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.

What is claimed is: 
     
         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;   a third inhibitor that is capable of lowering glycogen synthase kinase 3β (GSK3β) for activation of wingless (Wnt) signaling, and   instructions for inducing directed differentiation of a pluripotent stem cell into a differentiated neural crest lineage cell or a neuronal lineage cell in vitro, wherein said instructions comprise contacting said stem cell with said first and second inhibitors, and contacting said third inhibitor no later than 4 days from initial contact of said stem cell with said first and second inhibitors.   
     
     
         2 . The kit of  claim 1 , wherein said instructions comprise contacting said stem cell with said third inhibitor between 1 and 4 days from the initial contact of said stem cell with said first and second inhibitors. 
     
     
         3 . The kit of  claim 2 , wherein said instructions comprise contacting said stem cell with said third inhibitor 2 days from the initial contact of said stem cell with said first and second inhibitors. 
     
     
         4 . The kit of  claim 1 , further comprising a fourth inhibitor that is capable of lowering fibroblast growth factor (FGF) receptor family signaling. 
     
     
         5 . The kit of  claim 4 , wherein said FGF receptor family signaling comprises vascular endothelial growth factor (VEGF) receptor signaling, FGF receptor signaling, and platelet-derived growth factor (PDGF) tyrosine kinase receptor signaling. 
     
     
         6 . The kit of  claim 4 , further comprising a fifth inhibitor that is capable of lowering Notch signaling. 
     
     
         7 . The kit of  claim 6 , wherein said instructions comprise contacting said stem cell with said first and second inhibitors from day 0 through day 5, and contacting said stem cell with said third, fourth and fifth inhibitors from day 2 through day 10. 
     
     
         8 . The kit of  claim 1 , wherein said first inhibitor is SB431542. 
     
     
         9 . The kit of  claim 1 , wherein said second inhibitor is LDN193189. 
     
     
         10 . The kit of  claim 1 , wherein said third inhibitor is CHIR99021. 
     
     
         11 . The kit of  claim 4 , wherein said fourth inhibitor is SU5402 or PD173074. 
     
     
         12 . The kit of  claim 11 , wherein said fourth inhibitor is SU5402. 
     
     
         13 . The kit of  claim 6 , wherein said fifth inhibitor is DAPT or DAP-BpB (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-(S)-phenylglycine-4-(4-(8-biotinamido)octylamino)benzoyl)benzyl)methylamide). 
     
     
         14 . The kit of  claim 13 , wherein the fifth inhibitor is DAPT. 
     
     
         15 . The kit of  claim 1 , further comprising, one or more antibody used for the detection of expression of one or more protein(s) selected from the group consisting of nestin, OCT4, PAX6, TUJ1, SOX10, NTRK1, ISL1, POU4F1 (BRN3A), NEUROG2, NEUROG1, MAP2, OTX2, DLK1, DKK1, CUZD1, MSX1, ID2, AP2B, ETS1, FOXD3, NGN1, DCX, TUBB3, SYT4, STMN2, INA, GAP43, TAC1, VGLUT2, SLC15A3, and TRPV1. 
     
     
         16 . The kit of  claim 1 , further comprising, one or more pair of PCR primers for the detection of mRNA expression of one or more gene(s) selected from the group consisting of nestin, OCT4, PAX6, TUJ1, SOX10, NTRK1, ISL1, POU4F1 (BRN3A), NEUROG2, NEUROG1, MAP2, OTX2, DLK1, DKK1, CUZD1, MSX1, ID2, AP2B, ETS1, FOXD3, NGN1, DCX, TUBB3, SYT4, STMN2, INA, GAP43, TAC1, VGLUT2, SLC15A3, and TRPV1. 
     
     
         17 . The kit of  claim 1 , further comprising, one or more antibody used for the detection of expression of one or more protein(s) selected from the group consisting of Protachykinin-1 (TAC1), vesicular glutamate transporter 2 (VGLUT2) and solute carrier family 15, member 3 (SLC15A3). 
     
     
         18 . The kit of  claim 1 , further comprising, one or more pair of PCR primers for the detection of mRNA expression of one or more gene(s) selected from the group consisting of TAC1, VGLUT2 and SLC15A3. 
     
     
         19 . The kit of  claim 1 , wherein said instructions further comprise steps for making neural stem cell precursors and making nociceptor cells. 
     
     
         20 . The kit of  claim 1 , wherein said pluripotent stem cell is a human pluripotent stem cell. 
     
     
         21 . The kit of  claim 20 , wherein said human pluripotent stem cell is a human embryonic stem cell. 
     
     
         22 . The kit of  claim 20 , wherein said human pluripotent stem cell is a human induced pluripotent stem cell. 
     
     
         23 . The kit of  claim 20 , wherein said human pluripotent stem cell is a transgenic human pluripotent stem cell (hPSC) containing a bacterial artificial chromosome (BAC) comprising a SOX10 gene having an inserted reporter sequence encoding a green fluorescent protein. 
     
     
         24 . A method for inducing directed differentiation of a pluripotent stem cell, comprising
 a) providing, in vitro:
 a cell culture comprising human pluripotent stem cells; 
 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 wingless (Wnt) signaling; and 
   b) contacting said stem cell with said first and second inhibitors; and   c) contacting said stem cell with said third inhibitor no later than 4 days from initial contact of said stem cell with the first and second inhibitors, wherein a differentiated neural crest lineage cell or a neuronal lineage cell is produced.   
     
     
         25 . The method of  claim 24 , comprising contacting said stem cell with said third inhibitor between 1 and 4 days from the initial contact of said stem cell with said first and second inhibitors. 
     
     
         26 . The method of  claim 25 , comprising contacting said stem cell with said third inhibitor 2 days from the initial contact of said stem cell with said first and second inhibitors. 
     
     
         27 . The method of  claim 24 , further comprising providing a fourth inhibitor that is capable of lowering FGF receptor family signaling. 
     
     
         28 . The method of  claim 27 , wherein said FGF receptor family signaling comprises VEGF receptor signaling, FGF receptor signaling, and PDGF tyrosine kinase receptor signaling. 
     
     
         29 . The method of  claim 24 , further comprising providing a fifth inhibitor that is capable of lowering Notch signaling. 
     
     
         30 . The method of  claim 29 , comprising contacting said stem cell with said first and second inhibitors from day 0 through day 5, and contacting said stem cell with said third, fourth and fifth inhibitors from day 2 through day 10. 
     
     
         31 . The method of  claim 24 , wherein said first inhibitor is SB431542. 
     
     
         32 . The method of  claim 24 , wherein said second inhibitor is LDN193189. 
     
     
         33 . The method of  claim 24 , wherein said third inhibitor is CHIR99021. 
     
     
         34 . The method of  claim 27 , wherein said fourth inhibitor is SU5402 or PD173074. 
     
     
         35 . The method of  claim 34 , wherein said fourth inhibitor is SU5402. 
     
     
         36 . The method of  claim 29 , wherein said fifth inhibitor is DAPT or DAP-BpB (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-(S)-phenylglycine-4-(4-(8-biotinamido)octylamino)benzoyl)benzyl)methylamide). 
     
     
         37 . The method of  claim 36 , wherein the fifth inhibitor is DAPT. 
     
     
         38 . The method of  claim 24 , wherein a neural crest lineage cell is produced. 
     
     
         39 . The method of  claim 38 , wherein the neural crest lineage cell is differentiated into a peptidergic nociceptor cell. 
     
     
         40 . The method of  claim 39 , wherein said peptidergic nociceptor cell expresses a marker selected from the group consisting of OCT4, DLK1, PAX6, SOX10, POU4F1 (BRN3A), ISL1, NEUROG2, NEUROG1, NTRK1, RET, RUNX1, VGLUT2, TAC1, and TRPV1. 
     
     
         41 . The method of  claim 39 , wherein said peptidergic nociceptor cell expresses a marker selected from the group consisting of ISL1, POU4F1 (BRN3A), RET, RUNX1, and NTRK1. 
     
     
         42 . The method of  claim 39 , wherein said peptidergic nociceptor cell co-expresses Substance P and Calcitonin gene related peptide (CGRP). 
     
     
         43 . The method of  claim 39 , wherein said peptidergic nociceptor cell produces an action potential in response to external stimuli, wherein said external stimuli is an electrical current. 
     
     
         44 . The method of  claim 39 , wherein said peptidergic nociceptor cell is present within a highly enriched populations of neurons within 10-15 days after contacting said stem cell with said first and said second inhibitors. 
     
     
         45 . The method of  claim 24 , wherein said pluripotent stem cell is a human pluripotent stem cell. 
     
     
         46 . The method of  claim 45 , wherein said human pluripotent stem cell is a human embryonic stem cell. 
     
     
         47 . The method of  claim 45 , wherein said human pluripotent stem cell is a human induced pluripotent stem cell. 
     
     
         48 . A method of screening a biological agent in vitro, comprising:
 a) providing a nociceptor cell derived in vitro from directed differentiation of a pluripotent stem cell obtained from a method comprising:
 i) providing, in vitro,
 a cell culture comprising human pluripotent stem cells; 
 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; and 
 
 ii) contacting said pluripotent stem cell with said first and second inhibitors; 
 iii) contacting said third inhibitor no later than 4 days from initial contact of said stem cell with the first and second inhibitors; and 
   b) providing a test compound; and   c) contacting said nociceptor cell with said test compound and measuring nociceptor function, wherein said function is a measurement of an action potential.