Enhancing neuronal differentiation of ventral midbrain neural progenitor cells
OwnerDjordje Djordjevic, Jonathan Niclis +1inventors
InventorsDjordje Djordjevic, Jonathan Niclis, Josefine Raagaard Christiansen
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Abstract
The present invention relates to a method for directing differentiation of ventral midbrain NSCs into neurons comprising contacting a cell population comprising ventral midbrain NSCs with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling, wherein the ventral midbrain NSCs co-express the markers FOXA2, LMX1A, EN1, OTX2, 5 and SOX2.
Claims15
An in vitro method for directing differentiation of a cell population of PSCs into ventral midbrain neurons, comprising culturing the cell population of PSCs, contacting the cell population of PSCs with an inhibitor of SMAD protein signaling, an inhibitor of Wnt signaling, an activator of SHH signaling, an activator of FGF signaling, optionally ascorbic acid, and, optionally BDNF, to obtain a cell population comprising ventral midbrain NSCs, wherein the cell population comprising ventral midbrain NSCs is further contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling to direct differentiation into ventral midbrain neurons, and wherein the cell population is allowed to differentiate into ventral midbrain NSCs for 14 to 24 days prior to contacting the cell population with the inhibitor of MEK signaling and the inhibitor of NOTCH signaling.
An in vitro method for directing differentiation of ventral midbrain NSCs into neurons comprising contacting a cell population comprising ventral midbrain NSCs with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling, wherein at least 5% of the cell population comprising ventral midbrain NSCs co-express the markers FOXA2, LMX1A, EN1, OTX2, and SOX2, and wherein the cell population is allowed to differentiate into ventral midbrain NSCs for 14 to 24 days prior to contacting the cell population with the inhibitor of MEK signaling and the inhibitor of NOTCH signaling.
An in vitro method for directing differentiation of ventral midbrain NSCs into neurons comprising contacting a cell population comprising ventral midbrain NSCs with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling, wherein 40-60% of the cell population comprising ventral midbrain NSCs express ASCL1, 45-65% of the cell population comprising ventral midbrain NSCs express KI67, 10-15% of the cell population comprising ventral midbrain NSCs express INA, 2-5% of the cell population comprising ventral midbrain NSCs are INA+/SOX2−, and 80-95% of the cell population comprising ventral midbrain NSCs express SOX2.
An in vitro cell population comprising ventral midbrain NSCs, wherein the cell population when cultured in vitro for 5 days in a culture medium suitable for maintaining neural cells results in a cell population wherein at least 50% are INA+ or HuCD+, and less than 15% are K167+.
A composition comprising a cell population comprising ventral midbrain NSCs, an inhibitor of MEK signaling, and an inhibitor of NOTCH signaling.
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