Optimization of Human Embryonic Stem Cell Derivation Techniques and Production/Distribution of GMP-Grade Lines
The government has strict rules for producing cells that will be transplanted into patients. For example, these regulations discourage the use of animal products that could transmit diseases to humans. In this context, the high-quality and tightly regulated procedures that govern other cell-based therapies, e.g., bone marrow transplants, will be applied to regenerative-type clinical applications that employ human embryonic stem cells (hESCs). We need to produce these cells now so that they will be ready to use when research findings are translated into patient therapies. Our goal is to supply researchers in California and outside the state with the highest-quality hESCs. To achieve this goal, we will build on our previous work, published in the scientific literature, which includes deriving hESCs from intact embryos and their single-cell components. We also study the basic properties of embryos and hESCs so that we can formulate theories about how to improve the derivation process, which we then test in our laboratory. For example, adult humans need very precise levels of oxygen. Our work shows that the same is true for embryos and hESCs. We have also developed novel culture conditions that use defined components such as those that are required by the governmental agencies that set the standards for the production of cells used in therapeutic applications. Accordingly, we propose a two-phase approach. During the first two years, guided by advances made by our group and others, we will derive hESCs from embryos in a biologically relevant oxygen environment using defined, high-quality materials. We will also derive hESCs from single cells removed by biopsy from embryos at specific stages of development and/or from particular regions. We think that these lines might have more predictable properties than hESCs that arise randomly, the current practice. Thus, at the end of the first phase we will have produced and banked the next generation of lines, which will be derived under defined conditions that more closely comply with government regulations regarding the production of clinical-grade cells. In the second phase, year 3 of the project, we will use the conditions that best support hESC derivation/propagation to produce lines that can be transplanted into patients. Our efforts will benefit greatly from the infrastructure of our institution, which includes a government-approved facility for doing this work, and from colleagues with the requisite specialized expertise. With the resources provided, we think that we can generate and bank 12 to 20 cell lines; one-third will be produced in a manner that complies with government regulations pertaining to cell-based therapies. All will be widely distributed, as we believe that the pace of translational and clinical research depends on the availability of the highest-quality hESCs and on the important information about their fundamental properties that will emerge from this work.
California citizens were overwhelmingly in favor of proposition 71, due in large part to the public's belief that laboratory scientists working together with their clinical colleagues could develop therapeutic approaches that utilize human embryonic stem cells and their derivatives in regenerative medicine applications. The research teams are equally excited about cell replacement strategies for treating a variety of medical conditions, as in many cases a cure might be feasible. However, we know from the collective experience of the biotechnology industry that filling the pipeline that leads from basic research to clinical applications inevitably takes time. How do we start this process and shorten the timelines for delivering cell-based therapies to patients? Much of the work in the individual pipelines that focus on specific diseases/conditions can happen in a nonlinear fashion. For example, we need not wait until safe and robust strategies are devised for differentiating human embryonic stem cells into specific cell types to develop the lines that meet government regulations regarding the production of cells for transplantation into patients. There are many reasons that deriving these human embryonic stem cell lines now is crucial. For example, it is likely that production of the cells that will eventually be used in clinical applications will be an iterative process. That is, we will continue to make key discoveries about the fundamental properties of human embryonic stem cells, about which basic information is still needed to improve conditions for growing and deriving lines. This is particularly relevant to the production of clinical-grade cells, as it will be much easier to meet Food and Drug Administration requirements if cells are produced using defined materials that contain only human and recombinant components. It will also be important to know if clinical-grade cells, which for regulatory reasons must be derived under streamlined conditions, have the same properties as other human embryonic cell lines that are used for research purposes. Thus, extensive preclinical testing will be required before these cells are approved by the Food and Drug Administration for use in humans. Thus, accomplishing the major goals set forth in this application will be of enormous benefit to California's citizens. We envision that production of the next generation of human embryonic stem cells that can be used in clinical applications will speed the delivery of therapeutic applications to patients. Along the way these cells will have many other valuable applications. For example, they can be used to screen pharmacologically active compounds for both beneficial and detrimental effects. They will also be valuable tools for understanding the molecular etiology of disease processes. Accordingly, accomplishing the goals of this project will greatly benefit the people’s health and California’s economy.
The goal of our work is to derive new human embryonic stem cell lines for distribution to stem cell researchers. During the past year, we produced 11 new lines that we think will be valuable tools for all phases of work toward regenerative medicine therapies including discovery, translational, and eventually, early clinical applications. Along the way, this work is teaching us a great deal about early embryonic development and the specialized processes that distinguish critical steps in humans as compared to animal models.
What is the source of the embryos for our derivations? We use embryos that are left over after the conclusion of fertility treatments such as in vitro fertilization. In all cases, we obtain written informed consent from both individuals whose reproductive material was used to make the embryos. Additionally, researchers in our group have no direct contact with the couple making the donation. This process is handled by the University of California San Francisco Gamete and Embryo Bank. It is important to note that the embryos that we use would otherwise be destroyed, a point that is sometimes overlooked.
What are the methods that we used to derive these new human embryonic stem cell lines? The work that was carried out in the last 12 months employed two techniques. One line was derived using standard methods that have been employed for many years to produce stem cell lines in many species including humans. In general, early-stage human embryos are thawed and grown for a few days in the laboratory until a cluster of tightly packed cells, termed human embryonic stem cell colonies, emerges. Stem cell researchers can spot these special cells based on their appearance under a microscope. The advantage of this technique is that it is a straightforward method for reliably producing human embryonic stem cell lines. The disadvantage is that the progenitors we seek are exposed for extended periods of time to other cell types in the culture, which could impact their genetic makeup and, subsequently, their developmental potential.
Accordingly, we used a different method to derive the other ten embryonic stem cell lines that we produced during this grant period. The overall approach was to grow human embryos donated from a single couple until they contained 8-12 cells. Then we used specialized equipment to remove single cells from five sibling embryos. Each cell was allowed to develop in isolation. Four cells from one embryo gave rise to cell lines as did three cells from another embryo. The remaining three embryos each produced one cell line. This unique set of lines will enable stem cell researchers to understand the role that genetics plays in specifying the basic characteristics of human embryonic stem cells as compared to environmental signals that are inevitably transmitted as the lines are propagated in the laboratory. We are also interested in the concept that single cells that are extracted from very early stage embryos, which have not received signals from other cell types that are found at later stages of development, are more likely to be truly pluripotent as evidenced by their genetic and molecular signatures.
Why do we need additional human embryonic stem cell lines? Many of the most commonly used stem cells have been in culture for many years. This creates several problems. First, the extended length of time that the most commonly studied human embryonic stem cells have been in culture means that they are likely to have accumulated errors that are the result of mistakes made during the self-renewal process. This is all the more likely to occur because we do not yet understand the optimal conditions for growing the cells in the laboratory. Accordingly, ongoing studies in many groups, including our own, are designed to improve the environment for maintaining human embryonic stem cells, which we think will improve the fidelity with which they make carbon copies of themselves. Second, nearly all of the existing lines have been exposed to animal products, which raises the possibility that they could contain disease vectors. For this reason, the Food and Drug Administration makes it difficult (but not impossible) for cell-based therapies that are used in clinical applications to be approved once they have experienced these types of exposures. Therefore, all of our derivation work has employed solely human reagents, which we think will streamline their approval for use in clinical applications.
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.
The goal of our project is to improve methods for generating and banking human embryonic stem cells (hESCs), which are derived from human embryos. Since the federal government does not support work with human embryos, this research would be impossible without the funds we have received from the California Institute for Regenerative Medicine (CIRM). Additionally, our project has benefited greatly from CIRM Facilities Grants, which provided us with this specialized nonfederal laboratories that we need for experiments that employ human embryos.
Our project has three Specific Aims. The first is to derive hESC lines from intact human embryos that have been grown in the laboratory for approximately five days. This is the method that has been used to derive the overwhelming majority of the existing hESCs. In these experiments, cells that are destined to become hESCs emerge over several days. Thus, the precise origin and timing are unknown. Some investigators think that this element of randomness contributes to the significant differences that have been noted among hESCs. To date, we used this approach to derive one line, UCSF-4, which was approved by CIRM. Thus, other grantees of this agency can work with these cells. Since they were generated using standard techniques, UCSF-4 was approved by the National Institutes of Health. Therefore, researchers who have federal funding can also work with these cells.
The second Specific Aim proposed a new approach for deriving hESCs. We wanted to make lines from single cells that were removed from embryos at very precise stages of development. To date we have focused on the eight-cell stage, embryos that have been grown for three days in the laboratory. In these experiments, we used five embryos that were donated by a single couple. In all, we established 10 lines from individual cells. In other words, we derived multiple lines from the same embryo. Thus, this collection is unique because some of the lines are genetically identical and all have a high degree of genetic relatedness. This special property is important to stem cell biologists as we are trying to understand the changes that happened over time as the cells are maintained in the laboratory. Additionally, we have evidence that hESCs that are derived from embryos that have been grown for three rather than five days are more plastic in that they are better able to form the precursors of a broader range of cell types that comprise the human body.
Like UCSF-4, the ten lines that were derived from single cells removed from embryos were registered by CIRM. They were also submitted to the National Institutes of Health along with UCSF-4. However, we were notified that they do not fit the federal definition of an hESC line, which specifies derivation from an embryo that has been grown for five rather than three days in the laboratory. Accordingly, these lines are still pending approval, which will require changing the federal definition. Therefore, it is uncertain if they will ever be registered, a process that allows federally-funded scientists to work with hESC lines.
Our third Specific Aim was to derive a hESC line using Good Manufacturing Processes (GMP) as specified by the Food and Drug Administration. These guidelines have been established to make sure that cell-based therapies are safe for patients. For example, they are designed to prevent the spread of infectious agents that can contaminate animal cells to humans. One obstacle to producing GMP-grade cells is that hESC derivation requires a carpet of another cell type, which is thought to “feed” embryos the substances that are required for generation of hESC colonies. Thus these “feeder” cells must also be produced using GMP methods. To avoid this laborious step, we have developed methods for the “feeder-free” culture of hESCs. We plan to use the same approach to derive hESCs from intact embryos.
To date, with CIRM support, we have derived 11 hESC lines. Therefore, distributing these cells has become a significant part of this project. It is interesting to note that these lines are being used for a wide variety of experiments. For example, UCSF-4 is being studied by a Consortium that has been formed to understand how chemical modifications to genes control their expression. This is a particularly interesting question because the programs that specify the initial stages of human development are very poorly understood.
The other groups who are using our cells are focusing on a wide range of normal and disease processes. Several investigators are studying the mechanisms by which cells of the brain and spinal cord develop. One group hopes to use them as a therapeutic strategy for patients with amyotrophic lateral sclerosis or Lou Gehrig’s disease. Other investigators are studying formation of cardiac muscle, pancreatic beta cells and the liver. Finally, our lines are also being used for training purposes in our CIRM Shared Research and Teaching Facility.
The goal of our work has been to derive new human embryonic stem cells lines from embryos that are donated by couples at the completion of in vitro fertilization cycles. Many laboratories including our own, derived lines using the method that was originally described. Embryos that were grown for 5-6 days in the laboratory were transferred to a lawn of cells that supported the eventual outgrowth of human embryonic stem cells. Using standard methods to perform derivations, we were struck by ways in which we thought this method could be improved. For example, the cells of the embryo that are destined to form the placenta die. Therefore, it seemed likely that the products they release could have untoward influences on the stem cells. In addition, we observed the formation of morphologically distinct cell types before tightly packed “islands” of stem cells finally emerged in their midst. We speculated that these extraneous cells could also have undue effects. Finally, we examined, at very high magnification, embryos that were grown for 5-6 days. Although the cells that give rise to the offspring are supposed to be nearly equivalent at this stage, we saw that they no longer look alike. We took this as possible evidence of specialization, meaning they might already be starting down the road toward becoming one of the cell types of the body. Therefore, we began to consider new approaches for producing human embryonic stem cells. In particular, we wanted to derive lines from single cells of earlier stage embryos that might be less specified and more plastic in terms of their ability to differentiate into progeny that are difficult to obtain from conventional human embryonic stem cell lines.
In preliminary experiments, we worked with other scientists from the biotechnology sector to develop methods for deriving lines from single cells (termed blastomeres) that were removed from embryos that were grown for approximately 3 days in the laboratory. From the work of other scientists we knew that around this time the intrinsic genetic programs of the egg fade and those of the embryo start to unfold. Therefore, it was quite possible that human embryonic stem cell lines derived at this stage would be less specified than their counterparts, which were established by conventional methods. However, our initial work showed that this new approach was not very efficient. Although a few blastomeres formed lines, the majority did not. Therefore, we had to make this a more robust procedure. We took our clues for improving this method from the architecture of the embryo. Specifically, the cells are always grouped together and never found in isolation. Thus, to mimic this arrangement, we surrounded each blastomere with cells and proteins to simulate the geometry and molecular environment of the early embryo. The result was a much more efficient derivation procedure. Accordingly, we had developed the tools to perform the desired experiments.
For the derivation procedure, we used 5 embryos that were donated by a single couple. From single blastomeres removed at the 3-day stage we derived a total of 9 human embryonic stem cell lines. Thus, the lines were either genetically identical or related. First, we had to prove that they had the characteristics that researchers think are common to all human embryonic stem cells. Specifically, they expressed a panel of markers that are associated with this state. Also, in a laboratory dish, they made descendants of the 3 lineages that give rise to all the cell types in the body. The most rigorous test of the stem cell state is transplantation into a mouse where these cells are able to differentiate further. Like those derived by conventional means, our collection formed many structures such as bone, muscle and glands. Therefore, we concluded that they had the same general properties as human embryonic stem cells that were derived from later stage embryos.
Thus, we turned to the interesting question of whether they had unique properties. We characterized the cells at the genetic, epigenetic and functional level. At the level of genes, we compared the lines that were derived from a single cell of a 3-day old embryo to human embryonic stem cell lines that were derived by conventional means. There were striking differences in the gene expression patterns between the two. Epigenetic marks decorate DNA designating functional from nonfunctional areas in terms of gene expression. The lines that were derived from single cells had many fewer of these modifications suggesting less specialization. Finally, at a functional level, we showed that a subset of the lines we produced could spontaneously form cell types that are found in both the offspring and the placenta. Since these lineages diverge early in development, we took this as evidence of a greater degree of plasticity than is associated with the conventional lines. We think this property could be very useful for devising regenerative medicine therapies.
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