Induction of rat neural stem cells into oligodendrocyte precursor cells / 生理学报

We have previously established a culture method to isolate and cultivate neural stem cells (NSCs) derived from the rat embryonic brain and spinal cord. In the present study, we demonstrate that the spinal cord-derived NSCs can be induced to differentiate into oligodendrocyte precursor cells (OPCs) with a combined treatment composed of (1) conditioned medium collected from B104 neuroblastoma cells (B104CM) and (2) basic fibroblast growth factor (bFGF, 10 ng/ml). After induction, over 95% of the cells displayed bipolar or tri-polar morphology and expressed A2B5 and platelet derived growth factor receptor-alpha (PDGFR-alpha), markers that are specific for OPCs. Among PDGFR-alpha positive OPCs, only a few cells expressed glia fibrillary acidic protein (GFAP) and none expressed beta-tubulin III. In the presence of B104CM and bFGF, OPCs proliferated rapidly, formed spheres, expanded for multiple passages, and maintained their phenotypic properties. Upon withdrawal of B104CM and bFGF, these cells differentiated into either O4/GlaC-positive oligodendrocytes (OLs) or GFAP- and A2B5-positive type-2 astrocytes. Our results indicate that NSCs can be induced to differentiate into OPCs that possess properties of self-renewal and differentiation into oligodendrocytes and type-2 astrocytes, a property similar to that of O-2A progenitor cells. The OPCs can be maintained in an undifferentiated state over multiple divisions as long as both B104CM and bFGF are present in the medium. Thus, large quantity of OPCs can be obtained through this method for potential therapeutical interventions for various neurological degenerative diseases.

Isolation and cultivation of neural stem cells from the embryonic rat brain and spinal cord / 生理学报

The aim of this study was to establish the culture system of isolation and cultivation of the neural stem cells (NSCs) from the embryonic rat brain and spinal cord. The methods of microscopic dissection, cell culture and immunofluorescence cytochemistry were used. The results are as follows. (1) In the presence of fibroblast growth factor-2 (FGF-2) and epidermal growth factor (EGF), both brain- and spinal cord-derived stem cells proliferated and expanded in vitro for 8 - 10 passages (over 60 d). The period of expansion resulted in a 10(6)-fold increase in brain-derived NSCs and 10(5)-fold increase in spinal cord-derived NSCs. These proliferating cells expressed nestin. (2) In the medium containing 1% FBS, the two NSCs populations could be induced to differentiate into neurons, astrocytes and oligodentrocytes. The percentage of neurons (beta-tubulin III-ir) differentiated from brain-derived NSCs decreased rapidly from 11.95+/-2.5% at passage 2 (P(2)) to 1.97+/-1.16% at passage 5 (P5). Significant difference was shown between P(2) and P(5) (P<0.01). The percentage of oligodentrocytes (Rip-ir) differentiated from brain-derived NSCs remained mostly unchanged from 8.66+/-2.93% at P(2) to 9.12+/-1.13% at P(5). The same differentiation patterns were found in spinal cord-derived NSCs. All these results indicate that both embryonic rat brain- and spinal cord-derived NSCs can expand and proliferate in vitro through multiple passages, and retain the capacity to differentiate into all three major types of cells in the central nervous system.

Effects of embryonic neural stem cells and glial cell line-derived neurotrophic factor in the repair of spinal cord injury / 生理学报

The ability of implanted embryonic neural stem cells (NSCs) to improve survival, migration, and functional recovery following a compression spinal cord injury (SCI) was tested in adult rats. NSCs were isolated from E14-16 rat cerebral cortex and SCI was produced by using an aneurysm clip applicator applied to the 8th thoracic spinal cord according to method of Dolan and Tator. Two weeks after the injury, NSCs (4 microl of 1 x 10(4) cells/microl) were injected into the lesion site. The grafted NSCs were noted to survive and integrate with the host spinal cord 1 month after transplantation, which was demonstrated by the presence of Hoechst 33342 (a nuclear dye) pre-labeled NSCs within and surrounding the lesion site. Some of these cells remained undifferentiated and were stained with nestin, a marker for NSCs. Transplanted NSCs migrated for at least 3 mm from the injury epicenter towards both the rostral and caudal directions. Significant reduction in the lesion area (P<0.05) and improvement in inclined plane (P<0.05) and BBB locomotor rating scale (P<0.05) were found in the cases that received implantation of NSCs, as compared with those that received vehicle injection. More importantly, when glial cell line-derived neurotrophic factor (GDNF; 1.5 microg/microl) was added to the transplants, further reduction in lesion area (P<0.01) and improvement in the function were observed in the combined treatment group as compared with the vehicle infused group. Our results suggest that intraspinal treatment with NSCs and GDNF synergistically reduced lesion size and improved functional outcome after a compressive SCI in adult rats.