D_75N95019Q00277_Statement_of_Work.pdf

Version 11-30-2016-1 FAR Part 11—Describing Agency Needs

STATEMENT OF WORK (SOW)

Engraftment of human iPSC derived astrocytes into the forebrain of adult and neonatal wild type mice

I. BACKGROUND INFORMATION

The National Center for Advancing Translational Sciences (NCATS) at the National Institutes of

Health (NIH) has recently developed a protocol for differentiating induced pluripotent stem cells

(iPSC) into astrocytes. Astrocytes are the most numerous non-neuronal cells in the brain and play important role in normal brain development, synaptic function, and various pathological conditions (e.g. neurodegenerative diseases and opioid addiction).

Derivation of human astrocytes from a scalable source, such as induced pluripotent stem cells

(iPSCs), is an attractive approach for disease modeling and drug discovery. However, currently available protocols are variable, inefficient, and lengthy (lasting up to several months). The

NCATS protocol requires only 30 days to derive cells that express relevant astrocyte markers, show typical astrocyte morphology and have shown functionality in various in vitro assays.

NCATS requires the above-mentioned services to provide further functional validation of iPSC derived astrocytes and their ability to survive, integrate, and fully mature within the brain.

II. STATEMENT OF NEED AND PURPOSE

NCATS requires engraftment of human iPSC derived astrocytes into the forebrain of adult and neonatal wild type mice. Specifically, this service includes:

• Cell engraftment into the rodent forebrain

• Brain collection, processing and histological evaluation

III. PERIOD OF PERFORMANCE

The period of performance is 24 weeks and will begin upon receipt of cells from NCATS. The

Contractor will receive the cells within 30 days after order.

IV. SCOPE OF WORK

Independently and not as an agent of the Government, the Contractor shall furnish all the necessary services, qualified personnel, material, equipment, and facilities, not otherwise provided by the Government as needed to perform the general and specific requirements outlined below.

A. GENERAL REQUIREMENTS

The Contractor will prepare the cells according to the instructions below and inject 100,000 cells into the forebrain of 32 neonatal mice and 24 adult mice divided across four (4) treatment groups. After four (4) weeks, one-half of the animals will be sacrificed to collect brains, while the other half will be sacrificed after eight (8) weeks post-grafting. The

Contractor will perform subsequent histological evaluation with a set of predefined immunohistochemical markers for both groups, and statistically analyze the resulting data.

The Contractor will then make the data available to NCATS via FTP or HTML download.

B. SPECIFIC REQUIREMENTS

1. Neonatal Mice (engraftment). The Contractor will:

• Anesthetize mice via cryoanesthesia and dosed intracerebroventricular infusion

(ICV) with a dose volume of 1µl/g or no more than 2 µl of solution. A micro-liter calibrated sterilized glass micropipette is used that is attached to a 10uL Hamilton syringe.

• Use a fiber optic light to illuminate relevant anatomical structures used as a guide.

• Penetrate, perpendicularly, 2 mm into the skull, for ICV at a location approximately

0.25 mm lateral to the sagittal suture and 0.50-0.75 mm rostral to the neonatal coronary suture.

• Slowly dispense the dosing solution (about 1µl/sec), and slowly remove the needle

(about 0.5 mm/sec) to prevent back flow once the dose has been administered.

2. Adult mice (engraftment). The Contractor will:

• Anesthetize mice in an induction chamber using 3% Isoflurane in an air mixture. The mice are placed in stereotaxic apparatuses such that the 18o ear bars are in the ear canals and the incisors are in the tooth bar of the mouse adapter. Surgical planes of anesthesia are maintained with 2% Isoflurane by a nose cone fitted to the stereotaxic instrument.

• Prepare the scalp for surgery by cleaning the clipped injection site with Povidone iodine and a 70% ethanol rinse.

• Confirm sufficient anesthetic depth before an incision is made.

• Make an incision I the scalp, slightly off-center. Scrape the subcutaneous tissue and periosteum from the skull with sterile tipped applicators.

• Attach the guide cannula to the stereotaxic arm and position over Bregma. This coordinate is considered the zero point.

• Move the guide cannula to the appropriate anterior/posterior and medial/lateral coordinates.

• Infuse the mice with cells bilaterally in the cortex.

3. Collections. The Contractor will:

• Collect the brains of one-half of the mice at 4 weeks after engraftment, and the other half at 8 weeks after engraftment. Both of these groups will include 6 adult and 8 neonatal mice.

• Anesthetize mice and confirm sufficient anesthetic depth before an incision is made.

• Continuously administer ambient temperature phosphate-buffered saline.

• Perform perfusion with freshly prepared phosphate buffered 4% PFA.

• Harvest brains and post fixed overnight in freshly prepared phosphate buffered 4%

PFA. Brains will be kept refrigerated until shipped.

4. Histology and Analysis. The Contractor will:

• Perform histology in multiple milestones.

• Perform histological evaluation with a set of predefined immunohistochemical markers for both groups. The immunohistochemical markers include:

o STEM 121 (ms) + GFAP (gt) + PNM (rb) o STEM 123 (ms) + S100B (gt) + VIM (rb) o STEM 123 + Collagen IV (rb) + Oct4

• Statistically analyze the resulting data.

C. CELL PREPARATION

1. Shipping the cells. iPS Astrocytes will be plated at appropriate density on the surface of two T75 flasks prior to the shipping to Contractor. Flasks will be fully filled with the media and shipped to the Contractor in temperature-controlled environment.

Along with the cells, 100ml of the Astro maintenance medium will be shipped for

Contractor to replace the medium (20 ml per flask) upon arrival of the cells. Cells can be maintained up to a maximum of 3 days by the Contractor.

2. Preparing the grafting suspension: iPS Astrocytes should be dissociated with

Accutase (7ml per T75 flask. ).

Using Accutase is critical.

Incubate the flasks with 7ml of Accutase in the 37°C cell incubator for 4 mins.

Gently rinse the well using 10ml pipettor and make sure that all cells are detached before transferring the total volume to 50ml centrifuge tube.

Add 15ml of DPBS (Ca-; Mg-) to triple the volume of Accutase and centrifuge the cells at 300g for 4 minutes.

Prepare the grafting suspension:

• Group 1 and 2: Without the small molecule components.

• Group 3 and 4: Add small molecule components A,B,C in 1:10.000 ratio to the cell suspension. Add small molecule component D in 1:1.000 ratio.

* Small molecule components A,B,C,and D will be provided by NCATS

D. ADDITIONAL REQUIREMENTS

The Contractor will also be required to:

• Maintain animals in a controlled environment on a 12h dark/12h light cycle, with the room temperature and humidity continuously recorded in the holding room.

• Ensure the experimenter(s) are blind to the treatment distribution.

• Conduct all activities and procedures according to established protocols approved by the

IACUC committee and Contractor’s Standard Operation Procedures (SOP).

• Provide NCATS with periodic updates throughout the study and notify NCATS in the event of any unexpected finding or occurrence during the study.

E. GOVERNMENT RESPONSIBILITIES

NCATS will culture the biological material and ship 7 million cells plated in two T75 flasks, to the Contractor. NCATS will also send the Contractor the small molecule cocktail (SMC) -a protocol developed by NCATS to promote cell survival - to be administered to study groups 3 and 4.

NCATS will provide the Contractor with the following within 30 days of order:

1. A total of 7 million iPSCs derived astrocytes

2. 100ml of Astrocyte maintenance medium, sufficient for 2 media changes.

2. Four (4) small molecule cocktail components - A,B,C, and D – in the following volumes:

a) Component A: 0.5ml

b) Component B: 0.5ml

c) Component C: 0.5ml

d) Component D: 5ml

Cells will be provided ready for injection.

F. DELIVERY OR DELIVERABLES

The Contractor will provide NCATS a report via email within three (3) weeks of completion of the study plan, to an NCATS point of contact designated on the purchase order. The report will include the methods used, the analyzed data and interpretation/conclusion from the study along with microscopical images and graphic representation of the data. The report shall be emailed to the Point of Contact designated o the purchase order.

The report and supporting documentation will be provided in electronic form – PDF, MS

Excel sheets, MS Word, TXT files, PNG, TIFF figures, or other format based on the type of document.

V. OTHER CONSIDERATIONS

A. PUBLICATIONS AND PUBLICITIY

The Contractor may not publish any result of any work performed under this contract. The datasets derived by NCATS from these experiments will be part of scientific publications and they will be made available to the public on an NCATS public-facing internet website.

B. CONFIDENTIALITY

The Contractor will keep the results of the contract confidential.