 Hello, my name is Blair Gage and I'm a Ph.D. candidate in the lab of Dr. Timothy Kiefer here at the University of British Columbia. In this video abstract, I'm going to tell you about a CO1S cell line, which is a human embryonic stem cell line that's adapted for high throughput screening. This work was done in collaboration with Dr. Nicholas Caron in the lab of Dr. Jamie Paray, who's a corresponding author for the paper. Together we developed and characterized the human embryonic stem cell line that has the ability to be seeded from a single cell suspension without loss of pluripotency or induction of spontaneous differentiation. The reason for this work is based on some classical attributes of conventional human embryonic stem cells. Normally, human ES cells like H1 and the CA1 cells used in this study are passage by light enzymatic dissociation and mechanical disruption. This process results in large multicellular aggregates, which allow the cell line to be passage indefinitely. But upon passage, spontaneous differentiation and the induction of apoptosis means that if these cells were seeded into multi-well plates, a non-uniform cell population to start out your screen would result. This is the challenge we were trying to overcome in the paper talked about today. To overcome the classical problems of embryonic stem cells, CA1 human ES cells were treated with a series of enzymatic dissociation processes over the course of about 10 passages. Initial enzymatic dissociation resulted in poor culture viability of the CA1 cells when they were seeded. Over the course of the next subsequent three passages, this viability increased and eventually we were able to seed CA1 cells from single cell dissociated cultures at high viability without a loss of pluripotency and without the induction of spontaneous differentiation. This population was designated at CA1S with the S standing for single cell dissociatable CA1 cells. We next characterized the CA1S cells for the expression of pluripotency markers. We looked at oct4, SSA3, trial 160, and alkaline phosphatase activity and they were all there. As expected, the cells were still pluripotent. Transplantation of CA1S cells in immunocompromised mice resulted in the expected teratomas with well differentiated derivatives of all three embryonic germ layers. Using an in vitro differentiation protocol, we examined the ability of CA1S cells to differentiate in vitro into all three germ layers and we found the expected markers were able to be induced under simple conditions. Given the CA1S cells could follow simple differentiation cascades into all three germ layers, we were next interested to see if they could follow more complex sequential differentiation. To do this, we examined the pancreatic differentiation cascade outlined by Krunadel in 2008 in Nature Biotechnology. CA1S cells followed through expressing definitive endoderm markers, specifically CXCR4, and eventually expressed NKX6.1 and PDX1, suggesting pancreatic progenitors were present and these progenitors could form insulin and glupinome positive endocrine cells. Ultimately, we wanted to see if CA1S cells had the ability to be used in screening applications. The initial foray into this looked at a proliferation index assay to examine whether CA1S cells could decrease their proliferation in response to differentiation cues or altered media compositions. We saw that CA1S cells could change their proliferation in response to pH, rokinase inhibition, as well as increasing amounts of FVS, suggesting they could differentiate on cue. We continued our screening test using a high throughput version of flow cytometry looking for expression of CXCR4. The goal was to see if CA1S cells required both ACTIVAIN-A and WNT3A in order to induce CXCR4 expression. We found that CA1S cells only required ACTIVAIN-A and WNT3A was a minor player of any within the process. This was our primary screen. As a secondary screen, we used the conventional stem cell line H1 and examined a subset of conditions, only three. We found that H1 cells, as predicted by CA1S, were able to express CXCR4 in response to ACTIVAIN-A in the absence of WNT, but WNT3A did have a minor effect and was able to improve CXCR4 expression. This proved that CA1S are able to be a screening model, but importantly, each stem cell line likely has different levels of expression of various signaling molecules and should be treated independently. Taken together, we believe that the complexities of embryonic stem cell biology, as well as their ability to differentiate, will really only be examined effectively and understood using screening models. These screens are going to be onerous. There's a lot of information to actually dig out. And using an embryonic stem cell line like CA1S has the ability to empirically examine many combinations in a more rapid and high throughput manner than is currently achievable with conventional stem cell lines. Secondary screens with a select set of hit variables or specific pathways of interest can be applied to other stem cell lines or all the stem cell lines of interest with the ability to eventually understand what is the global regulators of differentiation. With that, I'd like to thank you very much for your attention and wish you happy reading.