Grantee Research Project Results

The goal of this project is to develop and implement a novel approach to assay developmental neurotoxicity of ToxCast chemicals using a human embryonic stem cell (hESC)-based model of neuronal development. For the first step, we proposed to investigate selective cytotoxicity towards either ventral or dorsal neural precursor cells (NPCs). In the proposed strategy, the partial or complete loss of a particular NPC population (e.g., ventral or dorsal NPCs) is a key event that precedes and predicts a specific adverse neurodevelopmental outcome both in vitro and in vivo. The objectives of this project are to:

 

Objective 1: Develop an HTS platform based on hESC-derived NPC to identify ToxCast phase I chemicals that selectively affect ventral or dorsal NPCs.

Objective 2: Adapt the human NPC-based assays to 384-well format and identify ToxCast phase II chemicals that selectively affect ventral or dorsal NPCs.

Objective 3. Investigate the cellular and molecular mechanisms of action of active ToxCast chemicals.

In the past year, we have made significant progress towards these objectives, which remain as originally proposed. However, we identified some technical limitations and developed an improved strategy to overcome these difficulties. The limitations are related to the heterogeneity of ESC-derived mixtures of NPCs, potential non-cell autonomous effects on NPC, and possible paracrine mechanism of action of ToxCast compounds. The remedies include the use of defined types of NPCs for screening of ToxCast compounds. The advantages of such an approach are three fold:

1. Derivation of dorsal and ventral NPCs and characterization of their differentiation potential.

 

Based on our previous expertise (2,3), we have refined the protocols for the derivation of ventral NPCs using early direct patterning with Super Sonic and Purmorphamine. Briefly, hESCs (H9 cells) were treated with Dispase then transferred to the ultra low attachment plates and grown in complete NPCs media containing bFGF, EGF, Insuline, B27, Nicotineamide, Super Sonic and Purmorphamine. Neurospheres were allowed to grow for 7-10 days and then plated on Matrigel coated plates to obtain monolayer cultures. One day prior to passaging, cells were treated with Stemgen cloning reagent (rock inhibitor). Cells were dissociated using three consecutive 5 minute incubations with Accutase. Dorsal NPCs were obtained similarly to that described above for the ventral NPCs except that Super Sonic and Purmorphamine were omitted from the NPC medium. Using immunofluorescent detection both dorsal and ventral NPCs were found to be uniformly positive for the two major markers of neural precursors, namely Sox2 and Nestin, thus confirming the neural precursor identity of both cell types (Fig. 1 A and B). The levels of Sox2 were variable, especially in the spontaneously formed clusters of ventral NPCs. Because ventral NPCs have to be patterned to acquire the ventral identity (in contrast to dorsal NPCs, which are derived by using standard NPCs medium), we used a set of specific markers to verify their ventral identity. Namely, ventral NPCs were found positive for FoxA2, Nurr1, Lmx1a, and Otx2, thus confirming the ventral nature of these cells (Fig. 1 C and D). Moreover, we found ventral NPCs to express the En1 (Engrailed) marker, which confirm the midbrain identity of these cells (Fig. 1 E). As expected, the dorsal NPCs did not express FoxA2, Nurr1, Lmx1a, Otx2, and En1 markers (not shown). To confirm the lineage properties of dorsal and ventral NPCs, these cell types were differentiated into neurons (using BDNF, GDNG, DAPT, and cAMP) as previously established in our laboratory (2,3). Immunostaining with a pan-neuronal marker MAP2 revealed that both types of NPCs can give rise to MAP2+ cells; however, only ventral NPCs were able to generate cells positive for TH, a classical marker of dopaminergic neurons (Fig. 1 F and G).

 

In summary, during the first year of this project we have identified some pitfalls (see above) and made improvements to our protocols to overcome these difficulties. Critically, for our second objective, during this first year we have developed the screening conditions for 384 well plate format. Further improvements of the platform will include the experiments aimed to increase homogeneity of the NPCs cultures and screening time/replicates optimization.

In the coming year, we plan to complete the characterization of the dorsal and ventral NPCs engineered with Fucci reporters and perform the screening of the phase I ToxCast library. We plan to perform secondary and orthogonal screens using antibodies to ventral and dorsal markers as originally proposed and characterize the lead compounds using titration series.

(1) Sakaue-Sawano A, et al. (2008) Visualizing spatiotemporal dynamics of multicellular cell-cycle progression. Cell 132(3):487-498.

 

(2) Cimadamore F, et al. (2011) Human ESC-Derived Neural Crest Model Reveals a Key Role for SOX2 in Sensory Neurogenesis. Cell stem cell 8(5):538-551.

 

(3) Cimadamore F, Amador-Arjona A, Chen C, Huang CT, & Terskikh AV (2013) SOX2-LIN28/let-7 pathway regulates proliferation and neurogenesis in neural precursors. Proceedings of the National Academy of Sciences of the United States of America 110(32):E3017-3026.

 

(4) Hardwick LJ & Philpott A (2014) Nervous decision-making: to divide or differentiate. Trends Genet 30(6):254-261.

 

(5) Nishitani H, Lygerou Z, & Nishimoto T (2004) Proteolysis of DNA replication licensing factor Cdt1 in S-phase is performed independently of geminin through its N-terminal region. The Journal of biological chemistry 279(29):30807-30816.