gms | German Medical Science

Objectives: It is now clear that neural stem cell identity in a subset of tumour cells is key to malignancy in high grade glioma, and probably also in lower grade lesions. Inducing stable therapeutic differentiation commitment in the glioma NS cells which persist after surgical resection offers the promise of effective treatment with very low toxicity. BMP4 is a potent driver of astrocyte differentiation, but differentiation therapies based on BMP4 have failed to deliver on their early promise, reflecting the capacity of glioma neural stem cells to escape differentiation commitment. We set out to develop an in vitro model of BMP4-induced differentiation commitment suitable for application to automated medium and high-throughput drug screening approaches.

Methods: We developed an in vitro model of BMP4-induced differentiation commitment. Mouse neural stem cells are first plated at low density in medium supplemented with BMP4. This drives uniform cell cycle exit and adoption of astrocyte morphology and marker expression. After 24 hours, BMP4 is replaced with the growth factors EGF and FGF in order to test astrocyte differentiation commitment. Escape from commitment in any individual cell results in reacquisition of NS cell marker expression and rapid cell cycle, resulting in formation of a colony. Neural stem cells and model glioma neural stem cells (EGFRvIII; INK4ARF-/-) were tested, and forced expression of the neural stem cell master regulator FOXG1 was used as a positive control to drive dedifferentiation to a neural stem cell state. As proof of principle for screening, the StemSelect Small Molecule Library 1 (Merck Millipore) was applied together with growth factors to BMP-treated NS cells in 96 well plates. After 7 days the plates were fixed and DAPI-treated, then automated counting of cell and colony numbers was performed using the Operetta High Content Imaging System (Perkin Elmer).

Results: We demonstrate stable differentiation commitment in BMP-treated normal NS cells on restoration of the growth factors EGF and FGF. As predicted, however, glioma NS model cells exhibit incomplete differentiation commitment, resulting in sporadic colony formation in this assay. Automated drug screening using a compound library of 303 compounds with known stem cell regulatory activity identified 3 hits with very marked propensity to induce dedifferentiation in committed astrocytes. These hits are each known to regulate the same key intracellular second messenger, lending validity to the approach and prompting further investigation of the role of this cascade in differentiation commitment.

Conclusion: Stem cells are key to the development and recurrence of glioma. We present an assay which allows for in vitro automated screening, to identify drugs which can modulate the escape from differentiation commitment which is the basis for glioma initiation and recurrence after treatment.