Grantee Research Project Results
2014 Progress Report: Human Stem Cell-Based Platform to Predict Selective Developmental Neurotoxicity
EPA Grant Number: R835552Title: Human Stem Cell-Based Platform to Predict Selective Developmental Neurotoxicity
Investigators: Terskikh, Alexey V. , Farhy, Chen
Current Investigators: Terskikh, Alexey V.
Institution: Sanford-Burnham Medical Research Institute
EPA Project Officer: Aja, Hayley
Project Period: September 1, 2013 through August 31, 2017
Project Period Covered by this Report: October 1, 2013 through September 30,2014
Project Amount: $800,000
RFA: Development and Use of Adverse Outcome Pathways that Predict Adverse Developmental Neurotoxicity (2012) RFA Text | Recipients Lists
Research Category: Chemical Safety for Sustainability , Human Health
Objective:
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.
Progress Summary:
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:
- This strategy allows us to prepare and store (freezing in liquid nitrogen) a large number of aliquots thus enabling reproducibility across multiple screening experiments.
- Although we originally considered mainly cell-autonomous mechanisms of chemical actions, the mixtures of NPCs with different identities complicate the interpretation of results due to potential paracrine signaling from the neighboring cells in culture and non-cell-autonomous mechanisms of action of ToxCast compounds.
- Finally, the possibility to screen individual types of NPCs enables a straightforward comparison across multiple types of NPCs and allows the implementation of live cell imaging.
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).
These results attest to the generation of desired NPC types from hESC enabling us to conduct the proposed screening of ToxCast chemicals. As previously observed in our laboratory, the level of marker expression varies within the populations, suggesting some degree of heterogeneity. In the next year of this project, one of our objectives is to understand the source of these variations and develop protocols that provide a more homogeneous expression of markers.
2. Live imaging of dNPCs and vNPCs under basal conditions and with Cyclopamine.
Although the originally proposed end point screening approach using antibodies remains in force, we have developed a more sensitive primary screen using live cell imaging enabling us to directly compare multiple types of NPCs. For this purpose, we have engineered both dorsal and ventral NPCs to express mCherry fluorescence protein fused to H2B histone variant (mCherry-H2B). The nuclear localization of such reporter protein allows straightforward live cell image tracing using the IC-200 fluorescent high content screening microscope, which is available at the Conrad Prebys Center for Chemical Genomics (CPCCG), a unique resource at Sanford-Burnham.
As a proof of principle for this approach, we have performed live imaging of mCherry-H2B engineered dorsal and ventral NPCs in the presence of Sonic Hedgehog and its inhibitor Cyclopamine. While dorsal NPCs showed a minimal response to the presence of Cyclopamine, ventral NPCs showed a significant dose-dependent reduction in proliferative potential in the presence of Cyclopamine (Fig. 2 A). These results are corroborated by the qPCR analysis of Sonic Hedgehog expression levels in dorsal and ventral NPCs. As expected, Sonic Hedgehog mRNA was almost undetectable in dorsal NPCs, while ventral NPCs show a robust expression of Sonic Hedgehog mRNA (~3000 fold difference).
These results provide a proof of principle for our screening protocol using live cell imaging. The ToxCast compounds, which selectively affect ventral or dorsal NPCs can change the total numbers of specific types of NPCs by different mechanisms including alteration to cell cycle and/or cell death. The live imaging step proposed here will not only identify selectively toxic compounds, but also will examine the kinetics of cellular changes thus discriminating between acute cytotoxicity and cytostatic effects. To further improve the potential of live cell imaging, we currently are engineering NPCs with Fucci reporter system that will be used to screen ToxCast chemicals.
3. Engineering of dorsal and ventral NPCs with the Fucci reporters to visualize the cell-cycle status.
The ToxCast compounds can selectively affect different types of NPCs by different mechanisms. For instance, ToxCast compounds can change the cell fate and induce cell differentiation. It has been shown that NPC differentiation is preceded by the slowing of the cell cycle (4). Moreover, slowing the G0/G1 phase of cell cycle is sufficient to induce neuronal differentiation in vitro and in vivo (4). We have adopted a Fucci reporter system (1) to visualize spatiotemporal dynamics and cell-cycle progression for dorsal and ventral NPCs using live cell imaging. Briefly, due to the cell cycle-dependent proteolysis, protein levels of both Geminin and Cdt1 cell cycle regulators oscillate inversely. The Cdt1 levels are highest during G1, while Geminin levels are highest during the S, G2, and M phases (5). The Fucci system takes advantage of the two genetically encoded indicators, Cdt1-GFP and Geminin-RFP, for visualization cell-cycle progression. Therefore, the cell-cycle status of the cells engineered with Fucci can be determined by live imaging of green and red fluorescent proteins (Fig. 3). Actively proliferating cells cultures will have a substantial proportion of green cells (S/G2/M phases of cell cycle), while quiescent cells are predominantly red (G0/G1 phase). We already have generated the ventral NPCs engineered with Fucci reporter (Fig. 3 B) demonstrating the feasibility of this approach. Determining the numbers of cells in G0/G1 vs S/G2/M phases of cell cycle (along with the total numbers of fluorescent cells) will enable a dynamic analysis of the effect of ToxCast chemicals on the cell numbers and cell fate of dorsal and ventral NPCs. In particular, increased numbers of cells in the G0/G1 phases of cell cycle will be indicative of the cytostatic effect and/or potential differentiation, while the increase of S/G2/M population will suggest the increase in cell proliferation. The decline in total number of fluorescent cells (both green and red) will indicate cellular toxicity.
Future Activities:
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.
- Generation of the desired NPC types from hESC enabling us to conduct the proposed screening of ToxCast chemicals.
- Obtaining a proof of principle for the live cell screening protocol enables us to not only identify selectively toxic compounds, but also examine the kinetics of cellular changes thus discriminating between acute cytotoxicity and cytostatic effects.
- Implementation of the Fucci reporting system allows us to determine the numbers of cells in G0/G1 vs S/G2/M phases of cell cycle (along with the total numbers of fluorescent cells), thus enabling a dynamic analysis of the effect of ToxCast chemicals on the cell numbers and cell fate of dorsal and ventral NPCs.
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.
References:
(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.
Supplemental Keywords:
exposure, risk assessment, human health, bioavailability, metabolism, vulnerability, sensitive populations, dose-response, teratogenProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.