Human late stage motor neuron progenitor cells produced from pluripotent stem cells
AssigneeCalifornia Stem Cell, Inc.
InventorAleksandra Jovanovic Poole
Life of this patent
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Abstract
Motor neuron progenitor (MNP) cells and populations of MNP cells, are provided, in particular, populations of human late stage MNP cells having a purity of greater than about 65% late stage MNP cells and high-purity populations of MNP cells having greater than 95% viable cells, as well as method of making and using the same, including deriving late stage MNP cells from pluripotent embryonic stem cells, producing high-purity populations of late stage MNP cells, producing populations of viable MNP cells, transporting viable MNP cells, and transplanting MNP cells.
Claims16
A method of generating a high-purity population of late stage motor neuron progenitor cells, wherein the population has a purity of greater than about 65% late stage motor neuron progenitor cells, comprising:
- (a) neuralizing a culture of pluripotent stem cells by culturing pluripotent stem cells in a growth media that contains beta fibroblast growth factor (bFGF) and that lacks bone morphogenic protein (BMP), and that is supplemented with one or more of albumin, a fatty acid, a thyroid hormone, a vitamin, a trace mineral, insulin and transferrin;
- (b) ventralizing the neutralized cell population of step (a) comprising culturing the cell population under non-adherent conditions in a motor neuron media supplemented with retinoic acid and beta fibroblast growth factor (bFGF) for at least about five days,
- (c) incubating the ventralized cell population of step (b) comprising culturing the cell population in growth media with bFGF without retinoic acid to promote formation of neurospheres,
- (d) selecting the neurospheres;
- (e) expanding neurospheres on an adherent substrate to form a layer of cells comprising a population of cells comprising greater than about 65% late stage motor neuron progenitor cells, wherein said late stage motor neuron cells express markers of the motor neuron HB9, Ach, Isl1, Lhx3, and Lim1, but lack a functional calcium channel.
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