Year 5 NCE

This is our final report. Our overall goal was to gain insights into the first few steps in human development, which begins with one cell and ends with formation of the entire body. This is a very complicated process that is difficult to understand if we only study the terminal steps. We theorized that by starting from the beginning, very close to the one-cell stage, we could reconstruct the molecular foundation on which the body plan is built. We think that this information is crucial for devising regenerative medicine therapies. We must be able to program human stem cells that are in the early stages of the developmental continuum into differentiated progeny that can integrate into fully formed organs and tissues for repair purposes. If we understand, at a molecular level, the processes that originally gave rise to the specialized cell types, then we can reproduce them in the laboratory, generating cells for transplantation into patients.

We accomplished all the goals that were set forth in our original application. We proposed a systematic analysis of the early steps in human development. First, we used high-powered microscopy techniques to conduct a detailed examination of human embryos. We let them develop for 6 days in the laboratory. At this point the round embryo, which is about the size of a pinhead, consists of 2 types of cells. One type, which is found at the surface, is fated to form the placenta, which physically connects the offspring to the mother and regulates the transfer of substances to the offspring. The other type, a small cluster of cells at one end of the interior of the embryo, goes on to form the entire body. When we examined the components of this cluster at very high magnification we saw that the individual cells looked quite different from one another and some had mature characteristic. This was a surprise because similar examinations of embryos at the same stage from other species have shown that these clusters are comprised of cells that all look alike with quite primitive structures, suggesting that they are functionally very similar. Thus, we concluded that the initial stages of human embryonic development may be on a faster track than observed in animal models.

How could we test this theory? Since we are working in the human species, there are a limited number of approaches available to us and we had to think of new ways to approach this question. Thus, we decided to take advantage of the fact that, working with colleagues, we helped to develop methods for deriving hESC lines from single cells of early-stage human embryos that were grown for only a few days in the laboratory. This method has many advantages as compared to the conventional approaches that have been used to establish nearly all the lines that scientists work with. For example, we know the precise cell that generated the line and its age (in days) at the time of derivation. This information is difficult to obtain when hESCs are derived from intact 6-day-old human embryos that we now think might be comprised of many kinds of cells. By making one change to this method, we made it very efficient. Specifically, we took into account the fact that the cells in the interior of the embryo reside in clusters. Therefore, when we cultured single cells that were removed from embryos we tried to recreate this environment by sandwiching them in molecules by which they are usually surrounded.

Using this method, we extracted 8-12 cells from each of 5 embryos that had been grown in the laboratory for 3 days. Ten of these cells gave rise to hESC lines. One embryo produced 4 lines (UCSFB1-4), a second embryo gave 3 lines (UCSFB5-7), and 3 embryos gave one line each (UCSFB8, UCSFB9 and UCSFB10). The 5 embryos were donated by one couple. Thus, the lines were either genetically related as are siblings or identical, i.e., triplets and quadruplets. The lines were registered with CIRM and are pending approval by NIH.

What did we learn from this collection of cells? First, we compared their gene expression patterns, a bar code consisting of about 25,000 elements that is unique to every cell type. We found that these cells had bar codes that were different from hESC lines that were derived by conventional methods. Very interestingly, each cell line had a different bar code even if they were established from cells that were removed from the same embryo. This suggested that the lines might have retained differences that were present from the beginning. To test this theory, we looked for the same differences in cell clusters of early-stage human embryos and found them. This is additional evidence in support of our fast track theory of human embryonic development and gives us insights into the molecules involved, i.e., the differentially expressed portions of the bar code. We think that this information will be very useful for guiding the production of specific cell types that will be used in regenerative medicine therapies.