Neurospheres

We investigated the efficiency of NSC expansion on surfaces with EGF-His immobilized in the correct orientation. NSCs were obtained from neurosphere cultures prepared from fetal rat striatum harvested on embryonic day 16. NSCs were cultured for 5 days on EGF-His-immobilized substrates prepared with mixed SAMs of different COOH-thiol contents. Cells adhered and formed network structures at a density that increased with the COOH-thiol content of the surface. As a control, cells were seeded onto surfaces without immobilized EGF-His. This resulted in poor cell adhesion during the entire culture period. In addition, when EGF-His adsorbed to SAMs with 100% COOH-thiol or SAMs with NTA-derivatized COOH that lacked Ni2+ chelation, we observed poor initial cell adhesion, and the cells formed aggregates within 5 days. Interestingly, the substrate used to covalently immobilize EGF-His with the standard carbodiimide chemistry was not a suitable surface for cell adhesion and proliferation. The control experimental results contrasted markedly with results from EGF-His-chelated surfaces. 

Table 1 Proliferation of neurosphere-forming cells in cultures with or without EGF-immobilized...

Determined for cells during the standard neurosphere culture... 

Fig. 7 Quantitative evaluation of NSC enrichments in cultures with different material surfaces. The fraction of NSC phenotypes in the cultures are shown for (open circles) nestin+ pill- or (closed circles) both nestin+ pin and nestin+ pill+. Data are also shown for neurosphere cultures (NS). The averages ( SEM) of five experiments are shown. Reproduced from Nakaji-Hirabayashi et al. [85] with permission from Elsevier, copyright 2007...

We also conducted experiments to compare our culture method with the standard neurosphere culture. In the standard neurosphere culture, cell number increased approximately nine times over 5 days. Immunostaining showed that the neurosphere cultures contained 54 5.3% nestin+ cells and 41 7.4% nestin+ pIIF cells. This demonstrated that the standard neurosphere culmring method was less efficient than EGF-immobilized substrates for selectively expanding NSCs. Thus, the EGF-immobilized substrates prepared from mixed SAMs with 10% COOH-thiol provided the most efficient method for selective NSC expansion. 

Some aspects of multiple sclerosis are reflected in the animal model experimental autoimmune encephalomyelitis, which is induced by immunization of susceptible animals with appropriate encephalogenic proteins or peptides. In these animals, if cultured adult stem cell neurospheres are injected into the bloodstream, injected cells can find their way to damaged portions of the nervous system and improve function in mice. How the injected cells augmented the recovery process is unclear. One possibility is that cells recruited to the lesions differentiated into oligodendrocytes and generated new myelin sheaths, but this seems unlikely in the face of ongoing cellular destruction. 

Pluchin, S., Quattrini, A., Brambilla, E. et al. Injection of adult neurospheres induces recovery in a chronic model of multiple sclerosis. Nature 422, 688-694, 2003. 

The Neurosphere Assay Applied to Neural Stem Cells and Cancer Stem Cells... 

Key words Neurosphere assay, Neural stem cell cultures, Cancer stem cell... 

Fig. 1. Only a fraction of cells within a GBM CSC-derived neurosphere express the EGFR. EGFR, greeiT, DAPI, light blue, nuclei. Magnification 200x (Mazzoleni and Galli, unpublished observations).

Seed the cells derived from the dissociation of clonal single neurospheres into uncoated 96-well plates in proliferation medium with or without the compound under testing. Count the cells in each well 2 h after plating, in order to obtain a baseline value to be used for establishing the efficiency of secondary sphere generation. 

Count the neurospheres generated in each well, 3-10 days after plating, depending on the cell type. Normalize the number of secondary neurospheres to the number of cells originally seeded in each well. 

Rodent NSCs from embryonic stages form neurospheres in about 2-3 days, whereas NSCs from postnatal stages require 5-7 days to give rise to neurosphere. 

Subculture neurospheres when their diameter is in the range of 200 and 500 pm. If neurospheres are too small when subcultured, the yield of cells will be very low if neurospheres are too large, the number of dead cells inside the spheres (that give rise to the dark core ) will be high, mechanical dissociation will be difficult, and viability of the culture will be very low. 

For achieving maximal disaggregation of neurospheres, slightly tilt the Pipetman and press tip against the bottom of the tube to generate a fair amount of resistance. Gently triturate pellet for 50-70 times for embryonic rodent cells and 60-80 times for adult rodent cells and cancer stem cells. 

Reynolds BA, Rietze RL (2005) Neural stem cells and neurospheres— re-evaluating the relationship. Nat Methods 2 333-336... 

Injection of adult neurospheres induces recovery in a chronic model of multiple sclerosis. Nature, 422, 688-94. 

Wu, S., Suzuki, Y., Kitada, M., Kataoka, K., Kitaura, M., Chou, H., Nishimura, Y., Ide, C. (2002). New method for transplantation of neurosphere cells into injured spinal cord through cerebrospinal fluid in rat. Neurosci Lett, 318, 81-4. 

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