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Induced Pluripotent

Stem Cells

 

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: https://stemcells.nih.gov/info/basics.htm

 

    1. 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.
    2. 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.
    3. 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.
    4. 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):

 

    1. 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.
    2. Immunostaining for pluripotency markers Tra-1-60, Tra-1-81, and SSEA4.
    3. Alkaline Phosphatase Staining as an indicator of pluripotency
    4. 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)
    5. Karyotype analysis.
    6. Re-expression of lamin A in differentiated cells
    7. 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 IDMutationGender and DonationAgeOriginating 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>TMale 8yr 5moDermal Fibroblasts HGADFN167167 PS 1J
HGADFN167 iPS 1Q LMNA Exon 11, 1824 C>TMale 8yr 5moDermal Fibroblasts HGADFN167167 iPS1Q
HGMDFN090 iPS 1B Mother of HGADFN167 (unaffected)Female 37yr 10moDermal Fibroblasts
HGMDFN090
090 iPS1B
HGMDFN090 iPS 1C Mother of HGADFN167 (unaffected)Female 37yr 10moDermal Fibroblasts
HGMDFN090
090 iPS1C
HGFDFN168 iPS1 D2Father of HGADFN167 (unaffected)Male 40yr
5mo
Dermal Fibroblasts HGFDFN168168 iPS1 D2
HGFDFN168 iPS1PFather of HGADFN167 (unaffected)Male 40yr
5mo
Dermal Fibroblasts
HGFDFN168
168 iPS1P

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 or questions, please contact Wendy Norris at: wnorris@lifespan.org or 401-274-1122 x 48063.

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 in frozen cryovials.  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.

The Human Pluripotent Stem Cell Facility (directed by Dr. Stanford) at the Ottawa Hospital Research Institute offers virtual one-on-one training on basics of iPSC culture techniques specific to the Progeria iPSC cell lines. Training options and format are flexible depending on the scientists’ level of experience.  For more information, please email hpscf@ohri.ca.

Step 4: The University of Ottawa will invoice you directly for each iPSC line plus courier costs, if any.

9.  HGPS and Control iPS Cell Culture Media Preparation

iPSC and ESCs need to be fed with mTeSR Plus from Stem Cell Technologies (cat# 5825). Please follow the supplier’s recommendations for storage.

10. Preparing Matrigel Plates

Note: All steps involving matrigel should be done as quickly as possible and stay as cold as possible.

  1. Thaw matrigel bottle at 4°C. Check the certificate of analysis on that lot to find its protein concentration.
  2. Add enough cold DMEM/F12 media to the thawed matrigel to reach a final concentration of 5mg/mL.
  3. Make 1mL aliquots of the prepared matrigel from step 2 in pre-chilled 15mL falcon tubes.
  4. Freeze and store all aliquots at -20°C.
  5. To make matrigel plates, remove one aliquot of matrigel (1mL) from -20°C and add 10mL of cold DMEM/F12. Mix well until pellet thaws (without creating bubbles, and always keep solution cold).
  6. Transfer to a 50mL tube, then add 20mL of cold DMEM/F12 (1mL of matrigel aliquot is diluted in 30mL of DMEM/F12), mix well.
  7. Plate (1mL/well for 6 well plate, 0.5mL/well for 12 well plate, 0.25mL/well for 24 well plate). Make sure that the solution is covering the entire surface area by shaking the plate gently. Do not re-freeze any leftover matrigel.
  8. If using the plate immediately, let the plate sit at room temperature for 1 hour (or 30 minutes at 37°C), observe matrigel under the microscope. Matrigel should be well dispersed and not “clumpy”.
  9. If using plates at another time, wrap the edge of the plate with parafilm and store at 4°C for up to 2 weeks.

Matrigel – BD/Fisher, cat#CB-40230

DMEM/F12 – Life Technologies, cat#11330-057

Dr. William Stanford-2022

11. Thawing ES or iPS cells (per cryo-vial)

  1. Take a matrigel plate out of 4°C and warm at room temperature for one hour, or make a fresh matrigel plate (see preparing matrigel plate protocol).
  2. Warm 4mL of mTeSR Plus in a 15mL falcon tube.
  3. Remove cells from the liquid nitrogen tank and swirl in a 37°C bath until only a small ice chunk is left. Vial should thaw in 1-2 minutes. This step must be done quickly.
  4. Ethanol cell tube and falcon media tube and place in the hood.
  5. Use a 1mL wide mouth tip to slowly add cells to 4mL of pre-warmed media (avoid mixing cell suspension).
  6. Spin at 130 rcf for 5 minutes.
  7. Remove supernatant.
  8. Add 2mL of PSC media, and with a wide mouth tip break up clumps gently. Transfer media to one well of a 6 well plate add 2uL of ROCK inhibitor (Y27632, final concentration of 10uM). Plate into one matrigel coated well (of a 6 well plate).
  9. Rock cells gently to evenly distribute cells, and place in a hypoxic incubator (5%O2, 10% CO2). Avoid plate disturbance for 24 hours post seeding.

    NOTE: It is very important to avoid excessive breaking down of clumps or aggressive pipetting. This can significantly reduce survival rate. The cells should remain in chunks of 100-300 cells big at the time of seeding. While being gentle, try working quickly once cells are thawed to minimize time they are in contact with cryoprotectant.​

  10. Remove media after 24 hours and add 2mL of PSC media (for a 6 well, 1mL for 12 well and 0.5mL for 24 well plate). See Harvesting and Caring for hESC/iPSC protocol.

    PSC media

    mTeSR Plus from Stem Cell Technologies (cat# 5825). Please follow the supplier’s recommendations for storage.

    What is ROCK Inhibitor Y27632?

    ROCK Inhibitor Y27632 is a selective inhibitor of the Rho associated kinase p160 ROCK. Treatment with ROCK Inhibitor Y27632 prevents dissociation induced apoptosis of human embryonic stem cells (hESC) and human induced pluripotent stem cells (iPSC), increasing the survival rate and maintaining pluripotency during subcultivation and thawing of hESCs and hiPSCs. ROCK Inhibitor Y27632 also has been shown to enhance the survival rate of stem cells during cryopreservation. Note that Rock inhibitor aliquots are sensitive to light and repetitive freeze thaw cycles. Make sure to use these aliquots within the supplier’s recommended shelf life.

    Dr. William Stanford-2022

    12. Harvesting and Caring for hESC/ iPSC

    1. The day after cells were thawed take a look at them under the microscope to determine survival rate. NOTE: it is normal to observe a high number of unattached cells. As long as some cells are attached, colonies can arise from them within 3 – 7 days.
    2. Remove media from the wells and pipette 2 mL (for a 6 well, 1mL for 12 well and 0.5mL for 24 wells plate) of fresh and warm PSC media per well. Return plate to incubator.
    3. Cells are fed until 60-70% confluent (follow supplier recommendations).
    4. As of day 2, cells should be observed and cleaned of any differentiated cells that might be growing.
    5. To clean cells, use the picking hood and scrape off differentiated cells with a pipette tip.
    6. Once cells are cleaned, change media as done in the above steps.

    (See Freezing hESC/iPSC or Passing hESC/iPSC)

    NOTE:

    PSC media was determined to be more efficient in a hypoxic environment. We have also observed that less differentiation occurs when cells are grown in hypoxic incubators versus normoxic. Lastly, seeded cells have a better survival rate when using a hypoxic incubator.

    Normoxic: 37°C, 21% O2, 5% CO2

    Hypoxic: 37°C, 5%O2, 10% CO2

    Dr. William Stanford-2022

    13. Passing hESC/iPSC

    1. Add 2mL of PSC media to a 6 well matrigel coated plate and set aside.
    2. Take the plate to be passaged and remove the media from the well and wash once with 1mL of PBS(-/-).
    3. Add 1mL of  the EDTA solution to the well and leave for 3-4 minutes at room temperature.  Don’t move the plate around as cells can start detaching.
    4. Remove EDTA solution and add 1mL of PSC media. Do not leave EDTA on the cells for more than 4 minutes as this will cause the cells to lift off.
    5. Scrape cells using a cell scraper and divide cells amongst the 6 wells of your plate containing PSC media. Avoid excessive breaking up of the colony pieces, and try to be gentle with scraping. Try to keep cells in large chunks. Use a wide mouth pipette tip to break up clumps if needed. Excessive breaking up of cells may cause cell death or excessive spontaneous differentiation following passaging.
    6. Incubate at 37°C after evenly distributing cells in each well (8 figure or L shape shaking). Avoid plate disturbance for 24 hours post passaging.

    NOTE: Once the cells have been scraped you want to transfer them to the new plate as soon as possible because the cells will reattach quickly (within 5 minutes).

    If EDTA is left on the cells for more than 4 minutes, the cells can start to detach. If this happens, simply collect the cells in a 15mL falcon with 4mL of PSC media. Spin cells at 130 rcf for 5 minutes. Resuspend the pellet with 1mL of media and divide evenly among a 6 well matrigel coated plate (160uL per well).

    EDTA solution:  Add 500uL of 0.5M EDTA (pH 8.0) into 500mL of DPBS (-/-). Add 0.9g of NaCl. Filter the solution to sterilize and store it at 4°C for up to 6 months.

    From the paper:

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

    Jeanette Beers,1 Daniel R. Gulbranson,2,3 Nicole George,4Lauren I. Siniscalchi,1 Jeffrey Jones,4,5 James A. Thomson,2,3,6 and Guokai Chen1,2

    14. Freezing hESC/iPSC

    1. Turn on Bio-Cool (Controlled Rate Freezer) and adjust temperature to -7°C.
    2. Remove cells from the incubator and observe confluency and morphology under the microscope.
    3. If the wells are 70% confluent, remove old media and wash once with PBS(-/-) then add 1 mL of EDTA solution (see passing with EDTA solution) per well.
    4. Incubate at room temperature for 3-4 minutes.
    5. Aspirate the EDTA solution and add 1 mL of cold mFreSR media (cat#05855, Stem Cell Technologies).
    6. Use a cell scraper to lift the cells gently. Keep cells in large chunks as much as possible and avoid pipetting up and down.
    7. Transfer cells/mFreSR to a cryotube using a wide mouth tip. Keep vials on ice until ready for step 8.
    8. Place tubes in Bio-Cool and incubate for 10 minutes.
    9. Get liquid nitrogen.
    10. After 10 minutes, seed the cells by dipping a spatula into liquid nitrogen and touching the side of the cryo-vial for approximately 10-30 seconds or until you see a crystal form on the side of the cryo-vial.
    11. Start program 1 by pressing “PROG” button and going through the program by pressing the button again and you should see the rate of 0.5°C/min. , then press “RUN”.
    12. Once temperature reaches -65°C the cryo-tubes can be transferred and stored in liquid nitrogen.

    Alternatives

    Another option is to place the cryo-tubes in a freezing container (Biocision-CoolCell) and store at -80°C overnight. Move the cryo-tubes to liquid nitrogen (liquid or vapor phase) on the following day.

    Dr. William Stanford-2022