An efficient method for generating murine hypothalamic neurospheres for the study of regional neural progenitor biology.

An efficient method for generating murine hypothalamic neurospheres for the study of regional neural progenitor biology. Endocrinology. 2020 Mar 10;: Authors: Nesan D, Thornton HF, Sewell LC, Kurrasch DM Abstract The hypothalamus is a key homeostatic brain region and the primary effector of neuroendocrine signaling. Recent studies show that early embryonic developmental disruption of this region can lead to neuroendocrine conditions later in life, suggesting that hypothalamic progenitors might be sensitive to exogenous challenges. To study the behavior of hypothalamic neural progenitors, we developed a novel dissection methodology to isolate murine hypothalamic neural stem and progenitor cells at the early timepoint of embryonic day (E) 12.5, which coincides with peak hypothalamic neurogenesis. Additionally, we established and optimized a culturing protocol to maintain multipotent hypothalamic neurospheres that are capable of sustained proliferation or differentiation into neurons, oligodendrocytes, and astrocytes. We characterized media requirements, appropriate cell seeding density, and the role of growth factors and sonic hedgehog (Shh) supplementation. Finally, we validated the use of fluorescence activated cell sorting of either Sox2GFPKI or Nkx2.1GFPKI transgenic mice as an alternate cellular isolation approach to enable enriched selection of hypothalamic progenitors for growth into neurospheres. Combined, we present a new tech...
Source: Endocrinology - Category: Endocrinology Authors: Tags: Endocrinology Source Type: research