Pluripotent stem cell (PSC) culture

April 20, 2014

This Session's Expert:

Dr. Nirupama Shevde

Company: Life Technologies

Job Title: Customer Training Manager

Introduction

Pluripotent stem cell (PSC) culture has evolved over the past decade, just as stem cell researcher needs have been changing and the market has been growing.  Initial culture conditions for stem cells used FBS and mouse embryonic feeders (MEFs).  The need for a more defined system brought about the introduction of KnockOut™ Serum Replacement (KSR) in 1997 for the culture of mouse embryonic stem cells (ESCs). Even today, KSR has remained an integral part of the feeder based workflow for both mouse and human PSC culture. The desire to move away from the variability and work required to maintain MEFs led to the development of feeder-free media systems.  There are a number of options for feeder-free based media and matrices; StemPro® hESC SFM and Essential 8™ media are two robust options available and both work with Geltrex® and Vitronectin matrices. As choice of media systems for PSCs grows, understanding which media to use for a specific application becomes an important consideration.

Join Dr. Nirupama (Rupa) Shevde, customer training manager at Thermo Fisher Scientific, to ask your questions regarding the culture of pluripotent stem cells. Questions can range from best culture options and technologies for deriving pluripotent stem cells to how to adapt cells to a new media and everything in between.

Question 1

My lab has begun to try and reduce the amount of serum used in our pluripotent cell culture media. Do you have a method you would recommend for weaning the cells or a lowest serum amount you think would work?

I am assuming that you are working with human pluripotent stem cells and you use Knockout serum replacement (commonly known as KSR). A lot of scientists have moved away from KSR as it is not defined and includes components that are of animal origin. We have a defined medium called Essential 8 medium that is serum free and is widely used by scientists to culture human pluripotent stem cells. I have attached the necessary protocols and a publication for your review. We have designed a protocol that will allow your cells to be weaned off the serum in a gentle way by changing one variable at a time in the culture conditions, so as to not stress the cells too much. Essential 8 medium has 8 components and has been successfully used to culture both human embryonic stem cells and human induced pluripotent stem cells. Cells cultured in this medium have shown to retain pluripotency, ability for trilineage differentiation and maintain a normal karyotype for more than 50 passages. Essential 8 medium can be used with substrates such as Geltrex or vitronectin.

The links below may also be helpful:

Culturing PSCs in Essential 8™ Medium - http://tools.lifetechnologies.com/content/sfs/manuals/feeder_free_PSCs_in_essential8_medium.pdf

Frequently Asked Questions – Essential 8™ Medium and Vitronectin - http://tools.lifetechnologies.com/content/sfs/manuals/FAQ_Essen8_Medium_vitronectin_man.pdf

Scalable expansion of human induced pluripotent stem cells in the defined xeno-free E8 medium under adherent and suspension culture conditions -http://www.ncbi.nlm.nih.gov/pubmed?cmd=Retrieve&dopt=AbstractPlus&list_uids=23973800

Authors: Wang Y, Chou BK, Dowey S, He C, Gerecht S, Cheng L,
Journal: Stem Cell Res (2013) 11:1103-1116

Question 2

We are having difficulty maintaining genome integrity in our ipsc in culture. We have seen several mutational changes when the cells are in culture for longer periods. Any suggestions on how to troubleshoot.

It is difficult to answer this question without having details of your culturing conditions. Following are the most common reasons for mutational changes and abnormal karyotypes in human pluripotent stem cell cultures-

1. Overgrowth of cells- if cells are routinely overgrown and not passaged when they are in log phase (actively growing), the stress can cause mutations

2. Use of a harsh dissociation agent- if a harsh dissociation agent such as trypsin is used for routine passaging and maintenance of human pluripotent stem cells, the cells get dissociated as single cells and will not be able to survive in cultures. IF they do survive, they will adapt under stress and have mutations

3. In general any culture conditions that will induce inadvertent stress or selective pressure will result in mutations after long-term passaging

4. Also, passaging the cells too soon (every two or three days) can induce stress

5. I would suggest to check culture conditions, dissociation agents, dissociation methods, passaging frequency as possible stressors

Question 3

What alternative methods exist for reprogramming adult cells into iPS cells, excluding methods that introduce foreign genetic material into the host cells? If there are any, what are the major limitations of these methods?

We currently offer several reprogramming products that are considered non-integrating, for a complete list of reprogramming options available, visit lifetechnologies.com/reprogramming.

CytoTune –iPS Sendai Reprogramming Kits - http://www.lifetechnologies.com/us/en/home/life-science/stem-cell-research/induced-pluripotent-stem-cells/sendai-virus-reprogramming.html?cid=fl-cytotune
The CytoTune™-iPS Reprogramming Systems use vectors based on replication-incompetent Sendai virus (SeV) to safely and effectively deliver and express key genetic factors necessary for reprogramming somatic cells into iPSCs. In contrast to many available protocols, which rely on viral vectors that integrate into the genome of the host cell, the CytoTune™ Reprogramming System uses vectors that are non-integrating and remain in the cytoplasm (i.e., they are zero-footprint) and divide out over time.

The CytoTune™ –iPS systems work with both fibroblast and blood samples in feeder and feeder free conditions. There are multiple publications that reference other cell types that have been successfully reprogrammed. Learn more at this link - http://www.lifetechnologies.com/us/en/home/life-science/stem-cell-research/induced-pluripotent-stem-cells/sendai-virus-reprogramming/cytotune-publications.html

We also have two different Episomal reprogramming systems:

Epi5™ Episomal iPSC Reprogramming Kit - http://www.lifetechnologies.com/order/catalog/product/A15960?ICID=search-product
The Epi5™ Reprogramming Vectors contain an optimized mixture of three episomal vectors with an oriP/EBNA-1 (Epstein-Barr nuclear antigen-1) backbone for delivering the reprogramming genes, Oct4, Sox2, Lin28, L-Myc, and Klf4. High transfection efficiency due to oriP/EBNA-1 mediated nuclear import and retention of vector DNA allows iPSC derivation in a single transfection (Yu et al.,2011). In addition, silencing of the viral promoter driving EBNA-1 expression and the loss of the episomes at a rate of ~5% per cell cycle due to defects in vector synthesis and partitioning allows the removal of episomal vectors from the iPSCs without any additional manipulation (Nanbo et al., 2007).

We have also shown that we can use Lipofectamine® 3000 (https://www.lifetechnologies.com/order/catalog/product/L3000015?ICID=search-product) for reprogramming with the Epi5™ kit and get excellent efficiency with fibroblast reprogramming.

Episomal iPSC Reprogramming Kit - http://www.lifetechnologies.com/order/catalog/product/A14703?ICID=cvc-reprogramming-c1t1
The Episomal iPSC Reprogramming Vectors are an optimized mixture of three vectors that can reprogram somatic cells to iPSCs without integration. The three episomal vectors have the oriP/EBNA-1 (Epstein-Barr nuclear antigen-1) backbone that delivers the reprogramming genes, Oct4, Sox2, Nanog, Lin28, L-Myc, Klf4, and SV40LT. This system has successfully demonstrated reprogramming of fibroblasts, CD34+ cells, and Peripheral Blood Mononuclear Cells (PBMCs). High transfection efficiency due to oriP/EBNA-1 mediated nuclear import and retention of vector DNA allows iPSC derivation in a single transfection.2 In addition, silencing of the viral promoter driving EBNA-1 expression and the loss of the episomes at a rate of ~5% per cell cycle due to defects in vector synthesis and partitioning allows the removal of episomal vectors from the iPSCs without any additional manipulation.

Current limitations to episomal systems are the need to use electroporation systems to enable the vectors to enter the cell. Keep in mind, the ability to use Lipofectamine 3000 with the Epi5™ reprogramming system does ameliorate some of this issue.

Question 4

How important is finding a reliable and economical source of recombinant proteins for the defined animal free culture of iPSCs?

Stem cell scientists use a variety of media systems based on their specific research needs. All of media systems are of the highest quality and are routinely purchased by scientists. Many publications also present data that is generated using these media systems. Since the needs are diverse, there are a lot of media systems spanning from feeder-based to completely defined. For your review, we have included a web link highlighting media used to culture human pluripotent stem cells. Lifetechnologies.com/pscculture

Question 5

As a student working on a startup project, I would like to learn more about the current needs and problems of researchers that are using iPS cells. I could use as much help as I can get. Thanks!

It’s a bit hard to make recommendations for your start-up without having a lot more information and specifics about what you are trying to accomplish within your research. Here are some resources that may help you learn more. Good luck in your start-up endeavor!

Life Technologies resource -

Stem cell resources – includes protocols, webinars, training opportunities and more
http://www.lifetechnologies.com/us/en/home/life-science/stem-cell-research/stem-cell-research-resources.html

Industry resource –

International Society for Stem Cell Research
http://www.isscr.org/

Question 6

How many passages can I expect to get out of my cells in long-term culture and on what days do you recommend splitting them? Also do you have any suggestions on how to increase the rate of expansion of cells?

Human pluripotent stem cells (both embryonic and induced pluripotent stem cells) can be successfully cultured and maintained for a very long time. Although they can be cultured for very long periods of time, most researchers and laboratories have guidelines as to how many passages they would use cells before they thaw a new vial (most guidelines suggest that scientists will use cells unto 50-75 passages before they will retire those cells and thaw a new vial). It is important to perform routine karyotype analysis to ensure that the cell line has not acquired an abnormal karyotype after long-term passaging.

There are no specific ways to increase the rate of expansion of cells. Most human pluripotent stem cell lines are routinely passaged on day 4 or day 5 and it is vital to keep the cells on a routine in order to avoid stress (passaging them too early or letting them overgrow will cause stress). Scientists have developed techniques to scale up the passaging by changing the split ratios and this is the optimal way to increase the yield. A good example is the use of EDTA as a dissociation agent when cells are cultured in a defined medium such as Essential 8 medium with a defined substrate such as Vitronectin. In this case, since the use of EDTA generates smaller colonies (compared to other dissociation agents such as collagenase and dispase) the passaging ratio can be adjusted to 1:8 1:10 or even 1:12.

We have attached the relevant protocols and a publication for culturing cells in Essential 8 medium on Vitronectin using EDTA.

Please find some links below which you may also find helpful:

Culturing PSCs in Essential 8™ Medium - http://tools.lifetechnologies.com/content/sfs/manuals/feeder_free_PSCs_in_essential8_medium.pdf

Frequently Asked Questions – Essential 8™ Medium and Vitronectin - http://tools.lifetechnologies.com/content/sfs/manuals/FAQ_Essen8_Medium_vitronectin_man.pdf

Scalable expansion of human induced pluripotent stem cells in the defined xeno-free E8 medium under adherent and suspension culture conditions

Authors: Wang Y, Chou BK, Dowey S, He C, Gerecht S, Cheng L, Journal: Stem Cell Res (2013) 11:1103-1116 - http://www.ncbi.nlm.nih.gov/pubmed?cmd=Retrieve&dopt=AbstractPlus&list_uids=23973800

Question 7

We are looking for a synthetic surface for our iPSCs. Do you have any suggestions and what should we look for when evaluating?

The most commonly used and commercially available synthetic substrate is Synthemax by Corning. This substrate has been used along with Essential 8 medium by many scientists. These are the following evaluations:

1. Test for pluripotency after culturing cells for a long period of time (50 passages or more).

2. Test for the ability to form Embryoid Bodies and potential for tri-lineage differentiation

3. Test for normal karyotype after long-term use of Synthemax

4. Test for the ability of the cells to differentiate into the cells of your interest

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