Adherent vs. Free-Floating Neural Induction by Dual SMAD Inhibition for Neurosphere Cultures Derived from Human Induced Pluripotent Stem Cells

Abstract

Keeping neural stem cells under proliferation, followed by terminal differentiation, can substantially increase the number of neurons generated. With regard to the usability of proliferating neurospheres (NSPHs) cultures, adherent induction protocols have not yet been studied in comparison to embryoid body (EB)-based protocols. To compare these proctocols, neural induction of human induced pluripotent stem cells was performed by dual SMAD inhibition under both adherent and free-floating EB culture conditions. After 10 days, we transferred cells to low-attachment culture plates and proliferated them as free-floating neurospheres. RNA was collected, transcribed to cDNA and analyzed for sonic hedgehog expression that plays an important role during proliferation process. NSPHs were analyzed by immunofluorescence imaging directly and upon continued differentiation. The EB-based approach yielded in higher numbers of cells expressing the neural stem cell marker Nestin, and showed in contrast to the adherent induction protocol increased expression levels of sonic hedgehog. Although improvements to culture consistency and reliability are desirable, the EB-based protocol appears to be superior to the adherent protocol for both, the proliferation and differentiation capacity.

MCF7 Spheroid Development: New Insight about Spatio/Temporal Arrangements of TNTs, Amyloid Fibrils, Cell Connections, and Cellular Bridges

“…The most recent studies point at three-dimensional (3D) spheroids as a good model for basic biological research on multicellular tumors [ 8 , 9 , 10 , 11 , 12 ] as well as for selection of new anti-cancer drug [ 13 , 14 , 15 ]. Cells spheroids gained a growing attention thanks to their ability to mimic in vitro avascular tumor, characterized by heterogeneous population of cells and, as such, represent a powerful tool to study their development, morphological changes, and metabolism [ 16 ].…”

SOX2, a stemness gene, induces progression of NSCLC A549 cells toward anchorage-independent growth and chemoresistance to vinblastine

“…Our hypothesis was that the growth behavior of an embryoid body in which pluripotency is enriched would be similar to that of a cancer cell aggregate detached from the substratum. 32 , 33 Over the past decade, 3D cultures of cancer cells in poly-2-hydroxyethyl methacrylate (poly-HEMA) hydrogel, which prevents cell spreading and cell attachment to the substratum due to its superhydrophilic nature, have received attention as valid models to recapitulate the anchorage-independent growth of cancer cell. 34 , 35 In this study, we report that SOX2 increases the growth of NSCLC A549 cell spheroids and increases the resistance to the anticancer drug vinblastine through AKT kinase signaling.…”

Visualizing the Synaptic and Cellular Ultrastructure in Neurons Differentiated from Human Induced Neural Stem Cells—An Optimized Protocol

“…Only the densely packed and rather small neurospheres reliably provided a sufficient ultrastructure. Neurospheres were generated by culturing iNSC under growth conditions until single spheres formed [27,28]. The growth of the spheres ceased after the induction of differentiation.…”