Work from our group points to the interesting conclusion that differentiation during the early stages of human embryonic development may occur before any morphological specializations are evident. Our first clue that this might be happening came from a series of high definition, high magnification microscopic analyses in which we examined in detail the cells of five-day-old human embryos. The results were surprising. Specifically, the cells in the interior of the embryo that go on to form the entire body do not have an identical appearance. Instead, they seem to have unique specializations. For example the size of the nucleus that contains a cell’s DNA is variable as is its extent of DNA condensation, which is an estimate of its activity.
These findings led us to suspect that cells of the early embryo might be unequal in their developmental potential, the primary theory that our project explores. During the current funding period, we made a great deal of progress toward testing this hypothesis. Our focus was on a set of 10 human embryonic stem cell lines that we derived from single cells of five sibling 8-cell human embryos. In two cases, multiple cells from the same embryo produced lines. Therefore, this collection consists of genetically identical or very genetically similar members. In one series of experiments, we profiled the genes that these lines express and compared them across the set. The results of this analysis suggested that some of the cells were at different stages of development when they were removed from the embryo for the purpose of line derivation. Interestingly, the differentially expressed genes included master regulators of development and important structural components that are linked to a cell’s identity. Our goal in the coming year is to determine if we can find early evidence of differentiation in human embryos, which would suggest that the changes that we are observing in cells that have been maintained in the laboratory are actually happening during normal early development.
We were also interested in determining if our lines, which were derived from single cells that were removed from very early-stage human embryos, function differently than the majority of human embryonic stem cell lines that were produce from later-stage embryos. To address this question, we studied the expression of molecules that are associated with the first differentiation process that establishes the fate of early embryonic cells as either contributing to the body or the placenta. The latter transient organ connects the offspring to the mother and supports its development before birth. Initial experiments showed that the lines differed in their expression of molecules that are associated with establishing a placental fate. These included human chorionic gonadotropin, which is assayed to determine if a woman is pregnant. This finding suggested that some of the lines are more able to contribute to placental development as compared to formation of the offspring.
To further investigate this possibility, we cultured lines that were derived from individual cells removed from a single embryo under conditions that stimulate placental development. We found that these human embryonic stem cells began to express markers that are normally associated with the first steps that establish placental identity. As differentiation continued, the cells began to express other placental factors that substantiated our theory that these lines were able to form both embryonic and placental structures. Thus, we think that deriving human embryonic stem cell lines from single cells that are removed during the very early stages of embryonic development yields lines that have an expanded developmental potential as compared to lines that are derived by conventional means from later-stage embryos.
We think that our findings have interesting implications. At a basic science level, the suites of genes that the new lines differentially express as compared to other human embryonic stem cell lines could give us important clues about the factors that are active during the crucial initial stages of human development. We are very interested in testing the function of these molecules, which we theorize play important roles. Our data also add new information with regard to differences among the developmental potential of existing human embryonic stem cell lines. For example, they can be multi-potent with a relatively limited potential, pluripotent with the ability to differentiate into all the cells of the body, or totipotent with the ability to form both the placenta and the offspring. Our results suggest that human embryonic stem cell lines derived from very early stage embryos are closer to a totipotent state, which could make them more amenable to subsequent differentiation into many cell types, a theory we will be explore during the coming year.