Method of nociceptor differentiation of human embryonic stem cells and uses thereof

Abstract

The present invention relates to the field of stem cell biology, in particular the linage specific differentiation of pluripotent or multipotent stem cells, which can include, but is not limited to, human embryonic stem cells (hESC), human induced pluripotent stem cells (hiPSC), somatic stem cells, cancer stem cells, or any other cell capable of lineage specific differentiation. Specifically described are methods to direct the lineage specific differentiation of hESC and/or hiPSC to nociceptors (i.e. nociceptor cells) using novel culture conditions. The nociceptors made using the methods of the present invention are further contemplated for various uses including, but limited to, use in in vitro drug discovery assays, pain research, and as a therapeutic to reverse disease of, or damage to, the peripheral nervous system (PNS). Further, compositions and methods are provided for producing melanocytes from human pluripotent stem cells for use in disease modeling.

1 . A kit comprising:
 a first inhibitor that is capable of lowering transforming growth factor beta (TGFβ)/Activin-Nodal signaling;   a second inhibitor that is capable of lowering Small Mothers Against Decapentaplegic (SMAD) signaling; and   a third inhibitor that is capable of lowering glycogen synthase kinase 3β (GSK3β) for activation of wingless (Wnt) signaling.   
     
     
         2 . The kit of  claim 1 , further comprising instructions for inducing in vitro directed differentiation of a stem cell into a differentiated neural crest lineage cell, a neural lineage cell, or a neuronal lineage cell, wherein the instructions comprise contacting the cell with the first, second and third inhibitors, wherein the initial contact of the cell with and the third inhibitor is no later than 4 days from the initial contact of the stem cell with the first inhibitor. 
     
     
         3 . The kit of  claim 1 , further comprising an inhibitor that is capable of lowering fibroblast growth factor (FGF) receptor family signaling. 
     
     
         4 . The kit of  claim 1 , further comprising an inhibitor that is capable of lowering Notch signaling. 
     
     
         5 . The kit of  claim 1 , further comprising a Bone Morphogenetic Protein (BMP) molecule. 
     
     
         6 . The kit of  claim 1 , further comprising an Endothelin (EDN) molecule. 
     
     
         7 . A method for inducing in vitro directed differentiation of a stem cell to a neural crest lineage cell, a neural lineage cell, or a neuronal lineage cell, comprising contacting the stem cell with a first inhibitor that is capable of lowering TGFβ/Activin-Nodal signaling, a second inhibitor that that is capable of lowering SMAD signaling, and a third inhibitor that is capable of lowering GSK3β for activation of Wnt signaling, wherein the contact of the cell with the third inhibitor is for at least 11 days. 
     
     
         8 . The method of  claim 7 , comprising initially contacting the cell with the third inhibitor no later than 4 days from the initial contact of the stem cell with the first inhibitor. 
     
     
         9 . The method of  claim 7 , further comprising contacting the cell with an inhibitor that is capable of lowering FGF receptor family signaling. 
     
     
         10 . The method of  claim 7 , further comprising contacting the cell with an inhibitor that is capable of lowering Notch signaling. 
     
     
         11 . The method of  claim 7 , further comprising contacting the cell with a BMP molecule. 
     
     
         12 . The method of  claim 7 , further comprising contacting the cell with an EDN. 
     
     
         13 . The method of  claim 7 , wherein the stem cell is a pluripotent stem cell. 
     
     
         14 . A method of screening a biological agent in vitro, comprising:
 a) contacting a nociceptor cell with a test compound, wherein the nociceptor cell is obtained by the method of  claim 7 ; and   b) measuring nociceptor function, which is a measurement of an action potential.   
     
     
         15 . A cell population comprising in vitro differentiated cells obtained by the method of  claim 7 . 
     
     
         16 . A composition comprising the cell population of  claim 15 . 
     
     
         17 . A cell population comprising in vitro differentiated cells, wherein at least about 10% of the cells express at least one marker, wherein the at least one marker is selected from the group consisting of BRN3A (POU4F1), ISL1, NEUROG2, NEUROG1, NTRK1, RET, RUNX1, VGLUT2, TAC1, TRPV1, and SLC15A3. 
     
     
         18 . (canceled) 
     
     
         19 . A cell population comprising in vitro differentiated cells, wherein at least about 10% of the cells express at least one marker, wherein the at least one marker is selected from the group consisting of SOX10, HMB45, c-kit, melanocyte transcription factor (MITF-M), tyrosinase (TYR), tyrosinase related protein 1 (TRP1), and dopachrome-tautomerase (DCT) (tyrosinase related protein 2). 
     
     
         20 . A composition comprising the cell population of  claim 19 . 
     
     
         21 . A composition comprising a cell population and at least one compound selected from the group consisting of inhibitors of SMAD signaling, activators of Wnt signaling, and combinations thereof, wherein the cell population comprises in vitro differentiated neural crest intermediate cells expressing SOX10.