Method for inducing cholinergic neurons from pluripotent stem cells
A technology of cholinergic neurons and pluripotent stem cells, applied in the biological field, can solve problems such as unsatisfactory clinical treatment and lack of
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Embodiment 1
[0167] Example 1. Establishment of pluripotent stem cells and iPSC-specific system for inducing cholinergic neurons
[0168] The high-efficiency Shh / BMP9 dual-factor mouse embryonic stem cells (ESC, also called ES) (or iPSC, also called iPS) established by the present invention are induced to differentiate into cholinergic neurons in a serum-free culture system, see figure 1a. The two important protein factors used in this induction method are Sonic hedgehog (Shh) and bone morphogenetic protein 9 (Bone Morphogenetic Proteins 9, BMP9). Shh is a protein factor secreted by the notochord located on the ventral side of the neural tube, and runs through the ventral side of the neural tube, forming a concentration gradient that gradually decreases from the ventral side to the dorsal side. Shh is the most critical factor in inducing various ventral neurons, and the neuroepithelium affected by different Shh concentrations will form different neurons, among which the cholinergic neuron...
Embodiment 2
[0178] Example 2. The cholinergic neurons obtained by the induction system are basal forebrain cholinergic neurons
[0179] It has been reported in the literature that cholinergic neurons are mainly distributed in the basal forebrain and spinal cord in the mammalian central nervous system, and the cholinergic neurons in the spinal cord are motor neurons. In order to detect whether the induced cholinergic neurons are basal forebrain cholinergic neurons or spinal motor neurons, Isl1 (basal forebrain cholinergic neurons and spinal cord motor neurons) were performed on the induced cholinergic neurons. Neuron Marker) and HB9 (motor neuron Marker) double staining.
[0180] The expression patterns of Isl1 and HB9 in vivo were verified by immunohistostaining. For staining results in 12.5 day mouse embryos see figure 2 A, Isl1 is expressed in both the basal forebrain and the spinal cord, while HB9 is only specifically expressed in the spinal cord, proving that the double fluorescent...
Embodiment 3
[0186] Example 3: The cholinergic neurons obtained by the induction system of the present invention can form functional synapses in vitro
[0187] In order to verify that the forebrain basal cholinergic neurons induced by the inventors are functional in vitro, the inventors did the following series of tests:
[0188] Utilize conventional ELISA analysis method, detect the content of acetylcholine and the activity of acetylcholinesterase in the cells of mouse embryonic stem cells differentiated to basal forebrain cholinergic neurons for 16 days, the results are as follows image 3 A, Both Shh / BMP9 and BMP9 were found to significantly increase the content of acetylcholine and the activity of acetylcholinesterase in cells, proving that the induced cholinergic neurons are similar to the mature cholinergic neurons in terms of acetylcholine synthesis and metabolism .
[0189] Whole-cell recordings were used to examine the electrophysiological properties of mouse embryonic stem cells d...
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