Addressing the Cell Purity and Identity Bottleneck Through Generation and Expansion of Clonal Human Embryonic Progenitor Cell Lines
Human embryonic stem (hES) cells and induced pluripotent (iPS) cells, such as reprogrammed skin cells, offer the potential to revolutionize medicine because they can replicate indefinitely and become virtually any cell in the body. They therefore have the potential to provide a limitless source of cells to replace cells lost to injury (spinal cord, skin wounds, etc.) or degenerative diseases like diabetes, Alzheimer’s, Parkinson’s, ALS, MS, and heart disease to name a few. Similarly they can be a source of cells that model human disease for improved drug development. As researchers develop new and better ways to make hES and iPS cells they are running into a bottleneck of how to provide enough cells of sufficient purity for clinical applications. Industrial scale production is needed and both hES and iPS cells are difficult and costly to manufacture in large quantities. Moreover, the manufactured cells must pass the scrutiny of the FDA. Purity and identity are essential qualities that are needed for any drug approval and are even more important for cell therapy because unlike a drug which may persist in the body for a matter of hours or days, a cell can persist in the body for a lifetime. Contamination of hES derived cells with the wrong cells could lead to toxicities resulting from normal but inappropriate tissue growth or tumor formation.
We therefore propose to develop a new type of cell, the embryonic progenitor (hEP) cell, from hES and iPS cells that is ideal for cell therapy because of its scalability and purity. The nature of hEP cells lies somewhere in between a hES cell and a fully mature cell like a nerve, heart muscle, or pancreatic cell. Many different specific types of hEP cells can be made for cell replacement of specific kinds of tissues. We have already begun to make over one hundred hEP cell lines. Because they divide using standard cell culture methods hEP cells could be readily grown in industrial scale quantities using standard bioreactors. Indeed, we propose here to optimize and standardize industrial scale up of hEPs lines. Importantly, hEP lines are clonal, meaning that they are derived from a single cell, and therefore have the potential to grow as a pure cell line. We propose here to map the surface markers on hEP lines so that we can identify a molecular signature specific to a given hEP line. The molecular signature will be key to assuring identity and reproducibility in preclinical and clinical studies and will facilitate purification of hEP cells from any hES or iPS line so that they can be easily and cost effectively obtained from cells of various genetic backgrounds. We will use our mapping technology us to identify antibodies and other cell purification reagents. The successful completion of our proposed project will provide well characterized hEP cells that are precursors of therapeutic cells such as nerve, blood vessel, heart muscle, and skin.
Safety is critical to the development of any new drug candidate and is even more essential when considering cellular therapies where cells can persist in the body for years. Thus, the primary benefit of our proposed project of generating well characterized cell lines and markers for their isolation is to provide a means of manufacturing sufficient quantities of cells with the needed purity to provide safer cell therapies. By providing California researchers with a bank of well characterized intermediate precursor cell lines and cell purification reagents we will help overcome the cell purity and identity bottleneck that is currently stalling the successful translation of basic stem cell research to clinical applications. A key beneficial outcome of our project will be to shorten the time it takes to get stem cell therapies from the research laboratory and into the hands of physicians to treat patients suffering from degenerative diseases and injuries. By accelerating the translation of research to drug approval more Californians that are currently in need of treatment will have the opportunity to benefit from stem cell therapies and Californians will see a more rapid return on their investment in the form of reduced health care costs.
Another significant benefit of our proposed project is in the application of pluripotent stem cells for modeling diseases. Our work will provide the cost effective means to purify well characterized precursor cells from various sources of embryonic stem cells including reprogrammed skin cells of different genetic backgrounds and disease states. This will reduce the need for animal models and the cost of disease models for drug discovery.
Finally, in addition to our cell bank and cell purification kits, we will provide Californians with a database of cell surface antigens that define the various intermediate cell types that occur during embryogenesis by their lineage and cell fate. This resource will provide California researchers with information that will help accelerate the pace of stem cell research. Thus, our proposed project will help bring stem cell therapies to Californians sooner by directly addressing the critical issue of cell safety and at the same time it will provide valuable resources for accelerating stem cell based drug discovery and our knowledge of human development.
CIRM Grant TR1-01276
Addressing the Cell Purity and Identity Bottleneck Through Generation and
Expansion of Clonal Human Embryonic Progenitor Cell Lines
Public Abstract: The ability of embryonic stem cells to differentiate into virtually any cell of the human body is both the source of their expansive therapeutic potential, as well as the basis of a substantial technical hurdle: how to produce a pure and homogenous cell therapy product from a cell type whose very nature is to differentiate continuously into complex mixtures of derivative cells? Ideally, a cell therapy product would be homogenous and well characterized but at present, most preparations are contaminated with a wide range of undesired cell types that can lead to inappropriate and potentially dangerous growth within the cellular graft. Our work addresses these bottlenecks through the development of embryonic progenitor (EP) cells. EP cell are derived from embryonic stem cells but have been isolated as highly purified populations and therefore have several advantages over most typical cell therapy preparations. Importantly, hEP lines are derived from a single cell and are therefore clonal; they have the potential to grow long term as a pure cell line and maintain stable biological potential. Because they divide using standard cell culture methods and can be conveniently stored, hEP cells could be readily grown in industrial scale quantities using standard bioreactors. We have developed a collection of over 140 clonal EP lines that each display unique biological potential. The overall aim of this grant is to develop tools and methods to allow for the reproducible derivation of any targeted EP cell line that displays the intended biological characteristics.
In our first year’s work, we performed long-term cultures of several EP cell lines to determine how well they can maintain both biological integrity, as measured by their ability to differentiate into a target cell type, and genomic integrity, as measured by the maintenance of a normal chromosomal count and composition. Our goal is to determine the extent to which EP cell lines can be expanded in standard tissue culture and yet still maintain all of the biological characteristics associated with a useful cell therapy preparation. Our initial analysis demonstrated that after expanding these cells to an extent consistent with commercial manufacturing processes, EP lines can maintain biological capabilities. However, extensive culturing can lead to the loss of biological capabilities and genomic integrity. Our goal is to now define more accurately the interval during which EP cell lines can be routinely expanded.
A second major aim of our work is to develop molecular reagents that will allow us to routinely isolate a desired EP cell line from embryonic or other stem cell populations. This effort takes two approaches. First, we are testing commercially available antibodies to identify those that will selectively bind to the target EP cell line but not to unrelated EP cell lines, nor the stem cell populations used to derive the EP lines. Antibodies provide high affinity and highly selective binding characteristics which make them ideal for use in cell sorting procedures. We surveyed over 200 antibodies to cell surface proteins and identified several promising candidates for use in cell line derivation. In addition, we are attempting to develop additional cell surface binding reagents using a process called phage display. In these efforts, large libraries of independent binding agents (peptides) are screened by exposing the entire library to the cell surface, then collecting those peptides with the highest affinity for the cell. We have identified lead peptide candidates and will now compare these peptides to the commercially available antibodies in tests to measure specificity and affinity.
The third aim of our project is to evaluate the range of biological capacity of our EP cell collection. EP cell lines represent a state of differentiation that is midway between the capacities of embryonic stem cells and fully differentiated adult cells in that EP cells can obtain a variety of cellular fates depending on the means by which the cell culture is manipulated. For example, placing certain EP cell lines in high density culture cause them to become cartilage producing cells, while other manipulations can cause them to develop into smooth muscle. We are developing large-scale screens in which EP cell lines are exposed to a wide range of biological growth factors, culture media and culture conditions to determine unique biological fates that may be available for these lines. We measure the biological fate of these treated lines using high capacity microarrays that provide an indication of cellular differentiation, and then apply bioinformatics techniques to identify unique biology induced by the treatments.
Our translation grant is intended to develop a safe and reliabel alternative to most stem cell based therapies. We have developed a large (>140) collection of purified embryonic progenitor cell lines that collectively, represent a wide swath of the biological diversity seen in human cells. Embryonic progenitor (EP) cell lines are derived from embryonic stem (ES)stem cells and represent cell lines that are mid-way between the pluripotent stem cells and fully differentiated adults cells. Our EP cell lines are clonally purified and thus display many advantages over ES cells in terms of commerical scale preparation and purification.
One of our primary study aims is to develop methods that will allow us to identify and isolate a specific EP cell type. One of the major challenges to accomplish this goal is to develop reagents and methods that can be used to purify a target EP cell line from the parental ES cell population from which it derived. For instance, we have identified several EP cell lines that can efficiently differentiate into cartilage producing cells. By assessing the protein markers that are expressed on the cell surface of these lines, we have developed a discrete collection of targets that can be used to develop affinity reagents(antibodies and peptides) that will allow us to purify these EP cell types from the parental ES cell populations. We have completed pilot experiments that show that antibodies to these cell surface targets can be used to efficiently purify EP cell lines from their ES source and plan in the final year of our studies to demonstrate this process for many additional EP cell types.
A second major aim of our study is to determine the overall biological diversity of our EP cell line collection. We have undertaken a large scale screen of these cell lines place under a broad range of differentiation conditions, including cell culture matrices, biological effectors and chemical inducers of differentiation. In the course of this activity, we have identified EP lines that effectively develop into cartilage, bone, nerves and fat. Further optimization of these differentiation regimens has allowed us to fine tune the process to produce products that can work effectively in animal-based models of human disease, such as cartilage deficit.
And finally, our research also aims to assess the ability to expand EP cell lines using methods and culture systems that are compatible with commercial production methods for cellular therapeutics. We have expanded EP cell lines for 20-30 doublings and have shown good stability of both the genome and biological capabilities of these lines. This, coupled with the clonal purity of the EP lines, can provide significant advantages with regards to the manufacturing of therapeutic products.
Our CIRM-funded project studied a novel method of isolating and expanding diverse cell types from human pluripotent stem cells in order to increase the diversity and purity while decreasing the cost and complexity of making such products. This novel strategy utilized the method of creating expandable lines of cells from a single cell (hence the line is said to be a "clonal" cell line), where that cell was no longer pluripotent, but instead had begun to differentiate into a body cell type. These novel cell lines are called "human clonal embryonic progenitor (hEP) cell lines." Under the grant, we extensively examined these lines to determine their potential for use in human therapeutic applications. One goal of the funded research was to determine whether their were molecules on the surface of the cells that would facilitate the repeated derivation of the lines should they prove valuable in medicine. We demonstrated that indeed, proteins could be identified that specifically recognized the lines thereby aiding in the identification of the lines. We also exposed the lines to diverse signals to explore the differentiation potential of hEP cells. In hundreds of such experiments where the cells were examined by looking at global gene expression, we discovered many novel and medically-important cell types could be made in this manner such as cartilage, bone, tendon, as well as many other cell types. In the case of the lines capable of making cartilage, we have published results showing their ability to effect the repair of damaged cartilage. An important advantage of the hEP cell lines is that they are clonally purified, therefore they represent a relatively homogenous population of cells. This is in comparison to standard ES-derived cell populations that most often comprise a widely heterogenous (mixed) population of cells. As such, we believe hEP lines have distinctive advantages, both in terms of safety (better defined biology without unintended admixture of diverse cell types) and in terms of manufacturing (better ability to provide well defined assays and measurements during the derivation and cell production process).