Directed Differentiation of Neural Cells from Human Umbilical Cord Stroma-derived Neural Stem Cells

Abstract

Objective to investigate the directed differentiation of human umbilical cord stromal-derived stem cells into neural cells. Methods: (1) Human umbilical cord stromal cells (HUMSC-NSCs) were induced to differentiate from primary human umbilical cord stromal cells, and two different methods were used to induce differentiation to neural cells, which were observed under electron microscope. (2) The neural cells induced to differentiate by different induction methods were identified by morphological differences, immunohistochemistry, and Western blot. Results: (1) Some of the suspended NSCs started to adhere to the wall 48-72 h after the addition of the inducer, and elongated protrusions could be seen in 5-7 days with the neurosphere as the center, and cells gradually migrated outward from the neurosphere. 10 days later, several cells with different morphologies could be seen. Some of the cells had increased refractive index and the length of the protrusions increased, showing bipolar growth. In some cases, the protrusions were short and dense, centered on the cytosol, and protruding in a discrete manner. Under electron microscopy, the neurospheres consisted of multiple clonal clusters and two different morphologies of cells, shaped like neurons and astrocytes, could be clearly observed. The number of bipolar neurons was significantly increased in the group with the addition of BDNF.(2) Before induction, 84.5 ± 1.6% and 88.62 ± 1.1% of HUMSC-NSCs expressed stem cell-specific markers: Stro-1 and nestin; neurospheres still expressed 62.7 ± 3.9% of HUMSC-NSCs positive for nestin after 5 d of culture in neuronal cell induction medium. After 10 days of induction, the percentage of immunohistochemically positive stained cells was counted. With the BDMF induction protocol, 38.6 ± 2.9% and 8 ± 1.9% Hoechst33342 positive cells expressed immature (β-tubulin III) and mature (MAP2ab) neuronal cell markers, respectively. Also, 15.8 ± 4.5% and 20.6 ± 4.6% of Hoechst33342-positive cells expressed GFAP (astrocyte marker) and GalC (oligodendrocyte marker). Comparison using a paired t-test revealed that the percentage of β-tubulin III (P<0.001) and MAP2ab (P<0.05) positive cells was significantly higher in the BDNF-induced group than in the general induction group. Western blot results confirmed the immunohistochemical data. Conclusion: After adding BDNF to induce differentiation in the culture group, the number of bipolar neurons was significantly increased, which could promote the induced differentiation of human umbilical cord-derived neural stem cells into neuronal cells.