9, K18, various others). Since some of these genes (K8, K18, K19) are repressed during neural progenitor cell development, the PI will also characterize the chromatin structure of the promoters by ChIP analysis of acetylated histones, comparing ES and NP cells derived from them. This should confirm active chromatin domains in the hESC lines. In Aim 2, a Cdx2:GFP gene will be transduced by lentivirus to generate reporter ESC lines that will later assist in monitoring trophoblast differentiation under a variety of conditions.
SIGNIFICANCE AND INNOVATION: The studies have relevance to the syndrome of preeclampsia, which occurs in hypoxic placenta and may often be due to poor development or maturation of trophoblast derivatives. The project is innovative at moving what is known in the mouse system into the hESC system, but the approaches are fairly well established. The observation from the preliminary data that at least one of these cytoskeletons seems to be expressed in a subset of cells could be significant. Thus, the proposed studies under Specific Aim 1 to look at the consistency of expression across different lines has the potential to yield important results regarding clonality and consistency. The second area of focus under Specific Aim 1 is to do a detailed analysis of the factors that regulate the expression of the proposed cytoskeleton proteins. The identification of negative regulatory proteins expressed in the neuronal progenitor cells could provide an important means to modulate the differentiation pathways. These are important questions to address. Regarding Specific Aim 2, assuming they can indeed differentiate the human ESC cultures into trophoblast, this area of investigation seems highly innovative and of (potentially) great significance towards addressing an issue that is of significant medical concern.
STRENGHTS: The PI is an expert in the biology of trophoblast cells and in particular with IF gene function and regulation. It is a major strength that he is already working with the hES cells and generating relevant preliminary data. The study is for the most part well focused, feasible, and appropriate in scope. The comparison of different ESC lines, while descriptive, may be enlightening and provide novel insight into functional capabilities. The environment is also excellent.
The investigators have supplied preliminary data to support the work -- preliminary studies on cytoskeleton expression by IHC show that for K7 there is heterogeneous expression among the culture. These data strongly support the proposed studies in Aim 1. Likewise, for Aim 2, the investigators provide preliminary data to show that the proposed approach of using reporter constructs that are Ets sensitive do work; they also have a number of other reagents already prepared for the various gene expression work they plan to perform. The investigator appears to have performed analogous studies looking at the role of Ets2 in other cell culture systems (mostly mammary tumors), and therefore, is knowledgeable in the pathway, allowing him to use well-defined strategies for modulating gene expression in this pathway, and to then look at the consequences on the differentiative state of the ESC cultures or its derivatives.
WEAKNESSES: The author’s work on the trophoblast stem cells is based on a published report that human ES cells can be driven to differentiate into trophoblasts with BMP4, but this is not their work and based on the preliminary data shown they have not repeated this work. Yet, they assume that they will have no problem seeing the same effect. Since all of the work in Aim 2 is based on their ability to drive differentiation into TS cells, this is the most risky thing about their proposal. What if they can't repeat the published study, either due to subtle differences in ES lines, or other unexplained/uncontrolled factors? It would be useful to comment on how the studies will proceed once the protocols for generating TS are optimized, and to see a consideration of benchmarks or definition of “optimized”.
Regarding the attempts to modulate the pathway, the investigators do not discuss any studies that might distinguish between whether the down-modulation of these particular cytoskeletons in neural progenitor cells is cause or effect. Also, it is unclear (i.e., reviewers could not find evidence) whether the expression array for negative regulatory proteins is in existence, or has been used previously (described under #2 for Research and Design Methods).
In general, the experiments analyzing promoter “repression state” in neural progenitor cells is a bit off target and may detract from productivity relevant to TS. While it will confirm the active state of the TS promoters, it is not clear why chromatin structure is an important issue, as long as the keratin genes are expressed.
DISCUSSION: This is a well-written proposal, and the applicant has thoughtfully considered what is known about trophoblast stem cell differentiation in the mouse and believes that expression in hESC of some of the genes characteristic of the trophoblast lineage that are not expressed in mESC may make hESC more amenable to differentiation along a trophoblast lineage. This is still very much a pilot project, and is a major risk in that its whole premise is based on a published report from another lab. There is no preliminary data to support the work with the applicant's conditions and cell line. Reviewers had trouble fully understanding the scientific aims. Aim 2 is descriptive, and the nature/availability of the arrays to be used is not described. Also, what is the negative control? As proposed, the repression state experiments are a tremendous amount of work and a distraction from the main project. One suggestion is to use the down-regulation of these cytoskeletal proteins in NPCs to address the question of whether the affect is direct or indirect.