A method of producing a
population of mature human pluripotent
stem cell-derived sensory neurons (hPSC-SNs) expressing a
target gene associated with at least one of nociceptive pain,
chronic pain, pruriception and a nociceptive- or pruriceptive- mediated condition, the method comprising introducing into a
population of human pluripotent stem cells (hPSCs) a composition comprising at least one site-directed
nuclease targeting
a site within the
target gene, and at least one
nucleic acid comprising a
nucleotide sequence encoding at least one screenable,
selectable marker that is flanked by (i) a
nucleotide sequence homologous with a region located upstream of the
target site within the
target gene and (ii) a
nucleotide sequence homologous with a region located downstream of the
target site within the target
gene, wherein the
target site is located downstream of the
open reading frame of the target
gene, and wherein the site-directed
nuclease cleaves the target site of the target
gene and the
nucleic acid encoding the screenable,
selectable marker is inserted at the target site; covering said
population of hPSCs under an
extracellular matrix comprising at least one
neuronal differentiation driver to produce a sensory committed
neural crest population; contacting said sensory committed
neural crest population with at least one
neuronal differentiation driver and at least one neurotrophic factor to produce a population of early sensory neurons (SNs); and contacting said population of early SNs with at least one neurotrophic factor to produce a population of mature hPSC-SNs expressing at least one of SN marker or one pan neuronal marker; isolating the cells expressing the at least one screenable,
selectable marker, wherein said population of mature hPSC-SNs expressed said one target gene associated with nociceptive pain,
chronic pain, pruriception a nociceptive- or pruriceptive- mediated condition, and wherein said population of mature hPSC-SNs responds to a nociceptive and pruriceptive stimulus.