Date Published: February 9, 2017
Publisher: Public Library of Science
Author(s): Anthony Flamier, Supriya Singh, Theodore P. Rasmussen, Majlinda Lako.
Teratogens are compounds that can induce birth defects upon exposure of the developing fetus. To date, most teratogen studies utilize pregnant rodents to determine compound teratogenicity in vivo. However, this is a low throughput approach that cannot easily meet the need for comprehensive high-volume teratogen assessment, a goal of the US Environmental Protection Agency. In addition, rodent and human development differ substantially, and therefore the use of assays using relevant human cells has utility. For these reasons, interest has recently focused on the use of human embryonic stem cells for teratogen assessment. Here we present a highly standardized and quantitative system for the detection and analysis of teratogens that utilizes well-characterized and purified highly pluripotent stem cells. We have devised strategies to mass-produce thousands of uniformly sized spheroids of human ESCs (hESCs) that can be caused to undergo synchronous differentiation to yield embryoid bodies (EBs) in the presence and absence of suspected teratogens. The system uses all human cells and rigorously controlled and standardized EB culture conditions. Furthermore, the approach has been made quantitative by using high-content imaging approaches. Our system offers distinct advantages over earlier EB systems that rely heavily on the use on mouse ESCs and EB aggregates of stochastic sizes. Together, our results show that thousands of suspected teratogens could be assessed using human EB-based approaches.
Annually, 3 to 5% live births in the United States are impacted by birth defects leading to over 100,000 occurrences of birth defects per year [1, 2]. Teratogens include environmental compounds to which women are exposed before or during pregnancy through food, drinking water, airborne sources, and through dermatological exposure. In addition, many pharmaceuticals have teratogenic activities. The U.S. Environmental Protection Agency has a long-standing mission to identify potentially toxic compounds through the Toxcast program which aims to assess thousands of chemicals of potential concern . However, methods to screen vast collections of compounds for teratogenicity are currently laborious and plagued by relatively low throughput, though recent computational approaches have identified a subset of ToxCast compounds, many with potentially teratogenic effects .
We developed and refined a human EB system that utilizes highly characterized cells of demonstrated pluripotency in such a way that the effects of teratogens can be evaluated and quantitated by numerical descriptors with statistical accuracy. Since teratogens impact the course of directed cellular differentiation during embryogenesis, we strived for utilizing only the very highest quality of cells to initiate EB cultures. To this end, we assessed hESCs rigorously for their pluripotency-associated marker expression. We found that TRA1-60 is a superior surface pluripotency marker as compared to SSEA4. Furthermore, we wished to initiate our EB cultures with spherical aggregates of hESCs, which we found could be mass-produced in aggrewell plates. To do this, we faced a challenge in that suspensions of single cells are required in order to seed aggrewell plates. This is complicated by the fact that cell-cell contacts (mediated by E-Cadherins in hESCs) are required to maintain the pluripotent state of hESCS. We therefore developed a strategy whereby feeder cell-free starter cultures of hESCs are disaggregated to single cells by Accutase treatment. The singularized cells were maintained in the presence of ROCK inhibitor, which can stabilize the maintenance of pluripotency of single hESCs during the disaggregation of hESC colonies to single cells. We also immunologically purified TRA1-60+ cells using magnetic beads and used these to seed aggrewell plates to prepare large numbers of uniform spheroids that could in turn be used to initiate EB cultures. We found that we can prepare large numbers of EBs by this method, which are of initial uniform size, and whose growth and proliferation occur synchronously. We found that by simply monitoring a cohort of EBs treated with teratogens (at doses that are sub-cytotoxic), that we could observe defects in EB growth characteristics that could be quantified precisely. We were able to reproducibly and quantitatively detect effects on EB development using a panel of three reference teratogens. This report builds on previous successes with human EBs, but our system makes use of highly standardized EBs of known pluripotency which are of standardized and uniform size and pluripotency prior to differentiation in the presence of potentially teratogenic compounds.