Directed Differentiation Of Embryonic Spinal Cord Stem Cells Into Cholinergic Neurons

Part one Isolation, proliferation and natural differentiation of spinal cord stem cellsObjective: To study the biological characteristics in proliferation and natural differentiation of spinal cord stem cells. Methods: The single cell suspensions, derived from rat embryos spinal cord at embryonic day 13, were plated into the serum-free medium containing DMEM/F12, EGF, bFGF and B27 supplement. One part of the neurospheres were labeled by immunofluorescence detection of Nestin. Another part of the neurospheres were carried to exam the natural differentiation effects in the medium containing 1% FBS. Use the distinctive marker for neuron (MAP-2) and astrocyte (GFAP) to determine the phenotype of the differentiated cells after 14 d. Results: After 2 weeks'culturing, spinal cord cell suspension emerges many cell neurospheres. The neurospheres exhibited Nestin positive. The natural differentiated cells expressed MAP-2 and GFAP respectively. Conclusions: The neural stem cells isolated from spinal cord possess the ability in proliferating and self-renewing, and have multipotential ability to differentiate into neurons and astrocytes.Part two The differentiation of spinal cord stem cells into cholinergic neurons in vitroObjective: To seek the factors of inducing spinal cord stem cells to differentiated into cholinergic neurons in vitro. Methods: Spinal cord stem cells clone spheres were divided into 4 groups and induced by different differentiation medium. The control group only uses the base differentiation medium. All groups were incubated in 95% air/5% CO2 humidified atmosphere at 37℃for 14 d. The cholinergic neurons were detected by using immunofluorescence of the choline acetyltransferase (ChAT), which is the distinctive marker for cholinergic neuron. To determine the number of cells expressing a particular antigen, 15 fields per sample were examined and totaled. Results are expressed as percentages for the numbers of positive cells for ChAT compared to total numbers of progeny of ESCSCs determined by counting Hoechst 33342 stained nuclei. Statistical analyses were carried out...