Our goal is to derive human embryonic stem cell (hESC) lines using new and improved methods. During the first two years of this project, we produced eleven new hESC lines. One line was derived from an intact six day-old human embryo that consists of 50-100 cells. Nearly all existing hESCs were derived using this approach. The other ten lines were derived using a new method that we helped to pioneer. This technique entails removing a single cell from an earlier stage human embryo that is comprised of 8 cells. Under the right laboratory conditions the cell makes copies of itself, a process that produces an hESC line. The advantage of this method over the more conventional approach is that we know when the founding cell was removed and where it came from. Additionally, the founder is isolated from the signals of other embryonic cells, which could erase aspects of the stem cell state. Finally, we derived multiple lines from individual embryos so subsets of the lines have the same genetic makeup, which will allow scientists to study the influence of the environment on basic stem cell properties.
During the current grant period, we registered the cells with the California Institute for Regenerative Medicine (CIRM). The process entailed submitting the paperwork that includes proof that the couples who donated embryos to this project gave informed consent. We also described the conditions we used for deriving and propagating these cells. Overall our methods emphasized the use of human materials, which avoids exposure to animal products that could be contaminated with infectious agents. These cells are now available for use by CIRM investigators and other scientists who have nonfederal research funds.
This year the National Institutes of Health (NIH) initiated a parallel process for registering hESCs. The goal was to greatly expand the number of hESC lines that investigators would be allowed to use in experiments that were paid for by federal dollars. To make the cells we derived with CIRM funds more widely available, we attempted to register our cells with the NIH, which required submitting paperwork that was very similar to the documentation CIRM required. The line derived by conventional methods was quickly approved.
However, the lines that were derived from earlier stage embryos have not yet advanced through the approval process. There were two problems. The first was that the Federal government defined an hESC line as coming from an embryo that consists of 50-100 cells. Our lines that came from 8-cell human embryos did not meet this criterion. Accordingly, the Government notified the public of the intent to change this definition. A comment period, which has now ended, ensued. However, the lines were still not approved. It then became apparent that a lawsuit contesting the use of federal funds for hESC research had been reinstated. Thus, ten of our lines are still listed as pending on the Federal Registry and we (and other investigators) cannot apply for (or use) Federal dollars to study them.
Our scientific progress included establishing a bank of the hESCs that we derived. The purpose of a bank is to stockpile cells at a particular stage for future use. The need for banks acknowledges that the basic properties of hESCs can change as a function of the amount of time they are propagated in the laboratory. To bank our cells, we expanded them to the point where we were able to store approximately a thousand vials that contained a million cells each. All were frozen after they had self-replicated only twelve times, which reduces the chance that untoward changes occurred. Thus, we are now in a position to distribute our cells to other investigators who want to use them in their work. Importantly, the size of the bank ensures that we and other investigators can return to this source to get additional vials of the same cells that had been cultured for the same length of time.
In additional experiments, we compared the properties of the ten lines that were derived from early embryos with conventional hESCs. In one series of experiments, we took a global approach in which we analyzed the fundamental properties of the cells. The results yielded data that suggested that the new lines had unique properties. We also found that lines that were derived from different embryos were more similar than lines that came from the same embryo, possible evidence that differentiation begins much earlier during the beginning stages of human development than was previously thought.
Finally, we asked whether the fundamental differences we observed between our new lines and conventional cells were mirrored at a functional level. These experiments are still in progress, but we have interesting evidence that supports this possibility. For instance, we found lines that came from the same 8-cell stage human embryo differed in their ability to make the kinds of neurons that are envisioned as cell-based therapies for Parkinson’s Disease.