AbstractThe in vitro study of neural progenitors is based around the idea that defined culture conditions can emulate an environment which maintains an undifferentiated state and self-renewal capability of explanted progenitor cells, culminating in the growth of neurospheres. Neurosphere culture systems are used to study cells of physiological origin as well as transformed cells. In the case of the cerebellar granule cell lineage, neurosphere cultures have been produced from medulloblastoma tumors, while non-transformed granule progenitors are reported to be transient in vitro. SHH signaling is a key signaling pathway with roles in morphogenesis and cell proliferation in the central nervous system, in particular, involved in mitogenic signaling within the context of postnatal cerebellar granule cell expansion. In this paper, I demonstrate that in addition to commonly used mitogens such as EGF and bFGF, the SHH pathway agonist SAG, as well as genetic activation of SHH signaling by PTCH1 deletion, can lead to the growth of neurospheres from postnatal day 7 (p7) cerebellar explants.I also show that these neurosphere adhere to the cerebellar GCP lineage.. Strikingly, mSS cells can be maintained indefinitely in culture, as demonstrated by extensive clonogenic capability, assayed over a period of 10 weeks. In the context of self-renewal, I assay gene expression of POU3f2, POU5f1, NANOG, and SOX2, genes associated with neural progenitors, which I find expressed in mSS cells. Importantly, mSS cultures are continuously dependent on SAG for their clonogenic potential and SHH pathway activation, assayed by expression of GLI1, PTCH1 and NMYC. In addition to the aforementioned extensive self-renewal capability, mSS neurosphere cultures also maintain the ability to differentiate. In vitro differentiation leads to formation of cells with typical granule cell morphology and which are positive for beta3-tubulin and express GABRA6.Overall, my work demonstrates that by applying culture conditions which are tailored towards biological characteristics of specific regions of the central nervous system the paradigm of the neurosphere can be expanded to include lineages not previously studied in this way. In particular, I apply this principle to unmask the property of cells from the GCP lineage as having extensive self-renewal capability in vitro.
ZFP91 zinc finger protein expression pattern in normal tissues and cancers
