Multipotency and stability of mouse embryonic stem cell derived neural stem cells
thesisposted on 27.02.2017, 23:41 by Zeng, Wendy Rong
Neuronal cell loss is a common feature of many neurological disorders, including stroke, Parkinson’s disease, Alzheimer’s disease and traumatic brain injury. Embryonic stem (ES) cells and ES cell-derived neural stem cells or progenitor (NS/NP) cells may provide a number of new ways for studying and treating diseases and injuries in the brain. NS/NP cells derived from embryonic stem cells, isolated from the fetal or adult central nervous system (CNS) are defined by their abilities to self-renew and differentiate into neurons, astrocytes and oligodendrocytes. Their growth and specialisation is dependent on environmental cues, such as media formulation and exposure to patterning growth factors. It is important to study the propagation and differentiation characteristics of ES cell derived NS/NP cells because contamination of ES cell derived cultures with pluripotent cells or unwanted phenotypes is a practical problem that may result in false positive hits in high through-put screening and tumour formations in replacement therapies. If NS or NP cells are used as a starting point, the likelihood of contamination is reduced. These cells potentially represent an unlimited source for neuron replacement therapies; they are also a suitable source of differentiated cells for studying functional genomics, proteomics or for drug screening; and they allow us to study early brain development. The first experimental chapter (Chapter 3) investigated the effect of altering the period of neural induction (NI) by exposing ES cells to the neural inducing media N2B27 for up to 10 days prior to forming neurospheres and growing cells in EGF and FGF2 conditions which are widely used to propagate NS cells. The ability of the ES cell-derived NS/NP cells to generate dopaminergic, serotonergic, cholinergic and gamma aminobutyric acid (GABA)ergic neurons was assessed using immunocytochemistry. Extending the NI period to 10 days, prior to the generation of neurospheres, and subsequent expansion as monolayer cultures, gave rise to more multipotent NS/NP cells that were able to generate phenotypically diverse neurons with very small numbers of residual NS/NP cells and astrocytes. The second experimental chapter (Chapter 4) explored the effect of long term maintenance of ES cell derived NS/NP cells in the presence of EGF and FGF2, and investigated their ability to give rise to catecholaminergic and GABAergic neurons after extended propagation. Lmx1a reporter cells were used to identify cells at the NP stage using flow cytometry. It was found that the presence of EGF and FGF2 was not sufficient to stabilise Lmx1a-positive NP cells in either monolayer or PA6 co-culture during long term maintenance. Although the neurogenic potential of these cells remained stable over 10 passages; the percentage of catecholaminergic neurons reduced dramatically. The last experimental chapter (Chapter 5) investigated the isolation and propagation of clonal NS/NP populations via neurospheres using low numbers of cells. Neurosphere formation in the presence of EGF and FGF2 was shown to be a poor method for the maintenance of Lmx1a-positive NP cells since the proliferation and neurosphere formation capability of these cells declined dramatically over the three passages. When neurospheres were formed with 10 cells, progressive loss of neurogenic potential with passaging and poor yield of neurons after exposure to patterning cues indicated that this culture method was unable to adequately support the propagation of NS/NP cells.