Purifying Immature Neurons from Differentiating Neural Stem Cell Progeny Using a Simple Shaking Method

Abstract

Objective: Enriched neuronal cell populations are valuable tools both for laboratory investigations and cell therapy applications. However, available cell purifying approaches demand costly equipment such as FACS or MACS that limits their universal accessibility. In this study, we developed an efficient method for purifying immature neuronal cells from differentiating neural stem cell (dNSC) progeny based on their differential substrate attachment properties without using any expensive cell separating tools. Methods: Neural stem cells were harvested from the ganglionic eminence of embryonic day 14 mice brains using the neurosphere assay. Neurospheres were then dissociated into single cells and differentiated employing the neuroblast assay method. Following a brief trypsinization, the dNSC culture was gently shaken at 150 rpm for 30 min to detach the top neuronal cell clusters from the underlying astrocytic cell monolayer. The neuronal purification yield, astrocyte contamination, and presence of dividing cells were compared to a MACS purification method using PSANCAM antibody. Results: While a neuronal yield of 97.1 ± 0.45% was achieved using MACS; it reached 97.9 ± 0.6% using the shaking method that was not significantly different.On the other hand, the percentage of astrocytes in the MACS approach was 1.18 ± 0.15%, but it significantly decreased to 0.6 ± 0.15% using the shaking method. Moreover, 4.41 ± 0.23% and 5.3 ± 0.4% of the isolated cells in the MACS and shaking methods, respectively, were Ki-67 immunoreactive dividing cells, of which 97.34 ± 1.6% and 97.9 ± 0.7% were co-expressing β III-tubulin, confirming their neuronal identity. Additionally, based on the neural-colony forming cell assay, the shaking method resulted in the generation of a homogenous neuronal cell population without any bona fide NSC contamination. Conclusions: The shaking purification method allows easy, low cost, efficient and large-scale separation of immature neurons from dNSC progeny, potentially benefiting both basic and clinical applications.