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.