Induced Pluripotent Stem Cells

The Progeria Research Foundation Cell & Tissue Bank
Human 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

Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007;131:861-872.
Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, Slukvin, II, Thomson JA. Induced pluripotent stem cell lines derived from human somatic cells. Science. 2007;318:1917-1920.
Liu GH, Barkho BZ, Ruiz S, Diep D, Qu J, Yang SL, Panopoulos AD, Suzuki K, Kurian L, Walsh C, Thompson J, Boue S, Fung HL, Sancho-Martinez I, Zhang K, Yates J, 3rd, Izpisua Belmonte JC. Recapitulation of premature ageing with iPSCs from Hutchinson-Gilford progeria syndrome. Nature. 2011;472:221-225.
Misteli T. HGPS-derived iPSCs for the ages. Cell Stem Cell. 2011;8:4-6.

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 Institute’s 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):

Mycoplasma Testing for each line: Dr. Stanford’s lab has performed mycoplasmaanalysis by PCR for each cell line. In addition, after expansion and prior to shipping cells, the lines will be retested for mycoplasma.
Immunostaining for pluripotency markers Tra-1-60, Tra-1-81, and SSEA4.
Alkaline Phosphatase Staining as an indicator of pluripotency
Embryoid body formation and subsequent immunostaining for markers of the three germ-layers. Markers tested were βIII-Tubulin (Ectoderm), Smooth-Muscle Actin (Mesoderm), and Gata4 or AFP (Endoderm)
Karyotype analysis.
Re-expression of lamin A in differentiated cells
Teratoma assays

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	Originating Cell Type Click here.	Supporting Data
HGADFN003 iPS 1B	LMNAExon 11, 1824 C>T	Male 2yr 0mo	Dermal Fibroblasts HGADFN003	003 iPS1B
HGADFN003 iPS 1C	LMNA Exon 11, 1824 C>T	Male 2yr 0mo	Dermal Fibroblasts HGADFN003	003 iPS1C
HGDFN003 iPS 1D	LMNA Exon 11, 1824 C>T	Male 2yr 0mo	Dermal Fibroblasts HGADFN003	003 iPS1D
HGADFN167 iPS 1J	LMNA Exon 11, 1824 C>T	Male 8yr 5mo	Dermal Fibroblasts HGADFN167	167 PS 1J
HGADFN167 iPS 1Q	LMNA Exon 11, 1824 C>T	Male 8yr 5mo	Dermal Fibroblasts HGADFN167	167 iPS1Q
HGMDFN090 iPS 1B	Mother of HGADFN167 (unaffected)	Female 37yr 10mo	Dermal Fibroblasts HGMDFN090	090 iPS1B
HGMDFN090 iPS 1C	Mother of HGADFN167 (unaffected)	Female 37yr 10mo	Dermal Fibroblasts HGMDFN090	090 iPS1C
HGFDFN168 iPS1 D2	Father of HGADFN167 (unaffected)	Male 40yr 5mo	Dermal Fibroblasts HGFDFN168	168 iPS1 D2
HGFDFN168 iPS1P	Father of HGADFN167 (unaffected)	Male 40yr 5mo	Dermal Fibroblasts HGFDFN168	168 iPS1P

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. Stanford’s 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

88% (v/v) DMEM – Invitrogen cat#11965092
10% (v/v) FBS – Invitrogen cat#12483020
1% (v/v) Glutamax – Invitrogen cat#35050061
1% (v/v) Penicillin/Streptomycin – Invitrogen cat#15140122

Store at 4˚C 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	Supplier	Cat#	Volume
Knock-Out DMEM	Invitrogen	10829-018	250ml
Knockout Serum Replacement	Invitrogen	10828‐028	45ml
Penicillin / Streptomycin	Invitrogen	15140‐122	3ml
Glutamax	Invitrogen	35050‐061	3ml
Beta Mercaptoethanol	Invitrogen	21985‐023	540μl
20μg/ml Human FGF basic	PeproTech or Invitrogen	100‐18B PHG0024	150μl of 20ug/ml stock (final 10ng/ml)

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:

0.25% Trypsin‐EDTA – Invitrogen cat#25200056
MEF media
0.2% (w/v) gelatin (Sigma G2500)
Plates to coat

Procedure:

Coat surface of wells to be used with 0.2% gelatin (approximately 1 ml per 10 cm² of surface area) and let stand for at least 20 minutes at room temperature. Alternatively, coat plates and store at 4˚C (up to 7 days).
Aspirate medium from MEF cells and add trypsin (spread it over the entire surface) then aspirate the trypsin (leaving a small amount) and incubate for 3 minutes at 37˚C.
After incubation period, add MEF media to neutralize trypsin and resuspend to dissociate cells; perform a cell count.
MEFs must be inactivated by irradiating with 2000 rads.
Irradiated MEFs are plated at the following concentrations: 5×10⁵ cells/T-25 flask; 2×10⁵ cells/well (6-well plate); 1.5×10⁶ cells/T-75 flask.
Allow MEFs to settle and attach for at least 4 hours but preferably overnight before seeding iPSCs onto feeders.
Irradiated MEFs can be used up to 10 days after seeding; be sure to feed them every 3rd day or so if not being used immediately.

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:

Culture medium (for Progeria lines is the Knock-Out DMEM mixture)
Plate with prepared layer of MEFs that has had the media changed at least an hour before using.
15ml tube x 2
ROCK inhibitor (y-27632 5mM solution Calbiochem 688001)

Procedure:

Warm media. Prepare a 15ml tube with 2ml of media per vial to be thawed and Rock inhibitor at a final concentration of 10mM.
Remove iPSCs from LN2 freezer
Thaw cells in cryovial by gently swirling in 37˚C water bath. Be sure not to dip above cap level. When small ice pellet remains, remove vial from water bath (this should take no more than 1-2 minutes).
Wash vial with 70% ethanol and wipe.
Pipette contents up and down once to mix; transfer to empty 15ml tube.
Take 5ml of media (no ROCK inhibitor) and add to cells in drop wise fashion so as to avoid shocking the cells.
Centrifuge for 3 minutes @ 800rpm, room temperature.
After centrifugation, aspirate media and resuspend cell pellet in 2ml of media with Rock Inhibitor.
Transfer suspension onto prepared MEFs surface containing 1-2ml of hiPSC media. Spread suspension on surface by moving in circular fashion. Incubate at 37˚C in 5% CO2.
Perform daily medium changes. Check for undifferentiated colonies that are ready to passage (dense centered) approximately 5 – 7 days after thawing.

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 Cells Updated 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 (I’ve 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:

iPSC culture media
1mg/ml Collagenase Type IV – Stem Cell Technologies cat#07909
Cell scrapers
Inactivated MEF-coated surfaces (changing media to iPS media at least one hour before passage)
Collagenase should be aliquoted and stored long term at -20˚C.

Procedure:

Aspirate old media and add 1mg/ml collagenase (1 ml per well of 6 well).
Incubate for at least 10 minutes at 37˚C.
Take inactivated MEF-coated surface and replace MEF media with feeder‐based hiPSC culture media in order to condition it (this can be done 2-3 hours before splitting if desired, step is not mandatory but recommended).
After incubation period is done, aspirate the collagenase from the cells and wash the layer of cells with 2ml PBS, aspirate off PBS. Then add some hiPSC media. Using a cell scraper, dissociate cells and pipette the suspension up and down approximately 10 times to create small clusters. Spin down the cells at 800 rmp for 4 minutes.
Resuspend the cell pellet gently with 6mls of media and passage 1ml into each new well of a six well plate containing inactivated MEF-coated surface (recommend 1:6 split).
Place freshly passaged cells into incubator, moving plate/flask side to side so as to spread the cells out as much as possible on the layer of feeders. Swirling the plate will cause all of the cells to settle into the centre of the well, resulting in overcrowding.

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.

Materials:

iPS Media
1mg/ml Collagenase Type IV – StemCell cat#07909
Freezing media (prepared at 2x concentration)

o 30% (v/v) hiPSC media

o 50% (v/v) ES qualified FBS

o 20% DMSO

Procedure:

In the iPSC culture to be cryopreserved, use a microscope to visually identify regions of differentiation. Mark these using a felt tip or lens marker on the bottom of the plate.
As in routine passaging, this selection should not exceed 20% of the well if the culture is of high quality.
Remove regions of differentiation by scraping with a pipette tip or by aspiration.
Aspirate media and add 1ml of collagenase type IV.
Incubate for at least 10 minutes at 37˚C.
Aspirate collagenase and wash with 2ml of PBS. Aspirate PBS and add fresh media. Using a cell scraper, dissociate cells from surface.
With a pipette, break up colonies by pipetting up and down approximately 5 times (if colonies are too small they will not survive well upon thawing).
Transfer suspension to a 15ml tube and centrifuge @ 800rpm for 3 minutes.
While cells are spinning, label cryovials with your initials, cell line, passage number and date.
When spinning is done, aspirate media and resuspend in half volume of media.
Add an equivalent amount of 2x freezing media (for a final concentration of 1x) dropwise so as to avoid stressing the cells. Note that the addition of DMSO will give an exothermic reaction. This heat may damage the cells so it is important to make the freezing media and then let it chill on ice before use.
Aliquot 1ml of suspension into each cryovial.
Store at -80˚C overnight in a cryobox; the following day transfer frozen cells to LN2.