What are Embryonic Stem Cells?

Embryonic Stem (ES) cells are derived from the Inner Cell Mass (ICM) or Epiblast of early embryos while they are little more than a ball of cells. The ICM is what gives rise to the actual embryo, while the trophectoderm (TE) cells become extra-embryonic tissues such as the placenta. In this article, unless otherwise stated, “ES cell” refers to embryonic stem cells derived from mice. ES cells were first isolated and cultured in the early 1980s.



What defines ES cells?

In culture ES cells can be identified through:

• Their immortality over many cell cycles.

• Their developmental potential:

o Their ability to fuse with a genetically distinct embryo to produce fertile chimeric offspring. (Note that this has yet to be demonstrated for primates and for ethical reasons this has not been attempted with human ES cells).

o Their ability to form teratomas consisting of cell types from all three germ layers (endoderm, mesoderm and ectoderm) when injected into adult tissues.

• The culture conditions required to maintain the population.

• The morphology of the cells.

• The transcriptional profile of the cells, measured using microarrays.

• The epigenetic profile of the cells.

Totipotency and pluripotency

A fertilised egg has the potential to form all of the cell types in an entire embryo – it is referred to as being totipotent. As this cell and its daughter cells divide their fate becomes increasingly restricted. The cells of the ICM can form any cell type from the embryo, but not of extra-embryonic tissues – they are referred to as being pluripotent. Because ES cells are derived from ICM cells they retain the characteristic of being pluripotent.

ES cells are also immortal – they can divide indefinitely in vitro, provided that they are supplied with the necessary growth factors. One reason that they can do this is because they express telomerase, an enzyme which repairs the telomeres at the end of chromosomes. Without telomerase, cells can only divide a fixed number of times before they become senescent and die. This is known as the Hayflick limit.

So ES cells are both pluripotent and immortal, but that does not mean that you can’t have one without the other. Cell lines have been derived from mice that are immortal, but nullipotent (they cannot give rise to any other cell types).



Table of Potencies



Immortality and cancer

In evolutionary terms, why is immortality a property of stem cells, but not normal somatic cells? One reason is so that tissues can grow and regenerate in a controlled way, for example the multipotent intestinal crypt stem cells divide to produce new cells to replace those lost from the microvilli structures. Another important reason is to prevent cancers. The Hayflick limit can be thought of as a self-destruct button for cells, preventing those that are likely to acquire mutations (for example skin cells exposed to UV) from surviving long enough to become cancerous.

Heterogeneity of derived cell lines

As the embryo develops, the cells of the ICM show increasing restriction of fate. They transition from being able to give rise to any cell type to being able to produce progressively fewer. Adult stem cells are generally limited to producing cells of a particular tissue. Stem cell lines derived from different developmental stages of mouse embryos appear to mimic these transitional states. For example mouse ES cells and mouse Epiblast Stem cells (EpiSCs) require different culture factors in vitro and show different pluripotency markers. This is important because it suggests that ES cells are not just an in vitro construct, but correspond to a real embryonic cell identity. It also means that an ES cell is not just a single cell type, but a continuum of states.

There are some transcription factors, such as OCT4/POU5F1, NANOG and SOX2, which act to maintain pluripotency in all these cell types.



hES cells and their correspondence to mES cells

The continuum of states is relevant when we consider human ES (hES) cells. Research on hES cells is comparatively new compared with mouse ES cells. Although humans and mice are very closely related when compared with other model organisms, features of their embryology are different. This means that it is not clear which human cell lines correspond to which mouse lines. It has been argued that human ES cells correspond to mouse EpiSCs. This is because:

• hES cells and EpiSCs both require nodal or activin expression, while mouse ES do not.

• Neither hES cells or EpiSCs are maintained by LIF, which mouse ES cells need.

• They show similarities in response to culture conditions, such as poor growth when dissociated.

However, they do show differences in cell surface markers and gene expression.

hES and their correspondence to human embryonic cells

Some of the markers expressed by human ES cells are also seen in the human ICM cells. This suggests they may have a similar surface phenotype and identity to cells of the pre-implantation epiblast. In general, the properties of cultured stem cells are thought to be due to a combination of where the cells originated and any adaptive changes that occur due to propagation in vitro.

ES cells are not germ cells

It is important to note that ES cells and germ cells are not equivalent. Germ cells give rise to the gametes, which give rise to an embryo after fertilisation, but germ cells are not stem cells because they can only produce gametes and not any other cell type of the body.



Culture conditions

Like all cells grow in vitro, ES cells require certain conditions in order to be cultured. In general, mammalian cells need conditions that mimic their in vivo environment, including Oxygen and Carbon Dioxide concentration, pH, temperature, glucose and other nutrient concentrations, and growth factors.

There are key signalling systems that maintain the stem cell state in vitro, which act to suppress differentiation and promote proliferation. These systems are extremely complex and include extracellular signalling molecules, membrane receptors, intracellular signalling cascades, and transcription factors.

ES cells require factors to inhibit differentiation and promote survival and division. Over time these factors have been refined to be more specific and increase the efficiency of ES cell derivation.

At first ES cells were grown on a feeder layer of mitotically inactivated fibroblasts along with foetal calf serum in the culture medium. Through fractionation of the culture medium, the small signalling cytokine Leukaemia Inhibitory Factor (LIF) was found to be secreted by the fibroblasts. This meant that the fibroblasts could be removed so long as LIF was added separately. LIF activates the JAK-STAT pathway. Note that LIF inhibits the differentiation of mouse ES cells, but is not required for the culture of human ES cells. LIF can also be removed if there is forced expression of the pluripotency-associated transcription factor NANOG.

Later the foetal calf serum was replaced with Bone Morphogenic Protein 4 (BMP4) to inhibit differentiation.

Activin and nodal are both members of the TGF-beta protein family, both signal through the same receptor, and both suppress differentiation in human ES cells.

More recently, it has become possible to culture ES cells without feeder cells, serum or cytokines using combinations of small-molecule inhibitors of particular pathways including FGF, Mek, Erk, Gsk3. These conditions are known as 2i or 3i, depending on the factors used, and the protocols for ES cell culture are still being improved. 2i/3i conditions have been successful in culturing ES from previously recalcitrant strains of mouse and from strains of rats. These conditions produce ES cells with much higher efficiency than before, which supports that view that the derivation of ES cell lines is not just selection of a few rare cells in culture.



Signalling pathways in stem cell maintenance

Stem cells, in vivo or in vitro, maintain pluripotency and ability to self-renew through the action of extrinsic and intrinsic signaling pathways. These pathways cause changes in gene expression through activation or repression of transcription factors, which alter gene expression. This can involve complex positive and negative feedback loops.

 

Notch

As both the notch receptor and most of its ligands are transmembrane proteins, notch signaling generally occurs between adjacent cells. Notch signaling involves many different ligands and receptors, and its effects strongly affected by cellular physiological contexts. In general though it seems that notch signaling often acts to inhibit differentiation. For example, notch signalling acts early in development to inhibit differentiation of neural stem cells (NSCs) in the brain and so maintain a pool of neural progenitor cells. However, later notch signalling can promote the differentiation of certain cell types. This highlights the tissue-specific nature of the signaling pathway.

 

Wnt/ß-catenin

The Wnt/ß-catenin pathway is involved in maintaining self-renewal of both mouse and human ES cells. However, there is evidence that activation of Wnt in ES cells can also promote differentiation. It appears that this discrepancy is due to the fact that ß-catenin can form different transcription regulating complexes in the nucleus. For example, it seems the ß-catenin/p300 complex promotes differentiation of ES cells, whereas the ß-catenin/CBP complex promotes self-renewal. Again, this shows how the effects of signaling pathways are dependent on context.Wnt signaling is also not just a single pathway, and can include ß-catenin (canonical) or not (non-canonical).

The Wnt and Notch pathways interact. For example, inhibiting Notch signaling in regenerating muscle causes increased Wnt activity and premature differentiation.

 

Oct4

Oct4 is a transcription factor that promotes self-renewal and is strongly expressed in ES cells. It is activated by Nanog. It has also been shown to interact with ß-catenin through co-immunoprecipitation.

Oct4 has been shown to regulate its own expression through negative feedback – preventing excessive up-regulation or down-regulation.

 

Sox2

Sox2 is another key transcription factor in the maintenance of pluripotency. It regulates multiple other transcription factors including ones that affect Oct4 expression.

Sox2, Oct4 and Nanog regulate many of the same target genes.

 

TFG- ß

This family includes TGF-ßs, bone morphogenic proteins (BMPs), growth and differentiation factors (GDFs), activins and nodal.

TGF-ß or activin signaling is necessary to maintain pluripotency in hES cells and EpiSC cells (through the regulation of Nanog), and is important in the proliferation of ES cells.

 

BMPs

In hES cells, BMP signaling promotes mesodermal and trophectodermal differentiation. However, in mouse ES cells BMP4 inhibits the Erk and p38 MAPK pathways and promotes self-renewal.

 

Oxygen concentration

Muscle cell differentiation is inhibited by hypoxia, which stabilises the transcription facter HIF1. Chondrocyte differentiation is promoted by hypoxia.

 

MAP Kinase

Mitogen Activate Protein (MAP) Kinase cascades are often triggered by Receptor Tyrosine Kinase (RTK) signaling. As RTK signalling is instrumental in embryological differentiation, blocking MAP kinase activity enhances stem cell maintenance.

ES cells vs iPSCs

In 2012 the Nobel Prize for Physiology or Medicine was award to John B. Gurdon and Shinya Yamanaka "for the discovery that mature cells can be reprogrammed to become pluripotent." Gurdon’s work in 1958 showed that an adult frog cell nucleus could be reprogrammed to a totipotent state by factors in an egg cytoplasm and give rise to adult clones of the original animal. In 2006 Yamanaka identified four transcription factors which, when expressed in adult mouse fibroblasts through viral vectors, caused those cells to dedifferentiate into a pluripotent state. Injection of these cells into early embryos gives rise to fertile chimeric mice.

These Induced Pluripotent Stem Cells (iPSCs) are similar to ES cells in that they are immortalised and can differentiate into any cell type of the body. Derivation of iPSCs has generated much excitement because:

1) They can be generated from adult cells, avoiding the destruction of human embryos.

2) Cells generated from patient-specific IPSC lines would avoid the immunological issues of ES cells if used for therapy.

3) IPSCs derived from individuals with genetic diseases can be used to generate cells of the affected tissue and study it in vitro.

However, there are some ways in which iPSCs are less useful than ES cells:

1) The epigenetic reprogramming of adult cells to iPSCs can often be incomplete, so these cells show biased or restricted differentiation behaviour.

2) The reprogramming process introduces mutations into the iPSC DNA, meaning that iPSCs have to be extensively characterised before they can be used in any study.

3) Currently the reprogramming factors (Oct4, Sox2, Klf4, c-Myc) are introduced through retroviral vectors. Therefore the IPSCs generated are transgenic.

So while iPSCs look set to become an important tool in biology in years to come, currently ES cells are more extensively characterised and thus, better understood.

Potential applications of ES cells

The potential applications of ES cells in science and medicine are huge; from studying development and disease states in vitro to generating cells and tissues for therapy and transplantation.This is because ES cells can not only be maintained indefinitely in culture, but that we are increasingly able to induce directed differentiation. By exposing the cells to particular culture factors and conditions in turn, often by mimicking what they would be exposed to in normal embryogenesis, we can theoretically produce any cell type in the body. With the right scaffolding it should also be possible to grow tissues and perhaps organs.

In one trial, neural support cells produced from hES cells were injected into patients with spinal injuries. Although this does not seem to have benefitted the patients, the trail has now been run for long enough to provide strong evidence that ES cell-based therapies could be safe.

Another trial has showed some evidence that ES cells can be used to partially rebuild the layer of photoreceptor-supporting cells in patients with certain kinds of blindness.

Moral controversy

Because the derivation of human ES involves the manipulation of human embryos, the field of ES cell research is the subject of a large amount of ethical debate.

It should be noted that human ES cells are generated from surplus or unsuitable embryos that are generated by in vitro fertilisation (IVF) during fertility treatments and donated with consent. As these embryos would otherwise be discarded, one could argue that practice prevents waste of precious materials that could further the understanding of disease and development.

There is a legal distinction between the derivation of new ES cell lines, which is heavily restricted, and working on existing cell lines, which does not involve the use of human embryos.

It has been shown to be possible to extract and culture human ES cells without destroying the embryo. Does this resolve the problem? If you bisected an early embryo and took one half for ES cell culture and allowed the other half to implant and develop into a whole foetus, would that still count as destruction of an embryo? Clearly this debate is very complex and points of view will most certainly differ on the subject now and in the future.

These ethical issues mean that human ES cell research is also a legal battleground. On the 23rd of August 2010, all federally funded ES cell research in the USA was shut down for 17 days due to a lawsuit filed by opponents to the research.  An appeals court suspended the injunction while the case went through the courts, and the case was eventually unsuccessful.

Further Reading

http://www.bioethicsinternational.org/blog/2013/01/15/high-court-ensures-continued-us-funding-of-human-embryonic-stem-cell-research/

http://www.nature.com/news/court-lifts-cloud-over-embryonic-stem-cells-1.12215

References

Evans M, Kaufman M (1981). Establishment in culture of pluripotent cells from mouse embryos. Nature 292 (5819): 154-156. Doi:10.1038/292154a0.

Williams RL, Hilton DJ, Pease S, et al (1989). Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature 336 (6299):684-687. Doi:10.1038/336684a0.

Rossant J (2001). Stem Cells from the Mammalian Blastocyst. Stem Cells 2001;19:447-482

Sakaki-Yumoto M, Katsuno Y, Derynck R (2013). TGF-β family signalling is stem cells. Biochimica et Biophysica Acta 1830 (2013) 2280-2296

Wray J, Hartman C (2012). WNTing embryonic stem cells. Trends in Cell Biology, Vol. 22, No. 3. Doi:10.1016/j.tcb.2011.11.004

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