The Progeria Research Foundation Cell & Tissue BankHuman Induced Pluripotent Stem Cells (iPSC)

1. iPSC Background information for the non-scientist Stem cells are immature cells that have not yet committed to becoming any one cell type. They are pliable because they have the potential to develop into many different types of mature cells in the body, such as cells that make up the heart or blood vessels, and other tissues and organs. In 2007, researchers discovered a strategy for creating stem cells in the laboratory by reprogramming mature adult cells that we commonly grow for research purposes.1, 2 . These artificially created stem cells are called Induced Pluripotent Stem Cells (iPSCs). For the field of Progeria, this is a huge breakthrough. For the first time, scientists can now make Progeria stem cells and ask questions about how stem cells function and develop in Progeria. Previously there was no source of human Progeria stem cells, and there was therefore a void of information about how Progeria stem cells function compared with stem cells from people without Progeria. In addition, scientists can re-program the Progeria stem cells to create, for the first time, mature Progeria blood vessels, heart cells, and other cell types. Until now, there was no source of human Progeria heart or blood vessel cells. We can now ask key questions about the heart disease that leads to early death in Progeria from heart attacks and strokes. We can compare these discoveries with the heart disease and aging in the general population and discover more about what influences aging in all of us. Already there have been several excellent studies published using Progeria stem cells.3-5 Our goal at The Progeria Research Foundation is to facilitate many more discoveries using this invaluable tool. For a primer on stem cells, please see this US government website: http://stemcells.nih.gov/info/basics/defaultpage.asp

2.Purpose of induced pluripotent stem cell (iPSC) generation and distribution by The Progeria Research Foundation The mission of The Progeria Research Foundation is to discover treatments and the cure for Hutchinson-Gilford Progeria Syndrome and its aging-related disorders. In 2009, PRF entered into a collaboration with an expert team of scientists at the University of Toronto, Canada, under the direction of William Stanford, PhD, to generate high quality Progeria iPSCs. Dr. Stanford is the Canada Research Chair in Integrative Stem Cell Biology. As of 2011, PRF continues to collaborate with Dr. Stanford at the University of Ottawa, Canada where he is Professor of Cellular and Molecular Medicine, Faculty of Medicine, and Senior Scientist at Ottawa Hospital Research Institutes Sprott Centre for Stem Cell Research.

Our goal is to provide this invaluable tool to researchers throughout the world. This new research tool will be used to generate new and innovative research in Progeria, as well as its relationship to heart disease and aging.

3. Generation of Hutchinson-Gilford Progeria Syndrome Induced-Pluripotent Stem Cells (iPSCs) Induced-Pluripotent Stem Cells (iPSCs) were derived using VSVG-pseudotyped retroviral transduction of four human factors, Oct4, Sox2, Klf4, and c-Myc into fibroblasts. iPSC colonies were derived on mouse-embryonic fibroblasts (MEFs). The procedure used was essentially as previously described but without the use of the EOS reporter (Nature Protocols 4: 1828-1844, 2009).

4. Quality Control: Validation and Characterization The lines that are currently available have undergone several validation steps (see downloadable PDFs below):

Additional validation in process: Some lines have completed teratoma assays as shown in supporting data. For all other lines, teratoma assays are in process and status will be updated as these assays are completed.

5. Original starting material from which these iPS cells were derived iPSCs were derived from PRF Cell & Tissue Bank non-transformed fibroblast cell lines.

The transduction method used for all iPS lines was Retrovirus MKOS.

iPSC Line ID

Mutation

Gender and Donation Age

HGADFN003 iPS 1B

LMNAExon 11, 1824 C>T

Male 2yr 0mo

Dermal Fibroblasts HGADFN003

HGADFN003 iPS 1C

LMNA Exon 11, 1824 C>T

Male 2yr 0mo

Dermal Fibroblasts HGADFN003

HGDFN003 iPS 1D

LMNA Exon 11, 1824 C>T

Male 2yr 0mo

Dermal Fibroblasts HGADFN003

HGADFN167 iPS 1J

LMNA Exon 11, 1824 C>T

Male 8yr 5mo

Dermal Fibroblasts HGADFN167

HGADFN167 iPS 1Q

LMNA Exon 11, 1824 C>T

Male 8yr 5mo

Dermal Fibroblasts HGADFN167

HGMDFN090 iPS 1B

Mother of HGADFN167 (unaffected)

Female 37yr 10mo

Dermal Fibroblasts HGMDFN090

HGMDFN090 iPS 1C

Mother of HGADFN167 (unaffected)

Female 37yr 10mo

Dermal Fibroblasts HGMDFN090

HGFDFN168 iPS1 D2

Father of HGADFN167 (unaffected)

Male 40yr 5mo

Dermal Fibroblasts HGFDFN168

HGFDFN168 iPS1P

Father of HGADFN167 (unaffected)

Male 40yr

5mo

Dermal Fibroblasts HGFDFN168

PRF AVAILABLE CELL LINES

6. Join our email list for future iPSC updates and new cell lines We are continuing to generate iPSC lines. If you would like periodic updates on iPSCs held in the PRF Cell & Tissue Bank,please join our emailing list by clicking here

7. Questions? Please contact Leslie Gordon, MD, PhD, Medical Director, with any questions or needs, at lgordon@progeriaresearch.org or 978-535-2594

8. Ordering iPS cell lines

In 2014, PRF instituted a policy of no changes to our MTA. This is the result of 12 years of contractual arrangements with 70 research teams working at institutions in 14 countries. PRF and its counsel have taken into consideration the issues that have arisen in that time period and edited the agreement accordingly, resulting in what we feel are fair and reasonable terms.

For U.S. Federal Government Institutions, please contact Joan Brazier, Research Study Coordinator, at joan_brazier@brown.edu or 401-863-9628.

Step 1: Complete an application and material transfer agreement Application and Agreement for Non-government Institutions

Material Transfer Agreement for Non-government Institutions*

Step 2: Return the completed application and material transfer agreement to PRF at info@progeriaresearch.org. Once approved, you will receive an email confirming your order and anticipated shipping date.

Step 3: Dr. Stanfords laboratory is currently distributing lines as live cultures. His laboratory will email you when the culture has been shipped, with shipping and tracking information. Inexperienced researchers are directed to obtain training at specialized courses essential to human embryonic stem cell/iPSCs work.

Step 4: The University of Ottawa will charge $84.00 per iPSC line plus courier costs, if any, and will send you a bill directly.

9. HGPS and Control iPS Cell Culture Media Preparation Culturing Progeria iPSCs requires the preparation of various kinds of media depending on the growth conditions of the cells and the experimental requirements. In addition to maintenance media, there is also supportive media for the MEFs. The HGPS iPSCs were derived using a Knock-Out medium containing Knock Out Serum Replacement (KOSR).

MEF medium

Store at 4C and use within 4 weeks. If purchasing untreated MEFs from Millipore it is recommended to increase the FBS concentration to 20% for better growth during expansion.

HGPS and Control hiPSC media

Reagent

We recommend Lot testing the Knockout Serum Replacement on established hES cells before being used for Progeria iPS cells.

10. Preparation of HGPS and Control iPSC Culture Surfaces To maintain high quality cells and colonies, it is imperative to passage onto appropriate surfaces. This surface could consist of inactivated mouse embryonic fibroblasts (MEFs, replication arrested through irradiation or mitomycin-c treatment). The protocol for inactivation of MEFs by irradiation follows. However MEFs can also be inactivated by treatment with mitomycin C if there is no access to an irradiator. Inactivated MEFs can be made in house or purchased through Millipore (cat# PMEF-CFL for MEFs that have not been mitotically inactivated or cat# PMEF-CFL for inactivated ones that are ready to use). A vial of untreated MEFs can be expanded and treated with Mitomycin C used immediately or frozen down for future use.

11. Inactivating (by irradiation) and plating MEFs

Materials:

Procedure:

12. Thawing HGPS and Control iPS cell lines One vial of hiPSCs should be thawed into one well of a 6-well plate containing inactivated mouse embryonic feeders cells (MEFs).

Have all tubes, warmed medium, and plates ready before starting the protocol to ensure that the thawing procedure is done as quickly as possible.

Materials:

Procedure:

Note: If only a few undifferentiated colonies are observed after thawing, it may be necessary to select only these colonies for passaging and replate them in the same size well on a new plate.

13. Routine Passaging and Maintenance of Undifferentiated HGPS and Control iPSCs

In order to assure healthy cells, it is important to change their media on a daily basis. This is a simple process of aspirating the old media and replacing it with fresh iPS media. After some time, usually 4-6 days after splitting, it will be necessary to split the cells once again. Splitting cells before they become too confluent will ensure a higher number of undifferentiated cells. Usually a 1:6 or 1:8 split will work well and allow 6-7 days between passages.

14. Suggested Protocol for Passaging iPS CellsUpdated September 4, 2014

The following protocol, obtained from Beers et al, 2012 has been giving excellent results for the team at the Human Pluripotent Stem Cell Facility of Ottawa Hospital Research Institute. According to this team, the protocol has dramatically helped to decrease the differentiated cells that might start to grow and it speeds up the passaging. Cells are often ready within 3 to 5 days instead of 5 to 7. Therefore this could save time and money on media.

EDTA solution: Add 500ul of 0.5M EDTA (pH 8.0) into 500ml of DPBS (-/-). Add 0.9g of NaCl and adjust the osmolarity to 340 mOsm. Filter the solution to sterilize and store it at 4C for up to 6 months. The goal is to create the least amount of disturbance for the cells during dissociation. Therefore the EDTA solution is at the same osmolarity as the E8 media.

Protocol

Add 2ml of E8 media to a 6 well matrigel coated plate.

Take the plate to be passaged and remove the media from the well and wash twice with 1ml of PBS(-/-).

Add 1ml of the EDTA solution to the well and leave for 4min at room temperature.

Once 4 min. is up remove EDTA solution and add 1ml of E8 media.

Scrape cells and divide cells amongst the 6 wells of your plate containing E8 media (Ive been taking 160ul into each well). Avoid breaking up the pieces as much as possible. Preferably use a wide mouth pipette tip.

Swirl and incubate at 37C.

NOTE: Once the cells have been scraped, transfer them to the new plate as soon as possible because the cells will re-attach quickly.

Source:

Passaging and colony expansion of human pluripotent stem cells by enzyme-free dissociation in chemically defined culture conditions

Jeanette Beers, Daniel R. Gulbranson, Nicole George, Lauren I. Siniscalchi, Jeffrey Jones, James A. Thomson, and Guokai Chen

Nat Protoc. 2012 Nov; 7(11):2029-40

15. Culturing HGPS and Control iPSCs on MEFs

Materials:

Procedure:

16. Cryopreservation of HGPS and Control iPSCs

Multiple passaging and expansion of iPSCs will result in a surplus of cells. Instead of disposing, it is good practice to freeze cells on occasion to build up a stock and give you cells you can go back to and thaw out for use in the future.

The protocols described below are based on iPSC cultures in 6-well plates where initial clump seeding is adjusted so that wells are 60 70% confluent at time of cryopreservation. Before cryopreservation, iPSCs should be of high quality (primarily undifferentiated with less than 20% of the cells being differentiated). Cryopreservation should be done approximately 1 day before the cells are ready to passage. iPSCs will have improved survival following thawing if cryopreserved as large clumps.

Link:
Induced Pluripotent Stem Cells | The Progeria Research Foundation