Induced Pluripotent Stem Cells

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

  1. Progeria iPSCs Background Information for the Non-scientist 
  2. Purpose of Induced Pluripotent Stem Cell Generation and Distribution by The Progeria Research Foundation  
  3. Generation of Hutchinson-Gilford Progeria Syndrome Induced-Pluripotent Stem Cells (iPSCs) 
  4. Quality Control: Validation and Characterization 
  5. Original Starting Material from which iPSCs Were Derived
  6. Join our Email List for Future iPSC Updates and New Cell Lines  
  7. Questions?  Contact us.
  8. Ordering iPSC Lines 
  9. HGPS and Control iPSC Culture Media Preparation 
  10. Preparing Madrigal Plates 
  11. Thawing hESCs and iPSCs
  12. Harvesting and Caring for hESCs and iPSCs 
  13. Passaging with EDTA solution
  14. Freezing hESCs and iPSCs

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 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
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 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.

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
iPSC and ESCs need to be fed with either TeSR-E8 from Stem Cell Technologies (cat# 05990) or Essential 8 medium from ThermoFisher Scientific (cat#A1517001). Please follow the supplier’s recommendations for storage.

10.Preparing Madrigal Plates

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

  1. Thaw matrigel bottle (10ml) at 4C.
  2. Take matrigel bottle and add 10ml of cold DMEM/F12 media.
  3. Aliquot 1ml of matrigel in pre-chilled 15ml falcon tubes.
  4. Freeze all aliquots at -20C
  5. To make matrigel plate, remove one aliquot of matrigel (1ml) from -20C and add 18ml of cold DMEM/F12. Let pellet thaw and once thawed mix well (without creating bubbles) and plate (1ml/well for 6 well plate, 0.5ml/well for 12 well plate, 0.25ml/well for 24 well plate). These plates are labelled as MG1.
  6. If using plate immediately, let plate sit at room temperature for 1 hour, observe matrigel under the microscope. Matrigel should be well dispersed and not “clumpy”.
  7. Once 1 hour is passed transfer matrigel to a second plate (this plate is labelled MG2). If using right away, incubate at room temperature for 1 hour.
  8. If using plates at another time, wrap edge of plate with paraffin and store at 4 degrees.


Matrigel – BD/Fisher, cat#CB-40230
DMEM/F12 – Life Technologies, cat#11330-057

Dr. William Stanford-2018

11. Thawing hESCs and iPSCs

Thawing ES or iPS cells (per cryo-vial)

  1. Take matrigel plate out of 4C and warm at room temperature for one hour or make fresh matrigel plate (see preparing matrigel plate protocol).
  2. Warm 4ml of PSC media (pluripotent stem cell media) in a 15ml falcon tube.
  3. Remove cells from freezer and swirl in 37 degree water bath until only a small ice chunk is left. 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 prewarmed media
  6. Spin at 850rpm for 5min
  7. Remove supernatant
  8. Add 2ml of PSC media and with a wide mouth tip break up clumps. Transfer media to one well of a 6 well plate add 2ul of ROCK inhibitor (Y27632, final concentration of 10uM)​.
  9. 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
TeSR-E8 from Stem Cell Technologies, catalogue # 05990 or Essential 8 Medium from ThermoFisher Scientific, catalogue # A1517001

What is ROCK Inhibitor Y27632?
ROCK Inhibitor Y27632 is a selective inhibitor of the Rhoassociated kinase p160ROCK. Treatment with ROCK Inhibitor Y27632 prevents dissociationinduced apoptosis of human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC), 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.

Dr. William Stanford-2018

12.Harvesting and Caring for hESCs and iPSCs

  1. Remove media from 4C and place in 37C water bath.
  2. The day after cells were thawed take a look at them under the microscope to determine survival rate. (If cells were frozen only with mFreSR the survival rate is appr. 20%, if cells were frozen using the Bio-Cool survival rate is appr. 60%).
  3. Remove media from the wells and pipette 2ml (for a 6 well, 1ml for 12 well and 0.5ml for 24 wells plate) of fresh PSC media per well.
  4. Place plate in 37C incubator (hypoxic condition: 10% CO2 and 5% O2).
  5. Cells are fed every day for the next 3-5 days until 80% confluent.
  6. Cells should be observed and cleaned of any differentiated cells that might be growing.
  7. To clean cells use the picking hood and “scrap off” differentiated cells with a pipette tip.
  8. Once cells are cleaned changed media as done in above steps.

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

NOTE:
PSC media was determine 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. Also when seeding cells there’s a better survival rate when using a hypoxic incubator.

Normoxic: 5% CO2
Hypoxic: 5%O2, 10% CO2

Dr. William Stanford-2018

13.Passaging with EDTA solution

  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 3-4min at room temperature.  Don’t move the plate around as cells can start detaching.
  4. Once 4 min. is up remove EDTA solution and add 1ml of PSC media. Do not leave EDTA on the cells for more than 4 min as this will cause the cells to lift off.
  5. Scrape cells (with cell scrapper) and divide cells amongst the 6 wells of your plate containing PSC media (I’ve been taking 160ul into each well). Avoid breaking up the pieces in very small pieces. Try to keep large chunks. Preferably use a wide mouth pipette tip.

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 5min).

If EDTA is left on the cells for more than 4mins. the cells can start to detach. If this happen simply scrap cells and collect in a 15ml falcon with 4ml of PSC media. Spin cells at 850rpm for 5 min. 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 and adjust the osmolarity to 340 mOsm. Filter the solution to sterilize and store it at 4C for up to 6 months. We want the least amount of disturbance for the cells during dissociation therefore the EDTA solution is at the same osmolarity as the PSC media.

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 hESCs and iPSCs

  1. Turn on Bio-Cool (Controlled Rate Freezer) and adjust SP temperature to -7C.
  2. Remove cells from incubator and observe confluency and morphology under the microscope.
    NOTE: Looking for ~80% confluency
  3. If confluent, remove old media and wash once with PBS(-/-) then add 1 ml of EDTA solution (see passaging with EDTA solution) per well.
  4. Incubate at room temperature for 3-4 minutes.
  5. Aspirate the EDTA solution and add 1 ml of mFreSR media (cat#05855, Stem Cell Technologies)
  6. Use cell scraper to detach the cells from the well (gently).
  7. Transfer cells/mFreSR to a cryotube using a wide mouth tip.
  8. Place tubes in Bio-Cool and incubate for 10min.
  9. Get liquid nitrogen.
  10. After 10min. seed the cells by dipping a spatula into liquid nitrogen and touching the side of the cryo-vial for approximately 10 seconds or until you see a crystal form on the side of the cryo-vial.
  11. Start program 1 by pushing “PROG” button and going through the program by pushing the button again and you should see the rate of 0.5C/min. , then press “RUN”.
  12. Once temperature reaches -65C the cryo-tubes can be transferred and stored in liquid nitrogen.

Alternatives
1-Another option is to place the cryo-tubes in a freezing container (Biocision-CoolCell) and store at -80C overnight. The next day store cryo-tubes in liquid nitrogen.

Dr. William Stanford-2018