Methods for drug discovery using stem cell-derived schwann cells

Abstract

The presently disclosed subject matter relates to use of Schwann cell precursors and Schwann cells derived from stem cells (e.g., human stem cells) for drug discovery in regeneration of peripheral nervous system (PNS) and/or central nervous system (CNS), prevention and/or repair myelin damages, and/or prevention and/or treatment of Schwann cell-related disorder (e.g., peripheral neuropathy diabetic peripheral neuropathy).

What is claimed is: 
     
         1 . An in vitro method of screening a compound that is suitable for regeneration of peripheral nervous system (PNS), for regeneration of central nervous system (CNS), for prevention and/or repair of myelin damage, and/or for preventing and/or treating a Schwann cell related disorder, comprising:
 (a) exposing a population of cells expressing one or more Schwann cell precursor marker to a glucose concentration of at least about 30 mM, wherein said cells are obtained from in vitro differentiation of stem cells,   (b) contacting said cells with a test compound after glucose exposure,   (c) measuring one or more of first sorbitol level, first glucose level, and first cell viability of said cells without treatment of said test compound;   (d) measuring one or more of second sorbitol level, second glucose level, and second cell viability of said cells treated with said test compound;   (e) comparing one or more of the followings:
 (i) said second sorbitol level with said first sorbitol level, 
 (ii) said second glucose level with said first glucose level, 
 (iii) said second cell viability with said first cell viability; and 
   (f) identifying a test compound that is suitable for regeneration of PNS and/or CNS, for prevention and/or repair of myelin damage, and/or for preventing and/or treating of a Schwann cell related disorder where one or more of the followings is present:
 (i) said second sorbitol level is lower than said first sorbitol level, 
 (ii) said second glucose level is lower than said first glucose level, and 
 (iii) said second cell viability is lower than first cell viability. 
   
     
     
         2 . An in vitro method of screening a compound that is suitable for regeneration of peripheral nervous system (PNS), for regeneration of central nervous system (CNS), for prevention and/or repair of myelin damage, and/or for preventing and/or treating a Schwann cell related disorder, comprising:
 (a) exposing a population of cells expressing one or more Schwann cell marker to a glucose concentration of at least about 30 mM, wherein said cells are obtained from in vitro differentiation of stem cells,   (b) contacting said cells with a test compound after glucose exposure,   (c) measuring one or more of first sorbitol level, first glucose level, and first cell viability of said cells without treatment of said test compound;   (d) measuring one or more of second sorbitol level, second glucose level, and second cell viability of said cells treated with said test compound;   (e) comparing one or more of the followings:
 (i) said second sorbitol level with said first sorbitol level, 
 (ii) said second glucose level with said first glucose level, 
 (iii) said second cell viability with said first cell viability; and 
   (f) identifying a test compound that is suitable for regeneration of PNS and/or CNS, for prevention and/or repair of myelin damage, and/or for prevention and/or treatment of a Schwann cell related disorder where one or more of the followings is present:
 (i) said second sorbitol level is lower than said first sorbitol level, 
 (ii) said second glucose level is lower than said first glucose level, and 
 (iii) said second cell viability is lower than first cell viability. 
   
     
     
         3 . The method of  claim 1 , wherein said cells expressing one or more Schwann cell precursor marker are obtained from in vitro differentiation of stem cells by a method comprising: contacting a population of stem cells with one or more inhibitor of TGFβ/Activin-Nodal signaling and contacting said cells with one or more Wnt activator, and further contacting said cells with one or more FGF activator for at least about 3 days. 
     
     
         4 . The method of  claim 2 , wherein said cells expressing one or more Schwann cell marker are obtained from in vitro differentiation of stem cells by a method comprising:
 contacting a population of stem cells with one or more inhibitor of TGFβ/Activin-Nodal signaling and contacting said cells with one or more Wnt activator,   further contacting said cells with one or more FGF activator for at least about 3 days to produce the population of differentiated cells expressing one or more Schwann cell precursor marker, and   subjecting said population of differentiated cells expressing one or more Schwann cell precursor marker to conditions favoring maturation of Schwann cells precursor cells into Schwann cells.   
     
     
         5 . The method of  claim 3 , wherein said method for differentiating the stem cells comprises (a) contacting said cells with said one or more FGF activator for about 14 days; and/or (b) initially contacting said cells with said one or more FGF activator no later than about 20 days, or between about 10 days and about 15 days, about 11 days from the initial contact of said stem cells with said one or more inhibitor of TGFβ/Activin-Nodal signaling. 
     
     
         6 . The method of  claim 3 , further comprising contacting said cells with one or more SC differentiation inducer to produce a population of cells expressing one or more SC precursor marker, optionally wherein said at least one SC differentiation inducer is selected from the group consisting of neuregulins, LIF, CNTF, Forskolin, TGFβ and FBS, optionally wherein said one or more SC differentiation inducer comprises NRG1. 
     
     
         7 . The method of  claim 6 , wherein said method for differentiating the stem cells comprises (a) contacting said cells with said one or more SC differentiation inducer for at least about 3 days to produce a population of differentiated cells that express one or more Schwann cell precursor marker, optionally contacting said cells with said one or more SC differentiation inducer for about 14 days;
 and/or (b) initially contacting said cells with said one or more SC differentiation inducer between about 10 days and about 15 days from the initial contact of said stem cells with said one or more one or more inhibitor of TGFβ/Activin-Nodal signaling.   
     
     
         8 . The method of  claim 3 , wherein said population of stem cells are differentiated into a population of differentiated cells that express one or more said Schwann cell precursor marker on or after about 25 days from the initial contact of said stem cells with said one or more inhibitor of TGFβ/Activin-Nodal signaling. 
     
     
         9 . The method of  claim 3 , wherein said method for differentiating the stem cells further comprises contacting said stem cells with one or more SMAD inhibitor, optionally wherein said one or more SMAD inhibitor is a small molecule selected from the group consisting of LDN193189, derivatives thereof, and mixtures thereof, optionally wherein said one or more SMAD inhibitor comprises a LDN193189. 
     
     
         10 . The method of  claim 3 , wherein said method for differentiating the stem cells comprises initially contacting said cells with said one or more Wnt activator no later than about 4 days from the initial contact of said stem cells with said one or more inhibitor of TGFβ/Activin-Nodal signaling, optionally initially contacting said cells with said one or more activator of Wnt signaling about 2 days from the initial contact of said stem cells with said one or more inhibitor of TGFβ/Activin-Nodal signaling. 
     
     
         11 . The method of  claim 3 , wherein
 (a) said one or more inhibitor of TGFβ/Activin-Nodal signaling is a small molecule selected from the group consisting of SB431542, derivatives thereof, and mixtures thereof, optionally wherein said one or more inhibitor of TGFβ/Activin-Nodal signaling comprises SB431542; and/or   (b); said one or more Wnt activator lowers glycogen synthase kinase 3β (GSK3β) for activation of Wnt signaling, optionally wherein said one or more Wnt activator is a small molecule selected from the group consisting of CHIR99021, Wnt-1, WNT3A, Wnt4, Wnt5a, derivatives thereof, and mixtures thereof, optionally wherein said one or more Wnt activator comprises CHIR99021; and/or   (c) said one or more FGF activator is selected from the group consisting of FGF1, FGF2, FGF3, FGF4, FGF7, FGF8, FGF10, FGF18, derivatives thereof, and mixtures thereof, optionally wherein said one or more FGF activator comprises FGF2.   
     
     
         12 . The method of  claim 1 , wherein said one or more Schwann cell precursor marker is selected from the group consisting of SOX10, GAP43, BLBP, MPZ, Dhh, P75NTR, CD49D, TFAP2, CDH19, CD44, ERBB3, POU3F1, GFAP, CALCB, GRP116, TSPYL5, ITPKA, SLC17A6, SYPL2, LOC100128252, ANGPTL7, LOC728978, ZNF502, SLC16A6, LPL, SLC30A2, and SLC10A4. 
     
     
         13 . The method of  claim 3 , wherein said stem cells are human stem cells, optionally wherein said human stem cells are selected from the group consisting of human embryonic stem cells, human induced pluripotent stem cells, human parthenogenetic stem cells, primordial germ cell-like pluripotent stem cells, epiblast stem cells, F-class pluripotent stem cells. 
     
     
         14 . The method of  claim 4 , wherein said conditions favoring maturation of Schwann cell precursor cells into Schwann cells comprise: contacting said cells with one or more FGF activator, and one or more Schwann cell differentiation inducer. 
     
     
         15 . The method of  claim 14 , wherein said conditions favoring maturation of Schwann cell precursor cells into Schwann cells comprise contacting said cells with the one or more FGF activator and the one or more Schwann cell differentiation inducer for at least about 3 days, optionally for about 10 days or for about 35 days. 
     
     
         16 . The method of  claim 14 , wherein said conditions favoring maturation of Schwann cell precursor cells into Schwann cells comprise further contacting the cells with one or more SC differentiation enhancer. 
     
     
         17 . The method of  claim 16 , wherein said conditions favoring maturation of Schwann cell precursor cells into Schwann cells comprise contacting the cells with the one or more SC differentiation enhancer for at least about 3 days, optionally for about 10 days or for about 35 days. 
     
     
         18 . The method of  claim 16 , wherein said one or more SC differentiation enhancer is selected from the group consisting of neuregulins, cyclic adenosine monophosphate (cAMP), Forskolin, LIF, and CNTF, optionally wherein said one or more SC differentiation enhancer comprises cAMP. 
     
     
         19 . The method of  claim 4 , wherein said conditions favoring maturation of Schwann cell precursor cells into Schwann cells comprise aggregating said cells into 3D spheroids; and contacting said 3D spheroids with said one or more FGF activator, and said one or more Schwann cell differentiation inducer, optionally wherein said conditions favoring maturation of Schwann cell precursor cells into Schwann cells further comprise culturing said 3D spheroids in adherent culture. 
     
     
         20 . The method of  claim 4 , wherein said one or more Schwann cell marker is selected from the group consisting LRRTM4, CDH1, FABP7, BDNF, UNCB5, SOSTDC1, OLIG1, PLAT, KCNJ10, SHH, NTN1, GDNF, ERBB3, GAP43, SOX10, 5100, GFAP, POU3F1, PMP22, MBP, AQP4, MPZ, NGFR, NFATC4, MOG, IFNG, MAL, NTF3, TGFB1, CD9, CD81, CD44, CD98, CD49E, CD49D, TYRP1, ENTHD1, NTSE, HTR2B, NOV, IL8, SLC16A6, CDKN2A, PLP2, S100A6, AQP9, and CDH19. 
     
     
         21 . The method of  claim 1 , wherein said glucose concentration is at least about 10 mM. 
     
     
         22 . The method of  claim 21 , wherein said glucose concentration is about 30 mM. 
     
     
         23 . The method of  claim 1 , wherein said measurement is performed at least about 12 hours from the initial exposure of said cells to said glucose. 
     
     
         24 . The method of  claim 23 , wherein said measurement is performed at about 72 hours from the initial exposure of said cells to said glucose. 
     
     
         25 . The method of  claim 1 , wherein the Schwann cell related disorder is selected from the group consisting of peripheral neuropathy, Schwannomatosis, Charcot-Marie-Tooth Disease, and Guillain-Barre Syndrome. 
     
     
         26 . The method of  claim 25 , wherein the Schwann cell related disorder is a peripheral neuropathy. 
     
     
         27 . The method of  claim 26 , wherein the peripheral neuropathy disorder is a diabetic peripheral neuropathy. 
     
     
         28 . A method of regeneration of peripheral nervous system (PNS), for regeneration of central nervous system (CNS), for prevention and/or repair of myelin damage, and/or for preventing and/or treating a Schwann cell related disorder in a subject, comprising administering an effective amount of a compound or a composition comprising thereof to the subject suffering from the Schwann cell related disorder, wherein the compound is selected from the group consisting of potassium channel blockers, norepinephrine-dopamine reuptake inhibitors, cyclopenthiazide, captopril, isradipine, condelphine, nimesulide, triamcinolone, salts thereof, solvates thereof, hydrate thereof, clathrates thereof, and prodrugs thereof, and combinations thereof.