iPSCs: How to achieve top quality cultures

In 2006 Shinya Yamanaka published his ground-breaking research showing that adult cells

could be re-wired into an embryonic like state. These so called ‘induced pluripotent stem

cells’ (iPSCs) had acquired the remarkable ability to become any cell type in the adult body.

Cells lost to disease or injury could now theoretically be replaced using a person’s own cells,

and human cell types could be grown in the lab like never before. Just 6 years after publishing his work, Shinya Yamanaka went on to win the 2012 Nobel Prize for physiology and medicine and the burgeoning field of iPSC research has continued to flourish ever since.

The history and applications of iPSC research

Pluripotent stem cells have the extraordinary capacity to become any cell type in the adult

body, but unfortunately only appear naturally for a very brief window during early embryonic development. In contrast, totipotent stem cells share this ability, yet can also contribute to the extra-embryonic tissues, such as the placenta and umbilical cord. At the other end of the spectrum, multipotent stem cells are found throughout development and adulthood, but can only produce a handful of different cell types.

Researchers have long been conscious of the potential applications of pluripotent stem cells.

Early work by Evans, Kaufman and Thomson showed that pluripotent stem cells could be

isolated from the inner cell mass of blastocyst stage embryos and cultured indefinitely in

vitro1,2. These were termed ‘embryonic stem cells (ESCs)’ and garnered a tremendous amount of controversy, since discarded embryos from IVF were used to make them. During this period federal funding for stem cell research in the USA was massively withdrawn3. This provided an impetus to find another, less ethically controversial, source of pluripotent stem cells.

Since the 1960’s, thanks to pioneering work by John Gurdon, it has been known that all adult

cells contain the complete genetic toolkit to make any other cell type. The only reason they

don’t is that they are epigenetically hard-wired through development to become a particular

specialised cell type. In the early 2000’s Shinya Yamanaka hypothesised, against all

conventional wisdom, that this wiring might be surprisingly simple to revert. He made an

educated guess of 24 genes that he thought might be able to do it, later describing this as like ‘buying a winning lottery ticket’. Through a process of elimination, he found that just four of these genes, when overexpressed, were enough to rewire adult cells back into a pluripotent state. These genes (C-Myc, SOX2, KLF4, and OCT4) are known as the ‘Yamanaka Factors’, and the cells became known as induced pluripotent stem cells (iPSCs)4.

Since Yamanakas breakthrough, the advances made using iPSCs have been unprecedented.

Clinical trials have already begun in humans using iPSC-based stem cell therapies to treat agerelated macular degeneration and Parkinson’s disease5,6.

Disease modelling and drug discovery has now become more sophisticated than ever. Human cell types that were previously impossible to obtain by other means can now be derived from patient iPSCs with disease specific genetic backgrounds. Developments in organoid technology and disease on chip technologies mean it is now possible to recreate tiny organ systems with different interacting cell types in a dish. Examples include miniature kidneys, modelling the blood brain barrier, and modelling the loss of nerve-muscle connectivity in motor neuron disease7-9.

This provides an unprecedented level of insight into disease and an innovative platform for drug discovery. In the future it might even be possible to grow entire organs from iPSCs that could be transplanted into humans10.

Achieving top quality iPSC cultures

Despite all the advancements in iPSC technologies, culturing iPSCs still remains a challenging

and complicated process. There are now more options than ever for reprogramming

methods, growth medias, cell attachment substrates, dissociation reagents, and passaging

techniques. Ultimately there is no one way to grow iPSCs.

One lab using MEF feeders, mTESR medium and colony passaging may have just as much success as another lab using feederfree attachment substrates, Essential 8 medium and single cell passaging. What is important is that you find an approach that suits you, and more importantly, that you know how to properly evaluate the quality of your cultures.

  1. Get to know your morphology

Observing the morphology of your iPSCs is the fastest and most effective way to monitor the

quality of your iPSC cultures. It is normal for iPSC morphology to change at different stages of a culture. The following is an example of a normal four-day iPSC culture using single cell

passaging, iPSbrew and laminin521 attachment substrate (Figure 1).

A After single cell passaging ROCK inhibitor is often used for up to 24h to improve cell

attachment and viability. This can have a dramatic effect on cell morphology; cells will not

form colonies and will take on a fibroblastic appearance (1a).

B Upon removal of ROCK inhibitor cells will shrink, cluster tightly, and form small colonies


C Mature colonies should have a clearly defined border, tightly clustered small cells with

uniform morphology (1c).

D It is important to passage the cells at about 70-80% confluency before large colonies

completely merge as this can lead to compromised viability and spontaneous differentiation


Looking out for signs of compromised cell viability and spontaneous differentiation is also

incredibly important, as both of these can severely impact the quality of your iPSCs.

A, Signs of compromised viability include cell shrinkage, cell rounding and excessive levels of

floating dead cells (Figure 2a).

B, Spontaneous differentiation can occur at the edge of the colony; fibroblastic looking cells

may start to grow outward and there will no longer be a clear colony border (2b).

C, Spontaneous differentiation can also happen at the center of the colony; cells will start to

grow on top of one another and may start to form patterned structures (2c).

  1. Monitor the growth of your iPSCs

It is important to keep an eye on the growth rates of your iPSCs.

- This can be done formally through cell counting/measuring colony size and plotting a

growth curve (Figure 3)

- Or it can be done more casually by noting the interval between passaging and the split

ratios used.

Either way it is important to monitor changes in the growth of your iPSCs as this can

indicate problems with the culture, from sub-optimal culture conditions through to

genetic/karyotypic abnormalities.

- Typically iPSCs double every 15-20h, however this may vary depending on the

reagents used and your cell line11.

- iPSCs should always be maintained at the log phase of growth.

  1. Monitor the pluripotency of your iPSCs

It is important to regularly immunostain/run a qRT-PCR for pluripotency markers to confirm

that you are properly maintaining your iPSCs as pluripotent stem cells. Some common

markers include:

- OCT4 (nuclear)

- SOX2 (nuclear)

- SSEA-4 (cell surface)

- Nanog (nuclear)

- TRA-1 60 (cell surface)

- KLF4 (nuclear & cytoplasmic

Immunostaining/flow cytometry is preferable over qRT-PCR as it allows you to identify the

percentage of pluripotent cells in the culture.

  1. Assess the differentiation potential of your iPSCs

In conjunction with assessing the pluripotency of your iPSCs it is also important to assess the

differentiation potential of them as well. iPSCs may be pluripotent but can be more or less

prone to differentiate down certain lineages due to epigenetic artifacts left over from

reprogramming. As such it is important to assess this. A common method is to generate

embryoid bodies (EBs) and stain for markers against the three germ-lineages:

- Common ectoderm markers: PAX-6 and SOX2

- Common endoderm markers: CSCR4 and SOX17

- Common mesoderm markers: SM22a and CD144

  1. Check your iPSCs for genetic/karyotypic abnormalities

Genetic and karyotypic abnormalities can occur at any stage of an iPSC culture. It is important to check for any abnormalities after initial reprogramming and at regular passaging intervals

– every 10 or so passages is a good benchmark. Such abnormalities can impact the growth

kinetics of your iPSCs and may negate any conclusions you can draw from your experiments.

G-banding is the most common way to assess the karyotype of your iPSCs, however more

recently ‘Karyostat’ genotyping has been used as an alternative since it can detect much

smaller chromosome abnormalities. Other genomic profiling approaches include HLA typing,

STR genotyping and CNV analysis.

  1. The silent killer: Mycoplasma

Mycoplasma can wreak havoc with any form of cell culture. Unlike most other bacterial or

fungal contaminations that are obvious within a matter of days, mycoplasma contaminations

can go completely undetected. Mycoplasma can severely compromise cell growth and proliferation, expose your cells to unwanted metabolites, and severely impact the levels of protein, RNA and DNA in your cultures negating the validity of your findings.

PCR detection tools are available and should be routinely carried out in iPSC labs. If a contamination is found it is crucial to dispose of all cultures with the contamination and get rid of associated reagents. Traditional antibiotics are ineffective against mycoplasma. However, some success can be achieved with long and intense exposure to plasmocin. This is not 100% effective12.


Culturing iPSCs is a challenging yet rewarding process. The ability to study any human cell

type from any genetic background is unparalleled and the breakthroughs made using iPSCs

will only continue to grow. While there may be many different ways to culture iPSCs, what is

important is that you know how to robustly assess and maintain the quality of your iPSCs.


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