How can embryonic stem cells be used as tools to understand how the brain develops before birth?

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How can embryonic stem cells be used as tools to understand how the brain develops before birth?

Introduction

Embryonic Stem Cells (ESCs) are cells isolated in the inner cell mass of a blastocyst. They are pluripotent with the advantage of giving rise to any cell in the body on mass (Reubinoff et al, 2001). ESCs have elucidated the information not only on our in utero development and all its intrinsic mechanisms, but also species-specific evolutionary traits and pathogenic precursors. Their ability to differentiate into any cell of the human body coupled with their potential to create a reservoir of unlimited donor cells only adds to their value as a model for research. This makes them a useful tool for studying neural development, specifically corticogenesis, in vitro as we can observe their diversity and complexity resulting from self-organisation (Anderson et al, 2014).

The key to modulating their pluripotency lies in microRNA (miRNA) which is a tiny molecule only 22 nucleotides long that regulate their cell fate. There has been a total of 600 miRNA discovered in humans so far. They work by repressing transcription factors namely Oct4, Sox2 and Nanog. This offers us the opportunity to use the miRNA signature as a means of distinguishing ESCs from differentiated cells (Reubinoff et al, 2001).

These studies hope to shine a light on the mechanisms that initiate and control the development of the brain. This will further our understanding of the causes of intractable disorders that plague us, and the evolutionary aspects surrounding them (Tadar and Struder, 2014)

Neurodevelopment via In vitro modelling

Corticogenesis is the process by which the brains most complex structure, the cerebral cortex, is created using in vitro technics with the goal of replicating the early patterning signals that take place during neural development. These technics involve both small molecule and morphogen based approaches to directed differentiation (Anderson et al, 2014).

Three direct induction technics have been formulated to this end namely embryoid body formation, co-culture on neural-inducing feeders and direct neural induction (figure 1).

Figure 1 Varying strategies in neuron cell induction

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Embryoid body formation involves triggering human embryonic stem cells (hESCs) differentiation in a serum free media using Rho-associated protein kinase inhibitor Y-27632 to improve formation of neural lineages. This is done by preventing cell death of cells that’s have dissociated from the hESC layer (Tadar and Struder, 2014). Neural rosettes are generally formed which are mechanically isolated using trypan blue staining (Reubinoff et al, 2001).

Stromal or astrocytes feeder based cultures generate populations of dopaminergic neurons due to their efficiently with cells that produce tyrosine hydroxylase which is involved in dopaminergic synthesis. There is however issues with this technic due to the heterogeneity of the cells produced. Causes for this may be due to the fact these feeders origins are generally non-human coupled with the lack of knowledge of the factors they secrete (Ambasudhan et al, 2014).

Direct neural induction involves signalling via the use of inhibitors such as transforming growth factor (TGF) and bone morphogenetic protein (BMP) wish is a dual SMAD inhibition. The benefit of this is an increased efficiency and homogeneity of the differentiated neural cells. This protocol allows for the derivation of many lineages pertaining to the nervous system. An example of the is cortical neuron diversity which has allowed us to generate forebrain neural precursors without the presence of morphogens. The morphogens are inhibited by the aforementioned TGF, BMP and Wnt signalling greatly enhancing the speed of neural induction (Tadar and Struder, 2014).

This dual SMAD inhibition yields a uniform population of early neural cells. If Sonic the Hedgehog (SHH) signalling is not added, the neural progenitors form dorsal cortical progenitors and glutamatergic cortical pyramidal neurons however if its added then ventral telencephalic progenitors will form differentiating into GABAergic and cholinergic neurons (Anderson et al, 2014). These subsequent changes further our knowledge of neural development ushering in a future of whole organ replacement.

Cell Cycle Control and Evolution

Using direct comparisons between mouse and human ESC grown using identical culture conditions revealed corticogenesis was significantly longer in the human ESC then those of the mouse. The times for maturation of the hESCs were in line with the protracted time for cortical neurogenesis in vivo with the cortical progenitors generating post mitotic neurons after 4 weeks. Corticogenesis then takes a further 10-15 weeks to complete after the appearance of radial glia like progenitors. This is a vast contrast with mouse ESC corticogenesis which is completed in 2-3 weeks (Anderson et al, 2014).

These results may relate to species-specific features as in the case with the human cortex having a larger diversity of neurons for example the outer radial glial which mice lack. These other types or progenitors add to the evolutionary changes that exist between species. Other factors include differential cell cycle control owing to the prolonged amount of time involved with maturation and the different intrinsic properties relating to self-renewal vs terminal differentiation. All serve to increase neural output and cortical expansion (Anderson et al, 2014).

This prolonged maturation can be seen in ESC-derived cortical neurons that have been transplanted into a mouse’s neonatal cortex taking upwards of 9 months to develop synaptic activity. This delayed maturation presents a relentless challenge for widespread application of this model as treatment for human diseases.

ESCs In vivo brain transplantation

At present we are beset by increasing numbers of neural disorders with no relative cure and only expensive palliative care to look forward to. ESCs offer us the opportunity to gain access to disease relevant cell type produced in large numbers for use in in vivo transplantation (Tadar and Struder, 2014).

This interest in transplantation has increased research into arealization and their underlining mechanisms so that area-specific cortical strategies can be devised (Anderson et al, 2014). An example of this is an experiment that was undertaken with human neural progenitors. They were transplanted into the brain of a new-born mouse and later it was found that they were widely distributed through the host’s parenchyma via migratory tracks. The neural progenitors had differentiated specific to their region, due to extrinsic cues from the host (Reubinoff et al, 2001).

Another experiment involved the transplantation of hESCs, which were not fully mature and specific to markers of the occipital cortex, to the frontal cortex of a mouse. After a time however these specification were lost again showing the significance of arealization. The cause for this can be put down to lack of maturation making the cortical progenitors more susceptible to extrinsic cue from the host (Anderson et al, 2014).

These experiments serve to highlight these determinants and allow us to harness them in pathological neurogenesis. Diseases that may benefit from this include Parkinson’s, Huntingtons and Timothy syndrome. In respect to Parkinson’s disease which is a neurodegenerative disease affecting the loss of dopamine neurons in the substansia nigra pars compacta, there has been previous methods used to treat the disease namely implantation of human fetal tissue (Ambasudhan et al, 2014). There however has been a subset of cases where some implantees have developed side effects namely graft induced dyskinesia (Anderson et al, 2014).

The reason for this can be found in the fact fetal stem cells lack DNA binding forkhead box protein A2 causing its efficacy in respect to grafting to be underwhelming. Due to the precise development of hESCs through a floor plate intermediate stage instead of a neuroepithelial intermediate, the dopamine neurons have both biochemical and physiological features in line with the native dopamine neurons. Factors adding to the fully defined nature of these neurons are the activation of Wnt signalling using a small molecule inhibitor glycogen synthase kinase (GSK) (Tadar and Struder, 2014).

The eventual goal from in vivo cell-fate conversion is to offer us an alternative approach to cell transplantation by allowing us to convert neurons using the very same extrinsic factors used in neural differentiation.

Conclusion

HESCs are an invaluable model for the study of corticogenesis amongst others. Its multiple disciplinary advantages have led to a greater understanding not only of the underlining mechanisms that causes our brains to function how they do but also evolutionary effects of those mechanisms.

Its use as a model for human neurodegenerative disorders is leading us toward a future where such occurrences will be but a forgotten memory. The discovery of novel genes coupled with growth and differentiation factors will help in IVF pre-screening with the eventual goal of eliminating degenerative neural diseases (Reubinoff et al, 2001).

References

Ambasudhan, R., Dolatabadi, N., Nutter, A., Maslieh, E., McKercher, S.R., Lipton, S.A., (2014) Potential for cell therapy in Parkinson’s Disease Using Genetically Programmed Human Embryonic Stem Cell-Derived Neural Progenitor Cells. The Journal of Comparative Neurology 522: 2845-2856

Anderson and Vanderhaegen (2014) Cortical neurogenesis from pluripotent stem cells: Complexity emerging from simplicity. Curr Op Neurobiology 27: 151-7

Leemput, J., Boles, N.C., Kiehl, T.R., Corneo, B., Lederman, P., Menon, V., Lee, C., Martinez, R.A., Levi, B.P., Thompson, C.L., Yao, S., Kaykas, A., Temple, S., Fasano, C.A., (2014). CORTECON: A Temporal Transcriptome Analysis of In Vitro Human Cerebral Cortex Development from Human Embryonic Stem Cells. Neuron 83: 51-68

Reubinoff, B.E., Itsykson, P., Turetsky, T., Pera, M.F., Reinhartz, E., Itzik A., Ben-Hur T., (2001) Neural progenitors from human embryonic stem cells. Nature Biotechnology 19: 1134-1140

Tabar, V. and Studer, L., (2014). Pluripotent Stem Cells in regenerative medicine: challenges and recent progress. Nature Reviews Genetics 15: 82-92

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