Year 1

A number of promising hESC derived cell products are or will be moving into pre-clinical studies over the next few years. In anticipation of this, we have been focusing on cell culture optimization in order to address the imminent requirement for cGMP-compliant scale-up manufacture of hESC parental cell banks. Current standard laboratory cell culture practice for hESC involves such components such as mouse fibroblast feeder layers, serum and poorly defined media and reagents that are not suitable for manufacture of precuts for human clinical use. In addition most laboratory work with hESC is performed at very small scales, and often processes effective at laboratory scale do not perform well when scaled up to support pre-clinical and clinical studies. Our goal has been to develop cell culture adaptation processes to allow for production of hESC cell banks in support of ongoing and imminent CIRM-funded pre-clinical and clinical projects throughout California. We have specifically focused on establishing adherent culture conditions that eliminate all animal derived factors (mouse feeder layers, serum) and poorly defined reagents (e.g. many of the available hESC growth media). In addition, scale up in adherent cell culture is limited by the ability to manipulate many, or large, cell culture flasks and plates. In order to achieve large scale banking capability, we have focused on the development suspension cell culture. However, hESC may be prone to genetic and epigenetic instability during long term culture or from stress induced by passage in defined animal-free conditions. To address this, we are developing sophisticated epigenetic monitoring technologies that will allow us to detect shifts in expression patterns before these changes are detectable with current technology. These techniques will allow us to monitor the stability of cell cultures during culture adaption and maintenance, but may also allow us to assess the purity, and thereby safety, of differentiated cell products derived from our banks.

The specific aims of this project include: 1: Adapt three hESC lines to feeder layer-free, serum-free suspension culture, 2: Establish and optimize propagation protocols for suspension culture adapted hESC lines and generate cGLP banks, and 3: Establish hESC profiling panel including epigenetic and genetic fingerprinting and correlate with pluripotency. We have made significant progress toward achieving each of these objectives. We have adapted multiple lines to feeder-free and xeno-free conditions and have successfully scaled up these cultures to produce development cell banks of up to 500 vials. Viability and growth rate are readily maintained at high levels and pluripotency is confirmed using a panel of QT-PCR and flow cytometric analysis assays developed in our program. In addition, we have demonstrated the feasibility of maintaining and expanding these lines in suspension culture by achieving over one million fold expansion of each line while maintaining adherent culture levels of growth rate, viability and pluripotency. Significant progress has also been made in developing epigenetic analysis of the hES cells maintained under different culture conditions and at various differentiation states. The preliminary result indicates that epigenetic profiles may be helpful in predicting the state of differentiation of the hES cells. Progress made in each area is summarized below under each specific aims.

Adaptation to feeder free and xeno-free culture conditions was readily achieved using commercially available, but well defined, reagents and media. While ROCK inhibitors, increasingly reported as beneficial in hESC cell culture, appear to provide increased survival of cells during passage and cryogenics, we have found them unnecessary during routine propagation of our adapted cultures. In contrast, we explored numerous conditions before successfully establishing a suspension culture condition that provided cell growth, viability and maintenance of pluripotency comparable to adherent cultures. Numerous growth factors, protectants, inhibitors and other factors were tested. In addition, we tested, and optimized, a variety of parameters before defining passage frequency, culture media and factors, enzyme choice and other factors that influence aggregate size and culture vitality. To achieve sustained viability and meaningful cell growth rates, and to maintain pluripotency, various additives were tested using a common culture medium as a starting point. Two approaches were tested to adapt hES cells in suspension. One was to grow cultures as single cells, or as small loose clusters, and the other approach was to maintain the cells as aggregates of defined size. Efforts to establish single cell suspension conditions were performed at both COH and in collaboration with our collaborators. Numerous conditions were evaluated but none proved effective. Efforts focused on suspension adaptation o