Full clinical potential of human ES (hES) cell therapy can be achieved when one can grow hES cells effectively while maintaining full pluripotency. We have focused on developing stem cell culture media by which we can maintain pluripotency of human ES (hES) cells. It is critical to determine and develop a chemically defined media that are animal product-free and feeder cell-free conditions so that the media can be standardized throughout stem cell research and in clinical situations.
One major recombinant protein component we will use in developing chemically-defined media is a set of TGF-beta signaling ligands, receptor domains, and ligand-specific antagonists. We have established a new method of generating a diverse array of these ligands, including BMPs, Activins, inhibin, and their heteromeric ligands of the BMP/Activin class ligands. Some of these heteromeric ligands possess their signaling properties unlike their homodimeric counterparts. These reagents include Noggin, BMP2, BMP3, BMP6, GDF6, BMP2/6 heterodimer, and their derivatives. These reagents have been engineered by chimeric recombination. They were also further modified by site-specific mutagenesis, and by combinatorial heterodimeric assembly to create and modify protein-specific binding affinity to their binding counterparts. Several of these reagents are now available as recombinant protein in sufficient quantity for large-scale screening for media composition.
To establish the functional characteristics and optimal culture combinations using these new reagents, we have used an established hES cell, H9. We have cultured H9 cells in various compositions of culture media containing some of the engineered reagent and followed expression of several differentiation markers to monitor for pluripotency of hES cells, and also for their differentiation-guiding and pluripotency-maintaining abilities. We have first examined effect of aforementioned reagents: Noggin, BMP2, BMP3, BMP6, GDF6, BMP2/6 heterodimer, BMP3 S28A mutant, in our standard culture media mTeSR condition, which does contain bFGF, for proliferation and differentiation of hES cells. In these assays, hES cell line H9 was cultured and reagents were added at varying concentration (1-100 ng per ml) over 1-5 days culture period. Reagents were added in new media during the course of cell culture. We have used morphological change and the presence of markers as a means to follow the differentiation. Ectoderm markers are Nestin, Cdx2; Mesoderm by Brachyury, HBZ; Endoderm markers by CXCR4, Sox17, Gata4, HBF4 alpha, Gata6, AFP. Two BMPs had pronounced effects in inducing cells to endoderm. We have followed up by analyzing the efficiency using FACS. Up to 60% of cells have undergone to endoderm-marked cells. With the availability of a cell sorter, we evaluated pluripotency by means of proliferation rate, morphology, fluorescent signal in the reporter lines by visual inspection and FACS, then we further characterized the factors by real-time PCR for stem cell markers and karyotyping.
It is known that high concentration of FGF can suppress the action of BMPs, so we planned to repeat the experiments in mTeSR media with lowered levels of FGF to re-evaluate the effects of BMPs on cell differentiation abilities. After these tests were completed, we established a protocol performing these assays in high-throughput manner. We are currently in the process of writing this work for publication (Valera et al., in preparation).
Towards the development of chemically defined culture media to maintain pluripotency, we have then tested various newly-engineered reagent to replace a protein component in TeSR media. We have established a combination of protein factors known to maintain established hES cells without using nonhuman products except human albumin, which include basic fibroblast growth factors (bFGF), and a bone morphogenetic protein derivative known as AB2008. We have termed this new media as CAV media. We are currently in the process of writing this work for publication (Valera et al., in preparation).