Sasha Bakhru: Introduction to Adult Neural Stem Cell Culture Methods

Neural Stem Cell (NSC) culture can be achieved through adherent or cluster (neurosphere) culture in vitro. Long term hNSC expansion was first accomplished under neurosphere culture conditions with EGF, FGF2, and LIF. A doubling time of 10-15 days was observed (Carpenter et al. 1999). Due to lack of nutrients and cytokines or growth factors at their core, neurospheres grew as heterostructures, with NSCs principally located on their periphery and differentiated cells (including Tuj1+, GFAP+) (Campos et al. 2004) towards the interior, along with cell necrosis at their core (Bez et al. 2003).

Differentiation of NSCs during neurospheretype culture limits the expansion speed, while the mixed cell population presents significant obstacle to their therapeutic use. In order to realize practical cell-based therapy for neurodegenerative diseases, such as Alzheimer's and Huntingdon's diseases, it is crucial to achieve an adequate number of NSCs in short period of time, stemming from the small number available from each biopsy source. Idiopathic Parkinson's Disease (PD), is characterized by loss of motor control, typically in geriatric patients. Neurons bridging the substantia nigra and striatum, termed nigrostriatal neurons, employ the neurotransmitter dopamine to transmit signals to the striatum. However, in PD, these nigrostriatal neurons (Fig. 2.3) degenerate due to unknown causes. This, in turn, triggers gradual loss of the control of body movement. Neural stem cells have offered the first glimpses of hope regarding cellular based therapy for the treatment of this disabling disease. If able to differentiate at the appropriate location, neural stem cells may replace the lost nigrostriatal neurons and restore motor control in way that no acellular therapy has proven capable to date (Bjorklund et al. 2000; Lindvall et al. 2004).

2.2 Stem Cell Microenvironment

The native cellular microenvironment of stem cells is termed the stem cell niche, of which there are three primary features: cell secreted factors, cell-cell interaction, and the extracellular matrix (Watt et al. 2000). Cell-secreted factors regulate many stem cell activities. For example, TGF-beta and Wnts, two families of common secretedfactors found across many species, are responsible for proliferation via asymmetric divisions. At least two types of TGF-beta were also found to be important in differentiation of neural crest stem cells. Although these factors are important, direct cell-to-cell contact is also necessary for other local signals that control stem cell activity. Examples of this are the two transmembrane proteins Notch and Delta.

In Drosophila, the progeny of the sensory organ precursor is regulated by Notch activity (Watt et al. 2000). In humans, it is responsible for correctly determining stem cell fate in skeletal muscles, blood, and retinal neuroepithelium. The binding between extracellular matrix components and integrin receptors on stem cells is another vital part of the stem cell niche (Watt et al. 2000). In epidermal stem cells, high expression of beta1 integrins is necessary for their survival. The loss of beta1 integrins can lead to cell death and migration of the cells away from the niche (Watt et al. 2000). The ECM proteins are responsible for the activation and maintenance of the beta1 integrins, and is required for the distribution of secreted factors among the stem cells (Campos et al. 2004; Watt et al. 2000).

Other Articles by Sasha Bakhru: