Requirements of human brain-derived neural stem cells for different storage, isolation and cultivation

Yang WANG; Zhifeng DENG; Xianliang LAI; Xueming HUANG

ABSTRACT

BACKGROUND:

Due to a new finding in the in vitro proliferation and differentiation of neural stem cells, neural stem cells are regarded as optimal materials for repairing and replacing the injured neural tissues. But how to enhance the proliferation of neural stem cells and how to induce neural stem cells to differentiate into certain phenotypes remain objects for discussing.

OBJECTIVE:

To investigate the frozen/nonfrozen storage techniques in isolation and cultivation of neural stem cells isolated from human brain and to discuss the conditions suitable for their differentiation.

DESIGN:

A single sample based on cells.

SETTING:

Urinology Institute of Jiangxi Medical University and Neurosurgery Department of Second Hospital Affiliated to Jiangxi Medical College.MATERIALS From December 2003 to June 2004, this study was conducted at the Urinology Institute of Jiangxi Medical College. Brain tissues removed from sixteen-week-old embryos were studied.

METHODS:

After being digested with trypsin, cells were separated from the brains of embryos. They were cultured in the serum-free media, stimulated by basic fibroblast growth factor and epidermal growth factor and were induced to differentiate using serum. Immunofluorescent cytochemical staining technique was adopted to detect the expressions of the Nestin and the neuron-specific enolase(the marker for mature neural cells) in these cells. The influence of basic fibroblast growth factor, epidermal growth factor and serum on the proliferation and differentiation of the neural stem cells were investigated.MAIN OUTCOME

MEASURES:

The main outcome measurements in this tification of the Nestin and the neuron-specific enolase in these cells.bryo brain successfully. The embryo brain cells in primary culture were clear and round. After 3 days some cells aggregated to form neurospheres and some others suspended. Two weeks later, the neurospheres enlarged and a part of cells in it expanded into folds with strong reflection and proliferation. The continuous cultured cells inherited these characteristics and kept the morphological properties. The clones were cultured in the medium with serum. Twenty-four hours later, most of the cells were attached to the bottom of the plates. Cells were dissociated from the neurospheres and became irregular. Forty-eight hours later, most of the cells differentiated into many scattered patches of astrocytes with various shapes and neurites and many isolated neural stem cells were continuously cultured in vitro and they expressed Nestin, the marker of neural stem cells. After being cultured in medium with serum for 48 hours, the differentiated cells mainly expressed freeze-thaw cells were alive. Their living status had no significant difference compared to that of the fresh isolated cells.

CONCLUSION:

Serum-free culture medium, together with basic fibroblast growth factor and epidermal growth factor, enables the in vitro culture, proliferation and purification of the neural stem cells. The feasibility of the method is confirmed. Meanwhile, the comparison between the cultures of freeze-thaw cells and fresh isolated cells indicated that frozen storage can be used as a way to preserve the human embryo brain cells, which will be available for study after thaw.