Modeling the impact of alcohol on cortical development in a dish: strategies from mapping neural stem cell fate

Abstract

During the second trimester period, neuroepithelial stem cells give birth to millions of new neuroblasts, which migrate away from their germinal zones to populate the developing brain and terminally differentiate into neurons. During this period, large numbers of cells are also eliminated by programmed cell death. Therefore, the second trimester constitutes an important critical period for neuronal proliferation, migration, differentiation and apoptosis. Substantial evidence indicates that teratogens like ethanol can interfere with neuronal maturation. However, there is a paucity of good model systems to study early, second trimester events. In vivo models are inherently interpretatively complex because cell proliferation, migration, differentiation, and death mechanisms occur concurrently in regions like the cerebral cortex. This temporal overlap of multiple developmental critical periods makes it difficult to evaluate the relative vulnerability of any individual critical period. Our laboratory has elected to utilize fetal rodent cerebral cortical-derived neurosphere cultures as an experimental model of the second-trimester ventricular neuroepithelium. This model has enabled us to use flow cytometric approaches to identify neuroepithelial stem cell and progenitor sub-populations and to show that ethanol accelerates the maturation of neural stem cells. We have also developed a simplified mitogen-withdrawal/matrix-adhesion paradigm to model the exit of neuroepithelial cells from the ventricular zone towards the subventricular zone and cortical plate, and their maturation into multipolar neurons. We can treat neurosphere cultures with ethanol to mimic exposure during the period of neuroepithelial proliferation and by using the step-wise maturation model, ask questions about the impact of prior ethanol exposure on the subsequent maturation of neurons as they migrate and undergo terminal differentiation. The combination of neurosphere culture and stepwise maturation models will enable us to dissect out the contributions of specific developmental critical periods to the overall teratology of a drug of abuse like ethanol.

Fig. 1

Schematic for modes of cell division observed in the stem cell beds. Symmetrical division (a) results in the generation of two daughter stem cells and permits the early expansion of the stem cell pool. Asymmetric division (b) in contrast, results in the generation of one stem and one more mature, progenitor daughter cell (e.g., in bone marrow, this results in the generation of a common lymphoid or myeloid progenitor, and a replacement stem cell). Asymmetric cell division therefore results in the maintenance of the stem cell pool. Subsequent cell divisions result in the clonal expansion of the progenitor pool (e.g., the transformation of a common myeloid progenitor to erythroid or myeloid CFUs). During the late period of maturation (c) symmetric cell division results in two daughter cells that are more mature, blast-type cells, resulting in a depletion of the parent progenitor pool (e.g., transformation of an erythroid CFU to a proerythroblast, resulting finally in the formation of an erythrocyte)

Fig. 2

Presumptive maturation program of neural stem cells. Neural stem cells give rise to progenitors, which give rise to blast-type cells (radial glia and astrocyte-type cells), which can give rise to neurons

Fig. 3

(a) Boxed area indicates the region from which neuroepithelial cells are isolated. (b and c) Neuroepithelial cells dispersed by trituration, and cultured in defined medium, generate nonadherent aggregates of cells referred to as neurospheres. The cellular composition of neurospheres is heterogeneous, and includes cells that express stem and progenitor markers (ABCG2, Sca-1, c-kit/CD117, CD133), nonselective markers of immature cells (nestin), markers for blast cells (Glial Fibrillary Acidic Protein, GFAP), as well as markers for early neuronal maturation (MAP-2). However, neurosphere cells do not express the neuron-specific marker, NeuN

Fig. 4

Schematic of the Mitogen-withdrawal, extra-cellular matrix addition paradigm. (a) The mitogenic condition results in an expansion of the nonadherent neurosphere population. Exposure to laminin in addition to mitogenic medium (b) results in neurospheres becoming adherent. However, migratory neuroepithelial cells retain an immature squamous epithelioid appearance. Withdrawal of EGF and LIF, and provision of laminin as an adhesion matrix (c), results in neurospheres becoming adherent to the culture dish, and the appearance of bipolar migratory cells. These cells express nuclear NeuN, but do not exhibit nestin immunoreactivity, showing that these cells have transformed into migratory neurons. The additional removal of FGF (d) results in the appearance of multipolar neurons between 24 and 72 h

Fig. 5

Gas chromatographic analysis of ethanol content in culture medium over a 5-d exposure period. Culture medium is replaced on day 3. The data show that ethanol concentrations remain stable over the period of the experiment