Attachment II NSA Task 1 SOW.pdf

TASK ORDER 1

STATEMENT OF WORK

NIEHS DIVISION OF THE NATIONAL TOXICOLOGY PROGRAM

HUMAN NEUROSPHERE ASSAY DATA COLLECTION

1.0. INTRODUCTION: Under this task order, the Contractor shall run the human neurosphere assay (NSA) and collect data on 115 environmental compounds. All services, labor, equipment, and supplies provided under this task order shall be performed under accordance with the terms and conditions of the contract Statement of Work (SOW) and task order SOW.

2.0. TASK: The contractor shall evaluate the neurodevelopmental effects of 115 environmental compounds using nine (9) neurodevelopmental endpoints in the NSA endpoints: 1) hNPC proliferation; 2) Viability (proliferating and differentiating hNPC); 3) Cytotoxicity (proliferating and differentiating hNPC); 4) Radial glia migration; 5) Neuronal differentiation; 6) Neuronal migration; 7) Neurite morphology; 8) Oligodendrocyte differentiation; and 9) Oligodendrocyte migration.

2.1. Chemicals: All test compounds and dimethylsulfoxide (DMSO) will be supplied by the NIEHS/DNTP blinded in a one-time shipment. All other assay controls shall be supplied by the contractor. The Contractor shall be responsible for supplying all other reagents, equipment and facilities needed to conduct the specific assays.

2.2. NSA data collection: The contractor shall employ a multiplexed approach in conducting the following assays: primary hNPC proliferation assay (NPC1), primary hNPC migration assay (NPC2a-c), primary hNPC neuronal differentiation assay (NPC3), primary hNPC neuronal morphology assay (NPC4), and primary hNPC oligodendrocyte differentiation assay (NPC5). Specific assay endpoint descriptions are described in section 3.3-3.7. NPC 1-5 shall be performed in a 96-well plate format with four replicants for each condition and eight conditions for each experiment. These conditions shall consist of one solvent control and seven concentrations of the test compounds. Test concentrations shall be prepared in a 1:3 dilution series from a starting concentration of 100 µM. Three independent experiments shall be performed. An independent experiment is defined as an experiment performed with a new compound on a different day with another cell passage of hNPCs derived from different individual donors. Specific assay endpoint descriptions are described in section 2.3-2.7. If cytotoxicity is noted in greater than three (>3) concentrations then the dilution series shall be repeated at a lower range.

All assays shall be conducted in a semi-automated experimental workflow using an automated liquid handling system (MICROLABSTAR M; Hamilton), plate reader and high content imaging (Cellomics ArrayScan VTI; ThermoFisher) readouts, a bioinformatic workflow utilizing artificial intelligence, Ominsphero Software (Schmuck et al., 2017)and a database-based biostatistics pipeline for unbiased sample evaluation and generation of concentration-response curves and respective benchmark concentrations (BMC).

2.3. Primary hNPC proliferation assay (NPC1): Proliferation shall be detected by direct quantitation of DNA synthesis by measuring incorporation of bromodeoxyuridine (BrdU) in neurospheres at 3 days in vitro (DIV) using a luminescence-based BrdU Assay. Neurospheres derived from primary hNPC of fetal origin (Lonza) shall be plated one per-well filled with proliferation media and experimental solutions in a 96 well plate and incubated at 37°C and 5% CO2. At 16 hours prior to 3 DIV, BrdU labeling solution shall be added to each neurosphere and assayed using a luminometer. At 3 DIV viability (Alamar Blue Assay) and cytotoxicity (Lactate Dehydrogenase assay) shall be assessed using a fluorescent plate reader.

2.4. Primary hNPC migration assays (NPC2a-c): Neurospheres derived from primary hNPC of fetal origin (Lonza) shall be plated one per-well filled with differentiation media and experimental solutions in a Poly-D-Lysine + laminin coated 96 well plate and incubated at 37°C and 5% CO2. Epidermal growth factor (EGF) and the src kinase inhibitor PP2 shall be included in each assay as endpoint-specific positive and negative controls. The plated spheres shall then be allowed to adhere to the laminin-coated well surface and migrate radially. At 7 DIV spheres shall be immunofluorescently labeled using the neuronal marker tubulin beta 3 class III (TUBB3) and the pro-oligodendrocyte marker O4. Nuclei present in the spheres shall be stained with Hoechst dye. High content image analysis (HCA) and the Omnisphero software analysis of brightfield images shall be quantify the migration of radial glial cells out of the Hoechst stained neurospheres (NPC2a). HCA and Ominsphero analysis of immunofluorescently labeled cells shall be used to quantify the migration of TUBB+ neurons (NPC2b) and O4+ oligodendrocytes from the neurosphere (NPC2c). At 7 DIV viability (Alamar Blue Assay) and cytotoxicity (Lactate Dehydrogenase assay) shall be assessed using a fluorescent plate reader.

2.5. Primary hNPC neuronal differentiation assay (NPC3): NPC3 shall be conducted as an extension of NPC2. Omnisphero software and a convolutional neural network (CNN) shall be used to identify the number of TUBB+ neurons present in the migration are of each neurosphere at 7 DIV. The number of TUBB+ neurons shall then be normalized to the number of Hoechst+ nuclei to calculate the percent neurons present in the migration area of each sphere.

2.6. Primary hNPC neuronal morphology assay (NPC4): NPC4 shall be conducted as an extension of NPC3. All neurons identified via the CNN in NPC3 shall be analyzed for their morphology using a skeletonization algorithm using the Omnisphero software. Neurite length and neurite are of TUBB3+ neurons shall be calculated.

2.7. Primary hNPC oligodendrocyte differentiation assay (NPC5): NPC5 shall be conducted as an extension of NPC2. Omnisphero software and a convolutional neural network (CNN) shall be used to identify the number of O4+ oligodendrocytes present in the migration are of each neurosphere at 7 DIV.

The number of O4+ oligodendrocytes shall then be normalized to the number of Hoechst+ nuclei to calculate the percent oligodendrocytes present in the migration area of each sphere.

3.0. DATA RIGHTS: See IDIQ contract.

4.0. DELIVERABLES AND REPORTING REQUIREMENTS: See IDIQ contract.

4.1. Required Reports: A report of completion for Task 1 shall provide a summary of the methods used and the results obtained in sufficient detail and quality to meet the requirements to demonstrate completion of the task. The report of completion shall be delivered in both hard and electronic formats (MS Word) for review and comment by the Contracting Officers Representative (COR). The report of completion shall include but not be limited to:

4.1.1. A detailed description of all experimental procedures

4.1.2. A full set of all raw data, summarized/normalized data

4.2. Contract Management Reports: See IDIQ contract.

4.3. Periodic Status Reports: See IDIQ contract.

4.4. Draft Final Report: See IDIQ contract.

4.5. General Reporting Requirements: See IDIQ contract.

4.6. Electronic Deliverables: See IDIQ contract.

5.0. PLACE OF PERFORMANCE: See IDIQ contract.

6.0. PERIOD OF PERFORMANCE: 12-14 months

7.0. Section 508 Requirements for Deliverables: See IDIQ contract.

8.0. CONTRACTOR FURNISHED MATERIALS: See IDIQ contract.